Ministero della Salute

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1 Ministero della Salute Direzione Generale della Ricerca e dell' Innovazione in Sanità ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO Cerebellar-cortical circuits in Autism Spectrum Disorders: new perspectives for treatment implementation. Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SAN GALLICANO TITOLO PROGETTO Can biological agents withdrawn in psoriatic arthritis (PsA) patients after a stable remission of arthritis is achieved? A clinical, radiological and ultrasonographic study. Area Expertise Musculoskeletal, Oral and Skin Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Associazione Oasi Maria SS TITOLO PROGETTO Novel Biomarkers for Improved Characterization, Disease Tracking and Outcome Prediction in Traumatic Brain Injury Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale HUMANITAS TITOLO PROGETTO Engineered regulatory T cell Adoptive Cell Therapy as a novel tool for the treatment of Cardiovascular diseases Area Expertise Cardiovascular and Respiratory Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

2 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale Calabria TITOLO PROGETTO New mtor inhibitors development and study of their effects on the central nervous system(mtorneu) Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Veneto TITOLO PROGETTO Longitudinal multi-parametric clinical, CSF and MRI study aimed at predicting disability progression in Multiple Sclerosis Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Emilia-Romagna TITOLO PROGETTO Diaphragmatic dysfunction in critically patients undergoing mechanical ventilation. Area Expertise Cardiovascular and Respiratory Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Innovative biotechnologies Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale NEUROMED TITOLO PROGETTO The role of authopagy as a protective mechanism in cardiovascular diseases: new insights into oxidative stress modulation and cardiovascular regeneration Area Expertise Cardiovascular and Respiratory Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

3 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale ISTITUTO TUMORI MILANO TITOLO PROGETTO Functional inhibition of HSP105 as a new therapeutic strategy for human aggressive B-cell non-hodgkin lymphomas Area Expertise TIPOLOGIA_RICERCA Oncology 1 - Basic Translational Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO Role of Lamin A/C-Polycomb crosstalk in Emery-Dreifuss Muscular Dystrophy (EDMD) Area Expertise Genes, Genomes and Genetics TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Piemonte TITOLO PROGETTO Prevention of cancer therapy-related heart failure through phosphoinositide 3-kinase gamma inhibition Area Expertise Cardiovascular and Respiratory Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Liguria TITOLO PROGETTO Estimating the risk for cardiac toxicity in patients undergoing anticancer therapies: a new dynamic risk algorithm integrating levels of cardiovascular serum biomarkers. Area Expertise Oncology 2 - Translational Clinical TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00

4 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale Toscana TITOLO PROGETTO Role of biological markers for cerebral bleeding risk stratification in patients with atrial fibrillation on oral anticoagulants for primary or secondary prevention of ischemic stroke Area Expertise Biology of Development and Aging TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO Deciphering the role of SETD5 in intellectual disabilities and setting a new gene-on therapeutic strategy. Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale CARLO BESTA TITOLO PROGETTO The risk-benefit profile of pharmacological treatments in secondary prevention for patients with ischemic stroke: a systematic review Area Expertise Healthcare Delivery and Methodologies TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO Comprehensive tracking of hematopoietic stem/progenitor cells dynamics and activity in vivo in humans by insertional barcoding Area Expertise Genes, Genomes and Genetics TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00

5 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale ISTITUTO TUMORI MILANO TITOLO PROGETTO Dissecting the role of unexplored mir-205 host gene as a basal cell-specific long non-coding RNA in prostate cancer development Area Expertise TIPOLOGIA_RICERCA Oncology 1 - Basic Translational Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale ISTITUTO TUMORI MILANO TITOLO PROGETTO Neutrophil traps in leukemia: from triggers of disease progression to vehicle for new vaccines Area Expertise TIPOLOGIA_RICERCA Oncology 1 - Basic Translational Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Puglia TITOLO PROGETTO Unravelling the pathogenetic pathways in an experimental model of Diabetic Encephalopathy by a omics-based platform: potential for novel diagnostic and therapeutic interventions Area Expertise Endocrinology, Metabolism, Nutrition and Reproductive Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale ISTITUTO DERMOPATICO DELL'IMMACOLATA TITOLO PROGETTO Targeting cutaneous anti-apoptotic PI3K/AKT signalling as a new therapeutic approach for treating skin inflammation Area Expertise Musculoskeletal, Oral and Skin Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00

6 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale CARLO BESTA TITOLO PROGETTO ULTRASENSITIVE DIAGNOSTIC TEST FOR DEGENERATIVE DEMENTIAS BASED ON AMPLIFICATION OF PERIPHERAL DISEASE-SPECIFIC BIOMARKERS FROM THE OLFACTORY MUCOSA Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale IZSPLV TITOLO PROGETTO Antimicrobial drug resistance in food-producing animals. Essential oils and supercritical fluid extraction: study the antimicrobial and immuno-stimulating activity in livestock for reducing the use of antibiotics and environmental pollution, improvement o Area Expertise Veterinary TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Food safety and animal safety Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Istituto Superiore di Sanita' TITOLO PROGETTO Inducible pluripotent stem (ips) cell-derived human astrocytes as a new disease model to shed light into the molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts (MLC). Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale I. O. V. TITOLO PROGETTO Setup of a multi-site radiation dose management system for the optimization of radiological procedures and the assessment of individual and collective radiation dose. Area Expertise Healthcare Delivery and Methodologies TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Finanziamento Assegnato euro Methodological, epidemiological, ,00 socio-economic, organizational, managerial emerging public health issues related to the above reported areas

7 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale I.E.O. TITOLO PROGETTO Prognostic and predictive values of tumor infiltrating lymphocytes in patients with ductal carcinoma in situ of the breast. Area Expertise Population Sciences and Epidemiology TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO A comprehensive preventive program for dementia tailored on the neuropsychological profile of persons with Mild Cognitive Impairment: cognitive stimulation, physical intervention and healthy nutrition, a randomized controlled trial. Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Molise TITOLO PROGETTO Economic crisis and adherence to the Mediterranean diet: possible impact on biomarkers of inflammation and metabolic phenotypes in the cohort of the MOLI-SANI study. Area Expertise Healthcare Delivery and Methodologies TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO Cardiac troponin after cardiac surgery Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

8 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale ISTITUTO DERMOPATICO DELL'IMMACOLATA TITOLO PROGETTO Ectodermal dysplasia syndromes by defective nectins (nectinopathies): molecular mechanisms and clinical implications Area Expertise Musculoskeletal, Oral and Skin Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Innovative biotechnologies Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SAN DONATO TITOLO PROGETTO Predicting post-surgery complications in patients undergoing coronary artery bypass graft through the assessment of perioperative cardiovascular control indices Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO Investigating insertional mutagenesis as a new mechanism of HIV-1 persistence in infected host Area Expertise AIDS and Related Research TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale HUMANITAS TITOLO PROGETTO CXCR4 mutants in Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) and Waldenstrom Macroglobulinemia (WM) pathogenesis: signalling properties and therapeutic opportunities Area Expertise Immunology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00

9 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale HUMANITAS TITOLO PROGETTO Natural killer cells and macrophages cross-talk in the pathogenesis of HIV-1 infection Area Expertise Immunology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale IRCCS AOU San Martino-IST (Genova) TITOLO PROGETTO Immunoregulatory role of innate immunity cells in the tumor microenvironment Area Expertise Immunology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO Role of muscle interstitial fibroadipogenic progenitors in the regulation of myofiber response to acute and pathological denervation Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Emilia-Romagna TITOLO PROGETTO DISCO TRIP - Disrupting Respiratory Complex I to Trigger Pseudonormoxia: an anticancer strategy Area Expertise TIPOLOGIA_RICERCA Oncology 1 - Basic Translational Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00

10 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale Lombardia TITOLO PROGETTO Optimization of extracorporeal carbon dioxide removal through blood acidification: development of new technologies Area Expertise Bioengineering Sciences and Technologies TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Innovative biotechnologies Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Friuli-Venezia Giulia TITOLO PROGETTO Molecular Nanodecoders for the Quantitative, Multiplexed, Layer-by-Layer Imaging of Disease-Associated Markers Area Expertise Bioengineering Sciences and Technologies TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Innovative biotechnologies Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Emilia-Romagna TITOLO PROGETTO BAP1-dependent Malignant Mesothelioma: mechanisms and early detection Area Expertise TIPOLOGIA_RICERCA Oncology 1 - Basic Translational Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale I. O. V. TITOLO PROGETTO Challenging the role of chemotherapy in HER2 positive early breast cancer: PI3KCA pathways alteration and response to neoadjuvant pertuzumab-trastuzumab plus letrozole or paclitaxel in Hormone Receptor positive-her2 positive breast cancer patients. Area Expertise Oncology 2 - Translational Clinical TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00

11 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale S.CAMILLO TITOLO PROGETTO Investigating the role of mitochondria-er interaction in CMT: new insights in the biology of peripheral neuropathy Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale S.CAMILLO TITOLO PROGETTO Mitochondrial DNA dynamics: Implications in neurodegenerative diseases Area Expertise Cell Biology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO CMR evaluation of myocardial inflammation persistence after acute myocarditis: prognostic relevance Area Expertise Cardiovascular and Respiratory Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Piemonte TITOLO PROGETTO Development and validation of a mathematical model for esteem of central blood pressure in normal subjects and in patients with proximal aorta dilatation Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

12 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale Emilia-Romagna TITOLO PROGETTO COMPOSITION OF GUT MICROBIOTA AND IMPACT ON IMMUNE SYSTEM RECOVERY AND ACUTE GRAFT VERSUS HOST DISEASE IN PEDIATRIC PATIENTS UNDERGOING HEMATOPOIETIC STEM CELL TRANSPLANTATION Area Expertise Oncology 2 - Translational Clinical TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Lazio TITOLO PROGETTO Brain Function Monitoring to Evaluate Efficacy and Safety of Palliative-Sedation-in-the- Last-Days-Therapy in Terminally Cancer Patients. Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale REGINA ELENA TITOLO PROGETTO Type I IFN-mediated induction of a chemoresistant niche of tumor cells: from animal models to humans Area Expertise TIPOLOGIA_RICERCA Oncology 1 - Basic Translational Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale BURLO GAROFALO TITOLO PROGETTO Novel therapeutic perspectives for angioedema associated with C1-inhibitor deficiency or angiotensin converting enzymeinhibitor treatment Area Expertise Vascular and Hematology TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

13 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO INDIVIDUAL PHENOTIPIC CHARACTERIZATION OF PATIENTS WITHIN THE FRONTOTEMPORAL LOBAR DEGENERATION/MOTOR NEURON DISEASE SPECTRUM USING MULTIMODAL MRI Area Expertise Biobehavioral and Behavioral Processes TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO "THE ROLE OF 124-IODINE/CG250 PET IMAGING TECHNIQUE IN DETECTING LYMPH NODE METASTASES IN RENAL CELL CARCINOMA PATIENTS" Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale RIZZOLI TITOLO PROGETTO Diagnostic accuracy and cost-effectiveness of Next Generation Sequencing (NGS) strategies in the genetic testing of Rare Orthopaedic Diseases Area Expertise Genes, Genomes and Genetics TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Finanziamento Assegnato euro Methodological, epidemiological, ,00 socio-economic, organizational, managerial emerging public health issues related to the above reported areas CODICE GR Destinario Istituzionale IRCCS AOU San Martino-IST (Genova) TITOLO PROGETTO The Stem cell secretome for doxorubicin-induced Cardiomyopathy REgeneraTion (SECRET) Area Expertise Cell Biology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

14 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale DON GNOCCHI TITOLO PROGETTO Upper limb impairment in Charcot-Marie-Tooth patients: evaluation of motor strategies and effectiveness of rehabilitation treatments by means of an innovative instrumental protocol. Area Expertise Musculoskeletal, Oral and Skin Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Istituto Scienze Neurologiche TITOLO PROGETTO Deep genetic and phenotypic characterization of Autism Spectrum Disorder (ASD) families: analysis of the nuclear and mitochondrial genome Area Expertise Genes, Genomes and Genetics TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale MAGGIORE TITOLO PROGETTO In vivo evaluation of the process of Epithelial-Mesenchymal and Mesenchymal-Epithelial transition (EMT-MET) in Hepatocellular Carcinoma (HCC) dissemination and metastasization. Area Expertise TIPOLOGIA_RICERCA Oncology 1 - Basic Translational Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Umbria TITOLO PROGETTO Biostatistical methods for meta-analyzing individual participant data in diagnostic and prognostic research, with application to the role of cerebrospinal fluid biomarkers in Parkinson's disease. Area Expertise Healthcare Delivery and Methodologies TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00

15 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale Toscana TITOLO PROGETTO RELATIONSHIP BETWEEN OSTEOPOROSIS AND CARDIOVASCULAR RISK IN PATIENTS WITH THALASSEMIA MAJOR Area Expertise Vascular and Hematology TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Sicilia TITOLO PROGETTO The addition of simvastatin portal venous infusion to cold storage solution of explanted whole liver grafts for facing ischemia/reperfusion injury in an area with low rate of deceased donation Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale CARLO BESTA TITOLO PROGETTO An Italian study on intermediate-lenght polyq-tract expansions: frequency, clinical variability, and brain morphometry of subjects at risk for late-onset neurodegenerative diseases. Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale BURLO GAROFALO TITOLO PROGETTO TRAIL, THE REGULATION OF BODY WEIGHT AND METABOLISM, AND ITS INTERACTION WITH THYROID HORMONES AND METFORMIN. Area Expertise Endocrinology, Metabolism, Nutrition and Reproductive Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

16 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale Emilia-Romagna TITOLO PROGETTO Endovascular therapy in acute ischemic stroke: an observational prospective study of an inter-provincial Hub and Spoke Model Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Finanziamento Assegnato euro Methodological, epidemiological, ,00 socio-economic, organizational, managerial emerging public health issues related to the above reported areas CODICE GR Destinario Istituzionale SAN RAFFAELE PISANA TITOLO PROGETTO Role of the adipocyte mineralocorticoid receptor in the development of sarcopenic obesity and comorbidities Area Expertise Endocrinology, Metabolism, Nutrition and Reproductive Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Istituto Scienze Neurologiche TITOLO PROGETTO Melanopsin retinal ganglion cells and circadian rhythms: function and dysfunction in Alzheimer's disease and aging. Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO Mapping spatial neglect symptoms in the brain: A study combining behavioural data, functional neuroimaging, and computational modelling Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00

17 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO Multimodal experimental and theoretical approach for the study of the Spinal Cord in healthy and diseased subjects Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale BURLO GAROFALO TITOLO PROGETTO Vitamin D in the prevention of subclinical and advanced atheromatosis. Area Expertise Vascular and Hematology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale I.E.O. TITOLO PROGETTO Immunogenic cell death as novel immune response mechanism to EGFR-targeted therapy in Colorectal Cancer Area Expertise Immunology TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Toscana TITOLO PROGETTO Peripheral blast clearance analysis during induction treatment in patients with acute myeloid leukemia Area Expertise Oncology 2 - Translational Clinical TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00

18 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale STELLA MARIS TITOLO PROGETTO The sense of number in Developmental Dyscalculia Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale RIZZOLI TITOLO PROGETTO Pain in knee osteoarthritis, a placebo-controlled randomized clinical trial for a new potential therapeutic approach: magnetic resonance guided focused ultrasound surgery (MRgFUS) Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SAN MATTEO TITOLO PROGETTO Identification and characterization of human broadly neutralizing antibodies against human coronaviruses and human parainfluenza viruses Area Expertise Infectious Diseases and Microbiology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SAN RAFFAELE PISANA TITOLO PROGETTO NEUROMASTER: NEUROnavigated MAgnetic STimulation in patients with mild-moderate Alzheimer disease combined with Effective cognitive Rehabilitation Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00

19 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale DON GNOCCHI TITOLO PROGETTO Neural manual vs. robotic assisted mobilization to improve motion and reduce pain hypersensitivity in hand osteoarthritis: a randomized controlled trial. Area Expertise Musculoskeletal, Oral and Skin Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Finanziamento Assegnato euro Methodological, epidemiological, ,00 socio-economic, organizational, managerial emerging public health issues related to the above reported areas CODICE GR Destinario Istituzionale CARLO BESTA TITOLO PROGETTO High-throughput mirnaome profiling in myasthenia gravis patients: towards the identification of pathogenic mechanisms underlying autoimmunity Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Toscana TITOLO PROGETTO Exploring the potentialities of lung ultrasound in pediatric cardiac surgery: an easy, fast, inexpensive, radiation-free, but still surprisingly underutilized diagnostic tool. Area Expertise Cardiovascular and Respiratory Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Lazio TITOLO PROGETTO Efficacy of lymphatico-venous anastomosis alone, autologous vascularized lymphnode transfer alone and combined lymphatico-venous anastomosis and autologous vascularized lymphnode transfer in the treatment of breast cancer-related lymphedema Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00

20 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale Calabria TITOLO PROGETTO RApid versus SLOw Withdrawal of antiepileptic monotherapy in two-year seizure-free adult patients with epilepsy (RASLOW Study): a pragmatic multicentre, prospective, randomized, controlled study. Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Friuli-Venezia Giulia TITOLO PROGETTO Cardiac preservation with normothermic donor heart perfusion compared to standard cold storage in heart transplantation: a new standard of care to face donor shortage? Area Expertise Cardiovascular and Respiratory Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale NEUROMED TITOLO PROGETTO Understanding neural mechanisms of Spatial Neglect by linking anatomical damage to resting state functional connectivity Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale I.E.O. TITOLO PROGETTO LOW ADIPONECTIN LEVELS AS INDIPENDENT RISK MARKER FOR BREAST CANCER IN HIGH RISK POSTMENOPAUSAL WOMEN Area Expertise Population Sciences and Epidemiology TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00

21 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO The neural markers of post-stroke recovery: tracking the early dynamics of cortical reorganization with a novel longitudinal multimodal approach. Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale IZSPB TITOLO PROGETTO DEVELOPMENT OF INNOVATIVE ANALYTICAL METHODS FOR MEAT PRODUCTS SAFETY AND QUALITY ASSURANCE Area Expertise Veterinary TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Food safety and animal safety Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Lazio TITOLO PROGETTO Innovative use of satellite data for exposure assessment to evaluate the health effects of extreme temperatures and air pollution in Rome Area Expertise Population Sciences and Epidemiology TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Finanziamento Assegnato euro Methodological, epidemiological, ,00 socio-economic, organizational, managerial emerging public health issues related to the above reported areas CODICE GR Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO Targeted genome editing of the CD40LG gene for the treatment of X-linked hyper-igm immunodeficiency Area Expertise Genes, Genomes and Genetics TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Innovative biotechnologies Finanziamento Assegnato euro ,00

22 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale NEUROLESI TITOLO PROGETTO Magnetic Resonance Spectroscopy as a marker of response to dopaminergic treatment in Parkinson's Disease Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale HUMANITAS TITOLO PROGETTO CD45RA-depleted donor lymphocyte infusions for the prevention of infections in patients affected by hematological malignancies after haploidentical stem cell transplantation and post-transplant cyclophosphamide Area Expertise Immunology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale OSPEDALE BAMBINO GESU' TITOLO PROGETTO Chimeric antigen receptor-redirected T cells targeting CD19 antigen: first-in-human Italian trial for children with chemotherapy-refractory Acute Lymphoblastic Leukemia. Area Expertise Oncology 2 - Translational Clinical TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Piemonte TITOLO PROGETTO The PRIORITY Study - PRedictIng long term Outcomes after Isolated coronary artery bypass surgery Area Expertise Healthcare Delivery and Methodologies TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

23 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale NEUROLESI TITOLO PROGETTO The predictive values of brain connectivity for the diagnosis of disorder of consciousness using a multimodal magnetic resonance imaging protocol Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO Targeting the lysosome to modulate mtorc1 function and treat obesity-associated metabolic disorders Area Expertise Endocrinology, Metabolism, Nutrition and Reproductive Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale SPALLANZANI TITOLO PROGETTO Autophagy impairment in patients with chronic HCV infection: impact on steatohepatitis and carcinogenesis. Area Expertise Infectious Diseases and Microbiology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00 CODICE GR Destinario Istituzionale Veneto TITOLO PROGETTO A new strategy for the detection of residual leukemia cells Area Expertise Oncology 2 - Translational Clinical TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00

24 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Giovani Ricercatori INDICE PROGETTI CODICE GR Destinario Istituzionale I.E.O. TITOLO PROGETTO Role of Thymic stromal lymphopoietin on immune cell polarisation and cancer development Area Expertise Immunology TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 SEGUONO ABSTRACT PROGETTI FINANZIATI

25 GR Cerebellar-cortical circuits in Autism Spectrum Disorders: new perspectives for treatment implementation. Clausi Silvia Clinical health care research/clinicoassistenziale Fondazione Santa Lucia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Cellular, anatomical, and systems-based studies of changes in the neural substrate and function of brain in response to disease Cerebellum, Autism spectrum disorders Neuroimage, Brain Stimulation Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Laboratorio per lo studio delle atassie General programming and coordination of the study; patients selection and clinical evaluation, definition of behavioral tasks, MRI data acquisition and images processing. Data analysis and interpretation. 2 Sapienza Università di Roma 3 Policlinico Universitario Campus Bio-Medico Dipartimento di Psicologia dei processi di sviluppo e socializzazione Neurology Department Responsable for ASDs patients and healthy subjects recruitment. Social cognition evaluation. Responsable for implementation, planning and execution of tdcs experiment. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Laghi Fiorenzo Sapienza Università di Roma ASDs patients and healthy subjects recruitment. Social cognition evaluation 27/11/ Ranieri Federico Policlinico Universitario Campus Bio-Medico Implementation of cerebellar tdcs protocol, planning and execution of tdcs experiment 24/05/ /07/ / 6

26 GR Cerebellar-cortical circuits in Autism Spectrum Disorders: new perspectives for treatment implementation. Clausi Silvia Clinical health care research/clinicoassistenziale Fondazione Santa Lucia Background and Significance Autism spectrum disorders (ASDs) are high prevalence developmental disorders (Levy et al, 2009), but the underlying pathogenesis is unclear. Recent findings have implicated the cerebellum in this syndrome. Post-mortem studies showed a cerebellar Purkinje cells loss in ASDs patients and neuroimage data evidenced cerebellar gray and white matter alterations in these patients (Amaral et al. 2008; Scott et al. 2009). The ASDs diagnosis is based on a pervasive social interaction disorder (World Health Organization 1993) and its main behavioral hallmark is an impairment in Theory of Mind (ToM), the ability to recognize and attribute mental states to others to explain and predict their behaviour (Baron-Cohen 1995). Intriguingly, ToM alterations have been reported in patients with spino-cerebellar ataxia (SCA) (Sokolovsky et al. 2010; D'Agata et al. 2011) and neuroimage studies showed a cerebellar activation during emotions recognition tasks (Habel et al. 2005). These evidences, supported by cerebellar connections with limbic and associative areas (Schmahmann, Pandya, 1997), suggest a role of cerebellum in ASDs mentalizing impairment. In the present study, for the first time, the ToM profile and neuro-anatomical substrate of SCA and ASDs patients will be directly compared. It will allow to elucidate the cerebellar involvement in specific ToM aspects (Coricelli, 2005) and clarify the role of cerebello-cerebral circuits in ASDs pathogenesis, opening new perspectives on ASDs treatment. Specific aims Aim 1: Aim 2: Aim 3: To characterize the abilities of SCA and ASDs patients in specific components of ToM. To investigate the neuro-anatomical substrate of ToM profiles as evidenced in SCA and ASDs patients by using advanced MRI methods to evaluate the morphological and functional alterations in cerebellar-cortical circuits. To verify the effect of cerebellar excitability modulation on different ToM components in healthy subjects and ASDs patients by using the transcranial direct current stimulation (tdcs). Hypothesis: Given the literature reported data (see background session), we hypothesize that the cerebellum may have a role in mentalizing impairments observed in ASDs. Moreover, considering the complexity of processing related to the social behavior we hypothesize that the behavioural alteration reported in both cerebellar and autistic patients could be a consequence of a complex alteration of the neuronal network in which the cerebellum acts. To investigate these hypothesis we will use an integrate behavioural, neuroimaging and neurophysiological approach. Furthermore, considering the emerging findings about the improvement effect of the cerebellar tdcs on cognitive functions (Pope, Miall 2014) and emotion recognition (Ferrucci et al 2012) we postulate that the tdcs modulation of the cerebellar excitability could influence the ToM ability. Preliminary data: In a pilot study, 15 patients with cerebellar pathologies and 15 healthy matched controls were tested by three ToM tasks: Emotion Attribution task (EA - Blair & Cipollotti, 2000), Fuax Pas (FP -Stone et al, 1998) and Advanced ToM task (AT - van Harskamp et al, 2005). Preliminary data indicate that the cerebellum acts in processing of different ToM components as showed by the significant differences between patients and controls in all tasks (EA: p=0,04; FP: p=0,04; AT: p=0,01). In a preliminary experiment on 8 of such patients, by using advanced neuroimage methods of voxel based morphometry (VBM) (Good et al. 2001) on the whole brain, we found a bilateral atrophy of the caudate nucleus, cingulate gyrus and orbitofrontal cortex, all known to be implicated in emotional processing (Phillips et al. 2003). Moreover, a preliminary cerebro-cerebellar functional connectivity analyses performed in 6 ASDs patients, using resting-state fmri, showed decreased functional connectivity between the left dentate nucleus and left and right precuneus, a core region of the Default Mode Network, known to be active during cognitive processes related to social deficits seen in ASDs (e.g. ToM) (Assaf et al., 2010). 10/07/ / 6

27 GR Cerebellar-cortical circuits in Autism Spectrum Disorders: new perspectives for treatment implementation. Clausi Silvia Clinical health care research/clinicoassistenziale Fondazione Santa Lucia Finally, in a preliminary tdcs experiment, we modulated cerebellar exitability in 26 healthy subjects to analyse its effect on mental states recognition ability. A digital version of the 'Eyes Test' was employed (Vellante et al 2012). Both anodal and cathodal tdcs improved selectively the mental states recognition processing as showed by the significant reduction of the RTs between the baseline and post-stimulation sessions (p< 0.05). Materials and Methods Thirty SCA patients, 30 ASDs adults and 60 matched healthy subjects will undergo a neurological and neuropsychological assessment, the Autism Spectrum Quotient (Ruta et al. 2011), and an MRI (3T) exam including conventional, diffusion MRI and resting-state fmri scans. We will use the VBM to evaluate the structural alteration in cerebellum and connected areas, the Diffusion Tensor Imaging (Basser et al. 1994) to reconstruct and analyse the cerebello-cortical tracts and resting-state fmri to evaluate the cerebellocortical functional connectivity. An ToM battery (Baron-Cohen, 2003; Blair, Cipollotti, 2000; Baron-Cohen, Knight, 1998) to assess specific components will be performed and all scores will be compared among groups and correlated with MRI data. To investigate the effect of the cerebellar modulation on specific ToM aspects the tdcs will be delivered over the cerebellum of 20 healthy subjects and 20 ASDs patients and ToM abilities will be tested before and after the tdcs. Impact and Translational Implications ASDs are highly prevalent development disorders, with complex etiology and high social cost, often associated with cerebellar injury. The anatomical and functional characterization of the cerebello-cortical alterations in SCA and ASDs patients related with ToM deficits will provide a crucial insight on the comprehension of ASDs pathogenesis. The demonstration of the modulatory effect the cerebellar tdcs on ToM abilities could be crucial to evaluate new treatment approaches in ASDs. 10/07/ / 6

28 GR Can biological agents withdrawn in psoriatic arthritis (PsA) patients after a stable remission of arthritis is achieved? A clinical, radiological and ultrasonographic study. Graceffa Dario Clinical health care research/clinicoassistenziale LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Infectious and immunological diseases Istituti fisioterapici ospitalieri - Istituto Dermatologico Santa Maria e San Gallicano Project Classification IRG: Project Classification SS: Musculoskeletal, Oral and Skin Sciences Arthritis, Connective Tissue and Skin - ACTS Project Keyword 1: Project Keyword 2: Project Keyword 3: Arthritis and Connective Tissue: Inheritable, inflammatory and degenerative diseases of joints and connective tissues. Psoriatic Arthritis Therapeutic algorithms Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Clinical Dermatology Dermatology and rheumatology assessment ; therapy management ; study coordination 2 Istituti fisioterapici ospitalieri - Istituto Dermatologico Santa Maria e San Gallicano Radiology Ultrasound assessment, radiology assessment. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Elia Fulvia Istituti fisioterapici ospitalieri - Istituto Dermatologico Santa Maria e San Gallicano 2 Ultrasound assessment, radiology assessment. 09/07/ Maiani Elisa stituti fisioterapici ospitalieri - Istituto Dermatologico Santa Maria e San Gallicano ; Clinical Dermatology Dermatology assessment 09/05/ /07/ / 5

29 GR Can biological agents withdrawn in psoriatic arthritis (PsA) patients after a stable remission of arthritis is achieved? A clinical, radiological and ultrasonographic study. Graceffa Dario Clinical health care research/clinicoassistenziale Istituti fisioterapici ospitalieri - Istituto Dermatologico Santa Maria e San Gallicano Background and Significance The prevalence of PsA in patients with psoriasis has been estimated to be up to 30%. PsA can erode and destroy affected joints leading to loss of functional abilities and a considerable decline in quality of life. Moreover, psoriasis is often associated with various comorbities; the most frequently reported are cardiovascular and dismetabolic disorders. TNF blockers provide to achieve absent or very low disease activity, also slowing the progression of related comorbidities. However, these therapies are associated with a marked increase in the direct costs of the treatment and patients are exposed to potentially life threatening adverse events. Unfortunately, few studies have been conducted addressing treatment algorithms in PsA. Furthermore, no standardized criteria for clinical remission in PsA patients are available. Cantini et al. (2008) have defined remission criteria for PsA, adapting the American College of Rheumatology (ACR) criteria for rheumatoid arthritis and showed that remission may be sustained in PsA despite treatment interruption. The use of ultrasound assessment in the early detection of disease recurrence could became necessary to prevent the relapse and direct the clinician s choice concerning a re-treatment regimen. Potential benefits of this approach would include prevention of drug side effects, removal of risks associated with prolonged immunosoppression and significant financial savings. Specific aims Aim 1: Aim 2: Aim 3: We will evaluate, in patients in stable clinical and ultrasonographic remission for at least 1 year, the maintenance of remission and relapse characteristics (timing and severity) after anti TNF discontinuation. We will assess whether the remission of PsA is associated with a stabilization of the principal comorbidities (cardiovascular and metabolic). Evaluation of the financial saving and reduction of the risks of possible side effects associated with anti TNF therapies and development of specific therapeutic algorithms for patients with PsA. Hypothesis: We hypothesize to clearly define remission criteria for PsA based on advanced clinical, radiologic and ultrasonographic assessments. In addition will be possible to validate specific treatment algorithms (including treatment interruptions and/or dosage adjustments) designed to reduce the potential side effects and the direct treatment costs associated with the anti TNF therapy. Preliminary data: Our previous studies evaluated the maintenance of efficacy and relapse characteristics after anti TNF interruption in patients who obtained clinical and serological remission of moderate to severe peripheral PsA. We followed 47 PsA patients after the treatment discontinuation and we analyzed the characteristics of the disease recurrence. Although a gradual worsening of disease was observed in almost patients, the length of the disease-free period was consistent, lasting a mean of weeks after treatment discontinuation. 11/07/ / 5

30 GR Can biological agents withdrawn in psoriatic arthritis (PsA) patients after a stable remission of arthritis is achieved? A clinical, radiological and ultrasonographic study. Graceffa Dario Clinical health care research/clinicoassistenziale Istituti fisioterapici ospitalieri - Istituto Dermatologico Santa Maria e San Gallicano Materials and Methods Prior informed consent, 60 patients (age range, years) diagnosed with PsA according to ClASsification criteria for Psoriatic Arthritis (CASPAR), treated with anti TNF, in stable clinical remission for at least 1 year, will be enrolled in the study. Patients enrolled will stop the treatment and will be followed for 2 years. In addition to clinical examinations, all patients will undergo a complete power Doppler ultrasound joint assessment at the time of drug discontinuation and every 4 weeks for 2 years. At the time of treatment interruption and every 4 months, the patients will also undergo to Contrast Enhanced Ultrasound (CEUS) of the more closely involved joint. Moreover, they will undergo a global cardiovascular risk assessment, supra aortic arteries color Doppler ultrasound and liver ultrasound. Serum markers of inflammation, blood glucose, lipid profile and transaminases will be obtained from each patient's laboratory test within 48 hours of each visit. Impact and Translational Implications Currently, no standardized remission criteria in PsA patients are available; the use of an advanced ultrasound assessment in the early detection of disease recurrence could allow to prevent the relapse of the disease and provide a useful tool to define more accurate remission criteria. If patients could remain in a true remission whilst experiencing some degree of treatment interruption, it would significantly reduce the treatment cost for PsA patients.

31 GR Novel Biomarkers for Improved Characterization, Disease Tracking and Outcome Prediction in Traumatic Brain Injury Mondello Stefania Biomedical/Biomedica Associazione Oasi Maria SS LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Acute Neural Injury and Epilepsy - ANIE Project Keyword 1: Project Keyword 2: Relevant disorders include stroke/ischemia, epilepsy, spinal cord injury and traumatic brain injury. Biomarkers Project Keyword 3: Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Laboratories DI 2 Gaetano Martino University Polyclinic Neurosciences/Neurosurgery and Laboratory of Neurosciences and Molecular Biology Collaborator 3 University of Pecs János Szentágothai Research Centre Scientific Consulting Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Kobeissy Firas Gaetano Martino University Polyclinic Co-Investigator Expertise: biomarkers, proteomics, systems biology 2 Czeiter Endre University of Pecs Co-Investigator Expertise: neurosurgery and neurotrauma 15/06/ /04/ Grillo Lucia Rosa Maria Oasi Maria SS/Department of Laboratories, Laboratory of Medical Genetics Co-Investigator Expertise: genetics and genomic bioinformatics/ genetic variation, disease genes 20/11/ /07/ / 6

32 GR Novel Biomarkers for Improved Characterization, Disease Tracking and Outcome Prediction in Traumatic Brain Injury Mondello Stefania Biomedical/Biomedica Associazione Oasi Maria SS Background and Significance Traumatic brain injury (TBI) is one of the greatest unmet needs in medicine and public health. Country based estimates of the annual incidence of hospitalization following TBI range from new cases/100,000 inhabitants. TBI is a major cause of death and disability, and leads to great personal suffering to victims and relatives as well as huge costs to society. In Europe, the financial burden of TBI has been estimated at over 33 billion per year. These data provide strong ethical, medical, social and economic reasons for implementing research and clinical interventions in TBI. TBI is considered "the most complex disease in our most complex organ". It is characterized by great heterogeneity in terms of etiology, mechanisms, pathology, severity, and treatment, with widely varying outcomes. While basic research has increased our knowledge of the mechanisms involved, clinical research in TBI remains particularly challenging due to its heterogeneity. Guidelines for the treatment are available, but the evidence underpinning these recommendations is weak. Moreover, current approaches to the characterization of disease severity and outcome have not undergone refinement for more than three decades. Recent advances in genomics, proteomics, and biomarker development provide unparalleled opportunities for refinements in clinical characterization, offering more accurate disease phenotyping. Such improved characterization and stratification will allow for more targeted therapies. Specific aims Aim 1: Aim 2: Aim 3: To establish a biospecimen repository and explore in depth the clinical value of novel brain damage biomarkers for a better characterization of TBI, with emphasis on tracking disease processes and predicting outcome. We will determine the magnitude and time course of biomarker concentrations, analyzing differences and relationships with type of injury, clinical presentation and outcome. Trends and changes in biomarker patterns will be explored and quantified. To characterize DNA from a cohort of TBI subjects to identify genetic predisposition to complications and outcome. We will perform DNA sequencing and variant detection to investigate genetic drivers of lesion incidence, pathophysiology, and progression, and to explore associations between genotype and outcome. To use proteomics to identify new candidates to complement existing TBI biomarkers. We will use differential neuroproteomic analysis based on multidimensional protein separations and capillary liquid chromatography/tandem mass spectrometry to identify and characterize TBI-associated changes in protein levels in blood and CSF. Systems Biology analysis will then be conducted to determine biological networks, functional associations of key factors and pathways involved in TBI. Hypothesis: We hypothesize that the use of emerging technologies (biomarkers, genomics and advanced proteomic techniques) will improve characterization, disease tracking and outcome prediction in TBI. Appropriate markers, alone and/or in combination, can provide information critical on pathophysiology as well as on possible genetic influence, risk factors, injury mechanism(s) and cellular and subcellular localization of injury. Improved characterization with these tools will facilitate targeted management for individual patients, thereby holding promise for personalized medicine. Preliminary data: We have conducted preliminary analyses of CSF and serum samples from patients with TBI. These analyses have provided strong support for the hypotheses to be tested in this proposal. Biochemical markers of brain damage were detectable in both CSF and serum for several days after injury. Consistent with our hypotheses, we have demonstrated the ability of temporal profiles of biomarker to track disease processes and predict TBI outcome and we have provided the first evidence that different patterns of brain injury biomarkers are related to different types of injury. Furthermore, our laboratory conducted a number of clinical studies assessing the role of APOE genotype and genetic variations as risk factors for neurodegeneration and adverse outcome. Finally, Drs. Mondello and 10/07/ / 6

33 GR Novel Biomarkers for Improved Characterization, Disease Tracking and Outcome Prediction in Traumatic Brain Injury Mondello Stefania Biomedical/Biomedica Associazione Oasi Maria SS Kobeissy have participated in the first proteomics-based program to systematically identify biochemical markers of TBI. These studies have identified a number of promising markers which provide information on proteolytic mechanisms of TBI (necrosis vs. apoptosis) as well as on the cell type and subcellular localization of injury. Materials and Methods The key driver of our research plan is an integrated approach combining biomarkers, genomics and proteomics to improve disease characterization that will facilitate targeted management and individualized approaches to improve outcomes in TBI. We will recruit a cohort of 100 moderate to severe TBI patients aged 18 or older admitted to the Neurosurgery Unit of University Polyclinic of Messina over 18 months. Controls will consist of age- and sex-matched subjects with no evidence of brain injury. Data collection will be prospective and longitudinal, up to 12 months. Blood samples and CSF, when available, will be collected from all patients during the acute phase and then up to a maximum of 10 days after injury. Samples will be analyzed for biochemical and genetic biomarkers, and employed for proteomic studies. The results will be linked to acute care, post-acute care and outcomes. We will create and maintain biorepositories that will be an important resource for legacy research. Impact and Translational Implications This project represents a systematic effort to advance the characterization of TBI, and improve understanding of disease processes that will lead to therapies that are better targeted and more individually oriented (Precision Medicine). We expect profound impacts in terms of treatment provision (guidelines and best practices), economy (reduced costs), health care policy, and most importantly, improved outcome and quality of life for patients. 10/07/ / 6

34 GR Engineered regulatory T cell Adoptive Cell Therapy as a novel tool for the treatment of Cardiovascular diseases KALLIKOURDIS MARINOS Biomedical/Biomedica Istituto Clinico Humanitas LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Cardiovascular and Respiratory Sciences Physiology and Pathobiology of Cardiovascular and Respiratory Systems - PPCR Project Keyword 1: Project Keyword 2: Project Keyword 3: Effects of immune processes and infection on target tissue physiology regulatory T cells cellular immunotherapy Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Istituto Clinico Humanitas Coordinator: coordination of experiments, execution of immunotherapy and immunoprofiling. 2 Istituto Clinico Humanitas Istituto Clinico Humanitas Partner 1 unit: cardiac disease model and studies 3 Università degli Studi di Milano Investigators, Institution and Role in the Project Department of Pharmacological and Biomolecular Sciences Partner 2 unit: atherosclerosis model and studies Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Elia Leonardo Istituto Clinico Humanitas Head of Partner 1 unit: cardiac disease model and studies 2 Tibolla Gianpaolo Università degli Studi di Milano Head of Partner 2 unit: atherosclerosis model and studies 03/02/ /12/ /07/ / 6

35 GR Engineered regulatory T cell Adoptive Cell Therapy as a novel tool for the treatment of Cardiovascular diseases KALLIKOURDIS MARINOS Biomedical/Biomedica Istituto Clinico Humanitas Background and Significance Cardiovascular (CV) disease is a major burden on the health service. Recent work has shown that the development of atherosclerosis, as well as the progression of dilated cardiomyopathy (DCM), which can lead to heart failure, are driven by inflammation, essentially a dysregulated immune response. Whilst the pathological role of this inflammation is becoming increasingly clear, clinical trials targeting soluble effectors of the immune system have yet to produce results in CV disease. Regulatory T (Treg) cells, a population of anti-inflammatory lymphocytes, have been shown, in mouse and human studies, to be key suppressors of this inflammation. The use of Treg cells as a treatment for many immunopathologies is an expanding area of research. Adoptive Cell Therapy (ACT), a procedure where a patient is treated with autologous T cells that have been expanded and modified in order to combat an immune-mediated ailment, is increasingly becoming a promising therapeutic strategy for several pathologies. A drawback of current ACT strategies is the lack of target-organ homing specificity in the adoptively transferred T cells, which greatly reduces the efficacy of the approach. We have shown that by identifying the chemokines selectively expressed by the target organs and by modifying the chemokine receptors of the transferred T cells so that they preferentially home to the selected target organ, this obstacle can be overcome. Specific aims Aim 1: Aim 2: Aim 3: Optimize transduction of Treg cells with chemokine receptors matching the chemokines expressed at sites of inflammation in the atherosclerotic plaque and the failing myocardium (the chemokines are well-characterized in models of atherosclerosis and we have identified them in models of DCM -see preliminary data). Optimize an inducible Foxp3 vector, to enable the conversion of pro-inflammatory T cells into immunosuppressive induced Treg (itreg). This additional strategy enables more specific suppression (see main proposal). (Coordinator with material from all units, m 1-12) Investigate the improved homing of chemokine receptor transduced Treg/iTreg to the heart in trans-aortic constriction(tac) model of DCM. Investigate the effect of control- or chemokine receptor-transduced Treg/iTreg transfer in disease progress. (Partner 1 and Coordinator, m 7-36) Investigate the improved homing of CCR2/CCR5-transduced Treg/iTreg to the atherosclerotic plaque in LDLR-KO mouse model of familial hypercholesterolemia(fh), a disease leading to severe atherosclerosis and premature death. Investigate the effect of modified Treg/iTreg therapy in disease progress (including analysis of the pathological/immune profile of the treated plaques). (Partner 2 and Coordinator, m 7-36) Hypothesis: We hypothesize that retrovirally transducing chemokine receptors that match the chemokines preferentially expressed at the site of inflammation in dilated cardiomyopathy and atherosclerosis will enable the directed homing of transduced Treg to the site of inflammation. The Treg should suppress the ongoing immune response and thus slow down/block disease progression. This will be performed on mouse models of the diseases, whilst we will assay chemokine expression in human patient samples to validate the translational potential of our approach. Our proof-of-concept study may assist the development of a clinically applicable strategy. We are focusing on the more severe forms of cardiovascular disease, though as cell therapy safety issues are being actively addressed by many groups, a wider application of our strategy may be eventually possible. Preliminary data: The coordinator has cloned retroviral vectors for most pro-inflammatory chemokine receptors and shown that Treg can be specifically directed to an organ by transducing them with a chemokine receptor matching the chemokine preferentially expressed at the site. The efficacy of this technique in modifying an immune response has been already demonstrated in two different in vivo contexts: maternal-fetal tolerance (Kallikourdis et al. PNAS 2007) and immunodeficiency (Kallikourdis et al., Blood 2013). The unit is already participating in a grant-funded project to treat inflammation-driven neurodegeneration with receptor-modified Treg. Coordinator and Partner 1 have performed full 08/07/ / 6

36 GR Engineered regulatory T cell Adoptive Cell Therapy as a novel tool for the treatment of Cardiovascular diseases KALLIKOURDIS MARINOS Biomedical/Biomedica Istituto Clinico Humanitas profiling of chemokine expression in DCM. Partner 2, in preliminary experiments with the Coordinator, has shown improvement of homing to the plaque achieved by CCR2 transduction. Materials and Methods Partner 1, an independent group in the Division managed by Prof Condorelli, have expertise in the TAC mouse model of DCM. Partner 2, an independent group in the department managed by Prof Catapano, have expertise in the LDLR-KO model of FH and atherosclerosis. Human tissue samples will be provided by external collaborators in Niguarda and Bassini hospitals, after informed consent. Chemokine expression from mouse and human tissue will be analyzed by qpcr and ELISA, standard assays in the Coordinator unit. Treg purification and transduction with chemokine receptor constructs will be performed by the Coordinator unit as previously (Kallikourdis et al. PNAS 2007). Treg homing and inflammatory cell profile at the endpoint of experiments will be examined by FACS, Immunohistochemistry(IHC) and qpcr. Atherosclerosis progression will be monitored by ELISA analysis of serum as well as qpcr and IHC of sections of the aorta. Heart failure progression will be monitored by in vivo echocardiography. Impact and Translational Implications We aim to provide proof of principle for a novel therapeutic approach for cardiovascular diseases. The innovation lies in using immunosuppressive cells to combat two pathologies with recently-identified immune-mediated components. Enhancing the precision of the treatment by chemokine receptor modification of the Treg should further boost the therapeutic efficacy. With continuous improvement of ACT strategies for clinical use, we hope to contribute towards a clinically-applicable novel therapy. 08/07/ / 6

37 GR New mtor inhibitors development and study of their effects on the central nervous system (mtorneu) Russo Emilio Biomedical/Biomedica Calabria LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Acute Neural Injury and Epilepsy - ANIE Project Keyword 1: Project Keyword 2: Project Keyword 3: Recovery of function/rehabilitation; neurological and functional evaluation of neural prostheses, electrical/magnetic stimulation, behavioral and pharmacological interventions, and physical therapy. Identification of new effective molecules in the treatment of Epilepsy Molecular brain functions Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Science of Health department, Clinical Pharmacology Unit 2 University of Messina Dipartimento di Scienze del Farmaco e Prodotti per la Salute 3 Mario Negri Institute for Pharmacological Research, Milan Laboratoty of Experimental Neurology Department of Neuroscience Coordinator, in vitro and in vivo experiments Chemistry and Pharmaceutical chemistry experiments in vitro and in vivo experiments Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 De Luca Laura University of Messina Chemistry and Pharmaceutical chemistry experiments 2 Balosso Silvia Maria Mario Negri Institute for Pharmacological Research, Milan 11/11/1975 in vitro and in vivo experiments 21/07/ /07/ / 5

38 GR New mtor inhibitors development and study of their effects on the central nervous system (mtorneu) Russo Emilio Biomedical/Biomedica Calabria Background and Significance The mammalian target of rapamycin (mtor) is an evolutionarily conserved serine/threonine protein kinase with multiple cellular functions influencing physiological and pathological neuronal processes. In the central nervous system (CNS), mtor has been associated with synaptic plasticity, memory function and neuronal repair mechanisms. Recent evidence suggests that an altered mtor pathway is implicated in several neurological disorders such as Alzheimer's and Parkinson's diseases as well as epilepsy and epileptogenesis. The effectiveness of rapamycin (RAP) and the involvement of mtor in epilepsy/epileptogenesis are noteworthy. Animal and clinical studies support the hypothesis that mtor pathway hyperactivation is highly correlated to the epileptogenic process leading to the development of spontaneous recurrent seizures (SRS) and that mtor inhibition possess disease modifying properties reducing neuronal damage and SRS development. Ongoing clinical trials are providing new insights into the efficacy of mtor inhibitors; however, the currently available drugs lacking of CNS selectivity are highly limited by their toxicity. Therefore, more research is certainly warranted on both drug development which might selectively act in the brain avoiding other side-effects associated with mtor inhibitors, mechanism of action identification and efficacy determination. Specific aims Aim 1: Aim 2: Aim 3: We will perform drug design studies through computational techniques to identify new chemical entities capable to inhibit mtor either with a rapamycin-like mechanism (binding to FKBP12) or at the catalytic ATP site influencing both mtorc complexes of the kinase. We will synthesize the most promising compounds using innovative ecofriendly methods and technologies. All synthesized compounds will undergo in vitro and in vivo evaluation. The biological activity of the synthesized compounds will be then completed by in vitro activity on cells and cytotoxicity screening on astrocytes and neuronal primary cultures. We will also evaluate the toxicity of active molecules both acutely injected at high dosages and after 10 days of repeated administration. To test selectivity for the CNS, the most promising molecules will be screened for their in vivo activity to inhibit mtor in the brain measuring after administration S6 phosphorylation degree To study the antiepileptogenic effects of mtor inhibition using quantitative EEG analysis of epileptic activity in a mouse model of drug-resistant mesial Temporal Lobe Epilepsy and in the WAG/Rij rat genetic model. As additional functional outcome measures, inflammatory response and cognitive performance will be also evaluated. To investigate a functional interaction between mtor and NMDA receptors, we will study whether mtor inhibitors modify neuronal responses evoked by proinflammatory stimuli and activation of NMDA receptors by using time-lapse single-cell Ca2+ imaging in primary cultures of mouse hippocamapl neurons. Hypothesis: Our hypothesis is that brain injuries leading to epilepsy activate mtor pathway contributing to epileptogenesis and to the occurence of spontaneous seizures. Thus, mice or rats treated with the identified CNS specific mtor inhibitor should either show a significant delay in seizure onset, or no seizures. Alternatively, we may detect fewer or less severe seizures in the chronic epileptic phase and/or protection against neuronal cell loss; either would be considered as clinically relevant disease-modifying effects. Preliminary data: Our research group has already published some relevant data on the effects of rapamycin in the WAG/Rij rat model with depressive-like comorbidity (Russo et al., 2013, 2014). We have published the effects of rapamycin in vivo against the synthesis of IL-1beta and TNF-alpha in the rat brain after treatment with LPS, further studying several components of the mtor pathway (Russo et al., 2014). Briefly, we found that rapamycin treatment would reduce the development of spontaneous seizures and this effect was associated with a strong modulation of inflammatory responses. 09/07/ / 5

39 GR New mtor inhibitors development and study of their effects on the central nervous system (mtorneu) Russo Emilio Biomedical/Biomedica Calabria Materials and Methods We will synthetize new compounds selectively interacting with mtor, which will be screened for their effects on the CNS. mtor inhibitor treatments will be performed during epileptogenesis on C57BL/6 mice treated with intra-amygdala kainate injection, a mouse model of TLE, or WAG/Rij rats. We will study treatment effects on: spontaneous seizures development; neuronal death; hippocampal neurogenesis; mossy fiber sprouting and behavioral comorbidity. The following behavioral tests will be performed with the support of ETHOVision: tail suspension and forced swimming test; open field test; Elevated plus maze, Morris water maze test, sucrose-consumption test. In all in vivo and in vitro models, samples will be analyzed by immunohistochemistry and western blotting with phospho specific antibodies for S6K, AKT, AMPK and mtor. Furthermore, samples will be evaluated for inflammatory response analyzing: histological damage, NF-kB activation, proinflammatory cytokine production, glia activation Impact and Translational Implications Our project will identify new molecules inhibiting mtor with a selective action on the CNS, defining the role of this kinase. Our results will also clarify the role of this process in models of epilepsy/epileptogenesis and related comorbidity possibly indicating new molecular targets and clarifying the potentiality of mtor inhibitors. mtor might represent a suitable target across several brain pathologies offering new perspectives for clinical therapy.

40 Longitudinal multi-parametric clinical, CSF and MRI study aimed at predicting disability progression in Multiple Sclerosis GR Calabrese Massimiliano Biomedical/Biomedica Veneto LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroimmunology and Brain Tumors - CNBT Project Keyword 1: Project Keyword 2: Project Keyword 3: The relevant diseases are multiple sclerosis, myasthenia gravis, inflammatory neuropathies and myopathies, infectious diseases of the nervous system, prion disease and nervous system tumors. Disability progression magnetic resonance imaging Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: GR X I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Azienda Ospedaliera Universitaria Integrata Verona, Sezione di Neurologia du 2 University of Padova Dept. of Information Engineer MRI analysis Study coordinator, Patients recruitment and follow up 3 Istituto Superiore di Sanita' Department of Hematology, Oncology and Molecular Medicine Section of Stem Cells and Endothelium CSF analysis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Enrico Grisan University of Padova MRI analysis 09/02/ ROSSI STEFANIA Istituto Superiore di Sanita' CSF analysis 21/03/ Vedovello Marcella Ospedale Papa Giovanni 23, UOC Neurologia Patients recruitment and follow up 19/07/ /07/ / 6

41 Longitudinal multi-parametric clinical, CSF and MRI study aimed at predicting disability progression in Multiple Sclerosis GR Calabrese Massimiliano Biomedical/Biomedica Veneto Background and Significance Studies on the natural history of Multiple Sclerosis (MS) have disclosed that its evolution from the relapsing remitting (RR) to the secondary progressive (SP) phase is a pivotal event in the disease evolution being characterized by the accumulation of irreversible physical and cognitive disability and often unresponsive to each therapeutic approach. Up to date an accurate prediction of the risk to enter the SP phase is not available yet Several studies have recently showed: (a) that the focal and diffuse grey matter damage is visible by the means of application of advanced MRI sequences such as Double Inversion Recovery, (b) that it is a frequent an early phenomenon in Multiple Sclerosis and (c) that the rate of physical and cognitive disability progression is more related to grey matter pathology than to the white matter lesion load (Calabrese et al 2012) and this is probably associated to the failure of the compensatory mechanisms of the brain network, and finally (d) that functional connectivity of Resting State Networks (RSNs) is different between RR and SP patients (Basile et al 2014). Morever grey matter (GM) pathology in MS has been recently associated with increased level of meningeal inflammation (Magliozzi 2007, Lucchinetti 2011). Factors released by immune cells circulating in the CSF and settling in lymphoid-like structures within the subarachnoid space could diffuse across the pial membrane, activate microglia and directly damage glia and neurons Specific aims Aim 1: Aim 2: Aim 3: Assessing the relationship between GM damage, modifications of effective brain connectivity and the disability progression Identifying clinical and MRI parameters that may predict the conversion to the SP phase and developing a prediction model for the risk of conversion to the SP phase Integrating the outcome of the developed prediction model with the available clinical tools to obtain a risk stratification that can be used to design class-specific care plans Hypothesis: Our working hypothesis is that a direct relationship between the accumulation of GM damage, the failure of the compensatory mechanisms of the brain network and the evolution towards the SP phase of the disease exists and that the neurodegenerative events affecting the cortical and deep GM, although subclinical, are already detectable by means of non conventional MRI methodologies) (Dalton et al., 2004 Calabrese et al., 2011, Lucchinetti et al. 2011) in the early RRMS. Moreover, it can be envisaged that CSF may consist of biomarkers of the GM pathology that can help to select a subgroup of MS patients with more severe cortical pathology, particularly active disease and poor prognostic factors, that could benefit from early treatment with second line, more aggressive drugs, or drug combinations. Preliminary data: The optimization of the MR sequences has been already done on the Philips Achieva TX 3T scanner available at Verona. Thus isotropic high resolution anatomical sequences (T1, T2, FLAIR, DIR) and non conventional MRI (DTI multi-shell, fmri for RSNs analysis, and Quantitative Susceptibility Mapping) sequences are ready to be used. Our preliminary results indicate that age and the volume of cortical lesions and of the cerebellar cortex have a significant impact in predicting the conversion to the SP phase. Morever among 27 chemo/cytokines/growth factors investigated on CSF of the first 4 patients (we chose 2 MS patients with and 2 without grey matter pathology) by using immunometric assays (Multiplex technology), high levels of IL-6, -8, IL1R antagonist and FGF were detected only in the patient with the highest GM pathology; high levels of TNF, IFN, IL-13, and GM-CSF were detected only in the two MS patients with GM pathology; high levels of the chemokine IP-10 were 11/07/ / 6

42 Longitudinal multi-parametric clinical, CSF and MRI study aimed at predicting disability progression in Multiple Sclerosis GR Calabrese Massimiliano Biomedical/Biomedica Veneto only detected in the MS patients without GM pathology. Moreover the proteomic analysis using the "Trident" method confirmed a precise inflammatory profile of CSF in association with the amount and presence of GM damage. In particular, a high number of molecules associated with neuronal/axonal structures were found in the MS patients with very high GM pathology, while immunoglobulins and antibody-associated molecules have been detected mainly in the MS patients without GM pathology. Materials and Methods 250 consecutive MS patients with at least 10 years of disease duration and at least 35 years old will be included in this 3- year longitudinal study. At study entry each patient will undergo a 3T MRI and will be asked to undergo a lumbar puncture. Neurological examination will be performed every 6 months. Advanced machine learning techniques will be used to develop a prediction model. Features included will be demographic and clinical data; MRI markers (regional distribution of cortical lesions and regional cortical thickness, volume of the deep GM structures, diffusion and susceptibility indices, and RSNs effective connectivity); A proteomic and molecular CSF characterization will be performed in a subset of MS patients whose CSF will be already available or who will accept to repeat the lumbar puncture for prognostic purposes. The proteomic facility at ISS is fully equipped for proteomic analyses using/maldi-tof, LC-MS/MS and immunometric assays (Multiplex technology). Impact and Translational Implications Early stratification of the risk of disease progression by identification of CSF/MRI parameters that mark the entry into progressive phase has significant (I) clinical (delaying physical and cognitive disability), (II) social (maintaining the quality of life) and (III) economical (reducing direct and indirect costs designing more effective patient-tailored therapeutic pathways that address the most active/expensive drugs for patients at high risk of disability progression) rebounds. 11/07/ / 6

43 Diaphragmatic dysfunction in critically patients undergoing mechanical ventilation. BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Spadaro Savino Clinical health care research/clinicoassistenziale Emilia-Romagna LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Innovative biotechnologies Project Classification IRG: Project Classification SS: Cardiovascular and Respiratory Sciences Respiratory Integrative Biology and Translational Research - RIBT Project Keyword 1: Project Keyword 2: Project Keyword 3: Neural control of breathing including central and peripheral chemoreceptors, central neural processes, airway receptors, and neural aspects of dyspnea. Neurally Adjusted Ventilatory Assist and Diaphragmatic Dysfunction Biomarker of diaphragmatic injury and ultrasound Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Morphology, Surgery and Experimental Medicine, Operative Unit Anesthesia and Intensive care 2 University Of Ferrara Department of Biomedical and specialist surgical sciences, section of medical Biochemistry, Molecular Biology and Genetics Study coordination and conduction of the clinical phase of the project Unit Responsible for lab processing and analysis of biologic samples Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Trentini Alessandro University Of Ferrara Laboratory analysis of biologic samples 24/11/ Zani Gianluca Clinical Investigator 26/07/ /07/ / 6

44 Diaphragmatic dysfunction in critically patients undergoing mechanical ventilation. BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Clinical health care research/clinicoassistenziale Spadaro Savino Emilia-Romagna Background and Significance Mechanical ventilation (MV) is life-saving in ICU patients but is associated with potential complications. The failure to wean patients from MV is a significant clinical problem that prolongs time on the ventilator, increases healthcare costs, morbidity and mortality. Prolonged MV results in diaphragmatic dysfunction (DD). Despite growing evidence that DD develops in critically patients and contributes to weaning failure, the respiratory muscles are poorly monitored in the ICU. The availability of sensitive tool to monitor damage of the respiratory muscles during MV is a relevant point. The Neurally Adjusted Ventilator Assist (NAVA) is a mode of assisted MV where the level of ventilatory assist is linearly proportional to electrical activity of the diaphragm (EAdi). The qualitative and quantitative monitoring of the EAdi signal might be helpful in the prevention of DD. More promising tools are ultrasonography to evaluate diaphragm movement and thickness. These sonographic diaphragmatic parameters can provide valuable information in the assessment of patients with DD, in terms of optimization ventilator management. Considering the important role of plasma markers in the diagnosis, could be reasoned that the detection of muscle-specific proteins in plasma would be a valuable tool to monitor damage of the respiratory muscles. The definition of skeletal muscle troponin I as marker of DD could suggest new diagnostic and therapeutic strategies. Specific aims Aim 1: Aim 2: Aim 3: The primary endpoint of this project is to determine the utility of fast and slow-twitch skeletal muscle troponin I (fstni and sstni, respectively) as biomarkers of diaphragm injury during assisted mechanical ventilation. Circulatory biomarkers for respiratory muscle injury are needed, and they will hopefully appear in the next few years. Recent experimental findings indicate that measuring serum levels of stni is more effective to detect injury of respiratory muscles. However, the relation between plasma markers of muscle damage and functional measures in critically ill patients has not been investigated yet. To achieve this objective we will perform a prospective study to evaluate the relationship between NAVA level or Pressure Support Ventilation (PSV) level assistance with levels of the proposed markers. The secondary endpoint will be to define if the application of NAVA, as opposed to PSV, assessed by diaphragmatic ultrasonography (US) may become helpful in identifying patients at high risk of difficult weaning. Our results can indicate diaphragmatic evaluation as a predictor of extubation outcome in the weaning process. An increase in the use of US is likely to lead to prompt identification of patients with diaphragmatic dysfunction. The final endpoint will be to define if the application of NAVA, as opposed to PSV, may reduce time of weaning, rate of reintubation, number of tracheotomy, reduction of costs for patient. Prolonged diaphragmatic inactivity might contribute to prolonged ventilation associated to a higher incidence of tracheotomy and last but not least, higher costs. Hypothesis: We hypothesize that fast and slow-twitch skeletal muscle troponin I could be useful to identify diaphragm injury during assisted mechanical ventilation. Furthermore, Ultrasonography in evaluating diaphragmatic contractility, in relation to a possible diaphragm injury, during assessed mechanical ventilation could be useful in diagnosing VIDD as a cause of difficult weaning and may lead to adjustments of ventilator setting. Moreover NAVA could minimize the risk of VIDD because it is based on the continuous coupling between ventilator assistance and the patients neural output. Further, NAVA could be able to reduce the number of the days of ventilation and ICU length of stay. This will be the first study that identifies markers of diaphragm injury with new diagnostic and therapeutic implications. Preliminary data: We enrolled 16 Patients previously ventilated in CMV for at least 72 hours, were randomized to receive NAVA (8) or PSV (8) for 48 hours. Neuro Ventilatory Efficiency, (VT/EAdi) and other physiological parameters were measured in periods of spontaneous ventilation (2 min each) at 05/07/ / 6

45 Diaphragmatic dysfunction in critically patients undergoing mechanical ventilation. BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Clinical health care research/clinicoassistenziale Spadaro Savino Emilia-Romagna baseline, 24 and 48h. As compared to PSV, NAVA assistance significantly improved NVE. We speculate that this was due to a better matching between diaphragm contraction and positive pressure assistance during NAVA. Materials and Methods This is a prospective, randomized, interventional study. We will consider eligible all intubated patients with acute respiratory failure undergoing MV for a period less than or equal to 48 hours. After randomization (NAVA or PSV), EAdi catheter will be positioned in both groups. In the NAVA group will be tested three NAVA levels, while in the PSV group three pressure support levels, lasting 20 minutes each. The US examination is performed concomitantly for each step in both groups. We will collect informations about diaphragm thickness and diaphragm displacement for both emidiaphragms at each step during NAVA and PSV. Serum markers of muscle damage will be measured. In particular, fast and slow stni will be measured by commercially available ELISA kits, CAIII will be measured by ELISA activity assay; aldolase will be determined by continuous spectrophotometric coupled enzymatic assay; CK-MM will be measured by commercially available ELISA kits. Impact and Translational Implications The project aims to define the utility of fast and slow skeletal muscle troponin I as well as CAIII, aldolase and CK-MM as biomarkers of diaphragm injury during assisted mechanical ventilation. Implementation of the use of NAVA and ultrasonography in assessment of diaphragmatic function into the clinical practice can already give information in ventilation management, and clinician has to learn how these data might be interpreted and used for tailoring strategies. 05/07/ / 6

46 GR The role of authopagy as a protective mechanism in cardiovascular diseases: new insights into oxidative stress modulation and cardiovascular regeneration Sciarretta Sebastiano Biomedical/Biomedica Istituto Neurologico Mediterraneo Neuromed LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Cardiovascular and Respiratory Sciences Cardiac Contractility, Hypertrophy, and Failure - CCHF Project Keyword 1: Project Keyword 2: Project Keyword 3: The basic molecular and cellular mechanisms underlying cardiac hypertrophy and failure: myocyte growth, proliferation, metabolism and apoptosis; receptor signaling; transcriptional pathways; inflammatory/ cytokine-mediated processes. Oxydative stress Authopagy Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: X I declare that the object/s of this application is/are under patent copyright Patent owner: Owner: "Sapienza" University of Rome. Patent number: WO Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Dipartimento di Angio-Cardio-Neurologia, Unita' di Basi Sperimentali e Cliniche dell'ipertensione Arteriosa 2 Regione Lazio, Policlinico Umberto I 3 "Sapienza" University of Rome Investigators, Institution and Role in the Project Department of Internal Medicine and Medical Specialties, Policlinico Umberto I Department of Medical Surgical Science and Biotechnology Unit 1. Principal investigator. Tasks: animal models, evaluation of autophagy and cardiac phenotypes, generation of lentiviral vectors; data analysis/dissemination. Unit 2. Tasks: oxydative stress assessment, platelet activation and endothelial function, data analysis/dissemination. Unit 3. Tasks: human and murine CPC isolation, in vitro and in vivo characterization of regenerative potential, data analysis/dissemination. Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Carnevale Roberto Regione Lazio, Policlinico Umberto I Coordinator Unit 2. Tasks: oxydative stress assessment, platelet activation and endothelial function, data analysis and dissemination of the results. 2 Chimenti Isotta "Sapienza" University of Rome Coordinator Unit 3. Tasks: human and murine CPC isolation, in vitro and in vivo characterization of regenerative potential, data analysis and dissemination of the results. 06/12/ /08/ /07/ / 6

47 GR The role of authopagy as a protective mechanism in cardiovascular diseases: new insights into oxidative stress modulation and cardiovascular regeneration Sciarretta Sebastiano Biomedical/Biomedica Istituto Neurologico Mediterraneo Neuromed Background and Significance Heart failure (HF) and stroke still represent major causes of disabilities and death. Thus, new therapies are needed to inhibit chronic maladaptive cardiac remodeling (CR) and dysfunction that usually follow ischemic and overload cardiac diseases, and lead to end-stage HF. Effective therapies should also reduce endothelial dysfunction (ED), oxidative stress (OxS) and platelet activity caused by several risk factors (e.g. metabolic syndrome), in order to avoid vascular damage and stroke. Activation of autophagy may represent a promising approach to treat CR, HF and ED. Autophagy is an intracellular degradation process, in which long-lived proteins and damaged organelles are sequestered by double-membrane vacuoles, termed autophagosomes, and delivered to lysosomes for degradation. Its activation mediates cellular adaptation to ischemia and OxS. Autophagy promotes also cardiomyocyte (CM) survival in response to acute cardiac stress. However, the importance of autophagy activation in reducing CR and ED still remains to be clarified. Our hypothesis is that autophagy activation would prevent CR and HF by reducing OxS and death in CMs. Autophagy could also promote survival of cardiac progenitor cells (CPCs), thereby preserving either resident and/or transplanted CPCs and supporting endogenous and/or exogenous repair of the remodeled heart. Finally, autophagy could prevent ED development in response to risk factors by reducing OxS and increasing nitric oxide availability. Specific aims Aim 1: Aim 2: Aim 3: To elucidate the role of autophagy activation during chronic CR and dysfunction. We will test whether: 1a) cardiac genetic disruption of autophagy by ATG7 deletion impairs the response to chronic myocardial infarction (MI) and pressure overload; 1b) trehalose, a natural disaccharide that strongly activates autophagy, reduces CR and dysfunction during chronic MI and pressure overload; and 1c) the beneficial effects of trehalose during CR are lost in mice with cardiac-specific ATG7 deletion. To investigate the role of autophagy in CPC survival and regenerative capacities during stress. We will test whether: 2a) CPCs isolated from ischemic hearts of patients with metabolic syndrome, a condition known to inhibit autophagy, show increased OxS, impaired autophagy and reduced survival as compared to CPCs from patients without metabolic syndrome; 2b) trehalose preserves resident CPC pool after MI; and 2c) autophagy activation by trehalose or ATG7 overexpression modulates CPC regenerative potential in vitro and in vivo. To clarify the role of autophagy in vascular protection. We will test whether: 3a) endothelial genetic disruption of autophagy by ATG7 deletion impairs endothelial function; 3b) autophagy activation by ATG7 overexpression or trehalose reduces OxS and preserves vascular integrity and function, and platelet activity in the presence of metabolic syndrome; and 3c) genetic or pharmacological NADPH oxidase inhibition preserves endothelial function and platelet activation in the presence of metabolic syndrome with additive beneficial effects to that of trehalose. Hypothesis: ATG7-mediated autophagy is required for CM survival and cardiac response to stress. Pharmacological activation of autophagy by trehalose reduces chronic CR, ventricular dysfunction and OxS. Autophagy activation preserves resident/transplanted CPCs and increases their survival and regenerative capacities. Autophagy activation preserves also cell integrity and endothelial function by reducing OxS, NADPH oxidase activity and maladaptive platelet-endothelial interaction. Preliminary data: Comprehensive preliminary data are reported in the figure in the full project form. CMs with adenoviral-mediated ATG7 knockdown show a significant inhibition of autophagy and reduction of survival (0.61-fold, p<0.05) after 12h of H2O2 treatment (200µM) in vitro. Mice with cardiac-specific ATG7 knockout also show disruption of autophagy. C57BL/6 mice were subjected to permanent left anterior descending coronary artery (LAD) ligation. After 4 weeks, mice treated with trehalose displayed a significant attenuation of LV dilation (LVEDD: 3.2±0.1 vs. 3.9±0.3mm, p<0.05) and LV systolic dysfunction with respect to placebo (FS: 39.2±2.0 vs. 28.8±5.1%, p<0.05). Trehalose administration markedly increased cardiac autophagy (LC3 puncta number 4.1-fold, p<0.05). 10/07/ / 6

48 GR The role of authopagy as a protective mechanism in cardiovascular diseases: new insights into oxidative stress modulation and cardiovascular regeneration Sciarretta Sebastiano Biomedical/Biomedica Istituto Neurologico Mediterraneo Neuromed Notably, when trehalose was administered to beclin-1 +/- mice, which present a defect in autophagy, the protective effects were attenuated (p=ns). Materials and Methods MI and pressure overload will be induced in mice by LAD ligation and transverse aortic constriction, whereas metabolic syndrome by 12 weeks of high fat diet, all under approved animal protocols. Echocardiograph analyses will be performed with Visualsonics Vevo 2100 and its offline software. Autophagy will be assessed by the evaluation of LC3-II and p62 levels, and mrfp-gfp-lc3 puncta. NOX2 activity will be evaluated by ELISA assay, whereas O2- and H2O2 concentration by DHE, DCF and Amplex Red assays. Platelet activity will be assessed by aggregation and recruitment test. CPCs will be isolated as cardiospheres from surgical biopsies of patients under approved clinical protocols and procedures, and from explanted murine hearts. For the in vivo model, cells will be acutely delivered in mice by direct intramyocardial injection. Endothelial function will be evaluated through the study of endothelial-dependent vascular relaxation and cerebral blood flow through two-photon microscopy. Impact and Translational Implications Our study may provide new insights into novel therapies for HF and stroke, potentially reducing their high social and medical costs. Our results may represent a translational basis to scale-up future trials in humans, testing the efficacy of autophagy activation to prevent cardiac remodeling and ED. Indeed, the proposed project may constitute an original, cutting-edge tool to counteract HF, promote cardiac regeneration, and reduce ED and vascular damage. 10/07/ / 6

49 GR Functional inhibition of HSP105 as a new therapeutic strategy for human aggressive B-cell non-hodgkin lymphomas Zappasodi Roberta Biomedical/Biomedica LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Project Classification IRG: Project Classification SS: Oncology 1 - Basic Translational Tumor Cell Biology - TCB Project Keyword 1: Project Keyword 2: Project Keyword 3: Pathways regulated by oncogenes and tumor suppressors that affect tumor cell phenotype and behavior, such as survival, proliferation, and death B-cell non-hodgkin lymphomas Heat Shock Proteins Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Medical Oncology Department, Immunotherapy and Anticancer Innovative Therapeutics Unit 2 Fondazione IRCCS Istituto Nazionale dei Tumori - Milano Experimental Oncology Department, Molecular Targeting Unit 3 University of Palermo, Italy Department of Health Science, Human Pathology Section Generation of HSP105 silenced B-NHL models, identification of biomarkers for HSP105 inhibition, in vitro functional screening of anti- HSP105 agents Anti-HSP105 mab characterization and in vivo studies of selected anti-hsp105 agents as monotherapies and in combination with available anti-lymphoma treatments Pathology imaging and analyses of primary human B-NHL tissues and tumor xenografts from in vivo preclinical studies of anti-hsp105 agents 11/07/ / 6

50 GR Functional inhibition of HSP105 as a new therapeutic strategy for human aggressive B-cell non-hodgkin lymphomas Zappasodi Roberta Biomedical/Biomedica Investigators, Institution and Role in the Project Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Castagnoli Lorenzo Fondazione IRCCS Istituto Nazionale dei Tumori - Milano Responsible for anti-hsp105 mab characterization and in vivo studies of selected anti-hsp105 agents as monotherapies and in combination with available anti-lymphoma treatments 17/11/ Guarnotta Carla University of Palermo, Italy Responsible for pathology imaging and analyses of primary human B-NHL tissues and tumor xenografts from in vivo preclinical studies of anti-hsp105 agents 26/09/ ruggiero giusi Fondazione IRCCS Istituto Nazionale dei Tumori - Milano Collaborator in the generation of HSP105 silenced B-NHL models, identification of biomarkers for HSP105 inhibition, in vitro functional screening of anti-hsp105 agents 27/01/1985 Background and Significance We showed that dendritic cell-based vaccination in relapsed B-cell non-hodgkin lymphomas (B-NHLs) patients achieved clinical efficacy strongly associated with multifaceted immunologic responses, including tumor-restricted humoral immunity (Di Nicola et al., Blood 2009). In particular, we found that responder patients specifically developed an antibody (Ab) response against heat shock protein (HSP) 105 (Zappasodi et al, Blood 2011). Moreover, we have demonstrated that HSP105 is expressed on the surface of B-NHL cells in function of their aggressiveness, and its targeting by a specific polyclonal Ab provides a significant therapeutic activity against human aggressive B-NHLs in vivo (Zappasodi et al., Blood 2011). This indicates that HSP105 constitutes a novel potential therapeutic target in these diseases. B-NHLs can rapidly induce HSPs in response to proteotoxic stress (Zappasodi et al., Cancer Research 2010), and use this mechanism to maintain protein homeostasis and ensure overexpression of key oncogenes (Cerchietti et al., Nature Medicine 2009). In light of these findings, we believe that targeting HSP105 might offer a new valuable therapeutic option to be combined with current anti-lymphoma treatment and improve the management of the many patients who still relapse or do not respond to standard chemoimmunotherapy. Specific aims Aim 1: Selection of HSP105-specific monoclonal(m)abs and molecular inhibitors Screening of hybridomas obtained by anti-hsp105 immunization and available libraries of HSP inhibitors for HSP105 specificity Aim 2: Anti-lymphoma activity of HSP105-specific mabs and inhibitors, and identification of relevant oncogenic pathways affected by HSP105 inactivation Analysis of the antitumor effects of the selected anti-hsp105 agents against human aggressive B-NHL models in vitro and in vivo and identification of the affected oncogenic pathways Aim 3: Identification of effective therapeutic combinations based on anti-hsp105 agents against human aggressive B- 11/07/ / 6

51 GR Functional inhibition of HSP105 as a new therapeutic strategy for human aggressive B-cell non-hodgkin lymphomas Zappasodi Roberta Biomedical/Biomedica Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano NHLs In vitro and in vivo study of the anti-lymphoma activity of the selected anti-hsp105 agents in combination with inhibitors of rational HSP105 co-targets identified in Aim 2 and/or standard anti-lymphoma treatments Hypothesis: Based on our previous work, we hypothesize that HSP105 is specifically involved in the stabilization of the expression of key oncoproteins in aggressive B-NHLs, which may thus result particularly sensitive to its targeting. The availability of anti-hsp105 agents will offer a new therapeutic strategy for B-NHLs as well as a mean to identify the pro-lymphomagenic pathways sustained by HSP105 overexpression and the most suitable co-targets for more effective anti-hsp105-based drug combinations. Preliminary data: To model the effects of HSP105 inhibition and understand the therapeutic potential of this strategy, we have studied the biomolecular consequences of HSP105 silencing in the human Burkitt lymphoma cell line Namalwa. In vitro and in vivo growth of silenced (sihsp105) cells was significantly delayed up to the complete loss of tumorigenicity when 10^4 cells were injected in mice. Interestingly, in sihsp105 cultures and xenografts the expression of BCL-6 and c-myc - the two oncoproteins most frequently involved in aggressive B-NHL pathogenesis - were significantly downregulated with respect to control cells. These preliminary results highlight the potential therapeutic value of HSP105 inhibition in aggressive B-NHLs, including those subtypes overexpressing c-myc and Bcl-6, which are characterized by an extremely unfavorable prognosis. In view of HSP105 cell-surface localization in aggressive B-NHLs and the promising anti-lymphoma activity of a commercial polyclonal anti-hsp105 Ab we reported, we believe that targeting HSP105 by specific mabs may be an effective anti-lymphoma therapeutic strategy. Therefore, we have started to immunize mice with a recombinant human HSP105 to develop specific mabs by hybridoma technology. So far, we have identified an early hybridoma clone whose Abs immunoprecipitate HSP105 from lymphoma cell lysates and react against HSP105+ but not HSP105- live NHL cells by flow cytometry. This is consistent with its ability to recognize the native form of HSP105 and suitability for passive immunotherapeutic approaches. Materials and Methods HSP105+ hybridoma supernatants will be selected by flow cytometry and immunoprecipitation. Screening of HSP105 inhibitors will be performed in collaboration with the Department of Pharmacology at Weill Cornell Medical College. HSP105 inhibitors will be tested against sihsp105 and MOCK B-NHL cell lines in standard proliferation/cytotoxicity assays. Compounds with preferential activity against MOCK cells will be studied in lymphoma-xenografted immunodeficient mice. Anti-HSP105 mabs will be studied both in vitro and in vivo to assess direct (non immune-mediated) and indirect (Fc mediated) anti-lymphoma effects. Gene expression microarray analyses and western blot of known B-NHL oncoproteins in HSP105-inhibited and control B- NHL cells will serve to identify biomarkers for HSP105 inactivation and suitable pathways to target in combination with HSP105 blockade. Combinations of selected anti-hsp105 agents (Aim 1, 2) and suitable drugs (Aim 2) will be investigated in vitro and in vivo. 11/07/ / 6

52 GR Functional inhibition of HSP105 as a new therapeutic strategy for human aggressive B-cell non-hodgkin lymphomas Zappasodi Roberta Biomedical/Biomedica Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Impact and Translational Implications Our research will provide novel anti-hsp105 agents and indication for the most rational anti-lymphoma therapeutic combinations based on HSP105 inhibition, paving the way for their clinical evaluation. In view of our preliminary data supporting that HSP105 favors the expression of the two most common oncogenes in aggressive B-NHLs, which are currently untargetable, the availability of anti-hsp105 agents might enable their indirect inhibition as an effective new therapy for these diseases.

53 Role of Lamin A/C-Polycomb crosstalk in Emery-Dreifuss Muscular Dystrophy (EDMD) BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Lanzuolo Chiara Biomedical/Biomedica Fondazione Santa Lucia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Genes, Genomes and Genetics Molecular Genetics - MGA Project Keyword 1: Project Keyword 2: Project Keyword 3: Chromatin: Chromatin structure function, remodeling and modification, epigenetic control mechanisms, DNA and histone modifications, gene silencing, functions of non-coding RNAs Polycomb, epigenetics, transcriptional regulation Lamin A/C, Emery Dreifuss Muscular Dystrophy Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Epigenetics and stem cells Project coordinator Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date Background and Significance Laminopathies are rare human degenerative disorders caused by mutations in Lamin A/C gene. The laminopathy with the highest frequency (1/ ) in human populations is the autosomal dominant Emery-Dreifuss muscular dystrophy (AD- EDMD). To date, despite the large number of identified mutations, it is difficult to correlate phenotype and genotype in EDMD, suggesting an involvement of the individual epigenetic background to the disease. Although recently has been emerging that changes in chromatin architecture are peculiar of several laminopathies, the epigenetic players and molecular mechanisms involved in laminopathy pathogenesis and progression are not described. Key epigenetic regulators of chromatin architecture are Polycomb group (PcG) of proteins, epigenetic transcriptional repressors of genes primarily involved in differentiation and development. In particular, during myogenesis, modulation of EZH2 levels, the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) ensures the correct muscle differentiation. In the nucleus, PcG proteins form microscopically visible foci and high-through-put data together with microscopy analysis revealed that their targets are organized in chromatin loops. We show in preliminary data, that Lamin A/C sustains PcG foci influencing PcG nuclear compartmentalization and modulating their repressive functions. We will use our expertise in epigenetics to identify and to characterize PcG dependent mechanisms involved in EDMD pathology. Specific aims 10/07/ / 5

54 Role of Lamin A/C-Polycomb crosstalk in Emery-Dreifuss Muscular Dystrophy (EDMD) BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Lanzuolo Chiara Fondazione Santa Lucia Aim 1: Aim 2: Aim 3: Dissection of the functional contribution of PcG proteins in EDMD pathology. Characterization of the role of Lamin A/C/PcG interplay in muscle regeneration Analysis of satellite cell pool dynamics in the pathogenesis and progression of EDMD. Hypothesis: Within this proposal we will test the hypothesis that in Emery Dreifuss Muscular Dystrophy altered PcG protein functions could lead to an exhaustion of satellite cell compartment which ultimately leads to an impaired muscle regeneration contributing to the progression of skeletal muscular dystrophy. Preliminary data: Laminopathies affects several physiological processes, such as differentiation and aging, where PcG proteins play a key role, suggesting a functional interplay between Lamin A/C and PcG proteins. In our unpublished data we demonstrate that Lamin A/C is required to modulate PcG function. During myogenesis, Lamin A/C depletion leads to an anticipated onset of muscle differentiation evidenced by the premature expression of PcG-regulated muscle genes. Transcriptome analysis revealed that among almost 4800 genes deregulated upon Lamin A/C depletion, more than half are similarly affected by EZH2 depletion. Gene ontology analysis of common upregulated genes revealed that most of them are involved in muscle differentiation and development, suggesting that Lamin A/C and PRC2 act on same pathways to regulate the onset of myogenesis. At the molecular level we show that Lamin A/C physically interacts with PcG proteins and these interactions are required for a correct muscle genes repression. Confocal microscopy supported by new algorithms for image analysis together with electron microscopy and proximity ligation assays reveal that Lamin A/C knockdown leads to PcG foci disassembly and PcG proteins dispersion. This causes EZH2 detachment from chromatin leading to impaired PcG repressive functions. (Described data are currently under consideration for publication). In the first four weeks of mouse life, satellite cells undergo multiple rounds of cell division to form the correct muscle mass, following the body growth. During adult life there is low myonuclear turnover so satellite cells become mitotically quiescent. These cells are characterized by Pax7 expression. After injury, satellite cells leave their niche becoming active and start to coexpress Pax7 and MyoD. One of the PcG targets affected by Lamin A/C depletion is Pax7, a key regulatory transcriptional factor involved in the maintenance of satellite cell pool. Using the mouse model for EDMD, the Lmna delta- 8-11, and analysing different time points after birth we found an higher number of Pax7/MyoD positive cells in mutant muscle fibers. These cells are incapable to enter the myogenic terminal differentiation program due to aberrantly high levels of Pax7. This is reflected into an exhaustion of the muscle stem cells pool, which we speculate might ultimately leads to the impaired muscle growth and Lamin A/C-dependent muscular dystrophy. 10/07/ / 5

55 Role of Lamin A/C-Polycomb crosstalk in Emery-Dreifuss Muscular Dystrophy (EDMD) BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Lanzuolo Chiara Fondazione Santa Lucia Materials and Methods This proposal will combine molecular and cellular biology and the use of high-resolution techniques, such as genome wide studies. Experiments will be performed in two mouse models for EDMD: the Lmna delta-8-11 and the knock in LmnaH222P. We will try to reproduce the dystrophic phenotype in the heterozygous Lmna delta-8-11 by performing multiple injury/regeneration experiments and evaluating the regeneration potential of satellite cells. Polycomb mediated epigenetic signatures during muscle regeneration will be analysed by using ChIP, IF, RT-PCR and FISH. To unravel the role of PcG dependent chromatin architecture in EDMD pathogenesis we will perform RNAseq, Chip-seq and DNA seq in Lmna delta-8-11 satellite cells. To evaluate their relevance in EDMD, selected PcG regulated pathways involved in EDMD pathogenesis will be also investigated in LmnaH222P. All proposed technologies, instrumental for the successful completion of the proposal were already set up in the laboratory. Impact and Translational Implications This study will uncover an unprecedented role of PcG proteins in development of muscular dystrophy. Identification of epigenetic defects that precede the onset of pathological phenotype could open up new scenarios and could indicate time frames in which it is possible to intervene to correct defective PcG dependent molecular pathways involved in EDMD pathogenesis. This should stimulate the development of new pharmacological approaches aimed at reverting epigenetic dysfunctions in EDMD.

56 GR Prevention of cancer therapy-related heart failure through phosphoinositide 3-kinase gamma inhibition Morello Fulvio Biomedical/Biomedica Piemonte LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Cardiovascular and Respiratory Sciences Cardiac Contractility, Hypertrophy, and Failure - CCHF Project Keyword 1: Project Keyword 2: Project Keyword 3: The basic molecular and cellular mechanisms underlying cardiac hypertrophy and failure: myocyte growth, proliferation, metabolism and apoptosis; receptor signaling; transcriptional pathways; inflammatory/ cytokine-mediated processes. heart failure cancer Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Città della Salute e della Scienza di Torino, Molinette Hospital, Emergency Department, S.C. Medicina d'urgenza Coordination, fund management, experimental design, interpretation of data, experimental procedures Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 LI MINGCHUAN Animal and cell work, experimental procedures 2 Ciraolo Elisa Animal and cell work, experimental procedures 20/02/ /07/ /07/ / 6

57 GR Prevention of cancer therapy-related heart failure through phosphoinositide 3-kinase gamma inhibition Morello Fulvio Biomedical/Biomedica Piemonte Background and Significance Cancer therapy is associated with increased risk of cardiovascular disease, leading to early discontinuation and to an excess of cardiovascular morbidity, mortality and hospitalization. In particular, both cytotoxic and targeted anti-cancer drugs (such as anthracyclines and trastuzumab) can cause the development of heart failure, especially in elderly patients and in patients with heart disease. As both the number of patients at-risk and patients surviving cancer are increasing, cancer therapy-related heart failure (CTHF) has become a relevant issue worldwide. Population ageing and aggressive use of cancer therapies will further enhance the impact of CTHF in the future. Clinical evaluation can identify patients at-risk, but there are currently no means to prevent CTHF. Thus, targeting of molecular mechanisms underlying iatrogenic cardiotoxicity provides a unique opportunity to reduce the cardiovascular burden of anti-cancer therapies. Phosphoinositide 3-kinase gamma (PI3Kg) is a pivotal signaling enzyme mediating the processes of inflammation and cardiac remodeling, and is implicated at-large in heart failure. We have obtained preliminary data that ablation of PI3Kg significantly reduces CTHF in mice. We therefore propose to test in preclinical studies the hypothesis that inhibition of PI3Kg activity can prevent CTHF, to explore the underlying molecular mechanisms, and to define preventive protocols targeting PI3Kg applicable to patients receiving cancer therapy. Specific aims Aim 1: Aim 2: Aim 3: Does genetic ablation of PI3Kg prevent CTHF? Experimental design: in vivo analyses of genetically modified and wild-type mice receiving anti-cancer therapies. Endpoints: mortality, cardiac size and function (systolic, diastolic), arrhythmogenesis. Does pharmacological inhibition of PI3Kg prevent CTHF? Experimental design: in vivo analyses of wild-type mice receiving anti-cancer therapies and PI3Kg inhibitors. Endpoints: see aim 1. What are the molecular mechanisms linking PI3Kg to CTHF? Experimental design: (1) ex vivo analyses of hearts from genetically modified and wild-type mice receiving anti-cancer therapies, and (2) in vitro analyses of mouse cardiomyocytes treated with anti-cancer agents. Endpoints: molecular signaling, apoptosis, autophagy, oxidative stress, inflammation, fibrosis. Hypothesis: Anti-cancer agents such as anthracyclines and trastuzumab cause cardiomyocyte damage through several mechanisms including oxidative stress, apoptosis and inflammation. Previous studies from this and other groups have demonstrated that myocardial stress and inflammation enhance PI3Kg activity, both in cardiomyocytes and in infiltrating cells. In turn, PI3Kg engages pathological responses leading to further oxidative burst, cardiomyocyte damage, myocardial fibrosis, and ultimately diastolic and systolic cardiac dysfunction. Of note, targeting of PI3Kg has been shown to protect from heart failure in different experimental models. Hence, our working hypothesis is that inhibition of PI3Kg can prevent CTHF. Preliminary data: We have taken advantage of genetically modified mice expressing a kinase-dead (KD) PI3Kg, modeling PI3Kg inhibition. High-dose doxorubicin (4 mg/kg i.p. weekly, cumulative dose 12 mg/kg) led to lower mortality in PI3Kg KD mice (20%, n=20) than in wild-type (WT) mice (50%, n=16, p<0.05) within 6 weeks. Echocardiography performed 8 weeks after the first doxorubicin injection showed preserved left ventricular (LV) systolic function in PI3Kg KD mice (fractional shortening FS=37±2%, -7% vs baseline, n=6), as compared to severe systolic dysfunction in WT mice (FS=21±1%, -40% vs baseline, n=6, p<0.001). PI3Kg KD mice had unchanged LV wall thickness and were protected against doxorubicin-induced cardiac cachexia, as evidenced by preserved cardiomyocyte cross-sectional area (PI3Kg KD 313±18 µm2, n=6 vs WT 243±9 µm2, n=7, p<0.01). Furthermore, TUNEL and picrosirius red stainings revealed reduced cardiomyocyte apoptosis (PI3Kg KD 29±2%, n=9 vs WT 37±3%, n=9, p<0.05) and 02/07/ / 6

58 GR Prevention of cancer therapy-related heart failure through phosphoinositide 3-kinase gamma inhibition Morello Fulvio Biomedical/Biomedica Piemonte fibrosis (PI3Kg KD 2.9±0.2%, n=16 vs WT 5.2±0.3%, n=14, p<0.001) in PI3Kg KD mice. Genetic inhibition of PI3Kg led to enhanced activation of protective autophagy after doxorubicin, with increased LC3II to LC3I ratio (marking autophagosomal formation), and decreased expression of the autophagy substrate p62. Taken together, these data suggest that PI3Kg inhibition markedly protects from CTHF by limiting cardiomyocyte proteotoxicity and by eventually reducing pathological cardiac remodeling and dysfunction. Materials and Methods Mice will be housed and manipulated in a dedicated facility. Systemic PI3Kg inhibition will be modeled genetically with locally available PI3Kg KD mice expressing a kinase-inactive PI3Kg. Control animals will be age-matched C57BL/6J mice. CTHF will be obtained by administration of doxorubicin (4 mg/kg i.p. weekly, cumulative dose 12 mg/kg), trastuzumab (2 mg/kg i.p., single dose) or both drugs. Pharmacologic inhibition of PI3Kg will be obtained by administration of AS (10 mg/kg i.p.) at every scheduled dose of anti-cancer agents. Non-invasive monitoring of cardiac morphology and function will be performed by echocardiography with Vevo 2100 Imaging System (Visual Sonics Inc.). Invasive pressure-volume analysis will be performed with a transducer catheter and instrumentation (Millar Instr.). Heart tissues will be analyzed by standard histological staining and molecular analyses of RNA and protein. Mouse neonatal cardiomyocytes will be harvested and plated with standard protocols. Impact and Translational Implications CTHF affects the morbidity, mortality and hospitalization of cancer patients even decades after cancer therapy. Hence, cardioprotective strategies preventing CTHF represent a so far unmet clinical need. Preliminary data define PI3Kg inhibition as a novel promising strategy to reduce CTHF. This strategy is feasible in humans, as drugs targeting PI3K enzymes (including orally administered compounds) are already available and undergoing clinical trial, and novel inhibitors are under development. 02/07/ / 6

59 GR Estimating the risk for cardiac toxicity in patients undergoing anticancer therapies: a new dynamic risk algorithm integrating levels of cardiovascular serum biomarkers. Puntoni Matteo Clinical health care research/clinicoassistenziale Liguria LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Oncology 2 - Translational Clinical Clinical Oncology - CONC Project Keyword 1: Project Keyword 2: Project Keyword 3: Pharmacologic and toxicologic studies of new modalities in patients and correlative studies relevant to therapeutic clinical trials. cardiovascular risk anthracyclines and target therapies Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION E.O. Ospedali Galliera/Scientific Directorate Coordinator: enrollment, design, data collection and analysis 2 IRCCS-IRST, Meldola (FC) U.O. Oncologia Medica Operative Unit: enrollment 3 Regione Toscana/Ospedale Misericordia USL9 Dipartimento di Oncologia/U.O.C. Oncologia Medica Operative Unit: enrollment Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Casadei Gardini Andrea IRCCS-IRST, Meldola (FC) Clinical Investigator 02/06/ Pastina Ilaria Regione Toscana/Ospedale Misericordia USL9 3 Provinciali Nicoletta E.O. Ospedali Galliera/Medical Oncology Clinical Investigator 22/04/1975 Clinical Investigator 06/06/ /07/ / 6

60 GR Estimating the risk for cardiac toxicity in patients undergoing anticancer therapies: a new dynamic risk algorithm integrating levels of cardiovascular serum biomarkers. Puntoni Matteo Clinical health care research/clinicoassistenziale Liguria Background and Significance The cardiac damage induced by antineoplastic drugs is one of the most common causes of discontinuation of treatment, as well as being a complication difficult to manage with appropriate treatment and high impact on quality of life. The current cardiac monitoring approach assessing the left ventricular ejection fraction (LVEF) detects significant variations only after extensive cardiac damage has occurred; moreover, persistently increased serum levels of troponin and natriuretic peptide during chemotherapy are predictive of cardiac damage and the early introduction of drugs such as ACE inhibitors and betablockers may have a favorable impact on the recovery of cardiac function in cancer patients. Therefore it is important to early identify patients at risk of cardiovascular (CV) events to prevent the development of left ventricular dysfunction. The existing algorithms to predict the risk of cardiac damage in cancer patients have low discriminant and predictive power, do not update the risk prediction during therapy, and do not use information coming from levels of specific serum biomarkers in patients. The purpose of this project is to develop a tool for "dynamic prediction" of the risk of heart disease in patients undergoing antineoplastic treatments in order to provide from one-size-fits-all to a more personalized cardiac risk estimation of antineoplastic treatments, considering also the levels of new biomarkers of CV risk recently emerged from literature data. Specific aims Aim 1: Aim 2: Aim 3: to determine the independent predictors of cardiovascular events, at baseline and during chemotherapy, through a multivariate statistical model. to develop and test the performance of a "dynamic model", using time-dependent variables, to predict the risk of cardiac damage during antineoplastic therapy, in comparison to currently available algorithms. to identify groups of patients at higher risk for cardiac events considering the levels of new predictive cardiac/metabolic biomarkers and type of anticancer therapy, through an explorative graphical statistic technique ("STEPP", see below). Hypothesis: A strong limitation of the current algorithms for the evaluation of risk of cardiotoxicity is that they estimate the risk at basal condition, before treatment. Therefore, we will: 1) create a "dynamic" model, that includes the modification of clinical variables during chemotherapy, in order to allow clinicians to update the estimates of risk and make the more appropriate therapeutic choice (i.e. continuing, discontinuing, changing chemotherapy avoiding the interruption of a potentially effective treatment); 2) Use the STEPP method (Subpopulation Treatment Effect Pattern Plot) to show how the risk of an event changes as a function of a covariate of interest (i.e. a biomarker), in its continuous scale, following the principle that categorizing the expression of a biomarker results in a loss of information, thus failing to identify the worth of the biomarker as a predictor of an effect (Stat Med 2006;25;127). Preliminary data: Evidence indicates that the early detection and treatment (i.e. ACE-inhibitors and beta-blockers) of cardiotoxicity may have a favorable impact on cardiac function and prognosis in cancer patients (J Am Coll Cardiol 2009;53:2231). LVEF is currently monitored during chemotherapy (J Clin Oncol 2010;28:3429; J Clin Oncol 1999;11:3596), but a decrease of LVEF certifies a cardiac damage already occurred, limiting the likelihood of a functional recovery. Troponins and natriuretic peptide (NT-proBNP) are now considered more specific biomarkers for early detection of cardiotoxicity induced by antineoplastic drugs (Br J Cancer 2011;105:1663). Preliminary data indicate that persistently increased serum levels of troponin and NT-proBNP during chemotherapy are predictive of cardiac damage and development of left ventricular dysfunction. In our institution a multidisciplinary CV risk assessment unit (mainly oncologists and internists) has been set up in order to perform every 3 months a set of assessments on patients undergoing high-risk CV anticancer therapies, including: physical examination, lipid profile, microalbuminuria, troponin, brain natriuretic 11/07/ / 6

61 GR Estimating the risk for cardiac toxicity in patients undergoing anticancer therapies: a new dynamic risk algorithm integrating levels of cardiovascular serum biomarkers. Puntoni Matteo Clinical health care research/clinicoassistenziale Liguria peptide (BNP) and LVEF. The early institution of appropriate therapy (antihypertensives, ACE inhibitors, lipid profile control by dietary recommendations) allowed physicians to avoid the interruption of anticancer treatment on all the first 50 patients involved. Materials and Methods Study population will be breast, lung and colorectal cancer patients with advanced disease, willingness to undergo cardiac monitoring, without clinically relevant cardiac disease and uncontrolled hypertension. The monitoring program will include CV biochemistry (lipid profile, microalbuminuria, troponin and BNP) and instrumental exams (echocardiography and blood pressure). Dose and duration of anticancer drugs will be registered and patients will be stratified by type of cancer. The main study outcome will be the functional heart disease, defined as a lower ejection fraction until the onset of heart failure symptoms or signs. 200 patients enrolled in 2 years +1 year of follow-up will be sufficient to observe 40 events of cardiomyopathy giving to the study the adequate power to test the combinations of risk factors associated with a relative risk of cardiotoxicity >2 (95%CI x/ 2) and to employ STEPP analysis. Impact and Translational Implications Towards the era of personalized medicine, the institution in the standard clinical practice of a multidisciplinary model of care for CV risk assessment adopting a predictive tool able to estimate the risk of cardiac damage during therapy ("dynamic" algorithm), including precise information (STEPP method) from new important biomarkers (troponin, BNP), could have a direct impact on the decision making of clinicians and on prognosis and quality of life of cancer patients. 11/07/ / 6

62 GR Role of biological markers for cerebral bleeding risk stratification in patients with atrial fibrillation on oral anticoagulants for primary or secondary prevention of ischemic stroke Poggesi Anna Clinical health care research/clinicoassistenziale Toscana LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Biology of Development and Aging Aging Systems and Geriatrics - ASG Project Keyword 1: Project Keyword 2: Project Keyword 3: Development and validation of biomarkers of biological health and aging. atrial fibrillation dementia and cognitive decline Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION SOD STROKE UNIT E NEUROLOGIA DAI CUORE E VASI AOU CAREGGI 2 ALMAMATER STUDIORUM DIPARTIMENTO DI INGEGNERIA UNIVERSITA' DI BOLOGNA DELL'ENERGIA ELETTRICA E DELL'INFORMAZIONE "GUGLIELMO MARCONI" 3 AZIENDA OSPEDALIERO UNIVERSITARIA CAREGGI Investigators, Institution and Role in the Project ATHEROTHROMBOTIC DISEASE UNIT DAI CUORE E VASI Study coordination, patient enrollment, clinical assessment at baseline and follow-up, data collection, dedicated database MRI protocol definition and image postprocessing biological biomarkers assessment Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Diciotti Stefano ALMAMATER STUDIORUM UNIVERSITA' DI BOLOGNA 2 Cesari Francesca AZIENDA OSPEDALIERO UNIVERSITARIA CAREGGI 3 Salvadori Emilia UNIVERSITA' DEGLI STUDI DI FIRENZE 4 Pescini Francesca AZIENDA OSPEDALIERO UNIVERSITARIA CAREGGI Biomedical Engineer: MRI image post-processing Biologist: biological biomarker assessment Neuropsychologist: neuropsychological evaluation and data analyses Neurologist: patient enrollment, clinical assessment at baseline and follow-up, data collection 04/09/ /11/ /10/ /10/ /07/ / 6

63 GR Role of biological markers for cerebral bleeding risk stratification in patients with atrial fibrillation on oral anticoagulants for primary or secondary prevention of ischemic stroke Poggesi Anna Clinical health care research/clinicoassistenziale Toscana Background and Significance Thromboprophylaxis with oral anticoagulation is effective in reducing stroke risk in patients with atrial fibrillation (AF). Benefits have to be balanced against the risk of bleeding, with intracranial hemorrhage being the most feared one. Stroke and bleeding risk stratification schemes are aimed at identifying patients who may benefit most from oral anticoagulation of different type (new vs old anticoagulants). Such schemes (for example CHADS2VASC2, and HASBLED scores) currently rely on just clinical information, validity of which remains controversial and need to be improved. In AF patients advanced imaging (MRI) technology has led to increased detection of asymptomatic brain changes, particularly those related to small vessel disease (SVD), which represent the pathologic substrate for primary intracerebral hemorrhage. These changes have been also demonstrated to strongly predict stroke risk (Inzitari D, Stroke 2003). Such imaging findings, obtained at baseline and over time (in terms of lesions progression) could be used as additional markers for risk stratification. Beside imaging, markers of coagulation activation, including prothrombin fragment 1+2, thrombin-antithrombin complex, D-dimer, time in therapeutic range for warfarin, and drug dosage for new anticoagulants, may be studied as cofactors. Preliminary data suggest that circulating biomarkers of endothelial dysfunction, hypercoagulable state, and inflammation, may further enhance risk prediction. Specific aims Aim 1: Aim 2: Aim 3: To investigate MRI markers (baseline and progression) of SVD in a cohort of patients with AF on oral anticoagulants as a surrogate marker for prediction of cerebral bleeding To investigate possible effect of circulating biomarkers studied in relation to occurrence or progression of SVD changes as cofactors of prediction To provide preliminary knowledge useful to design adequately sized studies aiming to estimate the incremental value of biological markers, including advanced cerebral neuroimaging, and circulating biomarkers, in the prediction of cerebral bleeding in anticoagulated AF patients in respect of the currently used clinical scores Hypothesis: Validity of models currently recommended for stratification of risk ratio between benefits and hemorrhage risk in anticoagulated AF patients is limited. Moreover, these models do not specifically address intracranial hemorrhage, which is indeed the most severe hemorrhagic complication. We hypothesize that biological markers, both circulating and imaging-based, and their possible interaction, might improve the prediction of bleeding risk in AF patients under treatment with any type of oral anticoagulants. Neuroimaging biomarkers of SVD, particularly microbleeds, may serve as a selective surrogate marker of such complication. Circulating biomarkers assessed together with imaging may further improve predictive potentials Preliminary data: Basis for this project development derive from the large experience accumulated over years by the proponent through observational studies of patients with biomarkers of brain SVD, including: 1. The LADIS (Leukoaraiosis And DISability) Study, supported by the European Union, coordinated by the proponent centre and involving 11 European highly qualified centers, that has investigated clinical and functional outcomes in 639 non-disabled elderly subjects with SVD changes. The project has produced more than 50 publications in major acknowledged international journals (LADIS group, 2011). In LADIS, AF independently predicted stroke risk and transition to functional disability (HR 1.69; 95%CI: ) (Inzitari, 2009). 2. The MILES study, sponsored by MIUR- PRIN 2006, and coordinated by the proponent centre, has evaluated biological markers associated with microvascular leucoencephalopathies, both of the genetic type and the sporadic age-related type. Results have shown an association between endothelial and circulating progenitor cells (EPC and CPC) in CADASIL, suggesting an important role of endothelial dysfunction in these patients (Pescini, 2010). 03/07/ / 6

64 GR Role of biological markers for cerebral bleeding risk stratification in patients with atrial fibrillation on oral anticoagulants for primary or secondary prevention of ischemic stroke Poggesi Anna Clinical health care research/clinicoassistenziale Toscana 3. The ongoing VMCI-Tuscany study, supported by Tuscany region, is evaluating the most advanced, circulating or imaging-based biomarkers as predictors of functional decline, stroke or dementia in patients presenting with mild cognitive impairment and SVD changes (Poggesi, 2011). This study includes a detailed assessment of brain microvascular changes, such as microbleeds, microstructural white matter changes and atrophic abnormalities (Mascalchi, 2012) Materials and Methods Three-hundred patients with AF (>70 years) will be thoroughly assessed by means of a detailed clinical protocol (history, dietary habits, neurological, functional, cognitive status, mood, gait) at baseline and after 12 and 24 months. On entry, brain MRI will be performed in all patients, according to a standardized protocol for the assessment of SVD changes. After 2 years follow-up brain MRI will be repeated, using the same protocol, in order to assess progression of SVD. Circulating biomarkers, assessed at baseline and follow-ups, will include: a) endothelial function biomarkers; b) pro-and antiinflammatory molecules, including metalloproteinases; c) markers of renal function; d) markers of coagulation activation, including prothrombin fragment 1+2, thrombin-antithrombin complex, D-dimer; f) time in therapeutic range (warfarin), and drug dosages (new anticoagulants). Main study outcome: SVD progression. Secondary outcome: stroke occurrence (ischemic or hemorrhagic). Impact and Translational Implications Results from the project may provide information about the possible contribution of biological markers, including neuroimaging and circulating biomarkers, and their possible interaction, in the individualized prediction of bleeding risk in AF anticoagulated patients, contributing to optimizing effectiveness of a preventive treatment such as anticoagulation for stroke prevention in AF patients, a treatment so important to reduce the burden of stroke in our health system 03/07/ / 6

65 GR Deciphering the role of SETD5 in intellectual disabilities and setting a new gene-on therapeutic strategy. Sessa Alessandro Biomedical/Biomedica Ospedale San Raffaele - Milano LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Developmental Brain Disorders - DBD Project Keyword 1: Project Keyword 2: Project Keyword 3: Developmental disorders: Mental retardation, learning disabilities, specific language impairment, dyslexia, autism, cerebral palsy, sudden infant death syndrome - SIDS, and other relevant disorders. Epigenetics gene-on rescue strategy Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION DIBIT/Neuroscience/Stem cells and neurogenesis Principal Investigator 2 Istituto Italiano di Tecnologia Neuroscience and Brain Technologies Electrophysiological analysis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Sacchetti Silvio Istituto Italiano di Tecnologia Responsible for electrophysiological analysis 02/04/ /07/ / 6

66 GR Deciphering the role of SETD5 in intellectual disabilities and setting a new gene-on therapeutic strategy. Sessa Alessandro Biomedical/Biomedica Ospedale San Raffaele - Milano Background and Significance Neurodevelopmental disorders represent a major area of unmet medical need, with a dramatic burden for society. Intellectual disability (ID) and autistic spectrum disorders (ASD) are the most common among such disorders (3-5% of the population). Despite they account for heterogeneous conditions, thereby posing an immense challenge for researchers and clinicians, ID and ASD are genetically very divergent, although in some cases can converge onto common pathways. In fact, the majority of the mutated genes regulate processes involved in neuronal morphology, synapse biology and regulation of protein synthesis and turnover. Loss of function mutations in one SETD5 allele have been recently identified as relatively frequent mendelian causes of both ID and ASD. Interestingly, SETD5 is deleted in the 3p25 microdeletion syndrome which is characterized by ID, microcephaly, cleft palate, congenital heart defects and other phenotypic findings. These findings suggest that SETD5 haploinsufficiency is sufficient to cause the disease and it is a rather frequent common cause of ID and ASD. SETD5 codifies for a putative, but yet uncharacterized, histone H3 methyltransferase highly expressed in the brain. Herein, we plan to analyze its mechanisms of action and to determine the effects of its silencing in neurons. Besides its biological characterization we will challenge new therapeutic pathways exploring the possibility to rescue in our models SETD5 level to minimize the neuronal phenotype. Specific aims Aim 1: Aim 2: Aim 3: Analysis of the biological functions of SETD5 SETD5 has been classified as methyltransferase on the basis of sequence homology to other SET (Su(var)3-9, enhancer-of-zeste, trithorax) domain proteins. In this aim we will determine its enzimatic activity and its importance for chromatin organization both in vivo and in vitro. In addition, we will map the epigenetic modifications in which it is involved on the genome. Since methylation of histone tails deeply impact on the regulation of gene expression, we will likely infer in this way the genetic pathways regulated by SETD5. Development of disease models Since SETD5 deletions or point mutations (nonsense or frameshifts) are recognized to cause ID or ASD through an haploinsufficient mechanism, we will experimentally model the pathogenic conditions lowering the level of SETD5 protein. We have generated effective shrnas against the murine Setd5 to knock it down in primary neurons. These cultures will be analyzed molecularly and functionally to identify neuronal defects possibly explaining the human disease. In addition, we will characterize the brain specific Setd5 conditional knock-out mice which are already available in the lab. This mouse model will give us the opportunity to investigate the phenotype into a whole animal as well as the possibility to obtain full knock-out neuronal cultures. Restoring SETD5 protein levels by means of CRISPR/Cas ON technology Theoretically, a disease due to haploinsufficiency should be successfully treated by restoring the normal levels of the protein. CRISPR/Cas (clustered, regularly interspaced, short palindromic repeats - CRISPR-associated), an adaptive immune system of bacteria and archea was recently adapted for genome editing or for regulation of gene expression in eukaryotes. We propose to employ this cutting edge technology to target Setd5 promoter inducing an increase of transcription from the wild-type allele; this will be likely sufficient to re-establish the physiological levels of the functioning protein. Hypothesis: Many genes between those encoding for epigenetic modifiers (e.g. MECP2, EHMT1, KMT2D, KMD6A, etc) are dosage sensitive and their haploinsufficiency is recognized to have a major contribution to the phenotype of ID and/or ASD. The identification of SETD5 as critical locus for ID/ASD-driving mutations offers the intriguing possibility to give new light on the epigenetic control of gene transcription as relevant cause of intellectual impairment. We have already demonstrated that Setd5 is expressed into neural cells both in cultures and in the 10/07/ / 6

67 GR Deciphering the role of SETD5 in intellectual disabilities and setting a new gene-on therapeutic strategy. Sessa Alessandro Biomedical/Biomedica Ospedale San Raffaele - Milano Preliminary data: brain with particularly high levels of the protein in the postmitotic neurons. Molecularly we were able to localize SETD5 with histone H3, sustaining the idea that it could act as methyltransferase on its tail. We have developed effective shrnas against Setd5 which causes a significant reduction on dendrite harborization when transduced in primary neuronal cultures. Materials and Methods SETD5 activity will be assessed by biochemical methylation assays in vitro and featuring its localization at cell level compared with majors H3 modifications. ChIP assay will be conducted to identify docking sites on DNA at genome scale. Putative Setd5 downstream pathways will be verified upon gain and loss of function experiments both in cell cultures and in vivo by in utero electroporation approach. We will evaluate the Setd5 haploinsufficiency and its total deplicion in CNS analyzing a mouse line harboring floxable Setd5 allele(s) (EMMA consortium) and brain specific Cre drivers. Neuronal functionality upon alteration of Setd5 will be assessed by electrophysiological analysis. We are identifying the Setd5 brain specific promoter in murine genome to design CRISPR/Cas ON strategy to increase its expression. We will apply the system to neuronal cultures with Setd5 +/- genetic background; moreover, using a viral vector based approach we will pursue in vivo correction of Setd5 dosage. Impact and Translational Implications The project here presented would be instrumental in multiple fashions for potential treatment of ID and ASD. Indeed our analysis on the functions of this critical gene will add priceless informations on the pathological processes; this could help to clarify the molecular basis of these diseases possibly opening new routes for therapies. In addition we do expect to find the proof of principle showing the rescue of SETD5 haploinsufficency could be beneficial at both cellular and organism level. 10/07/ / 6

68 GR The risk-benefit profile of pharmacological treatments in secondary prevention for patients with ischemic stroke: a systematic review Tramacere Irene Clinical health care research/clinicoassistenziale Fondazione Istituto Neurologico Carlo Besta LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Healthcare Delivery and Methodologies Biostatistical Methods and Research Design - BMRD Project Keyword 1: Project Keyword 2: Project Keyword 3: Data analysis and modeling: development of statistical theory, analytic methods and models, computational tools and algorithms for the analysis and interpretation of data from clinical studies, randomized trials, epidemiological studies, human genetic association studies, environmental studies, and complex surveys; methods to handle data features and anomalies such as correlation, clustering, missing and skewed data; risk prediction and forecasting methods; causal modeling; high dimensional data methods Comparative efficacy and safety of different treatment options through Cochrane systematic reviews and Network Meta-Analyses Secondary prevention for patients with ischemic stroke Project Request: Animals: Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Neuroepidemiology Unit 1: project coordination and cooperation 2 Università degli Studi di Modena e Reggio Emilia Diagnostic, Clinical and Public Health Medicine Unit 2: project cooperation and methodological support (in particular for statistical analyses) 3 Fondazione IRCCS Istituto Neurologico Carlo Besta Cerebrovascular Diseases Unit 3: project cooperation and clinical support Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Del Giovane Cinzia Università degli Studi di Modena e Reggio Emilia 2 Boncoraglio Giorgio Battista Fondazione IRCCS Istituto Neurologico Carlo Besta Co-worker of the PI, head of Unit 2 24/04/1979 Co-worker of the PI, head of Unit 3, in charge of one young neurologist that will be involved in the project 02/06/ /07/ / 6

69 GR The risk-benefit profile of pharmacological treatments in secondary prevention for patients with ischemic stroke: a systematic review Tramacere Irene Clinical health care research/clinicoassistenziale Fondazione Istituto Neurologico Carlo Besta Background and Significance Worldwide, stroke is the second most common cause of death after myocardial infarction and the leading cause of permanent acquired disability. Stroke survivors are at high risk for subsequent vascular events, including recurrent stroke, myocardial infarction, and death from vascular causes. In secondary prevention, three principal medical regimens appear to be appropriate for nearly all patients with ischemic stroke: anti-hypertensive drugs, statins, and antiplatelet therapy for noncardioembolic stroke, or anticoagulants for patients with cardioembolic stroke subtype. However, only few head-to-head trials directly addressed the role of these therapies in secondary prevention among patients with ischemic stroke, and for each regimen the choice of specific drugs, with the optimal drug dosage, should be clearly defined. The systematic reviews (SRs) and meta-analyses (MAs) of randomized clinical trials (RCTs) have been established as a tool to synthesize study results in order to have a summary of the best evidence for a given comparison. Recently, a novel methodology, called Network Meta-Analysis (NMA), has been developed, which summarizes evidence when many treatments are involved, using both direct and indirect comparisons across trials. We intend to apply this new methodology to the critical clinical question of secondary prevention in patients with ischemic stroke in order to define a ranking of the best available medical therapies. Specific aims Aim 1: Aim 2: Aim 3: To estimate the relative efficacy, acceptability and safety, and provide a ranking of anti-hypertensive drugs, statins, antiplatelet therapy and anticoagulants in secondary prevention of ischemic stroke. To provide results, and recommendations for planning future studies, to scientific societies and agencies developing best practice guidelines. To illustrate, through real clinical questions, an innovative framework based on the new methodology of the Network Meta-Analysis (NMA), to inform clinicians, researchers and policy makers in order to facilitate their decisions. Hypothesis: In patients with ischemic stroke, it has been estimated that at least 80% of recurrent events might be prevented with the use of a comprehensive approach that includes dietary modification, exercise, antihypertensive drugs, statins, and antiplatelet/anticoagulant therapy. Preliminary data: The role of blood pressure (BP) treatment in secondary prevention of ischemic stroke was postulated. However, although initiation of BP therapy appears to be indicated, with a relatively high level of confidence, for patients having an established BP >=140 mm Hg systolic or >=90 mm Hg diastolic, for those patients with BP <140 mm Hg systolic and <90 mm Hg diastolic, initiation of BP therapy is being debated because of uncertain benefit. In addition, the available data indicate that diuretics, angiotensin-converting-enzyme inhibitors, calcium channel blockers, ß-blockers, and angiotensin-receptor blocker are useful, but the choice of specific drugs, schedules and dosages, respect to the patient characteristics, need to be defined. Statin therapy with intensive lipid-lowering effects is recommended to reduce risk of stroke and cardiovascular events among patients with ischemic stroke who have evidence of atherosclerosis, independently of the low-density lipoprotein cholesterol (LDL-C) level and the presence of coronary heart disease. However, very limited populations of patients, in particular with low (<100 mg/dl) LDL- C level, were evaluated. Furthermore, the choice of specific drugs and LDL-C level target reduction need to be still defined. For patients with non-cardioembolic ischemic stroke, the use of antiplatelet agents is recommended, although various therapies, as aspirin monotherapy, the combination of aspirin and dipyridamole, and clopidogrel monotherapy, appear to be all acceptable options, in the absence of strong 10/07/ / 6

70 GR The risk-benefit profile of pharmacological treatments in secondary prevention for patients with ischemic stroke: a systematic review Tramacere Irene Clinical health care research/clinicoassistenziale Fondazione Istituto Neurologico Carlo Besta evidences on which is the optimal drug therapy. For patients with cardioembolic ischemic stroke, dose-adjusted warfarin has been the mainstay of therapy. Newer oral anticoagulant strategies, including direct thrombin inhibitors and factor Xa inhibitors, are now available and are likely to replace warfarin in many cases, although to date no conclusive evidence on the most successful alternative anticoagulant is available. NMAs are increasingly published in major medical journals embracing several medical fields, like oncology, rheumatology, neurology and it is expected to become even more popular. Materials and Methods Two Cochrane systematic reviews (SRs) of randomized controlled trials (RCTs) comparing the efficacy, acceptability and safety of anti-hypertensive drugs, statins, antiplatelet therapy and anticoagulants versus placebo or another active treatment commonly used for secondary prevention in patients with cardioembolic or non-cardioembolic ischemic stroke will be performed. Outcome measures will be all-cause mortality, cardiovascular mortality, recurrent stroke and myocardial infarction for efficacy; withdrawals due to adverse events (AEs) for acceptability, and serious AEs (SAEs) for safety. Subgroup analyses will be performed by age, gender, patients with hypertension, high levels of cholesterol, diabetes. The Network Meta-Analysis (NMA) will be performed to summarize the evidence and rank the treatments according to their efficacy, acceptability and safety. GRADE will be used to evaluate quality of the evidence. Aim 2 and 3 will be achieved using the developed framework. Impact and Translational Implications To provide the best available evidence on the risk-benefit profiles of competitive treatments for secondary prevention of ischemic stroke to optimize patient care, reduce inappropriate variation, and likely decrease treatment costs for the National Health System. To inform research priorities by highlighting areas where uncertainty exists. To provide training activities targeted to stakeholders to critically appraise and use results of systematic reviews (SRs) and Network Meta-Analyses (NMAs). 10/07/ / 6

71 GR Comprehensive tracking of hematopoietic stem/progenitor cells dynamics and activity in vivo in humans by insertional barcoding Biasco Luca Biomedical/Biomedica Ospedale San Raffaele - Milano LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Infectious and immunological diseases Project Classification IRG: Project Classification SS: Genes, Genomes and Genetics Gene Therapy and Inborn Errors - GTIE Special Emphasis Panel Project Keyword 1: Project Keyword 2: Project Keyword 3: Studies of transduction, integration, replication and repair, gene expression and gene silencing mechanisms in animal and human tissues and in animal models of diseases Hematopoietic stem cell biology In vivo clonal tracking Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) Principal Investigator 2 Harvard Medical School Department of Systems Biology Mathematical modeling Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Klein Allon Harvard Medical School Mathematical modeling 24/06/ Dionisio Francesca San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) 4 Scala Serena San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) Vector production insertion site retrieval and molecular barcoding Transduction / phenotypic and functional studies on transduced cell 20/10/ /05/ /07/ / 5

72 GR Comprehensive tracking of hematopoietic stem/progenitor cells dynamics and activity in vivo in humans by insertional barcoding Biasco Luca Biomedical/Biomedica Ospedale San Raffaele - Milano Background and Significance The hematopoietic system is composed by a variety of blood cell types covering a wide spectrum of different functions essential to human physiology. Hematopoietic stem\progenitor cells (HSPC) are endowed with the ability of producing all blood cells being capable of multipotency and self-renewal capacity (Seita and Weissman, 2010). For these reasons, treatments involving genetic engineering and transplantation of HSPC are being successfully employed for an increasing number of hematological diseases and disorders (Naldini et al, 2011). Still, dynamics, clonal composition and activity of HSPC in humans, during early hematopoietic reconstitution and years after transplant, remain uncovered. Hierarchical relationships among mature blood cell types and maintenance of hematopoietic reservoir in humans are also currently under intense investigations. Addressing these questions in vivo in patients would be of paramount importance for the development of novel and more efficacious therapeutic approaches. However, despite important advances in the field, human hematopoiesis is still mainly studied only through in vitro assays and humanized animal models (Doulatov et al, 2012; Dykstra et al, 2014). An ideal setting would require the possibility to univocally mark in vitro individual HSPC clones and to then track in vivo in humans the fate and dynamics of the infused HSPC and their progeny at single clone level. Specific aims Aim 1: Aim 2: Aim 3: To study HSPC survival and dynamics in humans To study clonal properties and molecular marking of infused HSPC To study clonal and hierarchical properties of genetically engineered T cells and other mature cell types Hypothesis: This study proposal is based on the concept that, upon retroviral gene transfer, each target cell becomes univocally tagged by an integration site (IS). Our gene therapy (GT) clinical trials for ADA (adenosine deaminase) deficient-scid (Aiuti et al, 2009) and Wiskott-Aldrich Syndrome (WAS) (Aiuti et al, 2013), based on genetically engineered HSPC, essentially generated a stable "transgenic" hematopoietic system where infused HSPC clones give rise to a blood cell progeny traceable in vivo in humans by IS analysis. Our general aim is to study hematopoietic system dynamics and hierarchical relationships at clonal levels in humans by IS-tracing and to analyze in details the insertional marking of the different subpopulations composing the infused cell products. The result of this work will have crucial implication for the development of novel gene- and cell-based therapies for hematological diseases and tumors. Preliminary data: Over the last years we collected IS from linear amplification mediated PCR combined with highthroughput sequencing on a variety of cell lineages purified from bone marrow and peripheral blood from GT treated patients. To date our IS database comprises a total of IS from 4 WAS patients up to 3 years after GT and IS from 4 ADA-SCID patients up to 6 years after GT. We confirmed the marking of multipotent HSPC, tracking overtime shared integrations between HSPC/colony forming units and PB lineages. We unraveled the timing and nature of short, intermediate and long term HSPC output showing that it occurs in distinct waves during the first 6-9 months after transplantation reaching a 'homeostatic equilibrium' only by 12 months after GT and that early reconstitution is markedly skewed towards myeloid production. We showed that identical IS are consistently detected at multiple lineages level even several years after GT, and collected evidence that HSPC 'awaken' from dormancy in vitro, due to gammaretroviral transduction protocol, can still retain in vivo long-term activity in humans. More recently we designed a strategy to purify and analyze the molecular IS-marking of 7 different types of CD34+ progenitors, isolated ex vivo from treated patients. We are extending this analysis to the infused cell product and to CD34+ cells transduced with different lentiviral and retroviral-based vector platforms. We are also studying genetically engineered T-cell subtypes from ADA-SCID GT patients receiving infusions of gene- 10/07/ / 5

73 GR Comprehensive tracking of hematopoietic stem/progenitor cells dynamics and activity in vivo in humans by insertional barcoding Biasco Luca Biomedical/Biomedica Ospedale San Raffaele - Milano corrected mature lymphocytes. We found that transduced T memory stem cells (TSCM) survived and retained their plasticity in vivo for >10 years after infusion. We showed that TSCM have been stably generated and enriched upon in vitro stimulation used for lymphocytes transduction. Materials and Methods We will purify from GT patients samples different lineages by FACS sorting/magnetic beads purification, and sort different HSPC populations ex vivo at different time points after GT and in vitro after transduction with different vector platforms. We will exploit LAM-PCR, a protocol well established in our group, for enrichment of vector-genome junctions and, to improve the quantification power, we will design new unbiased methods based on genome shearing. We will perform deep sequencing through state-of-the-art platforms (currently Illumina-MiSeq) and improve the bioinformatic pipelines for sequence processing. We will apply statistical and mathematical approaches to study population dynamics and relationships by Bayesian networks and deterministic/stochastic models. Additionally we will study the molecular and functional properties of infused cell products by performing phenotypic characterizations, barcoding with vector libraries, IS retrieval and in vitro differentiation assays. Impact and Translational Implications The present study will set new references for the definition of human HSPC and human hematopoietic hierarchy and help defining the potentiality of TSCM and other mature cell types as tools for cell-based therapies. Overall, data collected from this project will have a profound impact on the improvement of standard therapeutic approaches and on the design of advanced treatments based on gene- and cell-therapy for a broad spectrum of hematological diseases, including genetic disorders and tumors. 10/07/ / 5

74 GR Dissecting the role of unexplored mir-205 host gene as a basal cell-specific long non-coding RNA in prostate cancer development Gandellini Paolo Biomedical/Biomedica LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Project Classification IRG: Project Classification SS: Oncology 1 - Basic Translational Cancer Molecular Pathobiology - CAMP Project Keyword 1: Project Keyword 2: Project Keyword 3: Gene regulation including chromatin structure and remodeling, transcription, RNA processing and stability, and translation relevant to oncogenesis long non coding RNA prostate cancer Project Request: Animals: Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Dept. Experimental Oncology and Molecular Medicine- Molecular Pharmacology Coordination of the project. Cell and molecular biology experiments, functional analyses, gene expression profiling, analyses on clinical specimens 2 University of Milan Dept. Biosciences ChIP-Seq, ChIRP-Seq experiments and bioinformatics Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 DOLFINI DILETTA University of Milan ChIP-Seq, ChIRP-Seq experiments and bioinformatics 19/07/ /07/ / 6

75 GR Dissecting the role of unexplored mir-205 host gene as a basal cell-specific long non-coding RNA in prostate cancer development Gandellini Paolo Biomedical/Biomedica Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Background and Significance Prostate epithelium is composed of various cell lineages. Placed at the interface between luminal secretory cells and the basement membrane (BM), p63-positive basal cells govern tissue polarity and serve as stem cells of the organ, being their differentiation an inescapable step in prostate morphogenesis. In the human setting, only basal cells have been proven to act as the prostate cancer (PCa) cell of origin. Since their transformation leads to tumors with luminal cell phenotype, pathological basal-to-luminal differentiation, as induced by an inflammatory microenvironment, may favor tumorigenesis. Notably, loss of basal cell layer has long been serving as the major diagnostic criterion for PCa. Hence, the study of genes relevant for maintaining basal phenotype may have major implications for dissecting the mechanisms of PCa initiation and developing novel therapeutic strategies. In this regard, we showed that expression of mir-205 is enriched in prostate basal cells, where, together with p63, it participates in a network essential for maintenance of the BM. The sequence for human mir-205 is located within the intron of an uncharacterized gene, the function of which is still unknown. Our preliminary data suggest that mir-205 Host Gene (MIR205HG) may be devoid of protein-coding potential and act as a nuclear long noncoding RNA (lncrna). Aim of this project is to shed light on the role of MIR205HG as lncrna with potential huge role in the biology of human prostate epithelium. Specific aims Aim 1: Dissecting the functional role of MIR205HG in prostate basal cells and its involvement in basal-to-luminal differentiation or BM deposition as well as understanding if tumor microenvironment may influence basal-toluminal differentiation by modulating MIR205HG expression Aim 2: Elucidating the mechanism of action of MIR205HG as nuclear lncrna Aim 3: Assessing MIR205HG expression pattern in situ in a large series of human prostate clinical specimens and investigating its transcriptional and functional interplay with mir-205 Hypothesis: Despite the utmost importance of processes governing the function of basal cells during normal prostate development and tumorigenesis, the role of noncoding RNAs in basal cells is largely unexplored and, to our knowledge, no lncrna has been described thus far as regulator of basal features. The study of genes with relevant regulatory activities in the context of prostatic basal cell layer, as is the case of MIR205HG, may help elucidate the mechanisms of PCa initiation and allow the identification of novel targets or tools for anticancer therapies. Preliminary data: By interrogating gene expression profiles of a large series of prostatic normal, tumor and metastatic tissue specimens (GSE21032), we found that MIR205HG expression levels highly correlate with those of p63 mrna, reflecting a basal cell-specific expression. Accordingly, MIR205HG levels are significantly reduced in primary prostate tumors and even lower in metastatic samples compared to normal tissues. Interestingly, estimation of the area under ROC curve suggests MIR205HG having high capability to discriminate between tumor and normal samples, with a value comparable to that of p63 mrna. Silencing of p63 in RWPE-1 prostate epithelial cells resulted in reduction of MIR205HG expression, indicating that its transcription may be regulated by p63. Based on annotated NCBI RefSeq NM_ , the processed transcript of MIR205HG is 899-nt long with a predicted open reading frame (ORF) of 102 aminoacids. Generally, transcripts having ORFs< aminoacids are classified as non-coding. MIR205HG falls just out of this range and, actually, no protein product has been identified so far. To verify whether or not MIR205HG may code for a protein, two plasmid vectors, one containing the full-length primary and one the mature 10/07/ / 6

76 GR Dissecting the role of unexplored mir-205 host gene as a basal cell-specific long non-coding RNA in prostate cancer development Gandellini Paolo Biomedical/Biomedica Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano transcript of the gene, were produced to be used in an in vitro transcription/translation assay. No protein products were obtained with either plasmid, thus confirming that MIR205HG may have no protein-coding potential. As lncrnas predominantly localize to the nucleus, a further indication of MIR205HG aptitude to function directly as lncrna has been obtained by assessing nuclear/cytoplasmic distribution of its mature transcript in RWPE-1 cells. Whereas GAPDH mrna was more abundant in the cytoplasm (as expected for mrnas that need to be translated into proteins), MIR205HG displayed an opposite spatial expression pattern, supporting the hypothesis that MIR205HG may act as nuclear lncrna. Materials and Methods To functionally characterize MIR205HG, loss-of-function approaches (by using sirnas, locked nucleic acid (LNA)-modified or chimeric DNA/LNA antisense oligonucleotides to alternatively knock-down MIR205HG, mir-205 or both) will be used in prostatic basal cells, followed by phenotypic characterization (i.e. differentiation and BM production) and gene expression profiling. Interaction of MIR205HG mature transcript with relevant transcription factors (i.e., p63) will be checked through RNA-immunoprecipitation, whereas ChIRP- and ChIP-Sequencing will be instrumental to identify the genomic regions eventually bound by the lncrna with associated transcription factors. Quantitative RT-PCR and in situ hybridization will be used to assess MIR205HG expression pattern in matched tumor and adjacent non-neoplastic tissues collected from 98 patients subjected to radical prostatectomy. All specimens, together with clinical and patho-biological information are already available. Impact and Translational Implications The identification of genes regulating basal cell function may lead to a more exhaustive characterization of the mechanisms of PCa development and the identification of novel therapeutic targets. Given that lncrnas are gaining increasing respect as rulers of pivotal biological processes, MIR205HG, though never investigated, may represent a very promising candidate, as it hosts and may functionally interact with mir-205, which is recognized as one of the most relevant mirnas in PCa. 10/07/ / 6

77 Neutrophil traps in leukemia: from triggers of disease progression to vehicle for new vaccines BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Sangaletti Sabina Biomedical/Biomedica LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Project Classification IRG: Project Classification SS: Oncology 1 - Basic Translational Tumor Microenvironment - TME Project Keyword 1: Project Keyword 2: Project Keyword 3: Molecular and cellular aspects of bi-directional interaction between tumor and stromal cells (including fibroblasts, glial cells, epithelial cells, adipocytes, immune cells, inflammatory cells, vascular compartments, and bone marrow cells) during neoplastic progression, tumor angiogenesis, growth and metastasis, including studies of cancer stem cell niche and tumor cell dormancy Leukemia, Neutrophil Extracellular Traps (NET), Extracellular Matrix Bone-marrow stroma, SPARC, Matricellular Proteins Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Experimental Oncology and Molecular Medicine Dept - Molecular Immunology Unit 2 Univerisity of Palermo Department of Health Sciences, Human Pathology Section. University of Palermo Immunology Unit Set up of murine models, in vivo and in vitro experiments also with human leukemic blasts, vaccinations, GEP analysis, data collection and analysis Perform histopathological and immunophenotypical analyses on human and murine samples. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Tripodo Claudio Univerisity of Palermo Dr. Tripodo will coordinate and perform histopathological and immunophenotypical analyses on human BM samples. He will be responsible for correlating data from the above described analysis with archival follow-up data of relevant clinical events and survival data in order to retrospectively test the clinical impact of the studied variables. 07/07/ /07/ / 6

78 Neutrophil traps in leukemia: from triggers of disease progression to vehicle for new vaccines BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Sangaletti Sabina Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Background and Significance A correlation between chronic immune stimulation (infection, autoimmunity) and development of myeloid malignancies has been described but the pathogenic link remains obscure. Deregulated neutrophil activation and death towards neutrophil extracellular trap (NET) formation has pathogenic relevance in autoimmune diseases and in their transition towards malignancy. NET, decondensed chromatin threads decorated with antimicrobial proteins, exert anti-microbial function by entangling and killing microbes, yet they foster autoimmunity by stimulating IFN production in pdc and transferring selfantigens to dendritic cells (DC) for cross-presentation and auto-antibody production. NET are effective antigen vehicles for vaccination strategies. In hematopoietic neoplasms carrying specific mutations able to generate new antigens, such as acute myeloid leukemia with mutated nucleophosmin (NPM), where NPM re-localizes to the cytoplasm and induces the myeloproliferative phenotype, NET could display therapeutic relevance. Myelopoiesis and NET formation are both controlled by the extracellular matrix (ECM) through the collagen receptor LAIR-1. LAIR-1 engagement inhibits neutrophil activation and NETosis or blast proliferative responses, in line with bone marrow (BM) stromal niche collagens regulating hematopoietic homeostasis. Indeed, stromal signals are deeply rearranged upon autoimmune disease or neoplasm onset and genes coding for ECM proteins are frequently deregulated in such conditions. Specific aims Aim 1: Aim 2: Aim 3: To investigate the influence of autoimmunity on the onset and progression of myeloproliferative disorders. To investigate whether faulty ECM signals lead to NETosis, which is responsible for breaching the tolerance in the BM. To test whether mutated NPM re-localizing into the cytoplasm (NPMc) could be immunogenic as part of the NET thread and whether NET could be adopted in vaccination strategies to prevent myeloproliferation in a transgenic mouse model expressing human NPMc mutation. Hypothesis: We hypothesize that NET could be the pathogenic link between autoimmunity and myeloid malignancies and, at the same time, a vehicle for vaccination against leukemic cell-associated antigens. We also hypothesize that the down-modulation of the ECM protein SPARC, which occurs as part of the early BM stromal changes associated with pre-leukemic state, could favor loss of tolerance and NET formation in BM undergoing a myeloproliferative spur. Besides the role of NET in triggering the transition from autoimmunity to leukemia, we would investigate the hypothesis that DC-loaded with NET could be used as a vaccine against leukemia-associated antigens, such as the prototypical NPMc mutant. Mutant NPMc marks the cytoplasm of immature cells within the BM of NPMc transgenic mice, but not their mature progeny. Thus, the effective immunization using NPMc-derived NET should allow selective elimination of NPMc-expressing cells but not that of residual normal progenitors or mature myeloid cells. Preliminary data: 1. NET promote hematopoietic precursors proliferation. Hematopoietic precursors (LK cells) have been purified from normal BM and cultured with PMA-induced NET. We found that NET, but not naïve neutrophils, strongly promoted LK cells proliferation. 2. Stromal SPARC expression is inversely correlated with myeloid cell proliferation and NETosis. In human MDS evolving to myelofibrosis and in PMF, we found SPARC expression increased and associated with BM stromal cells. On the contrary, SPARC expression was markedly reduced in BM stroma infiltrated by AML cells. The transplantation of ApcMin BM, characterized by altered HSC quiescence, gave rise to myelodysplasia when transplanted into Sparc-/- recipients. Furthermore, BM neutrophils collected from ApcMin>Sparc-/- chimeras showed increased spontaneous NETosis than from ApcMin>wt counterpart. 3. Immature c-kit+ cells of NPMc transgenic mice undergo NETosis after PMA-treatment and re- 11/07/ / 6

79 Neutrophil traps in leukemia: from triggers of disease progression to vehicle for new vaccines BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Sangaletti Sabina Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano localize mutant NPMc onto NET structure. Immature blasts (GR-1+Kit+) have been sorted from the BM of NPMc transgenic mice, seeded onto poly-d-lysine coated glasses and treated with PMA. NET treads were stained with Abs to histone, mutant NPM and MPO. We found that NPM from mutant blasts localizes along NET thread in close proximity with MPO. Materials and Methods To investigate the influence of autoimmunity over myeloproliferation, pre-leukemic NPMc-transgenic mice will be injected with DC loaded with NET components, a strategy that we demonstrated to induce autoimmunity in naive mice. Expansion of BM myeloid precursors and early progenitors and development of circulating auto-antibodies will be analyzed. To investigate the influence of stromal Sparc deficiency over myeloproliferation and autoimmunity, NPMc-transgenic hematopoietic cells will be transplanted into Sparc-/- hosts. The loss of tolerance in Sparc-/- recipients will be analyzed as phenotype and activity of regulatory and effector T cells, and the NETosis of BM cells. To explore the possibility of adopting NET from NPMc mutant-leukemic blasts as vaccine to control the abnormal proliferation of NPMc mutant cells, NPMc transgenic mice will be injected with DCs co-cultured with NET from NPMc transgenic mice and the myeloproliferative phenotype will be characterized. Impact and Translational Implications Our studies offer precious insights into the dynamics linking autoimmunity and myeloid leukemogenesis. We can envisage the possibility to therapeutically modulate SPARC expression through drugs, such as lenalidomide, acting on the structural and immunological sides of stroma remodeling. Our original vaccination strategy using NET will allow to open the possibility of using autologous DC loaded with NET from mutant leukemic blasts as vaccine to control NPMc-mutant AML patients. 11/07/ / 6

80 GR Unravelling the pathogenetic pathways in an experimental model of Diabetic Encephalopathy by a omics-based platform: potential for novel diagnostic and therapeutic interventions Gnoni Antonio Biomedical/Biomedica Puglia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Endocrinology, Metabolism, Nutrition and Reproductive Sciences Cellular Aspects of Diabetes and Obesity - CADO Project Keyword 1: Project Keyword 2: Project Keyword 3: Insulin biosynthesis, trafficking, and secretion; insulin action,;mechanisms of insulin signaling; glucose transport; downstream signaling pathways in insulin action, including the actions of scaffold proteins, phospholipids, kinases, and phosphatases. Diabetic Encephalopathy high-throughput omics technologies Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Azienda Ospedaliera-Universitaria OO.RR. di Foggia/Dipartimento di Patologia Clinica/Laboratorio Biochimica- Clinica Universitario PO D'Avanzo proteomics, metabolomics and biochemical analysis 2 University of Bari Department of Basical Medical Sciences, Neurosciences and Sensory Organs samples preparation and two-dimensional gel electrophoresis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Taurino Federica University of Bari samples preparation and two-dimensional gel electrophoresis 2 16/03/ Gelzo Monica Azienda Ospedaliera- Universitaria OO.RR. di Foggia/Dipartimento di Patologia Clinica/Laboratorio Biochimica- Clinica Universitario PO D'Avanzo metabolomics analysis 13/05/ /07/ / 4

81 GR Unravelling the pathogenetic pathways in an experimental model of Diabetic Encephalopathy by a omics-based platform: potential for novel diagnostic and therapeutic interventions Gnoni Antonio Biomedical/Biomedica Puglia Background and Significance Diabetes mellitus (DM) is a metabolic disorder in which impaired insulin secretion induces a chronic hyperglycaemia and long-term complications that are the major health issues. Chronic hyperglycaemia is associated with a variety of long-term complications that damage retina, kidneys, cardiovascular systems, peripheral and central nervous system (CNS). The neurological complications targeting the CNS are referred to as "Diabetic Encephalopathy" (DE). The pathogenesis of DE has not been yet investigated systematically, however, it is likely to be a multifactorial process involving adverse effects of chronic hyperglycaemia on the brain. In recent years, high-throughput technological approaches, such as omics sciences, accelerate and improve the research studies aiming to discover or clarify metabolic and pathophysiological networks. The project will address a systematic analysis of proteins and metabolites involved in the development of experimental DE, at early and late stages in different brain regions (hippocampus, cerebral cortex, cerebellum and hypothalamus) of the animal model. Such a detailed mapping, reached through an integrated -omics-based platform (i.e. proteomics, metabolomics and bioinformatics), will enable to characterize, for the first time, both the brain area(s) and the molecular target(s), that determine or mainly contribute to DE. These findings will be used to screen for therapeutical compound(s) acting on the identified molecular pathways. Specific aims Aim 1: Aim 2: Aim 3: Spotting analysis of the brain area(s) mainly targeted in the early and late stages of the Diabetic Encephalopathy. Characterization of the protein and metabolite pathways involved in the pathogenesis at early and late stages of the Diabetic Encephalopathy. Identify proteins and metabolites as promising biomarker of the Diabetic Encephalopathy and or/diabetes Mellitus. Hypothesis: The neurological complications targeting the Central Nervous System are referred as Diabetic Encephalopathy encompasses characteristic biochemical, neurophysiological, morphological, and cognitive deficits in diabetic patients. Impairments in learning, memory, problem solving, and mental and motor speed are more common in diabetic patients than in the general population. Notably, the neurological changes induced by Diabetes are associated with increased risk of Alzheimer's disease, dementia, seizures and stroke. In particular, Alzheimer's disease is twice as prevalent in the diabetic population as in non-diabetic subjects. Brain regions, such as hypothalamus, cerebellum, hippocampus and cerebral cortex are extremely sensitive and responsive in different ways to glucose homeostasis alterations. These regions are involved in cognitive, motor, and neuroendocrine activities; thus, their impairments during Diabetes are relevant in the pathogenesis of the disease. In recent years, generation of biological high-throughput technological approaches, such as proteomics and metabolomics, led to acceleration and improvement of research studies aiming to discover or clarify metabolic and pathophysiological networks. Omics data are going to be even very useful and adopted to face the real challenge of collecting and integrating information about diseases as a whole, in order to outline enhanced strategies of therapy and drug design. In this context, omics data concerning the CNS physiology as well as nervous pathological onsets linked to metabolic diseases could represent a reference point for the following up of the disease. Preliminary data: PI's recent works inspire the proposed research programme. We first investigated the changes of the mitochondrial proteome, from total brain, in the early phase of DE. It was found that, in diabetic rats, the proteome was stable except for severe decrement of the Ndufs3, core subunit of complex I; moreover, a severe impaired catalytic activity of the complex I was highlighted. We next demonstrated that DE affects in a tissue-specific way the expression of some regulatory molecules belonging to s family proteins, proposing them as a new class of molecular targets in DE. 10/07/ / 4

82 GR Unravelling the pathogenetic pathways in an experimental model of Diabetic Encephalopathy by a omics-based platform: potential for novel diagnostic and therapeutic interventions Gnoni Antonio Biomedical/Biomedica Puglia Materials and Methods Diabetes will be induced by a single injection of streptozotocin (STZ) to adult rats; clinical hallmarks of Diabetes will be monitored. The rat experimental groups are the following: a)short-term non-diabetic Control, b) Short-term Diabetic (3 weeks after STZ injection), c) Short-term Diabetic treated with insulin (3 weeks after STZ injection); d) Long-term nondiabetic Control; e) Long-term Diabetic (12 weeks after STZ injection); f) Long-term Diabetic treated with insulin (12 weeks after STZ injection). For each experimental condition, total lysate (TL) and mitochondrial (M) fractions from different brain areas (hippocampus, cerebral cortex, cerebellum and hypothalamus) will be prepared. Then, proteomics and metabolomics analyses will be carried out. Bioinformatics study of the obtained data using suitable software for metabolic network analyses will be performed. On the basis of the obtained results, biochemical, structural and bioenergetics assays will be carried out. Impact and Translational Implications The project will give a complete overview of the molecular networks involved in the early stage and in the progression of the DE. Hence, it will be clear which area(s) of the brain and which stage of the disease are involved. Such information will provide a rationale, at pre-clinical level, proposing specific molecular target (or a set), as promising biomarker for DM and/or DE.

83 Targeting cutaneous anti-apoptotic PI3K/AKT signalling as a new therapeutic approach for treating skin inflammation GR Madonna Stefania Biomedical/Biomedica Istituto Dermopatico dell'immacolata (IDI) LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Infectious and immunological diseases Project Classification IRG: Project Classification SS: Musculoskeletal, Oral and Skin Sciences Arthritis, Connective Tissue and Skin - ACTS Project Keyword 1: Project Keyword 2: Project Keyword 3: Skin and Cutaneous Biology: Disorders of skin and skin appendages, such as inflammatory, preneoplastic, and hyperproliferative disorders, as well as systemic diseases with significant cutaneous involvement. PI3K/AKT pathway psoriasis Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Laboratory of Experimental Immunology PI will be involved in the study of the effects of PI3K/AKT inhibitors in keratinocytes and T cells in vitro and in two mouse models of psoriasis. PI will study the cutaneous phenotype of a new transgenic mouse model overexpressing AKT in the epidermis and will be involved in the coordination of the activities. Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 10/07/ / 6

84 Targeting cutaneous anti-apoptotic PI3K/AKT signalling as a new therapeutic approach for treating skin inflammation GR Madonna Stefania Biomedical/Biomedica Istituto Dermopatico dell'immacolata (IDI) Background and Significance Psoriasis is an immune-mediated skin disease driven by infiltrating T cells, which locally release pro-inflammatory cytokines responsible for the activation of the epidermal keratinocytes (KC). Recently, our group demonstrated that the pro-survival PI3K/AKT pathway, strongly expressed in lesional psoriatic skin, contributes to the reduced susceptibility of psoriatic KC to apoptosis by activating the anti-apoptotic and pro-inflammatory NF-kB cascade and inhibiting the pro-apoptotic BAD function. Interestingly, peculiar resistance of psoriatic KC to pro-apoptotic stimuli could be strictly associated to their senescence phenotype, known to be characterized by an enhanced expression of specific senescence markers and the secretion of various inflammatory mediators. In the skin, PI3K/AKT pathway also regulates adaptive immune cell activation. Recent studies showed that PI3Kgamma and PI3Kdelta blockade ameliorates imiquimod (IMQ)-induced psoriasis-like disease correlating with decreased IL-17A-producing gamma-deltat cells. A second mouse model of psoriatic-like disease in which RAS is targeted to suprabasal layers of the skin has been developed and is likely to be a useful model to study effects of PI3K/AKT inhibition, since this pathway is directly downstream of RAS. Due to the key role of PI3K/AKT in cutaneous homeostasis, this study will investigate the efficacy of PI3K/AKT targeting in ameliorating the anti-apoptotic and inflammatory processes occurring in psoriatic skin. Specific aims Aim 1: Aim 2: Aim 3: To investigate whether PI3K/AKT blockade influences the senescent phenotype of psoriatic KC, by evaluating the secretion of specific inflammatory factors and the expression of specific senescence markers. In parallel, the effects of PI3K/AKT blockade on the recruitment, survival, differentiation and functioning of T cell populations involved in the disease expression will be investigated. To study the effects of the pharmacological inhibition of the pro-survival PI3K/AKT pathway in IMQ-induced and RAS-induced psoriasis-like dermatitis models. Both models reproduce the epidermal structure changes and lymphocytic infiltrate, including IL-17A-secreting Th17 and gamma-delta T cells common to psoriasis, with IL17A+IFN-g+CD8+ T cytotoxic cells playing a key role in determining the psoriasiform phenotype in RAS-induced psoriasis-like dermatitis model. to assess whether the overexpression of a permanently activated AKT form in the suprabasal epidermis of transgenic mice can result in a psoriasiform phenotype. Hypothesis: PI3K and its AKT effector play a crucial role in skin homeostasis by regulating the pro-survival processes both in the epidermal KC and in adaptive immune cells, including infiltrating T lymphocytes. We hypothesize that targeting of the intracellular PI3K and AKT molecules have a strong therapeutic potential to modulate the inflammatory and anti-apoptotic processes associated to the senescent phenotype occurring in psoriatic keratinocytes, and, in parallel, to influence the recruitment, activation and survival of T lymphocytes with a pathogenetic role in psoriasis. Preliminary data: In recent studies, we observed that the senescence p16 marker is strongly expressed in suprabasal layers of lesional psoriatic skin in vivo and in activated psoriatic KC in vitro. p16 staining in psoriatic epidermis correlates with the expression of a number of pro-survival markers, including p-akt and the downstream anti-apoptotic p-p65, p-bad and BCL-XL molecules, as well as of pro-inflammatory chemokines (i.e. MCP-1 and IP-10), in terms of localization within the psoriatic epidermal compartment. Interestingly, we observed that the release of these pro-inflammatory chemokines analyzed in serial passages of primary cultures of lesional activated KC directly correlated to the expression of the p16 marker in these culture conditions, suggesting an intriguing link between senescence phenotype and inflammatory responses in psoriatic KC. Finally, our preliminary data show that the topical application of the chemical PI3K inhibitor, Ly294002, significantly reduced the 10/07/ / 6

85 Targeting cutaneous anti-apoptotic PI3K/AKT signalling as a new therapeutic approach for treating skin inflammation GR Madonna Stefania Biomedical/Biomedica Istituto Dermopatico dell'immacolata (IDI) epidermal thickening, and the inflammatory infiltrate associated with IMQ induced psoriasis-like dermatitis. Materials and Methods Primary KC cultures from healthy or psoriatic donors will be treated with PI3K/AKT inhibitors or subjected to RNA interference. The expression of inflammatory molecules and senescence markers will be evaluated by ELISA and Real-time PCR analysis.t cell lines and clones obtained from psoriatic skin and/or peripheral blood will be treated with specific PI3K/AKT inhibitors and functionally characterized. Migration of skin T cells towards treated or untreated KC will be studied in a trans-well systems. IMQ-induced and RAS-induced psoriasiform mouse models will be treated topically with PI3K and AKT inhibitors.the epidermal thickening and the inflammatory infiltrate will be analyzed by IHC and FACS. CD8+ T cell recruitment and KC-T cell cross-talk will be investigated. A mouse model overexpressing a tissue- and stage-specific permanently activated AKT form will be generated by the facility of Mouse Modeling at the IGB-ABT-CNR, Naples and characterized phenotypically by IHC and RT-PCR. Impact and Translational Implications This study will provide clear-cut data on the efficacy of the PI3K and AKT pharmacological blockade in counteracting the senescence phenotype of psoriatic keratinocytes and, in parallel, the survival, recruitment and differentiation of infiltrating T cells having a pathogenetic role in psoriasis. Our results will support the rationale for new therapeutic approaches in psoriasis. 10/07/ / 6

86 ULTRASENSITIVE DIAGNOSTIC TEST FOR DEGENERATIVE DEMENTIAS BASED ON AMPLIFICATION OF PERIPHERAL DISEASE-SPECIFIC BIOMARKERS FROM THE OLFACTORY MUCOSA GR Moda Fabio Biomedical/Biomedica Fondazione Istituto Neurologico Carlo Besta LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Alzheimer s disease and other dementias. protein misfolding early diagnosis Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Divisione di Neuropatologia e Neurologia- - Synthetic Abeta and recombinant tau protein 5 production for RT-QuIC assay - Collection of olfactory mucosa specimens from patients with AD, LBD and FTD. - Abeta and tau aggregation assay (RT-QuIC) using brain homogenates and olfatory mucosa samples. 2 Università degli Studi di Verona Policlinico G.B. Rossi 3 Scuola Internazionale Superiore di Studi Avanzati (SISSA) Investigators, Institution and Role in the Project Dipartimento di science neurologiche e del movimento Laboratorio di biologia dei prioni - Alpha-synuclein aggregation assay (RT-QuIC) usign brain homogenates and olfactory mucosa samples. - Recombinant Alpha-synuclein production and purification Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 bongianni matilde Università degli Studi di Verona Policlinico G.B. Rossi 2 Narkiewicz Joanna Scuola Internazionale Superiore di Studi Avanzati (SISSA) Researcher involved in RT-QuIC test using brain homogenates and olfactory mucosa samples. Researcher involved in production and purification of recombinant Alphasynuclein. 14/06/ /06/ /07/ / 6

87 ULTRASENSITIVE DIAGNOSTIC TEST FOR DEGENERATIVE DEMENTIAS BASED ON AMPLIFICATION OF PERIPHERAL DISEASE-SPECIFIC BIOMARKERS FROM THE OLFACTORY MUCOSA GR Moda Fabio Biomedical/Biomedica Fondazione Istituto Neurologico Carlo Besta Background and Significance Alzheimer's disease (AD), Lewy body dementia (LBD) and Frontotemporal dementia (FTD) are the most common form of dementias and their definite diagnosis is difficult at early stages. All these disorders are characterized by the accumulation of misfolded proteins in the central nervous system, i.e. amyloid-ß (Aß) and tau proteins in AD, alpha-synuclein (as) in DLB and hyperphosphorylated tau in a subset of FTD patients. The mechanism by which natively folded proteins undergo misfolding and aggregation is unclear. Upon misfolding, the proteins aggregate and form assemblies of different sizes. Interestingly, these assemblies are able to seed their own aggregation in vitro, a feature which has been exploited to create an ultrasensitive assay called Real Time Quacking Induced Conversion (RT-QuIC) assay where soluble recombinant proteins are used as substrate. When added to the reaction, the disease-associated aggregates trigger the clump up of soluble proteins and lead to the formation of fibril-like structures which can be monitored with the thioflavin T (ThT) fluorescence dye. Without seeds the aggregation occurs but is much slower. Recent studies showed that the olfactory mucosa (OM) of patients with AD, LBD, FTD and Creutzfeldt-Jakob disease (CJD) contains aggregates of Aß, as, tau and PrP respectively. The development of RT-QuIC assay to detect disease-specific aggregates in OM would allow to make a differential diagnosis at the early stage of dementias. Specific aims Aim 1: Aim 2: Aim 3: 1. We will assess the RT-QuIC detection limits using different dilutions of brain homogenates obtained from patients with AD, LBD and FTD-tau. 2. We will analyze the ability of RT-QuIC to specifically detect aggregates in the OM of patients with AD, LBD and FTD-tau. 3. We will evaluate the appearance of aggregates in the OM of patients with genetic forms of the disease at the pre-symptomatic stage. Hypothesis: The presence of disease-specific aggregates in the olfactory mucosa (OM) of patients with AD, Parkinson and CJD has been extensively reported. In particular, their accumulation seems to occur earlier during disease progression. The structure of these aggregates is similar to that in the brain and this is not surprising since OM is part of the nervous system. Brain derived aggregates are able to act as seed in the RT-QuIC assay. As results, the kinetic of protein aggregation (monitored with ThT) is accelerated when brain-derived seed are added to the RT-QuIC reaction. Our hypothesis is that OM-derived aggregates might behave as the brain ones thus accelerating the kinetic of protein aggregation in RT-QuIC assay. Even more important the fact that they can be collected at early stages of through a simple procedure and might allow the differential diagnosis among degenerative dementias. Preliminary data: Recent data from the literature have shown the ability of RT-QuIC to detect misfolded prion protein in the CSF of patients with Creutzfeldt-Jakob disease (CJD) with 91% sensitivity and 98% specificity. Our laboratory found similar result using the CSF of patients with AD in which Aß oligomers were detected with a sensitivity of 90% and specificity of 92%, thus providing an ultrasensitive diagnostic tool that can help for an intravitam diagnosis of specific dementias. Since the collection of CSF is an invasive procedure, we will apply the same technology for detection of misfolded protein in samples of OM which are less invasive to collect. Recently, Zanusso and colleagues (2014) successfully applied RT-QuIC for the detection of misfolded prion protein in the OM of patients with the sporadic forms of CJD. We have recently optimized the RT-QuIC conditions for in vitro aggregation of recombinant alpha-synuclein, tau and synthetic Aß that will be used as substrate to test whether the OM-derived aggregates might act as seed in RT-QuIC. Preliminary results from our laboratory showed that brain homogenate of patients with LBD was able to seed the aggregation of 11/07/ / 6

88 ULTRASENSITIVE DIAGNOSTIC TEST FOR DEGENERATIVE DEMENTIAS BASED ON AMPLIFICATION OF PERIPHERAL DISEASE-SPECIFIC BIOMARKERS FROM THE OLFACTORY MUCOSA GR Moda Fabio Biomedical/Biomedica Fondazione Istituto Neurologico Carlo Besta recombinant as in RT-QuIC. Materials and Methods Human recombinant as and tau will be expressed in E. coli and purified for the RT-QuIC aggregation assay while Aß will be sinthetically produced. In aim 1, brain homogenates from patients with AD, LBD and FTD will be diluted from 10-2 to in PBS and used as seed in RT-QuIC assay for Aß, as and tau, respectively, in order to assess the limit of detection. A final volume of 200 µl will be prepared and different concentrations of monomers will be used for each assay, particularly: Aß [4µM], tau [20µM] while as [70µM]. The aggregation will be monitored at various time points by reading ThT fluorescence at 485 nm after excitation at 444 nm using a plate spectrofluorometer. For aim 2 and 3, OM from patients with different diseases (AD, LBD and FTD) will be collected from the mid-part of the inferior turbinate by brushing with cotton swab and resulting cells will be used to seed RT-QuIC assay. The kinetic of aggregation in the presence or absence of OM aggregates will be compared. Impact and Translational Implications There is an urgent need for tests that can allow the correct differential diagnosis among different dementias at early stages. The presence of Aß, as and tau in the olfactory mucosa of patients with AD, LBD and FTD respectively, at early stages of the disease will be exploited in RT-QuIC assay. This test is able to detect and amplify trace amount of disease-associated proteins in OM, providing a fundamental contribution to the diagnostic field. 11/07/ / 6

89 Antimicrobial drug resistance in food-producing animals. Essential oils and supercritical fluid extraction: study the antimicrobial and immuno-stimulating activity in livestock for reducing the use of antibiotics and environmental pollution, improvement of animal welfare and food safety. In vitro and in vivo efficacy evaluation of Ocimum basilicum essential oil extracted by supercritical carbon dioxide in rainbow trout (Oncorhynchus mykiss) affected by Lactococcus garvieae. Validation of protocols for administering. GR Barbero Raffaella Biomedical/Biomedica LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Food safety and animal safety Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'aosta Project Classification IRG: Project Classification SS: Veterinary Animal Health - ANHE Project Keyword 1: Project Keyword 2: Project Keyword 3: Study and identification of molecules with immuno-modulating properties or of natural origin, with the objective of reducing the use of antibiotics and/or other antimicrobials, with particular regard to the validation of protocols for administering these substances that are compatible with good zootechnical and aquaculture practice and aimed at reducing environmental residues The use of essential oils in medicated feed in livestock as a valuable strategy to reduce the use of antimicrobials and environmental pollution and to improve animal welfare and food safety. Use supercritical fluid extraction with carbon dioxide: mild technology for temperature unstable natural compounds extraction. Development a rapid process at low temperature, moderate pressure, nontoxic, non-flammable, chemically stable and inexpensive. Essential oils extracted from Ocimum basilicum: antimicrobial efficacy and immuno-stimulating activity evaluation. Experimental study on trout (Oncorhynchus mykiss) infected by Lactococcus garvieae, a major aquaculture problem worldwide. Validation of protocol for administering. Reduce the use of antimicrobial in fish livestock and environmental pollution. Improve animal welfare and food safety. Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright 10/07/ / 7

90 Antimicrobial drug resistance in food-producing animals. Essential oils and supercritical fluid extraction: study the antimicrobial and immuno-stimulating activity in livestock for reducing the use of antibiotics and environmental pollution, improvement of animal welfare and food safety. In vitro and in vivo efficacy evaluation of Ocimum basilicum essential oil extracted by supercritical carbon dioxide in rainbow trout (Oncorhynchus mykiss) affected by Lactococcus garvieae. Validation of protocols for administering. GR Barbero Raffaella Biomedical/Biomedica Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'aosta Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Serology and Animal Welfare Project Coordination; Essential oil and supercritical fluid extraction; medicated feed production; experimental study on infected trouts; chemical and hematological analysis; oxidative stress biomarker determination 2 University of Turin - Department of Veterinary Sciences Department of Veterinary Sciences- Immuno-stimulating activity evaluation; nonspecific immunity and Vanilloid Receptors Division of Pharmacology and Toxicology determination; environmental pollution study 3 Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana National Center of Reference for Antimicrobial-Resistance In vitro and in vivo efficacy evaluation of essential oils antimicrobial activity with microbiological methods Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Vercelli Cristina University of Turin - Department of Veterinary Sciences 2 Alba alderete Patricia Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana 3 Foglini Claudio Istituto Zooprofilattico Sperimentale dell Piemonte, Liguria e Valle d'aosta Grantholder-Investigator. Immunostimulating activity evaluation; nonspecific immunity and Vanilloid Receptors determination; environmental pollution study Grantholder-Investigator. In vitro and in vivo efficacy evaluation of essential oils antimicrobial activity with microbiological methods Grantholder-Investigator. Management of experimental study on trouts, treatment, medicated feed and drug administration, sampling 14/12/ /06/ /10/ /07/ / 7

91 Antimicrobial drug resistance in food-producing animals. Essential oils and supercritical fluid extraction: study the antimicrobial and immuno-stimulating activity in livestock for reducing the use of antibiotics and environmental pollution, improvement of animal welfare and food safety. In vitro and in vivo efficacy evaluation of Ocimum basilicum essential oil extracted by supercritical carbon dioxide in rainbow trout (Oncorhynchus mykiss) affected by Lactococcus garvieae. Validation of protocols for administering. GR Barbero Raffaella Biomedical/Biomedica Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'aosta Background and Significance Despite the fact that the use of antimicrobials is crucial for food-producing animals and public health protection, irrational and irresponsible drug use may cause antimicrobial resistance. Sometimes, antibacterial drugs may be the only option after preventive actions have failed. For these reason, it is crucial that are employed prudently and rationally, in order to maintain their efficacy for the therapy against major pathogens, whenever necessary, and as last resort. In aquaculture, this process has resulted in the emergence of antibiotic-resistant bacteria in aquatic environments, in the increase of antibiotic resistance in fish pathogens, in the transfer of these resistance determinants to other bacteria in the ecosystem, to animal and to human pathogens, and in alterations of the bacterial flora both in sediments and in the water column. In order to minimize the spread of antibiotic resistance, National and European authorities recommend to improve strategies to cut and/or limit the use of antibiotics in food-producing livestock. Several essential oils extracted from aromatic plants are known to generate biological activity; in particular they exhibit strong antibacterial and antioxidant effects. The aim of this project is to study the antimicrobial and immuno-modulating activity of essential oil extracts in livestock, to reduce the use of antimicrobials for bacterial diseases and the validation of protocols for administering the aforementioned compounds. Specific aims Aim 1: Aim 2: Aim 3: a)development and standardization of a method for Ocimum basilicum essential oil extraction with supercritical fluid b)extraction of essential oil Ocimum basilicum with supercritical carbon dioxide method c)analysis of chemical composition of essential oils of Ocimum basilicum with HPLC-MS/MS d)in vitro efficacy evaluation of Ocimum basilicum essential oil versus Lactococcus garvieae e)in vitro efficacy evaluation of Ocimum basilicum essential oil versus antimicrobial drugs generally used in rainbow trout infected by Lactococcus garvieae (e.g.erythromycin, florfenicol and beta-lactams) a)production of medicated feed with essential oil of Ocimum basilicum b)oral administration of antimicrobial drugs (erythromycin, florfenicol and beta-lactams) in rainbow trout experimentally infected by Lactococcus garvieae and efficacy evaluation c)oral administration of essential oil of Ocimum basilicum in rainbow trout experimentally infected by Lactococcus garvieae and in vivo efficacy evaluation d)oral administration of medicated feed with essential oil in rainbow trout naturally affected by Lactococcus garvieae e)evaluation of animal welfare markers in all groups of treated animals (reference values of specific haematological and serum biochemical parameters and biomarkers of oxidative stress) f)evaluation in all groups of treated animals of essential oil immuno-stimulating activity (receptors expression evaluation, specifically Vanilloid TRPV receptors expressed in inflammation, infection and endotoxiemic conditions) and non-specific immunity a)study of Minimum Inhibitory Concentration of essential oil vs Lactococcus garvieae b)study and validation of the Efficacy Dose of of essential oil c)protocol validation of feed medicated oral administration in rainbow trout farms affected by Lactococcus garvieae d)evaluation of environmental pollution using cell lines derived from gills of trout using MTT method to evaluate cytotoxicity Hypothesis: Non-rational use of veterinary antimicrobials may result in pressure selection of antimicrobial resistant 10/07/ / 7

92 Antimicrobial drug resistance in food-producing animals. Essential oils and supercritical fluid extraction: study the antimicrobial and immuno-stimulating activity in livestock for reducing the use of antibiotics and environmental pollution, improvement of animal welfare and food safety. In vitro and in vivo efficacy evaluation of Ocimum basilicum essential oil extracted by supercritical carbon dioxide in rainbow trout (Oncorhynchus mykiss) affected by Lactococcus garvieae. Validation of protocols for administering. GR Barbero Raffaella Biomedical/Biomedica Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'aosta pathogens, which may endanger both animals and public health. Therefore, research aimed at a possible therapeutic use of new active compounds from natural origin is advisable. The use of essential oils in medicated feed in livestock could represent a valuable strategy to reduce the use of antimicrobials and environmental pollution and to improve animal wellbeing and food safety Preliminary data: Several essential oils extracted from aromatic plants are known to exhibit strong antibacterial effects (Król, 2013), particularly the essential oil from Ocimum basilicum (Opalchenova, 2003). Supercritical fluid extraction (SFE) was used with success to obtain extracts (Filip, 2014; Fornari, 2012). Preliminary results of our research group show the activity of Ocimum basilicum essential oils extracted by SFE on Vibrio alginolyticus in breeding fish species. The antimicrobial and immunostimulating activity was evaluated with encouraging results.unpublished data Materials and Methods Development of an engineering prototype with high level of automation for supercritical CO2 extraction Evaluation of essential oil chemical composition by HPLC-MS/MS Medicated feed production by pellet machine In vivo treatment of trout infected by Lactococcus garvieae with extracts of Ocimum basilicum vs antimicrobial drugs In vitro and in vivo efficacy evaluation (e.g.mic determination) of essential oil antimicrobial activity Haematological and blood chemical analysis (automated laser cell counter and photometer) Non specific immunity: determination of serum lysozyme, serum bactericidal activity, serum electrophoretic pattern with agarose gel electrophoresis system Evaluation of Vanilloid Receptor levels (Western blotting) in tissues Biomarkers of oxidative stress in trouts after treatments In vitro evaluation of cytotoxicity of oil and drugs on trout cell lines (e.g. RTgill-W1) Impact and Translational Implications The identification and use of natural origin molecules with antimicrobial activity and immuno-stimulating properties such as essential oil of Ocimum basilicum have great importance for reducing the use of antimicrobials in livestock and environmental pollution and, in general, to improve animal wellbeing and food safety.translational implications concern the administration with medicated feed of other essential oils obtained by SFE for different bacterial diseases in food-producing species. 10/07/ / 7

93 Inducible pluripotent stem (ips) cell-derived human astrocytes as a new disease model to shed light into the molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts (MLC). GR Lanciotti Angela Biomedical/Biomedica Istituto Superiore di Sanita' LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Cellular, anatomical, and systems-based studies of changes in the neural substrate and function of brain in response to disease astrocytes Ion homeostasis Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Istituto Superiore di Sanità (ISS), Rome, Dept. Cell Biology and Neurosciences 2 Operative Unit 2 (OU2) Pediatric Hospital Bambino Gesù (OPBG), Rome, Dept. Neuromuscular and neurodegenerative diseases Dept. Neuromuscular and neurodegenerative diseases PI; Aim: Generation of human ipsc-derived astrocytes as a pathological model for MLC disease; biochemical and functional studies. Aim: MLC1 mutation analysis; collection of skin and blood samples; generation of fibroblast and macrophage cultures from control donors and MLC patients. 3 Operative Unit 3 (OU3) University of Perugia, Dept. Experimental Medicine Dept. Experimental Medicine Aim: Electrophysiological studies of MLC mutation-induced defects in human cell-based MLC models. 10/07/ / 6

94 Inducible pluripotent stem (ips) cell-derived human astrocytes as a new disease model to shed light into the molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts (MLC). GR Lanciotti Angela Biomedical/Biomedica Investigators, Institution and Role in the Project Istituto Superiore di Sanita' Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Aiello Chiara Operative Unit 2 (OU2) Pediatric Hospital Bambino Gesù (OPBG), Rome, Dept. Neuromuscular and neurodegenerative diseases 2 Servettini Ilenio Operative Unit 3 (OU3) University of Perugia, Dept. Experimental Medicine 3 Veroni Caterina ISS, Rome, Dept. Cell Biology and Neurosciences 4 Caiello Ivan OPBG, Rome, U.O.Rheumatology PI of OU2; mutation analysis of MLC patients; collection of skin biopsies and set up of fibroblast cultures. PI of OU3; analysis of mutation-induced defects on ion channel and volume regulation properties of MLC pathological models. Collaborator of OU1; biochemical characterization of human pathological models for MLC disease. Collaborator of OU2; set up of monocyte/macrophage (M/M) cultures from controls and MLC patients. 11/03/ /09/ /07/ /07/1984 Background and Significance Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare, incurable leukodystrophy that is caused in most patients by mutations in the MLC1 gene encoding a protein of still unknown function. MLC patients develop macrocephaly, ataxia, spasticity, motor and cognitive impairment; aggravation of clinical conditions may occur after trauma or fever. Subcortical cysts, brain edema, myelin vacuolation and reactive astrocytosis are the MLC histopathological hallmarks. No genotype-phenotype correlation is known. Because MLC1 protein is mainly expressed in brain astrocytes, elucidating MLC1 physiological function and astrocyte functional alterations caused by pathological MLC1 mutations is the key to understand MLC pathogenesis and find a cure. Recent studies, including our own, indicate that MLC1 mutations alter astrocyte ability to regulate ion exchange and cell volume in response to neuronal activity and osmotic stress. We have also hints that MLC1 might play a role in the modulation of signaling pathways involved in the cellular response to inflammatory and oxidative stress, raising the possibility to target these pathways for therapeutic purposes. Recapitulation of MLC phenotype in the disease-relevant cell types through generation of induced pluripotent stem cells (ipscs) from patients and differentiation of these ipscs into astrocytes likely represents the best strategy to understand MLC pathophysiology and identify novel drug targets. Specific aims Aim 1: Aim 2: Aim 3: To perform mutation analysis in Italian MLC patients with a well characterized clinical phenotype, collect control and patient skin biopsies and set up of fibroblast cultures for the generation of ipscs (see Aim 2) (OU2). To model MLC disease by 1) generating ipsc cultures from control and MLC patient fibroblasts and differentiating them into astrocytes, the disease-relevant cells (OU1); and 2) establishing monocyte/macrophage (M/M) cultures from control donors and MLC patients, as a possible easily accessible and fast cell system for defective pathway validation (OU2). To study MLC1 mutation-induced defects in patient ipsc-differentiated astrocytes and M/M focussing on: i) the activity of specific ion channels responsible for cell volume regulation; ii) the postulated channel properties of MLC1 protein itself; and iii) the regulation of intracellular signaling pathways controlling astrocyte reactivity and 10/07/ / 6

95 Inducible pluripotent stem (ips) cell-derived human astrocytes as a new disease model to shed light into the molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts (MLC). GR Lanciotti Angela Biomedical/Biomedica Istituto Superiore di Sanita' volume changes in response to stress signals (combined effort of OU1, OU2 and OU3, based on the specific expertises). Hypothesis: The hypotheses to be tested in this project are: 1. MLC1 mutations alter the response of astrocytes to stress stimuli (i.e., inflammatory, oxidative, osmotic) and the underlying pathways, resulting in the clinical features of the disease; 2. restoring astrocyte function(s) by targeting MLC1 mutation-induced defective pathways can correct MLCassociated brain abnormalities. Preliminary data: Using human astrocytoma cells overexpressing wild-type or mutated MLC1 and patient-derived monocytes/macrophages, OU1/OU2 demonstrated that MLC1 is involved in the control of cell volume after hyposmotic stress and that this function is impaired by different MLC1 mutations (Brignone, Lanciotti et al., 2011; Lanciotti et al., 2012; Brignone, Lanciotti et al., 2014, Petrini et al., 2013). In particular, by interacting with Na, K-ATPase and the TRPV4 channel, MLC1 favors intracellular calcium increase after hyposmotic treatment, leading to activation of the regulatory volume decrease needed to rescue hyposmosis-induced cell swelling. Preliminary experiments also indicate that MLC1 regulates the astrocyte response to inflammatory/oxidative stimuli by downmodulating the extracellular regulated kinase (ERK) and NF-kB activation, opening new avenues for the identification of drug targets. Because MLC patients aggravate after mild trauma and/or infection (both conditions inducing astrocyte activation and volume changes), altogether the above findings suggest that MLC1 mutations affect astrocyte activation in response to different stress stimuli causing brain damage. As to disease modeling, OU1 in collaboration with the CECS/I-Stem Institute (Evry, France) has recently generated ipscs by reprogramming human fibroblasts from one control subject and two MLC patients carrying different MLC1 mutations (splice-site mutations); to date, 2 ipsc clones from the control subject and 5 ipsc clones from the MLC patients are being expanded. 10/07/ / 6

96 Inducible pluripotent stem (ips) cell-derived human astrocytes as a new disease model to shed light into the molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts (MLC). GR Lanciotti Angela Biomedical/Biomedica Istituto Superiore di Sanita' Materials and Methods 1) Mutation analysis of MLC1 gene in peripheral blood cells from MLC patients using an ABI Prism 3130 xl automatic sequencer (OU2). Patients will be clinically assessed at the Pediatric Hospital Bambino Gesù, a national reference centre for genetic screening of MLC patients. 2) Fibroblast cultures from skin biopsies, PBMC separation, monocyte isolation and in vitro macrophage differentiation (OU2). 3) Development of ipscs by reprogramming of control and patient fibroblasts using episomal vectors and differentiation of ipscs into astrocytes using established protocols (OU1). 4) Analysis of the biochemical and functional properties of wild-type and mutated MLC1 in the human cell-based disease models using biochemical, proteomic and immunocytochemical techniques (OU1/OU2). 5) Electrophysiological studies of the activity of specific ion channels responsible for volume regulation and putative ion channel function of MLC1 in ipsc-derived astrocytes using the patch clamp technique (OU3). Impact and Translational Implications The most important outcomes of this project are a better understanding of MLC molecular pathogenesis and identification of targets for pharmacological intervention. The recent demonstration of MLC1 involvement in the response of astrocytes to stress stimuli already hints at specific drugs to be tested in the human disease models set up in this project. The results obtained could be translated to other neurodegenerative diseases characterized by abnormal astrocyte reactivity and brain edema. 10/07/ / 6

97 Setup of a multi-site radiation dose management system for the optimization of radiological procedures and the assessment of individual and collective radiation dose. GR Gabusi Michele Clinical health care research/clinicoassistenziale Istituto Oncologico Veneto LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Methodological, epidemiological, socio-economic, organizational, managerial emerging public health issues related to the above reported areas Project Classification IRG: Project Classification SS: Healthcare Delivery and Methodologies Biomedical Computing and Health Informatics - BCHI Project Keyword 1: Project Keyword 2: Application of advanced computing architectures to questions in biomedical and clinical information and knowledge management. Integrated system for radiation dose monitoring, patient dose tracking and communication Project Keyword 3: X-ray imaging modalities (Computed Tomography, Interventional Radiology, Mammography and Planar x-ray) Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Medical Physics Unit Project management, data analysis 2 Istituto Oncologico Veneto Radiology Unit Data analysis, clinical expertise Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 pintacuda giovanna Istituto Oncologico Veneto Clinical Support 07/10/ Bezzon Elisabetta Istituto Oncologico Veneto Clinical Support 06/03/ roggio antonella Istituto Oncologico Veneto Medical Physics Support 10/05/ /07/ / 6

98 Setup of a multi-site radiation dose management system for the optimization of radiological procedures and the assessment of individual and collective radiation dose. GR Gabusi Michele Clinical health care research/clinicoassistenziale Istituto Oncologico Veneto Background and Significance As reported by the International Atomic Energy Agency (IAEA), radiation is in everyday life. The term radiation is very broad, but hereinafter it refers to ionizing radiation, i.e. radiation which releases ionization energy per mass unit (dose) in human tissues and, as such, has the stochastic potential of inducing cancers. Medical exposures from radiological imaging contribute to individual and collective dose levels. In recent years, the growing use of X-ray imaging has raised concern about low-dose cancer effects. In 2014 EuroSafe Imaging Campaign was launched to promote safe and up-to-date radiological practices and to improve the communication among patients, healthcare professionals and institutions. Nowadays, all modern radiological equipment can provide radiation dose indices. In Italy, most of hospitals and healthcare sites do not separate storage of dosimetric data from other radiological data like images. Therefore, dosimetric data can be manually retrieved and summarized on request only. Software packages exist to automatically extract radiation dose data and use them to populate a dedicated database. Our Institution provides quality assurance and dosimetric services to multiple radiological units, connected to the same image archive and distributed over a wide geographical area. This makes the IOV the ideal candidate to kick-off a wide-area dosimetric project and supervise the overall radiation dose monitoring and communication process. Specific aims Aim 1: To fill an anonymous registry of patient exposures, stratified by imaging equipment, clinical protocols, patient age/sex, body part. Reference dose levels will be set in order to compare local dose levels with National and European standards, to detect possible equipment misuse or obsolescence and to ground epidemiological studies focused on the stochastic effects of low-dose radiation procedures. Aim 2: To track the individual dose history of patients within and across radiological sites. A precise knowledge of individual cumulative dose may help medical physicists to improve the accuracy of dose assessment, while setting the base for organ dose estimation. Dose history may also support clinicians and radiologists to tailor the most appropriate radiological procedure for each patient and clinical task, and to evaluate alternative pathways for specific clinical cases. Aim 3: To provide a comprehensive, quantitative information based on clinical data about radiation dose levels in radiology, facing a sometimes misperception of the risks associated to radiation exposure. Hypothesis: 1) The availability of an automatic software for the systematic extraction of dosimetric data provided by radiological equipment is a strict requirement to build and populate the dosimetric database. 2) Feeding the database by multi-vendor equipment installed in radiological divisions with different missions and specific clinical tasks, makes the dataset representative of a large population and radiation dose assessment more reliable. 3) There is an increasing need of improving awareness and communication about radiation protection. The right information on radiation dose should increase people consciousness about the balance between benefits and risk of medical exposures. Preliminary data: Ad-hoc routines have been already developed to fetch data from images and reports manually retrieved, in order to check the completeness of dosimetric information provided by the eligible radiological equipment (26 different modalities). This preliminary test was performed on computed tomography (CT), interventional angiography (XA) and mammography images. The analysis have 11/07/ / 6

99 Setup of a multi-site radiation dose management system for the optimization of radiological procedures and the assessment of individual and collective radiation dose. GR Gabusi Michele Clinical health care research/clinicoassistenziale Istituto Oncologico Veneto shown that in 2013 more than 55,000 CT examinations have been performed within the radiological network under surveillance. About 25% of patients with more than one CT examination was imaged in different sites. Patients with more than two CT scans were about 9%, with the highest effective doses observed for abdomen CTs. Materials and Methods Year1: Connection and setup of all imaging modalities (CT, interventional angiography, mammography) feeding the database of radiation dose data. Statistical analyses and development of scorecards and dashboards, to provide radiologists summary results about dose distributions per modality, acquisition protocol, patient age and sex. Year2: Development of an individual dose tracking tool for patients accumulating dose from multiple radiological procedures, because of repeated examinations and follow-up protocols within the radiological network. Such tool will be designed to support radiologists in the application of the Justification principle of radiation protection. Year3: Launch an informative campaign, including several initiatives (web, press, flyers, etc.) addressed to patients, general population, referral physicians, general practitioners, aiming to convey the right information about health risks from dose exposures from radiological procedures. Impact and Translational Implications -The expertise achieved by managing a complex dosimetric network represents a strategic resource for a future Regional dose tracking system -The evaluation of equipment obsolescence/miscalibration will provide objective criteria to ground decisions for instrumentation replacement -The most effective communication techniques implemented in the project, including the key educational messages, can be reused and further developed by other institutions with minimum effort 11/07/ / 6

100 GR Prognostic and predictive values of tumor infiltrating lymphocytes in patients with ductal carcinoma in situ of the breast. LAZZERONI MATTEO Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Population Sciences and Epidemiology Epidemiology of Cancer - EPIC Project Keyword 1: Project Keyword 2: Project Keyword 3: Elucidation of the determinants of cancer and biomarkers of cancer by assembling groups of individuals to determine systematically whether the risk of disease/condition is different for individuals who are exposed or not exposed to specific factors (or combinations of factors) of interest. These may be either risk or protective factors and include genetic, epigenetic, molecular, behavioral, and environmental factors. tumor infiltrating lymphocytes Ductal Carcinoma In Situ of the breast Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Division of Cancer Prevention and Genetics DI - Applicant Institution Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 10/07/ / 6

101 GR Prognostic and predictive values of tumor infiltrating lymphocytes in patients with ductal carcinoma in situ of the breast. LAZZERONI MATTEO Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia Background and Significance Ductal carcinoma in situ (DCIS) of the breast, recently renamed ductal intraepithelial neoplasia (DIN) is a clinically and molecularly heterogeneous disease with different malignant potentials. About half of the women with a local recurrence develop a new DIN and the other half an invasive carcinoma. Although women with a primary DIN have a very good prognosis, those with a subsequent invasive carcinoma have an increased risk of dying from breast cancer (BC). One of the major goals of the treatment of DIN is to prevent invasive disease. Our recently published data suggest that the expression of estrogen receptors (ER), progesterone receptors (PgR), human epidermal growth factor receptor-2 (HER2), and Ki-67 labelling index (LI) may be a useful prognostic and predictive adjunct in DIN patients. However, little is known about factors associated with the risk of developing invasive BC. Evasion of host immunity is thought to be critical for cancer growth and progression and tumor-stromal interactions involve continuous crosstalk among different cell types. Tumor infiltrating lymphocytes (TILs) are important components of these tumor-stromal interactions. While BC has not traditionally been considered an "immunogenic" tumor type, TILs have been correlated with a good outcome in a number of cohorts of patients with invasive BC but, to our knowledge, no data are available about TILs in pure DIN patients. Specific aims Aim 1: Aim 2: Aim 3: to investigate the relationship between quantity and location (stromal, epithelial) of lymphocytic infiltrate at diagnosis of DIN and clinical outcome in a cohort of 1667 consecutive patients operated for DIN in a single institution from 1996 to Disease free BC related cumulative incidence, in-situ cumulative incidence and invasive cumulative incidence stratified by adjuvant treatment - radiotherapy (RT), hormonal therapy (HT) - will be studied. TILs will be evaluated according to molecular phenotype of DIN in order to explore possible subgroups associated with a more favorable immune microenvironment. to investigate the characteristics of the immune infiltrate on patient's outcome. The density of total CD3, CD4, CD8, CD20, PD-1, PD-L1 and CTLA-4 positive lymphocytes will be evaluated by immunohistochemistry (IHC). The results will be correlated with tumor recurrence, patient's survival, and response to RT and HT. to analyze the immune signature of DIN by the Molecular Score which evaluates the relative expression of genes involved in immunity and cell cycle progression. We will compare DIN immune signature with TILs and the prevalence of lymphocyte subtypes as evaluated by IHC. Hypothesis: we hypothesize that higher levels of TILs present at diagnosis are associated with decreased risk of BC recurrence. We intend to investigate whether TILs could be useful as stratification or adjustment factor in decision making regarding the treatment of DIN, as well as providing the rationale for evaluating immunotherapeutic approaches. Preliminary data: In our published series of 1171 patients, according to literature, about half of all new ipsilateral events after a primary DIN are invasive carcinoma. Interestingly, this distribution is also maintained between the low-grade lesions. Recently, TILs have been significantly associated with prognosis and therapeutic efficacy in two subtypes of invasive breast cancer (triple negative and HER2 positive subtypes). In particular, the potentially most novel finding was the association between TILs and increased benefit from the addition of trastuzumab to chemotherapy in HER2+ invasive BC. In a Swedish study on 266 women with a primary DIN from two source populations, all with a known ipsilateral event, HER2+ primary DIN tumors with a known recurrence were associated with a halved risk of the recurrence being invasive. We speculate that this molecular phenotype might be associated with a more favorable immune microenvironment. A fraction (roughly 500) of the cases included in our present study will be investigated by next generation sequencing techniques at the Ontario Institute for Cancer Research (PI Dr. John Bartlett), and compared to the histological and 10/07/ / 6

102 GR Prognostic and predictive values of tumor infiltrating lymphocytes in patients with ductal carcinoma in situ of the breast. LAZZERONI MATTEO Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia phenotypical characteristics. Materials and Methods Histopathologic analysis of the lymphocyte infiltrate will be performed on hematoxylin and eosin stained sections. ER, PgR, Ki-67 LI and HER2 expressions will be evaluated by immunohistochemistry. FISH Test (Fluorescence In Situ Hybridization) will be performed if required (HER-2 2+). The Molecular Score has been developed by Myriad Genetics and validated by our own group in a series of 700 invasive breast cancers with a Luminal B intrinsic subtype (SABCS 2014, abstract submitted). Statistical methods: the prognostic and predictive roles of TILs will be evaluated with a multivariable Cox regression model in a cohort of 1667 consecutive patients operated for DIN in a single institution from 1997 to Impact and Translational Implications The aim of this study is to look for patient and primary tumor characteristics that might be associated with progression from in situ to invasive breast cancer. If we can better categorize DIN patients and predict who has a low risk for an invasive recurrence, this might help us to individualize type of surgery and adjuvant treatment (RT, HT), providing also the rationale for evaluating immunotherapeutic approaches. 10/07/ / 6

103 A comprehensive preventive program for dementia tailored on the neuropsychological profile of persons with Mild Cognitive Impairment: cognitive stimulation, physical intervention and healthy nutrition, a randomized controlled trial. GR DI SANTO SIMONA GABRIELLA Clinical health care research/clinicoassistenziale Fondazione Santa Lucia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Alzheimer s disease and other dementias. Prevention programs Cognitive stimulation, physical exercise, dietary interventions Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Clinical and Behavioural Neurology Recruitment and evaluation of participants, cathegorization/inclusion/treatment of subjects, follow up visits. Study design and data collection, monitoring, and management, statistical analyses, interpretation of results. 2 National Research Council (CNR) - ITIA 3 National Research Council (CNR) - IBFM Institute of Industrial Technologies and Automation (ITIA) Institute of Molecular Bioimaging and Physiology (IBFM) Main developer of the virtual environment as an effective and usable tool for intervention on trial participants. Ideation of a physical exercise program to be administered to the participants in the trial. Neuroimaging data analysis. 11/07/ / 6

104 A comprehensive preventive program for dementia tailored on the neuropsychological profile of persons with Mild Cognitive Impairment: cognitive stimulation, physical intervention and healthy nutrition, a randomized controlled trial. GR DI SANTO SIMONA GABRIELLA Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Fondazione Santa Lucia Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Arlati Sara National Research Council (CNR) - ITIA Definition and implementation of a virtual environment for supporting users (people at risk of dementia) to train their cognitive abilities for prevention purposes with a program of specific cognitive stimulation. 16/12/ Rastelli Fabio National Research Council (CNR) - IBFM Responsible for design of an exercise program suitable for the psychophysical characteristics of the participants and for the diagnostic work related to neuroimaging investigations of the subjects enrolled in the study. He will quantify the damage in different brain areas in persons with initial cognitive damage and in those developing AD during follow-up visits. 02/04/ Nicoli Marco IRCCS Fondazione Santa Lucia Management of neuropsychological assessments, data collection/entry. 27/06/1988 Background and Significance Alzheimer Disease (AD) is a neurodegenerative progressive disorder causing in advanced stages cognitive impairment and dementia. Persons with MCI (pmci) have an increased risk to develop AD dementia (ADd) and thus represent target population for interventions aimed at halting/reducing AD progression. A significant number of pmci remain stable or return cognitively normal over time, hence it is crucial to identify the subject's characteristic associated to progression of AD. An early and precise diagnosis of AD in pmci is an important requirement, allowing to intervene when many cognitive/functional abilities are still preserved. Data from observational studies indicate that physically and cognitively active lifestyles and Mediterranean diet are protective against AD. In randomized controlled trials (RCT) physical exercise (PE) was effective in enhancing cognitive performance in healthy and MCI persons, whereas promising but inconclusive data exist on the efficacy of cognition-based interventions. PE and proper nutrition are expected to exert an action over the general disease mechanisms underlying neurodegeneration, while cognitive stimulation (CS) is aimed to restore impaired (or strengthen residual) functions. Since heterogeneity exists in cognitive presentations of MCI, it seems reasonable to consider that pmci could benefit from a patient-specific CS tailored on the subjects' neuropsychological profiles. Specific aims Aim 1: Aim 2: AD is a progressive disorder: prior to developing dementia, affected subjects show for long no symptoms or only a mild cognitive impairment (MCI) that do not affect significantly their functional independence. Memory compromission is specific to ADd but some patients show 'variant' presentations with prominent impairment of language (LAN), visuospatial (VS) and executive (EXE) functions. First aim of this project is to evaluate if subtypes of MCI with predominant MEM/VS/LAN or EXE deficits could be identified that will have different probabilities of developing ADd over time. At present, drug treatments for AD have only symptomatic effects and preventive measures centered on modifiable risk/protective factors for ADd could represent the preferred intervention/strategy to delay the onset of 11/07/ / 6

105 A comprehensive preventive program for dementia tailored on the neuropsychological profile of persons with Mild Cognitive Impairment: cognitive stimulation, physical intervention and healthy nutrition, a randomized controlled trial. GR DI SANTO SIMONA GABRIELLA Clinical health care research/clinicoassistenziale Fondazione Santa Lucia symptoms and/or reduce patients' disabilities. Cognitive training might improve cognitive skills and mood in pmci. The study aims therefore to verify: - if specific cognitive stimulation (CS) focused on the subject's neuropsychological profile may slow the cognitive decline of pmci or/and reduce the risk of dementia; - if a CS tailored on the worst affected domain(s) is more effective than aspecific CS or than an 'enhancing' CS focused on residual cognitive abilities; - if the possible effects are only short-term or persist after 12-months follow up. Aim 3: Since there is some evidence that physical exercise (PE) may be protective against AD, third aim is to further verify the effectiveness of PE in reducing the risk for dementia, which of CS and PE is the most effective intervention, if PE and CS exert an independent action or if any effect of interaction may be observed. Hypothesis: We hypothesize that a large number of pmci will progress to ADd, even if their neuropsychological profile does not involve primarily memory. We hypothesize that pmci who undergo an intervention consisting of CS and/or PE have a reduced risk of developing dementia and reduced rates of cognitive and functional decline compared to controls. Preliminary data: In a previous Study we observed that 4 subgroups of ADd patients with preminent MEM, VS, LAN or EXE impairment could be identified. These subgroups recalled cognitive presentation of typical and ADd variants. It was also observed that in ADd the dominance of impairment in one cognitive domain seems to persist over time and it may be regarded in terms of earliness/entity of involvement of the pertaining cerebral areas in the progressive degenerative process. This questions the characterization of ADd as a disease of memory. Materials and Methods A cohort of 300 persons impaired in 1 or more cognitive domains and without significant functional impairment will be identified (pmci). At enrollment, clinical and neuropsychological data, a blood sample for APOE genotyping and structural brain MRI for morphological investigation will be collected. Participants will be categorized according to number/types of affected domains. A randomized controlled trial with factorial design will be conducted to compare the separate and combined effect of a CS tailored on the subject's cognitive profile and PE. CS will be conducted in a virtual environment designed applying innovative ICT. Indications for Mediterranean diet will be provided to all participants. The cohort will be evaluated every 6 months up to 3 years for the occurrence of AD according to NIA-AA criteria. Primary Outcome: progression to AD diagnosed clinically and confirmed by positivity of at least one biomarker (CSF evaluation of Aß, tau, p- tau, MRI, FDG and amyloid PET). Impact and Translational Implications The more precise definition of the cognitive profile of early typical and atypical AD will give new insight for early diagnosis, prevention and disability compression (i.e. the reduction of the time period of disability associated with cronic disorders). This is one of the best public health strategies in terms of cost-effectiveness, with a wide range of benefits covering physical and mental health as well as those closely related to the consequences of the disease. 11/07/ / 6

106 GR Economic crisis and adherence to the Mediterranean diet: possible impact on biomarkers of inflammation and metabolic phenotypes in the cohort of the MOLI-SANI study. Bonaccio Marialaura Biomedical/Biomedica Molise LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Healthcare Delivery and Methodologies Health and Health Related Behavior of Individuals and Populations Fellowship - F16 Project Keyword 1: Project Keyword 2: Project Keyword 3: Population-based studies of health promotion and environmental change Nutrition metabolic risk factors Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Dipartimento di patologia neuro e cardiovascolare. Unita' di epidemiologia e prevenzione Coordinator, population identification and recruitment, laboratory analysis, data management and analysis Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date Background and Significance The Mediterranean diet has been associated with reduced risk of developing major chronic diseases and metabolic disorders and its beneficial effects have been mainly ascribed to its anti-inflammatory properties. Late evidence suggests that people from Mediterranean areas are drifting away from this dietary pattern in favour of less healthy diets associated with an increased risk of developing major chronic disease. National data on Italian food expenditure recently revealed a decrease in the purchase of key-foods of the Mediterranean diet such as pasta (-5%), fish (-7%), extra virgin olive oil and fresh vegetables (-4%). Unhealthy diets are mainly adopted by disadvantaged groups and cluster with other unhealthy lifestyles leading to a social gradient in mortality and morbidity. Previous studies suggest that diet quality follows a socioeconomic gradient showing a linear relationship between food cost, eating patterns and obesity. So far, possible unfavourable health outcomes deriving from the current economic downturn have only been predicted on the basis of what happened during previous recessions. There is reason to believe that the economic downturn that contributes to the increase in quality food cost is going to determine unfavourable effects on the dietary lifestyle of Italian people, especially among the less wealthy groups. It is likely that the abandon of the Mediterranean diet could have detrimental health outcomes and increase social gaps across the population. 10/07/ / 5

107 GR Economic crisis and adherence to the Mediterranean diet: possible impact on biomarkers of inflammation and metabolic phenotypes in the cohort of the MOLI-SANI study. Bonaccio Marialaura Biomedical/Biomedica Specific aims Molise Aim 1: Aim 2: Aim 3: To identify population groups differently affected by the economic crisis within the population-based cohort of the MOLI-SANI study recruited in the years (before economic crisis). This aim will be achieved by a new assessment of self-reported economic difficulties possibly emerged after the recruitment. To estimate possible changes in dietary and health-related behaviours (with particular focus on the adherence to the Mediterranean diet) in subjects identified in the previous aim as highly or poorly affected by the economic crisis. Inflammatory status and metabolic phenotypes will be assessed in the two groups, recalled in a suitable proportion, to establish a possible link between shifting from the Mediterranean diet and adverse health outcomes. Quality of life and stress status will also be evaluated. To evaluate in the group more affected by economic constraints whether nutrition knowledge and mass media exposure would account for the decline in the adherence to the Mediterranean diet and consequent changes in inflammatory status and/ or metabolic phenotypes. Hypothesis: The hypothesis is that the current economic crisis is contributing to the observed rapid decrease in the adherence to Mediterranean diet, a protective factor against the development of major chronic diseases. Elevated dietary-related inflammation biomarkers or increase in metabolic phenotypes should represent the biological account for the decline in the adherence to Mediterranean diet within the group most affected by the economic crisis. This project will also test whether in the most economically affected people cultural resources could somehow attenuate the impact of material circumstances on lifestyle changes attributable to the economic crisis. Preliminary data: MOLI-SANI is a population-based cohort study that recruited, between 2005 and 2010, 24,325 men and women at random from individuals aged 35 years or older resident in the Molise region, to investigate genetic and environmental risk factors for cardiovascular, cerebrovascular and tumour diseases. In this cohort we have collected preliminary data suggesting that low income is inversely associated with adherence to a Mediterranean- like eating pattern and positively linked to higher prevalence of obesity, independently of other components of socioeconomic status. Nutrition knowledge and information level were related to a significantly higher adherence to healthy diets. Adherence to the Mediterranean pattern changed over time falling from over 30% ( ) to 18% ( ). Among participants recruited in the years , major socioeconomic indicators were not associated with a Mediterranean behaviour, whereas in people recruited in the period socioeconomic determinants began to play a major role in explaining the association with the Mediterranean diet. We hypothesized that the economic downturn could be on the causal pathway, but longitudinal investigations are needed. 10/07/ / 5

108 GR Economic crisis and adherence to the Mediterranean diet: possible impact on biomarkers of inflammation and metabolic phenotypes in the cohort of the MOLI-SANI study. Bonaccio Marialaura Biomedical/Biomedica Molise Materials and Methods Baseline information on the cohort recruited before economic crisis is already available. A questionnaire will be administered to evaluate present lifestyle and possible changes on dietary habits attributable to the economic downturn. Groups differently affected by economic crisis will be identified. A statistically suitable proportion of these groups will be called for a second visit to assess changes in dietary habits by a semi-quantitative food frequency questionnaire. Inflammatory biomarkers (hs-crp, IL-6, IL-18, TNF, PAI-1, adhesion molecules, adiponectin, platelet and leukocyte counts) will be measured. Metabolic phenotypes will be defined as variation in the prevalence of hypertension, diabetes, obesity, metabolic syndrome, elevated blood lipid profile or in the levels of glucose, insulin, cholesterol, HDL, LDL, triglycerides, blood pressure, hip and waist circumferences. Nutrition knowledge, exposure to mass media, quality of life and stress status will also be assessed. Impact and Translational Implications The project will evaluate the effects of economic crisis on adherence to Mediterranean diet, inflammatory markers and health outcomes in Italy where healthy dietary habits have long been followed against major diseases. It will help identify new high-risk population groups for which targeted prevention strategies could be required. By the interaction of cultural and material circumstances, tools will be found to identify modifiable risk factors and reduce socioeconomic inequalities in health.

109 Cardiac troponin after cardiac surgery BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Monaco Fabrizio Clinical health care research/clinicoassistenziale Ospedale San Raffaele - Milano LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Surgical Sciences, Biomedical Imaging, and Bioengineering Surgery, Anesthesiology, and Trauma - SAT Project Keyword 1: Project Keyword 2: Project Keyword 3: Tissue, organ and systemic injury responses to surgery, trauma, burn, sepsis, hemorrhage, ischemia-reperfusion, or resuscitation, including integrating pathways and signals. cardiac surgery postoperative vascular events Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Anesthesiology and Intensive Care Project Management, Patients' enrolment Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date NICELLI ELISA Ospedale San Raffaele/Anesthesiologist 4 cama eliodoro Ospedale San Raffeale/Anesthesiologist Contributor, patients' enrolement 08/03/1982 Contributor, patients' enrolement 05/01/ /07/ / 5

110 Cardiac troponin after cardiac surgery BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Clinical health care research/clinicoassistenziale Monaco Fabrizio Ospedale San Raffaele - Milano Background and Significance Worldwide over 1 million adults undergo cardiac surgery annually. Although cardiac surgery provides important procedural benefits, it is associated with major adverse vascular events. There is promising but inconclusive evidence that troponin measurements after surgery may identify prognostically important myocardial injury after cardiac surgery. Furthermore, the majority of important myocardial injuries are likely undetected without perioperative troponin monitoring. The proposed threshold to define an important myocardial injury in the latest guidelines are arbitrary and not informed by high-quality observational data. Identifying those who suffer aprognostically important myocardial injury after cardiac surgery will have immense implications for improving the postoperative outcomes in a vulnerable patient population. Additionally, the change in the patient population undergoing cardiac surgery (older and greater comorbidity), modifications in surgical and anesthetic techniques, and limitations of previous research contribute to uncertainty regarding the current incidence of major vascular events in patients undergoing cardiac surgery. We propose a prospective cohort study to address these issues. Specific aims Aim 1: Aim 2: Our primary objective is to determine the relationship between troponin measurements and the 1-year risk of death. Our second objective is to determine the incidence of major vascular events (i.e., a composite of vascular death, nonfatal myocardial infarction, nonfatal stroke, and mechanical assist device) at 30 days and 1-year after surgery. Aim 3: - Hypothesis: The literature clearly indicates that early myocardial injury markedly impacts short-term, intermediate-term, and long-term survival in post-cardiac surgery patients. There is a shift in practice patterns away from CK- MB towards laboratories using high-sensitivity troponin. Therefore, there is a need to define the prognostically relevant high-sensitivity troponin threshold(s) after cardiac surgery. Our proposed study will utilize the contemporary measure of high-sensitivity troponin in order to determine the distribution of peak troponin values and change from preoperative values. We will then be able to determine the prognostically relevant troponin thresholds that significantly impact 1-year mortality. This will support a uniform approach and specific definition of myocardial injury, which can then be used to inform the definition of perioperative myocardial infarction. Preliminary data: A patient-level data meta-analysis (n=7234) suggested that only age and peak troponin ratio within 24 hours after coronary artery bypass grafting (CABG) surgery were independent predictors of 30- day mortality. Another meta-analysis of 8 studies included 3363 patients of whom 144 died at 12 months after cardiac surgery. The summary estimates of the association (odds ratio and 95% confidence intervals) of troponin above versus below the cut-off in an individual study and the occurrence of death after adult cardiac surgery was 5.46 (95% CI: ) for intermediate-term (=>12 months) all-cause mortality and 6.57 (95% CI: ) for short-term (<=30 days) all-cause mortality. The results were not influenced by the type of cardiac surgery (i.e., CABG surgery, CABG and valvular surgery, or isolated valvular surgery). Although these studies provide encouraging evidence that elevated troponin measurements after cardiac surgery predict mortality, meta-analyses have several limitations. The third universal definition of myocardial infarction was recently revised in a 2012 consensus statement of the European Society of Cardiology, American College of Cardiology Foundation, American Heart Association, and the World Heart Federation and the authors acknowledged in this guideline that their proposed definition of an elevated troponin measurement in the first 48 hours after CABG surgery was based on, "arbitrary convention". A large high-quality study including a 11/07/ / 5

111 Cardiac troponin after cardiac surgery BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Clinical health care research/clinicoassistenziale Monaco Fabrizio Ospedale San Raffaele - Milano representative sample of patients (i.e., CABG, valvular, combined surgeries, along with those undergoing elective or emergent/urgent surgeries) is urgently needed to address these important issues. Materials and Methods This is a prospective cohort study of a representative sample of 1,000 adult patients undergoing cardiac surgery in San Raffaele Hospital. Study personnel will evaluate patients prior to surgery, throughout their hospitalization, and contact patients at 30 days and 1 year after surgery. All patients will have a troponin measured within the 4 hours prior to surgery, 3 to 12 hours postoperatively, and on the first, second, and third day after surgery. Impact and Translational Implications This study will establish the role of perioperative high-sensitivity troponin measurements in identifying prognostically important myocardial injury after cardiac surgery. Knowing the current burden of perioperative vascular events will inform clinicians, administrators, and granting agencies about resources required to address the problem and this will have profound public health implications. 11/07/ / 5

112 GR Ectodermal dysplasia syndromes by defective nectins (nectinopathies): molecular mechanisms and clinical implications Brancati Francesco Biomedical/Biomedica Istituto Dermopatico dell'immacolata (IDI) LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Innovative biotechnologies Project Classification IRG: Project Classification SS: Musculoskeletal, Oral and Skin Sciences Arthritis, Connective Tissue and Skin - ACTS Project Keyword 1: Project Keyword 2: Project Keyword 3: Skin and Cutaneous Biology: Disorders of skin and skin appendages, such as inflammatory, preneoplastic, and hyperproliferative disorders, as well as systemic diseases with significant cutaneous involvement. Ectodermal dysplasia Cell-adhesion Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Laboratory of Molecular and Cell Biology Project coordination; patient recruitment; data collection and interpretation; genomic studies; in vitro and in vivo studies 2 Istituto Dermopatico dell'immacolata 3 Sapienza University of Rome Investigators, Institution and Role in the Project Laboratory of Molecular and Cell Biology In vitro and in vivo studies using primary keratinocytes and skin/hair follicle cultures Laboratory of Dermatology, Department of Neuroscience, Mental Health and Sensory Organs ChIP-seq analysis; methylation/acetylation studies Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Cordisco Sonia Istituto Dermopatico dell'immacolata In vitro and in vivo studies using primary keratinocytes and skin/hair follicle cultures 25/06/ Botti Elisabetta Sapienza University of Rome ChIP-seq analysis; methylation/acetylation studies 16/07/ /07/ / 6

113 GR Ectodermal dysplasia syndromes by defective nectins (nectinopathies): molecular mechanisms and clinical implications Brancati Francesco Biomedical/Biomedica Istituto Dermopatico dell'immacolata (IDI) Background and Significance Nectin cell-adhesion proteins are essential to form adherens junctions (AJs) and regulate cellular activities such as movement, proliferation, survival, differentiation and polarization. The dynamic regulation of nectins is pivotal in morphogenesis and in skin, hair and tooth integrity and renewal. Indeed, PVRL1 (nectin-1) mutations cause cleft lip/palate ectodermal dysplasia (ED) syndromes with digit, tooth and hair defects, while PVRL4 (nectin-4) is mutated in ED-syndactyly syndrome, with hair and tooth anomalies. Notably, clinical overlap exists between phenotypes resulting from nectin mutations (i.e. nectinopathies) and those associated to p63 and IRF6 transcription factors mutations. p63-morphopathies feature ectodermal defects, cleft lip/palate and limbs malformations, while IRF6 mutations cause variable phenotypes with cleft lip/palate or a syndrome of cutaneous syndactyly and distal limb involvement. More interestingly, p63 and IRF6 have been shown to participate in a regulatory feedback loop, while p63-transcriptional regulation of major cell-adhesion molecules, cadherins, was recently documented. Here, we aim at defining the role of nectins in keratinocytes and hair follicle homeostasis and renewal and in the crosstalk between nectins and regulatory transcription factors. Together with an in-depth clinical-molecular characterization of nectinopathies, this project will advance knowledge of cell-adhesion physiopathology and improve patient care. Specific aims Aim 1: Aim 2: Aim 3: To define the clinical-molecular spectrum of nectinopathies and genotype-phenotype correlations To characterize the role of nectins in skin and hair development and renewal To outline the transcriptional regulation of nectins by p63 and IRF6 transcription factors and transcriptional consequences of nectin defects Hypothesis: The molecular and cellular mechanisms by which nectins contribute to tissue morphogenesis, skin and adnexa maintenance and renewal are still largely undetermined. Previous studies on AJs function envisage a role for nectins in proliferation and collective cell migration through crosstalk between cell-cell and cellextracellular matrix adhesion, transduction of intracellular signals and control of cell polarity axis. Clarifying these mechanisms will shed light on how nectins contribute to the diverse clinical manifestations of nectinopathies, from hair anomalies to craniofacial (lip/palate) and digit defects. In this respect, cultured keratinocytes and skin biopsies from patients affected by nectinopathies represent an ideal study model. As hair anomalies and progressive alopecia are constant features in nectinopathies and nectins are expressed in distinct hair structures and layers, we predict a relevant role for nectins and their regulatory factors in hair development and cycling. Finally, since mutations in genes encoding p63 and IRF6 transcription factors (TFs) lead to human disorders mirroring nectinopathies, we hypothesize that nectin expression may be modulated by such TFs. Preliminary data: Clinical-molecular characterization of six available families indicates that defective nectin-4 (N4) can also lead to cleft lip/palate further pointing to the strict nectin-1 (N1)/N4 interplay and attesting the N1/N4 phenotypic overlaps. Primary fibroblasts and keratinocytes from 3 different patients, two with N4 and one with N1 mutations, are also available. In previous studies, we demonstrated that a synergistic action of N1/N4 induces the activation of Rac1, a member of the Rho family of small GTPases, and regulates E-cadherin-mediated AJs formation. Preliminary data indicate that mutant N4 results in defective wound closure in an in vitro scratch assay. In mouse hair, N1/N4 are most prominently expressed in the transient amplifying cells of anagen bulb and in different layers of outer and inner root sheath, similarly to what previously reported by our group for N4 in human hair follicle. Overall, nectin expression appears reduced 10/07/ / 6

114 GR Ectodermal dysplasia syndromes by defective nectins (nectinopathies): molecular mechanisms and clinical implications Brancati Francesco Biomedical/Biomedica Istituto Dermopatico dell'immacolata (IDI) during catagen and telogen phases in mouse. Together with published expression data showing increased N4 in mouse bulge stem cells as compared to the hair germ, these data point to a functional role of nectins in hair cycling. IRF6 ChIP-seq on differentiating primary keratinocytes revealed preferential binding to adhesion and differentiation genes at sites that also contain activator protein-1 responsive element. PVRL1 and PVRL4 genes bind IRF6 at enhancer marked regions. Materials and Methods Aim1. The clinical-demographic data of ED patients will be collected through national and international collaborations in a dedicated database. Sequencing of the PVRL1 and PVRL4 genes will be performed. If no mutation is identified, a next generation sequencing panel study will be run. Aim2. The effects of nectin altered expression on cell proliferation and migration, Rac activity, cytoskeleton organization and AJs distribution will be investigated in vitro by using N1/N4-defective patient keratinocytes, and viral-transduced keratinocytes and organotypic skin/hair cultures. N1/N4 expression will be studied in mouse and human hair. Aim3. ChIP-seq data will be generated from samples of keratinocytes derived from N1/N4-defective patients and healthy controls using: a) anti-irf6 and anti-p63 antibodies to identify promoter occupancy; b) histone methylation antibodies to characterize active promoters and enhancers; c) anti-acetylated histone-antibody for transcriptionally active chromatin. Impact and Translational Implications - Increased clinical and genetic knowledge of nectin-related disorders will translate into earlier diagnosis, more appropriate disease management and counselling and, overall, improved quality of life of patients and their families. - Definition of the role of nectins and their transcriptional regulation in morphogenesis, epithelial tissue homeostasis and renewal will set the basis for the identification of druggable molecules causing specific defects for future therapeutic interventions. 10/07/ / 6

115 GR Predicting post-surgery complications in patients undergoing coronary artery bypass graft through the assessment of perioperative cardiovascular control indices Bari Vlasta Clinical health care research/clinicoassistenziale Policlinico San Donato LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Surgical Sciences, Biomedical Imaging, and Bioengineering Surgery, Anesthesiology, and Trauma - SAT Project Keyword 1: Project Keyword 2: Project Keyword 3: Tissue, organ and systemic injury responses to surgery, trauma, burn, sepsis, hemorrhage, ischemia-reperfusion, or resuscitation, including integrating pathways and signals. Monitoring autonomic nervous system during general anesthesia Predicting atrial fibrillation and acute kidney injury Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Cardiothoracic - Vascular Anesthesia and Intensive Care Unit Biomedical signal proccessing and data analysis; patients' enrollment and data acquisition in critical care unit Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date Marianello Daniele IRCCS Policlinico San Donato, Staff Anesthesiologist Patients' enrollment, clinical parameters evaluation 22/07/ /07/ / 6

116 GR Predicting post-surgery complications in patients undergoing coronary artery bypass graft through the assessment of perioperative cardiovascular control indices Bari Vlasta Clinical health care research/clinicoassistenziale Policlinico San Donato Background and Significance A significant percentage of patients undergoing coronary artery bypass graft (CABG) runs into post-surgery complications such as atrial fibrillation (20%) and moderate (10%) to severe (2-5%) acute kidney injury. The prevention of these unfavorable events might lead to a better outcome after surgery, to an improvement of the patient's management and to a reduction of the hospitalization duration and health care costs. Cardiovascular control is performed by multiple, simultaneously interacting, regulatory mechanisms activated through the action of the vagal and sympathetic branches of the autonomic nervous system (ANS). An impairment of cardiovascular control might trigger atrial fibrillation and concur to renal failure. Cardiovascular control and ANS state can be assessed through the continuous evaluation of the beat-to-beat changes of cardiovascular variables such as heart period, systolic arterial pressure, ventricular repolarization duration and cerebral blood flow. During surgery an inadequate peripheral tissues perfusion might cause an impairment of the cardiovascular control and, consequently, an adverse post-surgery outcome. Perioperative monitoring of the cardiovascular regulation, cerebral blood flow, ANS state and microcirculatory perfusion might, thus, be helpful to predict and prevent adverse post-surgery events. Specific aims Aim 1: Aim 2: Aim 3: The first aim is to assess the state of the autonomic nervous system (ANS) before and after induction of general anesthesia in 200 patients undergoing coronary artery bypass graft (CABG) surgery through the evaluation of microcirculatory perfusion and oxygenation of tissues and through the computation of indices derived from the beat-to-beat variabilities of heart period, systolic arterial pressure, ventricular repolarization duration and cerebral blood flow. The second aim is to evaluate the statistical correlation between indices describing the state of the autonomic nervous system and those describing the condition of specific districts such as cardiovascular, renal and cerebral ones before and after the induction of general anesthesia in patients undergoing CABG surgery. The third aim is to develop and validate a statistical prediction model that relates post- surgery adverse outcomes, such as atrial fibrillation and renal failure, with the extracted perioperative parameters. The model will be built by considering perioperative indices showing the highest correlation with clinical markers of adverse outcome. The validity of the model will be tested through the receiving operating curves (ROC) technique. The perioperative monitoring of such indices will be helpful in developing the best strategy in order to reduce patient's risk of adverse post-surgery outcomes. Hypothesis: Post operative complications, such as arrhythmias or kidney failure, can be linked to an impairment of the autonomic nervous system, of baroreflex sensistivity or of tissue perfusion and oxygenation, that can be evaluated by means of perioperative monitoring of different variables. We hypothesize that there is a link between an impaired cardiovascular control before surgery and the occurrence of post-surgery unfavorable events such as atrial fibrillation and renal failure in patients undergoing coronary artery bypass graft, thus allowing the prediction and the prevention of these complications based on the assessment of perioperative cardiovascular control indices. Preliminary data: The feasibility of the project was verified over 34 patients undergoing coronary artery bypass graft surgery. The ability of the extracted perioperative indices to describe cardiovascular control and ANS function before and after the induction of general anesthesia in such patients was confirmed in [Porta et al, JAP,115(7):1032:42,2013]. Indices related to ANS activity resulted also correlated with the peak serum creatinine level after cardiac surgery, as well as with the duration of hospitalization. Baroreflex sensitivity index was chosen to develop a preliminary prediction model of renal failure, showing a good performance in terms of area under the ROC curve, thus suggesting a relation of the 10/07/ / 6

117 GR Predicting post-surgery complications in patients undergoing coronary artery bypass graft through the assessment of perioperative cardiovascular control indices Bari Vlasta Clinical health care research/clinicoassistenziale Policlinico San Donato autonomic nervous system state with the post-surgery condition of the renal district and outcome. Materials and Methods ECG and invasive arterial pressure signals will be continuously monitored and acquired using an A/D board (sampling rate: 1 KHz) connected to patient's monitor. Cerebral blood flow velocity (CBFV) will be monitored with a transcranial Doppler device. Microcirculatory tissue perfusion will be assessed through sidestream-darkfield images at the level of the sublingual vasculature. Recording sessions of 10 minutes will take place before and after general anesthesia induction with propofol and remifentanil. Heart period, ventricular repolarization duration, systolic arterial pressure and mean CBFV will be extracted on a beat-to-beat basis. Indices assessing cardiovascular control derived from variability series and perioperative markers of blood oxygen saturation and microcirculatory perfusion will be correlated with postoperative clinical indexes. ROC analysis will be adopted to develop a multivariate statistical prediction model to forecast the occurrence of postsurgery adverse events. Impact and Translational Implications This project stresses the need of monitoring perioperative indices describing cardiovascular control, brain perfusion, microcirculatory regulation and ANS state in patients undergoing CABG surgery for the prediction of postoperative adverse events. The demonstration of the ability of these indices in predicting post-surgery complications would lead to a faster patient's recovery, an improvement of short- and long-term outcome and a reduction of patients' hospitalization and health care costs. 10/07/ / 6

118 GR Investigating insertional mutagenesis as a new mechanism of HIV-1 persistence in infected host Cesana Daniela Biomedical/Biomedica Ospedale San Raffaele - Milano LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Infectious and immunological diseases Project Classification IRG: Project Classification SS: AIDS and Related Research AIDS Molecular and Cellular Biology - AMCB Project Keyword 1: Project Keyword 2: Project Keyword 3: Molecular and biochemical mechanisms of virus entry, genome integration, proviral transcription, and viral particle assembly and release. HIV-1 mediated insertional mutagenesis HIV-1 persistence Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Division of Regenerative Medicine, Stem Cells and Gene Therapy; Safety of gene therapy and insertional mutagenesis research unit 2 Ospedale San Raffaele Division of Regenerative Medicine, Stem Cells and Gene Therapy; Autoimmune genetic diseases unit PI Research collaborator, Immunological studies 3 Ospedale San Raffaele Department of Infectious Diseases Research collaborator, Managment of clinical samples Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Santoni de Sio Francesca Romana Ospedale San Raffaele Research collaborator, she will take care of the immunological studies of the work 25/05/ Chiappetta Stefania Ospedale San Raffaele Research collaborator, she will provide important clinical know-how and management of the samples that will be obtained from HIV-infected patients 28/03/ /07/ / 6

119 GR Investigating insertional mutagenesis as a new mechanism of HIV-1 persistence in infected host Cesana Daniela Biomedical/Biomedica Ospedale San Raffaele - Milano Background and Significance Oncoretroviruses can leads to cancer in mammals through insertional activation of proto-oncogenes. It was commonly thought that HIV-1 does not take advantage of insertional activation mechanisms, since the rapid cytopathicity of infection kills transformed cells. Nevertheless, we and others have demonstrated that HIV-1-derived lentiviral vector integrations are able to interfere with the cellular transcriptional machinery leading to gene activation/inactivation and aberrantly spliced viral/cellular chimeric mrnas. Furthermore, a significant enrichment of integrations targeting specific genes, STAT5B and BACH2, was observed in T-cells from HIV-1 infected patients under Anti-Retroviral Therapy (ART) with respect to the other lentiviral based datasets. The narrow clustering and peculiar disposition of HIV-1 integration sites within those genes resemble that of retroviral integrations that activate oncogenes by promoter insertion mechanisms in tumors arising from infected mice. Main goal of this grant application is to assess if HIV-1 integration interferes with cell functions to its own advantage and to shed light on the molecular mechanisms of this phenomenon. By this approach we will uncover new molecular mechanisms exploited by HIV-1 to promote viral escape to the immune system, latency and ultimately persistence in the host. Hence, our work will help identifying novel important therapeutic targets for HIV-1 eradication. Specific aims Aim 1: Aim 2: Aim 3: To generate an annotated database of genes over-targeted by HIV-1 integrations and of virus/host chimeric mrna sequences in different hematopoietic cell subsets of HIV-1 infected patients. To characterize the functional impact of BACH2 and STAT5B for their possible involvement in the regulation of the HIV-1 life cycle. We will assess if HIV-1-mediated deregulation of these genes promotes HIV persistence in the host cells by overexpressing them in naive CD4+ T-cells and test their effect on HIV expression and immune cell function. To characterize the functional impact of selected candidate genes significantly targeted by HIV-1. We will perform the functional studies described in Aim2 on candidate genes selected in Aim1. Hypothesis: HIV-1 could exploit insertional mutagenesis mechanisms to deregulate specific target genes and modulate the behavior of the infected cells to promote its persistence in infected host. Preliminary data: We have recently demonstrated that HIV-derived lentiviral vectors can deregulate the expression of targeted genes and promote tumor onset acceleration in tumor prone mice through insertional mutagenesis mechanisms (Cesana, JCI 2012 and Mol Ther 2014). Independent studies observed clustered HIV-1 integrations within BACH2 and STAT5B in T-cells from ART-treated patients (Maldarelli, Science 2014 and Ikeda, JID 2007) and suggested activation of these genes by insertional mutagenesis. Interestingly, both genes are transcription factors playing important roles in the function of T cells, tolerance induction and HIV-1 transcriptional silencing. This might suggest that under ART pressure cell clones harboring these HIV-1 activating integrations are selected. We aim to confirm i) the enrichment of integrations targeting STAT5B or BACH2, ii) if these integrations are able to activate target genes and iii) if target gene deregulation has a functional role in modulating the phenotypic behavior of CD4+ T-cells in additional patient cohorts and cell lineages. A preliminary integration site analysis (N=199) from peripheral blood mononuclear cells from 103 HIV-1 infected patients under ART from our institution confirmed that STAT5B and BACH2 were the most targeted genes. We also identified and sequenced validated chimeric HIV-1/STAT5B fusion transcripts from the same samples. Still, we observed that forced expression of STAT5B in naïve CD4+ T-cells from healthy donors improved their differentiation towards a T-regulatory phenotype and increased their proliferation rates. These preliminary data suggest a wider role for insertional mutagenesis in HIV-1 persistence with other genes over-targeted and/or deregulated in HIV-1 infected patients. By combining integrome and transcriptome data from HIV-1 infected patient cells, 10/07/ / 6

120 GR Investigating insertional mutagenesis as a new mechanism of HIV-1 persistence in infected host Cesana Daniela Biomedical/Biomedica Ospedale San Raffaele - Milano we aim to find a list of candidate genes that could have a role in defining HIV-1 persistence in infected host. Materials and Methods We will characterize HIV-1 integration profile and transcriptome by high-throughput linear amplification mediated-pcr and mrna next-generation sequencing (Illumina) on different T cell subsets and monocyte cells from different cohorts of infected patients. Available and dedicated bioinformatics pipelines will be used to map the genomic sequences and to identify the virus/host chimeric transcripts. Functional studies to address the putative role of the candidate culprits (i.e BACH2 and STA5B) in favoring HIV-1 persistence will be performed. By using reverse genetic approaches we will test the role of the candidate genes in controlling HIV-1 expression and immune cell function. Specific in vitro assays will be design on the basis of the nature of the candidate gene and the cellular subset where it was found deregulated (i.e. apoptosis, proliferation, differentiation) Impact and Translational Implications The characterization of the HIV-integrome and transcriptome in different hematopoietic cell types from infected patients will allow us identifying molecular targets and previously unknown molecular mechanisms modulating viral escape to the immune system, latency and the overall persistence of HIV-1 in the host. Since these processes are the main causes of the failure of current HIV-1 therapies, our results will significantly help in progressing towards better therapies for HIV-1 eradication. 10/07/ / 6

121 CXCR4 mutants in Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) and Waldenstrom Macroglobulinemia (WM) pathogenesis: signalling properties and therapeutic opportunities GR Borroni Elena Monica Biomedical/Biomedica Istituto Clinico Humanitas LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Infectious and immunological diseases Project Classification IRG: Project Classification SS: Immunology Innate Immunity And Inflammation - III Project Keyword 1: Project Keyword 2: cytokines, chemokines, lipid mediators, other autocoids, and their receptors G-protein coupled receptors Project Keyword 3: Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Immunology/Laboratory of Leukocytes Biology Principal Investigator 2 seconda unità - Azienda Ospedaliera Spedali Civili di Brescia Department of Pediatric Research Unit Responsible Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Dotta Laura seconda unità - Azienda Ospedaliera Spedali Civili di Brescia Research Unit Responsible 03/05/ /07/ / 6

122 CXCR4 mutants in Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) and Waldenstrom Macroglobulinemia (WM) pathogenesis: signalling properties and therapeutic opportunities GR Borroni Elena Monica Biomedical/Biomedica Istituto Clinico Humanitas Background and Significance CXCR4 is a chemokine receptor that controls leukocytes survival and mobilization from bone marrow. Mutations in the C- term of this receptor cause WHIM syndrome, a rare immunodeficiency characterized by neutropenia, and have also recently been identified in 30% WM patients. As for other chemokine receptors, CXCR4 signalling results from balanced activation of G proteins, which support chemotactic activity, and ß-arrestin/Rac1 pathway, supporting receptor internalization/desensitization, actin polarization, and other presently unknown signals. Interestingly, CXCR4 mutants show increased activation of G proteins but altered interaction with ß-arrestins in vitro and neutrophils from WHIM patients display defective Rac1 activation, leading to the hypothesis that signalling of CXCR4 mutants results from unbalanced activation of G proteins and ß-arrestins. The CXCR4 inhibitor AMD3100 has some therapeutic potential in WHIM patients but this drug is complex to handle. The development of new treatments is therefore desirable, but requires a more precise understanding of signalling properties of CXCR4 mutants and the role that different signalling pathways may have in WHIM and WM pathogenesis. This project will deeper investigate the signalling properties of presently known and newly identified CXCR4 mutants and will screen a panel of CXCR4 binding peptides seeking new molecules able to normalize signalling properties of CXCR4 mutants as new candidates for WHIM and WM therapy. Specific aims Aim 1: Aim 2: Aim 3: Role of G protein and ß-arrestin-mediated signalling pathways in WHIM CXCR4 mutants Functional characterization of novel CXCR4 mutants identified in WHIM patients Investigation of new CXCR4 antagonists effective in the treatment of myelokathexis in a murine model of WHIM Hypothesis: WHIM syndrome is caused by nonsense/frameshift mutations that result in truncation of the C-terminal domain of CXCR4 and because this region is involved in receptor desensitization, they lead to abnormally increased receptor signalling activity. Among WHIM mutations, the most common and best studied is the CXCR4-R334X (also carried by WM patients), but recently a new missense, charge-changing mutation in the C-terminal end (E343K) has been identified in a family with autosomal dominant inheritance of WHIM syndrome. Interestingly, Unit 2 have also recently identified WHIM patients carrying a novel CXCR4 mutation leading to loss of CXCR4 intracellular tail. As our findings extend our knowledge about CXCR4 gain-of-function mutations, we hypothesized that deeper characterization of CXCR4-317 may provide new knowledge about the biochemical basis of complex WHIM syndrome phenotypes. It is well established that ß-arrestins function as signalosome adaptor/scaffolding proteins that interact with receptor C-terminus and regulate both receptor internalization/desensitization and activation of signalling pathways. It has been reported that WHIM CXCR4 shows an altered interaction with ß-arrestins. Moreover, published data and our preliminary results indicate that WHIM CXCR4 displays defective activation of ERK1, AKT and Rac signalling pathways that are known to be regulated by ß-arrestins. We hypothesize that WHIM CXCR4 retains defective ß-arrestin signaling and its downstream normalization may represent an innovative treatment for WHIM and WM diseases. Preliminary data: U1 has obtained evidence that CXCR4 is able to bind both ß-arrestin 1 and 2, has confirmed that CXCR4-R334X is not internalized after CXCL12 stimulation, and has obtained preliminary evidence that it exhibits a constitutive activation of Rac1, suggesting that WHIM CXCR4 exhibits an altered ß- 2arrestin signaling. U2 has identified a new CXCR4 mutation causing the loss of 40 intracellular residues, which is presently not functionally characterized. Because of the clinical heterogeneity of WHIM syndrome, the study of CXCR4 signalling in these patients will contribute to understand the disease pathogenesis. Finally, three CXCR4 binding peptides have been identified which in wild-type mice induce a rapid increase in the peripheral neutrophil count, and differently from AMD3100 have 11/07/ / 6

123 CXCR4 mutants in Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) and Waldenstrom Macroglobulinemia (WM) pathogenesis: signalling properties and therapeutic opportunities GR Borroni Elena Monica Biomedical/Biomedica Istituto Clinico Humanitas prolonged efficacy (up to 24 h) and do not induce significant organ damage. The potential of these peptides to revert myelokathexis will here be tested in our murine WHIM model. Materials and Methods ß-arrestins association to CXCR4 is evaluated by BRET technology in HEK293 cells transfected with CXCR4 WT/WHIMluciferase and ß-arrestin1/2-YFP. ß-arrestins role in CXCR4 is assessed by transfecting CXCR4 WT/WHIM HEK293 cells with sirna targeted ß-arrestins or constitutive active mutants (Arr1-R169E) and analysing receptor membrane expression and internalization by FACS. PI3K and Rac activation are studied with standard methods in CXCR4 WT/WHIM HEK293 cells, whereas ERK1/2 activation is performed by Western blotting using anti perk antibody. Leukocytes from WHIM/WM patients are collected by using Ficoll gradient purification. The WHIM animal model is a knock-in mouse strain harboring a WHIM-associated heterozygous mutation of the CXCR4 gene (CXCR4/1013). Neutrophils mobilization in mice is evaluated by FACS (CD45+/CD11b+/Ly6G+ cell) in blood collected 1, 2, 6 and 24 h after subcutaneous injection of peptides (5 or 30 mg/kg) or AMD3100 (3 or 30mg/kg). Impact and Translational Implications The discovery of new CXCR4 binding peptides able to mobilize neutrophils and with reduced toxicity as compared to AMD3100 would represent a new therapeutic option in WHIM management. Further, it may be exploited in other disorders where AMD3100 is already FDA-approved (non-hodgkin's lymphoma, multiple myeloma), and it might represent a new therapeutic tool in WM, where resistance to anticancer agents caused by WHIM-like CXCR4 mutations is successfully reverted by AMD /07/ / 6

124 Natural killer cells and macrophages cross-talk in the pathogenesis of HIV-1 infection BANDO 2013 PROGETTI GIOVANI RICERCATORI GR savino benedetta Biomedical/Biomedica Istituto Clinico Humanitas LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Infectious and immunological diseases Project Classification IRG: Project Classification SS: Immunology Immunity and Host Defense - IHD Project Keyword 1: Project Keyword 2: Project Keyword 3: host defense: innate and acquired immune responses that protect the host from deleterious effects of pathogens, including basic mechanisms of immune responses to limit pathogen invasion and toxicity, and development of animal models of potential bioterrorism agents. NK cells Macrophages Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Laboratory of Leukocyte Biology conception and design, realization of experiments of murine settings and KS model 2 Istituto Clinico Humanitas Laboratory of Clinical and Experimental Immunology conception and design, realization of experiments on autologous NK cellsmacrophages and HIV infection Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Roberto Alessandra Istituto Clinico Humanitas experiments on autologous NK cellsmacrophages and HIV infection 2 01/04/ Torre Alessandro Antonino Ospedale Luigi Sacco / Malattie Infettive III divisione responsible of recruiting HIV-1 positive individuals, to collect blood draw and to organize a clinical database 04/07/ /07/ / 5

125 Natural killer cells and macrophages cross-talk in the pathogenesis of HIV-1 infection BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica savino benedetta Istituto Clinico Humanitas Background and Significance HIV infection is an urgent global health problem, in particular in developing countries, and the development of an effective vaccine so far has proved to be a difficult task. Innate immune mechanisms also may support beneficial effects against HIV- 1, but a number of infection-related abnormalities hamper its potential in vivo. Natural killer (NK) cells have been proven to control HIV-1 infection through different mechanisms (direct lysis of infected cells, ADCC, secretion of antiviral and proinflammatory factors), but their functions are ultimately markedly impaired in patients with high levels of HIV-1 viremia due to preferential expansion of an highly anergic CD56neg subset. Similarly, rather than developing a classic (M1) activation sustaining antiviral immune responses, in late stages of the HIV-1 infection macrophages have been shown to preferentially undergo alternative (M2) polarization and converted in important viral reservoir. Furthermore, infiltration of pro-tumor M2 macrophages secreting immune-modulatory mediators may hamper the ability of NK cells to eliminate malignant cells in Kaposi's sarcoma (KS), a vascular tumor diagnosed at higher frequency in patients affected by acquired immunedeficiencies. To possibly develop novel and alternative therapeutic approaches, the present proposal will investigate the molecular mechanisms dictating dysfunctions of these innate immune cells and their relevance in HIV-1 infection and KS progression. Specific aims Aim 1: Aim 2: Aim 3: Analysis in vitro of the molecular mechanisms involved in NK/macrophage cross-talk and influence of HIV-1 infection. Ex vivo analysis of the cross-talk between NK and macrophages from HIV-1 infected patients: impact of M2 polarization on NK cell antiviral immune response. Effect of NK-macrophage cross-talk in an experimental model of KS. Hypothesis: Several reports have demonstrated that HIV-1 markedly affects functions of both NK cells and macrophages, thus favoring the establishment of viral reservoirs, disease progression, and the onset of opportunistic infections. Furthermore, our recent publications and preliminary evidence here provided strongly suggest that the interaction between dysfunctional NK cells and HIV-infected polarized macrophages likely play a key role in hampering the antiviral potential of these innate immune cells, similar to what we have previously demonstrated for autologous dendritic cells. A better understanding of mechanisms underlying the expansion of the dysfunctional CD56neg NK cell subset and the preferential M2 macrophage polarization occurring in HIV-1 infected patients would allow us to design new strategies to reverse this vicious circle and re-establish an optimal NK cell-macrophage cross-talk able to boost anti-viral and anti-tumoral immune responses even in advanced stages of the infection. Preliminary data: We have recently characterized cell-cell contacts and soluble factors supporting priming of autologous NK cells functions (cytoxicity, IFN-gamma secretion) by M1 macrophages, which in turn are activated by NK-derived IFN-gamma. We have also collected evidence indicating that this positive feed-forward loop, potentially relevant for the antiviral immune response, is severely compromised in M2 polarized macrophages. In addition, we have recently described molecular mechanisms underpinning the role of tumor-associated macrophages (TAM) in the progression of KS lesions. In particular, we reported that a KS experimental model (based on the injection of the human cell line KS-IMM in nude mice) required the infiltration of TAM with M2 phenotype sustaining angiogenesis and tumor growth. Similarly, at diagnosis patients with rapid progressing KS tumors were characterized by lesions with increased infiltration of CD68+/CD163+ (M2) macrophages as compared to slow progressing tumors. Reports by several groups, including ours, indicate that HIV-1 infected patients are characterized by macrophages with preferential M2 polarization and an high 11/07/ / 5

126 Natural killer cells and macrophages cross-talk in the pathogenesis of HIV-1 infection BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica savino benedetta Istituto Clinico Humanitas frequency of circulating dysfunctional CD56neg NK cells, we hypothesize that HIV-1 may have a significant impact on the NK cell-macrophage cross-talk, contributing to the compromised innate antiviral immune response and potentially sustaining KS progression. Materials and Methods Human blood NK cells will be co-cultured with autologous monocyte derived macrophages infected with both X5 and R5 HIV strains. We will enroll HIV-1 infected patients in several stages of diseases. Clinical data of the patients will be collected and correlated with our experimental findings. We will use techniques of both cellular (i.e. cell isolation and cultures, flow cytometry and cell sorting, ELISA, in vitro infections) and molecular (Q-PCR) immunology. Co-culture will be conduct in presence of NK cells isolated from spleen of Rag2-/- mice and macrophages (BMDM) differentiated from bone marrow cells of wt mice. Gene expression and cytokine production will be studied. In vivo model of Kaposi s sarcoma will be performed injecting sc the cell line KS-IMM in CD-1 nude mice, as highly vascularized tumors. The effect of coadministration of macrophages and NK in KS tumors will be verify on tumor growth and on phenotype of tumor-associated macrophages analyzed by FACS. Impact and Translational Implications The disclosure of the interactions between NK cells and macrophages in HIV-1 infection is highly relevant to understand the physiopathology of innate immune responses that are not able to control the infection. Our findings will likely be used to target novel molecules with the final aim to improve the anti-viral immune responses. Moreover, data from the model of KS will support the knowledge on the pathways regulating the tumor growth in order to develop alternative therapeutic strategies. 11/07/ / 5

127 Immunoregulatory role of innate immunity cells in the tumor microenvironment BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Vacca Paola Biomedical/Biomedica Azienda ospedaliera universitaria San Martino - IST - Istituto nazionale per la ricerca sul cancro LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Immunology Transplantation, Tolerance and Tumor Immunology - TTT Project Keyword 1: Project Keyword 2: Project Keyword 3: Tumor Immunology: immune surveillance, mechanisms of immune evasion, or immune suppression in the tumor microenvironment, in both humans and animal models; identification of new tumor associated antigens; early stage development and testing of tumor vaccines in animal models. Innate Immunity NK cells/ilc Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION terapie oncologiche integrate/immunologia 2 IRCCS AOU San Martino IST terapie oncologiche integrate/oncologia molecolare e angiogenesi Coordination of the project. Experimental development of AIM1:identification and characterization of ILC subsets in various tumors. Experimental development of AIM2: Study the cross-talk between ILCs and tumor-associated mesenchymal stromal or tumor cells. 3 IRCCS AOU San Martino IST terapie oncologiche integrate/bioterapie Experimental development of AIM3: Investigation of the effect of soluble factors present in TM on ILC, TA-MSC, and tumor cells. 10/07/ / 6

128 Immunoregulatory role of innate immunity cells in the tumor microenvironment BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Investigators, Institution and Role in the Project Vacca Paola Azienda ospedaliera universitaria San Martino - IST - Istituto nazionale per la ricerca sul cancro Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Venè Roberta IRCCS AOU San Martino IST she will define the interactions between ILC and TM, focusing on isolation/culture/analysis of TA-MSC and tumor cells. 2 Carbotti Grazia IRCCS AOU San Martino IST she will analyze the role of TM soluble factors in human cell cultures and in mouse tumor models. 3 Montaldo Elisa University of Genoa she will perform ex vivo characterization of tumor ILC, focusing on ILC isolation/culture/analysis as well as on the interaction beween ILC and TM. 4 Carrega Paolo Giannina Gaslini Institute he will assess in vivo localization of ILC and ELS in human tumors; he will investigate the features of ILC in mouse tumor models. 03/10/ /01/ /02/ /12/1980 Background and Significance In solid tumors, the function of the newly described Innate Lymphoid Cells (ILC) is poorly defined. Despite the well-known antitumor activity of NK cells, an ILC1 subset, their effective exploitation in therapy is far from being achieved. ILC3 are involved in tissue remodeling and lymphoid organogenesis. Interestingly, ILC3, similarly to NK cells, express receptors that recognize tumor cells. Moreover, ILC3 produce TNF/IL8/IL17 proinflammatory cytokines and IL22 that promotes tissue integrity. Thus, ILC may play role in antitumor immune responses and influence the Tumor Microenvironment (TM). In this context, it is worth to investigate if the frequency of tumor-infiltrating ILC may have prognostic potential. Tumor-associated Ectopic Lymphoid-like Structures(ELS) correlate with a better prognosis in many tumors. ILC3 promote lymphoid organogenesis through the interaction with Mesenchymal Stromal Cells(MSC). Thus, the cross-talk of ILC3 and tumorassociated(ta) MSC may trigger ELS formation. ILC function is influenced by IL12 cytokines (i.e.il12/23/27/30/35) secreted by myeloid cells. These cytokines can act on TM cells inducing the release of immune regulatory factors. Thus, addressing the effects of IL12 cytokines directly on ILCs, as well as, their indirect regulatory potential in TM, may improve our understanding of immune responses at the tumor site. In conclusion, the knowledge of ILC role in tumor immune surveillance/promotion would help to develop novel therapies. Specific aims Aim 1: To analyze the presence of ILC subsets in various human tumors and its correlation with progression we will i) investigate the presence and frequency of ILC subsets in tumors; ii) study whether the functional features (cytokine release/lti activity/cytotoxicity) of ILCs in tumors differ from those of ILC present in healthy tissues; iii) correlate the frequency of ILC subsets with the tumor stage and clinical outcome; iv) analyze the association between ILC3 with the occurrence of ELS; v) verify the role of ILC in mouse tumor models. Aim 2: Aim 3: To study the cross-talk between ILCs and tumor-associated mesenchymal stromal cells (TA-MSC) or tumor cells we will i) investigate whether TA-MSC or tumor cells affect ILC function; ii) assess whether cytokines released by ILC affect tumor cell growth, iii) verify whether ILC may regulate TA-MSC mediated immune suppression. To evaluate the effect of soluble factors present in TM on ILC, TA-MSC, and tumor cells we will: i) identify soluble 10/07/ / 6

129 Immunoregulatory role of innate immunity cells in the tumor microenvironment BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Vacca Paola Azienda ospedaliera universitaria San Martino - IST - Istituto nazionale per la ricerca sul cancro factors able to influence ILC function, e.g. IL12 family cytokines released by TA-myeloid cells; ii) verify whether the modulation of ILC function by IL12 cytokines may correlate with tumor progression; iii) study whether IL12 cytokines could affect tumor cell growth and TA-MSC functional activities; iv) assess, in vivo, the presence and the role of IL12 family cytokines in mouse tumor models. Hypothesis: ILC regulate inflammatory processes, lymphoid tissue organization, epithelial cell differentiation and neoangiogenesis. Based on our preliminary data, it is conceivable that ILCs present in the TM may influence tumor immunesurveillance/progression. On the other hand, TA-MSC and TA-myeloid cells may interfere with ILC recruitment and function. We also hypothesize that targeted cancer therapy may modify TM, thus interfering with ILC recruitment, function, and ILC/TM cross-talk. The characterization of these processes may help to develop new anti-tumor therapies and reagents for diagnosis and prognosis. Preliminary data: Our Lab has long been focused on the role of NK cells in solid and hematopoietic cancers. We demonstrated that NK cells infiltrate the majority of human solid tumors and that NK cells endowed with anti-tumor activity are present in malignant pleural effusion (mpe). Notably, we recently dissected the various ILC subsets that populate decidual tissues, an immunosuppressive microenvironment that closely resembles TM. Based on these data, we are now extending our analysis to other ILC subsets in tumors. Our preliminary data indicate that ILC3 are present in Non-Small Cell Lung Cancer (NSCLC) samples and that ILC3 interact with TA-MSC. Moreover, we have evidence that ILCs, other than NK cells, are present in mpe. On the other hand, we observed that TA-MSC from Colorectal Carcinomas (CRC) produce immunoregulatory cytokines (i.e.tgfß) that inhibit the immune response. In addition, we showed that IL27, produced by tumor-infiltrating myeloid cells, drives the expression of immunesuppressive molecules. Materials and Methods To perform this project we will: Identify ILC infiltration, ILC intra- or peri-tumoral localization, and presence of ELS by IHC analysis of tumor specimens. Isolate ILC by cell sorting or immunomagnetic enrichment and culture them in vitro with different stimuli. Analyze features of fresh and cultured tumor ILCs by: flow cytometry (surface/intracellular/intranuclear staining), molecular (RT-qPCR, WB) and functional (cytokine production, LTi activity, cytotoxicity/degranulation) assays. Isolate TA-MSC and tumor cells; establish primary cell lines. Evaluate interactions between ILC and TA-MSC, TA-myeloid cells, and tumor cells by trans-well or cell-to-cell contact cocultures. Characterize soluble factors in TM by ELISA multiplex assay, RT-qPCR, WB, and IHC and their influence on ILC, TA-MSC, and tumor cells. Characterize ILC and soluble factors in mouse tumor models. Analyze the statistical correlation between ILC infiltration and/or presence of ELS within the tumor with clinical data. 10/07/ / 6

130 Immunoregulatory role of innate immunity cells in the tumor microenvironment BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Vacca Paola Azienda ospedaliera universitaria San Martino - IST - Istituto nazionale per la ricerca sul cancro Impact and Translational Implications Studying tumor infiltrating ILCs and their interaction with other cells of the TM would offer new insights on immune-mediate mechanisms that control cancer and cancer-related immune-regulatory circuits. The expected results may pave the way to innovative immunotherapy strategies and new diagnostic tools. These results will impact on the scientific community, through the sharing of new data, and on the general healthcare and welfare, through the development of novel cancer therapies.

131 GR Role of muscle interstitial fibroadipogenic progenitors in the regulation of myofiber response to acute and pathological denervation Madaro Luca Clinical health care research/clinicoassistenziale Fondazione Santa Lucia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Amyotrophic Lateral Sclerosis (ALS) and related motor neuron disorders. Muscle Atrophy Next-generation sequencing Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Laboratory of Epigenetic and Regenerative Medicine 2 University of Siena Department of Medicine/Surgery and Neurosciences Hosting Lab species and animal facility for most of the experiment Collection and molecular analysis of human blood samples and muscle biopsy Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Benigni Michele University of Siena Director unit2: Collection and molecular analysis of human blood samples and muscle biopsy 09/11/ /07/ / 5

132 GR Role of muscle interstitial fibroadipogenic progenitors in the regulation of myofiber response to acute and pathological denervation Madaro Luca Clinical health care research/clinicoassistenziale Fondazione Santa Lucia Background and Significance Neurogenic muscle atrophy occurs in consequence of traumatic spine injuries and neuromuscular disorders and is characterized by loss of muscle mass. Despite of the discovery of signaling pathways that control muscle mass, by regulating protein synthesis and breakdown, the transcriptional network that orchestrates the expression of genes leading to muscle atrophy remains largely unknown. A critical issue regards the identity of cell types that mediate the atrophic response and the functional interplay among different muscle cell populations during compensatory or pathogenic responses of skeletal muscle to atrophic stimuli. This gap of knowledge has prevented the identification of effective interventions toward countering neurogenic muscle atrophy. Growing evidence is pointing to histone deacetylases (HDACs) as pivotal chromatin-modifying enzymes that regulate muscle growth and atrophy suggesting that pharmacological blockade with HDAC inhibitors (HDACi) can counter muscle atrophy (Moresi 2010; Judge 2014). We will focus on the role of purified muscle cell populations in the atrophic response in denervation and in a mouse model of Amyotrophic Lateral Sclerosis (ALS). In particular, we aim at characterizing the interplay among these cells types and their responsiveness to treatment with the HDACi, in order to identify molecular markers to be targeted for selective intervention. Moreover, we propose to validate candidates biomarkers on human muscle biopsy of denervated muscle. Specific aims Aim 1: Aim 2: Aim 3: Identification of transcriptional signatures that underlie neurogenic muscle atrophy in mouse models of traumatic nerve injury (denervation) and in a mouse model of Amyotrophic Lateral Sclesosis (G93A-SOD1 mice). Both untreated and HDAC inhibitor treated mice will be analyzed. In vitro characterization of mechanisms regulating the crosstalk among different cell populations isolated from mouse models of traumatic spine injury (denervation) and ALS treated or not with HDAC inhibitor. Validation of prognostic biomarkers derived from previous genome wide experiments in human samples of denervated muscle cryosections, blood samples and freshly isolated cells from muscle biopsies. Hypothesis: An almost unexplored issue regards the identity of cell populations that are implicated in the regulation of myofibers atrophy upon denervation, and the cell type-specific networks that coordinate the functional interplay by which these cells contribute to compensatory or pathogenic/maladaptive responses of skeletal muscle to denervation. Our hypothesis is that interstitial cells activate a specific gene program during denervation participating actively to this process. Moreover, we speculate that genes activated in these cells during acute denervation or ALS can be used in human as predictive/prognostic biomarkers. Preliminary data: Our preliminary data indicate that reciprocal interactions among interstitial cells, such as Fibro Adipogenic Progenitors (FAPs), macrophages (MP), and muscle satellite cells (MuSCs) determine the ability of myofibers to activate compensatory regeneration or maladaptive protein breakdown and fibrosis in a mouse model of muscle atrophy induced by denervation. A first pilot experiment revealed a global transcriptome change in FAPs during denervation. We found an increased number of FAPs with induced expression of genes including IL6, IGF1 previously implicated in anabolic muscle growth, in denervated muscles. Accordingly, in a mouse model of ALS (G93A-SOD1 mice) we identify a similar gene expression pattern during the disease progression. We collect data indicating that treatment with histone deacetylases inhibitors counteracts denervation-induced muscle atrophy at both morphological and molecular level, inspiring the overall goal of the proposed project. 11/07/ / 5

133 GR Role of muscle interstitial fibroadipogenic progenitors in the regulation of myofiber response to acute and pathological denervation Madaro Luca Clinical health care research/clinicoassistenziale Fondazione Santa Lucia Materials and Methods We will use genome-wide analysis of gene expression in whole muscles and in FAPS, MuSC and MP to compare RNA- Sequencing profiles from individual cell types isolated from both denervated and ALS mice. This approach will enable us to capture cell types specific "molecular signatures" of compensatory versus maladaptive responses of atrophic muscles exposed or not to HDACi. RNA-seq data will be validated by qpcr analysis on freshly isolated cells and histochemical analysis on muscle sections. We will combine transwell co-culture experiments and RNA interference technology to target relevant networks detected in FAPS, MP and MuSC to unravel the molecular determinants of pro- and anti-atrophic responses. We will use histological investigation to confirm the presence of selected biomarkers in human samples of denervated muscles. Cytokine detection will be validated in blood samples from human patients. FACS-sorted cells from human biopsies will be employed to validate cell-specific markers Impact and Translational Implications This study will provide the first identification of new cellular determinants of the atrophic response in denervated muscles and the characterization of functional networks that coordinate this process in mouse and human models. HDACi are currently used in clinical trials highlighting the close proximity of this project to medical applications, while warranting their safety and ethical pertinence.

134 GR DISCO TRIP - Disrupting Respiratory Complex I to Trigger Pseudonormoxia: an anticancer strategy Gasparre Giuseppe Biomedical/Biomedica Emilia-Romagna LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Oncology 1 - Basic Translational Tumor Cell Biology - TCB Project Keyword 1: Project Keyword 2: Pathways regulated by oncogenes and tumor suppressors that affect tumor cell phenotype and behavior, such as survival, proliferation, and death Project Keyword 3: Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Az.Osp.S. Orsola-Malpighi, Dip. Salute della Donna, U.O. Genetica Medica. Principal Investigator, study design and supervision; data analysis and management; scientific papers writing Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date Kurelac Ivana Az.Osp.S.Orsola-Malpighi, Dip. Salute della Donna, U.O. Genetica Medica. Post-doc fellow. 4 Iommarini Luisa University of Bologna, Dip. Farmacia e biotecnologie (FABIT). Post-doc fellow. Post-doc fellow - in vivo experiments and molecular biology and genetic studies. Collaborating fellow with the Drosophila group. Post-doc fellow - Biochemical studies - optimization and performance of biochemistry-related experiments. 09/10/ /05/ /07/ / 5

135 GR DISCO TRIP - Disrupting Respiratory Complex I to Trigger Pseudonormoxia: an anticancer strategy Gasparre Giuseppe Biomedical/Biomedica Emilia-Romagna Background and Significance Mitochondria-coded genes have been shown to play an active part in tumorigenesis. Although association between somatic mtdna mutations and cancer has been demonstrated, their functional impact has been scarcely investigated, especially with respect to the genetic features of mtdna, namely hetero-versus homoplasmy and the threshold effect. The PI's group has demonstrated that, following a threshold-effect model, homoplasmy of damaging mtdna mutations causes aggressive tumors to switch to a low-proliferating, non-invasive behaviour. We have reported that respiratory complex I (CI) is essential to induce the Warburg effect and demonstrated a strong association between CI disruption and chronic destabilization (pseudonormoxia) of hypoxia inducible factor 1alfa (HIF1a), a critical player in the metabolic adaptation of cancer cells during their progression to a malignant state. Accumulation of a-ketoglutarate, substrate of the prolyl-hydroxylase-mediated reaction of HIF1a degradation, was shown to occur in mtdna-mutated cell models, which explained the lack of metabolic adaptation occurring in tumors that display such mutations, allowing them to escape malignancy. Such mechanisms are strongly supportive of the use of adjuvant anti-cancer therapies targeting the oxidative respiration capacity of cancer cells, as it has been suggested for several tumors, and may underlie the emerging role of respiratory complexes inhibitors such as Metformin in cancer therapy. Specific aims Aim 1: Aim 2: Aim 3: Dissect the molecular mechanisms linking CI disruption to pseudonormoxia and to tumor growth arrest Use a Drosophila model of tumorigenesis in which epithelial and nervous tissues showh high malignant phenotypes to specifically switch off CI expression and test its effect on cell/mass behaviour Assess the toxicity/efficacy profiles of compounds targeting CI function as an adjuvant anti-cancer therapeutic strategy Hypothesis: MtDNA CI disruptive mutations may positively contribute to tumor growth reduction, from which derives our recent functional definition of oncojanus. Disruption of mitochondrial CI would induce pseudonormoxia, preventing the tumor to acquire a Warburg phenotype and progress to malignancy. CI disruption may be induced by targeted drugs to arrest tumor growth. Preliminary data: We showed that CI function was markedly reduced only in cells bearing a high load of m.3571insc mutation in MTND1. CI disassembly caused an imbalance of the akg/sa ratio, which prevented hypoxic adaptation, a process regulated by HIF1a, leading to pseudonormoxia and to a lowproliferating indolent tumor phenotype. Complementation of MTND1 recovered tumorigenic potential, a lower akg/sa ratio and, in turn, the ability to respond to hypoxia. Moreover, recovery of CI ensured that tumors acquired a typical Warburg gene expression profile. Investigation of biochemical mechanisms showed that CI disassembly implicated the lack of NADH oxidation, leading to its accumulation. NADH Inhibition of akg dehydrogenase determines accumulation of akg and decrease of SA, respectively the substrate and allosteric inhibitor of PHD, which regulates HIF1a degradation. These findings were confirmed in cells harbouring different mtdna mutations disrupting CI, whereas those slightly altering function of the complex and fostering NADH consumption induced an opposite phenotype. Preliminary data obtained in different cancer and non-cancer cell lines by using rotenone, a CI irreversible inhibitor, suggest that non-cancer cells (LC50 527nM) are significantly more resistant in comparison to their cancer counterparts (LC50 155nM). Last, we have generated several osteosarcoma clones knockout for NDUFS3 via the ZNF approach, showing CI complete disruption. 10/07/ / 5

136 GR DISCO TRIP - Disrupting Respiratory Complex I to Trigger Pseudonormoxia: an anticancer strategy Gasparre Giuseppe Biomedical/Biomedica Emilia-Romagna Materials and Methods Metabolites will be measured by gas-chromatography/mass spectrometry; enzymatic activity will be evaluated by spectrophotometric analyses. Hypoxic experiments will be performed using Invivo300 chamber. HIF1a stabilization will be verified by western blot analysis. Prolyl-hydroxylase activity will be measured with an ODD-GFP construct. Zinc-fingers against NDUFS3 will be used to generate cell lines with stable CI disassembly from breast and lung cancer-derived cells. Non-degradable HIF1a will be generated upon cloning and site-directed in vitro mutagenesis on the recognition domain. Stable transfectant and controls will be injected subcutaneously in Crl:CD-1-Foxn1nu/nu mice. CI-disassembling compounds atoxic for non-cancer cells will be used to treat nude mice intra-xenograft. Drosophila CI knockout will be carried out via RNAi. NDUFS3 orthologue will be knocked and hallmarks of malignancy such as 3D growth, basement membrane discontinuity and metastatic index assessed. Impact and Translational Implications Proof that certain mtdna CI mutations imply reduction of tumor growth will contribute to evaluate the prognostic power of mitochondrial genotyping. Results will contribute to determine the feasibility of adjuvant therapies targeting respiratory complexes, following evaluation of toxicity/efficacy profiles and selection of appropriate doses of CI-disassembling candidate drugs. The Drosophila model will set the basis for the development of CI knockout transgenic conditional/inducible mouse models.

137 Optimization of extracorporeal carbon dioxide removal through blood acidification: development of new technologies GR Zanella Alberto Biomedical/Biomedica Regione Lombardia - Direzione Generale Sanità LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Innovative biotechnologies Project Classification IRG: Project Classification SS: Bioengineering Sciences and Technologies Instrumentation and Systems Development - ISD Project Keyword 1: Project Keyword 2: Analytical instrumentation: novel methods for improving throughput in analytical techniques; optical methods; chemical methods; spectroscopy; microfluidics; hardware and computer systems. modular extracorporeal support for severe respiratory failure and acute renal failure Project Keyword 3: Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: X I declare that the object/s of this application is/are under patent copyright Patent owner: Università degli studi Milano-Bicocca Patent number: WO Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Dipartimento di Emergenze e Urgenza, Azienda Ospedaliera San Gerardo Monza 2 Università degli Studi di Milano Bicocca 3 Università degli Studi di Milano Dipartimento di Scienze della Salute Dipartimento di Scienze Veterinarie e Sanità Pubblica principal investigator technological development coordinator animal model coordinator Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Salerno Domenico Università degli Studi di Milano Bicocca technological development coordinator 13/04/ Pirrone Federica Università degli Studi di Milano animal model coordinator 27/09/ /07/ / 6

138 Optimization of extracorporeal carbon dioxide removal through blood acidification: development of new technologies GR Zanella Alberto Biomedical/Biomedica Regione Lombardia - Direzione Generale Sanità Background and Significance Extracorporeal CO2 removal (ECCO2R) is a technology designed to prevent ventilator induced lung injury (VILI) in patients with acute respiratory distress syndrome (ARDS), and nowadays it finds application also in COPD patients or in managing patients waiting for lung transplantation. The commonly employed ECCO2R techniques, veno-venous bypass featured with a membrane lung (ML), require extracorporeal blood flow (BF) higher than 1 L/min to remove a substantial fraction (higher than 50%) of total body CO2 production. This BF can be challenging in standard clinical application. Moreover acute renal failure is frequently associated to respiratory failure and, complicating the clinical scenario, increases mortality, morbidity and health care system costs. In the last years our group developed and tested in a swine model a technique, based on infusion of lactic acid into the extracorporeal blood, able to increase, up to 70%, the ECCO2R performance. Such technique was feasible and safe but was burdened by systemic side effects that have limited the clinical use. Aims of this project are to reduce such side effects so as to make this method more secure, efficient, and transfer this technique on human patients. Specific aims Aim 1: Aim 2: Aim 3: The first aim will be to develop an extracorporeal system able to remove a conspicuous fraction of the infused acid anion. Therefore a smaller amount of acid will reach the systemic circulation, reducing the systemic adverse effects. This system of acid removal, based on ultrafiltration / dialysis technology, will also allow to perform a continuous renal replacement therapy (CRRT). The development of adequate replacement fluids, with specific anions concentration, allows to safely remove the loaded acid without reducing the efficiency of the extracorporeal therapy The second aim is to evaluate different types of metabolizable acids. We will evaluate acids with reduced systemic impact, high clearance and favorable characteristics. We will focus on the use of citric acid which may chelate calcium and therefore may allow to obtain an enhanced extracorporeal CO2 removal and a selective regional anticoagulation. Systemic coagulation, often contraindicated and not free of severe complications, will be no longer needed. The third aim is to evaluate non-metabolizable acid. Hydrochloric acid seems to be the most beneficial, since chloride ions are the most represented anions in human blood, it is not toxic and will be easily removed. Infusion of hydrochloric acid will require a system of acid removal, which is the aim #1 of the presented project. Hypothesis: The hypothesis is to optimize a technique of extracorporeal support we developed and patented (WO A2, applicant UNIMIB). Implementing this extracorporeal support with a system able to perform a CRRT and remove a conspicuous fraction of the infused acid should decrease the metabolic and systemic side effects related to acid infusion while maintaining hydro-electrolytic homeostasis, even for long period of treatment. Preliminary data: We recently published several article on this topic, demonstrating: 1- Lactic acid infusion highly increased extracorporeal CO2, up to 48 hours, without haemolysis, or any organ injury. 2- Highly concentrated acid may be safely infused in a closed dialysis circuit 3- Acid infusion may allow to move the membrane lung from the blood stream to the dialysis stream, thus decreasing extracorporeal blood volume, and the non-biological surface exposed to blood, hence minimizing biocompatibility and clotting problems. 4- Lactic acid slightly increases total CO2 production. 5- Citric acid is effective in increasing extracorporeal CO2 removal Zanella A, et. Crit Care Nov 11;17(6):R /07/ / 6

139 Optimization of extracorporeal carbon dioxide removal through blood acidification: development of new technologies GR Zanella Alberto Biomedical/Biomedica Regione Lombardia - Direzione Generale Sanità Cressoni M, et al. Crit Care Med Sep;37(9): Zanella A, et al. Anesthesiology Feb;120(2): Zanella A, et al. J Heart Lung Transplant May;33(5): Zanella A, et al. Intensive Care Med Aug;35(8): Scaravilli V, et al. ASAIO J May-Jun;60(3): Materials and Methods Each of the three aims will be tested in two phases: the first in-vitro and the subsequent in-vivo. The in-vivo study is intended to collect the preliminary data to evaluate the hypothesis and obtain the optimal setting to be tested in-vivo. The invivo phase will be tested in a healthy swine model. Such model has been already developed by our group and will allow to evaluate the feasibility, efficiency and safety. Once we have obtained an optimized extracorporeal support, we will test the developed technique in a swine model of respiratory failure and multi organ failure. This final tests will allow to confirm the preliminary hypothesis and will allow to translate this achievements on human patients. Impact and Translational Implications The technology developed in this project would allow to reduce the occurrence of VILI in mechanically ventilated patients and to reduce endotracheal intubation in patients with COPD, asthma, and on the list for lung transplantation. These results would reduce days of mechanical ventilation, ICU stay and possibly mortality. The number of patients who would benefit from this treatment may be as high as one third of patients admitted to ICU. 11/07/ / 6

140 GR Molecular Nanodecoders for the Quantitative, Multiplexed, Layer-by-Layer Imaging of Disease-Associated Markers Castronovo Matteo Biomedical/Biomedica Friuli-Venezia Giulia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Innovative biotechnologies Project Classification IRG: Project Classification SS: Bioengineering Sciences and Technologies Nanotechnology - NANO Project Keyword 1: Project Keyword 2: Project Keyword 3: Design, synthesis, and development of nanostructures, nanodevices, and nanosystems. immunofluorescence imaging in cells and tissues Functional DNA nanotechnology Project Request: Animals: Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Academic Hospital of Udine, Regional Coordinator Centre for Rare Diseases To coordinate the project. To develop and apply Nanodecoders, with specific expertise in functional nucleic acids (NA) nanotechnology, rare disease clinical/biological research and imaging of cultured cells. 2 University of Rome "Tor Vergata" Department of Chemistry To support Nanodecoders development with expertise in functional NA nanotechnology, and specialized instrumentation for Nanodecoders synthesis and characterization. 3 Azienda Ospedaliera- Universitaria "Ospedali Riuniti" Trieste Pneumology Unit To assist Nanodecoders development, and apply prototypes, with specific expertise in lung cancer clinical research and analysis of archival tissues samples. 11/07/ / 7

141 GR Molecular Nanodecoders for the Quantitative, Multiplexed, Layer-by-Layer Imaging of Disease-Associated Markers Castronovo Matteo Biomedical/Biomedica Investigators, Institution and Role in the Project Friuli-Venezia Giulia Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Ricci Francesco University of Rome "Tor Vergata" To support the PI in developing fluorescently labelled clamp-like DNA probes responding to specific On/Offcodes, with expertise in self-assembly of nucleic acids (NA) nanostructures, NA thermodynamics, fluorescence-based NA sensors, and analytical chemistry. 01/01/ Cifaldi Rossella Azienda Ospedaliera- Universitaria "Ospedali Riuniti" Trieste To assist the development of Nanodecoders by (i) preparing tissue section of paraffin-embedded, Lung Cancer samples from patients with complete clinical information; (ii) applying Nanodecoders prototypes and characterizing their performances; (iii) contributing to data analysis. 11/09/1981 Background and Significance The analysis-by-imaging of progressive changes in protein or nucleic acid amounts is the current gold standard in evaluating diseases associated with tissue or cellular alterations. This approach informs on disease identification, its progression, and response to therapy. However, the level of change can only be semiquantitatively established by visual scoring, thus leading to inherently limited results [1]. The long-range goal of this project is to develop a molecular nanodevice (Nanodecoder) for the multiplexed, quantitative imaging of biomarkers in cultured cells and tissue samples with immunofluorescence optical imaging (IFI). The nanodecoder technology will focus initially on the protein profiles of two etiologically distinct diseases: (i) glycogenosis type II (GII) - a rare genetic lysosomal storage disease - and (ii) non-small cell lung cancer (NSCLC), which is currently among the leading causes of mortality in the world. We will analyse cellular models and archival tissues, respectively, as highly relevant testing workbenches for the nanodecoder technology. The value of this approach is based on two premises. First, the multiplexed, quantitative detection of a specific collection of disease-associated proteins would provide accurate disease evaluation, and second, a cost-effective approach to biomarker imaging in clinical settings should help the National Health System avoid the otherwise necessary acquisition of expensive instrumentation items. Specific aims Aim 1: Aim 2: To develop and apply Nanodecoders for the multiplexed, quantitative IFI of disease-related proteins in cultured cells from patients affected by GII. The biochemical characteristics of GII include (i) a deficit of alpha-glucosidase (GAA) activity, (ii) lysosomal glycogen accumulation, and (iii) severe impairment of the autophagic process. We aim to apply the nanodecoder to study in vitro the effect of new therapeutic approaches on both GAA expression and autophagy [2]. To develop and apply Nanodecoders for the multiplexed, quantitative IFI of disease-related proteins in paraffinembedded archival samples of lung cancer. We aim to use antibodies for the diagnosis for NSCLC [3], and to achieve a unprecedented, quantitative examination of markers for accurate prognosis and response to therapy [4]. 11/07/ / 7

142 GR Molecular Nanodecoders for the Quantitative, Multiplexed, Layer-by-Layer Imaging of Disease-Associated Markers Castronovo Matteo Biomedical/Biomedica Friuli-Venezia Giulia Aim 3: - Hypothesis: The use of a different dye for each biomarker currently limits immunofluorescence optical imaging (IFI) to the semi-quantitative co-localization of a maximum of four biomarkers in the same sample. This is due to the paucity of fluorochromes with non-overlapping absorption/emission spectra [1,5]. The central hypothesis of this project is that a novel nanotechnological approach can overcome these limits [5]. Each nanodecoder will > consist of a self-assembled DNA nanostructure that can emit a fluorescent signal in response to hybridization of a nucleic acid (NA) sequence ("On-code"), and can be deactivated upon hybridization of an alternative NA sequence ("Off-code"); > contain a single type of fluorescent dye; > be chemically coupled to an antibody that uniquely recognizes a specific disease biomarker; > be used as a probe for detecting disease biomarker distributions in cells and tissues, in conjunction with IFI under identical imaging conditions, and in a layer-by-layer fashion. For example, the detection of ten biomarkers will use ten Nanodecoders that recognize separate On-/Off-codes. Preliminary data: We have already identified a promising Nanodecoder with a virtually unlimited number of designable On-/Off-codes. The nanodecoder consists of a clamp-like DNA probe that binds a complementary 10-base target as On-code, thus forming a ph-sensitive, triplex DNA structure [6]. At ph 6 there is excellent discrimination efficiency (no evidence of binding to a target with a single-base mismatch). Also the clamp-like ssdna probes can be labelled with a Förster Resonance Energy Transfer (FRET) dye pair, that generate fluorescence only if the triplex DNA structure forms [7]. The Off-code will work in a two-step manner: (i) the On-code and nanodecoder elements form a linear duplex at ph 8, the On-code can be displaced by an invading ssrna molecule that strongly binds the nanodecoder, (ii) the RNA strand of the DNA-RNA hybrid can then be degraded by the action of the hybrid-specific enzyme, RNase H. We have already characterized the robust catalytic action of RNase H (human and bacterial) on surface-confined RNA-DNA heteroduplexes in highly dense, spatially-confined nanostructures. 11/07/ / 7

143 GR Molecular Nanodecoders for the Quantitative, Multiplexed, Layer-by-Layer Imaging of Disease-Associated Markers Castronovo Matteo Biomedical/Biomedica Materials and Methods Nanodecoders will Friuli-Venezia Giulia >be fashioned by hybridizing several short ssdnas to non-consecutive regions of a long ssdna scaffold [5]; >serve as a molecular "breadboard" for incorporating multiple clamp-like ssdna probes, labelled with the same FRET-pair [7]; >be coupled to marker-specific Antibodies using a biotin-streptavidin strategy [5]; >be applied to (Aim 1) cultured human fibroblasts from healthy controls (N=10) and GII-affected patients (M=10), in order to quantify GAA protein and different markers of autophagy, and (Aim 2) paraffin-embedded tissues from 50 NSCLC-affected patients and 50 healthy controls for diagnosis with multiplexed IFI, and evaluation of prognosis/response-to-therapy with quantitative IFI; >be validated using standard IFI (Aim 1, 2), western-blotting (Aim 1), and immunohistochemistry (Aim 2). References: 1) /PAI ) /nar/gkt987 3) /chest ) /chest ) /nmeth ) /nn404305e 7) /ac501418g Impact and Translational Implications The successful development of Nanodecoders will greatly advance biomarker imaging. There is a current lack of a highthroughput, convenient method for the in situ quantification of biomarkers in cells or tissues. The implications of this project extend well beyond the specific aims, as this technology will use standard imaging equipment to detect and characterize tissue-related diseases. In turn this will minimize cost, in addition to improving diagnosis and monitoring of therapy. 11/07/ / 7

144 BAP1-dependent Malignant Mesothelioma: mechanisms and early detection BANDO 2013 PROGETTI GIOVANI RICERCATORI GR GIORGI CARLOTTA Biomedical/Biomedica Emilia-Romagna LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Oncology 1 - Basic Translational Cancer Molecular Pathobiology - CAMP Project Keyword 1: Project Keyword 2: Project Keyword 3: Cell death pathways (both apoptotic and non-apoptotic) and autophagy in cancer Tumor suppressor and signal transduction in cancer calcium Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Azienda ospedaliera Universitaria di Ferrara Dipartimento Diagnostica per immagini e medicina di laboratorio, U.O. Programma di ricerca e sviluppo della diagnostica della malattie autoimmuni e infiammatorie Coordination and experimental execution of the project 11/07/ / 6

145 BAP1-dependent Malignant Mesothelioma: mechanisms and early detection BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Investigators, Institution and Role in the Project GIORGI CARLOTTA Emilia-Romagna Key Personnel Institution/Org./Pos. Role in the project Birth Date Missiroli Sonia Università di Ferrara dipartimento di morfologia, chirurgia e medicina sperimentale Sezione di patologia, oncologia e biologia sperimentale Phd student Involved in all the aims 17/01/ federica poletti Università di Ferrara dipartimento di morfologia, chirurgia e medicina sperimentale Sezione di patologia, oncologia e biologia sperimentale Tecnico Involved in all the aims 20/02/1984 Background and Significance The malignant mesothelioma (MM) is a highly aggressive asbestos-related cancer. Recent estimations indicate that onequarter million people will die of this neoplasm in Europe in the next three decades. There is no standard curative therapy for MM that is largely unresponsive to conventional chemotherapy and radiotherapy. As a consequence, new targets for effective therapeutic strategies are needed for MM, as well as new biomarkers for an early diagnosis. Early detection is indeed associated with better responses to therapy and prolonged survival. Recently it has been discovered that genetics influence the risk of developing MM. Germline mutations of BAP1 confer increased susceptibility for the development of MM with a penetrance close to 100% by age of 65. How BAP1 causes mesothelioma and other malignancies is unclear. BAP1 is a tumor suppressor that is believed to mediate its effects possibly through via the ubiquitin-proteasome system. BAP1 is indeed a member of the ubiquitin C-terminal hydrolase (UCH) subfamily of deubiquitinating enzymes (DUBs). Despite the evidence to suggest that BAP1 is a major tumor suppressor inactivated in various cancers, it remains unknown the manner in which BAP1 protects against cancer development. Here we want to investigate how reduced levels of BAP1 influence the high incidence of cancer and MM and aid in the identification of new targets for cancer therapy. Specific aims Aim 1: Aim 2: To determine whether BAP1 mutations increase resistance to apoptosis by modulating Ca2+ homeostasis. Here we propose to investigate a novel extranuclear activity of BAP1 and its possible link to cell death. In view of the localization of BAP1 at the ER, our goal is to elucidate if Ca2+ could be involved in the onset/progression of mesothelioma tumor highlighting the Ca2+ processing as a potential target in the pharmacological treatment of tumors. To investigate whether BAP1 protein is involved in the autophagic process and its relevance in chemoresistance. 11/07/ / 6

146 BAP1-dependent Malignant Mesothelioma: mechanisms and early detection BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica GIORGI CARLOTTA Emilia-Romagna This aim is specifically focused on the investigation of the role of autophagy in the context of MM BAP1- dependent. Moreover, we want to furnish initial clues to the relevance of autophagy manipulation in the treatment of mesothelioma. In particular, we will modulate autophagy to rescue sensitivity to chemotherapeutic agents in MM. Aim 3: To determine whether HMGB1 can be used as a biomarker for early detection of MM in individuals with germline BAP1 mutations. It has been recently reported that transformed mesothelial cells are addicted to a damage-associated protein HMGB1, and that MM cells actively secrete HMGB1, which is required for MM growth and invasion. The secretion of HMGB1 by MM cells suggested that HMGB1 might be a serological biomarker for MM diagnosis and prognosis. We hypothesize that BAP1 mutations predispose to MM also through an alteration of HMGB1 release. Hypothesis: In recent years, many oncosuppressor proteins, such as PML, PTEN, Bax and Bak, BOK, PERK and ERO1-alpha have been demonstrated to be localized to the endoplasmic reticulum (ER), where they modulate cell death in a Ca2+-dependent manner. In particular, release of the ER Ca2+ pool through the type 3 IP3R (IP3R3) appears to induce a sensitization of cells to apoptotic stimuli. Accumulating evidence suggests that Ca2+ has an essential role not only in apoptosis but also in the regulation of autophagy. Autophagy is a lysosomal degradation pathway used by tumor cells to tolerate metabolic stress and is involved in nearly all stages of tumorigenesis and tumor progression. We hypothesize that BAP1 mutations, which predispose individuals to MM, influence intracellular Ca2+ homeostasis and in turn sensitivity to chemotherapeutic agents through de-regulation of apoptosis and autophagy. Preliminary data: Our main preliminary results are: i) BAP1 was detected in the ER via immunofluorescence analysis and subcellular fractionation. ii) We identify a de-regulated Ca2+ homeostasis in MM cell lines and in human fibroblasts derived from BAP1+/- carriers. iii) BAP1 protein specifically interacts with the IP3R3. iv) BAP1 down-regulation is associated with a higher level of autophagy. Materials and Methods To study MM that develops in a background of germline heterozygous BAP1 mutations (BAP1+/-), we have assembled a unique cohort and set of reagents, thanks to an active collaboration with Prof. Michele Carbone (University of Hawaii Cancer Center). We have enrolled the only existing cohort of germline BAP1+/m families, from which we have collected tumor biopsies and a variety of biospecimens, including plasma and primary fibroblasts cell cultures we established from punch biopsies of BAP1+/- mutant carriers and wild-type (wt) controls of these families. At this time, we have enrolled 70 individuals, aged 7-87 years old, from two BAP1 mutant families. For all of them, we have acquired written informed consent according to Institutional Review Board guidelines. These will allow us to study how BAP1 mutations influence different cellular mechanisms in a unique human model. Impact and Translational Implications The goals of the Project are expected to have a definitive impact in the field of mineral fiber carcinogenesis, MM diagnosis and possibly MM management. In this proposal we will investigate the mechanisms that may account for the very high incidence of MM and other malignancies in BAP1+/- carriers. We also propose to test whether HMGB1 can help us monitor these individuals for early cancer detection and the improved MM prognosis. 11/07/ / 6

147 Challenging the role of chemotherapy in HER2 positive early breast cancer: PI3KCA pathways alteration and response to neoadjuvant pertuzumab-trastuzumab plus letrozole or paclitaxel in Hormone Receptor positive-her2 positive breast cancer patients. GR Guarneri Valentina Clinical health care research/clinicoassistenziale Istituto Oncologico Veneto LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Oncology 2 - Translational Clinical Clinical Oncology - CONC Project Keyword 1: Project Keyword 2: Project Keyword 3: Clinical therapy trials including surgical intervention, chemotherapy, radiation therapy and radiopharmaceuticals, combined modality therapy, immunotherapy (antibody and cellular), vaccine and gene therapy, and therapy with biological response modifiers. PIK3CA mutations genome sequencing Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Medical Oncology 2 Principal investigator, is responsible for collection and quality of patient data from the clinical part of the study. She will be responible for the coordination of tissue samples collection from the centers participating the clinical study. 10/07/ / 6

148 Challenging the role of chemotherapy in HER2 positive early breast cancer: PI3KCA pathways alteration and response to neoadjuvant pertuzumab-trastuzumab plus letrozole or paclitaxel in Hormone Receptor positive-her2 positive breast cancer patients. GR Guarneri Valentina Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Istituto Oncologico Veneto Key Personnel Institution/Org./Pos. Role in the project Birth Date Dieci Maria Vittoria Istituto Oncologico Veneto IRCCS, Medical Oncology 2, Attending Physician She will supervise the molecular Studies for PIK3CA mutational analisys and immunohystochemistry for PTEN protein expression, and will coordinate the interplay between different laboratories involved in the project 04/12/ Baretta Zora Istituto Oncologico Veneto IRCCS, Medical Oncology 2, Attending Physician She will be responsible for clinical and biological data collection, and will coordinate the collection of frozen samples. 17/07/1982 Background and Significance Approximately half of hormone receptor (HR) positive breast cancer over-expresses HER2 receptor, and important bidirectional crosstalk occurs between the two pathways. Neoadjuvant trials in HER2+ breast cancer patients have consistently shown that the probability of achieving a pathologic complete response is lower in HR positive tumors as compared to HR negative tumors. The PI3K/AKT pathway is frequently disrupted in cancer, and somatic mutations of the PIK3CA gene are reported in up to 40% of breast cancers. In HER2 positive preclinical models, PTEN loss and/or PIK3CA deregulation have been associated with trastuzumab resistance. In the neoadjuvant setting, the presence of PIK3CA mutations were associated with a lower probability of achieving a pathologic complete remission after chemotherapy plus anti-her2 agents. In the metastatic setting, PIK3CA mutations are associated with a worse prognosis in patients treated with docetaxel/trastuzumab or docetaxel/trastuzumab/pertuzumab. Moreover, PI3K has been implicated as a key mediator of hormone resistance in preclinical models. However, several retrospective clinical studies have shown that activating mutations in PIK3CA paradoxically predict for a better long-term prognosis in HR positive patients treated with hormonal therapy. A prospective evaluation of the predictive role of PIk3CA mutations in HR+/HER2+ early breast cancer patients treated with anti-her2 therapy and chemotherapy or hormonal therapy is still lacking Specific aims Aim 1: Aim 2: Aim 3: To evaluate the prevalence of PIK3CA mutations (exon 9 and 20) and PTEN loss in 68 HER2 positive/ hormone receptor positive breast cancer patients undergoing neoadjuvant therapy in the PerElisa study, and the relation of PIk3/PTEN deregulation with tumor histotype, degree of expression of hormone receptor and tumor proliferation. To evaluate the Ki67 inhibition after a 2-weeks window therapy with letrozole according to the PIk3/PTEN deregulation in patients enrolled in the perelisa study, and the relation of PIk3/PTEN deregulation with breast and axillary pathologic complete remission (pcr) after the neoadjuvant perelisa protocol (trastuzumab plus pertuzumab associated with letrozole or paclitaxel) To identify additional genomic alterations that might be predictors of the activity of dual anti-her2 blockade combined with hormonal therapy. Exome sequencing of selected oncogenes and tumor suppressors will be applied in a subset of the patients enrolled in the PerElisa Study 10/07/ / 6

149 Challenging the role of chemotherapy in HER2 positive early breast cancer: PI3KCA pathways alteration and response to neoadjuvant pertuzumab-trastuzumab plus letrozole or paclitaxel in Hormone Receptor positive-her2 positive breast cancer patients. GR Guarneri Valentina Clinical health care research/clinicoassistenziale Istituto Oncologico Veneto Hypothesis: The perelisa protocol described in the following section is designed to evaluate whether a 2 weeks window neoadjuvant therapy with letrozole would select patients with different sensitivity to hormonal therapy on the basis of Ki67 inhibition. The hypothesis of this research project is that the PIk3/PTEN deregulation (PI3KCA mutations and/or PTEN loss) would refine our ability to identify the subgroup of patients that can be optimal candidates to combined endocrine manipulation and HER2 signaling blockade. Additional potential genomic markers will be explored for their potential impact on tumor response. Preliminary data: Our research team have extensively studied the predictive and prognostic role of PI3KCA mutations breast cancer neoadjuvant studies. We evaluated the role of dual HER2 targeting with trastuzumab+lapatinib combined with chemotherapy in a phase II randomized study in 121 HER2 positive patients (Guarneri et al, J Clin Oncol 2012). In this study, the prevalence of PI3KCA mutations in exon 9 or 20 was 20.2%. The median PTEN expression was 80% (0;100). In the whole population, pcr rates are similar in PIK3CA wild type (wt) and PIK3CA mutated (mut) patients (33.3% vs 22.7%; p= 0.34). However, for patients receiving trastuzumab plus lapatinib (n=41) the probability of achieving a pcr is higher in case of PIK3CA wt (48.5% vs 12.5%; p= 0.06) (to be presented ESMO 2014). In 92 postmenopausal patients with HER2 negative and hormone receptor positive disease, the objective response rate to letrozole-lapatinib was significantly higher in case of PI3KCa mutations vs PI3KCA wild-type. No differences in the objective response rate according to PI3KCA status was observed in the letrozole-placebo arm (Guarneri et al, J Clin Oncol 2014). The PerElisa study (EUDRACT , PI Valentina Guarneri) represents the clinical Platform of this study. Eight Italian centers have already received approval from Ethical Committes, and three patients already started treatment. Materials and Methods The Per-ELISA study is a phase II multicentric study, coordinated by the Istituto Oncologico Veneto. In this trial, HER2+/HR+ stage II-IIIA post-menopausal patients receive 14 days of neoadjuvant letrozole, and undergo a new biopsy for KI67 measurement. According to the relative change of Ki67 from baseline, patients will be classified as molecular responders or non-responders. Molecular responders will start therapy with the anti-her2 MoAb trastuzumab and pertuzumab in combination with letrozole. Molecular non-responders will stop letrozole and will receive chemotherapy with paclitaxel plus trastuzumab and pertuzumab. A total of 68 patients will be enrolled. Primary aim is the pcr rate in the two cohorts. PIK3CA status will be evaluated on tissue samples from the primary tumor. Analyses of PIK3CA mutational hot spot regions in exon 9-20 will be performed by Sanger Sequencing. PTEN protein status will be examined by IHC. Exome sequencing will be performed by MiSec Illumina. 10/07/ / 6

150 Challenging the role of chemotherapy in HER2 positive early breast cancer: PI3KCA pathways alteration and response to neoadjuvant pertuzumab-trastuzumab plus letrozole or paclitaxel in Hormone Receptor positive-her2 positive breast cancer patients. GR Guarneri Valentina Clinical health care research/clinicoassistenziale Istituto Oncologico Veneto Impact and Translational Implications To prospectively establish the predictive role of PI3KCA mutations would provide relevant clinical information, in view of newer anti-her2 agents available. Indeed, this pathway is a key druggable pathway, and PIk3 inhibitors are already in clinical development. Moreover, the possibility to identify molecular predictors of a specific sensitivity to hormonal blockade plus anti-her2 agents would allow a better treatment selection, potentially sparing unnecessary toxicity from chemotherapy

151 GR Investigating the role of mitochondria-er interaction in CMT: new insights in the biology of peripheral neuropathy Giacomello Marta Biomedical/Biomedica Ospedale San Camillo LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Amyotrophic Lateral Sclerosis (ALS) and related motor neuron disorders. inter-organellar communication mitochondria Project Request: Animals: Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Centro Studi sulle Malattie Neuromuscolari Principal Investigator Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date Background and Significance The term Charcot Marie Tooth (CMT) refers to a group of hereditary neuropathies. Although the genetic origins of CMT forms have already been identified, the molecular mechanisms at the basis of motor/sensory neurons degeneration are yet not clear, and no cure for these devastating disorders is available. Shedding light on the processes that lead to peripheral nerves degeneration or impaired maintenance of myelin sheaths is fundamental to develop effective therapeutical interventions. Interestingly it has been shown in model cells that knockdown of two proteins associated with two forms of CMT, i.e. Mitofusin2 and GDAP1 (mutations respectively linked to CMT2A and CMT4A) affects the proximity between mitochondria and Endoplasmic Reticulum (ER). The interface between these organelles controls both Ca2+ handling (through the formation of microdomains of high Ca2+ concentration on the surface of mitochondria) and lipid homeostasis (since at patches of ER membranes associated to mitochondria are located proteins involved in lipid biosynthesis/metabolism): two processes that, if altered, contribute to axon degeneration and demyelination. Although these evidence suggest that impaired mitochondria-er interactions contribute to CMT, this aspect has so far not been studied (probably due to lack of suitable tools to monitor mitochondria-er communication in live cells). Addressing this point will not only clarify the etiology of CMT, but also highlight new potential druggable targets. Specific aims Aim 1: Determine the correlation between impaired mitochondria-er interactions and type of CMT. Even if not all CMT 10/07/ / 5

152 GR Investigating the role of mitochondria-er interaction in CMT: new insights in the biology of peripheral neuropathy Giacomello Marta Biomedical/Biomedica Ospedale San Camillo forms are due to proteins directly involved in the tethering, mitochondrial dysfunctions found in CMT models could also affect their interaction with ER. We will unravel this point in cells taken from CMT patients and correlate the type/phenotype of neuropathy (i.e. axonal or demyelinating/ age of onset and severity) to the extent of mitochondria-er interaction and mitochondrial defects. Aim 2: Aim 3: Role of mitochondria-er tethering defects in axonal degeneration. Intra-axonal ER Ca2+ release plays a role in the onset of axonal degeneration. Thus, regulation of mitochondrial Ca2+ uptake is crucial for motor neurons wellbeing. We will study if the presence of mutated Mfn2/GDAP1 contributes to axonal degeneration or to the susceptibility of motor neurons to stress or apoptotic stimuli. Effects of mitochondria-er contacts alterations on lipid homeostasis. In the sites of juxtaposition between the two organelles there are proteins acting as checkpoint of lipid metabolism/biosynthesis. Defective mitochondria-er interactions can cause depletion of lipid precursors essential for myelin sheaths, leading to demyelination. We will generate Schwann model cells expressing CMT mutations and unravel if myelin production is affected by their presence. Hypothesis: We hypothesize that defective mitochondria-er association is a general hallmark of CMT. Altered mitochondria-er interactions can affect Ca2+ homeostasis and lipid handling, leading to axon degeneration in motor neurons and lipid dyshomeostasis in Schwann cells, thus accounting for different forms of peripheral neuropathy. Preliminary data: Up to now studying interorganellar communication was difficult due to lack of appropriate methods. The currently available techniques have some drawbacks: classical co-localization analysis of two fluorescent proteins suffers of inaccuracy due to microscopy resolution limits; electron microscopy suffers of artefacts due to sample fixation. We thus decided to develope a new probe to monitor the proximity between mitochondria and ER. Its progenitor is a FRET-based indicator composed of a CFP fusion protein on the ER surface (CFP-ER) and a YFP targeted to the outer mitochondrial membrane (OMM-YFP, provided by prof. G.Hajnockzy, USA). In this system alterations of the FRET Ratio mirror changes of the mitochondria-er proximity. The main drawback of the original probe was the high variability of the starting FRET value due to different expression levels of the two proteins. We solved this issue by means of a viral peptide generating multiple polipeptides from a single cistron (courtesy of prof. M. Ryan, UK): we successfully engineered a vector to express a Red fluorescent protein (Kate), CFP-ER and OMM-YFP. Not only all the proteins are simultaneously expressed in each transfected cell, but they are also perfectly localized in their subcellular compartment and able to work as FRET biosensor. Materials and Methods Mitochondria-ER contacts will be analysed in cells of CMT patients (collaboration with prof. C.Angelini, director of the Neuromuscular Tissue Bank) with of our new indicators. We will monitor Ca2+ homeostasis and redox changes with genetic probes (Cameleons, rogfp) and ATP content through bioluminescence assays. To get insights on the mechanisms leading to CMT, we will generate by genome editing model cells (based on the commercially available Motor Neuron NSC-34 and Schwann SW10 cell lines) expressing Mfn2 and GADP1 mutants. We will study the extent of axonal fragmentation and cell death (TUNEL assay) induced by excitotoxic stimuli as well as composition of whole lipid extracts (Thin Layer Chromatography, mass spectrometry) and cholesterol content (Filipin stain). To verify also if defective mitochondria-er interactions affect myelin structure we will perform Electron Microscopy studies in mutant SW10 cells co-cultured with NSC /07/ / 5

153 GR Investigating the role of mitochondria-er interaction in CMT: new insights in the biology of peripheral neuropathy Giacomello Marta Biomedical/Biomedica Ospedale San Camillo Impact and Translational Implications The outcomes of this project will improve our knowledge in the field of inter-organellar communication and provide insights on the processes at the basis of the motor/sensory neurons degeneration typical of CMT, which so far have not been understood. Since peripheral neuropathy is an hallmark not only of CMT, but also of other pathological conditions, our results will be relevant for a broader spectrum of human disorders and will shed the basis for new therapeutical strategies.

154 Mitochondrial DNA dynamics: Implications in neurodegenerative diseases BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Soriano Garcia Cuerva Maria Eugenia Biomedical/Biomedica Ospedale San Camillo LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Cell Biology Nuclear and Cytoplasmic Structure/Function and Dynamics - NCSD Project Keyword 1: Project Keyword 2: Project Keyword 3: Organelle biogenesis (for example mitochondria, chloroplasts, peroxisomes and lysosomes/vacuoles, Golgi), including organelle maintenance, proliferation, segregation, and dynamics mitochondrial DNA cristae remodelling Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION At present: Department of Biology University of Padua In case of funding the DI will decide the department/division PI Unit Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 11/07/ / 6

155 Mitochondrial DNA dynamics: Implications in neurodegenerative diseases BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Soriano Garcia Cuerva Maria Eugenia Ospedale San Camillo Background and Significance Mitochondrial dysfunction is main cause of neurodegenerative disorders. A frequent marker of dysfunction is the altered mitochondrial ultrastructure and network, which are in part regulated by the so called fusion and fission proteins. Mutations in fusion proteins are associated with neurological disorders such ADOA (OPA1 mutations) and Charcot Marie Tooth (MFN2 mutations). On the other hand, the importance of fission in the neurological development and function is demonstrated by a de novo mutation of Drp1 found in a lethal syndromic central nervous system pathology. In addition, mitochondrial diseases are also characterized by mtdna instability showing during the pathology a decrease in mtdna copy number and deletions. Recently has been shown that mitochondrial fusion is a necessary event in mitochondrial DNA stability and in the maintaining of heteroplasmy under the pathologic threshold. In spite of it all, very few is known about the mtdna dynamics but is clear that MtDNA instability has dramatic consequence for the cell. MtDNA is organized in complexes of DNA and proteins named nucleoids which composition is very debated. It is assumed that nucleoids are associated to the inner boundary membrane as occurs in low eukariotics, but it needs to be further clarified. This research proposal wants to characterize the dynamics of mtdna inside mitochondria in normal and dysfunctional mitochondria, to further understand the implications in neurodegenerative diseases. Specific aims Aim 1: Aim 2: Aim 3: To identify the location of mtdna inside mitochondria (associated to inner boundary, cristae membrane or in matrix) and to clarify whether the localization of mtdna is important for replication. We will study how changes in mitochondrial ultrastructure affects localization and replication and since it has been published that mitochondrial fusion is important in mtdna maintenance we will clarify the contribution of fusion and fission to mtdna location and replication. To study the relevance of Aim1 in the onset and progression of neurological diseases. MtDNA dynamics will be studied in Opa1flx/flx and Drp1flx/flx mouse adult fibroblast (MAFs) after protein acute deletion. Besides, we will study whether the recovery of the mitochondrial ultrastructure and/or fusion-fission events (genetic approach) are able to recover mtdna location, function, and mtdna copy number. The same studies will be performed in vivo in Opa1 or Drp1 flx/flx mice crossbreed with mice hemizygous for the Camk2a-CreERT2 transgene (inducible brain specific), and we will correlate the onset of the neurodegenerative disease with the loss of mtdna copy number and deletions, which are also a sign of mtdna instability. The in vivo experiments will clarify whether the alterations in mtdna dynamics precede or follow the onset of the disease. We will identify the key regulators implicated in mtdna maintenance. We propose a dynamic approach by performing comparative proteomics of purified nucleoids and isobaric labelling of two different situations: (i)normal versus cristae remodelling or (ii)resting versus mtdna replication. I emphasize that we will not use models with mutations in any of the proteins directly implicated in the mtdna replication, but models in which what we modify is mitochondrial ultrastructure and/or mitochondrial morphology. The proteomic approach will give us some possible therapeutic targets to enhance mtdna stability. Hypothesis: We hypothesize that changes in mitochondrial ultrastructure affects mtdna position and replication. Even if very little is known about the mtdna dynamics, we expect that changes in mtdna localization affect the functioning and stability, leading to loss of mtdna copy number and probably mtdna deletions. Preliminary data: We have observed by proteomic analysis that during cristae remodelling there is disruption of complexes containing proteins implicated in mtdna replication, translation and transcription. Remarkably,OPA1 knockout cells, which show alterations in mitochondrial ultrastructure have a reduced number of mtdna copy number, while OPA1 transgenic cells show a slight increase in mtdna copy number. Moreover,OPA1 deletion or Mitofilin silencing, both important in the 11/07/ / 6

156 Mitochondrial DNA dynamics: Implications in neurodegenerative diseases BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Soriano Garcia Cuerva Maria Eugenia Ospedale San Camillo maintaining of mitochondrial ultrastructure,result in a decrease of mitochondrial protein synthesis that leads to a bioenergetics defect and sensibility to cell death under stress conditions. All these data suggest a direct link between mitochondrial ultratructure and mtdna maintenance and stability. Materials and Methods Our cellular models will be Opa1flx/flx and Drp1flx/flx MAFs. OPA1 or DRP1 deletions will be induced by retroviral expression of CRE recombinase.to dissect the contribution of mitochondrial ultrastructure and morphology to mtdna stability we will use Opa1 KO versus wildtype and Mfn2 KO (normal ultrastructure but fragmented).in vivo experiments will be done in Opa1 flx/flx and Drp1 flx/flx mice both cross bred with Camk2a-CreERT2 mice. MtDNA location and detection of replicative mtdna will be performed by 3D reconstruction of electron microscopy tomograms where mtdna will be immunolabelled with TFAM antibody detected with nanogold in pre-embedding conditions. Replicative mtdna will be detected by growing cells in presence of BromodeoxyUridine immunodetected with a HRP antibody and revealed with diaminobenzidine. MtDNA copy number and mt protein synthesis will be done as described in Cogliati et al2013. Comparative proteomics will be done after isobaric labelling (itraq) Impact and Translational Implications The proposed research will clarify whether and how the mtdna dynamics inside the mitochondria may affect the onset and progression of neurodegenerative diseases. Moreover it will identify proteins implicated in mtdna maintenance as potential therapeutic targets in neurodegenerative diseases. 11/07/ / 6

157 GR CMR evaluation of myocardial inflammation persistence after acute myocarditis: prognostic relevance Esposito Antonio Clinical health care research/clinicoassistenziale Ospedale San Raffaele - Milano LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Cardiovascular and Respiratory Sciences Clinical and Integrative Cardiovascular Sciences - CICS Project Keyword 1: Project Keyword 2: Project Keyword 3: Human clinical studies (and appropriate translational animal studies): including pediatric populations, mechanisms and consequences of disease: Investigations may include: coronary physiology and pharmacology, cardiac electrophysiology, regional circulations, hemodynamic studies, cardiac mechanics, and genetic considerations in cardiovascular studies. Disease states can include: cardiac or vascular ischemia, hypertension, diabetes, thyroid disease, atherosclerosis, general inflammation, or hypercholesterolemia. Myocarditis, Myocardial Inflammation Cardiac Magnetic Resonance Imaging (CMR) Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Unità di Ricerca Clinica Radiologica / Centro di Imaging Sperimentale / Ospedale San Raffaele 2 Regione Lazio Dipartimento di Scienze Radiologiche Oncologiche e Anatomo-Patologiche, Università di Roma "La Sapienza" / UOC di Radiologia A / Policlinico Umberto I 3 Regione Piemonte Istituto di Radiologia / AOU Città della Salute e della Scienza di Torino Principal Investigator Collaborator Collaborator 11/07/ / 6

158 GR CMR evaluation of myocardial inflammation persistence after acute myocarditis: prognostic relevance Esposito Antonio Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Ospedale San Raffaele - Milano Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Galea Nicola Regione Lazio Collaborator: Responsible for the enrollment of about 1/3 of the patients in Rome. Responsible for the assessment of the prognostic role of new CMR parameters (T2-rt, T1-rt, ECV) 24/04/ Faletti Riccardo Regione Piemonte Collaborator: Responsible for the enrollment of about 1/3 of the patients in Turin. Central assessment of image quality. 3 GODINO COSMO Ospedale San Raffaele Collaborator: Central collection and evaluation of clinical data of enrolled patients 05/10/ /06/1976 Background and Significance In patients with acute myocarditis (AM), spontaneous improvement can be observed in most of cases, but progression toward a dilated cardiomyopathy is a not rare outcome ( 20% of patients) (Feldmann NEJM 2000;343: ). Differences in the course of disease may reflect course of underlying viral infection. In a large series of patients with AM submitted to two endo-myocardial biopsies (EMBs) in few months, Kuhl demonstrated that viral clearance was associated with spontaneous ejection fraction (EF) improvement, while EF did not improve or even deteriorated in patients with viral and myocardial inflammation persistence (Kühl Circulation 2005;111: Kühl Circulation 2005;112: ). However, repeated EMBs cannot be proposed in the clinical routine and, hence, non-invasive detection of the subgroup of patients with inflammation persistence should have important implications. Today, Cardiac Magnetic Resonance (CMR) is recognized as an accurate non-invasive imaging tool to diagnose acute myocarditis, because of its ability to detect myocardial inflammation and necrosis (Friedrich JACC 2009;53: ). However, although the role of CMR parameters of inflammation and necrosis is fairly well established in diagnosis of AM, any study investigated the potential value of these parameters in the assessment of inflammation persistence after acute phase. We aspect the assessment of early changes in CMR parameters at a second imaging study may have great value in outcome prediction Specific aims Aim 1: To assess the prognostic value of the early changes (at one-month follow-up) in CMR parameters (% of gre, T2- ratio and LGE amount) reflecting inflammation activity in terms of myocardial oedema, hyperemia and tissue necrosis, in order to identify the subgroup of patients with greater risk to develop dilated cardiomyopathy (DCM). Aim 2: Aim 3: To evaluate the prognostic value of individual CMR parameters of myocardial inflammation measured at baseline or at one-month follow-up, and to compare them to the performance of its percentage variation between the two CMR studies. To assess the prognostic value of new quantitative CMR parameters of structural tissue alteration, such as T1 relaxation time (T1 mapping), extracellular volume (ECV) and T2 relaxation time (T2 mapping). Hypothesis: The persistence of a subtle myocardial inflammation is an important factor in determining a negative outcome in patients with acute myocarditis; moreover, CMR demonstrated to assess myocardial inflammation with high sensitivity and, hence, has the potentiality to follow the myocardial inflammation over 11/07/ / 6

159 GR CMR evaluation of myocardial inflammation persistence after acute myocarditis: prognostic relevance Esposito Antonio Clinical health care research/clinicoassistenziale Ospedale San Raffaele - Milano time. Our hypothesis is that a short term monitoring with CMR (1 month) may allow differentiating patients evolving toward a complete healing from patients with persistent myocardial inflammation, which may have a greater risk to progress toward DCM Preliminary data: A previous study from our group, performed on patients with chronic inflammatory cardiomyopathy, demonstrated as the positivity of CMR parameters of necrosis (LGE) and inflammation (Edema) is significantly higher in patients with an active inflammation at EMB, compared to patients with borderline histological criteria (De Cobelli 2006 JACC;47: ). These data suggest the possibility to detect subtle myocardial inflammation persistence with CMR in patients with chronicization of an acute myocarditis. Moreover, Wagner and Colleagues demonstrated, in a small group of patients, a correlation between CMR evidence of myocardial hyperemia persistence 4 weeks after the onset of acute myocarditis and the negative LV-remodeling 30 months later (Wagner A 2003 MAGMA;16:17-20). Currently, the three Unit involved in this project are collecting a common register of all patients submitted to a CMR study for the assessment of an acute myocarditis; this work collecting imaging features of several patients (> 100) allows to compare and standardize the protocols of acquisition and post-processing of CMR studies. Materials and Methods This is a prospective multicenter cohort study. 80 patients with diagnosis of acute myocarditis (AM) will be enrolled. All patients admitted to Hospital with suspect of AM will be submitted to: collection of detailed anamnesis and physical examination, 12-lead ECG, laboratory exams, transthoracic echocardiography, coronary catheterization or coronary CT angiography when an ischemic cause of symptoms need to be excluded and CMR imaging within 3-5 days. All patients with clinically or EMB confirmed diagnosis of AM will be enrolled and will undergo a second CMR study 1 month later. Percentage of change in global Relative Enhancement (gre), T2-ratio and amount of LGE between the two CMR studies will be assessed. All patients will undergo to a clinical/instrumental follow-up including: CMR assessment of LV ejection fraction and enddiastolic after at least 1 year from diagnosis; registration of all-cause mortality, cardiac death and aborted cardiac sudden death in patients with ICD. Impact and Translational Implications Myocarditis is characterized by significant heterogeneity of long-term evolution. CMR could play a key role in the noninvasive and early identification of patients with persistent myocardial inflammation, at high risk to evolve toward an irreversible post-myocarditis heart failure. So CMR may help to design tailored management of patients. In this perspective, invasive characterization of damage mechanism and aetiology might be reserved to patients with CMR evidence of inflammation persistence 11/07/ / 6

160 GR Development and validation of a mathematical model for esteem of central blood pressure in normal subjects and in patients with proximal aorta dilatation MILAN ALBERTO Clinical health care research/clinicoassistenziale Piemonte LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Surgical Sciences, Biomedical Imaging, and Bioengineering Medical Imaging - MEDI Project Keyword 1: Project Keyword 2: Project Keyword 3: Applications of imaging systems and modification of diagnostic methods for use in: screening; characterizing physiological effects,, and assessing risk. Central blood pressure Aortic dilatation Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION AO Città della Salute e della Scienza di Torino - Dpt of General and specialistic Medicine - Division of Internal Medicine - Hypertension Unit PI - Principal Investigator, design of the study, recruitment patients and haemodynamic approach and interpretation of the data and its findings. 2 Politecnico di Torino Department of Fluid Mechanics Model refinement and development 3 ASL CN1 Ospedale Santa Croce Mondovì Pronto Soccorso (D.E.A.) Echocardiography 11/07/ / 6

161 GR Development and validation of a mathematical model for esteem of central blood pressure in normal subjects and in patients with proximal aorta dilatation MILAN ALBERTO Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Piemonte Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Camporeale Carlo Vincenzo Politecnico di Torino Mathematical Model refinement and validation 2 Corrado Magnino ASL CN1 Ospedale Santa Croce Recruitment patients and haemodynamic Mondovì approach and interpretation of the data and its findings 3 Bertello Chiara AO Città della Salute e della Scienza di Torino - Division of Internal Medicine - ypertension Unit - Assistant in Internal Medicine Recruitment patients and haemodynamic approach and interpretation of the data and its findings 4 Guala Andrea Politecnico di Torino Mathematical model development and refinement 02/03/ /03/ /07/ /02/1986 Background and Significance Dilatation of proximal Aorta (pad) and hypertension represent the two main risk factors for aortic dissection. pad is frequent in hypertensive patients, with an average prevalence of 11%. The general population in Piedmont is of roughly inhabitans, 80% of which are over 20 years of age. This leads to an esteem of a minimum of people in Piedmont affected by aortic dilatation. Such preliminary epidemiologic remarks underline the importance of an accurate approach to the diagnosis and follow up of this disease. A consistent association between aortic remodelling and central pressure values has been observed. Today, evaluation of central pressure is made through expensive instruments, mostly available for research purposes only. Engineering models are being developed to give an esteem of blood pressure values along different tract of the vascular tree in the human body. The definition of such a mathematical model aimed at evaluating central blood pressure may grant precise haemodynamic informations for patients affected by pad. This may lead to a better risk stratification for this subgroup of patients and a consistent saving in terms of instruments and know hows. Specific aims Aim 1: Aim 2: Aim 3: Development of a complex mathematical model for the non invasive evaluation of central blood pressure Vallidation of the mathematical model on healthy subjects Validation of the model on patients with aortic dilatation Hypothesis: The central blood pressure is significantly associated with proximal aortic remodelling. The development of a model which allows to estimate the central pressure would grant a more accurately assess patients with aortic dilatation and make this assessment more accessible Preliminary data: In a joint project with the Turin Engineering School we are developing and refining a patient-specific complex mathematical model. This model is based so far on healthy male young volunteer - aged 25 - and considers the ventricular performance, the aortic dynamic and distal circulation. For every subject the results of the calibrated model has been compared to the non invasive measurements made with Sphygmocor, the actual gold standard for non invasive central pressure evaluation. Preliminary results of the model on the first healthy subjects (n= 7) how a mean error in the esteem 11/07/ / 6

162 GR Development and validation of a mathematical model for esteem of central blood pressure in normal subjects and in patients with proximal aorta dilatation MILAN ALBERTO Clinical health care research/clinicoassistenziale Piemonte of central pressure value in the range of -1% to +5%. Materials and Methods The mathematical model will be targeted to obtain central blood pressure values esteem from simple parameters: age, gender, brachial blood pressure and left ventricular volumes systolic and diastolic. Further stepwise development will follow: phase 1) model development, and 2) analysis and calibration of the model in a group of healthy subjects of different age range, and subsequent validation in an indipendent group. Phase 3) analysis and calibration of the model in patients affected by pad. In this latter phase, the model will be evaluated against non invasive and invasive measurements ( i.e. catheterization), in patients that independently have clinical indication to cardiac catheterization. In every phase involved subjects will undergo transthoracic echocardiogram and non invasive study of central haemodynamic through validated instruments Sphygmocor - Accuracy of the model will be evaluated through correlation, regression and Bland Altmann analysis. Impact and Translational Implications Non invasive evaluation of central haemodynamic has already showed prognostic significance. The development of a mathematical model that could determine central pressure values, drastically reducing instrument related costs, would grant a wider use of such parameters. This would lead to a better risk stratification with clinical implication in the management of hypertensive patients and moreover, in those who, carrying a proximal aortic dilatation, are at higher risk for aortic dissection. 11/07/ / 6

163 COMPOSITION OF GUT MICROBIOTA AND IMPACT ON IMMUNE SYSTEM RECOVERY AND ACUTE GRAFT VERSUS HOST DISEASE IN PEDIATRIC PATIENTS UNDERGOING HEMATOPOIETIC STEM CELL TRANSPLANTATION GR Masetti Riccardo Biomedical/Biomedica Emilia-Romagna LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Oncology 2 - Translational Clinical Cancer Immunopathology and Immunotherapy - CII Project Keyword 1: Project Keyword 2: Project Keyword 3: Hematopoietic stem cell transplantation and other adoptive cellular therapies with immune cells as cancer treatment Gut microbiota Acute graft versus host disease Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Azienda Ospedaliero Universitaria di Bologna, Dipartimento Salute della Donna, del Bambino e dell'adolescente. U.O. Pediatria 2 University of Bologna, Dipartimento di Farmacia e Biotecnologie - FABIT Dipartimento di Farmacia e Biotecnologie - FABIT. Laboratorio di Microbiologia e Biotecnologie 3 IRCCS Ospedale Pediatrico Bambino Gesù, Dipartimento Onco_Ematologia Pediatrica e Medicina Trasfusionale Dipartimento Onco_Ematologia Pediatrica e Medicina Trasfusionale Principal investigator Investigator Investigator 11/07/ / 6

164 COMPOSITION OF GUT MICROBIOTA AND IMPACT ON IMMUNE SYSTEM RECOVERY AND ACUTE GRAFT VERSUS HOST DISEASE IN PEDIATRIC PATIENTS UNDERGOING HEMATOPOIETIC STEM CELL TRANSPLANTATION GR Masetti Riccardo Biomedical/Biomedica Investigators, Institution and Role in the Project Emilia-Romagna Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Candela Marco University of Bologna, Dipartimento di Farmacia e Biotecnologie - FABIT 2 Merli Pietro IRCCS Ospedale Pediatrico Bambino Gesù, Dipartimento Onco_Ematologia Pediatrica e Medicina Trasfusionale 3 Zama Daniele Azienda Ospedaliero Universitaria di Bologna, Dipartimento Salute della Donna, del Bambino e dell'adolescente. U.O. Pediatria 4 Di Nardo Giovanni Azienda Ospedaliero Universitaria di Bologna, Dipartimento Salute della Donna, del Bambino e dell'adolescente. U.O. Pediatria MD, PhD. Dedicated to the phylogentic and functional carachterization of the gut microbiota MD, Dedicated to the immune recovery charachterization after HSCT MD, PhD Dedicated to the clinical activities related to HSCT MD, Assistant Professor. Pediatric endoscopist performing gut biopsies 06/10/ /09/ /08/ /07/1977 Background and Significance Allogeneic hematopoietic stem cell transplantation (HSCT) provides a curative treatment option for children with high-risk hematologic malignancies and inherited hematopoietic disorders. The main complications of HSCT are represented by infections and acute graft versus host disease (agvhd) whose pathophysiology results from a donor T-cell activation against a genetically disparate recipient. Human beings have been recently defined as metaorganisms, because of a symbiotic relationship with the gut microbiota (GM). The immunological interaction with the GM has been demonstrated as crucial for the development and functionality of our immune system. In the light of the GM potential to modulate the host immunological phenotype, we hypothesized that a mutualistic GM layout can be a strategic driver for a successful outcome of HSCT in terms of onset of agvhd and infections. Very few studies on the characterization of GM involving a longitudinal approach and including pediatric patients have been published. The present project will provide for the first time a longitudinal monitoring of GM starting before HSCT and evolving during the recovery of IS and the onset of agvhd, particularly gut agvhd, in children undergoing HSCT. These results can identify a panel of profiles of the GM associated with specific patterns of immunological recovery and agvhd and lay the foundations to temptatives of diet intervention aimed to modulate GM before HSCT towards a more protective configuration. Specific aims Aim 1: Aim 2: Characterization of the compositional and functional modifications of GM in children undergoing allogeneic HSCT. In particular fecal GM trajectories will be characterized before and periodically after HSCT in all patients enrolled in the study. In addition to the fecal GM characterization, a mucosal-associated microbiota in gut biopsies will be charcterized in patients developing gut agvhd. Characterization of specific patterns of immunological recovery in patients developing or not infections and agvhd and correlation with GM composition. A wide panel of lymphocyte subpopulations and cytokines will be studied periodically on peripheral blood in all patients and addiotionally on biopsies of patients developing gut 11/07/ / 6

165 COMPOSITION OF GUT MICROBIOTA AND IMPACT ON IMMUNE SYSTEM RECOVERY AND ACUTE GRAFT VERSUS HOST DISEASE IN PEDIATRIC PATIENTS UNDERGOING HEMATOPOIETIC STEM CELL TRANSPLANTATION GR Masetti Riccardo Biomedical/Biomedica Emilia-Romagna agvhd Aim 3: Development of strategies of diet intervention aimed to modulate GM before HSCT towards a more protective configuration. Hypothesis: The study provides for the first time a monitoring of the compositional and functional changes occurring on GM during the immune recovery of children after HSCT. GM regulates the adaptive immune response, exerts a key role in the education of IS to tolerance and influence the functionality of the intestinal epithelium. Moreover, GM can impact the host susceptibility to enteropathogen infection by exerting the well know barrier effect. In this research project we will determine whether specific phylogenetic and/or functional profiles of the GM can modulate the immune recovery after HSCT, eventually predisposing to or protecting from gut agvhd or infections. These results may build the sientific basis for temptatives of diet intervention aimed to modulate GM before HSCT towards a more protective configuration. Preliminary data: We recently carried out the first longitudinal study to follow the GM trajectory, from both the phylogenetic and functional points of view, in pediatric patients undergoing HSCT. 10 pediatric patients suffering from hematological disorders and undergoing HSCT, 5 of which developed agvhd after transplantation were enrolled. GM trajectories and SCFA production profiles were followed starting from before HSCT and through the 100 days after HSCT by sampling feces. HSCT procedures showed to temporarily cause a structural and functional disruption of the gut microbial ecosystem, describing a trajectory of recovery during the following 100 days. The onset of agvhd is associated with specific GM signatures both along the course of the GM reconstruction and, most interestingly, prior to HSCT. Our data open the perspective to manipulate the pre-hsct dietary habits to favor these mutualistic persisters with immunomodulatory properties in the gut. The revised paper is now back under review in Gut (ID gutjnl ). Materials and Methods We will enroll 50 patients. For each subject, structure and function of GM, lymphocyte subsets characterization and dosage of cytokines will be studied at 6 defined time points: before HSCT, engraftment and every two weeks for two months. Gut biopsies will be studied in patients developing gut GvHD. GM structure: 16S rrna gene fragments containing the V3 and V4 hypervariable regions will be sequenced by using MiSeq platform with multiplex. Sequences will be processed and assigned by following QIIME pipeline. Functional GM metatranscriptomics: cdna will be sequenced by using Illumina MiSeq platform with multiplex. The metatranscriptomes will be analyzed by combining assembling and direct mapping of the reads. Faecal metabolomic analysis will be carried by solid phase microextraction followed by GC-MS analysis to detect the volatile metabolites. Subsequent derivatization or alternative to GC-MS, orthogonal separation techniques, like LC-MS, will be used Impact and Translational Implications The clinical relevance of this study become evident considering the increasing number of disease potentially curable with allogeneic HSCT and the relevance of agvhd and infections as life-threatening complications limiting the procedure. In adults, a low diverse GM has been demonstrated to be an independent predictor of mortality in HSCT recipients. Our results will allow identifying dysbiosis of the GM associated to infections and agvhd and thus impacting on the outcome transplanted children. 11/07/ / 6

166 GR Brain Function Monitoring to Evaluate Efficacy and Safety of Palliative-Sedation-in-the- Last- Days-Therapy in Terminally Cancer Patients. Gian Paolo Spinelli Clinical health care research/clinicoassistenziale Lazio LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Surgical Sciences, Biomedical Imaging, and Bioengineering Medical Imaging - MEDI Project Keyword 1: Project Keyword 2: Project Keyword 3: Prediction, selection, and monitoring of therapeutic response based on imaging studies, with or without exogenous agents, using one or more modalities, especially for multi-temporal investigations to measure changes relative to a pretreatment baseline. Palliative care Palliative-Sedation-in-the- Last-Days-Therapy Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION ASL LATINA /DISTRETTO APRILIA/UOC ONCOLOGIA Principal Ivestigator Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 10/07/ / 6

167 GR Brain Function Monitoring to Evaluate Efficacy and Safety of Palliative-Sedation-in-the- Last- Days-Therapy in Terminally Cancer Patients. Gian Paolo Spinelli Clinical health care research/clinicoassistenziale Lazio Background and Significance The terminally cancer patient (TCP) is defined as a patient for whom death appears imminent, inevitable and predictable, due to an irreversible disease which is not responsive to active treatments. As proposed by European Association for Palliative Care (EAPC), Palliative-Sedation-in-the- Last-Days-Therapy (PSILD) is the use of nonopioid drugs for TPC, when relieving intolerable suffering is mandatory. Several issues concerning PSILD are still under debate for its ethical, sociocultural, and decision-making implications. To date, PSILD has been emerged as generally effective with rare adverse effects in several retrospective studies. However, some limitations are linked to the retrospective nature of these studies, particularly issues in assessing intention, monitoring, application of protocols and data collection. Indeed, no standardization for timing, medications, doses, and administration routes to be used for PSILD is nowadays available. In addition, there are no specific scales supporting the management of PSILD. Some scales are available for assessing the level of sedation-agitation such as the Ramsay and Richmond scale, but they have not been validated for PSILD. Thus prospective studies are needed to evaluate the efficacy and safety of PSILD by monitoring objective parameters such as the brain functions. Specific aims Aim 1: Aim 2: Aim 3: Brain Monitoring:The first aim is to monitor brain function real-time insight into the depth of patient's anesthesia and sedation during PSILD, calculating patient's measure of brain activity that reflects the level of sedation/anesthesia Efficacy and Safety:The second aim is to explore the efficacy and safety of PSILD by monitoring brain function, according to the parameters defined in the aim1. This will allow to identify the factors contributing to inadequate symptom relief and complications by sedation. Cost-effectiveness analysis:the last aim is to establish the costs for palliative care service in a low-resource setting, and to elucidate possible consequential quality-of-life improvements and potential cost savings. Hypothesis: We believe that PSILD is an important therapeutic intervention that should be offered to TCPs in an optimized manner, to avoid both over and under treatments. However, no available evaluation scale is enough simple, applicable, objectivable and reproducible in clinical practice. Moreover no consensus has been reached about the starting degree of sedation to be implemented and how to maintain it. The optimization of PSILD can be achieved by monitoring objective and/or objectivable parameters. The best candidate to be used in this setting can be brain function study. Here we propose our research to analyze the grade of sedation in TCP, using brain electrical activity by electroencephalogram (EEG). The hypothesis is to monitor brain function with Quantitative EEG (QEEG): - to Understand the effects of sedation on brain activity - to improve the care of TCP under sedation - to enable more individualized therapy finally optimizing the assistential costs and proposing more accurate guidelines. Preliminary data: Our Research Unit has a long lasting experience in the oncology field, with a particular attention to palliative therapy in TPC. The reference territory is the ASL Latina (over 200,000 residents). A consolidated collaboration network among Oncologists, Palliative care Specialists, General Medicine Doctors and Psychologists is active in our Unit. The four Palliative Care Units in the area assist about 10/07/ / 6

168 GR Brain Function Monitoring to Evaluate Efficacy and Safety of Palliative-Sedation-in-the- Last- Days-Therapy in Terminally Cancer Patients. Gian Paolo Spinelli Clinical health care research/clinicoassistenziale Lazio 160 patients. A monthly Disease Management Team is carried out to discuss all the oncological cases and the decisions regarding TPCs. This partnership has already resulted in major scientific publications [i.e. "evaluating the appropriateness of palliative care in terminal cancer patients " by Stati et al. at the congress of the Italian Society of Palliative Care (SICP) 2014]. In addition, an important Cancer Pain Treatment Service and a Simultaneous Care Service have already been activated for about a year; during these activities PSILD is emerging as an important medical, socio-economic and ethical issue. All these consolidated contexts allow the identification and the enrollment of a large number of patients for the study, creating the conditions for the realization of the proposed project. Materials and Methods Patients (>18 years of age) with histologically confirmed diagnosis of cancer for whom the occurrence of acute events lead to an mminent death will be eligible for the study. the patient's life expectancy must be assessed between a few hours and a few days. For the Brain function monitoring an algorithm based on extensive EEG records, to determine the Patient State Index(PSI) value (along a scale of 0 to 100) as a measure of sedation depth will be used. Regarding the induction and maintenance of PSILD, in our study, the following factors will be considered:(1)the sedation will be implemented with midazolam (1st choice) and, as an alternative, levomepromazine, chlorpromazine or barbiturates.(2) Doses of midazolam may vary from 5 to 1200 mg/day.(3)the sedation will be implemented at low initial doses, increasing them until it reaches the level of sedation is useful and monitored by EEG.(4)The subcutaneous or intravenously administration routes will be used. Impact and Translational Implications The evaluation of PSILD represents an important challenge in terms of understanding the effects of sedation on brain activity in terminally cancer patients. Here we propose a study to improve the care of terminally cancer patients under sedation. This prospective study could have a critical impact in order to individualize more accurate sedation therapy, with several advantages in patients' Quality of Life, proposing more accurate guidelines and consequently optimizing the assistential costs. 10/07/ / 6

169 GR Type I IFN-mediated induction of a chemoresistant niche of tumor cells: from animal models to humans SISTIGU ANTONELLA Biomedical/Biomedica Istituti fisioterapici ospitalieri - Istituto Regina Elena LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Oncology 1 - Basic Translational Tumor Microenvironment - TME Project Keyword 1: Project Keyword 2: Project Keyword 3: Molecular and cellular aspects of bi-directional interaction between tumor and stromal cells (including fibroblasts, glial cells, epithelial cells, adipocytes, immune cells, inflammatory cells, vascular compartments, and bone marrow cells) during neoplastic progression, tumor angiogenesis, growth and metastasis, including studies of cancer stem cell niche and tumor cell dormancy cancer stem cells Type-I interferons Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION UOC Experimental Oncology Principal Investigator 2 Gustave Roussy Cancer Campus INSERM Apoptosis, cancer and immunity Collaborator Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Vacchelli Erik Gustave Roussy Cancer Campus 2 Provision and analysis of human specimens 12/07/ Vitale Ilio IRCCS-IFO ISTITUTO NAZIONALE TUMORI REGINA ELENA - UOC Experimental Oncology 4 Manic Gwenola IRCCS-IFO ISTITUTO NAZIONALE TUMORI REGINA ELENA - UOC Experimental Oncology Execution and design of the experiments 28/04/1977 Execution and design of the experiments 05/09/ /07/ / 5

170 GR Type I IFN-mediated induction of a chemoresistant niche of tumor cells: from animal models to humans SISTIGU ANTONELLA Biomedical/Biomedica Istituti fisioterapici ospitalieri - Istituto Regina Elena Background and Significance Successful chemotherapy accounts for both tumor-related factors and host immune responses. Compelling evidence suggests that some chemotherapeutic agents (e.g. anthracyclines) can induce an immunogenic type of cell death stimulating tumor-specific immunity. Our unpublished data support the hypothesis that anthracycline-based immunogenic chemotherapy phenocopies viral infection leading to a cancer-cell autonomous Toll-like receptor (TLR)3>Type I interferon (IFN) fingerprint indispensable for inducing a protective anticancer immune response. We promote such viral mimicry a hallmark of successful chemotherapy. Even when chemotherapy kills most cells in a tumor it could spare or paradoxically select for cancer stem cells (CSCs) behind. CSCs are thought to represent a minority of cells within a tumor particularly resistant to therapy, able to self-renew, to differentiate and thus to reproduce a tumor. Conventional anthracycline-based chemotherapy can induce tumor regression while simultaneously selecting for a subset of drug-resistant cells that subsequently fuels tumor regrowth. Moreover TLR3>Type I IFN signaling appears to favour an open chromatin state which increases cell plasticity, transdifferentiation and malignant transformation. We are intended to investigate the dual role of immunogenic chemotherapy leading to cancer-cell autonomous TLR3>IFN in tumor suppression and promotion with the aim to design patient-tailored, combined therapies targeting differentiated and CSCs. Specific aims Aim 1: Aim 2: Aim 3: To verify a correlation between cancer cell-autonomous TLR3-IFNAR axis with the appearance of CSCs following immunogenic chemotherapy. To portray the molecular signaling downstream of TLR3/IFNAR axis which elicits CSC-mediated tumor escape mechanisms. Translational side of research: to correlate CSC markers pre- and post-chemotherapy with tumor relapse and overall survival (OS) in response to neoadjuvant anthracycline-based chemotherapy in several cohorts of breast cancer (BC) patients. Hypothesis: We postulate that viral mimicry by chemotherapy beyond being indispensable to elicit a protective anticancer immune response paradoxically favours the appearance of a subset of pluripotent tumor cells resistant to therapy. We believe that this "cancer-chemo editing" phenomenon is due to evolutionary conserved cellular responses phenocopying those of viral infection (e.g. unfolded protein response UPR and autophagy) that paradoxically could promote tumor progression/relapse either by selecting for tumor cells that are more fit to survive or by establishing conditions within the tumor microenvironment that facilitate tumor outgrowth. Preliminary data: Our unpublished data (Sistigu et al., manuscript in revision) suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such a "viral mimicry" constitutes a hallmark of successful chemotherapy. However, even tumors that show dramatic initial responses to therapy frequently relapse as chemoresistant malignancies that strongly correlate with decreased survival in patients. Compelling evidence suggests that conventional anthracycline-based chemotherapy can induce tumor regression while simultaneously selecting for a subset of drugresistant cells that subsequently fuels tumor regrowth. Of interest a surprising link between viral-like innate immune pathways and nuclear reprogramming has been recently reported. Based on those premises, the present project will investigate whether and how viral mimicry by chemotherapy could favour a subset of pluripotent tumor cells resistant to therapy. 11/07/ / 5

171 GR Type I IFN-mediated induction of a chemoresistant niche of tumor cells: from animal models to humans SISTIGU ANTONELLA Biomedical/Biomedica Istituti fisioterapici ospitalieri - Istituto Regina Elena Materials and Methods MCA205 sarcomas, AT3 mammary, LLC lung, CT26 colon carcinomas defective or not for cardinal points of the IFN pathway (TLR3, TICAM, IFNAR) will be in vivo treated with anthracyclines and at different time points analysed to track a phenotypic, transcriptional and cytogenetic portrait of induced CSCs. In vitro and in vivo clonogenic assays will confirm the tumorigenic potential of induced CSCs. A large collection of breast, colon and lung CSCs derived from different primary/metastatic neoplasms (CSC Biobank) will be used as positive control. Following an hypothesis-based strategy we selected some inhibitors of the UPR and/or autophagy to be combined with immunogenic chemotherapy for blocking the induction of CSCs. IHC analysis of IFN-related and CSC-related markers will be performed in paraffin embedded tumor tissue from BC patients before and after chemotherapy and correlated with the OS. Pairwise statistical comparisons will derive from unpaired 2-tailed Student's and Log-rank test. Impact and Translational Implications If our results will corroborate our hypothesis they will contribute to shed light on this cancer chemo-editing dogma dissecting the mechanisms by which chemotherapy plays a dual role in cancer suppression and cancer promotion. That could have potential positive consequences for the clinical management of cancer: i) to guide the choice of the drug according to its immunogenic potential, ii) to consider the combination of cytotoxic agents with targeted therapeutics against CSCs. 11/07/ / 5

172 GR Novel therapeutic perspectives for angioedema associated with C1-inhibitor deficiency or angiotensin converting enzyme-inhibitor treatment Bossi Fleur Clinical health care research/clinicoassistenziale Ospedale infantile Burlo Garofolo LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Vascular and Hematology Vascular Cell and Molecular Biology - VCMB Project Keyword 1: Project Keyword 2: Project Keyword 3: Vasomotor activity, including vasocontraction and relaxation, leukocyte trafficking, adhesion molecules; chemokines, cytokines; intercellular signaling; reactive oxygen and nitrogen species; Endothelial barrier function; extracellular matrix-mediated signaling. Endothelial cells Angioneurotic edema Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: GR X I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Diagnostic collection of human samples, analysis of the samples Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date Background and Significance Angioedema (AE) is a condition characterized by significant localized swelling of deeper layers of the skin or submucosal tissues that affects at least 20% of the population. AE is due to increased vascular permeability, leading to massive dermal and subcutaneous edema, becoming particularly life threatening if it occurs in the upper respiratory tract. The hereditary form of AE is an autosomal dominant disorder due to a decreased serum level of C1 inhibitor (C1-INH) or to a secretion of a non functional protein. The acquired form of AE is associated with the high consumption of C1-INH or to the presence of autoantibodies or induced by treatment with Angiotensin Converting Enzyme (ACE) inhibitors. The mediator that seems to be the main responsible for edema formation is bradykinin (BK), but the real contribution of other factors such as complement components or peptides derived from BK catabolism (e.g. des-arg9-bk) or from renin-angiotensin-aldosterone system remained to be defined. Furthermore AE episodes are localized at particular vascular sites and the mechanisms that maintained confined the edema has not been yet understood. In spite of a lifelong stable C1-INH deficiency or of a continuous ACE inhibitor treatment, patients with these conditions are only seldom symptomatic, with huge variation in frequency and severity of symptoms and the mechanisms responsible for such variability is completely unexplained and the therapeutic tools are still not completely satisfying. 10/07/ / 4

173 GR Novel therapeutic perspectives for angioedema associated with C1-inhibitor deficiency or angiotensin converting enzyme-inhibitor treatment Bossi Fleur Specific aims Clinical health care research/clinicoassistenziale Ospedale infantile Burlo Garofolo Aim 1: Aim 2: Aim 3: To identify the mediators responsible for increased vascular permeability in AE patients analyzing the role of kinins, of the soluble terminal complement complex and of renin-angiotensin-aldosterone system in inducing endothelial leakage. To investigate the factors that maintain the event localized to a specific vascular site studying the tissue distribution of gc1q and bradykinin receptors and the stimuli that induce their membrane expression. To analyse the enzymatic systems that promote AE episodes. Hypothesis: Available evidence suggests that the nonapeptide bradykinin (BK) may be largely responsible for the vascular permeability seen in acute attacks of AE but recent data collected in our laboratory indicate that other molecules could be also involved. For this reason we will analyze a large series of patients representing different clinical variants and different phases of the disease (spontaneous remission, attack, drug-induced remission) in order to better identify the factors responsible for the edema. In particular will be investigated the role of des-arg9-bk and the potentiating effect of BK by angiotensin 1-7. Furthermore, since gc1qr has been shown to be a major site for the assembly and activation of the kinin system and the kinins released bind in turn to specific B1 and B2 receptors, and the edema developed during acute attacks is confined to particular vascular sites, we will analyse the different receptor expression between edematous and normal tissues. Our hypothesis is that blockade of both BK receptors and blockade of gc1qr can attenuate the vascular permeability and prevent the generation of BK. Since des-arg9-bk is produced from BK by carboxypeptidase M along cell surface, we hypothesize that the blockade of the enzyme may also reduce the vascular leakage being a new potential target. The aim of the proposal is to thoroughly identify the mechanisms leading to the enhanced vascular permeability in clinical settings of AE by focusing not only on C1-INH deficiency as a primary disease model but also on ACE-inhibitor related AE in order to identify new possible therapeutic tools. Preliminary data: We have previously shown (Bossi et al JACI) that plasma from patients in AE attack phase but not in remission phase contains factors that enhance vascular permeability using both in vitro transwell system and in vivo intravital microscopy models. We have also demonstrated that the effect of these factors depends on the tissues from which the ECs are derived, indeed dermal ECs are more responsive than HUVECs. Using the same in vitro and in vivo models we showed that B1 receptors and gc1qr appear to play a predominant role in the attack plasma-mediated permeability since antagonists for B1 receptor, and monoclonal antibodies to gc1qr are able to inhibit vascular permeability. Furthermore we tested the effect of IL-1 beta finding that it could be one of the factors that induces the receptor membrane expression. Materials and Methods The human plasma samples will be collected and quickly frozen at -80 C. The quantification of the permeabilizing factors and cytokines present in plasma will be performed by ELISA or by chemiluminescent assay. The in vitro leakage assay will be run using a transwell system with the insert covered by a confluent monolayer of primary endothelial cells (from human skin or umbilical cord). The in vivo intravital microscopy will be performed on Wistar rats following the European (2010/63/UE) and Italian (D.L. 26/14) laws as previously described (Bossi et al JACI). The in vitro and in vivo models will be used to investigate the vascular leakage and the inhibitory effect of the different compounds. The expression of B1, B2 and gc1q receptors will be evaluated on endothelial cells (HUVECs, human dermal and pulmonary microvasculature) by QPCR, FACS analysis and ELISA and the expression of these receptors in paraffin-embedded human tissue samples will be documented by IHC and IF. 10/07/ / 4

174 GR Novel therapeutic perspectives for angioedema associated with C1-inhibitor deficiency or angiotensin converting enzyme-inhibitor treatment Bossi Fleur Clinical health care research/clinicoassistenziale Ospedale infantile Burlo Garofolo Impact and Translational Implications AE causes significant personal and occupational disability and exposes patients to the risk of death. Because the pathogenic mechanisms are not completely understood, progress in diagnosis and treatment has been very slow. The results obtained from these studies will elucidate the factors involved in the onset of vascular permeability during the acute attacks, and will provide us a sound rationale to design therapeutic approaches for the prevention and treatment of the disease.

175 INDIVIDUAL PHENOTIPIC CHARACTERIZATION OF PATIENTS WITHIN THE FRONTOTEMPORAL LOBAR DEGENERATION/MOTOR NEURON DISEASE SPECTRUM USING MULTIMODAL MRI GR Canu Elisa Clinical health care research/clinicoassistenziale Ospedale San Raffaele - Milano LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Biobehavioral and Behavioral Processes Adult Psychopathology and Disorders of Aging - APDA Project Keyword 1: Project Keyword 2: Project Keyword 3: Disorders of Aging: Diagnosis, etiology, comorbidity, and course in deficits and disorders associated with aging, including dementia; mild cognitive impairment, Parkinson s disease, and Alzheimer s disease. FRONTOTEMPORAL LOBAR DEGENERATION MAGNETIC RESONANCE IMAGING Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Neurology/ Division of Neuroscience 2 Spedali Civili di Brescia Azienda Ospedaliera Dipartimento di Scienze Neurologiche e della Visione/ UO Neurologia 2 Principal investigator; Patient recruitment and evaluation; MRI analysis. Patient recruitment and evaluation Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Cosseddu Maura Spedali Civili di Brescia Azienda Ospedaliera 2 Patient recruitment and evaluation; MRI acquisition 14/10/ Agosta Federica Ospedale San Raffaele Milano/ Neuroimaging Research Unit/Neurologist (MD, PhD) Patient evaluation; MRI acquisition; MRI analysis 01/04/ /07/ / 6

176 INDIVIDUAL PHENOTIPIC CHARACTERIZATION OF PATIENTS WITHIN THE FRONTOTEMPORAL LOBAR DEGENERATION/MOTOR NEURON DISEASE SPECTRUM USING MULTIMODAL MRI GR Canu Elisa Clinical health care research/clinicoassistenziale Ospedale San Raffaele - Milano Background and Significance Frontotemporal lobar degeneration (FTLD) encompasses a wide spectrum of clinical syndromes ranging from the behavioral variant of frontotemporal dementia (bvftd) to primary progressive aphasia (PPA) and progressive supranuclear palsy (PSP). Motor neuron diseases (MND) share clinical and neuropathological features with FTLD-related syndromes. Distinguishing between FTLD phenotypes is important with the emergence of future therapies targeted to specific disease mechanisms. The integration of different MRI modalities (i.e., structural, diffusion tensor [DT] and resting state [RS] functional MRI) would allow to identify commonalities and differences of these syndromes. Structural and functional connectivity studies are emphasizing the idea that FTLD/MND represents a specific neural system disease. However, the results of these studies have had a minimal clinical impact since they reported results at group rather than at individual subject level. For MRI to be useful in the clinical setting, studies on large, independent, clinically and biologically wellcharacterized FTLD populations are needed in order to improve our understanding of the clinical heterogeneity of the disease and determine multimodal MRI markers to be applied for individual patient classification. Specific aims Aim 1: Aim 2: Aim 3: In two independent, previously recruited cohorts of FTLD/MND patients, to identify the patterns of GM atrophy, WM tract damage and functional connectivity alterations reflecting the FTLD/MND continuum, as well as the phenotype-specific brain signatures using the complementary information derived from 3D T1 images, DT MRI and RS fmri. To define common and distinct structural and functional MRI features in FTLD familial compared to sporadic cases to provide the in vivo patterns of damage occurring with different genetic backgrounds. To define a common MRI protocol to be applied in a multicenter, longitudinal setting in a large sample of new FTLD/MND cases in order to validate the MRI markers obtained at aims 1 and 2 and to determine their accuracy in classifying different clinical syndromes and predict disease progression. Hypothesis: We hypothesize that the frontal cortex involvement, together with its main WM connections, would represent the common target of neurodegeneration in the FTLD/MND spectrum. Our multimodal MRI approach would also allow to define the MRI signatures responsible for the different clinical manifestations in each phenotype. Finally, structural and functional MRI features defined in independent FTLD populations would provide a useful classification tool for discriminating new FTLD cases and predicting their progression. Preliminary data: UO1 is extensively applying MRI to the study of FTLD. Using structural and DT MRI, GM and WM damage were explored in patients with bvftd, PPA (Brain 2009,2011; Cereb Cortex 2012), PSP (J Neurol 2014; NBA 2012; Mov Disord 2011a,b; Eur J Neurosci 2010a) and ALS (ALS 2009; JNNP 2009; AJNR 2010,2011a,b; HBM 2007,2014; Eur J Neurosci 2010b; PLosOne 2012,2013). These findings suggested that a combination of GM and WM MRI measures improve the characterization of FTLD phenotypes. In addition, UO1 made an internationally-recognized contribution to the in vivo demonstration that ALS is a multisystem disease associated not only with GM and WM damage within the motor network, but also with a widespread extramotor involvement in relation with cognitive deficits. Using RS fmri, UO1 also showed that: functional network alterations occur in bvftd (Neurology 2013; Cortex 2013) and PPA (NBA 2014a); ALS is characterized by altered RS functional connectivity within the motor network (Cereb Cortex 2011; NBA 2014b), as well as cognitive-related networks with a pattern similar to that observed in bvftd (NBA 2013a,2014b). The UO2 is involved in national and international multicentre studies aimed at the identification of novel genetic risk loci associated with FTLD (Lancet Neurol 2014) and at investigating the effect of known 11/07/ / 6

177 INDIVIDUAL PHENOTIPIC CHARACTERIZATION OF PATIENTS WITHIN THE FRONTOTEMPORAL LOBAR DEGENERATION/MOTOR NEURON DISEASE SPECTRUM USING MULTIMODAL MRI GR Canu Elisa Clinical health care research/clinicoassistenziale Ospedale San Raffaele - Milano and recently discovered FTLD-linked genes on clinical (NBA 2014c,d,e) and pathological (Acta Neuropathol 2014) phenotypic variability, as well as brain structure (Dement Geriatr Cogn Dis Extra 2014; NBA 2013b) and functional connectivity (J Nucl Med 2013; JAMA Neurol 2014). Materials and Methods Samples: Retrospective. MRI scans have been already obtained from two independent patient cohorts: 1) UO1: 20 MND, 20 MND-plus, 20 bvftd, 15 semantic variant (sv)ppa, 15 nonfluent variant (nfv)ppa, 20 PSP, 40 healthy controls; 2) UO2: 80 bvftd, 10 svppa, 30 nfvppa, 25 PSP, 35 FTLD familial cases, 40 healthy controls. Prospective. MRI scans will be obtained at study entry and every year for 3 years from: 1) UO1: same sample size as retrospective study; 2) UO2: 40 bvftd, 15 svppa, 15 nfvppa, 30 PSP, 40 healthy controls. Clinical evaluations (both retrospective and prospective samples): neurological, neuropsychological and behavioral examinations. Genetic study: GRN, MAPT, C9ORF72, SQSTM1, TMEM106B, TREM2. MRI: 3D T1, DT MRI and RS fmri to obtain patterns of cortical thickness/volume, WM tract damage, and RS functional connectivity using state of the art computational brain MRI tools (Freesurfer,SPM,FSL). Support vector machine and random forest analyses will be used to classify MRI data. Impact and Translational Implications The MRI protocol proposed in this study could be useful for new cases classification with a potentially high level of clinical translation. The intense search for diagnostic, prognostic and monitoring markers of the FTLD/MND continuum may also provide evidence for disease mechanisms that might aid the discovery of novel targets for therapeutic intervention and for testing pharmacological treatments that are being developed. 11/07/ / 6

178 GR "THE ROLE OF 124-IODINE/CG250 PET IMAGING TECHNIQUE IN DETECTING LYMPH NODE METASTASES IN RENAL CELL CARCINOMA PATIENTS" capitanio umberto Clinical health care research/clinicoassistenziale Ospedale San Raffaele - Milano LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Surgical Sciences, Biomedical Imaging, and Bioengineering Medical Imaging - MEDI Project Keyword 1: Project Keyword 2: Project Keyword 3: In vivo strategies and methods for characterizing tissue, and distinguishing between normal and pathologic states, based on estimates of biophysical, biomechanical, bioelectrical, biochemical, metabolic, perfusion/diffusion, or other properties. renal cell carcinoma lymph node invasion Project Request: Animals: Humans: Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Urology and Nuclear Medicine Principal Investigator (Urology) and Support Unit (Nuclear Medicine) Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 08/07/ / 4

179 GR "THE ROLE OF 124-IODINE/CG250 PET IMAGING TECHNIQUE IN DETECTING LYMPH NODE METASTASES IN RENAL CELL CARCINOMA PATIENTS" capitanio umberto Clinical health care research/clinicoassistenziale Ospedale San Raffaele - Milano Background and Significance Renal cell carcinoma (RCC) represents the third most frequent cancer in urological setting, after bladder and prostate tumors. In RCC patients, lymph node metastases detection and treatment are, to-date, the most critical issues in daily clinical decision-making. Indeed, conversely to other oncological settings, a) nodal status imaging, b) sentinel node technique and c) standard lymphadenectomy have been demonstrated inadequate in the staging and management of patients with renal cancer. Many of those difficulties resulted from several peculiarities regarding anatomy, pathophysiology and natural history of renal cancers. All the aforementioned aspects make of crucial importance the development of new imaging modality, which might accurately identify those patients who are at higher risk of having nodal metastases, to accurately plan the best management. As regards novel imaging techniques in RCC setting, a recent study suggested combining 124-Iodine PET with CT scanning in the detection of labelled antibodies against the carbonic anhydrase IX (CAIX) antigen, which has been recently proposed as a promising marker in the diagnostic process. In conclusion, a novel, accurate, standardized imaging technique is urgently needed in RCC setting to detect macro and micro nodal invasion. We propose to test 124-Iodine PET with CT scanning in detecting nodal metastases to fill such a crucial gap in the management of patients with renal cancer. Specific aims Aim 1: -To test the performance of 124-Iodine/cG250 PET technique in detecting nodal metastases in RCC patients. Aim 2: -To confirm and externally validate the role of 124-Iodine/cG250 PET technique in detecting clear cell RCC. Aim 3: -To explore the potential role of 124-Iodine/cG250 PET technique in detecting distant RCC localization (bone and lung metastases, etc). Hypothesis: 124-Iodine/cG250 PET technique is expected to improve the limited performance of available technique imaging in detecting and staging lymph node metastases in RCC setting. Preliminary data: Antibody cg250 reacts against carbonic anhydrase-ix, which has been demonstrated to be overexpressed in clear-cell RCC. CAIX is ubiquitously expressed in more than 90% of clear-cell RCC (ccrcc) but not in normal kidney. In addition, high expression of CAIX appears to be a marker of poor prognosis in ccrcc. The antibody G250 has been studied in ccrcc both as a murine antibody and a chimeric antibody (cg250). Materials and Methods Twenty patients candidates to radical nephrectomy and extended lymphadenectomy for clinical T4 cancers or renal masses with evidence of lymphadenopathies at preoperative CT scan or larger tumor (diameter>10 cm) will be included. As previously described, RCC candidates to surgery will receive a single intravenous infusion of 185 MBq/10 mg of 124IcG250 over 20 min. Surgery will be scheduled within 1 week after infusion. PET and CT scanning of the abdomen will be planned before surgery (nephrectomy plus extended lymphadenectomy). Relying on final pathological examination, accuracy, sensibility and sensibility, negative and positive predictive value of 124-Iodine/cG250 PET technique in detecting lymph node metastases will be calculated. 08/07/ / 4

180 GR "THE ROLE OF 124-IODINE/CG250 PET IMAGING TECHNIQUE IN DETECTING LYMPH NODE METASTASES IN RENAL CELL CARCINOMA PATIENTS" capitanio umberto Clinical health care research/clinicoassistenziale Ospedale San Raffaele - Milano Impact and Translational Implications The effect of the study is thus represented by a significant reduction on the costs related to a) accurate prognosis and surgical planning; b) avoiding of unnecessary lymphadenectomy and lymphadenectomy-related surgical sequelae c) programming of a cost-effective follow-up and potential benefit from targeted adjuvant therapies. RCC represent the third most frequent urological cancer and those direct and indirect cost effects will be beneficial for thousand RCC patients yearly.

181 GR Diagnostic accuracy and cost-effectiveness of Next Generation Sequencing (NGS) strategies in the genetic testing of Rare Orthopaedic Diseases Pedrini Elena Clinical health care research/clinicoassistenziale Istituto Ortopedico Rizzoli LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Methodological, epidemiological, socio-economic, organizational, managerial emerging public health issues related to the above reported areas Project Classification IRG: Project Classification SS: Genes, Genomes and Genetics Genomics, Computational Biology and Technology - GCAT Project Keyword 1: Project Keyword 2: Project Keyword 3: Generation, analysis, and mining of large data sets: genetic, epigenetic, biochemical, gene expression, metabolic, proteomic, microarrays, genome sequencing, comparative genomics. Next Generation sequencing Health Technology Assessment Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Medical Genetics Recruitment of patients and molecular screening (either with NGS technologies and standard techniques). 2 Regione Emilia Romagna Agenzia Sanitaria e Sociale Regionale, Regione Emilia-Romagna - ASSR RER /Osservatorio Regionale per l'innovazione - ORI Investigators, Institution and Role in the Project Health Technology Assessment analysis Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 trimaglio fabio Regione Emilia Romagna HTA analysis 06/11/ Ponti Francesca IRCCS - ISTITUTO ORTOPEDICO RIZZOLI/Department of Medical Genetics/Biologist NGS analysis and HRM-Sanger Sequencing analyses in Ehlers Danlos genetic testing 19/02/ caputo fabio Regione Emilia Romagna HTA analysis 29/01/ /07/ / 6

182 GR Diagnostic accuracy and cost-effectiveness of Next Generation Sequencing (NGS) strategies in the genetic testing of Rare Orthopaedic Diseases Pedrini Elena Clinical health care research/clinicoassistenziale Istituto Ortopedico Rizzoli Background and Significance Traditionally, Sanger sequencing has been considered the gold standard in mutation detection and it is still the method of choice for most diagnostic labs. However, this technology is not ideal for the screening of large sets of genes involved in molecularly heterogeneous disorders, including inherited orthopedic rare diseases. The coming of Next Generation Sequencing (NGS) technologies leads to the ability to analyze simultaneously a large amount of sequences, with documented advantages and reduced costs respect to Sanger sequencing, providing then new appealing approaches for diagnostic testing. Compared to the necessity in Sanger method to obtain a single homogeneous DNA fragment per each sequencing reaction, NGS procedures can provide the opportunity for parallel reactions. To make genetic testing faster, Department of Medical Genetics (IOR, Bologna) adopted the Ion Torrent NGS Technology, based on a semiconductor chip which would result in a simple, fast and cost-effective sequencing approach compared to both Sanger sequencing and other available technologies. Despite this documented technical improvement, its use for routine diagnostic purposes requires specific certification in terms of reliability, as well as an economic evaluation; if its accuracy in mutation detection will be confirmed, as well as its cost-effectiveness, Ion Torrent PGM platform could become the first approach in genetic testing, demonstrating its clinical utility to diagnose rare genetic diseases Specific aims Aim 1: Aim 2: Aim 3: Evaluation of diagnostic reliability: targeted next generation sequencing with PGM Technology will be performed to explore the accuracy of this new technique to identify disease-causing mutations in patients affected by Multiple Osteochondromas, Osteogenesis Imperfecta and Ehlers-Danlos Syndromes. HTA analysis: it is performed to evaluate the real effectiveness of medical interventions, their proper use, access criteria and qualitative improvements, the clinical and organizational benefits, therefore suggesting how to manage, promote and discourage them. Evaluate the pertinence for routine molecular diagnosis. Hypothesis: With the advances in next generation sequencing (NGS) technologies, the ability to analyze simultaneously a large amount of sequences provides new appealing approaches for the diagnostic testing however, before using NGS platform for routine diagnosis purposes, an accuracy validation is required in a high number of samples. To test the feasibility of using the Ion Torrent Personal Genome Machine (PGM) in clinical diagnosis, we will assess its performance to detect point mutations and big rearrangements previously identified with standard techniques (Sanger Sequencing, HRM, DHPLC, MLPA) in 600 patients affected by Multiple Osteochondromas, Osteogenesis Imperfecta and Ehlers Danlos Syndrome. Results comparison obtained by NGS platform and standard methods will grant us to evaluate the diagnostic reliability of the new technique, considering either sensitivity and specificity. To demonstrate the pertinence of the use of this innovative technology in health care, we will also consider economic, medical and social implications according with Health Technology Assessment (HTA), a multidisciplinary field of policy analysis applied to many different health care technologies before their diffusion and use. Preliminary data: Genetic testing in MO patients To perform molecular screening in MO patients we investigated the presence of mutations in either EXT1 and EXT2 genes using a DHPLC/MLPA multistep combined screening protocol; in case of negative results we looked for the presence of big deletion/insertion with MLPA technique. Using this molecular screening more than 1000 MO affected patients have already been analyzed. 10/07/ / 6

183 GR Diagnostic accuracy and cost-effectiveness of Next Generation Sequencing (NGS) strategies in the genetic testing of Rare Orthopaedic Diseases Pedrini Elena Clinical health care research/clinicoassistenziale Istituto Ortopedico Rizzoli Genetic testing in OI patients Diagnostic protocol used in our unit to detect autosomal dominant defects in COL1A1 or COL1A2 genes is based on HRM which permit the identification of either point mutations and copy number variations in the same reaction. Department of Medical Genetica (IOR) have already analyzed more than 500 OI affected patients.recessive autosomal defects were investigated in 20 selected patients analyzing CRTAP, LEPRE1 and PPIB genes by direct sequencing. Genetic testing in EDS patients Diagnostic protocol for mutational screening in EDS patients consists in HRM/Sanger Sequencing analysis of Col5A1/A2 and Col3A1, depending on the clinical features. More than 200 patients have already been analyzed by our genetic unit. Materials and Methods 600 selected subjects previously screened for the causative mutation using DHPLC, HRM, Sanger sequencing techniques and MLPA will be re-analyzed using the Ion Torrent PGM platform. 4 custom panels will be created by the Ion AmpliSeq Custom Designer and designed to cover UTR and coding regions for MO, genes for dominantly inherited OI, those related to recessive OI forms and genes related to the dominant inherited ED. Results obtained will be then compared to evaluate sensitivity and the specificity. An HTA analysis will be carried out to assess value of NGS in clinical practice, combining data from the diagnostic accuracy study with data from available literature. Analytical an clinical validity, clinical utility and cost effectiveness of the new technology compared with standard gene sequencing will be analyzed. Outcomes will include: sensitivity and specificity; failure rate, turnaround time and disease specific clinical outcomes; costs for testing and treatment. Impact and Translational Implications If validated in feasibility, the Ion Torrent PGM will enable a strong reduction in time and cost of genetic testing, resulting in faster diagnosis and better clinical management, making it the method of choice in clinical laboratories. In addition to enabling the identification of causative mutations for rare orthopedic genetic disorders, the clinical use of this technology with its broadest applications - will offer the possibility to enhance genetic understanding of skeletal diseases. 10/07/ / 6

184 The Stem cell secretome for doxorubicin-induced Cardiomyopathy REgeneraTion (SECRET) BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Tasso Roberta Biomedical/Biomedica Azienda ospedaliera universitaria San Martino - IST - Istituto nazionale per la ricerca sul cancro LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Cell Biology Intercellular Interactions - ICI Project Keyword 1: Project Keyword 2: Project Keyword 3: Cell migration, cell adhesion, cell organization and morphogenesis as related to tissue organization and development including stems cells and tumor cells. Cardiotoxicity chemotherapy Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Experimental Medicine / Laboratory of Regenerative Medicine Study of the cardioprotective potential of the stem cell secretome. 2 IRCCS AOU San Martino - IST, Genova Department of Internal Medicine (DiMI) / Unit of Cardiovascular Diseases Translation of the results to the animal model. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Ameri Pietro IRCCS AOU San Martino - IST, Genova 2 In vivo validation of experimental results. 31/07/ Bollini Sveva IRCCS AOU San Martino - IST, Genova Analysis of adult stem cells secretome regenerative potential on cardiac progenitor cells. 15/06/ /07/ / 6

185 The Stem cell secretome for doxorubicin-induced Cardiomyopathy REgeneraTion (SECRET) BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Tasso Roberta Azienda ospedaliera universitaria San Martino - IST - Istituto nazionale per la ricerca sul cancro Background and Significance Doxorubicin is an effective antineoplastic agent used for the treatment of a variety of cancers, including common tumors such as lymphoma and breast cancer. Unfortunately, it damages the heart in a considerable proportion of patients. Doxorubicin-induced cardiotoxicity most often results in a chronic cardiomyopathy (DIC - Doxorubicin-Induced Cardiomyopathy) which typically occurs several months or even years after treatment. Despite the numerous efforts made to address this problem, DIC remains a major health concern for oncological patients treated with doxorubicin. Accurate identification of novel therapeutic strategies is still highly demanded. Although the precise mechanism of DIC is still unclear, strong evidence suggests that cardiomyocyte and cardiac progenitor cells (CPC) senescence and apoptosis play a major role in the development and progression of the disease. A large number of studies have recently shown that mesenchymal stem cells (MSC) can mediate cardiac repair and regeneration through the modulation of the microenvironment, by inhibiting fibrosis, stimulating proliferation and activation of CPC, sustaining angiogenesis via the secretion of specific bioactive factors. This modality of action is defined as paracrine effect and the composite set of the cytokines, chemokines and growth/trophic factors orchestrating biological activities, secreted from the stem cells or shed into microvesicles (MV) or exosomes from their membranes, is here defined as secretome. Specific aims Aim 1: Aim 2: Aim 3: To define the paracrine effect of the human adult mesenchymal stem cells (MSC) secretome. To determine whether the human MSC secretome protects both the terminally differentiated cardiovascular cells and the CPC against doxorubicin toxicity in vitro. To determine whether the human MSC secretome is protective against doxorubicin cardiotoxicity in vivo. Hypothesis: We propose to exploit the stem cells paracrine potential to counteract doxorubicin cardiotoxicity and prevent/inhibit DIC. Despite recent literature has showed growing interest towards the therapeutic application of stem cells for the treatment of specific heart disease, little is known on the molecular aspects involved in this process and even less on the effect of the adult stem cells secretome on DIC. Human adult MSC have been widely described as an appealing source of trophic factors with tremendous pro-survival and stimulatory effects on both mature cardiovascular cells and CPC, especially following ischemic injury. Hence, given that resident cardiomyocytes and CPC represent the cellular target responsible for DIC, we want to investigate the regenerative potential of the human adipose tissue-derived mesenchymal stem cells (hasc) secretome on these cells, in order to define a novel approach for future paracrine therapy of DIC. To contrast the toxic effects of the doxorubicin on the cardiac tissue, we aim at harnessing the hasc paracrine potential to specifically achieve two distinct results: i) preventing mature cardiomyocyte and cardiovascular cell apoptosis and senescence, in order to secure cardiac function and maintain tissue viability following chemotherapy and ii) preserving the resident endogenous reservoir of CPC, by inhibiting their senescence to protect their functional properties, together with their regenerative potential in orchestrating the endogenous cardiac repair programme. We believe that this approach will cover several significant scientific, technological and clinical aspects, representing a more translational model to obtain stem cell-mediated regenerative effects but via cell-free delivery of paracrine soluble molecules/mvs. Preliminary data: Our preliminary data based on pilot in vitro viability studies are indicating that the secretome of hasc can decrease doxorubicin-induced cardiomyocyte senescence and apoptosis of about 50% and 80% 10/07/ / 6

186 The Stem cell secretome for doxorubicin-induced Cardiomyopathy REgeneraTion (SECRET) BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Tasso Roberta Azienda ospedaliera universitaria San Martino - IST - Istituto nazionale per la ricerca sul cancro respectively. These results are currently being validated in order to better define the key regulatory element driving the cardioprotective effect within the hasc paracrine potential. A detailed analysis of both the total hasc-conditioned medium and the hasc-derived microvesicles (MVs) is presently on going. Given the growing interest towards stem cell-derived MVs as a specific tool in paracrine intercellular communication, we have developed and optimized specific isolating protocols to characterise hasc-mvs by flow cytometry, transmission electron microscopy and molecular analyses, in order to analyse their role as carrier of paracrine factors. Materials and Methods WORK PACKAGE 1 (WP1) In vitro analysis of human adult adipose tissue-derived MSC (hasc) secretome by: - Characterisation of hasc in different culture conditions; - Comprehensive proteomic, genomic and functional analysis of the hasc conditioned medium and MVs. WORK PACKAGE 2 (WP2) In vitro assessment of the hasc secretome to: - Antagonize the senescence and apoptosis of cardiomyocytes induced by doxorubicin; - Preserve the endogenous pool of local CPC exposed to doxorubicin. WORK PACKAGE 3 (WP3) In vivo assessment of the hasc secretome to: - Inhibit the apoptosis of cardiomyocytes and cardiac vascular cells and stimulates CPC regenerative activity after exposure to doxorubicin; - Inhibit the structural changes and the impairment of cardiac geometry and function caused by doxorubicin, by histological analysis and echocardiography. Experimental model: adult C57Bl/6J and nu/nu mice treated for a week with 4 mg/kg doxorubicin every 48h and the hasc secretome in between treatments. Impact and Translational Implications This project represents a timely and novel clinically oriented study to provide a future therapeutic approach for the cancer survivors suffering from a dramatic side effect of doxorubicin-based chemotherapy with devastating consequences on the patients life quality and expectancy. By investigating a treatment based on the stem cells secretome, the present study may also provide the basis for the application of a new form of stem cell therapy to the clinical setting. 10/07/ / 6

187 GR Upper limb impairment in Charcot-Marie-Tooth patients: evaluation of motor strategies and effectiveness of rehabilitation treatments by means of an innovative instrumental protocol. Pazzaglia Costanza Clinical health care research/clinicoassistenziale Fondazione Don Carlo Gnocchi LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Musculoskeletal, Oral and Skin Sciences Musculoskeletal Rehabilitation Sciences - MRS Project Keyword 1: Project Keyword 2: Project Keyword 3: Mechanisms of exercise in relation to disability. Charcot-Marie-Tooth upper limb impairment Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Neurorehabilitation Patient identification and recruitment. Movement analysis assessment. Standard and robotic rehabilitation. Results Analysis. 2 Fondazione IRCCS Istituto Neurologico Carlo Besta 3 IRCCS Azienda Ospedaliera San Martino IST Investigators, Institution and Role in the Project Department Of Clinical Neurosciences/(SOSD) Central and Peripheral Degenerative Neuropathies Clinic Department of Neurosciences Patient identification and recruitment. Movement analysis assessment. Standard and robotic rehabilitation. Patient identification and recruitment. Movement analysis assessment. Standard and robotic rehabilitation. Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Piscosquito Giuseppe Fondazione IRCCS Istituto Neurologico Carlo Besta 2 MORI LAURA IRCCS Azienda Ospedaliera San Martino IST Study design, patients recruitment and analysis of results Study design, patients recruitment and analysis of results 05/03/ /11/ /07/ / 6

188 GR Upper limb impairment in Charcot-Marie-Tooth patients: evaluation of motor strategies and effectiveness of rehabilitation treatments by means of an innovative instrumental protocol. Pazzaglia Costanza Clinical health care research/clinicoassistenziale Fondazione Don Carlo Gnocchi Background and Significance Charcot-Marie-Tooth (CMT) disease is a group of inherited sensorimotor neuropathies characterized by loss of motor and sensory function of the distal limbs. The most part of the studies concern the distal lower limb function, although recently Researches investigated also the spared function of proximal segment in order to assess possible motor strategies aimed to recover the distal loss of function (Ferrarin et al., 2011). This approach could give important information also for the upper limb function that according to a recent Italian multicenter study (Padua et al., 2014), focused on rehabilitation and patients needs, stated that also the disability of upper limb function has a great impact on daily and working life of CMT patients. Despite of attempt of studies on drug therapy (Pareyson at al., 2011) currently rehabilitation is the only available treatment, but the optimal approach remains unknown. This happens both because researchers are still looking for the best standardized rehabilitation program both because there is a lack of monitoring tools for detecting short term changes. Being CMT a rare disease recently multicenter studies were performed in order to acquire consistent data. For this reason the Italian CMT study group was formed in All units participating to the current proposal are part of the Italian CMT study group. Specific aims Aim 1: Aim 2: Aim 3: To evaluate the prevalence of upper limb disability in CMT patients (with previously validated multidimensional assessment tools and a sensor engineered glove), differences among CMT subtypes, and impact on patient s Quality of Life. To describe the upper limb motor pattern in CMT patients by developing an ad-hoc instrumental protocol (with an optoelectronic system, a surface electromyography and sensor engineered glove); To evaluate possible clinical and instrumental changes in upper limb disability and motor patterns after traditional and robotic rehabilitation therapy. Hypothesis: Upper limb disability causes significant morbidity in CMT. Knowledge of adaptive arm motor patterns will lead to development of tailored rehabilitative strategies. Preliminary data: Ninety-eight percent of CMT1A patients report limitations in upper limb functioning, particularly affecting the dominant hand (Videler et al, 2009). They perceive impairment in work, family roles, and outdoor activities. Last year, we performed a pilot study to evaluate upper limb disability, using a novel sensor engineered glove to quantify motor function. This new device's suitability for detecting hand dysfunction in CMT patients as compared to healthy controls was assessed. Twenty-six patients (18 females, 8 males with mean age 49.8 ±14.5 years, range: 20-80, mean CMTNS ±4.9) participated, most (88%) with a molecular diagnosis of CMT1a. Eight dominant hand glove protocols were analyzed and compared between CMT patients and healthy controls. The glove protocol that best differentiated the groups was a sequence of oppositions of thumb to index, medium, ring and little fingers (SEQ). SEQ discriminated all CMT patients from controls and further differentiated CMT patients with and without hand involvement as detected by the Charcot-Marie-Tooth Neuropathy score (CMTNS). Some nerve conduction study parameters also correlated with SEQ: the ulnar and median nerve distal motor latencies correlated with touch duration (p= and p= respectively). No significant correlation existed between SEQ results and testing with the 9-hole peg test (9HPT) or dynamometer. 9HPT and dynamometry testing were unable to differentiate between patients with and without hand involvement. In conclusion the pilot study showed SEQ obtained from the sensory engineered glove was promising 10/07/ / 6

189 GR Upper limb impairment in Charcot-Marie-Tooth patients: evaluation of motor strategies and effectiveness of rehabilitation treatments by means of an innovative instrumental protocol. Pazzaglia Costanza Clinical health care research/clinicoassistenziale Fondazione Don Carlo Gnocchi for assessing hand function, providing accurate quantitative spatio-temporal data on finger opposition movements. Materials and Methods Aim 1: 75 CMT patients will undergo: 1) validated patient-oriented questionnaire (DASH), 2) SEQ testing with sensor engineered glove (Bove M et al., 2007), 3) MRC muscle strength and hand grip dynamometry, 4) evaluation of sensory loss, 5) 9-HPT, 6) CMTNS (Murphy et al 2011), 7) Quality of Life questionnaire (SF36), 8) Actigraph unit, 9) Sollerman test Aim 2: 75 CMT patients in Rome, Genoa and Milan, and 50 age-matched controls will have kinematic (motion capture system) and functional (surface electromyography) assessment of pointing, reaching and grasping, and writing tasks. Patients will perform the instrumental protocol in a follow-up visit. Aim 3: All CMT patients will ask to perform traditional and arm robotic rehabilitation for 6 months. All patients who will refuse will be considered for natural course and re-evaluated in a follow-up visit (after 6 months). Possible changes will be assessed by using Aim 1 and Aim 2 outcome measures. Impact and Translational Implications Quantification of upper extremity needs, disabilities, motor adaptive strategies is essential for developing tailored upper limb rehabilitation for CMT patients. Appropriate rehabilitation therapy may also improve patient Quality of Life and reduce Social and Health costs. 10/07/ / 6

190 GR Deep genetic and phenotypic characterization of Autism Spectrum Disorder (ASD) families: analysis of the nuclear and mitochondrial genome Rochat Magali Jane Biomedical/Biomedica Istituto delle Scienze Neurologiche - Bologna LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Genes, Genomes and Genetics Genetics of Health and Disease - GHD Project Keyword 1: Project Keyword 2: Project Keyword 3: Discovery of genes and genetic variation for human health, disease, and disease susceptibility: Complex and Mendelian diseases such as psychiatric, neurological, ophthalmological, auditory, endocrinological, cardiovascular, developmental, reproductive, oncological, autoimmune, urological, respiratory; use of sophisticated genetic and genomic methods to identify candidate genes, single nucleotide polymorphisms, haplotypes, and copy number variation. Autism Spectrum Disorder Mitochondrial DNA Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION UOC Neuropsichiatria Infantile PI, Recruitment, diagnosis and deep phenotypic characterization of Autism Spectrum Disorder families and deep phenotyping 2 IRCCS Istituto delle Scienze Neurologiche di Bologna UOC Clinica Neurologica, Laboratorio di Neurogenetica Genetic studies: deep sequencing of the mitochondrial genome and whole exome sequencing 3 Università di Bologna Dipartimento di Biotecnologie (FaBiT) Genetic analysis: SNP microarray and CNV studies, analysis of exome sequencing data Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Maresca Alessandra IRCCS Istituto delle Scienze Neurologiche di Bologna Coordinator of RU2, Genetic studies: deep sequencing of the mitochondrial genome and whole exome sequencing 18/10/ Bacchelli Elena Università di Bologna Coordinator of RU3, Genetic analysis: SNP microarray and CNV studies, analysis of exome sequencing data 16/06/ /07/ / 6

191 GR Deep genetic and phenotypic characterization of Autism Spectrum Disorder (ASD) families: analysis of the nuclear and mitochondrial genome Rochat Magali Jane Biomedical/Biomedica Istituto delle Scienze Neurologiche - Bologna Background and Significance Autism spectrum disorders (ASD) are complex neuro-developmental disorders characterized by deficits in social interaction, communication, and repetitive behaviors. Despite the strong genetic component, most cases still remain of unknown etiology, with devastating consequences on both prognosis and health-care assistance. While linkage and genome-wide association studies have failed to identify specific common variants with a substantial impact on risk, whole genome microarray studies and exome sequencing (WES) have demonstrated that a substantial proportion of ASD risk resides in rare variants, either submicroscopic structural variants (Copy Number Variants, CNVs) or single nucleotide variants (SNVs) which alter a broad and diverse pool of genes, highlighting a highly heterogeneous genomic landscape. A large number of genes have already been causally associated to ASD, but, according to recent estimates, many more still need to be discovered. Recent studies have highlighted the potential role of mitochondrial dysfunction in ASD. However, only scattered information is available to date, and a systematic mtdna analysis in ASD is still lacking. We propose to apply high-throughput technologies (dense SNP microarrays and WES) to detect potentially pathogenic CNVs and SNVs, and to perform deep mitochondrial genome analysis, with the aim of further expanding our knowledge through the identification of new genes and genetic mechanisms involved in ASD pathogenesis. Specific aims Aim 1: Recruitment, diagnosis and deep phenotypic characterization of 100 families with ASD (~25 multiplex and ~75 simplex families). This will allow to identify possible correlations with deep genetic data obtained through Aim 2 and 3. Aim 2: Aim 3: To perform deep sequencing of the entire human mtdna genome in ASD individuals and in a control group. This approach will allow to identify rare variants and to evaluate the burden of heteroplasmic variants possibly distinguishing ASD from controls. To carry out genome-wide analysis by DNA microarrays and WES in ASD families. Genotyping all family members using a high-resolution SNP array will allow to scan the whole genome for structural variation, thus identifying potentially pathogenic CNVs. Exome sequencing will lead to the detection of rare SNVs with a possible impact on disease. The most promising identified CNVs and SNVs will be validated experimentally and a potential association with clinical features will be investigated. Hypothesis: Despite high heritability of ASD, its genetic etiology remains largely unknown. It is becoming increasingly apparent that rare high-impact genomic variation, both CNVs and point mutations, have a major role in the pathogenesis of ASD. Given the extreme genetic heterogeneity of ASD, high-resolution genome-wide approaches such as microarray and next generation sequencing, are warranted for the identification of new rare variants and new genes implicated in ASD. Mitochondrial dysfunction represents an emerging mechanism possibly associated to ASD development, suggesting that mitochondrial variants and the burden of inherited heteroplasmy could play a role in ASD susceptibility and/or in the occurrence of specific endophenotypic features. NGS technologies represent the highest resolution approach to evaluate low frequency mtdna variants and their segregation along the maternal lineages. 10/07/ / 6

192 GR Deep genetic and phenotypic characterization of Autism Spectrum Disorder (ASD) families: analysis of the nuclear and mitochondrial genome Rochat Magali Jane Biomedical/Biomedica Istituto delle Scienze Neurologiche - Bologna Preliminary data: RU1 is a nationally accredited center for ASD, with a longstanding experience (25 years) in diagnosis, neurobiological assessment and treatment of ASD, and with links to parents' association. More than 100 families are annually assessed by RU1 for diagnostic protocol or follow up evaluations. As part of the Autism Genome Project consortium, our previous studies (RU3) reinforced the role of rare CNVs and SNVs as an important source of ASD risk, thus supporting the proposed strategy to investigate rare variants in both the nuclear and the mitochondrial genome. RU2 has a consistent experience in studying mtdna genetics in relation to mitochondrial biology and diseases. Moreover, through the Autism Genome Project consortium, we will have access to large international collaborations that will be crucial for interpretation, replication and follow-up of the results. Materials and Methods ASD diagnosis (DSM-5 criteria) and phenotypic aspects of behaviour will be assessed through a set of internationally recognized tools focusing on discrete and basic clinical signs. This approach will allow to cluster signs and patients into specific clinical subgroups. Deep sequencing of mtdna will be carried out with 10000x coverage and using the Illumina MiSeq instrument, available at the Laboratory of Neurogenetics (RU2). WES on ASD probands will be performed using the Illumina HiSeq2500 platform. The appropriate filtering strategies will be applied in order to identify variants possibly involved in ASD, and to generate a list of candidate genes. CNV analysis will be performed on all ASD probands and available parents, using the HumanOmniExpress chip (Illumina), that includes > genome-wide SNPs. Regions of copy number variation will be detected using two CNV calling algorithms, namely QuantiSNP and PennCNV, to obtain high-confidence call. Impact and Translational Implications The identification of genetic mechanisms involved in ASD has important translational implications: a) achieving a molecular diagnosis in a larger number of cases can improve genetic counselling b) identifying genetic risk factors will allow increased surveillance and early intervention in younger siblings sharing similar genetic factors; c) understanding the molecular pathways involved in ASD is a crucial first step to develop drug treatments for the "core" autism symptoms. 10/07/ / 6

193 GR In vivo evaluation of the process of Epithelial-Mesenchymal and Mesenchymal-Epithelial transition (EMT-MET) in Hepatocellular Carcinoma (HCC) dissemination and metastasization. Colombo Federico Simone Biomedical/Biomedica Fondazione Ca'Granda - Ospedale Maggiore Policlinico LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Oncology 1 - Basic Translational Tumor Progression and Metastasis - TPM2 Project Keyword 1: Project Keyword 2: Project Keyword 3: In vitro and in vivo models of tumor metastasis and angiogenesis including genetics and imaging analysis Hepatocellular carcinoma Epithelial mesenchymal transition Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Central Laboratory of Chimico-Clinical analyses and Microbiology/Flow Cytometry Service - Experimental hepatology laboratory Project coordination, experimental planning, in vitro cell culture experiment, mirna expression and in vivo tumorigenesis and metastasization experiments' execution. Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 10/07/ / 6

194 GR In vivo evaluation of the process of Epithelial-Mesenchymal and Mesenchymal-Epithelial transition (EMT-MET) in Hepatocellular Carcinoma (HCC) dissemination and metastasization. Colombo Federico Simone Biomedical/Biomedica Fondazione Ca'Granda - Ospedale Maggiore Policlinico Background and Significance Due to its chemoresistance and high rate of recurrence and metastasization, hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Many efforts have been made to contrast HCC progression and metastasization but the biological and molecular mechanisms underlying this phenomenon is still largely unknown. Innovative therapies are now being designed to hit new therapeutic targets such as the epithelial to mesenchymal transition (EMT). EMT, mainly studied in vitro, involves the loss of cell polarity and a cytoskeletal reorganization ending with the change from an epithelial differentiated morphology to a fibroblast-like, motile, and invasive cell behavior. Nevertheless, growing evidence seems to suggest that the maintenance of an epithelial phenotype will promote aggressiveness and stemness of cancer and even facilitate its dissemination. Moreover, pathologists hardly find signs of EMT in anatomical specimens and usually metastases, both in animal models and humans, show the phenotype and morphology of the primary tumor and not a mesenchymal phenotype. With our project we want to define what is the in vivo role of EMT in HCC metastasization and to investigate which mirna/mrna are modified during the EMT process. The establishment of new therapeutic strategies targeting HCC metastasization is of primary importance to ameliorate patients outcome, thus unraveling the biological mechanisms underlying this process is mandatory. Specific aims Aim 1: Aim 2: Aim 3: to generate two fluorescent HCC cell lines with epithelial (green) and mesenchymal (red) characteristics for the evaluation of in vitro EMT-Mesenchymal to Epithelial Transition (MET) process. to investigate the in vivo role of EMT and MET in HCC tumor and metastases after HCC cell lines transplantation in a mouse model by intravital microscopy. to identify which mirna/mrna are involved during the in vivo tumor formation and metastasization process. Hypothesis: Although in vitro experiments indicated EMT as a major driver in tumor metastasization, pathologists usually do not find EMT signs in diagnostic specimens, neither in the primary tumor nor in the metastatic lesions. To overcome this discrepancy, researchers proposed the reversion of EMT by cancer cells after metastasization, i.e. the MET, but in vivo evidences are still lacking. Our project is aimed at evaluating EMT role in HCC metastasization in a mouse model. We will start from the generation of two HCC cell lines stably expressing the fluorescent proteins GFP and mcherry under the control of E-cadherin and Slug promoter, respectively. Thus the epithelial cell line will be green, whereas the mesenchymal one will be red, but they will be able to switch their color upon the EMT-MET induction. These HCC cell lines will be injected in mice to generate tumors and intravital two-photon microscopy (IVM) will be used to monitor cell fluorescence during the metastatic process. This approach will help to understand if epithelial and/or mesenchymal tumor cells are responsible for blood vessel invasion and metastasization and the in vivo role of EMT-MET process. Preliminary data: Two HCC cell lines, routinely used in our laboratory, one Slug negative/e-cadherin positive (HepG2) and the other one Slug positive/e-cadherin negative (SNU475) will be used. We have previously characterized the biological and molecular properties of these two cell lines before the in vitro EMT- MET induction. HepG2 showed an epithelial feature (polygonal shape, EpCAM and E-Cadherin expression) whereas SNU475 had a mesenchymal feature (elongated shape, CD44, CD90, Vimentin and ZEB1 expression). Moreover, HepG2 showed stem-like properties, i.e. ATP binding cassette-b1 (ABCB1), NANOG and OCT4 (embryonic stem cell markers) expression, while SNU475 lacked these genes. Finally, after the induction of EMT by cell incubation with TGFb-1 for 30 hours, cell motility was increased in both cell lines, as shown by wound healing assay. However, SNU475 was able to close the scratches almost completely, due to its original mesenchymal properties. These two HCC 10/07/ / 6

195 GR In vivo evaluation of the process of Epithelial-Mesenchymal and Mesenchymal-Epithelial transition (EMT-MET) in Hepatocellular Carcinoma (HCC) dissemination and metastasization. Colombo Federico Simone Biomedical/Biomedica Fondazione Ca'Granda - Ospedale Maggiore Policlinico cell lines with different characteristics will be useful for the development of our project. Materials and Methods HepG2 and SNU475 will be transfected using two lentiviral vectors and cell migration/invasion, 2D-3D colony formation assay and side population detection wiil be performed to evaluate their invasive and stem properties under EMT or MET conditions. Tumor cells will be injected into the flank of 10 NOD/SCID mice; they will be sacrificed at day 7, 14, 21, 28 and 35 and tumors and metastases will be evaluated. For IVM, a Cascade Blue-dextran solution will be used to visualize blood vessels surrounded by cancer mass. Images will be taken every 10 minutes for 4 hours to visualize cancer cells intravasation. To view metastases formation, 5 new mice will be injected with HCC cells and IVM will be used to monitor cancer cell trapping in the liver sinusoids. Once identified, cells will be monitored by IVM for 14 days to understand if EMT- MET transition happens. mirna and mrna expression assays will be performed on tumor and metastases dissociated tissues. Impact and Translational Implications Our work will investigate in vivo the role of EMT activation and reversion in promoting HCC metastasization. Moreover, our study will pave the way to new therapeutic strategies. In fact, if MET will be proven to be essential for metastases formation by mesenchymal cancer cell, new treatments could be implemented to maintain cancer cells in a mesenchymal state, therefore avoiding their proliferation and the formation of metastases. Finally, our model could be used for other epithelial tumors. 10/07/ / 6

196 Biostatistical methods for meta-analyzing individual participant data in diagnostic and prognostic research, with application to the role of cerebrospinal fluid biomarkers in Parkinson's disease. GR Eusebi Paolo Clinical health care research/clinicoassistenziale Umbria LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Healthcare Delivery and Methodologies Biostatistical Methods and Research Design - BMRD Project Keyword 1: Project Keyword 2: Project Keyword 3: Data analysis and modeling: development of statistical theory, analytic methods and models, computational tools and algorithms for the analysis and interpretation of data from clinical studies, randomized trials, epidemiological studies, human genetic association studies, environmental studies, and complex surveys; methods to handle data features and anomalies such as correlation, clustering, missing and skewed data; risk prediction and forecasting methods; causal modeling; high dimensional data methods Methods for data analysis including: Numerical, statistical, mathematical and theoretical approaches to design and interpretation of large-scale studies such as methods for pattern discovery, data mining, sequence prediction, biomarker identification, therapeutic drug design and high throughput analyses; computational methods for organizing, maintaining, and integrating biological datasets and for large scale data modeling and simulations. Parkinson s disease and other movement disorders (Huntington s, Dystonias, Ataxias). Project Request: Animals: Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION University of Perugia - Department of Medicine - Neurologic Clinic Principal Investigator Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 11/07/ / 6

197 Biostatistical methods for meta-analyzing individual participant data in diagnostic and prognostic research, with application to the role of cerebrospinal fluid biomarkers in Parkinson's disease. GR Eusebi Paolo Clinical health care research/clinicoassistenziale Umbria Background and Significance Diagnostic test meta-analyses aim at summarising evidence regarding the performance of any diagnostic technology, including the biomarkers. In contrast with standard meta-analyses, individual patient data (IPD) meta-analysis can improve the quality of data and the reliability of the conclusions, having also the potential to establish the value of test combination. IPD meta-analyses are usually more costly and time-consuming than conventional meta-analyses of aggregate data. Statistical aspects in such meta-analyses are challenging and need substantial improvement, especially if the IPD approach is applied to biomarker validation, where complicated evaluations drive complex decision in healthcare delivery. The goal of the project is to provide a new methodology for the IPD meta-analyses. Latent variable models will be implemented to address frequent problems of these analyses, such as missing data and heterogeneity. The new models will be applied in an IPD meta-analysis of the diagnostic and prognostic value of cerebrospinal fluid (CSF) biomarkers in Parkinson's disease. Biomarkers are urgently needed, particularly in the early stages. This would give an early and cost-effective diagnosis, and would increase the quality of life of the patients that, tipically, show a good response to symptomatic therapies. Furthermore, such biomarkers would help in the search for disease-modifying therapies, as well as could be used as surrogate markers in clinical trials. Specific aims Aim 1: Aim 2: Aim 3: New biostatistical models will be provided, in order to ameliorate the methodology for IPD meta-analyses in diagnostic and prognostic research. Based both on simulated and real data, the performance of new biostatistical models will be compared with the standard ones. An IPD meta-analysis will be performed for investigating the diagnostic and prognostic value of CSF biomarkers in diagnosing Parkinson's disease. A cost-effectiveness analysis will be performed for evaluating the clinical utility of CSF biomarkers for the diagnosis of Parkinson's disease. Hypothesis: The improvements of biostatistics methods for IPD diagnostic meta-analyses will lead to more robust results and effective conclusions. In particular, this is expected in the assessment of the diagnostic and prognostic value of CSF biomarkers in Parkinson s disease, where the majority of studies are observational and clinical heterogeneity exists in terms of CSF biomarkers selection, assays, disease spectrum. The results of more informative IPD meta-analysis and subsequent cost-effectiveness study would allow for the delivery of better diagnostic and therapeutic strategies in Parkinson s disease. Preliminary data: In a joint work with Reitsma JB and Vermunt JK, we have introduced the Latent Class Bivariate Model [Eusebi et al, BMC Med Res Methodol 2014 in press], extending the standard Bivariate Model for the diagnostic meta-analyses of aggregate data [Reitsma et al, J Clin Epidemiol 2005]. As pointed out by one reviewer: the approach may be a solution towards solving the high amount of variability in diagnostic accuracy meta-analyses. Latent variable models can be also introduced in IPD metaanalyses. This new methodological framework can be of help if hidden clustering exists in the data (both at individual and study level), helping in the control of unobserved sources of heterogeneity. In fact, preliminary results show that these models perform well in terms of power, confidence and bias. Furthermore, latent variable models can be implemented as imputation model for handling missing data in multilevel data structure, as IPD are, overcoming some limitations in traditional methods. We applied these models to real-data example, but we have to assess its performance with extensive simulations. The models can be fitted both within the frequentist framework and within the Bayesian 11/07/ / 6

198 Biostatistical methods for meta-analyzing individual participant data in diagnostic and prognostic research, with application to the role of cerebrospinal fluid biomarkers in Parkinson's disease. GR Eusebi Paolo Clinical health care research/clinicoassistenziale Umbria one. The neurological research group in which I work as biostatistician has obtained several data confirming the potential role of CSF biomarkers in the diagnosis and prognosis of Parkinson s disease [Parnetti L et al. Mov Disord. 2011, Van Dijk K et al. Mov Disord. 2013, Parnetti L et al. Mov Disord. 2014, Parnetti L et al. Front Aging Neurosci 2014]. After a preliminary MEDLINE search, we found 865 published studies about CSF biomarkers in Parkinson s disease (517 on the last 5 years). However, despite the tremendous increase in published studies, we didn't found any meta-analytic evaluation. Materials and Methods Original biostatistics methodology will be proposed for diagnostic IPD meta-analyses. In particular we will propose: multilevel latent class models for multiple imputation of missing data; general latent class models for obtaining the final estimates. The models will be fitted with LatentGOLD, WinBUGS and R software. Potential benefit of these biostatistics methods will be evaluated in the field of CSF biomarkers for the diagnosis of Parkinson s disease. Studies are considered for inclusion, the first authors of the included studies will be invited to participate and share original data. After assessment of validity and completeness the acquired datasets are merged. Based on these data, a series of analyses will be performed, including: a comparison with conventional methodology for IPD meta-analysis and our contributed methods; a comparison with conventional IPD meta-analysis and non-ipd metaanalysis. Impact and Translational Implications The strongly motivated innovation in the biostatistics methods is expected to be vastly implemented on future meta-analytic studies. The new methodology will support an IPD meta-analysis for assessing the diagnostic and prognostic value of CSF biomarkers in Parkinson s disease. Validated CSF biomarkers would allow for early and accurate diagnosis and would help in the search for disease-modifying therapies, as well as could be employed as surrogate markers in clinical trials. 11/07/ / 6

199 GR RELATIONSHIP BETWEEN OSTEOPOROSIS AND CARDIOVASCULAR RISK IN PATIENTS WITH THALASSEMIA MAJOR Meloni Antonella Clinical health care research/clinicoassistenziale Toscana LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Vascular and Hematology Clinical Hematology Special Emphasis Pane -SECHl Project Keyword 1: Project Keyword 2: Project Keyword 3: Applications proposing single-site clinical studies related to inherited blood disease (including gene therapy or genetics), or relating to red blood cell/monocyte/leukocyte biology, hematopoiesis, hemostasis and thrombosis, or inflammation. Multi-center clinical trials are not appropriate for this panel cardiovascular risk osteoporosis Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Fondazione G. Monasterio CNR-Regione Toscana, CMR Unit Principal Investigator Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 10/07/ / 6

200 GR RELATIONSHIP BETWEEN OSTEOPOROSIS AND CARDIOVASCULAR RISK IN PATIENTS WITH THALASSEMIA MAJOR Meloni Antonella Clinical health care research/clinicoassistenziale Toscana Background and Significance Thalassemias are the most common genetic disorder worldwide and are most prevalent in Mediterranean Europe. Italy has more than ß-thalassemia major (TM) patients in which the prognosis is opening thanks to an optimal standard care by transfusions, chelation and the availability of magnetic resonance imaging (MRI) scans to quantify organ iron non-invasively. Anyway cardiovascular disease (CV) remains the main cause of mortality and osteoporosis represents a prominent cause of morbidity in this ageing population. An increasing number of recent evidence suggests a link between osteoporosis and CV disease through mechanisms linked to common risk factors and common pathophysiological mechanisms. Oxidative stress, age, menopause, dyslipidemia, hypertension and diabetes mellitus, known determinants for CV disease, have been associated with reduced bone mineral density (BMD). Conversely, biochemical markers of bone turnover have been implicated in the pathogenesis and development of CV disease. In TM patients, iron accumulation remains the main determinant of increased CV risk. By contrast, many pathological aspects of bone changes which occur in TM remain to be clarified and there are scarce data on circulating levels of biochemical markers of bone turnover and no evidences about their possible relationship to CV risk. Aim of this study is to evaluate the relationship between bone changes assessed by instrumental and laboratory biomarkers and CV risk in TM patients. Specific aims Aim 1: Aim 2: Aim 3: To evaluate the bone modification by DEXA, measurements of different biochemical turnover markers and bone marrow iron by T2* MRI in TM. To evaluate CV risk profile by integrated analysis of data anamnesis, different biochemical, physiological biomarkers and functional tests, and quantification of cardiac iron by T2* MRI, atrial and ventricular function by cine images and myocardial fibrosis by late gadolinium enhancement technique in TM patients. To study the onset and development of bone changes by using multiple technologies and laboratory biomarkers, and their possible relationship with the integrated assessment of CV risk profile in TM patients. Hypothesis: A role for bone biomarkers as potential contributors for CV complications in TM patients. Preliminary data: Our research and clinical units, belonging to Fondazione Toscana G Monasterio (FTGM) and Institute of Clinical Physiology, CNR of Pisa, have matured a very long know-how and experience in interdisciplinary fields concerning cardiovascular biology, with particular emphasis placed on the study of biomarkers of cardiometabolic risk. Our biochemical laboratory has deep expertise in the field of colorimetric and immuno-enzymatic assays, molecular biology, and cell culture to monitor levels of different biochemical and vasoactive factors and markers of oxidative stress in biological samples. Specifically, our previous studies evaluated the association between oxidative stress and inflammatory biomarkers and vitamin D levels with different cardiometabolic risk factors and cardiovascular disease. Moreover, we have previously reported the hypothesis of a close interrelationship between osteoporosis and CV risk, based on findings of shared risk factors and underlying common mechanisms, and the role of recognized biomarkers for bone turnover in CVD. Our MRI Unit at FTGM in Pisa has a ten-year experience in the management of TM patients, by the multi-organ quantification of iron load using the T2* technique and using homemade, previously validated and with international patent software (HIPPO MIOT ). The MRI Unit is the Cor Lab for a network of 9 MRI centers and 70 thalassemia centers around Italy with one of the largest clinical and instrumental web based database in the world as recognized by international independent observers. Gender-differences of iron deposition have been found for the bone marrow and we hypothesize that 10/07/ / 6

201 GR RELATIONSHIP BETWEEN OSTEOPOROSIS AND CARDIOVASCULAR RISK IN PATIENTS WITH THALASSEMIA MAJOR Meloni Antonella Clinical health care research/clinicoassistenziale Toscana this difference may be due to the fact that the male sex is associated with severely low bone mass, which can influence the T2* values. However further studies are needed to better characterize the relationship between bone marrow T2* values and BMD. Materials and Methods TM patients consecutively undergone to a scan at FTGM MRI Lab in Pisa for clinical purposes in order to quantify heart, liver and pancreatic iron, atrial and ventricular function and myocardial fibrosis, will be evaluated for the following parameters: - bone mineral density by DEXA (Dual X-ray Absorptiometry) to assess osteopenia or osteoporosis extent; - bone marrow MRI T2* on 1.5T scan, T2* images will be analyzed using the HIPPO MIOT software; - integrated assessment of cardiovascular risk profile; - biochemical parameters: hemocrome, creatinine, uric acid, iron, transferrin, ferritin, NTproBNP, troponin, C-reactive protein, interleukin 6; indices of oxidative stress: hydroperoxides and total antioxidant capacity; parameters of bone turnover: vitamin D, PTH, osteocalcin, bone alkaline phosphatase, amino-terminal peptide of type I procollagen (PINP), carboxy-terminal peptide of type I collagen (CTx); lipid profile: total cholesterol, HDL, LDL, triglycerides and glycemia. Impact and Translational Implications This study may help to identify early biomarkers and common molecular and cellular mechanisms of CV and bone involvement in TM. Accordingly, patients with bone involvement would benefit from a detailed CV assessment, whereas patients with CV risk may require BMD and bone-related parameter assessment. This holistic approach may allow to develop integrated approaches for multiple-purpose preventive and therapeutic interventions in order to further improve quality of life and prognosis of TM. 10/07/ / 6

202 GR The addition of simvastatin portal venous infusion to cold storage solution of explanted whole liver grafts for facing ischemia/reperfusion injury in an area with low rate of deceased donation PAGANO DUILIO Clinical health care research/clinicoassistenziale Sicilia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Surgical Sciences, Biomedical Imaging, and Bioengineering Surgery, Anesthesiology, and Trauma - SAT Project Keyword 1: Project Keyword 2: Project Keyword 3: Tissue, organ and systemic injury responses to surgery, trauma, burn, sepsis, hemorrhage, ischemia-reperfusion, or resuscitation, including integrating pathways and signals. Organ shortage and extended-criteria donors Liver transplantation and Statins Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION ISMETT, Department of Surgery Management of the selection and care of patients requiring liver transplant, Coordination for cadaveric donor, Operating room activities, Clinical, Diagnostic and Therapeutic Services, Management and distribution of pharmaceuticals 2 Fondazione Ri.MED Regenerative Medicine and Advanced Biotechnologies Unit Laboratory and research activities, Data management and statistical analysis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Oliva Elisabetta Fondazione Ri.MED Research activities, Data recording, assessment, management and statistical analysis. 27/12/ /07/ / 5

203 GR The addition of simvastatin portal venous infusion to cold storage solution of explanted whole liver grafts for facing ischemia/reperfusion injury in an area with low rate of deceased donation PAGANO DUILIO Clinical health care research/clinicoassistenziale Sicilia Background and Significance Liver transplantation (LT) is the only life-saving therapy for most types of end-stage liver disease. In Italy, the overall rate of cadaver donation is 22.2 donors per million per year, and in some regions, as Sicily, the rate is 12.6 donors per million per year. This shortage of organs led to use steatotic liver grafts from extended-criteria donors, even if these livers are much susceptible to ischemia/reperfusion (I/R) injury and have poorer outcome than non-steatotic livers. It has been demonstrated by preclinical studies that these negative effects of I/R can be prevented adding simvastatin to the cold preservation solution shortly before procurement to protect both parenchymal and endothelial components of the liver after warm reperfusion. The proposed project wants to be a first prospective randomized clinical trial to prove the efficacy of simvastatin in preventing I/R injury and expand the donor pool. Specific aims Aim 1: Aim 2: Aim 3: To evaluate the efficacy of simvastatin addition to cold-storage in preventing ischemia/reperfusion injuries. To study whether the simvastatin, which is a very cheap vasoprotective compound, might be a rapid and useful pharmacological support to donor surgeons for increasing the viability of the harvested organ from deceased donors. To provide an evidence-based research to demonstrate which strategies might reduce health care costs and prevent the primary organ failure, retransplantation in urgency, and any complications related to high mortality. Hypothesis: The hypothesis behind the present study is that a single intravenous injection of simvastatin will be more effective in improving liver graft metabolism than the alone physical reduction of temperature obtained with the cold storage. This early clinical trial might represent a crucial bridge between preclinical drug study and the definitive regulatory hurdle for drug approval in multi-organ harvesting procedures from deceased donors. The incorporation of scientifically and analytically validated clinical effects of simvastatin into this rationally designed hypothesis-testing clinical trial could offer a promising way forward to achieve prevention I/R injury and expand the donor pool. Preliminary data: The shortage of organ donors is a problem worldwide, with approximately 15% of adult patients with life-threatening liver diseases dying while on the waiting list (Gridelli B. Nat Rev Gastroenterol Hepatol. 2013). ISMETT is dedicated to care patients with end-stage vital organ disease and steadily to increase the number of transplants, despite a consistently low donation rate, thanks to marginal donors, living donors and split liver transplantation (Gruttadauria S, Parikh V, Pagano D, et al. Liver Transpl. 2012). The use of simvastatin during the harvesting procedures of liver grafts from deceased donors is an attempt to correct metabolic defects, or to support liver function as a bridge to liver transplantation and, as such, has raised a number of expectations (Gracia-Sancho J, García-Calderó H, Hide D, J Hepatol. 2013). Preclinical published data show that simvastatin is effective in reducing oxidative stress, upregulating autophagy and inducing endothelial protection during I/R injury. Statins have been previously showed to protect donor livers, with a strong hepato- and vasoprotective effects in livers from healthy, cirrhotic and obese animals (Russo L, Gracia-Sancho J, García-Calderó H, et al. Hepatology. 2012). Simvastatin is the more powerful statin formulation due to its higher hepatic vasoprotective effects in comparison to other FDA-approved statins. 11/07/ / 5

204 GR The addition of simvastatin portal venous infusion to cold storage solution of explanted whole liver grafts for facing ischemia/reperfusion injury in an area with low rate of deceased donation PAGANO DUILIO Clinical health care research/clinicoassistenziale Sicilia Materials and Methods This prospective randomized double-blind clinical trial will include a total of 100 adult deceased donors, divided in two groups. Fifty cases will receive a single dose of portal venous infusion of simvastatin (1 mg/kg) in addition to cold storage Celsior Solution(CS).The other fifty explanted whole liver grafts will be treated solely with cold storage with CS. LTs with standard technique will be performed and all of the recipients will be routinely evaluated during the hospitalization with an intraoperative post-reperfusion liver biopsy, intensive care monitoring for the early post-operative course, daily laboratory and Doppler ultrasound examinations. The study phases will be: 1) enrollment and post-operative period: months 0-21; 2) follow-up period months 12-33; and 3) statistical analysis, and verification of results with peer-review committee, months Graft loss because of technical failure, acute rejection or death, within the first three months post-lt will be included. Impact and Translational Implications Characterization of the metabolic effect of a new pharmacological approach will be useful to prevent I/R injury and therefore the primary failure of the transplanted liver. Even if the results will not favor simvastatin, the study will contribute to ameliorate the deceased donor harvest procedures for avoiding I/R injury induction. This clinical trial might be extended to the other solid organs transplants studies.

205 GR An Italian study on intermediate-lenght polyq-tract expansions: frequency, clinical variability, and brain morphometry of subjects at risk for late-onset neurodegenerative diseases. Nanetti Lorenzo Clinical health care research/clinicoassistenziale Fondazione Istituto Neurologico Carlo Besta LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Neuroimaging, functional, biochemical, and neuropathological studies to assess the onset, progression, treatment, and development of biomarkers for brain disorders. Polyglutamine disorders CAG-Intermediate-lenght alleles Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Diagnostics and Applied Technology/Unit of Genetics of Neurodegenerative and Metabolic Diseases 2 Sapienza University of Rome 3 University of Naples Federico II Department of Neurosciences, Mental Health and Sensory Organs/ School of Medicine and Psychology Department of Neurosciences, Reproductive SCiences and Odontostomatology Coordination; enrolment of the subjects; clinical, neuroradiological, and genetic investigations Selection and enrollment of subjetcs, and clinical investigation Selection and enrollment of subjetcs, and clinical investigation Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Romano Silvia Sapienza University of Rome Selection and enrollment of subjetcs, and clinical investigation 2 Salvatore Elena University of Naples Federico II Selection and enrollment of subjetcs, and clinical investigation 07/02/ /04/ /07/ / 6

206 GR An Italian study on intermediate-lenght polyq-tract expansions: frequency, clinical variability, and brain morphometry of subjects at risk for late-onset neurodegenerative diseases. Nanetti Lorenzo Clinical health care research/clinicoassistenziale Fondazione Istituto Neurologico Carlo Besta Background and Significance A group of inherited adult-onset neurodegenerative disorders are caused by CAG repeat expansions in the causative genes encoding for polyglutamine tracts (poly-q). The number of CAG is relevant for age at onset and clinical manifestation. The poly-q diseases are untreatable disorders and include Huntington Disease (HD) and a subgroup Spinocerebellar Ataxias (SCAs). Clinically, HD is characterized by involuntary movements and dementia, while SCAs by progressive disequilibrium and motor incoordination. HD and poly-q SCA are the most frequent in Italian patients, with an estimate prevalence of 3-12/ CAG expanded tracts are dynamic and can modify their length through the generations. CAG expansion above the define threshold are invariably associated with disease manifestation, while intermediate alleles (IAs) are associated with reduced penetrance, and meiotic instability. Recent studies on HD have estimated a high frequency of IAs (1/17). Poly-Q IAs could represent a reservoir for fully-expanded alleles, and in SCA1-SCA2 patients cause different clinical phenotypes such as Parkinson Disease and Amyotrophic Lateral Sclerosis. In HD, IAs have been hypothesised to have a role in brain development and grey matter structure. We propose a multi-centric genetic survey in a large Italian cohort to identify the frequency of IAs for HD and SCAs and to correlate, in the carries subjects, clinical phenotypes, brain morphology and cognitive performances. Specific aims Aim 1: Aim 2: Aim 3: To estimate the frequency of poly-q intermediate alleles in a multi-centric study including a large cohort of Italian at-risk subjects and unrelated control subjects. To characterize the cohort of poly-q intermediate allele carrier subjects with clinical, cognitive, and neuroradiological examinations, to improve early diagnosis, ameliorate clinical care and genetic counselling for family members, and identify clinical biomarkers useful for future therapeutic trials. To study possible poly-q intermediate allele implication in brain development and aging. Intermediate allele carriers and sex-age matched healthy controls will undergo longitudinal brain morphometry and cognitive evaluations. Hypothesis: A high frequency of polyq-intermediate length alleles in Caucasian population has been recently demonstrated. Several literature data suggested the possibility that polyq-intermediate length alleles may contribute in the variability in the disease manifestation and also in brain development. In this study we will test two major hypotheses. The first is that polyq-intermediate length alleles may be associated with signs or symptoms of neurodegeneration, whose identification may contribute in the study of pathophysiologic mechanisms of disease onset and progression. The second hypothesis is that elongation of poly-q tract below threshold length may affect brain structure, and in particular grey matter volume. Preliminary data: The Neurological Institute Carlo Besta in Milan is one of the Italian referral centre for clinical and genetic diagnosis of hereditary neurodegenerative diseases. In the past decades, molecular diagnosis has been performed in more than 2000 HD patients and at risk family members and in approx SCAs families with different poly-q genotype. In addition, DNA samples from more than1000 healthy unrelated individuals have been analysed and stored for research purposes. The applicant participated in the European longitudinal observational studies on HD (EHDN) and SCA (EUROSCA and RISCA), and contributed in several studies on biomarkers in HD patients and premanifest HD and SCA individuals, (Mariotti, 2010; Minati, 2011; Ferraro, 2014; Tezenas, 2014). We confirmed in different cohort of subjects the role of SCA2 polyq-intermediate alleles as risk factor for Amyotrophic Lateral Sclerosis (Nanetti, 2009;Gellera, 2012), and we recently performed a preliminary survey in our cohort of families to define the role of 42 and 43 CAG alleles in SCA17 pathogenesis. These previous studies, allowed us to identified approximately 110 subjects carrying 11/07/ / 6

207 GR An Italian study on intermediate-lenght polyq-tract expansions: frequency, clinical variability, and brain morphometry of subjects at risk for late-onset neurodegenerative diseases. Nanetti Lorenzo Clinical health care research/clinicoassistenziale Fondazione Istituto Neurologico Carlo Besta polyq-intermediate alleles on HD or SCAs genes. In order to have reliable access to a sufficiently large cohort of participants, we design an Italian multi-centre study involving two Centres in Rome and Naples with a similar experience in poly-q diseases. The PIs of the collaborating Centres have already worked together in previous observational and interventional trials on neurodegenerative disorders. Materials and Methods We plan to enroll 40 subjects carrying intermediate-length HD alleles, 40 subjects carrying intermediate-length SCAs alleles, and 40 healthy controls carrying normal-length alleles. Blood samples will be obtained to measure CAG repeat number. Genetic counseling will be offer to all participants and at-risk family members. Cognitive, motor, functional, and behavioral scales will be used. MRI protocol: we will perform cortical thickness analysis with Free surfer and a voxel based morphometry on the segmented gray matter and white matter of 3D T1-weighted images. MRI connectivity study: participants will be scanned on a state-of-the-art 3 Tesla system with a 32-channel head coil; the resting-state imaging sequences will cover whole brain, having a TR of approximately 2s and lasting for 6 min. We plan to perform a baseline evaluation and a 1-year follow-up analysis for all enrolled subjects. In addition, in a subgroup of subjects, a 2-year follow-up will also been obtained. Impact and Translational Implications The frequency of polyq intermediate-length allele carriers in the Italian population is not known. The identification of the subjects at risk for late-onset neurodegenerative diseases, could represent an opportunity for studying disease pathogenesis in patients presenting a very slowly progressive neurodegeneration, and improving the understanding of the phenotype-genotype correlation in these diseases, permitting an earlier clinical recognition and diagnosis and the definition of biomarkers. 11/07/ / 6

208 GR TRAIL, THE REGULATION OF BODY WEIGHT AND METABOLISM, AND ITS INTERACTION WITH THYROID HORMONES AND METFORMIN. Bernardi Stella Clinical health care research/clinicoassistenziale Ospedale infantile Burlo Garofolo LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Endocrinology, Metabolism, Nutrition and Reproductive Sciences Molecular and Cellular Endocrinology - MCE Project Keyword 1: Project Keyword 2: Project Keyword 3: Molecular mechanisms of polypeptide, steroid/thyroid hormone action and peptide hormone synthesis, processing, secretion, signaling, and trafficking. TRAIL Metabolism Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: X I declare that the object/s of this application is/are under patent copyright Patent owner: Operative Units Istituto di Ricovero e Cura a Carattere Scientifico Materno-Infantile Burlo Garofolo Patent number: WO patent 2,012,117,336 INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Dipartimento di Diagnostica Study conception, experiment design, data acquisition and interpretation, patient management (selection and treatment), writing. Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 10/07/ / 6

209 GR TRAIL, THE REGULATION OF BODY WEIGHT AND METABOLISM, AND ITS INTERACTION WITH THYROID HORMONES AND METFORMIN. Bernardi Stella Clinical health care research/clinicoassistenziale Ospedale infantile Burlo Garofolo Background and Significance Prevalence of obesity with its associated diseases has reached epidemic proportions and its management has become a major public health challenge. New therapies against obesity are desperately needed. TNF-related apoptosis-inducing ligand (TRAIL) is a soluble protein that induces apoptosis in transformed cells through activation of specific death receptors. In non-transformed cells, however, the actions of TRAIL are less well characterized. Recent studies suggest that TRAIL may have an important role in the treatment of obesity and its associated metabolic disturbances. High-fat diet (HFD) fed TRAIL-knockout mice display significant weight gain as compared to control mice (Di Bartolo, 2011). Consistent with this, we have shown that TRAIL delivery significantly reduces fat gain in HFD fed mice (Bernardi, 2012). In this study, TRAIL significantly increased fatty acid muscular oxidation, improving insulin sensitivity and reducing the levels of glucose and insulin. Given that altered fatty acid metabolism is involved in the development of obesity, our data suggests that TRAIL reduces weight gain by increasing fatty acid oxidation. Nevertheless, its mechanisms of action remain to be defined. The present application proposes to characterize the mechanisms underlying TRAIL effects on body weight and metabolism, and to study the relationship between TRAIL and conditions stimulating weight loss and lipid oxidation, such as hyperthyroidism and metformin treatment. Specific aims Aim 1: Aim 2: Aim 3: To determine: (i) TRAIL effects on adipocyte differentiation and apoptosis, (ii) TRAIL targets on cells involved in lipid metabolism, such as adipocytes, hepatocytes, and myocytes. To determine the relationship between TRAIL and thyroid hormones and whether TRAIL mediates and/or promotes thyroid hormones peripheral effects. To determine the relationship between TRAIL and metformin and whether TRAIL mediates and/or promotes metformin peripheral effects. Hypothesis: TRAIL prevents fat gain not only by inhibiting adipocyte differentiation and promoting adipocyte apoptosis, but also by promoting fatty acid oxidation. TRAIL is upregulated by hormones and drugs that induce weight loss and fatty acid oxidation and it mediates/facilitates part of their peripheral effects. Preliminary data: 1. We have recently shown that TRAIL reduces the accumulation of fat mass and increases fatty acid oxidation in HFD fed mice, improving insulin resistance (Bernardi, 2012). In these mice, TRAIL changed adipose tissue expression of genes related to apoptosis and adipocyte differentiation. This work forms the basis of our therapeutic patent (WO patent ). 2. By contrast, we have shown that OPG, which is TRAIL decoy receptor, has opposite actions to those of TRAIL (Toffoli, 2011; Bernardi, 2014). 3. In parallel studies we have found that TRAIL expression is influenced by thyroid hormones, which are well known regulators of fat tissue and metabolism. In particular, serum TRAIL significantly increased in 39 untreated hyperthyroid (29.3 ± 5.9 pg/ml) and decreased in 24 untreated hypothyroid (8.4 ± 1.2 pg/ml) patients, as compared to 39 controls (18.0 ± 4.2 pg/ml). Once euthyroidism had been restored TRAIL normalized. Overall, between TRAIL, ft3 (r=0.48, p<0.01), and ft4 (r=0.50, p<0.01) there was a significant direct correlation. There was no correlation between OPG, ft3, and ft4. Moreover, in hyperthyroid patients TRAIL gene expression was two-fold higher, whereas in hypothyroid patients it was two-fold lower, as compared to the controls (p<0.05). In order to evaluate whether it was T3 or T4 that stimulated TRAIL release, purified PBMC were cultured for 20 hours in the presence of either T3 or T4 at two different concentrations (10-7M, 10-9M). T3 10/07/ / 6

210 GR TRAIL, THE REGULATION OF BODY WEIGHT AND METABOLISM, AND ITS INTERACTION WITH THYROID HORMONES AND METFORMIN. Bernardi Stella Clinical health care research/clinicoassistenziale Ospedale infantile Burlo Garofolo significantly increased TRAIL release in a dose-dependent manner, while T4 did not have any effect on it. In particular, T3 at the dose of 10-7M elicited a greater response than IFNgamma (our positive control), as T3 stimulated a 3-fold increase, while INFgamma stimulated a 2-fold increase of TRAIL release. These treatments did not induce any OPG release. 4. Interestingly, preliminary experiments in purified PBMC show that also metformin stimulates TRAIL expression in a dose dependent-manner. Materials and Methods 1. Cells will be exposed to TRAIL with and without its inhibitor. TRAIL effects on adipocyte differentiation and apoptosis will be studied. Fatty acid metabolism key enzymes (e.g. ACC1/2, AMPK) and other molecular targets (e.g. CD36, CPT1A/B, PGC-1alpha, PPARalpha/gamma, RXRalpha) will be evaluated. 2. TRAIL and OPG will be measured in hyperthyroid and hypothyroid patients, before and after their treatments, and in euthyroid patients. In vitro, we will analyze T3 and T4 effect on TRAIL and OPG expression; T3 effect on the expression of molecular targets (e.g. CPT-1) with or without TRAIL inhibitor; TRAIL effect on TRalpha and TRbeta expression. 3. TRAIL and OPG will be measured in patients before and after metformin treatment. In vitro, we will analyze metformin effect on TRAIL and OPG expression; metformin effect on the activation of AMPK with or without TRAIL inhibitor; TRAIL effect on OCT1 expression. Techniques involved: cell culture, ELISA, RT-PCR, Western Blot. Impact and Translational Implications Based on our preliminary findings we expect to clarify whether TRAIL actions on body weight can be ascribed to its effects on adipocyte differentiation and/or apoptosis. Moreover, we expect to understand how TRAIL regulates lipid metabolism, whereby it possibly mediates thyroid hormones/metformin effects. These results will shed light on the mechanisms underlying TRAIL anti-adipogenic effects and open new therapeutic possibilities against weight gain, obesity, and its associated diseases. 10/07/ / 6

211 GR Endovascular therapy in acute ischemic stroke: an observational prospective study of an interprovincial Hub and Spoke Model Zini Andrea Clinical health care research/clinicoassistenziale Emilia-Romagna LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Methodological, epidemiological, socio-economic, organizational, managerial emerging public health issues related to the above reported areas Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Acute Neural Injury and Epilepsy - ANIE Project Keyword 1: Project Keyword 2: Project Keyword 3: Relevant disorders include stroke/ischemia, epilepsy, spinal cord injury and traumatic brain injury. Stroke Endovascular therapy Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION AUSL Modena - Department of Neuroscience - Nuovo Ospedale Civile "S.Agostino-Estense" - Stroke Unit- Neurology Clinic 2 REGIONE EMILIA- ROMAGNA 3 IRCCS Arcispedale - Istituto in tecnologie avanzate e modelli assistenziali in oncologia - Reggio Emilia AUSL Modena - Department of Neuroscience - Nuovo Ospedale Civile "S.Agostino-Estense" - Neuroradiology Unit IRCCS Arcispedale Santa Maria Nuova - Reggio Emilia - Stroke Unit- Neurology Unit Principal Investigator Biographical Sketch Contributor Biographical Sketch Contributor Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Menetti Federico REGIONE EMILIA-ROMAGNA Biographical Sketch Contributor 14/12/ Zedde Marialuisa IRCCS Arcispedale - Istituto in tecnologie avanzate e modelli assistenziali in oncologia - Reggio Emilia Biographical Sketch Contributor 28/06/ /07/ / 6

212 GR Endovascular therapy in acute ischemic stroke: an observational prospective study of an interprovincial Hub and Spoke Model Zini Andrea Clinical health care research/clinicoassistenziale Emilia-Romagna Background and Significance Reperfusion of the ischaemic brain is the most effective therapy for acute ischaemic stroke (AIS) reducing the extension of the final infarct by enabling a better clinical outcome. In addition to the admission in Stroke Unit, intravenous thrombolysis (IVT) is the only effective therapy for AIS, but its efficacy is limited, mainly among patients with large-vessel occlusions. Indeed the lytic potential of IVT is linked to artery recanalization that is only achieved in less than 1/3 of proximal occluded cerebral arteries. Consequently, in spite of proven clinical efficacy in large series, >50% of the patients remain disabled or die. Furthermore many patients with AIS are left untreated because of contraindications to IVT or delayed hospital arrival. Endovascular therapies (EVT) may be utilized to improve the recanalization rate. For these reasons, despite lack of positive evidence from published RCTs EVT has become widespread in routine practice and also recommended by international and national guidelines in selected patients in experienced stroke centers with qualified interventional neuroradiologists.the lack of well-estabished evidence has prompted close monitoring EVT. On the other hand the quest for guaranteeing equity of access to EVT for all stroke patients who need this treatment and for fulfilling the principle of rationalization of healthcare costs calls for new organizational model. Specific aims Aim 1: Aim 2: Aim 3: To assess clinical outcome and safety measures after endovascular therapy in acute ischemic stroke patients Outcome measures: modified Rankin Scale (mrs) at day 90.A good outcome is established by a mrs<or=2. Mortality at day 90. Complete recanalization rate defined as TICI score 2b-3 Safety measures: symptomatic intracerebral haemorrhage (sich) as defined per SITS-MOST protocol Outcome and safety measures will be compared with published cohorts of EVT patients, and with controls selected from the Italian Registry of Endovascular Stroke Treatments as control groups. All EVT candidate patients who eventually fail to undergo to EVT are registered. To assess any improvements in EVT occurred over a three-year period, in term of number of treated patients, outcome and safety reports, and process measures. Applying the "Time is brain" concept both to IVT and EVT all process measures are linked to timing, monitoring stroke onset to door time, door to needle time (for patients treated previously with IVT), door to door time for transferred patients, door to groin puncture time, groin puncture to recanalization. A Cost-effectiveness analysis will be performed observing the health benefits and costs associated with EVT comparing with IVT alone or untreated patients. All probabilities, quality-of-life factors (QALY), and costs will be estimated from the published literature. Hypothesis: By implementing synergy and cooperation between the local health authorities of two neighboring provinces (Modena and Reggio Emilia) with a single center (Sant'Agostino Estense Hospital in Modena) collecting all patients candidates to EVT, according to a Hub&Spoke model, we hope to demonstrate the efficacy and safety of EVT in AIS patients (who clinically failed IVT or could not underwent IVT because of contraindications to IVT). Preliminary data: DRGs databank (year 2013) registered a total of 2,015 ischemic strokes in Modena and Reggio Emilia provinces. During the same time frame 90 and 150 patients underwent IVT at the IRCCS Arcispedale S.Maria Nuova Hospital and at Modena Hospital, respectively. In addition 55 patients underwent EVT patients in Modena (Italian Registry of Endovascular Stroke Treatment: In a two-center retrospective analysis conducted at the Sant'Agostino Estense Hospital in Modena and at the Karolinska University Hospital in Stockholm, a group of 52 consecutive AIS patients treated with EVT after clinically failed IVT was compared with a control group consisting of 260 consecutive patients treated with IVT alone. 10/07/ / 6

213 GR Endovascular therapy in acute ischemic stroke: an observational prospective study of an interprovincial Hub and Spoke Model Zini Andrea Clinical health care research/clinicoassistenziale Emilia-Romagna In the EVT group recanalization was achieved in 92% with a sich rate of 9.6%. EVT patients admitted with a severe stroke (median baseline NIHSS=17) had a significantly better outcome at 90 days compared to patients with the same score but treated with IVT alone (mrs0-2: 58.3% vs 34.4% respectively). Reference: Menetti F, Verganti L, Zini A et al. Interv Neuroradiol Jun 30;20(3): Materials and Methods We will build up a large inter-provincial stroke network according to a Hub&Spoke model (provinces of Modena and Reggio Emilia) with a single center (Sant'Agostino Estense Hospital in Modena), collecting all stroke patients candidates to EVT. The network will cover a population of about 1,200,000 residents. The Hub Hospital will offer a 24-hour service with an onsite neurologist and neuroradiologist.the interventional team will be on-call 24h. The stroke code activation will be achieved by directly preallerting the Hub neurologist. Criteria for EVT have been shared under a common protocol. EVT candidates will be selected according to clinical criteria (NIHSS>10) and to multimodal neuroimaging demonstration of vessel occlusion by CT angiography and of ischemic penumbra by CT perfusion. All clinical, neuroradiological and angiographic variables will be collected. Statistical analysis will be performed comparing all variables, outcome and process measures with those of the control group. Impact and Translational Implications The realization of a sinergy between two neighboring provinces will create a macro-area network for EVT in AIS patients. Positive results of the study might encourage spreading the organizational model to all the Country. With appropriate geographic range and population size, this approach is aimed at guaranteeing equity of access to EVT for all candidates. Concentrating EVT in experienced high-volume stroke centers will possibly attain better outcomes and help rationalizing healthcare costs. 10/07/ / 6

214 GR Role of the adipocyte mineralocorticoid receptor in the development of sarcopenic obesity and comorbidities Feraco Alessandra Biomedical/Biomedica Casa di cura San Raffaele Pisana LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Endocrinology, Metabolism, Nutrition and Reproductive Sciences Integrative Physiology of Obesity and Diabetes - IPOD Project Keyword 1: Project Keyword 2: Project Keyword 3: Cytokine, adipokines and inflammatory regulation of metabolic and energy control: effects on insulin action in adipose, liver, muscle and neural processes; cellular and molecular responses to changes in inflammation, cytokine and adipokines levels. mineralocorticoid receptor sarcopenic obesity Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Laboratory of Caridovascular Endocrinology 2 Sapienza University of Rome 3 Foro Italico University of Rome Experimental Medicine Department, Medical Pathophysiology, Food Science and Endocrinology Section, Food Science and Human Nutrition Research Unit Movement, Human and Health Sciences Department, Section of Health Sciences Generation of animal models, in vivo experiments, cellular studies, analysis and interpretation of the data Evaluation and characterization of human subjects, analysis and interpretation of the data in human subjects Cellular studies, evaluation of biochemical markers in serum patients, analysis and interpretation of the data Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Greco Emanuela Sapienza University of Rome Plan human studies, data analysis and interpretation 10/10/ PAPA Vincenza Foro Italico University of Rome Plan and perform cellular studies 02/07/ /07/ / 6

215 GR Role of the adipocyte mineralocorticoid receptor in the development of sarcopenic obesity and comorbidities Feraco Alessandra Biomedical/Biomedica Casa di cura San Raffaele Pisana Background and Significance Sarcopenic obesity (SO) is a disorder of body composition characterized by the presence of increased fat mass (FM) and reduced fat free mass (FFM). Several studies, including ours, have demonstrated a correlation between obesity and/or sarcopenia and disability. Moreover obesity and sarcopenia are both related to metabolic disturbances (i.e. glucose and lipid metabolism) and inflammation. Finally sarcopenia and obesity appear related to osteoporosis. Importantly, causal link between obesity and sarcopenia has not been yet characterized and, thus, requires further studies. It has been proposed that pro-inflammatory adipokines, released from the visceral adipose tissue, generate ectopic fat deposition and catabolic effects on skeletal muscle leading to loss of muscle mass and strength. Furthermore, several reports indicate that, in experimental murine models of obesity, pharmacological blockade of the mineralocorticoid receptor (MR) is able to restore altered proinflammatory adipokines expression with potential beneficial effects on adipose tissue, glucose metabolism, muscle and bone mass. SO represents an important risk factor for comorbidity, affecting the quality of life of patients with significant increase in health care costs. MR antagonists, widely used for the treatment of hypertension and congestive heart failure, may find novel application in the treatment of SO. Specific aims Aim 1: Aim 2: Aim 3: To investigate whether adipocyte-specific MR modulates expression of adipokines affecting skeletal muscle loss, we plan to evaluate mass, lipid accumulation and expression of markers of apoptosis, proteolysis and autophagy in the skeletal muscle of aged (2 years-old) adipocyte-specific MR knock-out (Adipo MR KO) mice fed a high fat diet for 120 days to induce obesity. Analysis of body composition, glucose control and insulin sensitivity, bone mineral density will be performed. To investigate whether adipokines transcriptionally regulated by MR might affect muscle and bone function, osteoblasts, myoblasts and satellite cells purified from wild-type mice will be treated with the conditioned media of primary adipocyte cultures derived from Adipo MR KO mice. In myogenic cells, expression of markers of differentiation, apoptosis, proteolysis and autophagy will be evaluated. Osteoblast differentiation markers expression will be analysed in MC3T3-E1 cells. Cell cultures incubated with conditioned media from adipose cells will be treated with neutralizing antibodies against specific adipokines to precisely identify the adipokines mediating the detrimental effects on skeletal muscle and bone. In order to evaluate if the observations conducted in mice and cell cultures on the role of the adipose MR in sarcopenic obesity can be translated to human sarcopenic obesity, obese and obese/sarcopenic subjects will be evaluated by MRI, Dual Energy X-ray Absorpiometry (DXA) and Bioimpedentiometry (BIA). Quality of life will be also determined in these subjects. MR protein presence and activity in adipose tissue of these obese patients will be tested to further characterize the role of MR in the pathophysiology of obesity comorbidities. In addition, serum of patients will be used to culture mesenchimal stem cells and evaluate potential effects in cell differentiation pattern induced by the serum of obese and obese/sarcopenic subjects. Hypothesis: Our hypothesis is that over-activation of adipose MR in obesity, promoting expression of pro-inflammatory adipokines, might stimulate loss of muscle and bone mass. Preliminary data: In a pilot study conducted on 79 obese (BMI > 30 kg/m2) subjects admitted to the day hospital facility at the Department of Experimental Medicine during a 1 year period the prevalence of SO was 54.4%. Sarcopenia was present not only among older obese individuals but also among younger obese subjects, and was related to inflammatory status, reduced functional performance and poorer psychological status and quality of life. 10/07/ / 6

216 GR Role of the adipocyte mineralocorticoid receptor in the development of sarcopenic obesity and comorbidities Feraco Alessandra Biomedical/Biomedica Casa di cura San Raffaele Pisana In addition, our recently published data indicate that the serum of obese or obese sarcopenic subjects significantly alter bone cells homeostasis in vitro. In particular, osteoblasts exposure to the serum of obese sarcopenic patients with/without osteoporosis significantly affects osteoblast differentiation by mechanism(s) linked to an alteration of Wnt/b-catenin pathway (Wannenes et al 2014). Materials and Methods Adipocyte-specific MR deficient mice will be generated crossing the floxed MR mouse with the adipocyte-specific adiponectin-cre mouse. Preadipocytes will be isolated from adipose tissue of the transgenic mice. Body composition will be analyzed using MRI and glucose tolerance test will be performed. For histological analysis, muscle sections will be stained with hematoxylin and eosin. Gene expression and Western blot analysis will be also performed in skeletal muscle excised from mice. MR protein expression will be evaluated in subcutaneous abdominal adipose tissue biopsies of obese subjects. In obese subjects, analysis of body composition and skeletal muscle quality will be performed (DXA, BIA, MRI and muscle biopsy at cross sectional area of the quadriceps level), as well as evaluation of muscle strength and function: acceloremeters/metabolic Holter, hand-grip strength test, sit to stand test, 6-minute walk test, test for obesity-related disabilities by the Italian Society of Obesity. Impact and Translational Implications SO, due to its negative impact on metabolic and cardiovascular functions, is becoming an urgent health problem in the developing word. The aim of this study is to understand the role of adipose MR in promoting the observed deterioration, in obese subjects, of skeletal muscle and bone. Indeed the results of this study might lead to identify MR as a potential novel pharmacological target to design innovative therapies against sarcopenic obesity and its comorbidities. 10/07/ / 6

217 GR Melanopsin retinal ganglion cells and circadian rhythms: function and dysfunction in Alzheimer's disease and aging. La Morgia Chiara Biomedical/Biomedica Istituto delle Scienze Neurologiche - Bologna LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Alzheimer s disease and other dementias. Circadian rhythms Aging Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION UOC Clinica Neurologica Recruitment of patients, in-vivo studies (cognitive and ophthalmologic evaluation, optical coherence tomography, actigraph, pupillometry and melatonin rhythm) and data collection. 2 Azienda Ospedaliero- Universitaria di Bologna MR Functional Unit 3 University of Bologna Department of Experimental, Diagnostic and Specialty Medicine (DIMES) Investigators, Institution and Role in the Project fmri studies using light paradigms to specifically stimulate melanopsin retinal ganglion cells. Genetic studies (genetic variants of clock and melanopsin genes in Alzheimer's patients and controls). Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Zanigni Stefano Azienda Ospedaliero- Universitaria di Bologna fmri studies using light paradigms to specifically stimulate melanopsin retinal ganglion cells. 2 SANTORO AURELIA University of Bologna Genetic studies (genetic variants of clock and melanopsin genes in Alzheimer's patients and controls). 09/07/ /04/ /07/ / 6

218 GR Melanopsin retinal ganglion cells and circadian rhythms: function and dysfunction in Alzheimer's disease and aging. La Morgia Chiara Biomedical/Biomedica Istituto delle Scienze Neurologiche - Bologna Background and Significance Circadian rhythms, characterized by a period of about 24 hours, are driven by the hypothalamic suprachiasmatic nucleus (SCN). Light is the main "zeitgeber", synchronizing these rhythms to the day-night cycle. Circadian photoentrainment is provided by melanopsin retinal ganglion cells (mrgcs), a subset of intrinsically photosensitive RGCs. The mrgc system can be selectively stimulated by blue light and specific activation/deactivation of brain areas has been shown by functional MRI in controls. Circadian dysfunction may occur in aging and in Alzheimer's disease (AD). This is pleomorphic, affecting sleep, cognition, mood and longevity, and may be counteracted by light therapy. An age-related loss of RGCs, including mrgcs, has been documented by optical coherence tomography and histological studies. In addition, optic neuropathy and specific SCN neurodegeneration are described in AD. Finally, a single nucleotide polymorphism (SNP) in the gene Clock has been associated to AD susceptibility. Overall, this proposal will extensively characterize with a multimodal and innovative approach the unexplored connection between circadian dysfunction and mrgc system in AD and aging, and in particular in successful aging. Our results will provide the basis for designing light therapeutic trials aimed at realigning circadian rhythms in AD patients and elderly. Furthermore, we will assess genetic variation in an extended panel of clock genes as contributors to AD susceptibility and longevity. Specific aims Aim 1: Aim 2: Aim 3: To evaluate in vivo and postmortem the mrgcs system and optic nerve pathology in correlation to circadian functions and cognition in AD and aging. To assess the contribution of a robust mrgcs system and circadian photoentrainment to longevity. To investigate the genetic variability of clock and melanopsin genes in AD compared to controls and in the extreme longevity phenotype. Hypothesis: This proposal is based on three hypothesis: 1) Melanopsin retinal ganglion cell (mrgc) loss and SCN pathology may contribute to circadian dysfunction in AD and aging. This hypothesis is based on the knowledge that mrgcs have a crucial role in circadian photoentraiment and cognitive functions, and that they are lost with aging. 2) A robust mrgcs system and the consequent circadian photoentrainment associate to and predict successful aging, i.e. longevity. This hypothesis is based on the knowledge that circadian misalignment and indirectly sleep dysfunction may lead to reduced life expectancy, increased risk of cancer and dementia. 3) Genetic variants of clock and melanopsin (OPN4) genes may influence circadian functions and be associated with susceptibility to AD or longevity. This hypothesis is supported by the pleomorphic role of clock genes and circadian functions on cognition and healthiness. Preliminary data: The PI conducted a pilot study, based on OCT and histological investigations in AD, which showed an age-related optic neuropathy and age-independent, possibly primary, loss of mrgcs in AD patients compared to age-matched controls. This is strongly supported by the evidence of abundant amyloid deposition in postmortem AD retinas. Actigraphic recordings showed a fragmentation of restactivity circadian rhythm (increased intradaily variability and reduced relative amplitude) in AD patients compared to controls (La Morgia et al., PhD thesis, manuscript submitted). Moreover, the PI has also evidence that a paradoxically higher number of mrgcs characterizes both AD patients and controls older than 90 years compared to individuals from the previous decade (80s). Overall, these preliminary results suggest that loss of mrgcs may contribute to circadian dysfunction in AD. Furthermore, most intriguingly, the strong complement of mrgcs in individuals over 90s supports 11/07/ / 6

219 GR Melanopsin retinal ganglion cells and circadian rhythms: function and dysfunction in Alzheimer's disease and aging. La Morgia Chiara Biomedical/Biomedica Istituto delle Scienze Neurologiche - Bologna the hypothesis that this may contribute to longevity. Unit 2 has already set the system and the protocol for running functional MRI studies under specific light paradigms including selective stimulation of mrgcs, in collaboration with the world-leading research group for fmri studies of the mrgcs system in humans (Cyclotron Research Center, University of Liège). This protocol is based on the exploration of the mrgc system by either a pure visual paradigm and a combined visual/cognitive paradigm. Materials and Methods Aim 1: -In vivo studies (50 AD and 50 age-matched controls): a) Cognitive, neurologic and ophthalmologic evaluation b) OCT measurement of retinal nerve fiber layer, rest-activity by actigraph and melatonin rhythm, pupillometric c) Brain morphological and fmri studies with blue light stimulation (visual and visual/cognitive paradigm) -Postmortem studies (collaboration with Doheny Eye Institute, UCLA, Los Angeles): a) evaluation of the presence of optic neuropathy and mrgcs in retinas and optic nerve cross-sections of 15 AD and 15 age-matched controls. b) evaluation of hypothalamic specimens from 5 AD and 5 age-matched controls. Aim 2: Comparison of the parameters detailed in aim 1 in ultranonagenarians compared to the previous decade. Aim 3: The genetic variability of clock and melanopsin (OPN4) genes will be assessed on a cohort of about 500 AD patients and 500 age-and sex-matched controls including nonagenarians, centenarians and semi-super centenarians (>105 years of age). Impact and Translational Implications This project evaluates the biological basis of circadian dysfunction in AD and aging investigating the mrgc system through an innovative multi-layered approach. We will also evaluate if a strong circadian synchronization is a biomarker of longevity. Overall, this will pose the theoretic basis to design light trials to counteract circadian dysfunction in AD and aging and, if our hypothesis will turn to be correct, this may translate into change of designing lightening conditions in daily living. 11/07/ / 6

220 GR Mapping spatial neglect symptoms in the brain: A study combining behavioural data, functional neuroimaging, and computational modelling Nardo Davide Clinical health care research/clinicoassistenziale Fondazione Santa Lucia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Brain Injury and Neurovascular Pathologies - BINP Project Keyword 1: Project Keyword 2: Project Keyword 3: Identification of novel therapeutic targets, neuroprotective agents and therapeutic strategies to prevent and treat neural injury. Spatial neglect fmri Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Neuroimaging Laboratory Patients' selection and recruitment; neuropsychological assessment; behavioural data analysis; fmri data collection; fmri data analyses; scientific communications. 2 Consiglio Nazionale delle Ricerche, Istituto di Scienze e Tecnologie della Cognizione Laboratory of Autonomous Robotics and Artificial Life Development and training of neural networks; computational modelling; simulations of humans behavioural performance in artificial systems. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Simione Luca Consiglio Nazionale delle Ricerche, Istituto di Scienze e Tecnologie della Cognizione 2 Development and training of neural networks; computational modelling; simulations of humans behavioural performance in artificial systems. 18/05/ Bordier Cecile Santa Lucia Foundation Non-conventional fmri analyses (Independent Component Analysis; Inter- Subject Correlations). 22/11/ /07/ / 6

221 GR Mapping spatial neglect symptoms in the brain: A study combining behavioural data, functional neuroimaging, and computational modelling Nardo Davide Clinical health care research/clinicoassistenziale Fondazione Santa Lucia Background and Significance Brain damage following stroke is one of the main causes of invalidity in western countries. A common consequence of stroke in the right hemisphere is spatial neglect, a complex syndrome associated with a reduced ability to orient toward the contralateral side of space. It is becoming increasingly clearer that neglect is not a monolithic disorder with a unique neural substrate, but rather a collection of symptoms (e.g., spatial bias towards the ipsilateral space, impairment in spatial orienting in presence of multiple stimuli, perceptual extinction, etc.) with different neural correlates. In clinical practice, neglect is routinely assessed with simple but well-proven tests. Some studies have shown that these tests assess different symptoms of neglect, which seem to rely on different brain regions. Standard tests typically include cancellation tasks, which relies on the visual search of target-stimuli among distractors, and bisection tasks, which relies on an intact internal representation of space. These tests are normally used jointly in order to make a general diagnosis of neglect, and to characterize its severity as a whole syndrome. However, insofar neglect might be conceived as a collection of different symptoms (which might be present to a certain extent), the assessment of the various symptoms should be done by considering each test (or group of tests) separately. This would improve the effectiveness of neuropsychological assessment and therapeutic intervention. Specific aims Aim 1: Aim 2: Aim 3: The present project aims at combining behavioural measures (scores on well-proven neuropsychological tests routinely used in clinical practice), functional neuroimaging (fmri), and computational modelling to investigate the neural substrate of distinct neglect symptoms. Our first aim will be the clustering of various neuropsychological tests according to the specific neglect symptoms they measure. Our second aim will be the identification of brain regions underlying distinct neglect symptoms in patients, and groups of neuropsychological tests in controls. This will be done by using task-related fmri. Moreover, restingstate fmri will be used to investigate any alteration in the functioning of key brain networks in neglect patients as compared to controls. Both standard hypotheses-driven, and non-conventional data-driven fmri data analyses will be used. Third, we will use computational modelling to complement the fmri results, and test specific hypotheses on neglect symptoms and their neural circuitry. This will imply two steps: 1) using Neural Computational Modelling (i.e., a "top-down" computational approach) to simulate the "neural architecture" identified by fmri results; 2) training an Artificial Neural Network (i.e., a "bottom-up" computational approach) with the same cognitive tasks performed by humans, to make comparisons with both patients and controls' performance. Hypothesis: Standard neuropsychological tests should be clustered as related to at least three different neglect symptoms: 1) spatial bias towards the ipsilateral space (e.g., line bisection, Wundt-Jastrow test, o'clock test, etc.); 2) impairment in orienting towards the contralateral space in presence of competing stimuli (e.g., letter cancellation, bells test, copying task, etc.); 3) visual extinction. The scores of the clustered tests will tend to correlate together, but will not necessarily correlate with scores of tests belonging to different clusters. The spatial bias towards the ipsilateral space will be related to lesions located in the superior parietal cortex and/or in perisylvian regions. The impairment in orienting towards the contralateral space in presence of competing stimuli will be associated with damages to the inferior frontal cortex. Visual extinction will be associated with lesions located in the inferior parietal cortex and/or in the temporo-parietal junction. As compared to controls, neglect patients will show significant alterations in resting state networks activity. Both computational modelling approaches will be capable of reproducing aspects of the behavioural performance of neglect patients and controls. More specifically, the removal or inhibition of the superior parietal, inferior frontal, or inferior parietal nodes simulate the presence of different neglect symptoms in 10/07/ / 6

222 GR Mapping spatial neglect symptoms in the brain: A study combining behavioural data, functional neuroimaging, and computational modelling Nardo Davide Clinical health care research/clinicoassistenziale Fondazione Santa Lucia patients: spatial bias towards the ipsilateral space, impairment in orienting towards the contralateral space in presence of competing stimuli, or visual extinction, respectively. Preliminary data: N/A Materials and Methods Neglect patients, brain-damaged, and healthy controls will be assessed with standard neuropsychological tests. Participants will undergo two fmri sessions: resting-state, and cognitive-task in which they will be presented with computerized versions of the tests. fmri data will be analysed with both hypotheses- and data-driven techniques. Independent Component Analysis separates sources from a complex mixture of signal and noise, identifying different resting state networks. Inter-Subject Correlation (designed for cognitive-task data) reveals different sets of functionallyrelated areas during a tasks. fmri analyses will be complemented by computational modelling to test hypotheses on neglect symptoms and their neural correlates. Neural Computational Modelling will be used to simulate the neural architecture inspired by fmri activity patterns. Artificial Neural Networks will be trained with the same cognitive tasks performed by our subjects, to make comparisons with humans performance. Impact and Translational Implications The project has a strong clinical relevance and uses an innovative integrated methodological approach with a major impact on diagnostics and on the identification of new biomarkers and predictors of therapeutic outcome in neglect. This should result in an improved and more effective assessment capability, useful in orienting clinical decisions on therapeutic intervention, for instance by focussing rehabilitation protocols on specific symptoms, instead of treating neglect as a whole. 10/07/ / 6

223 GR Multimodal experimental and theoretical approach for the study of the Spinal Cord in healthy and diseased subjects Fratini Michela Biomedical/Biomedica Fondazione Santa Lucia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Surgical Sciences, Biomedical Imaging, and Bioengineering Medical Imaging - MEDI Project Keyword 1: Project Keyword 2: Project Keyword 3: In vivo strategies and methods for characterizing tissue, and distinguishing between normal and pathologic states, based on estimates of biophysical, biomechanical, bioelectrical, biochemical, metabolic, perfusion/diffusion, or other properties. Spinal Cord fmri Vascular Model Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Magnetic Resonance for Brain Investigation Laboratory (MARBILab) MAIN UO (PI). Neuroimaging: definition of experimental protocols, acquisition and analysis of spinal cord images both by functional Magnetic Resonance (fmri) and by X-ray phase contrast tomography. Development of new analytical approaches and diagnostic tools for the study of behavior and physiological mechanisms that cause dysfunction and disease. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Di Nuzzo Mauro COLLABORATOR for the development of algorithms for the analysis of the vascular network and of vascular Models for the Spinal Cord BOLD response. 21/03/ /07/ / 5

224 GR Multimodal experimental and theoretical approach for the study of the Spinal Cord in healthy and diseased subjects Fratini Michela Biomedical/Biomedica Fondazione Santa Lucia Background and Significance Functional magnetic resonance imaging (fmri) is the tool of choice for the study of brain activity. Spinal cord (SC) fmri is technically more challenging than brain fmri, which prevented the characterization of the relation between SC physiology and the blood-oxygenation level dependent (BOLD) signal. The present project aims at overcoming some of the limiting factors for the use of SC-fMRI in clinical practice. The project will tackle the study of the SC vascular network (VN) by exploiting Synchrotron X-ray Phase Contrast µ-tomography (SXrPCµT). Detailed VN morphology in the SC will be extracted developing algorithms capable of exploiting high-resolution three-dimensional SXrPCµT data previously acquired on ex-vivo mouse SC. The obtained structural information will be incorporated into physiological models of neurovascular and neurometabolic coupling, in order to provide an accurate spatial estimate of the SC hemodynamic response function. Refining the currently available models of brain fmri will provide useful insights to predict the SC BOLD response to specific afferent/efferent activations. These models will be used to design fmri experiments on human healthy subjects as well as on patients suffering of various neurodegenerative diseases, such as multiple sclerosis (MS), where secondary vascular effects have been implicated. This experimental approach will aid preclinical investigations of SC pathologies and/or specific medical treatments. Specific aims Aim 1: Aim 2: Aim 3: We plan to develop algorithms that detects the vessels, neurons and axons by means of their characteristic geometry from the high resolution SXrPCµT images. SXrPCµT is able to reveal the micro-architecture of the SC with unprecedented spatial resolution and without the use of contrast agents, a hopeless task with conventional X- ray techniques. The images have been obtained as preliminary data (see below). The algorithms will allow extracting individual vessel features, such as length, diameter and tortuosity. The purpose of this study is to further delineate the micro-vascular and VN along the entirety of the mouse SC. In particular, the attainment of a 3D imaging and the quantification of the vascular network of the SC should considerably improve our knowledge of the effects of pathological processes, such as SC injuries and neurological diseases, where vascular secondary effects have been implicated. We plan to improve the classical model of the BOLD signal. The latter is based on several assumptions that only hold for the specific geometry of the brain (i.e. cerebral cortex) vasculature and whose validity may not be preserved in the case of the SC. The BOLD signal is the result of vascular and metabolic processes that strongly depend on the features of the tissue. We sought to incorporate structural information into previously published models of neurovascular and neurometabolic coupling. By performing explicit theoretical calculations, based on realistic modeling of the SC VN properties, we will greatly improve our understanding of the BOLD signal of the SC. We plan to transfer the knowledge about the SC physiology to experimental SC fmri. By detailing the biophysical features of the BOLD signal, we plan to perform a controlled motor task to study parametrically the relationship between stimulation strength and functional response in the SC of healthy and diseased subjects. This approach will be useful for the regular application of SC fmri in the clinical practice. SC fmri could be of immediate application in neuroradiology, in particular for the assessment and follow-up of spinal injuries, pain, and neurodegenerative diseases (e.g. multiple sclerosis), as well as in the development and evaluation of new therapies. Hypothesis: The central hypothesis of the present project is that the characterization of the microvascular structure of the SC, within an accurate theoretical account of tissue physiology, will pave the way for the routine use of SC fmri in the clinical practice. 11/07/ / 5

225 GR Multimodal experimental and theoretical approach for the study of the Spinal Cord in healthy and diseased subjects Fratini Michela Biomedical/Biomedica Fondazione Santa Lucia Preliminary data: By X-ray phase tomography, we obtained a simultaneous advanced imaging of the threedimensional vascular and neuronal network of the mouse SC at scales spanning from millimeters to tens of nanometers. We validated the images with conventional histological sections as well as with corresponding images obtained with a commonly used contrast agent. We also obtained preliminary fmri data in the spinal cord during isometric motor task stimulation with varying strength. Materials and Methods The project includes three different methodological phases, supported by subcontracts for technical and informatics support. First, new constraint-based segmentation algorithms will be developed to detect vessels, neurons and nerve fibers by means of their characteristic geometry from the high resolution SXrPCµT images, already obtained in healthy and diseased model mouse SC samples. Second, the morphology of the SC VN, so obtained in the previous phase, will be employed to refine a classic BOLD model developed only for a specific geometry of the VN (i.e. cerebrum). To this end, neurophysiologic and metabolic features of the SC will be incorporated in integrative models of neurovascular and neurometabolic coupling, in order to estimate the SC hemodynamic response function and make predictions on BOLD changes during stimulations. Finally, the models developed so far will be applied to in vivo fmri studies of the human SC physiology in both healthy and diseased subjects. Impact and Translational Implications The present project will provide a multidisciplinary framework combining X-ray advanced imaging techniques, fmri and modeling tools, thereby establishing a novel multimodal method for basic research as well as for clinical applications. The possibility to model and accurately interpret the SC BOLD signal using a realistic vascular geometry will be relevant to better understand several central nervous system disorders like MS, SC traumatic diseases as well as to aid the treatment of SC injuries. 11/07/ / 5

226 Vitamin D in the prevention of subclinical and advanced atheromatosis. BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Arcidiacono Maria Vittoria Biomedical/Biomedica Ospedale infantile Burlo Garofolo LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Vascular and Hematology Atherosclerosis and Inflammation of the Cardiovascular System - AICS Project Keyword 1: Project Keyword 2: Project Keyword 3: Therapeutic strategies; cellular and and animal models for atherosclerosis, restenosis, hyperlipidemia, vascular inflammation, vascular injury, diabetes, autoimmune myocarditis, abdominal aortic aneurysms and vascular calcification Vitamin D Atheromatosis Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Molecular Medicine and Laboratory The PI will be in charge of the animal models for atheromatosis setting and laboratory analyses. The DI laboratories will provide the equipment to perform the planned analyses of circulating (plasmatic) markers, flow cytometry, histological evaluations and statistical analysis. The DI has access to the CBM animal facility for the management of the in vivo murine models. Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 08/07/ / 5

227 Vitamin D in the prevention of subclinical and advanced atheromatosis. BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Arcidiacono Maria Vittoria Ospedale infantile Burlo Garofolo Background and Significance Cardiovascular disease (CVD) is the leading cause of death worldwide and in chronic kidney disease (CKD) patients the risk of mortality from CVD is 15 times over that in the general population. Vitamin D (VD) deficiency (serum 25(OH)vitaminD<15ng/ml) is a newly recognized risk factor for CV lesions in health and a predictor of CV mortality in the CKD population. Indeed, not only it increases vascular calcification through the elevation of serum PTH and PTH-driven mineral/skeletal abnormalities, causing ectopic calcium deposition, but more significantly, it is associated to atheromatous disease. During the atheromatous process, the increase in adventitial vasa vasorum (VV) promotes endothelial dysfunction (early stages) and plaque instability (advances stages). Importantly, the active form of VD, besides suppressing secondary hyperparathyroidism in CKD patients, was reported to suppress neoangiogenesis, endothelial cells proliferation and to counteract the increase/activation of metalloproteinase TACE (TNFa converting enzyme). In this context, by using a murine model of atheromatosis (ApoE-/- mice), the main goal of this proposal is to delineate the cause/effect link between systemic VD status, TACE activation, neovascularization, arterial wall thickening, plaque formation and instability in both health and kidney disease. These studies will contribute to identify key therapeutic targets and optimal VD status to prevent the onset and the progression of atheromatous disease. Specific aims Aim 1: Aim 2: Aim 3: To comparatively analyze the time course for the onset and progression of atheromatosis in ApoE-/- mice characterized by (a) normal or (b) deficient VD levels. Specifically, we will examine the association between markers of systemic inflammation, secondary hyperparathyroidism, classical risk factors for atheromatosis and VD status with the increase in adventitial/intraplaque VV and plaque formation/composition. To determine the effect of VD deficiency on the onset and progression of atheromatous disease in a murine model of CKD. To this end, we will assess the association between markers of renal dysfunction, systemic inflammation, secondary hyperparathyroidism, classical risk factors for atheromatosis and VD status with the increases in adventitial/intraplaque VV, plaque formation/composition. To identify the optimal VD intervention able to prevent/delay the onset and/or progression of atheromatous disease. For this purpose, by using the ApoE-/- mouse models, we will assess the efficacy of supplementation with (a) nutritional VD3 (cholecalciferol) or (b) nutritional plus active VD treatment. Hypothesis: Vitamin D deficiency, both in the population with normal kidney function or in patients with CKD, promotes the increases of the arterial adventitial vasa vasorum, as well as systemic inflammation, inflammatory vascular infiltration and foam cell formation that lead to plaque formation and instability. Therefore, correction of vitamin D deficiency in health and nutritional/active VD in CKD could prevent the onset or slow the progression of atheromatous disease. Preliminary data: The PI's previous studies provided the rational for the interest about the effect of VD deficiency on the onset/progression of atheromatous disease. Indeed, the recent results of the NEFRONA study (2445 CKD patients) demonstrated that levels of 25D<16ng/ml were associated with higher number of carotid and femoral artery plaques. Moreover, a different study of the PI pointed out that the increase in carotid adventitial VV, assessed by contrast-enhanced ultrasound, is an early marker of subclinical atheromatosis. In addition, for the purpose of this project it is important to underline that in previous studies and/or in preliminary experiments, the PI has already established: (a) a protocol for the induction of VD deficiency in balb/c mice, resulting in serum 25D<10 ng/ml; (b) evidence of the vascular damage upon nephrectomization of FV/B mice; (c) a protocol of VD replacement therapy (25D+paricalciol), at the moment, in a uremic rat model. Moreover, the PI has already assessed, in murine settings, markers of (a) systemic inflammation (i.e., monocyte TACE, VCAM, TNFa); (b) secondary hyperparathyroidism (i.e., PTH, calcium, phosphorous); (c) classical risk factors for 08/07/ / 5

228 Vitamin D in the prevention of subclinical and advanced atheromatosis. BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Arcidiacono Maria Vittoria Ospedale infantile Burlo Garofolo atheromatosis (i.e., cholesterol, triglycerides). Materials and Methods The murine models will include ApoE-/- mice: (i) with normal or deficient VD levels fed with a western-type diet (1.25% cholesterol, 16.5% fat) and with normal renal function; (ii) upon induction of 75% nephron reduction (nephrectomy, Nx). Deficient VD levels will be achieved in 3 weeks by a VD deficient diet in addition to endogenous 25D degradation trough i.p. injections of paricalcitol (2 weeks). For Nx, the left kidney will be decapsulated and the upper and lower poles removed by cauterization, leaving half of whole kidney weight. One week later, the right kidney will be removed. Mice will be analyzed at different time points (4, 8, 15, 20 weeks) for assessment of: VV increases, plaque formation and composition (fibrous plaque, unstable vs calcified plaque). Aorta, carotid arteries and kidney will be histologically analyzed. Blood will be drawn for assessment of soluble and cellular markers. Impact and Translational Implications The full delineation of the cause/effect link between systemic vitamin D status and the onset of subclinical atheromatosis, and/or its progression to cause death, is essential to support the therapeutic use of VD interventions for the general population and for chronic kidney disease patients in order to prevent CVD. Moreover, the results deriving from this project will allow the identification of the most sensitive early markers to monitor the efficacy of personalized VD interventions. 08/07/ / 5

229 GR Immunogenic cell death as novel immune response mechanism to EGFR-targeted therapy in Colorectal Cancer Pozzi Chiara Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Immunology Cellular and Molecular Immunology A - CMIA Project Keyword 1: Project Keyword 2: Project Keyword 3: the cellular, biochemical, structural and biophysical, and extra- and intracellular molecular events of T and B lymphocytes and other cells (dendritic and mast cells) involved in the adaptive and innate immune responses. Immunogenic cell death Cetuximab Project Request: Animals: X Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Experimental Oncology D.I. Applicant Institution 2 Istututo Europeo di Oncologia Unità Oncologia Medica Gastrointestinale e Tumori Neuroendocrini Operative Unit 2 Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Ravenda Paola Simona Istututo Europeo di Oncologia Principal Investigator - Operative Unit 2 18/12/ /07/ / 6

230 GR Immunogenic cell death as novel immune response mechanism to EGFR-targeted therapy in Colorectal Cancer Pozzi Chiara Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia Background and Significance Colorectal cancer (CRC) is the third most common cancer worldwide. About 40-50% of newly diagnosed CRC patients will develop subsequent distant metastases. The median overall survival of metastatic CRC (mcrc) patients has recently improved due to the introduction of new targeting agents. These include treatment with an anti-egfr antibody (Cetuximab) in combination with chemotherapy FOLFIRI (5-fluorouracile, leucovorin, irinotecan). Cetuximab is proposed to mcrc patients with a wild type form of KRAS. However, this therapy is effective only on a small proportion of patients. Some conventional chemotherapeutics are able to induce immunogenic cell death (ICD): they stimulate the immune system against cancer, inducing a long-lasting protective anti-tumor response. The first event of ICD is an Endoplasmic Reticulum (ER) stress characterized by the phosphorylation of eif2a and the translocation of calreticulin (CRT) to the plasma membrane of dying cells. Here, CRT serves as an eat me signal facilitating the engulfment of tumor cells by dendritic cells that prime tumor specific cytotoxic T cells. Other secondary signals stimulate antigen processing for anticancer immunity: in particular, a non-histone chromatin-binding nuclear protein (HMGB1). Based on our preliminary data, we propose that treatment with Cetuximab in combination with FOLFIRI (but not FOLFIRI alone) induces ICD. Our aim is to assess whether Cetuximab-induced ICD is responsible for treatment efficacy in mcrc patients. Specific aims Aim 1: Aim 2: Aim 3: We want to confirm that Cetuximab alone or in combination with FOLFIRI induces ICD in tumor cell lines in vitro (Task 1.1) and that this is leading to effective antitumor immunity in vivo (Task 1.2). From our preliminary data we know that Cetuximab induces a peculiar activation of EGFR. In this aim we want to assess whether partial EGFR phosphorylation is responsible for the induction of ICD. Task 2.1: we will identify the phosphorylation profiles of kinases and their protein substrates in treated tumor cells. Task 2.2: we will evaluate whether inhibition of EGFR phosphorylation correlates with inhibition of ICD. Task 2.3: we will investigate whether mutations in the EGFR signalling pathway may also interfere with Cetuximab-induced ICD using an isogenic cellular platform. We will analyse ICD induction in ex-vivo organ culture of Cetuximab-treated colon biopsies from newly diagnosed KRAS wt mcrc patients. We will correlate ICD induction with clinical outcome to evaluate whether it can be responsible for clinical efficacy. Hypothesis: We speculate that one of the mechanisms of action of Cetuximab treatment in mcrc patients is via the induction of immunogenic cell death and that when ICD is induced Cetuximab treatment leads to clinical benefit. Preliminary data: Our preliminary results, performed on 12 CRC cell lines (9 KRAS wt and 3 KRAS mut), provide evidence, for the first time, that an anti-egfr antibody treatment (Cetuximab) alone or in combination with a chemotherapeutic regimen (FOLFIRI) directs dying cells towards an ICD process characterized by 3 main mandatory modules: ER stress response (eif2a phosphorylation), phagocytosis by monocyte-derived dendritic cells (modcs) and alarmin secretion, in particular HMGB1. However, not all cell lines wt for KRAS displayed the same response. Importantly, Silac analysis performed on Cetuximab treated tumor cells confirmed that the eat me signal Calreticulin (CRT) is translocated to the cell surface in combination with other molecules, including ERp57. To further confirm that Cetuximab induced ICD in vivo, we generated a mouse CRC cell line (CT26) expressing human EGFR that, in response to Cetuximab plus FOLFIRI, undergoes ICD in vitro. Our preliminary data indicate that these dying cells can be used to successfully immunize mice, but these experiments have to be repeated also in an orthotopic transplantation model. Finally, we observed that in cell lines that undergo ICD, Cetuximab blocked the pathways leading to Akt and ERK1/2 10/07/ / 6

231 GR Immunogenic cell death as novel immune response mechanism to EGFR-targeted therapy in Colorectal Cancer Pozzi Chiara Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia phosphorylation, while inducing EGFR phosphorylation. Hence Cetuximab is uncoupling the activation of the signaling pathway induced via EGFR phosphorylation, suggesting that Cetuximabinduced ICD may be the effect of an alternative activation of the EGFR. Materials and Methods CRC cells will be treated with Cetuximab,Folfiri or the combination of the two for 4h and also with AG1478, PP2 or SU6656. Phospho-eIF2a will be analysed by WB. Translocation of CRT and ERp57 in cell lines and in ex vivo drug treated colon biopsies from newly diagnosed KRAS wt mcrc patients will be analysed by FACS. Phagocytosis: drug treated cells will be stained with DDAO and incubated for 2h with CFSE labeled modcs.the percentage of DDAO positive modcs will be measured. HMGB1 will be tested in supernatants by ELISA. Phosphorylation profiles in drug treated CRC cells will be analysed by human phospho-kinase antibody array. Balb/c mice will be vaccinated s.c. with drug treated hegfr-ct26 cells. After 10 days CT26 cells will be inoculated in the contralateral flank or into the distal colon. Tumor growth and survival will be analyzed. RNAseq analysis of ex vivo drug treated colon biopsies and PBMCs collected from the same mcrc patients before and during the therapy will be performed. Impact and Translational Implications Since there is a significant clinical need for the identification of biomarkers that better predict patient response to anti-egfr therapy, this study has strong relevance to cancer treatment. The overall impact of this study, characterized by a strong translational value, is expected to be of immediate clinical benefit.it will allow the clinicians to plan better therapeutic protocols combining or not Cetuximab with chemotherapy and stratify patients avoiding an ineffective and expensive therapy. 10/07/ / 6

232 GR Peripheral blast clearance analysis during induction treatment in patients with acute myeloid leukemia Mannelli Francesco Clinical health care research/clinicoassistenziale Toscana LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Oncology 2 - Translational Clinical Clinical Oncology - CONC Project Keyword 1: Project Keyword 2: Project Keyword 3: Clinical therapy trials including surgical intervention, chemotherapy, radiation therapy and radiopharmaceuticals, combined modality therapy, immunotherapy (antibody and cellular), vaccine and gene therapy, and therapy with biological response modifiers. acute myeloid leukemia peripheral blast clearance Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Azienda Ospedaliero-Universitaria Careggi, DAI Oncologia - SOD Ematologia Conception and design, clinical management of patients, flow cytometry, data collection and interpretation Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date Background and Significance Acute myeloid leukemia (AML) is an heterogeneous disease with wide differences in biology and prognosis. The treatment consists of 2 phases: induction and consolidation. The primary target of induction is complete remission (CR), a requisite for long term survival; once obtained, the consolidation phase deals with lowering the relapse risk. The gold standard of induction is the so-called "3+7" course, able to produce a CR rate of 55-65%. The lack of early prognostic factors has prompted physicians to adopt intensified induction, in order to improve the results of "3+7" course. Main clinical trials have showed that: i) it determines a benefit only in subgroups of patients due to an higher leukemic cells killing counterbalanced by higher toxicity; ii) there is an "intensification limit" beyond which there is increasing toxicity without efficacy. Thus, intensification is an effective tool but, given the heterogeneity of AML, if uniformly applied it is not appropriate. In order to assess AML chemosensitivity in a relevant clinical time, we evaluated the clearance of peripheral blasts (PBC) during induction chemotherapy. Preliminary data by our experience have shown that PBC provides a prediction of CR achievement and duration, being a sort of "in vivo" chemosensitivity test. Specific aims 10/07/ / 5

233 GR Peripheral blast clearance analysis during induction treatment in patients with acute myeloid leukemia Mannelli Francesco Clinical health care research/clinicoassistenziale Toscana Aim 1: Aim 2: PBC-driven treatment of patients affected by AML. PBC is the biologic platform for the future phase II clinical trial by Northern Italy Leukemia Group (NILG) for the treatment of AML patients. The primary aim is to improve the global prognosis, as expressed by overall survival. Cut-off refinement and subgroup analysis. The availability of a large cohort of patients will provide: a) identification of PBC cut-offs beyond which the patient has good prognosis; these patients might avoid highly toxic therapies; b) identification of PBC cut-offs below which the patient has poor prognosis and thus might be immediately switched to experimental treatment. Aim 3:. Hypothesis: By the application of PBC, we conceive to customize therapeutic strategy. Patients with low PBC would be immediately switched to intensification of induction and very early allocated to allogeneic transplant. Such an approach is expected to determine an high CR rate (at least comparable with the rate of an intensified induction) but with lower overall toxicity. Furthermore, this approach would provide some crucial information about chemosensitivity and relapse risk. Preliminary data: PBC study has been embedded within multi-center Northern Italy Leukemia Group (NILG) AML/02-06 protocol (Eudract code: ). Since 2007 to 2012, 151 out of 178 enrolled patients (84.8%) were assessed. PBC was expressed as the ratio between absolute leukemia-aberrant immune-phenotype (LAIP) bearing cells at baseline and at day 4 converted to a logarithmic scale. Median PBC was 1.9 ( ). Out of 151 patients, 108 (71.6%) achieved CR after one single induction course, 36 (23.8%) were refractory; in 7 cases (4.6%) an early death occurred. According to response, we observed no difference as concerns LAIP+ cells at baseline between responders and refractory patients, whereas the latter ones showed a significantly higher amount of LAIP+ cells at day 4 and consequently a markedly lower PBC value (1.0 logs) compared to the former patients (2.3 logs; p<0.0001). In order to define the cut-off that could discriminate patients' outcome at best, ROC curves were drawn and the most accurate value was 1.5 logs. By the application of this cut-off, we separated patients in two groups: 55 patients had PBC </=1.5 (PBC-low); 96 patients showed PBC > 1.5 (PBC-high). The outcome (as expressed by CR rate and event-free survival) of these two categories of patients was significantly different. The study of PBC within NILG AML protocol leads us to the following statements: a) PBC is a novel, powerful and very early predictor of CR achievement and outcome; b) the cut-off PBC value that best separates patients with significantly different prognosis is set at 1.5 at day 4 of therapy; c) PBC platform is reliable and feasible in a multicenter setting, making information available within a routine clinical context. Materials and Methods Patients: About 400 patients with a diagnosis of untreated AML are expected to be recruited by NILG in 3 years. Flow cytometry: The analysis of the immunophenotype will enable each NILG center to establish for each patient the most useful LAIP. We plan to quantify LAIP+ cells at 2 time-points during induction: day 1 (baseline value, immediately before therapy) and day 4. Each center will transmit Flow Cytometry Standard (FCS) files by a web-based procedure to coordinating center (Firenze) which will provide PBC data analysis in real time. Web-based transfer of files, remote data-entry (RDE) on electronic case report forms and statistical analysis will be outsourced. Treatment: patients having low PBC will be immediately switched to intensification of induction based on high doses cytarabine. Furthermore, PBC would allow a very early planning of allogeneic transplant, whose outcome is largely known to be influenced by timing. Patients showing high PBC would continue standard therapy. 10/07/ / 5

234 GR Peripheral blast clearance analysis during induction treatment in patients with acute myeloid leukemia Mannelli Francesco Clinical health care research/clinicoassistenziale Toscana Impact and Translational Implications A PBC-driven approach allows to optimize currently available therapies for AML cure. Specifically it will imply an increase of CR rate after a single cycle and consequently an higher proportion of patients receiving an allogeneic transplant in first CR. As such, we can expect an overall reduction of chemotherapy courses and consequently of global toxicity. Therefore, it is likely to obtain on one hand an improvement of patients' outcome and on the other hand a reduction in healthcare costs.

235 The sense of number in Developmental Dyscalculia BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Anobile Giovanni Clinical health care research/clinicoassistenziale Fondazione Stella Maris LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Developmental Brain Disorders - DBD Project Keyword 1: Project Keyword 2: Project Keyword 3: Developmental disorders: Mental retardation, learning disabilities, specific language impairment, dyslexia, autism, cerebral palsy, sudden infant death syndrome - SIDS, and other relevant disorders. Developmental Dyscalculia Numerosity perception Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Fondazione Stella Maris, Department of Developmental Neuroscience 2 Fondazione Stella Maris, Department of Developmental Neuroscience Fondazione Stella Maris, Department of Developmental Neuroscience DI fictitious unit, system bug (FAQ B 457) Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Gasperini Filippo Fondazione Stella Maris, Department of Developmental Neuroscience 2 3 Cicchini Guido Marco Neuroscience Institute, National Research Council, Pisa, Italy; researcher 4 Frijia Francesca Fondazione Toscana G. Monasterio MRI Unit researcher collaborator 24/11/1975 researcher collaborator 23/01/1976 researcher collaborator 13/09/ /07/ / 6

236 The sense of number in Developmental Dyscalculia BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Clinical health care research/clinicoassistenziale Anobile Giovanni Fondazione Stella Maris Background and Significance Developmental dyscalculia (DD) is a severe disorder, which disrupts the learning of mathematical concepts and quantities representation, despite normal intelligence. Its prevalence is 5-7%, similar to dyslexia. While dyslexia has been largely investigated, DD has received very little attention, even though its consequences are as severe as those of dyslexia. Figures for Italy are not available, but it is estimated that dyscalculia costs the UK 2.4 billion per year. From 2000 to 2010, the NIH spent $107 million funding dyslexia research but only $2.3 million on dyscalculia. Dyscalculia is clearly socially and economically important, and grossly under-funded. In recent years much progress has been made in understanding how humans process numbers, using imaging and psychophysical tools. One of the more powerful techniques has been to study mapping number onto space, using the numberline task. Mathematical ability has been shown to correlate with logarithmiclike non-linearities in the numberline, with dyscalculics performing extremely poorly. Our group has recently suggested a novel explanation for the non-linear numberline, suggesting that it reflects recalibration by the system, and this emerges as serially dependencies (Cicchini et al, PNAS, 2014). We aim to adapt the techniques to study DD within Italian school-age children. We expect the results to open new perspectives on the development of numerical cognition, with benefits for DD identification and treatment. Specific aims Aim 1: Aim 2: Aim 3: We aim to assess whether children with dyscalculia show typical behavioural and neural (imaging) signatures of numerosity adaptation (Burr et al, Current Biology, 2008), and examine how this relates to the acquisition of math skills. The second aim is to measure serial-dependencies in numerosity perception (Cicchini et al, PNAS, 2014) during typical and atypical development (DD) of math achievement. We also aim to characterize this phenomenon at brain level by imaging techniques. The third aim is to design and pilot-test novel rehabilitation training targeted at improving perceptual coding of numerosity. We aim to assess the effect of training on numeracy, and its generalization to every-day quality of life (e.g. academic performance). Training-induced modifications will also be tracked at brain level by imaging techniques with a pre-post procedure. Hypothesis: Adaptation and serial-dependencies are both signatures of a well-tuned (Pellicano et al, Current Biology, 2007), adaptive and efficient (Cicchini et al, PNAS, 2014) neural system. We expect to find behavioral and brain (imaging) abnormality on both of these phenomena in DD children. Preliminary data: We measured numerosity adaptation, with a paradigm similar to Burr (Current Biology, 2008), on 15 typical developing children (mean age 11, 7-14 y/old) and on a single child with dyscalculia (12.6 y/old). Control children, after been exposed for several second (3 secs) to a stimulus containing a high number of items (80), highly underestimate the second presented stimulus (mean effect 38%, ranging from 20 to 56%). However, the dyscalculic child showed no adaptation at all! In a recent study we demonstrated strong serial dependencies in numerosity perception in children (68 typical children, 8-11 y/old) (Cicchini et al, PNAS, 2014). When asked to place a numerical quantity (set of objects) on a line demarcating a numerical range (numberline), the responses strongly depend on the stimuli time history, with a strong relationship (r=0.20) between numberline mapping of the current trial and the numerosity of the previous trial. This strategy is advantageous, as results in reducing errors. It is important to examine this effect in dyscalculia: differences in serial dependency could provide strong insights into the mechanisms underlying developmental dyscalculia. 10/07/ / 6

237 The sense of number in Developmental Dyscalculia BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Clinical health care research/clinicoassistenziale Anobile Giovanni Fondazione Stella Maris Materials and Methods Psychophysical tasks will be child-friendly, non-invasive and computer-based. Numerosity adaptation will be tested with a comparison task (Burr et al, Current Biology, 2008) asking subjects to judge which of two briefly presented ensembles contain more items, both in baseline conditions and when preceded with a numerous adaptation stimulus. Serial dependencies will be measured with a numberline task (Cicchini et al,pnas, 2014). Children will decide where a numerosity have to be placed on a line demarcating a number range. We will statistically measure if the responses to the actual stimulus depend on the stimuli presented before. Math skills and IQ will be measured by standardized neuropsychological batteries (e.g. WISC). To assess neural activities, MRI scanning (3 T) will be proposed. We plan recruiting at least 100 DD participants (7-14 y/old) and 100 controls (pre-school to 14 y/old). Trainings will make use of games-like activities targeting numerosity perception. Impact and Translational Implications On 100 school-age children, 5 to 7 show difficulties in learning math. Low numeracy is a substantial cost to individuals and nations ( 2.4 billion per year in the UK). Improving numeracy could dramatically improve economic performance (OECD, Paris, 2010). Thus we expect that our research could have a profound socio-economic impact. Our project can also open new lines of neuroscience research on numeracy development, leading to the set-up of tools for DD early identification and treatment. 10/07/ / 6

238 GR Pain in knee osteoarthritis, a placebo-controlled randomized clinical trial for a new potential therapeutic approach: magnetic resonance guided focused ultrasound surgery (MRgFUS) Bazzocchi Alberto Clinical health care research/clinicoassistenziale Istituto Ortopedico Rizzoli LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Infectious and immunological diseases Project Classification IRG: Project Classification SS: Surgical Sciences, Biomedical Imaging, and Bioengineering Medical Imaging - MEDI Project Keyword 1: Project Keyword 2: Project Keyword 3: Image-guided interventions in integrated diagnostic and therapeutic systems. High-Intensity Focused Ultrasound Ablation, MRgFUS Pain, Osteoarthritis Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Diagnostic and Interventional Radiology Coordination, enrollment, imaging assessment, and treatment 2 The Rizzoli Orthopaedic Institute 3 The Rizzoli Orthopaedic Institute Investigators, Institution and Role in the Project Laboratory of Immunorheumatology and Tissue Regeneration Biological and laboratory analysis Laboratory of Biomechanics and Enrollment, clinical evaluation and management Technology Innovation, 2nd Orthopaedic and Traumatologic Clinic Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Manferdini Cristina The Rizzoli Orthopaedic Institute biological and laboratory analysis; collection, analysis and publication of data 05/04/ Di Martino Alessandro The Rizzoli Orthopaedic Institute patients enrollment, management and follow-up; clinical evaluation; collection, analysis and publication of data 11/01/ /07/ / 6

239 GR Pain in knee osteoarthritis, a placebo-controlled randomized clinical trial for a new potential therapeutic approach: magnetic resonance guided focused ultrasound surgery (MRgFUS) Bazzocchi Alberto Clinical health care research/clinicoassistenziale Istituto Ortopedico Rizzoli Background and Significance A major source of chronic pain is osteoarthritis (OA). Pain caused by OA is of huge impact, in terms of quality of life, social and economic burdens. The ageing of the population is even going to augment the problem. The knee is one of the most affected joint (20/30% prevalence in adults). The risk of mobility disability attributable to knee OA alone, affecting general well-being and performance at work, is greater than that due to any other medical condition in people aged 65 and over. The management of knee OA is still a matter of debate. OA disease is heterogeneous and characterized by failure of the synovial joint organ. The determinants of pain in OA are not well understood, but are believed to involve multiple interactive pathways and inflammatory mediators contribute to sensitize nociceptors. An important component of the biological contribution to pain comes from the multitude of tissues within the joint that contain nociceptive fibres and these are the likely sources of pain in OA. Magnetic resonance guided focused ultrasound (MRgFUS) is an emerging technology which enhances the potential efficacy of focused ultrasound with the safety and accuracy of treatment planning and monitoring by MRI. MRgFUS demonstrated to be safe and effective in ablating tissues and in treating pain caused by several medical conditions, including cancer involving bone (bone metastases), with preliminary results coming from benign focal bone lesions (e.g. osteoid osteomas), and OA. Specific aims Aim 1: Aim 2: Aim 3: The primary aim is to assess the efficacy of MRgFUS in the treatment of pain caused by knee OA in patients candidate for unicompartmental medial arthroplasty, by comparing the outcome of the procedure vs. sham treatment with primary endpoint defined as significant clinical improvement at 3 months follow-up according to visual analogue scale (VAS) pain score. The secondary endpoints include: a) to evaluate the outcome in terms of algometric parameters, function and quality of life; b) to evaluate the timing of potential pain drop after MRgFUS, in the first month after treatment; c) to evaluate the pain-free survival and quality of life after MRgFUS in a 12- month follow-up period, with potential cues for development of better patients selection criteria; d) to evaluate the deferred time to arthroplasty; e) to analyze patient-, lesion- and technical-related parameters in order to set rules for treatment approach and future protocols in MRgFUS and to provide new elements in the definition of treatment planning and management; f) to consider the comfort/discomfort experienced by patients during MRgFUS. To evaluate the effects of MRgFUS on the joint organ and to identify potential changes in imaging and biological features in response to treatment. Blood/urine release of inflammatory, bone/cartilage turnover and pain markers will be assessed to establish a correlation with MRgFUS treatment. To understand the impact of selective ablation of nociceptors on OA pain. Analysis of the association between clinical/imaging data and histological samples of resected joint tissues after arthroplasty with histological and immunohistochemistry evaluation of morphology and specific pain/nociceptors and angiogenic markers, useful to establish in situ the real biological effects of the treatment. Hypothesis: Areas of denuded cartilage are related to symptoms, but other bone-related causes of pain include periostitis associated with osteophyte formation, subchondral microfractures, bone attrition and bone angina due to decreased blood flow and elevated intraosseous pressure (reflected on imaging as bone marrow lesions). Synovial causes of pain also include irritation of sensory nerve endings within the synovium from osteophytes and synovial inflammation. MRgFUS may ablate sensory nerve endings (FUS) in a safe, precise, controlled and localized manner (MRI). Preliminary data: MRgFUS has been recently applied for the treatment of pain related to facet joint syndrome and to knee OA. Preliminary clinical experience has been described in two pilot studies, demonstrating the 10/07/ / 6

240 GR Pain in knee osteoarthritis, a placebo-controlled randomized clinical trial for a new potential therapeutic approach: magnetic resonance guided focused ultrasound surgery (MRgFUS) Bazzocchi Alberto Clinical health care research/clinicoassistenziale Istituto Ortopedico Rizzoli safety of the procedure and the potential efficacy while achieving satisfying results in terms of pain relief. Pain molecules and inflammatory factors has been demonstrated as good markers of ongoing pain in knee OA patients. Moreover, sprouting of nerve fibers occurs in subchondral bone and ostheophytes and nociceptors, mainly activated by inflammatory factors or joint movement due to mechanical activation, seem associated to OA pain. Materials and Methods Eighty adult patients with a diagnosis of medial knee OA, symptomatic and candidate for unicompartmental arthroplasty, will be enrolled. Eligible participants will be randomly assigned to interventional (MRgFUS) and control groups (1:1). All patients will undergo imaging examinations (X-ray, MRI, CT and US), VAS, SF12, KOOS, IKDC, Tegner, physical and algometric examinations at different time points during a 12-month follow-up. A rescue medication will be provided. Serum from venous blood and urine will be collected before MRgFUS and at follow-up time points. Inflammatory (IL6, IL8, MCP-1, VCAM-1), bone turnover (DKK-1, leptin, osteocalcin), cartilage turnover (collagen degradation: CTX, COMP) and pain (i.e. substance P, NGF, Neuropeptide) markers will be assessed and correlated with clinical outcomes. Joint tissues will be fixed and processed for histological and immunohistochemical analysis of pain, nociceptor and angiogenic markers (i.e.s-100, NGF, substance P, VEGF, CD31, CD146). Impact and Translational Implications Effective and lasting non-invasive approaches are missing for OA pain. MRgFUS is one of the most advanced tools available today, and seems to be candidate to hold a prominent position in the next clinical generation. This new minimally invasive imaging-guided technique may represent a valid solution for pain caused by degenerative disease, such as knee OA while providing several advantages (no incisions, no radiation exposure, precise and safe imaging guidance; potentially repeatable procedure). 10/07/ / 6

241 GR Identification and characterization of human broadly neutralizing antibodies against human coronaviruses and human parainfluenza viruses Piralla Antonio Biomedical/Biomedica Fondazione Policlinico San Matteo LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Infectious and immunological diseases Project Classification IRG: Project Classification SS: Infectious Diseases and Microbiology Small Business: Non-HIV Anti-Infective Therapeutics - SBN Project Keyword 1: Project Keyword 2: Project Keyword 3: Development and/or testing of novel anti-infective agents or of therapeutic process to fight infective agents using culture systems or animal models. Human broadly neutralizing antibodies Respiratory virus infections Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Microbiology and Virology Department/Molecular Virology Unit 2 Institute for Research in Biomedicine, Bellinzona, Switzerland Immune Regulation Unit project coordination,recruitment of patients, virological analyses Immunological analyses Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Corti Davide Institute for Research in Biomedicine, Bellinzona, Switzerland Identification of neutralizing antibodies; investigation of the seroprevalence and conformational specificity of the human neutralizing antibody response to human coronaviruses and human parainfluenza viruses; development of animal models used to testing the prophylactic and therapeutic efficacy of neutralizing antibodies 26/07/ /07/ / 5

242 GR Identification and characterization of human broadly neutralizing antibodies against human coronaviruses and human parainfluenza viruses Piralla Antonio Biomedical/Biomedica Fondazione Policlinico San Matteo Background and Significance Human Coronaviruses (hcovs) and Parainfluenza viruses (hpivs) are responsible of life-threatening condition such as acute respiratory distress syndrome (ARDS), particularly in immunocompromised patients. Among these two groups of viruses some new emerging viruses such as hcov-sars (Ksiazek et al., NEJM, 2003), hcov-nl63 (van der Hoek et al., Nat Med, 2004), hcov-hku1 (Woo et al., JVI, 2005), and hcov-mers (Zaki et al., NEJM, 2013) have been identified. Unfortunately, there are no specific antiviral therapies available for PIVs and hcovs other than hcov-sars (Ison et al., 2013). In addition, one of the main drawbacks for the success of antiviral therapy is the fast emergence of virus resistance as described for influenza (Piralla et al., JCV, 2013; Samson et al., Antivir Res, 2013). At present, the use of broadly neutralizing antibodies (bnabs) could be represents a valid alternative on the treatment of respiratory infections as demonstrated for influenza, hcov-sars, respiratory syncytial virus (RSV), and metapneumovirus (hmpv) (Corti et al., Science, 2011 and Nature, 2013; Dilillo et al., Nat Med, 2014; Ulbrandt et al., J Virol, 2006; Zhu et al., PNAS, 2007). Indeed, the problematic development of antiviral agents with clinical activity against other respiratory viruses raises the importance on these new biologic antivirals in the treatment of wide range of virus respiratory infections. Specific aims Aim 1: Aim 2: Aim 3: Understand the incidence and clinical relevance of hcovs and PIVs infection in patients hospitalized with acute respiratory distress syndromes. The isolation and characterization of bnabs reactive against hcovs and PIVs species produced by memory B cells of patients during the infection and post-infection (convalescence) period. Development of animal model used to testing the prophylactic and therapeutic efficacy of the neutralizing antibodies discussed in the aim 2. Hypothesis: As previously showed for RSV and hmpv (Corti et al., Nature, 2013; Schuster et al., JID, 2014) the existence of neutralizing antibodies with cross reaction over different viral species was assessed. Despite the variability of hcovs and PIVs some conserved regions are present in the proteins surface. Therefore, the present study will investigate the presence of cross-neutralizing antibodies in two different virus family Coronaviridae and Paramyxoviridae (Genera Rubulavirus and Respirovirus). In addition, the isolated neutralizing antibodies will be also used to understand the seroprevalence and conformational specificity of the human neutralizing antibody response to hcovs and PIVs surface glycoproteins and for structureassisted vaccine design. Preliminary data: bnabs reactive against several and even all variants of the same viral species have been recently described for influenza (Corti et al., Science, 2011), RSV/hMPV (Corti et al., Nature, 2013) HIV-1 (Walker et al., Science, 2009), Dengue virus (Dejnirattisai et al., Science, 2010), hcov-sars (Rockx et al. J. Virol. 2008) and West Nile virus (Nybakken et al., Nature, 2005). Importantly, bnabs fail to select viral escape mutants, a characteristic that is compatible with their development as a universal anti-viral therapeutics. The increased efficient isolation of antibodies combined with novel screening strategies have promoted the development of bnabs for a wide range of viruses. At present, only one licensed humanized monoclonal antibody, palivizumab (PZ), is available in clinical practice against RSV infection (Subramanian KN et al., Ped Inf Dis J, 1998). However, increasing use of PZ in immunocompromised patients such as hematopoietic stem transplant recipient provides opportunities for the emergence of PZ-resistant RSV variants (Zhao X, et al., Virol, 2004). Therefore, the effective use of bnabs in practical clinic remains to be determined together with the study of the potential development of resistance and the surveillance of appearance of these mutants in in the human population. 09/07/ / 5

243 GR Identification and characterization of human broadly neutralizing antibodies against human coronaviruses and human parainfluenza viruses Piralla Antonio Biomedical/Biomedica Fondazione Policlinico San Matteo Materials and Methods Respiratory specimens of patients hospitalized at Fondazione IRCCS Policlinico San Matteo with acute respiratory syndrome will be tested for respiratory viruses by a panel of real-time RT-PCR. Patients positive for hcovs (-OC43, -229E, -NL63, and HKU1) and PIVs (types 1-4) will be included in the present study and blood samples will be collected at 7 and 21 days after onset of symptoms. Respiratory samples will be inoculated in cell culture to isolate the virus strains and complete genome characterization will be performed with next generation sequencing. IgG+ memory B cells will be isolated and tested their ability to neutralize the hcovs and PIVs infection in a microneutralization assay and positive cultures will be collected and expanded. Germline sequences of the isolated antibodies will be determined with reference to the IMGT database. The evaluation of the prophylactic and therapeutic efficacy will be performed in vitro as well as in vivo in an suitable animal models. Impact and Translational Implications The development of bnabs as a biologic antiviral open a new era in the treatment of respiratory virus infections, especially against viruses in which the vaccine or efficacious antivirals are not available. The expansion of treatment options for respiratory viruses is needed to fill the therapeutic gap. The bnabs could be used for prophylaxis of hcovs and PIVs infections in immunocompromised patients or as therapeutic approach in patients admitted to intensive care unit with severe ARDS.

244 GR NEUROMASTER: NEUROnavigated MAgnetic STimulation in patients with mild-moderate Alzheimer disease combined with Effective cognitive Rehabilitation Vecchio Fabrizio Clinical health care research/clinicoassistenziale Casa di cura San Raffaele Pisana LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Alzheimer s disease and other dementias. non-invasive brain stimulation cortical plasticity Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Brain Connectivity Laboratory Principal Investigator - Operative Unit Coordinator 2 Policlinico Universitario A. Gemelli Dipartimento di Geriatria, Neuroscienze, ortopedia (Istituto di neurologia) Operative Unit Coordinator 3 IRCCS -San Giovanni di Dio- Fatebenefratelli Laboratorio di Epidemiologia e Neuroimaging Operative Unit Coordinator Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Vita Maria Gabriella Policlinico Universitario A. Gemelli 2 Parapini Maura Maria Luisa IRCCS -San Giovanni di Dio- Fatebenefratelli Operative Unit Coordinator; she will lead the Operative Unit#2 to perform neuropsychological tests, MRI, EEG recording and TMS to participants. Operative Unit Coordinator; she will lead the Operative Unit#3 to perform neuropsychological tests, MRI, EEG recording and TMS to participants 14/08/ /11/ /07/ / 5

245 GR NEUROMASTER: NEUROnavigated MAgnetic STimulation in patients with mild-moderate Alzheimer disease combined with Effective cognitive Rehabilitation Vecchio Fabrizio Clinical health care research/clinicoassistenziale Casa di cura San Raffaele Pisana Background and Significance The global prevalence of dementia is as high as 24 million worldwide and has been predicted to quadruple by the year AD is the most common cause of dementia. It is characterized by a progressive decline in cognitive function, above all in memory domain. Although medications can temporarily slow the worsening of symptoms and improve quality of life, there is no effective cure to stop or significantly modify the disease progression. Moreover, available pharmacological treatments for AD have limited effectiveness, are expensive, and with significant side effects. For these reasons, the scientific community is investing many resources in order to identify alternative or complementary adjuvant therapeutic strategies. Starting from the hypothesis that AD presents synaptic dysfunctions and altered neuroplasticity, it becomes also clear that the study of the brain plasticity represents one of the most important goals of research in this field and the starting point to develop neurorehabilitation strategies. In particular, the development of novel non-invasive methods of brain stimulation has increased the interest in neuromodulatory techniques as potential therapeutic tool for cognitive rehabilitation in AD. Modern neurophysiological technique allow us to induce and detect cortical plasticity changes. In fact, non-invasive approaches of rtms induce prolonged functional changes in the cerebral cortex while graph theory approaches could reveal this neuroplasticity modulation. Specific aims Aim 1: Aim 2: Aim 3: This project aims to develop a new, non-invasive and entirely innovative therapy for AD. Further specific objective of this project are to evaluate the effects of a TMS combined with cognitive computerized rehabilitation (NeuroAD) in mild-moderate AD patients, compared to both placebo and healthy subjects. The project is also aimed to test the persistence of the effect (in both cognitive and neurophysiologic markers) of this cognitive rehabilitation up to 12 months follow-up. The final aim is to test the incremental effect of TMS compared with cognitive rehabilitation alone using multomodal approach, in both cognitive and neurophysiologic markers. Hypothesis: We wish to explore the long-term offline improvement in overall cognitive functions and neuroplasticity in patients with AD after repeated treatment sessions of a combination of high-frequency neuronavigated rtms delivered over specific brain networks and cognitive training (rtms-cog) involving the same networks, compared to control groups or placebo controlled. We hypothesize that non-invasive brain stimulation modulates neuroplasticity in pathological human brain. The "dual stimulation" (magnetic stimulation and cognitive tasks) could improve and enhance long-term potentiation (LTP) process via synaptic convergence which strengthen the connection between the brain networks especially for learning and memory. We also expect to find in the mentioned biomarkers, a continuum from elderly, mild to moderate AD. Preliminary data: Experimental evidences suggest that TMS can interfere with cognitive performance and learning by influencing synaptic plasticity in the brain (Rossi et al., J Neurosci. 2004). Some proof-of-principlestudies indicate that a computerized cognitive rehabilitation primed by rtms neuronavigated for selective stimulation of task related brain areas for the treatment of AD (NeuroAD system, Neuronix Ltd., Israel) can be successfully used to treat AD patients (Bentwich et al., J Neural Transm 2011, Rabey et a., J Neural Transm 2013). Non-invasive approaches of rtms of the brain induce prolonged functional changes in the cerebral cortex. Several previous studies have shown that the therapeutically use of rtms to improve cognitive performances in AD patients induced short-duration beneficial effects and were not adequately powered to demonstrate therapeutic efficacy (Nardone et al., Int J Alzheimers Dis 2012), but could suggest the use of this "dual stimulation" procedure including rtms and cognitive training. 10/07/ / 5

246 GR NEUROMASTER: NEUROnavigated MAgnetic STimulation in patients with mild-moderate Alzheimer disease combined with Effective cognitive Rehabilitation Vecchio Fabrizio Clinical health care research/clinicoassistenziale Casa di cura San Raffaele Pisana Materials and Methods This is a placebo-controlled randomized clinical trial on the efficacy of the NeuroAD system (Neuronix Ltd, Israel) which delivers computerized cognitive rehabilitation combined with rtms in AD. 72 participants (healthy, mild and moderate AD) will sit in a custom chair fitted with a computer and neuronavigated coil capable of magnetic stimulation to specific brain areas based on the personalized MR template, and will undergo both behavioral and neurophysiological measures (high density EEG analyzed by a modern approach based on the graph theory) before and after each task and after 12 months of follow-up. The protocol will include stimulation and cognitive rehabilitation of 3 cortical areas per day for 6 weeks. rtms will be delivered on the neural network involved in each task. The "dual stimulation" (rtms and cognitive tasks) could improve and enhance long-term potentiation (LTP) process via synaptic convergence which strengthen the connection between the brain networks. Impact and Translational Implications Our proposal would contribute developing new therapeutic options for a chronic and disabling disease such as AD. Integration of multimodal data will provide further evidence on neural substrate of cognitive functions, and on the neuroplasticity enhanced by a non-pharmacological approach. Moreover, this new approach will not interfere -but eventually potentiate- pharmacological therapies and could be applicate to other Central Nervous System diseases, both neurodegenerative and not.

247 GR Neural manual vs. robotic assisted mobilization to improve motion and reduce pain hypersensitivity in hand osteoarthritis: a randomized controlled trial. VILLAFANE JORGE HUGO Clinical health care research/clinicoassistenziale Fondazione Don Carlo Gnocchi LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Methodological, epidemiological, socio-economic, organizational, managerial emerging public health issues related to the above reported areas Project Classification IRG: Project Classification SS: Musculoskeletal, Oral and Skin Sciences Musculoskeletal Rehabilitation Sciences - MRS Project Keyword 1: Project Keyword 2: Project Keyword 3: Rehabilitation strategies related to neural control of movement (including stroke, spinal cord injury, Parkinson's disease) and function (including carpal tunnel syndrome, repetitive stress injuries, low back pain) as well as strategies to prevent additional disabilities. Robotic inervention to restore limb function Mechanisms of exercise in relation to disability Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Unità Operativa di Recupero e Rieducazione Funzionale 2 Università degli Studi di Verona Dipartimento di Riabilitazione Data collection. Clinical and reliability study Data collection. Neurophysiological study Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Picelli Alessandro Università degli Studi di Verona Data collection. Neurophysiological study. 18/07/ Pè Pierpaolo Data collection. 01/04/ Battaglioli Aluena Data collection. 19/06/ imperio grace Data collection. Clinical and reliability study. 29/01/ /07/ / 6

248 GR Neural manual vs. robotic assisted mobilization to improve motion and reduce pain hypersensitivity in hand osteoarthritis: a randomized controlled trial. VILLAFANE JORGE HUGO Clinical health care research/clinicoassistenziale Fondazione Don Carlo Gnocchi Background and Significance Osteoarthritis (OA) is an important cause of pain, neurological symptoms and functional limitation of the hand (Dieppe, 2005). Pain leads to disability for the individual, and for society the disease has significant economic implications (Robinson et al, 2004). Pain in OA is considered a complex integration of sensory and cognitive processes involving several abnormal cellular mechanisms at peripheral and central levels of the nervous system (Dieppe et al, 2007). Furthermore, patients with hand OA who underwent functional brain neuroimaging (fmri) analysis, showed increased activation in the thalamus, cingulate, frontal and somatosensory cortex as a consequence of central sensitization (Sofat et al, 2013). Several studies have reported the outcomes of multimodal approach (Villafane et al, 2012 and 2013) to treatment and presence of generalized pressure pain hypersensitivity in patients with different chronic pain conditions such as fibromyalgia, whiplash associated disorders, lateral epicondylalgia, carpal tunnel syndrome, shoulder or carpometacarpal (CMC) OA. Recent technologies have facilitated the use of robots as tools to assist patients in the rehabilitation process thus maximizing patient outcomes (Bishop et al, 2013). Several groups have developed robotic tools for upper limb rehabilitation of the shoulder and elbow (Reinkensmeyer et al, 1999). These robotic tools assist the patient with carrying out exercise protocols and may help restore upper limb mobility. Specific aims Aim 1: Aim 2: Aim 3: To examine the effects of radial, ulnar and median nerves mobilization vs. robotic assisted on pain in sensitivity and central pain processing as well as on nerve function, analyze the quantitative and qualitative movement of hand in subjects with hand osteoarthritis. To investigate whether bilateral deep tissue hypersensitivity is a feature of patients with hand OA. To investigate the reliability of pressure algometry and manual digital palpation of the radial, ulnar and median nerves checked against ultrasonography and report normative values as compared to healthy controls. Hypothesis: Pain in OA cannot be attributed solely to peripheral nociception, and that modulation by nociceptive processing contributes to the pain experience. The peripherally directed therapies may modulate pain perception bilaterally. This hypothesis opens avenues for future research in the modulation of pain pathways, perhaps offering targets to optimize peripheral manual and physical therapies for pain management in osteoarthritis. Study 1. Neurodynamic techniques are a form of manual therapy directed to the neural structures through positioning and movement of multiple joints (Butler et al, 1991). The robotic tools assist the patient with carrying out movement protocols. We hypothesized that these patients would show hypoalgesia of neural mobilization as compared to robotic assisted mobilization. Study 2. The presence of widespread pressure pain hypersensitivity and the absence of correlation between symptoms and radiological findings suggest that central sensitization mechanisms may play an important role in OA related-pain (Arendt-Nielsen et al, 2010). We hypothesized that these patients with hand OA demonstrate a widespread hypersensitivity to mechanical pressure stimuli. Study 3. There are reports of the reliability of nerve palpation in relation to nerves of the upper limb (Schmid et al, 2009) and very limited data exist in relation to the reliability of lower limb nerve palpation. This further study aimed at approaching the diagnostic accuracy of the examination. Preliminary data: The clinical efficacy of neurodynamic techniques have been demonstrated in patients with thumb CMC OA (Villafane et al, 2012; Villafane et al, 2013). We performed a kinematic quantitative and qualitative analysis of the motor strategies employed by stroke patients when reaching and drinking from a glass as a tool towards advanced neurorehabilitation strategies (Aprile et al, 2014). Prior work 10/07/ / 6

249 GR Neural manual vs. robotic assisted mobilization to improve motion and reduce pain hypersensitivity in hand osteoarthritis: a randomized controlled trial. VILLAFANE JORGE HUGO Clinical health care research/clinicoassistenziale Fondazione Don Carlo Gnocchi we have been examined the injuries to the insula, the thalamus, the basal ganglia, and white matter tracts were significantly associated with severe upper limb post stroke spasticity with fmri (Picelli et al, 2014). We also verified the presence of bilateral widespread pressure pain hypersensitivity in individuals with unilateral symptomatic thumb CMC OA, suggesting that central pain processing mechanisms might be a feature of this pain population (Chiarotto et al, 2013). Materials and Methods Study 1. RCT. 72 patients, aged 18 to 90 years old of both genders, with hand OA will receive a neurodynamic intervention plus exercise or a robotic assisted passive mobilization (Gloreha) plus exercise for 12 sessions over 4 weeks. Outcomes: pain, fmri, electroneurography and motion analysis of hand. All outcome measures will be collected at baseline, immediately following the intervention, and 1- and 3- months following the end of the intervention. Study patients with hand OA and 20 healthy controls will be recruited. Pressure pain thresholds (PPTs) will be assessed bilaterally over the hand, the C5-C6 zygapophyseal joint, the median, ulnar and radial nerves, and tibialis anterior muscle. Study 3. The 3 upper limb nerves will be palpated in 20 patients with hand OA and 20 healthy subjects using PPTs and manual digital palpation checked against ultrasonography-guided localization. Impact and Translational Implications Neural mobilization is a relatively new therapeutic technique that can be easily applied by physiotherapists in different clinical settings in the Health National Service. PPTs in patients with thumb CMC OA could determinate the presence of central sensitization mechanisms. Understanding the mechanisms of pain and motion analysis of hand OA can lead to improve treatment approaches, focusing in the best way therapies. 10/07/ / 6

250 GR High-throughput mirnaome profiling in myasthenia gravis patients: towards the identification of pathogenic mechanisms underlying autoimmunity Cavalcante Paola Biomedical/Biomedica Fondazione Istituto Neurologico Carlo Besta LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroimmunology and Brain Tumors - CNBT Project Keyword 1: Project Keyword 2: Project Keyword 3: The relevant diseases are multiple sclerosis, myasthenia gravis, inflammatory neuropathies and myopathies, infectious diseases of the nervous system, prion disease and nervous system tumors. Autoimmunity Human and viral micrornas Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Clinical Neurosciences/ Neurology IV - Neuroimmunology and Neuromuscular Diseases Unit/ Laboratory of Molecular and Cellular Immunopathology Coordination of the project; collection of biological samples and clinical data; molecular analyses; in vitro and in vivo functional studies; statistical analysis; data elaboration and dissemination 2 Istituto Superiore di Sanita' (ISS) Department of Infectious, Parasitic and Immune-mediated Diseases/Antiinfectious Immunity Unit Virological analyses; data elaboration and dissemination 10/07/ / 6

251 GR High-throughput mirnaome profiling in myasthenia gravis patients: towards the identification of pathogenic mechanisms underlying autoimmunity Cavalcante Paola Biomedical/Biomedica Investigators, Institution and Role in the Project Fondazione Istituto Neurologico Carlo Besta Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 RIZZO FABIANA Istituto Superiore di Sanita' (ISS) 2 Coordination of Unit 2; molecular analyses to assess EBV DNA load and viral transcripts; analysis of viral micrornas and their predicted mrna targets 19/09/ Bonanno Silvia Fondazione IRCCS Istituto Neurologico Carlo Besta/ Resident in Neurology 4 Galbardi Barbara Fondazione IRCCS Istituto Neurologico Carlo Besta/ Bioinformatics researcher Collection of clinical data; expression analysis of human micrornas 07/02/1985 Statistical and bioinformatics analysis 11/04/1976 Background and Significance Myasthenia gravis (MG) is a prototypic B-cell-mediated autoimmune disease. Autoantibodies against the acetylcholine receptor (AChR) are present in more than 80% of patients and cause muscle weakness and fatigability. Current treatments for MG do not lead to complete remission, highlighting the need to better understand the molecular events underlying the disease, in order to develop more specific and effective therapies. A wealth of data implicates the thymus as the main site of autosensitization in MG patients, who frequently present thymic abnormalities, including hyperplasia and thymoma. However, the exact etiologic processes leading to MG are still unknown. Recently, we showed that an active Epstein-Barr virus (EBV) infection is a common feature of MG hyperplastic thymuses, suggesting that EBV might contribute to the immunological dysfunctions causing MG [Cavalcante et al Ann Neurol 2010]. MicroRNAs (mirnas) are key gene expression regulators, whose role in human diseases is rapidly emerging. Viruses, including EBV, are known to drive aberrant expression of human mirnas and encode mirnas that are able to target host genes, thus altering several intra-cellular pathways. MiRNAs have been implicated in innate and adaptive immune system modulation, inflammation, germinal center response and plasma cell differentiation. All these mechanisms are involved in MG, but the contribution of mirnas to the pathogenesis of the disease has never been thoroughly explored. Specific aims Aim 1: Aim 2: Aim 3: To identify thymic and peripheral human mirna/mrna target pairs implicated in immune dysregulation and development of chronic autoimmunity in MG patients. To identify virus-encoded mirnas, and to study their possible effects on host gene targets, in the thymus and/or peripheral blood of MG patients, thus disclosing potential virus-mediated mechanisms of autoimmunity in MG. To test in vivo a mirna-based treatment by targeting selected MG-associated mirnas in the experimental autoimmune myasthenia gravis (EAMG) animal model. Hypothesis: The leading hypothesis of the present project is that a dysregulated mirnaome might underlie the pathogenic mechanisms triggering chronic autoimmunity in MG. Specifically, we hypothesize that the expression profile of human and/or viral mirnas is altered in the thymus and peripheral blood of MG patients, and that the aberrant expression of specific mirnas/mrna targets may contribute to the initiation and perpetuation of AChR-specific autoimmunity. 10/07/ / 6

252 GR High-throughput mirnaome profiling in myasthenia gravis patients: towards the identification of pathogenic mechanisms underlying autoimmunity Cavalcante Paola Biomedical/Biomedica Fondazione Istituto Neurologico Carlo Besta Preliminary data: Growing data indicate that mirna dysregulation is implicated in the pathogenesis of autoimmune diseases [Ceribelli et al. Curr Opin Immunol 2012]. Recent studies showed mirna expression changes in peripheral blood mononuclear cells (PBMCs) and serum of MG patients, suggesting a key role of some mirnas (e.g. hsa-mir-145; hsa-mir-146a; hsa-mir150-5p; hsa-mir21-5p) in the immunopathogenesis of MG [Lu et al. Neurosci Lett 2013; Punga et al. Ann Clin Transl Neurol 2014; Wang et al. J Neuroimmune Pharmacol 2013]. Recently, we obtained preliminary evidence of mirna involvement in MG. Specifically, deep RNA-sequencing on PBMCs of AChR-MG patients and healthy controls, followed by validation of mature mirnas using qpcr, allowed us to identify a significant up-regulation of mir-612, mir-3651 and mir-3654 in MG patients compared to controls (unpublished data). Ingenuity Pathway Analysis (IPA) revealed functional interactions between the identified mirnas and MG-related genes (e.g. CHRNA1, CTLA4, IL-1ß), suggesting a specific link between the three dysregulated mirnas and MG etiology. Moreover, we recently found that two EBV-encoded mirnas, ebv-mir-bart1-5 and -BHRF1-1, were highly expressed in EBV-positive hyperplastic MG thymuses compared to EBV-negative normal thymuses (unpublished data). Interestingly, according to the RepTar prediction program, one of the targets of ebv-mir-bart1-5 is Fas, a gene up-regulated in MG thymocytes which are enriched in autoreactive cells [Moulian et al. Blood 1997]. Moreover, a potential target of ebv-mir-bhrf1-1 is TLR7, key member of innate immunity, that we found significantly up-regulated in B cells of EBV-positive hyperplastic MG thymuses in our recent investigations [Cavalcante et al. Autoimmun Rev 2013 and unpublished data]. Although preliminary, our data strongly support the idea that a global mirnaome profiling in the thymus and peripheral blood of MG patients could provide important new insights into the molecular events underlying autoimmunity in MG. Materials and Methods Subjects: The study will include 100 MG patients, 50 non-mg thymoma patients and 50 healthy controls (for discovery and validation phases). Plasma, PBMCs and thymic fragments will be collected from all patients and controls, with controls being adult cardiopathic donors. Cell suspensions will be obtained from 10 MG and 10 control thymuses. Virological studies: qpcr will be performed to assess EBV DNA load and transcripts in thymuses and PBMCs. MiRNA/mRNA profiling: Small RNA sequencing will be performed on thymuses and plasma by the TruSeq kit using the Illumina MiSeq platform; qpcr will be applied to validate dysregulated mirnas and analyze their predicted mrna targets. In vitro and in vivo functional studies: Functional effects of selected MG-associated mirnas will be analyzed in thymic cell culture models and EAMG rats by using specific mirna mimics or inhibitors. Data analysis: Statistical models will be applied using the R software environment ( 10/07/ / 6

253 GR High-throughput mirnaome profiling in myasthenia gravis patients: towards the identification of pathogenic mechanisms underlying autoimmunity Cavalcante Paola Biomedical/Biomedica Fondazione Istituto Neurologico Carlo Besta Impact and Translational Implications The overall outcomes of this investigation promise to significantly enhance our knowledge on MG etiology, thus opening new scenarios in which host and/or viral mirnas could represent novel targets of more effective therapeutic strategies for MG patients. Our findings could also have relevant implications for understanding the pathogenesis of other B-cellmediated EBV-associated autoimmune diseases, in which targeting critical mirnas could be a valuable therapeutic option.

254 GR Exploring the potentialities of lung ultrasound in pediatric cardiac surgery: an easy, fast, inexpensive, radiation-free, but still surprisingly underutilized diagnostic tool. Giordano Raffaele Clinical health care research/clinicoassistenziale Toscana LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Cardiovascular and Respiratory Sciences Clinical and Integrative Cardiovascular Sciences - CICS Project Keyword 1: Project Keyword 2: Project Keyword 3: Human clinical studies (and appropriate translational animal studies): including pediatric populations, mechanisms and consequences of disease: Investigations may include: coronary physiology and pharmacology, cardiac electrophysiology, regional circulations, hemodynamic studies, cardiac mechanics, and genetic considerations in cardiovascular studies. Disease states can include: cardiac or vascular ischemia, hypertension, diabetes, thyroid disease, atherosclerosis, general inflammation, or hypercholesterolemia. lung ultrasound congenital heart disease Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Fondazione G. Monasterio CNR-Regione Toscana, Pediatric Unit 2 Institute of Clinical Physiology Investigators, Institution and Role in the Project Institute of Clinical Physiology, National Research Council, Pisa, Leader of The Project Responsible of the supervision of Lung Ultrasound Examinations and of staysitical analysis Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Gargani Luna Institute of Clinical Physiology Responsible of the Supervision of Lung Ultrasound Examinations. 2 20/03/ Cantinotti Massimiliano Fondazione G. Monasterio CNR- Clinical responsible of The Project 15/07/1979 Regione Toscana, Pediatric Unit 4 Remoli Ettore Fondazione G. Monasterio CNR- Responsible of x-ray examinations 16/08/1976 Regione Toscana, Pediatric Unit 10/07/ / 6

255 GR Exploring the potentialities of lung ultrasound in pediatric cardiac surgery: an easy, fast, inexpensive, radiation-free, but still surprisingly underutilized diagnostic tool. Giordano Raffaele Clinical health care research/clinicoassistenziale Toscana Background and Significance Lung ultrasound (LUS) is gaining consensus as a non-invasive, radiation-free tool for the diagnosis of many acute and chronic pulmonary diseases at all the ages (1-21). LUS is an easy technique (10,11) that may be performed during routine echocardiography thus reducing time for pulmonary evaluation and implementing the diagnostic process. Pulmonary complications are very common in pediatric cardiac surgery (22-26), representing an important cause of morbidity, prolonged hospitalization and need for repeated examinations. Despite this the use of LUS in cardiac surgery is still limited (7,18,21-24) and its systematic use has never been proposed. So far LUS has been employed only in specific conditions (i.e. evaluation of pleural effusion, diaphragmatic excursion) with several limitations (i.e. lack of cut-off values, quantitative estimation of effusion volume). In contrast, x-ray examinations are over-employed, despite controversies on their real utility in some conditions (i.e. after drainage removal to check pneumothorax). The excessive use of radiography has an important impact in term of radiation exposure and costs (25-26), especially in children where dangerous consequence of radiation have been widely demonstrated (25-26). Therefore, our aim is to test whether the systematic use of LUS may ameliorate the diagnostic accuracy of pulmonary complications in pediatric cardiac surgery with the advantages to reduce times, costs and radiation exposure. Specific aims Aim 1: Aim 2: Aim 3: To evaluate the accuracy of LUS in the diagnosis and disease severity estimation of common complications after pediatric cardiac surgery. To compare diagnostic accuracy of LUS versus traditional X-ray techniques. To establish age and CHD (congenital heart disease) specific parameters to judge pulmonary disease severity by LUS (i.e. cut-off values for the diagnosis of diaphragmatic paralysis; diameters/formulas to correctly estimates pleural effusion volume in children). To evaluate other applications/potentialities of LUS in pediatric cardiac surgery. To evaluate feasibility of LUS at different ages and the effect of various confounders (age, different CHD, associated anomalies, type of surgery). Hypothesis: The extensive application of LUS may improve the diagnostic evaluation of common post-surgical pulmonary complications in pediatric cardiac surgery, reducing times, costs and limiting the X-ray examinations. Furthermore the use of LUS may contribute to a deeper knowledge of physio-pathological mechanisms underlying pulmonary diseases after pediatric cardiac surgery leading to a more prompt and appropiate treatment of such complications. Thus the use of LUS may help to ameliorate the outcome of children undergoing cardiac surgery and rationalize SSN resource, limiting unnecessary, expensive and dangerous x- RAY examinations (29-32). Preliminary data: From December 2013 we started a learning program of LUS to evaluate most common pulmonary complications after pediatric cardiac surgery including pleural effusion, diaphragmatic excursion abnormalities, pneumothorax, obstruction atelectasis, pneumonia, and pulmonary edema. LUS was performed with a linear 9 MHz probe (Philip IE-33) by conventional approaches. Results of LUS has been compared with X-ray that in our Center is usually performed daily in ICU at least for the first 3 post-surgical days. LUS was also performed at every time after drainage removal to check pneumothorax. At the same time X-Ray was also performed (as routine). LUS has been performed in 100 subjects (mean age 5,7 range 0-18 years) after cardiac surgery for CHD. LUS had a good diagnostic accuracy in the diagnosis of diaphragmatic paralysis (AUC=0,93). LUS tended to overestimate pleural effusion severity in compare to X-Ray, however good correlations among volumes estimated by echo (k=0.85) (calculated by max diameter x length) has been found in 9 children undergoing thoracenthesis. Accuracy of LUS in the diagnosis of 10/07/ / 6

256 GR Exploring the potentialities of lung ultrasound in pediatric cardiac surgery: an easy, fast, inexpensive, radiation-free, but still surprisingly underutilized diagnostic tool. Giordano Raffaele Clinical health care research/clinicoassistenziale Toscana pneumothorax was not satisfactory in the first 3 months (AUC=0.70) of our experience but ameliorated in the last 3 months (AUC=0.89). Similar results were obtained also for the evaluation of pneumonia and atelectasis. A new potential application of LUS (never described so far) in the diagnosis of retrosternal clots/hematomas has been also incidentally discovered. LUS allowed us new diagnosis of retro-sternal clots in 6 patients and a better delineation/quantification of clots suspected at echocardiography in other 6 cases. Materials and Methods Design: Observational cross sectional Setting: Single Center. Population: 700 consecutive patients (0-18 yrs) undergoing cardiac surgery at FTGM, Massa in the next 3 years. Inclusion criteria: all subjects with adequate acoustic window. Exclusion criteria: insufficient acoustic window; patients with thoracic deformities, pre-existing pulmonary or diaphragm pathology. Procedure: LUS will be performed pre and post-operatively daily in the ICU and when clinically indicated. LUS will evaluate common pulmonary complications after pediatric cardiac surgery. Results of LUS will be compared with X-ray that is commonly performed daily in ICU at least for the first 3 post-surgical days. LUS will be also performed after drainage removal to check pneumothorax. At the same time X-Ray will be also performed as routine. Statistical analysis: Cohen's kappa coefficient (k) and the ROC AUC statistic to compare LUS and X-Ray examinations. Local ethic Committee approved the protocol. Impact and Translational Implications LUS is gaining consensus as a low-cost, easy, fast and radiation free tool for the diagnosis of many common lung disease. Pulmonary complications are very common in pediatric cardiac surgery. Surprisingly the use of LUS in pediatric cardiac surgery so far has been extremely limited(1).the extensive use of LUS may help to improve the standard of diagnosis and treatment in pediatric cardiac surgery, and to optimize and rationalize resources, by reducing expensive and dangerous X- Ray examination. 10/07/ / 6

257 Efficacy of lymphatico-venous anastomosis alone, autologous vascularized lymphnode transfer alone and combined lymphatico-venous anastomosis and autologous vascularized lymphnode transfer in the treatment of breast cancer-related lymphedema GR Visconti Giuseppe Clinical health care research/clinicoassistenziale Lazio LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Surgical Sciences, Biomedical Imaging, and Bioengineering Surgery, Anesthesiology, and Trauma - SAT Project Keyword 1: Project Keyword 2: Project Keyword 3: Surgical approaches to organ/tissue-specific disease, injury, or repair including minimally invasive and transluminal surgical approaches breast cancer-related lymphedema lymphaticovenous anastomosis and vascularized autologous lymph node transplant Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION UOC Chirurgia Plastica e Ricostruttiva - Policlinico Universitario "Agostino Gemelli" - Università Cattolica del Sacro Cuore - Roma 2 UOC Chirurgia Senologica, Policlinico Universitario "Agostino Gemelli" - Roma UOC Chirurgia Senologica- Policlinico Universitario "Agostino Gemelli" - Università Cattolica del Sacro Cuore - Roma Study conception, methodology, computation, formal analysis, investigation (performing surgeries/ data collection), data management and data analysis Investigation (data collection), data management and analysis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 di leone alba UOC Chirurgia Senologica, Policlinico Universitario "Agostino Gemelli" - Roma data collection, management and analysis 15/02/ Accetta Cristina data collection, management and analysis 13/04/ BARONE ADESI Liliana UOC Chirurgia Plastica e Ricostruttiva, Policlinico Universitario "Agostino Gemelli" - Roma investigation (surgeries and data collection) 15/06/ /07/ / 6

258 Efficacy of lymphatico-venous anastomosis alone, autologous vascularized lymphnode transfer alone and combined lymphatico-venous anastomosis and autologous vascularized lymphnode transfer in the treatment of breast cancer-related lymphedema GR Visconti Giuseppe Clinical health care research/clinicoassistenziale Lazio Background and Significance Breast cancer-related lymphedema (BCRL) is a debilitating and usually underestimated complication affecting from 4 to 41% breast cancer patients. BCRL most often appears after a period of latency. Besides risk factors (obesity, radiation, axillary clearance) which may increase its occurrence, we still cannot predict who and when will develop this disease. Lymphedema patients are usually counseled to start combined physical therapy (CPT) with the aim of alleviating symptoms without curative intents. However, physical therapy are life-long lasting, requires a rigorous self-care to be effective and in cases on inadequate disease management, lymphedema may evolve to a chronic and irreversible status where fluid accumulation is replaced by fibrosis and fat. In the past, different attempts of surgical treatment of extremity severe lymphedema have been reported with unsatisfying results. Recently, the promising early results of new derivative (i.e. lymphaticovenous anastomosis,lva) and reconstructive (i.e. vascularized lymphnode transfer, VLNT) minimal-invasive microsurgical techniques have caught the attention of the surgical community on the treatment of not-irreversible lymphedema. However, the little evidence available limits the understanding of the successes and insuccesses of these techniques. Prospective randomized clinical studies are needed to rule out the indications, to refine surgical strategies in order to establish standard surgical therapy. Specific aims Aim 1: Quantitative assessment (at 6, 12 and 24-month f-up) compared to preop values and contralateral healthy arm by using standard circumferential measurements, perometer and 3D imaging upper limb volume analysis (Vectra XT, Canfield) to evaluate the impact of treatments on limb volume and discontinuation rates of conservative treatment. Aim 2: Aim 3: Qualitative assessment (at 6, 12 and 24-month f-up) compared to preop values and contralateral healthy arm by using near-infrared indocyanine green lymphography to evaluate the functional impact of treatments on lymph flow. Quality-of-life (QoL) assessment (at 6, 12 and 24-month f-up) by using QoL questionnaire and by recording lymphedema-related symptoms (heavy arm feeling, limitation in range-of-motion, dermatitis/cellulitis episodes, neuropathic pain) to assess the effect of treatment on symptoms relief and by recording the days off work/month to estimate the economic burden. Hypothesis: BCRL is a low-output lymphedema caused by an impairment of arm lymphatic drainage after axillary node surgery. LVA are reported to be effective in treating arm lymphedema as the microsurgical bypasses performed from impaired lymph collectors to veins are able to reduce the lymphatic dermal backflow and thus to clinically ameliorate/solve lymphedema. According to promising clinical experiences, VLNT seems also effective in ameliorating the lymph flow in lymphedematous arm. However, it is still not clear how the transplanted nodes may favour this process. It seems that, by stimulating lymphogenesis, VLNT favours spontaneous reconnection between the ligated axillary arm collectors, themselves and axillary veins (lvlnt pedicle and axillary vein), thus restoring the arm lymph flow. Moreover, very recently, it has been advanced the hypothesis that lymphnode seems also to have an active role in lymph flow by acting as pump. In this setting, VLNT may also play a role in active lymph drainage. The aim of this study is to analyze if and when (patients' features) by combining in one surgical procedure lymphaticovenous bypass (LVA) and by favouring the anatomical and functional restoration of the lymphatic flow (VLNT) is more effective than performing these two treatments alone. The baseline control will be the cases randomized for no surgery- 09/07/ / 6

259 Efficacy of lymphatico-venous anastomosis alone, autologous vascularized lymphnode transfer alone and combined lymphatico-venous anastomosis and autologous vascularized lymphnode transfer in the treatment of breast cancer-related lymphedema GR Visconti Giuseppe Clinical health care research/clinicoassistenziale Lazio CPT. Preliminary data: Microsurgical treatment of BCRL is an emerging surgical curative option. In literature there are only 10 case series on LVA and VLNT for BCRL treatment but no randomized clinical trials or comparative studies. The important methodological shortcomings (small number of patient per case series (6 to 24 patients) except Yamamoto's report on 100 LVA), very variable f-up time (6 to 132 months) as well as patients' inclusion criteria, indications and outcome assessment) are compensated by the promising clinical results (limb volume/circumference reduction from 2 to 50% and CPT discontinuation rate from 33 to 100%) which are reasons to further investigate these techniques with methodologically superior studies. Materials and Methods Breast Unit patients (treated by the same group of breast surgeons) with history of breast-cancer treatment including axillary clearance, at least 6 month after completing the adjiuvant radio and/or chemotherapy (if indicated) and free-ofdisease, will be evaluated at the Plastic Surgery Outpatient Clinic with quantitative (standard circumferential measurements, perometer and 3D imaging arm volume analysis (Vectra XT, Canfield), qualitative (near-infrared indocyanine green lymphography) and QoL assessments to stage the lymphedema according to International Society of Lymphology (ISL) and arm dermal backflow (ADB) staging. ISL 1-2 and ADB 1-3 will be randomized for treatments (no surgery-cpt, LVA, VLNT, simultaneous LVA and VLNF). Quantitative, qualitative and QoL assessments will be performed at 6, 12and 24-month follow-up by collaborators blind for patients' history and treatment and statistically interpolated with breast-cancer treatment history and lymphedema risk factors. Impact and Translational Implications Besides promising clinical experiences reported for the treatment of not fibrotic lymphedema with the new derivative (LVA) and reconstructive (VLNT) methods, there is lack of clinical evidence. The impact of this trial is to study with a higher level of evidence the efficacy of these new microsurgical treatments. The translational implications are to understand the indications for this surgery based on patients' data in order to rule out surgical standard of care for BCRL. 09/07/ / 6

260 RApid versus SLOw Withdrawal of antiepileptic monotherapy in two-year seizure-free adult patients with epilepsy (RASLOW Study): a pragmatic multicentre, prospective, randomized, controlled study. GR Ferlazzo Edoardo Clinical health care research/clinicoassistenziale Calabria LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Acute Neural Injury and Epilepsy - ANIE Project Keyword 1: Project Keyword 2: Project Keyword 3: Relevant disorders include stroke/ischemia, epilepsy, spinal cord injury and traumatic brain injury. epilepsy antiepileptic withdrawal Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Centro Regionale Epilessie, Azienda Bianchi-Melacrino-Morelli, Reggio Calabria, Italia 2 Istituto di Ricerche Farmacologiche Mario Negri Dipartimento di Neuroscienze, Laboratorio di Malattie Neurologiche Study Coordinator, patients' recruitment Site start up, remote monitoring, patient randomization, data analysis and interpretation 3 Università di Messina, AOU Policlinico "G. Martino" Dipartimento di Medicina Clinica e Sperimentale, UOSD Farmacologia Clinica Dosage of antiepileptic drugs plasma levels Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Giussani Giorgia Istituto di Ricerche Farmacologiche Mario Negri 2 Italiano Domenico Università di Messina, AOU Policlinico "G. Martino" 3 Gasparini Sara Centro Regionale Epilessie, Azienda Bianchi-Melacrino- Morelli, Reggio Calabria, Italia Collaborator 06/01/1984 Collaborator 26/12/1979 Collaborator 04/06/ /07/ / 5

261 RApid versus SLOw Withdrawal of antiepileptic monotherapy in two-year seizure-free adult patients with epilepsy (RASLOW Study): a pragmatic multicentre, prospective, randomized, controlled study. GR Ferlazzo Edoardo Clinical health care research/clinicoassistenziale Calabria Background and Significance Antiepileptic drug withdrawal may be an option for patients who have been seizure-free for some years. Relapse rate of seizures is highest in the first year following withdrawal, and cumulative probability of maintaining remission is about 40-55% (1). The best withdrawal rate is questionable; in particular, it is unknown whether "rapid" withdrawal is associated with a higher risk of relapse as compared to "slow" withdrawal. Some studies have been conducted only in children, with variable timelines. Hence, we aim to establish if a slow or a rapid withdrawal schedule of antiepileptic monotherapy influences relapse rate in adult patients with focal or generalized epilepsy who have been seizure-free for at least two years. 1) Specchio LM, Beghi E (2004). Should Antiepileptic Drugs Be withdrawn in Seizure-Free Patients? CNS Drugs 18:1-12. Specific aims Aim 1: Aim 2: To establish whether a slow or a rapid withdrawal schedule of antiepileptic monotherapy influence relapse rate in adult patients with focal or generalized epilepsy who have been seizure-free for at least two years. To evaluate differences between groups (slow vs rapid withdrawal schedule) in terms of compliance with withdrawal schedule and of occurrence of status epilepticus, seizure-related injuries and death during patients' follow-up (12 months). Aim 3: - Hypothesis: There are no differences between groups (slow vs rapid withdrawal schedule) in terms of seizure relapse, compliance with withdrawal schedule and occurrence of status epilepticus, seizure-related injuries and death during patients' follow-up (12 months). Preliminary data: A Cochrane review of randomized controlled studies (1) identified a single study of suboptimal quality, that compared a rapid withdrawal schedule (six weeks) with a slow one (nine months) in seizure-free children (2). That study (2) failed to identify significant differences in terms of relapse between the groups. Unit 2 contributed to guidelines by Italian League Against Epilepsy on antiepileptic drug withdrawal (3). Those guidelines conclude that, due to the lack of evidence in literature, further studies are needed to know the optimal withdrawal regimen both in children and adults. 1)Ranganathan LN, Ramaratnam S (2009). Rapid versus slow withdrawal of antiepileptic drugs (Review). Cochrane Database of Systematic Reviews, Issue 3. 2)Tennison M, Greenwood R, Lewis D, Thorn M (1994). Discontinuing antiepileptic drugs in children with epilepsy. A comparison of a six-week and a nine-month taper period. New England Journal Of Medicine;330: )Beghi E, Giussani G, Grosso S, Iudice A, La Neve A, Pisani F, Specchio LM, Verrotti A, Capovilla G, Michelucci R, Zaccara G (2013). Withdrawal of antiepileptic drugs: guidelines of the Italian League Against Epilepsy. Epilepsia 54(S7): /07/ / 5

262 RApid versus SLOw Withdrawal of antiepileptic monotherapy in two-year seizure-free adult patients with epilepsy (RASLOW Study): a pragmatic multicentre, prospective, randomized, controlled study. GR Ferlazzo Edoardo Clinical health care research/clinicoassistenziale Calabria Materials and Methods Adult subjects with a clinical diagnosis of focal or generalized epilepsy, seizure-free on monotherapy for at least two years, will be included. Inclusion will be subordinate to the clinicians' judgment as well as to the patients' consent. Exclusion criteria will be: epilepsy associated with progressive neurologic conditions; history of psychogenic seizures, status epilepticus or of seizure relapse after discontinuation of treatment. Included subjects will be randomized to AED discontinuation following one of two schedules: 1)Rapid withdrawal: reduced by 25% of initial dosage every 10 days until complete discontinuation (total withdrawal time: 30 days); 2)Slow withdrawal: reduced by 20% of initial dosage every 30 days until complete discontinuation (total withdrawal time: 5 months). A 1:1 central randomization will be stratified for type of epilepsy (generalized vs. focal). A sample size of 330 participants has been calculated. The whole duration of the study will be of 36 months. Impact and Translational Implications The demonstration that seizure relapse, compliance with withdrawal schedule and occurrence of status epilepticus, seizurerelated injuries and death do not differ in patients who undergo rapid vs slow AED withdrawal would provide a scientific basis in support of a rapid discontinuation and at the same time would be cost-effective for the National Health System.

263 GR Cardiac preservation with normothermic donor heart perfusion compared to standard cold storage in heart transplantation: a new standard of care to face donor shortage? Sponga Sandro Clinical health care research/clinicoassistenziale Friuli-Venezia Giulia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Cardiovascular and Respiratory Sciences Cardiovascular Sciences Small Business SECS Project Keyword 1: Project Keyword 2: Project Keyword 3: Cardiology, cardiac function, heart failure, myocardial ischemia, reperfusion injury, stents, thrombosis/thrombolysis, imaging, monitoring systems Cardiac transplantation Normothermic donor heart perfusion Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Cardiac Surgery Unit Clinical developement, patients enrollment, donor and recipients evaluation, organ transplantation with OCS or standard cold storage, biopsies, clinical data collection 2 University of Udine Departement of Medical and Biological Sciences Molecular biology and metabolic profiling of blood samples by proton (1H) and phosphorous (31P) Nuclear Magnetic Resonance (NMR). 3 University of Udine Departement of Experimental and Clinical Structural and ultrastructural analyses, Medicine immunohistochemical analyses in light and electron microscopy 11/07/ / 6

264 GR Cardiac preservation with normothermic donor heart perfusion compared to standard cold storage in heart transplantation: a new standard of care to face donor shortage? Sponga Sandro Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Friuli-Venezia Giulia Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Bonetti Antonella University of Udine Structural and ultrastructural analyses, immunohistochemical analyses in light and electron microscopy 2 Mimmi Maria Chiara University of Udine Metabolic profile of blood samples by proton (1H) and phosphorous (31P) Nuclear Magnetic Resonance (NMR). 3 CARAGNANO ANGELA University of Udine Cardiac stem cell culture, immunofluorescence, mycroscopy and molecular biology (analyses for markers of myocardial damage, such as ST2, mir1, mir133a, mir133b, mir499-5p, mir122 and mir375) 31/12/ /11/ /08/1976 Background and Significance Despite advances in mechanical circulatory support and stem cell therapy, heart transplantation (HTx) remains the gold standard treatment of advanced heart failure.however, the shortage of cardiac donors has led to a dramatic drop in the HTx performed worldwide over the last decade.in Italy, HTx decreased from 390 in 1995 to 219 in 2013 and, in the last year, 5.8% of patients died in waiting list.to recruit more organs, less restrictive selection criteria (older patients, longer ischemic time and high inotropic support) are now routinely considered with a higher risk of early mortality. Although universally adopted, cold ischaemic preservation is suboptimal because of low levels of anaerobic metabolism continue to deplete ATP stores and acidosis and myocardial oedema tend to increase beeing cause of diastolic dysfunction.an alternative to cold ischemia is a new method of normothermic donor heart perfusion: the Organ Care System (OCS) developed by TransMedics Inc.The ex vivo preservation of donor hearts in a beating status has potential advantages: it reduces time dependent ischaemic injury, it may resuscitate suboptimal hearts and recruit long distance donors, thus expanding the donor pool, it may allow to perform coronary angiography or echocardiography on donor organs, and it adds a time safety buffer in case of a suspected donor malignancy.although better cardiopreservation using ex vivo perfusion has been demonstrated in animal models, this evidence lacks in patients. Specific aims Aim 1: To ascertain whether the OCS procedure can warrant better myocardium preservation evaluating (i) strustructural and ultrastructural tissue features, (ii) expression rate of markers of myocardial injury, inflammation, and cell death, and (iii) molecular profiling of cardiomyocytes. Aim 2: Aim 3: Monitoring the metabolic profile of blood samples obtained from the coronary sinus to assess the functional state (generic metabolic processes, impaired myocardial energetics, specific cardiac metabolism) after OCS and standard cold ischaemic transportation, by Nuclear Magnetic Resonance (NMR). To demonstrate better outcome of patients transplanted with organs managed with OCS instead of standard cold storage, in terms of perioperative death, clinical, haemodynamic and echocardiographic performance. Hypothesis: We hypothesize that ex vivo normothermic donor heart perfusion with the OCS could offer better myocardial protection by reducing the ischemic time, by providing the metabolic substrate, by reducing the production of 11/07/ / 6

265 GR Cardiac preservation with normothermic donor heart perfusion compared to standard cold storage in heart transplantation: a new standard of care to face donor shortage? Sponga Sandro Clinical health care research/clinicoassistenziale Friuli-Venezia Giulia reactive oxygen free radicals, by avoiding the effect of cooling and rewarming, and by improving the microvascular blood flow. Preliminary data: The overall experience made by our institution with OCS system consist of 17 cases and our center was the only Italian to be part of the PROCEED 2 trial. Results from our experience and from 4 clinical trial (PROTECT 1 AND 2 AND PROCEED 1 AND 2) demonstrated the non inferiority in term of both safety and efficacy of ex vivo normotermic preservation compared to standard cold storage. Our group has a great experience on heart transplantation, organ preservation, and donor expansion selection criteria as presented in many publications. Materials and Methods Thirty heart transplants will be randomized 1:1 employing standard cold storage vs OCS perfusion. Cold storage patients will be treated with St Thomas cardioplegia. In the OCS group after connection of the aorta and the pulmonary arteries to the OCS cannulae, the heart will be perfused with donor blood mixed with a priming solution. Blood returning from the coronary sinus through the right heart will flow in an oxygenator and delivered into the aortic root by a pulsatile pump. Cardiac preservation will be assessed by the aortic pressure, coronary flow and lactate profile.clinical, hemodynamic, echocardiographic, laboratory data will be recorded. Myocardium will be sampled from the septum: at time of organ harvesting, before implantation and after reperfusion. Histological, immunohistochemical, EM and cell culture studies will be performed. Blood samples, collected before organ retrieval and at declamping time from coronary sinus, will be analyzed by NMR spectroscopy, RT-PCR and ELISA. Impact and Translational Implications In case of better myocardial preservation with OCS, this system might be considered the gold standard in all patients undergoing heart transplantation to prevent primary graft failure, early mortality and possibly expand the donors pool through the recruitment of marginal donors. From an economical point of view the widespread use of OCS will reduce the cost of the device and of the expensive treatment of primary graft failure and low cardiac output syndrome. 11/07/ / 6

266 GR Understanding neural mechanisms of Spatial Neglect by linking anatomical damage to resting state functional connectivity Baldassarre Antonello Biomedical/Biomedica Istituto Neurologico Mediterraneo Neuromed LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Neuroimaging, functional, biochemical, and neuropathological studies to assess the onset, progression, treatment, and development of biomarkers for brain disorders. Spatial Neglect Resting State Functional Connectivity Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION UOC di Stroke Unit Enrollment and neuropsychological evaluation of stroke patients. Collecting behavioral and neuroimaging data. Analyses of behavioral and neuroimaging data. Writing scientific papers. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Strappini Francesca Analyses of behavioral and neuroimaging data. Writing scientific papers. 09/03/ /07/ / 5

267 GR Understanding neural mechanisms of Spatial Neglect by linking anatomical damage to resting state functional connectivity Baldassarre Antonello Biomedical/Biomedica Istituto Neurologico Mediterraneo Neuromed Background and Significance Spatial neglect is a neurological syndrome characterized by the inability in detecting and responding to stimuli in the visual field contralateral to the side of the lesion, usually right hemisphere (1). Severity of SN is correlated with limitation in activities of daily living (ADL) (2). The neural correlates of spatial neglect remain controversial (3). Recent lines of evidence indicate that spatial neglect is associated to abnormal patterns of resting state functional connectivity (FC) MRI (4-5). Specifically, in a large-group (N=84) study (5), we found that core deficits of spatial neglect at acute stage were associated with two correlated patterns of FC of dorsal attention/sensory-motor networks: i. bilateral reduction of inter-hemispheric FC; ii. increased FC with fronto-parietal/default mode networks in the right hemisphere. However, it is unknown whether these patterns of behaviorally relevant FC are associated to specific lesion topography, and whether they are prognostic at chronic stage of spatial neglect outcome and ADL. Here we aim to identify in a large sample of right hemisphere stroke patients the pattern of lesions, which may lead to abnormal changes in FC associated to spatial neglect. We also aim to use behaviorally relevant FC at acute stage as a predictor of spatial neglect deficits and ADL at 3 months after stroke. 1.Haligan et al., Katz et al., Corbetta & Shulman, He et al., Baldassarre et al., under revision at Brain Specific aims Aim 1: Aim 2: Aim 3: To identify the patterns of resting-state functional connectivity (FC) MRI associated with the severity of spatial neglect in a large-group of right hemisphere stroke patients at acute stage (within 2 weeks of stroke onset). To investigate whether patterns of neglect-related resting-state functional connectivity (FC) MRI are associated with specific patterns of lesion topography in a large-group of right hemisphere stroke patients at acute stage. To investigate whether patterns of neglect-related resting-state functional connectivity (FC) MRI at the acute stage are predictive at the chronic stage (3 months after stroke onset) of spatial neglect outcome and activities of daily living (ADL) in a large-group of right hemisphere stroke patients. Hypothesis: Based on our study on a large-group of stroke patients under revision at Brain (5) we hypothesize to identify neglect-related patterns of FC involving attention/sensory-motor resting state networks, namely reduced inter-hemispheric FC and increased FC with fronto-parietal/default mode resting state networks in the right hemisphere (Aim 1). Based on preliminary results on 42 right-hemisphere stroke patients, we hypothesize that behaviorally relevant patterns of FC will be associated with a pattern of lesions involving the anterior and posterior portion of the superior longitudinal fasciculus in the right hemisphere (Aim 2). Finally, based on preliminary results, we hypothesize that patients showing highly abnormal patterns of FC associated with neglect at the acute stage will exhibit pathological neglect outcome and limitations in ADL at chronic stage (Aim 3). Preliminary data: In a large-group (n=84) study currently under revision at Brain (5), we found that core deficits of neglect were associated with two correlated patterns of resting state functional connectivity (FC) MRI of dorsal attention/sensory-motor networks: i. bilateral reduction of inter-hemispheric FC; ii. increased FC with fronto-parietal/default mode RSNs in the right hemisphere. Moreover, in a sub-analysis on right hemisphere damaged patients (n=42) we conducted a logistic regression analysis to identify the lesion topography associated with neglect-related patterns of FC. The uncorrected FC:Lesion map showed that lesions in the anterior and posterior portion of the superior longitudinal fasciculus tend to induce FC patterns associated with spatial neglect. Finally, in a second sub-analysis (n=65) we 10/07/ / 5

268 GR Understanding neural mechanisms of Spatial Neglect by linking anatomical damage to resting state functional connectivity Baldassarre Antonello Biomedical/Biomedica Istituto Neurologico Mediterraneo Neuromed correlated the scores of abnormal neglect-related FC at the acute stage with neglect scores and Functional Independence Measure (FMI) at the chronic stage. A correlation was found between acute FC and chronic neglect and FMI scores. Materials and Methods A sample of stroke patients will undergo behavioral evaluation of spatial neglect and activities of daily living (ADL) as well as anatomical and resting functional MRI sessions within 2 weeks after stroke (acute stage). Spatial neglect and ADL will be evaluated again at 3 moths after stroke (chronic stage). Using a newly developed method to correlate the FC with behavioral deficits (5) we will identify the patterns of abnormal FC of multiple resting state networks (RSNs), which are related to neglect deficits. Then, associations between lesion and behaviorally relevant FC will be investigated with logistic regression. Scores of abnormal FC at the acute stage will be correlated with lesion topography across patients. Finally, the strength of abnormal FC at the acute stage will be used as a predictor of behavioral measures of neglect and ADL at the chronic stage. Impact and Translational Implications Identifying the site of lesions leading to neglect-related functional connectivity (FC) is important for developing early intervention after stroke. Based on an anatomical scan it would be possible to predict which distant areas would be functionally affected and therefore targeted by manipulations of cortical excitability, e.g., with TMS or tdcs, which improve spatial neglect. Early intervention would also facilitate the recovery process, since acute FC would be related to chronic outcome. 10/07/ / 5

269 GR LOW ADIPONECTIN LEVELS AS INDIPENDENT RISK MARKER FOR BREAST CANCER IN HIGH RISK POSTMENOPAUSAL WOMEN Macis Debora Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Population Sciences and Epidemiology Epidemiology of Cancer - EPIC Project Keyword 1: Project Keyword 2: Project Keyword 3: Elucidation of the determinants of cancer and biomarkers of cancer by assembling groups of individuals to determine systematically whether the risk of disease/condition is different for individuals who are exposed or not exposed to specific factors (or combinations of factors) of interest. These may be either risk or protective factors and include genetic, epigenetic, molecular, behavioral, and environmental factors. adiponectin breast cancer Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Division of Cancer Prevention and Genetics DI APPLICANT INSTITUTION Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 11/07/ / 6

270 GR LOW ADIPONECTIN LEVELS AS INDIPENDENT RISK MARKER FOR BREAST CANCER IN HIGH RISK POSTMENOPAUSAL WOMEN Macis Debora Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia Background and Significance Adiponectin is an insulin-sensitizing hormone produced by adipocytes with anti-inflammatory, anti-atherogenic, antiangiogenic and antidiabetic properties. It has a major role in energy metabolism and adipose tissue health. Circulating low levels of adiponectin are associated with a variety of conditions that compromise women's health including type 2 diabetes, obesity, metabolic syndrome, coronary artery disease, polycystic ovarian syndrome and cancer. Circulating adiponectin is generally inversely correlated with body mass index (BMI), fat mass, visceral fat mass, and measures of insulin resistance. Recent evidence has indicated that low adiponectin levels is associated with the risk and prognosis of postmenopausal breast cancer. Many other circulating biomarkers such as leptin, homeostasis model assessment (HOMA) as an index of insulin resistance, sex hormone binding globulin (SHBG), high sensitivity C-reactive protein (hs-crp), insulin-like growth factor-i (IGF-I) and insulin-like growth factor-binding protein1 (IGF-BP1), other than mammographic breast density have been shown to be related to postmenopausal breast cancer risk. It has been hypothesized that these risk biomarkers may have a mediation effect on the obesity-postmenopausal breast cancer association. However, prospective studies with blood samples taken prior to breast cancer diagnosis, in order to avoid the bias due to the reverse causation effect, have been inconclusive. Specific aims Aim 1: Aim 2: Aim 3: to evaluate the role of baseline serum adiponectin as an indipendent risk marker for breast cancer in a prospective nested case-control study in the cohort of women included in the placebo arm of an international phase III prevention trial (IBIS-II). to conduct a mediation analysis in order to investigate whether low adiponectin levels have a direct effect on breast cancer risk through the mediation role of other breast cancer risk biomarkers, such as leptin, HOMA index, SHBG, hs-crp, IGF-I and IGF-BP1, and/or mammographic breast density. to evaluate the role of baseline adiponectin level as an indipendent risk marker for breast cancer in a prospective nested case-control study in the cohort of women included in the anastrozole arm of the international phase III prevention trial IBIS-II. Hypothesis: We hypothesize that the proportion of women who developed breast cancer during the IBIS-II trial (cases) are more frequent in the lowest baseline adiponectin quartile compared to the highest quartile. Preliminary data: We have previously shown that low adiponectin levels are associated with a history of early stage breast cancer and with a higher risk of second breast neoplastic events in premenopausal high risk women. Therefore, we conducted a meta-analysis of 15 observational studies that confirmed the inverse association between adiponectin and breast cancer risk. Overall, we showed a 34% reduction in breast cancer risk for the "highest" versus the "lowest" adiponectin levels. We found a significant inverse association in postmenopausal women and an indication of inverse association also in premenopause. Recently, we measured baseline serum adiponectin, leptin and HOMA index, in 534 postmenopausal women enrolled in 16 Italian centers and randomized in one of the two international phase III trials for breast cancer prevention -the IBIS-II (Prevention) and IBIS-II (ductal carcinoma in situ - DCIS) trials. We found that low serum adiponectin levels in postmenopausal women are more frequent in DCIS patients compared to healthy at risk subjects independently of BMI, HOMA index and age. In the IBIS-II Prevention trial, 3864 postmenopausal women at increased risk for breast cancer from 18 countries were randomized to receive 1 mg/day oral anastrozole or matching placebo for 5 years. After a median follow-up of 5.0 years, 40 women in the anastrozole group (2%) and 85 in the placebo 11/07/ / 6

271 GR LOW ADIPONECTIN LEVELS AS INDIPENDENT RISK MARKER FOR BREAST CANCER IN HIGH RISK POSTMENOPAUSAL WOMEN Macis Debora Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia group (4%) had developed breast cancer (hazard ratio 0.47, 95% CI , p<0.0001). We have the opportunity to design a prospective nested case-control study within the cohort of the IBIS-II Prevention trial, in which baseline blood samples of postmenopausal women at increased risk for breast cancer have been collected prior to breast cancer diagnosis. Recent report indicated that 3 months aromatase inhibitor treatment in 68 postmenopausal ERpositive breast cancer patients, did not alter non-fasting adiponectin levels. Materials and Methods We will design a prospective nested-control study in the cohort of women included in the IBIS-II prevention trial (3 controls matched to 1 case, by age and country). We expect to include nearly 100 cases in the placebo arm and 50 cases in the anastrozole arm. Serum samples that have been centrally collected in London, will be dug out and shipped to our centre. Serum baseline adiponectin, leptin, insulin, glucose, hs-crp, SHBG, IGF-I and IGF-BP1 will be measured with commercially available immunoassays. Conditional logistic regression model will be used to estimate odds ratios, adjusting for residual confounding variables. Adiponectin will be analyzed as a continuous estimate and categorized in quartiles, adjusting multivariable models for confounders. In order to investigate the direct effect of adiponectin, we will assess whether the effects of adiponectin on breast cancer are mediated by HOMA index, SHBG, BMI, other biomarkers, and/or mammographic density, by a mediation analysis. Impact and Translational Implications The validation of low adiponectin levels as an indipendent risk marker for breast cancer will be useful for a better risk stratification of postmenopausal women at increased risk for breast cancer. The mediation analysis could be effective in shedding further light on the role of adiponectin and other biomarkers in the aetiology of breast cancer. Raising adiponectin levels might be an attractive target for breast cancer prevention, notably for overweight or insulin-resistant subjects. 11/07/ / 6

272 GR The neural markers of post-stroke recovery: tracking the early dynamics of cortical reorganization with a novel longitudinal multimodal approach. Bonnì Sonia Clinical health care research/clinicoassistenziale Fondazione Santa Lucia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Acute Neural Injury and Epilepsy - ANIE Project Keyword 1: Project Keyword 2: Project Keyword 3: Recovery of function/rehabilitation; neurological and functional evaluation of neural prostheses, electrical/magnetic stimulation, behavioral and pharmacological interventions, and physical therapy. Stroke recovery Transcranial Magnetic Stimulation Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Clinical and Behavioural Neurophysiological and behavioural data Neurology/Non invasive Brain Stimulation acquisition, processing and analysis. Unit 2 IRCCS Centro San Giovanni di Dio - Fatebenefratelli Cognitive Neuroscience Section/ Neurophysiological Unit Analysis and elaboration of electrophysiological measures. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Pellicciari Maria Concetta IRCCS Centro San Giovanni di Dio - Fatebenefratelli Coordinator of Unit 2; EEG andtms-eeg analysis 10/09/ /07/ / 6

273 GR The neural markers of post-stroke recovery: tracking the early dynamics of cortical reorganization with a novel longitudinal multimodal approach. Bonnì Sonia Clinical health care research/clinicoassistenziale Fondazione Santa Lucia Background and Significance Since the early days after stroke, the brain undergoes a complex reorganization to allow compensatory mechanisms to promote functional recovery. It is known that cerebral networks reorganize their structural and functional anatomy in a dynamic manner over time. When a motor impairment occurs, the first weeks after stroke play a crucial role in determining a successful recovery. However, it is still debated which neurophysiological mechanisms are at the basis of recovery processes and how interconnected brain areas change their dynamics of functional communication. These discrepancies are due to the high variability among stroke patients and to a lack of systematic tracking of cortical reorganization after stroke, usually assessed with a specific isolated method. Therefore these inconsistent results make difficult to differentiate which patterns of cortical reorganization will result in beneficial or detrimental changes. We propose a multimodal approach that integrates the Transcranial Magnetic Stimulation (TMS), combined with electroencephalography (EEG) and neuroimaging tools to track longitudinally the neurophysiological, electrophysiological and neuro-anatomic mechanisms of functional recovery following stroke. This multimodal approach could provide specific markers to correlate to specific motor recovery patterns and it also will provide insight into how neuromodulatory interventions might target pathological networks configuration associated with dysfunctional recovery. Specific aims Aim 1: Aim 2: Aim 3: To track longitudinally the motor cortical reorganization following stroke in terms of: i) intra-cortical inhibitory and excitatory motor circuits, cortical plasticity and functional connectivity using TMS protocols; ii) cortical oscillations, reactivity and topographical connectivity using co-registration TMS-EEG; iii) structural and functional changes in cerebral networks connectivity using Resting State Functional Magnetic Resonance imaging (RS-fMRI) and Diffusion Tensor Imaging (DTI). The assessments of these measurements will be carried out at specific timepoints after stroke, in the affected and unaffected hemisphere. These measurements will be paralleled by behavioral motor changes and functional disability assessed by behavioral and clinical evaluations. To correlate the neurophysiological, electrophysiological and neuroanatomic measurements and behavioral motor changes in stroke patients to target the critical dysfunctional cerebral networks and the temporal windows in which specific cortical reorganization patterns can be prognostic of beneficial or detrimental functional outcome. - Hypothesis: We hypothesize that applying a multimodal approach (TMS, EEG and fmri) could to track the complex neural mechanisms and the time course of stroke-related cortical changes occurring in both the affected and unaffected hemisphere during the early and late phases of motor recovery. This longitudinal analysis will allow us to differentiate through neurophysiological, electrophysiological and neuroanatomical data, the specific patterns of the post stroke cerebral reorganization. In particular we expect that both the affected and unaffected hemispheres would present specific temporal dynamics of cortical reorganization that would be evident even when tested within intervals of few weeks, involving in particular the GABAergic intracortical circuits and the cortical oscillations within the alpha and beta band. We hypothesize that specific neurophysiological markers recorded at baseline or at specific time points during rehabilitation would be associated with a better or worse recovery and could therefore be used to track the rehabilitation strategies and to obtain prognostic markers of a potential recovery. Preliminary data: Preliminary data collected in a group of eight ischemic stroke patients demonstrate that is possible to track longitudinally the cortical changes following stroke, by means our proposed multimodal approach. Our data acquired in the laboratory of PI and the relative analysis carried out with Unit 2 show not only modulations of excitatory/inhibitory GABAergic motor intra-cortical circuits but also 11/07/ / 6

274 GR The neural markers of post-stroke recovery: tracking the early dynamics of cortical reorganization with a novel longitudinal multimodal approach. Bonnì Sonia Clinical health care research/clinicoassistenziale Fondazione Santa Lucia topographically-specific cortical reactivity and oscillatory activity (alpha band) changes, in several time points of longitudinal evaluation in both hemispheres. Notably these changes occur quickly at 40 and 60 days after stroke, but seem to be stabilized at 180 days, indicating that crucial mechanisms of cortical reorganization occur in this short time window. Materials and Methods We will recruit 60 patients in the sub-acute phase of an ischemic cortical and/or subcortical stroke in the territory of the middle cerebral artery admitted at the Santa Lucia Foundation for standard rehabilitation. Each patient will undergo 4 experimental sessions at distinct time points after stroke: within 20 days, after 40, 60 and 180 days. In every time point, we will assess in both hemispheres i) intracortical excitatory and inhibitory circuits, cortical plasticity and cortico-cortical connectivity of sensory-motor networks using paired pulse TMS and repetitive TMS protocols; ii) cortical reactivity and functional connectivity using co-registration of TMS-EEG; iii) anatomical lesion volume, the integrity of the white matter pathway and the cortical resting state networks using structural and functional MRI. At every time point, these measures will be paralleled by a battery of behavioral and clinical motor scales in order to monitor the associated individual clinical improvement. Impact and Translational Implications Stroke is the major cause of long term disability and represents an important economic/social cost. Despite the developing of new treatment, the level of rehabilitation is still unsatisfactory. Our project will allow us to identify neural markers, as critical predictors of functional recovery. This knowledge could be used as background to address neuro-rehabilitative research towards novel interventions based on the dysfunctional cerebral networks and applicable in specific temporal windows. 11/07/ / 6

275 GR DEVELOPMENT OF INNOVATIVE ANALYTICAL METHODS FOR MEAT PRODUCTS SAFETY AND QUALITY ASSURANCE Iammarino Marco Biomedical/Biomedica Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Food safety and animal safety Project Classification IRG: Project Classification SS: Veterinary Food Safety - FOSA Project Keyword 1: Project Keyword 2: Project Keyword 3: Development of innovative methods aimed at ensuring high level of safety of processed foods of animal origin. Mechanically separated meats Nitrosamines Project Request: Animals: Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION National Reference Centre for the Detection of Radioactivity in Feed and Foodstuff 2 University of Foggia Department of Agriculture, Food and Environmental Sciences Coordination. Development of analytical methods.statistical elaboration and final survey on commercial samples Development of analytical methods for Nitrosamines/Biogenic amines and Red Food Dyes in meat products. Study of combination: meat product / technological treatment that brings about the higher development of biogenic amines and nitrosamines. 3 Istituto Zooprofilattico Sperimentale della Lombardia e dell'emilia Romagna Chemistry applied to Food Technologies Development of analytical methods for Nitrosamines in meat products and of Mechanically Separated Meats. 10/07/ / 6

276 GR DEVELOPMENT OF INNOVATIVE ANALYTICAL METHODS FOR MEAT PRODUCTS SAFETY AND QUALITY ASSURANCE Iammarino Marco Biomedical/Biomedica Investigators, Institution and Role in the Project Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Mentana Annalisa University of Foggia Development of analytical methods for the determination of Nitrosamines/Biogenic amines and Red Food Dyes in meat products. Study of combination: meat product / technological treatment that brings about the higher development of biogenic amines and nitrosamines. 08/06/ bobba marco Istituto Zooprofilattico Sperimentale della Lombardia e dell'emilia Romagna Development of analytical methods for the determination of Nitrosamines in meat products and of Mechanically Separated Meats. 14/02/1978 Background and Significance Fresh meats and meat products are largely consumed foods worldwide. WHO and FAO now discourage a large consumption of these products due to some harmful effects on humans, especially after cooking. Moreover, in the last years the attention of scientific community has been focused on the safety and quality of meat products obtained from Mechanically separated meats (MSM). Despite these aspects, meat consumption increased worldwide, also in developing countries, from to million tons from 2000 to From these data it results evident that appropriate strategies should be developed in order to minimize the risks associated to meat consumption. This project is collocated in this contest. The research centres involved in this project, will make available them specific skills and expertise in order to develop innovative analytical methods, more efficient in comparison to those currently available, which will allow a correct evaluation of three aspects of meat products food safety: 1) MSM, 2) Nitrosamines and Biogenic amines and 3) Food dyes. The results should contribute to fill some analytical gaps that have been recently underlined by European Food safety Authority and European Commission, regarding MSM and food dyes. Another study, focused on the identification of the combination: meat product/heat treatment that brings about the higher development of Nitrosamines/biogenic amines, will give new significant knowledge about this food safety aspect of meat products. Specific aims Aim 1: Aim 2: Aim 3: Optimization of three analytical methods (Sr-90 by Liquid Scintillation Counting, Electronic Spin Resonance (after sample irradiation) and detection of Ca and P by X-ray diffraction) for the identification of MSM. Comparison of methods performances and identification of the most reliable one. Optimization of two analytical methods (GC-NCD and HPIC-CD) able to quantify Nitrosamines and Biogenic amines in meat products. Comparison of methods performances and identification of the most reliable one. Identification of the combination: meat product / heat treatment that brings about the higher development of nitrosamines and biogenic amines in meat products. Optimization of an analytical method by HPLC-UV-DAD for the determination of red food dyes (banned and authorized) that may be added in meat products and monitoring on commercial samples, as contribution to risk assessment. 10/07/ / 6

277 GR DEVELOPMENT OF INNOVATIVE ANALYTICAL METHODS FOR MEAT PRODUCTS SAFETY AND QUALITY ASSURANCE Iammarino Marco Biomedical/Biomedica Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata Hypothesis: The skills of the three research centres participating in the project are an important starting point in the perspective of achieving the expected results. In particular, IZS-PB and UNI-FG collaborate, since 2005, on the development and optimization of analytical methods by ion chromatography with conductivity detection for the determination of the most important precursors of nitrosamines in meat products (nitrites, nitrates and biogenic amines ). These methods are actually employed to carry out the routinely control activities on meat products at IZS-PB. The National Reference Centre for the Detection of Radioactivity in Feed and Foodstuff of IZS-PB has developed and accredited two analytical methods which involve the detection of bony fragments in meat products (Radiochemical detection of Sr-90 in solid matrices by Liquid Scintillation Counting and detection of irradiated meats by Electronic Spin Resonance). IZS-LER has also developed an analytical method, for the same purpose, which employs X-ray diffraction by allowing the quantification of Ca and P. Preliminary data: MSM: A preliminary test, carried out on two samples, one composed of not MSM (chicken fresh meat) and another one of MSM (wurstel of chicken origin) provided encouraging results. Indeed, a not negligible difference has been registered (20 mbq Kg-1 and 70 mbq kg-1 of Sr-90 for not MSM and MSM, respectively). The analytical method for the detection of irradiated meats by Electronic Spin Resonance may be also exploited for this purpose since it can detect up to 50mg of bony fragments in meats. IZS-LER published a preliminary study (M. Franceschini et al. 2011, Industria Conserve 2(86). Nitrosamines/Biogenic amines: UNI-FG and IZS-PB published several articles about Nitrosamines precursors (Biogenic amines, nitrites and nitrates): 1) C. Palermo, et al. (2013) Analytical and Bioanalytical Chemistry, 405 (2-3): ; 2) M. Iammarino et al. (2012) Food Chemistry, 140: ; 3) M. Iammarino et al. (2012) Int. J. Food Sci. & Tech., 47(9): ; 4) M. Muscarella et al. (2005) Vet. res. Comm. 29(2): Materials and Methods MSM: by quantification of Sr-90 (Liquid Scintillation Counting), Electronic Spin Resonance (after sample irradiation) and X- ray diffraction (quantification of Ca and P). Statistical comparison of methods performances. Nitrosamines/Biogenic Amines: by High Performance Ion Chromatography coupled with conductivity detectio and by Gas Chromatography coupled with Nitrogen Chemiluminescence Detection. Statistical comparison of methods performances. Study of different combinations: meat product / heat treatment and consequent quantification of Nitrosamines and biogenic amines. Red Food Dyes: by High Performance Liquid Chromatography coupled with UV-Diode Array Detector. Monitoring on commercial samples. 10/07/ / 6

278 GR DEVELOPMENT OF INNOVATIVE ANALYTICAL METHODS FOR MEAT PRODUCTS SAFETY AND QUALITY ASSURANCE Iammarino Marco Biomedical/Biomedica Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata Impact and Translational Implications The development of reliable methods for the determination of MSM, Nitrosamines/biogenic amines and red food dyes will give a significant support for control Organisms by allowing a substantial improvement of safety and quality of meat products. The study of Nitrosamines/biogenic amines development after cooking, together with a monitoring on commercial samples, will give new significant knowledge about these food safety aspects of meat products.

279 GR Innovative use of satellite data for exposure assessment to evaluate the health effects of extreme temperatures and air pollution in Rome Stafoggia Massimo Clinical health care research/clinicoassistenziale Lazio LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Methodological, epidemiological, socio-economic, organizational, managerial emerging public health issues related to the above reported areas Project Classification IRG: Project Classification SS: Population Sciences and Epidemiology Social Sciences and Population Studies - SSPS Project Keyword 1: Project Keyword 2: Project Keyword 3: Population and the environment; interrelationships between population processes and the physical environment mortality and morbidity heat and air pollution, satellite data Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Epidemiology SSR Lazio Coordination, definition of study protocols and design for exposure assessment using satellite and ground monitoring data, epidemiological analyses, health and satellite dataset, analysis, reports, publications 2 ARPA Lazio Servizio Tecnico - Divisione Atmosfera e Impianti Ad hoc monitoring campaign for validation of temperature exposure, meteorological and air pollution dataset, PM dispersion modelling, reports, publications 09/07/ / 7

280 GR Innovative use of satellite data for exposure assessment to evaluate the health effects of extreme temperatures and air pollution in Rome Stafoggia Massimo Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Lazio Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Barberini Silvia ARPA Lazio Coordinator of UO2, data collection on temperature and air pollution, PM dispersion modelling, validation study, publications 2 21/12/ de'donato Francesca Department of Epidemiology SSR Lazio 4 Narducci Stefano ARPA Lazio Servizio Tecnico - Divisione Atmosfera e Impianti Collaborator of UO1, temperature-satellite study design, epidemiological analysis, publications Collaborator UO2, data collection air pollution, temperature monitoring campaign, validation study 10/03/ /09/1983 Background and Significance The acute health effects of high temperatures and air pollution in urban and densely inhabited areas of Italy are well documented. The evidence of health effects, however, is limited for people living in suburban and rural areas and it is not clear how the effect varies within metropolitan areas. In addition, information is sparse on vulnerable subgroups of population due to chronic health conditions, poor socio-economic status and/or area of residence. In recent years, satellite data with improved spatial and temporal resolution have become a valuable tool as an alternative method for exposure assessment in epidemiological studies. These innovative methods overcome the limitations of data from monitoring networks as they are unable to capture the spatial heterogeneity of temperatures and air pollutants especially within urban areas. Modern exposure assessment methods together with advanced epidemiological strategies allow to better estimate the short-term and long-term effects of environmental exposures at the individual level. The aim of the project is to provide evidence of the differential health effects of heat and air pollution in suburban and rural areas of Lazio and in areas of Rome due to the urban heat island (UHI) intensity and the heterogeneous air pollution distribution. The use of satellite data and the Rome Longitudinal Study will offer the possibility to estimate differential health effects at individual level in over 1 million subjects residing in Rome. Specific aims Aim 1: Aim 2: Aim 3: To estimate satellite-derived spatio-temporal exposure to PM10/PM2.5, and to investigate joint short and longterm effects on mortality and hospital admissions at subject level within the Rome Longitudinal Study (RLS). To estimate satellite-derived spatio-temporal exposure to air temperature, to investigate short-term health effects on mortality and hospital admissions in rural, suburban and urban areas and to define the urban heat island intensity in Rome and the health effects in vulnerable subgroups using RLS. To define an integrated air pollution and heat warning system for Rome graded by areas at risk, using current dispersion models, heat watch warning systems and satellite-derived exposures to PM and UHI. Hypothesis: Air pollution and temperatures vary across space and time at a fine scale resolution. These differences are associated with heterogeneous effects on health outcomes in urban, suburban and rural settings. Furthermore, within large metropolitan areas there are subgroups of the population most at risk due to 09/07/ / 7

281 GR Innovative use of satellite data for exposure assessment to evaluate the health effects of extreme temperatures and air pollution in Rome Stafoggia Massimo Clinical health care research/clinicoassistenziale Lazio individual characteristics (socio-economic and chronic diseases) and area of residency (high UHI intensity, proximity to high traffic roads). We expect that results from the spatio-temporal analysis will produce more accurate health estimates and identify the relative contribution of the different susceptibility factors. Preliminary data: Strong evidence on the short-term effects of PM and high temperatures on mortality/hospitalizations comes from Italian and international studies set in urban contexts, and has identified the elderly with pre-existing chronic conditions and subjects living in disadvantaged areas among the most susceptible subgroups. Furthermore, it has been shown that air pollution and high temperatures act synergistically on health, thus representing a relevant public health problem. A preliminary study on the urban heat island of Rome using single satellite images has shown the differences in temperatures within the city with UHI intensities of +4 C in central areas compared to rural points. Data available on aerosol optical depth (AOD) from the NASA Terra satellite have shown large differences in particles scattering within Rome, with central and eastern parts of the city displaying, as annual average, 2- to 3-times higher values compared to other areas. The RLS is a Rome cohort with more than 1 million subjects 30+ years of age followed-up since October Individual data are available on age, gender, education, marital status, occupation and clinical conditions. Moreover, all residential addresses (around 150,000) have been georeferenced. Long-term effects of air pollution have been already investigated within the RLS, with clear evidence of associations of PM2.5 and NO2 on all-cause mortality, lung cancer incidence and cardio-vascular morbidity. 09/07/ / 7

282 GR Innovative use of satellite data for exposure assessment to evaluate the health effects of extreme temperatures and air pollution in Rome Stafoggia Massimo Clinical health care research/clinicoassistenziale Lazio Materials and Methods Materials -Observed air temperature and PM concentrations (regional networks, ad-hoc monitoring campaign); -PM dispersion model; -Satellite data for temperature (MODIS LST and NDVI 1x1km, LANDSAT 120m) and PM (MODIS AOD 1x1km); -Land use data (CORINE) -Population density (census 2001); -Mortality/hospitalization data in Lazio; -Rome Longitudinal Study. Methods A 3-step statistical model will be developed using satellite, observed and land use data to derive daily air temperature and PM10/PM2.5 by 1km in Lazio, Cox and case-crossover models will be fit to estimate short-term effects of temperature, short/long-term effects of PM on mortality and morbidity in Rome, and to identify vulnerable areas and population subgroups using RLS. Previous results, pollution dispersion models and heat warning systems will be integrated to forecast areas most at risk to PM and heat in Rome. The project will benefit from the collaboration with Harvard School of Public Health, Boston, USA. Impact and Translational Implications The project will provide estimates of the health effects of heat and air pollution in urban, suburban and rural settings, and will identify areas and populations most at risk within Rome. The integrated approach can be replicated in other locations. Results from the project will enable policy makers to: a) promote individual and community adaptation and mitigation strategies, b) reduce anthropogenic emissions, c) indorse social equity by targeting vulnerable subgroups. 09/07/ / 7

283 GR Targeted genome editing of the CD40LG gene for the treatment of X-linked hyper-igm immunodeficiency Genovese Pietro Biomedical/Biomedica Ospedale San Raffaele - Milano LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Innovative biotechnologies Project Classification IRG: Project Classification SS: Genes, Genomes and Genetics Gene Therapy and Inborn Errors - GTIE Special Emphasis Panel Project Keyword 1: Project Keyword 2: Project Keyword 3: Studies of transduction, integration, replication and repair, gene expression and gene silencing mechanisms in animal and human tissues and in animal models of diseases Site specific gene correction Primary immunodeficiencies Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION TIGET, Division of Regenerative Medicine 2 Ospedale San Raffaele - Milano TIGET, Division of Regenerative Medicine 3 Erasmus MC - Rotterdam Department of Immunology, University Medical Center Development and optimization of the CD40LG gene corection strategy in human and mouse HSC. Coordination of the work. Contribute to the analyses of transplanted mice Support in B cell analysis and provide primary cells from genotyped HIGM1 patients Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Castiello Maria Carmina Ospedale San Raffaele - Milano Contribute to test functional reconstitution of the immune response in the transplanted mice 2 van Zelm Menno C Erasmus MC - Rotterdam Support in B cell analysis and provide primary cells from genotyped HIGM1 patients 09/06/ /06/ /07/ / 6

284 GR Targeted genome editing of the CD40LG gene for the treatment of X-linked hyper-igm immunodeficiency Genovese Pietro Biomedical/Biomedica Ospedale San Raffaele - Milano Background and Significance Hematopoietic Stem Cells (HSC) gene therapy has provided clinical benefits in several patients affected by a variety of genetic diseases. However, the use of semi-randomly integrating vectors poses the risk of insertional mutagenesis and ectopic/unregulated transgene expression. The latter issue is particularly relevant when dealing with tightly regulated genes active on cell proliferation, like the CD40LG gene which expression on activated T cells leads to contact-dependent B cell activation and proliferation. Since its mutations cause the X-linked immunodeficiency with hyper-igm (HIGM1), HSC gene transfer was proposed as potential treatment for this syndrome. Although even few amounts of transduced cells restored humoral and cellular immune function in a HIGM1 mouse model, constitutive expression of CD40LG in tymocytes or peripheral T cell led to lymphoproliferations, most of which progressed in frank lymphomas (Brown et al, 1998; Sacco et al, 2000). Gene repair strategies that preserve physiologic expression control of the corrected gene might therefore represent a more promising approach for treatment of HIGM1. We recently developed a strategy based on engineered nucleases that enables efficient and specific targeted gene correction in human HSC while preserving their repopulation capacity (Genovese et al, 2014). Within this project we will capitalize our previous achievements to directly fix CD40LG mutations and model disease correction on mouse and human HIGM1 cells. Specific aims Aim 1: Aim 2: Aim 3: To select the best performing nuclease architecture and donor template design for the correction of the CD40LG gene To optimize our current gene targeting protocol on long-term repopulating human and murine HSC To provide proof-of-efficacy of HIGM1 disease correction Hypothesis: Targeted correction of the CD40LG gene while preserving its endogenous regulation allows safe and effective rescue of HIGM1 syndrome Preliminary data: In previous studies we showed that targeted integration (TI) in the most primitive hematopoietic stem/progenitor cells (HSPC) is constrained by poor gene transfer efficiency and a bias against the use of the homology directed DNA repair pathway in these cells. By combining Integrase Defective Lentiviral Vectors (IDLV) for donor template delivery and mrna transfection to drive a transient spike of ZFN expression and using culture conditions tailored to activate and expand HSPC, we overcame these barriers and provided stringent evidence of TI in human HSPC. Thus, we enabled TI of transgene expression cassettes into the AAVS1 "safe harbor" site or of a corrective cdna into a mutational hotspot of IL2RG gene with high efficiency (average 6%) and specificity (>95%) in cord blood or bone marrow-derived HSPC. Upon xeno-transplantation in NSG mice, the edited HSPC sustained normal hematopoiesis giving rise to both myeloid and lymphoid lineages as well as early progenitors for several months post-transplant, indicating successful targeting of long-term repopulating HSC. Importantly, HSPC targeted with the corrective IL2RG cdna were capable of generating polyclonal functional T cells that physiologically expressed IL2RG and, when tested ex vivo, were indistinguishable from wild-type cells in terms of proliferation, cytokine secretion and signal transduction. Finally, we demonstrated the therapeutic potential of our strategy by correcting the IL2RG gene in HSPC from a genotyped SCID-X1 patient, in which we found similar TI efficiency and confirmed restoration of g-chain expression in the targeted cells. (Genovese et al, 2014). More recently, we optimized the electroporation conditions, mrna construction and timing of ZFN and donor template administration in order to reproducibly achieve high levels of nucleases activity (average 35% NHEJ) and efficient TI of a corrective donor (average 6%, n=26) into a mutated IL2RG gene of murine Lin- HSPC. We validated this gene editing protocol by in vivo transplantation, 11/07/ / 6

285 GR Targeted genome editing of the CD40LG gene for the treatment of X-linked hyper-igm immunodeficiency Genovese Pietro Biomedical/Biomedica Ospedale San Raffaele - Milano demonstrating engraftment and multilineage differentiation of the targeted murine HSPC (Genovese, Schiroli et al, unpublished results). Materials and Methods TALENs (Golden Gate TALEN Kit 2.0), Cas9 and grna (Sigma-Aldrich) and ZFNs (Sangamo BioSciences) targeting the first intron of the human and mouse CD40LG genes will be delivered into activated human CD34 cells (cord blood or bone marrow derived) or Lin- cells from Cd40lg-/- mice (B6.129S2-Cd40lgtm1Imx/J) by mrna electroporation. Donor DNA templates containing a splice acceptor, a corrective cdna and a reporter expression cassette will be delivered by IDLV or AAV6/2 transduction. The reporter cassette will allow monitoring gene targeting efficiency in vitro and upon transplantation into Cd40lg-/- mice or xenotransplantation of the human cells into NSG mice. Disease correction will be evaluated in vitro by B cell co-stimulation assays using activated CD4 T cells and in vivo by evaluating humoral and cellular immune responses after challenging with mouse pathogens/vaccines. Impact and Translational Implications If efficacy of gene correction will be demonstrated, this study will provide a safe and effective therapeutic option for the treatment of HIGM1 disease. Considering that GMP manufacturing of lentiviral vectors and mrna electroporation are already established, we could envisage prompt clinical translation of this approach, which may benefit the vast majority of HIGM1 patients while sparing them the risks associated with allogeneic bone marrow transplantation. 11/07/ / 6

286 GR Magnetic Resonance Spectroscopy as a marker of response to dopaminergic treatment in Parkinson's Disease CIURLEO ROSELLA Clinical health care research/clinicoassistenziale Centro Neurolesi Bonino Pulejo LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Neuroimaging, functional, biochemical, and neuropathological studies to assess the onset, progression, treatment, and development of biomarkers for brain disorders. Magnetic Resonance Spectroscopy Parkinson's Disease Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Neurobioimaging Laboratory Project Coordination, Patient Recruitment, Neurological Assessment, MRI and 1H-MRSI Examinations, 1H-MRSI Data Analysis, Statistical Analysis, Result Dissemination 2 University of Messina Department of Clinical and Experimental Medicine, Movement Disorders Unit Patient Recruitment, Neurological Assessment Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Morgante Francesca University of Messina Patient Recruitment, Neurological Assessment 24/02/ /07/ / 6

287 GR Magnetic Resonance Spectroscopy as a marker of response to dopaminergic treatment in Parkinson's Disease CIURLEO ROSELLA Clinical health care research/clinicoassistenziale Centro Neurolesi Bonino Pulejo Background and Significance Parkinson's Disease (PD) is the most common neurodegenerative movement disorder. PD is characterized by progressive degeneration of dopaminergic neurons in substantia nigra pars compacta (SNc). This loss of dopaminergic neurons results in decreased levels of dopamine in the putamen of the dorsolateral striatum, leading to dysfunction of direct and indirect pathways of movement control that involve cortico-basal-thalamo-cortical loops (Obeso JA et al, 2000). Currently, PD treatment is symptomatic. One of the major challenge in PD is the development of disease-modifying therapies, such as neuroprotective or cell-based restorative agents. Few dopaminergic drugs, among which monoamine oxidase B (MAO-B) inhibitors and even levodopa, have been proposed to have a disease-modifying effect (Fahn S et al, 2006; Olanow CW et al, 2009; Pålhagen S et al, 2006). So far, the main outcome measures used in clinical trials to detect disease progression has been the motor part of the Unified Parkinson's Disease Rating Scale (UPDRS-III), which does not allow to distinguish between symptomatic and disease-modifying drug effects. Therefore, there is an unmet need to use an objective biomarker to evaluate disease progression. Magnetic Resonance Spectroscopy (MRS) is a non-invasive imaging technique for exploration in vivo of intracellular metabolic status, and may provide a neuroimaging biomarker of normal biological and pathological processes or response to therapeutic intervention in PD. Specific aims Aim 1: Aim 2: Aim 3: To evaluate the influence of dopaminergic therapy on metabolic profile of cortico-basal ganglia loops in de novo patients with PD by using proton MRS Imaging (1H-MRSI). To evaluate if possible brain metabolic changes induced by dopaminergic therapy correlate positively with the clinical improvement, as revealed by UPDRS-III scores. To evaluate which of study drugs (levodopa, MAO-B inhibitors and no-ergot dopamine agonists) is more effective in restoring the neurometabolite profile. Hypothesis: In vivo 1HMRS may provide an useful non-invasive tool for detection of neurometabolite changes in patients with PD (Marino S et al, 2012; Baglieri A et al, 2013). As suggested by other studies (Taylor-Robinson S et al, 1999; Clarke CE et al, 2000; Lucetti C et al, 2001; Camicioli RM et al, 2007; Gröger A et al, 2001; Levin BE et al, 2014; Griffith HR et al, 2008; Oz G et al, 2006), a first hypothesis is that the concentrations of main neurometabolites detectable by 1H-MRS in cortico-basal ganglia networks are different between PD patients and healthy control subjects. Furthermore, we speculate that dopaminergic therapy may partially re-establish the normal neurometabolite levels as result of drug-induced restoration of normal pattern of cortico-basalthalamo-cortical functions that in turn may induce a clinical improvement. Finally, it's not excluded that some dopaminergic drugs are more effective than others in restoring neurometabolite levels in a long-term assessment, supporting their potential disease-modifying effect. Preliminary data: So far, few studies evaluating possible brain metabolic changes in PD patients before and after dopaminergic therapy have been reported (Clarke CE et al, 1997; Lucetti C et al, 2007). We have studied 20 de novo drug-naïve PD patients before and after therapy with ropinirole by using multivoxel 1H-MRSI (Ciurleo R et al, submitted). At baseline, the 1H-MRSI findings indicated in PD patients reduced NAA and increased Cho levels compared to healthy controls. After 10 months therapy with ropinirole, PD patients showed a significant increase of NAA levels from motor cortex and an highly significant clinical improvement, as showed by UPDRS motor sub-score. No changes in Cho/Cr were found. The results of our study showed a clear correlation of NAA increase with clinical improvement of motor symptoms providing an important incentive to expand the research to the study of metabolic changes induced by other dopaminergic drugs in all brain structures involved in motor control. 10/07/ / 6

288 GR Magnetic Resonance Spectroscopy as a marker of response to dopaminergic treatment in Parkinson's Disease CIURLEO ROSELLA Clinical health care research/clinicoassistenziale Centro Neurolesi Bonino Pulejo Materials and Methods This prospective, rater-blinded pilot study will be conducted in de novo PD patients who are going to start dopaminergic treatment. Twenty levodopa-treated, 20 ropinirole-treated, 20 pramipexole-treated, 20 rotigotine-treated, 20 rasagilinetreated, 20 selegiline-treated de novo PD patients, and 60 age- and sex-matched healthy volunteers will be recruited. Drugtreated groups of PD patients will be matched for age. At baseline, drug-naïve PD patients will undergo clinical assessment by using UPDRS and, MRI and 1H-MRSI examinations by using a MR Scanner operating at 3 T. The healthy volunteers will undergo MRI and 1H-MRSI examinations only. For each PD patient in treatment with dopaminergic drug, the study period will consist of 18 months and will include 3 follow-up visits at 6, 12 and 18 months from baseline. During follow-up visits, PD patients will undergo MRI/1H-MRSI examinations and neurological assessment in which UPDRS scale will be performed in order to verify the outcome. Impact and Translational Implications 1H-MRSI could provide a non-invasive, quantitative and objective evaluation of the efficacy of dopaminergic therapy. In addition, this 1H-MRSI study could add new evidence about the potential disease-modifying effect of some dopaminergic drugs. Overall, the results of this research could preliminarily ascribe to 1H-MRSI an important role as a neuroimaging biomarker of response to current and future PD treatments resulting in an improvement of therapy management and its costs. 10/07/ / 6

289 CD45RA-depleted donor lymphocyte infusions for the prevention of infections in patients affected by hematological malignancies after haploidentical stem cell transplantation and posttransplant cyclophosphamide GR CROCCHIOLO ROBERTO Biomedical/Biomedica Istituto Clinico Humanitas LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Immunology Transplantation, Tolerance and Tumor Immunology - TTT Project Keyword 1: Project Keyword 2: Project Keyword 3: Transplantation: studies of transplant rejection when the major focus is on the immune response to the transplanted organ and immune mechanisms behind immunosuppressive drugs and therapies for prevention of transplant rejection; solid organ and hematopoietic transplant tolerance, in both small and large animal models as well as clinical studies and clinical trials; mechanisms of development and strategies for prevention of graft vs. host disease, including promotion of graft vs. tumor/leukemia effects. donor lymphocyte infusion haploidentical stem cell transplantation Project Request: Animals: Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Oncology Department/Hematology and Bone Marrow Transplantation 2 Istituto Clinico Humanitas Unit of Clinical and Experimental Immunology Project supervision, accrual of patients, clinical care, data analysis, publication of results Phenotypic and functional analysis of the patients' immune system Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Lugli Enrico Istituto Clinico Humanitas Phenotypic and functional analysis of the patients' immune system, laboratory experiments, fellows supervision, data analysis, publication 04/07/ /07/ / 6

290 CD45RA-depleted donor lymphocyte infusions for the prevention of infections in patients affected by hematological malignancies after haploidentical stem cell transplantation and posttransplant cyclophosphamide GR CROCCHIOLO ROBERTO Biomedical/Biomedica Istituto Clinico Humanitas Background and Significance Hematopoietic stem cell transplantation is potentially curative for blood cancers but is associated with profound immunodeficiency which exposes the patient to life-threatening infections. A platform of T-cell replete haploidentical (haplo) HSCT using post-transplant cyclophosphamide (P-Cy) has shown high reproducibility and acceptable safety in HSCT patients lacking a HLA-identical donor. P-Cy avoids ex-vivo graft manipulation as the allogeneic T cells, responsible for the pathogenesis of graft-versus-host disease (GvHD), are depleted following activation in vivo. Unfortunately, P-Cy also depletes donor CD45RA- memory T cells directed against pathogens, thus preventing the transfer of cellular immunity to the patient. Donor lymphocyte infusions (DLIs) are administered to HSCT patients, including those receiving haplo-hsct and P-Cy, with the double aim of enhancing anti-tumor immunity and better protect from infections. However, rates of acute GvHD, ranging from 10 to 38%, were reported after haplo-dli, thus counteracting their intended beneficial effect. Preclinical models revealed that CD45RA+ naïve T cells, but not CD45RA- memory T cells contained in the DLI are mostly responsible for GvHD, since they rapidly activate and proliferate in the presence of patient's alloantigens. We will evaluate the clinical and biological impact of DLIs depleted of CD45RA+ naïve T cells to promote the transfer of T cell immunity to the patient and to reduce viral infections and GvHD. Specific aims Aim 1: Aim 2: Aim 3: to evaluate the efficacy of CD45RA-depleted haplo-dlis (modified DLIs) in the setting of patients receiving haplo- HSCT and P-Cy, in terms of incidence of viral infections in the post-transplant period to evaluate the impact of the modified DLIs on the occurrence of GvHD, relapse (graft-versus-tumor effect) and other types of infections to evaluate the reconstitution and functions of major lymphocyte populations, including of donor memory T cells specific for opportunistic pathogens Hypothesis: through the negative selection of CD45RA+ cells, the modified DLIs will contain very low amounts of naïve T cells (that are CD45RA+), thus reducing the risk of GvHD after DLI. On the other hand, given the preferential depletion of memory T cells by P-Cy, we hypothesize that the transfer of such DLIs (enriched in memory T cells) will decrease the incidence of opportunistic infections Preliminary data: Up to April 2014, we analyzed 40 patients receiving haplo-hsct: 28 of them (70%) had at least one viral infection with a peak incidence between +30 and +100 after transplant. Of note, 75% of all viral episodes and 100% of fungal infections occurred before day +180, indicating that post-hsct lymphopenia is associated with the highest risk of infection. As far as our experience with haplo-dli is concerned, we infused 8 patients using unmanipulated DLIs to treat or prevent relapse after transplant. Haplo-DLIs were planned 30 days apart: the first with 5 x 10e5 CD3+/kg being transferred, while the second and the third with 1 and 5 x 10e6 cells, respectively. On a total of 14 infusions, we observed 1 acute GvHD occurring after the 2nd infusion. Published data used similar doses (the most frequent at 1 x 10e6 cells/kg) and reported a 25% incidence of acute GvHD (Zeidan AM, Biol Blood Marrow Transpl, 2014 Mar;20(3):314-8.), thus suggesting that haplodlis are associated with acceptable toxicities. In collaboration with Dr. E. Lugli, we studied primary specimens from haplo-hsct patients and revealed that in vivo P-Cy preferentially depletes donor CD45RAmemory T cells, while sparing those naïve T cells that are not allospecific. The former include both alloreactive T cells that differentiated from the transferred CD45RA+ naïve compartent in response to alloantigen recognition, and pathogen-specific memory T cells (manuscript submitted). In vitro incubation of FACS-purified naïve and memory T cells with mafosfamide, an active metabolite of Cy, 10/07/ / 6

291 CD45RA-depleted donor lymphocyte infusions for the prevention of infections in patients affected by hematological malignancies after haploidentical stem cell transplantation and posttransplant cyclophosphamide GR CROCCHIOLO ROBERTO Biomedical/Biomedica Istituto Clinico Humanitas comfirmed the increased tendency of memory T cells to undergo apoptosis. Accordingly, pathogenspecific memory T cells could not be detected in the post-transplant setting despite they were adoptively-transferred with the graft, thus suggesting that donor T cell memory does not persist in the transplanted patient, most likely due to P-Cy. Materials and Methods CD45RA+ naïve T cell depletion: we will use CE-marked reagents/devices approved for clinical use (CliniMACS, Miltenyi, Germany). Anti-CD45RA beads deplete CD45RA+ cells, thus the modified DLIs will contain mostly effector and central memory T cells. DLI schedule: the 1st modified DLI at +50 post-hsct (5x10e5 CD3+/kg of recipient); in the absence of adverse events, a 2nd and 3rd CD45RA-depleted DLIs at +80 and +120 respectively, at increasing doses of 1 and 5 x 10e6 CD3+/kg. Laboratory analysis: 18-color FACS sorting and molecular/cellular technologies (T cell receptor sequencing) to track donor pathogen-specific T cells in the recipient. Statistics: with the hypothesis that viral infections between +30 and +100 will lower from 70% to 45%, a sample size of 25 patients is needed. If a minimum of 13 treated patients will not present any viral infection in this timeframe, the hypothesis is accepted with an alpha error=0.05 and beta=80% (A'Hern RP, Stat Med Mar 30;20(6):859-66) Impact and Translational Implications The proposed clinical trial is highly translational in nature, as it is based on recent laboratory findings. CD45RA-depleted DLIs administered after haplo-hsct will confer increased protection against infections and thus lower their incidence. Moreover, the risk of GvHD is expected to be lower than that of conventional DLIs. The present intervention will lead to decreased morbidity and mortality and to inferior costs of supportive care (pharmacological prophylaxis and hospitalization) 10/07/ / 6

292 GR Chimeric antigen receptor-redirected T cells targeting CD19 antigen: first-in-human Italian trial for children with chemotherapy-refractory Acute Lymphoblastic Leukemia. Quintarelli Concetta Clinical health care research/clinicoassistenziale Ospedale pediatrico Bambino Gesù LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Oncology 2 - Translational Clinical Cancer Immunopathology and Immunotherapy - CII Project Keyword 1: Project Keyword 2: Project Keyword 3: Hematopoietic stem cell transplantation and other adoptive cellular therapies with immune cells as cancer treatment Chimeric Antigen receptor Acute Lymphoblastic Leukemia Project Request: Animals: X Humans: X Clinical trial: X The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Pediatric Hematology and Oncology 2 Ospedale San Raffaele Regenerative Medicine, Stem Cells and Gene Therapy/Leukemia Immunotherapy Unit 3 Istituto Superiore di Sanità Dipartimento di Biologia Cellulare e Neuroscienze/Reparto di Terapia Genica e Cellulare Investigators, Institution and Role in the Project DI/ UO1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Casucci Monica Ospedale San Raffaele PI UO2 14/06/ TIRELLI VALENTINA Istituto Superiore di Sanità PI UO3 15/12/1979 UO2 UO3 10/07/ / 6

293 GR Chimeric antigen receptor-redirected T cells targeting CD19 antigen: first-in-human Italian trial for children with chemotherapy-refractory Acute Lymphoblastic Leukemia. Quintarelli Concetta Clinical health care research/clinicoassistenziale Ospedale pediatrico Bambino Gesù Background and Significance Chimeric antigen receptors (CARs) are synthetic receptors that target surface molecules in a native conformation. The generations of CARs typically refer to the intracellular signaling domains. Recent clinical trials have shown the importance of at least one costimulatory domain in the CAR to measure a significant antitumor response. Moreover, these studies have highlighted the importance of infusing gene-modified T cells at early differentiation stages, which are endowed with increase performancies in vivo. The goal of this proposal is to develop a T-cell based therapy with greater efficacy and lower toxicity than conventional therapy for Acute Lymphoblastic Leukemia (ALL), the most common malignancy of childhood. In particular, patients with relapsed ALL have a poor prognosis despite the use of aggressive therapies, including allogeneic hematopoietic stem cell transplantation (HSCT). CAR modified T cells specific for CD19 antigen have been reported to be effective in adults with CLL and B-cell lymphomas. Our proposal benefits from more than five years of clinical experience based on CD19 specific CAR modified T cells, and we will apply this cell-mediated antitumor immune-based approach in a first-in-human Italian study enrolling at least 10 children with chemotherapy-refractory ALL, relapsed after HSCT, for whom a second transplant may not be possible or efficacious; these patients may benefit from CAR T-cell products that potentially offer durable remissions. Specific aims Aim 1: Aim 2: Aim 3: (UO1) to manufacture retroviral vectors carrying: 1) a third generation of CD19 CAR, characterized by different combinations of costimulatory domains (CD28/4-1BB or CD28/OX40); 2) interleukin-15 (IL15) gene and 3) an inducible caspase-9-based suicide gene (ic9/car.19/il15). The antitumor activity and persistence of CAR.19 modified polyclonal T cells will be in vitro and in vivo tested to select the construct with the best pre-clinical function to be employed in preparation for a clinical trial. (UO1-UO2) to optimize in vitro methods of T cell stimulation and expansion to generate highly active anti-cd19- CAR-expressing T cells from the blood of patients with ALL. (UO1-UO2-UO3) to design a Phase I study enrolling a total of 10 ALL patients relapsed after HSCT, applying an immunotherapeutic CD19-CAR approach. Production of the CAR-T cell lines will start in March 2017 in the GMP facility of the Bambino Gesù Children's Hospital in Rome approved by the National Regulatory Agencies. Hypothesis: Although great strides have been made in the improvement of outcome for pediatric and adult ALL, the prognosis for relapsed leukemia has lagged behind significantly. Hence, development of therapies for patients failing HSCT is a desirable objective and the adoptive transfer of tumor-specific T cells is promising. Clinical trials based on CD19-CAR T cells for different malignancies have revealed some potential drawbacks, including limited persistence and expansion of genetically modified T cells. To maximize benefits from CAR-based therapies, we will construct a third generation CAR vector carrying either CD28/4-1BB or CD28/OX40 costimulatory domain, to explore in vitro but also in vivo the best costimulatory signal for CAR persistence over time. In vivo persistence and antitumor activity will be further boosted by IL15 production upon T cell stimulation. Safety issue will be covered by including ic9 in the gene construct. In particular, we will evaluate whether sorting of naïve precursors before T-cell activation would results in an increased efficacy compared to activation of total PBMCs Preliminary data: We previously proved that in vivo survival, expansion and anti-lymphoma activity of CAR.19+ T cells remain suboptimal even when the CAR contains a CD28 co-stimulatory endodomain; we have already generated a construct that also incorporates the IL15 gene and an ic9 suicide gene (ic9/car.19/il15). We found that compared to CAR.19+ T cells, ic9/car.19/il15+ T cells had: (i) greater numeric expansion upon antigen stimulation; (ii) reduced expression of the programmed death 1 (PD-1) receptor; (iii) improved anti-tumor effects in vivo. 10/07/ / 6

294 GR Chimeric antigen receptor-redirected T cells targeting CD19 antigen: first-in-human Italian trial for children with chemotherapy-refractory Acute Lymphoblastic Leukemia. Quintarelli Concetta Clinical health care research/clinicoassistenziale Ospedale pediatrico Bambino Gesù We proved that in vitro activation with CD3/CD28 beads and interleukin IL-7/IL-15 was required for antitumor efficacy in vivo. Moreover, OU2 has recently developed an in vitro protocol to specifically expand and genetically engineer stem-memory T lymphocytes starting from naive precursors, using reagents that are currently manufactured under GMP quality conditions. Materials and Methods AIM1: CD19 single chain antibody, the CD28 endodomain and the CD3z chain will be cloned into SFG to generate CAR.19. Then we will add, through 2A sequences, the cassettes encoding IL15 and the ic9 suicide gene (ic9/car.19/il15). We will generate a third generation CAR adding OX40 (ic9/car.19_ox40/il15) or 4-1-BB (ic9/car.19_4-1-bb/il15) molecule. The two final vectors will be characterized by a differential hinge to in vivo follow transduced T cells. AIM2: Naïve-sorted T cells will be stimulated with anti-cd3/cd28 ab-conjugated cell-sized beads and IL-7 and IL-15, transduced and cultured with IL-7/IL-15 in order to expand CAR-T cells with a stem-memory phenotype. The persistence and antitumor activity of these cells will be evaluated in animal models. AIM3: We will manufacture the two producing cell lines according to standard protocols. Children with regrowing ALL after HSCT will be enrolled, after approval from National Competent Authorities (Istituto Superiore di Sanità and AIFA). Impact and Translational Implications We will design the first-in-human Italian trial based on an immunotherapeutic approach exploiting T cells redirected to tumor by the expression of CD19-CAR. Moreover, we will define the best strategy for increase their in vivo efficacy and persistence, that could also be applied to similar immunotherapeutic clinical trials. This approach has a high translational impact, since it can be extended to other childhood and adulthood CD19+-malignancies with a poor prognosis. 10/07/ / 6

295 GR The PRIORITY Study - PRedictIng long term Outcomes after Isolated coronary artery bypass surgery BARILI FABIO Clinical health care research/clinicoassistenziale Piemonte LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Healthcare Delivery and Methodologies Health Services Organization and Delivery - HSOD Project Keyword 1: Project Keyword 2: Project Keyword 3: Healthcare quality, effectiveness, and outcomes; clinical practice guidelines; treatment and prevention outcomes; patient and provider satisfaction; health status and outcomes assessment; evidence-based practice; health-related quality of life; medical decision-making. Coronary Artery Bypass Graft (CABG) Long term outcomes Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION S. Croce Hospital - Department of Cardiac Surgery 2 Istituto Superiore di Sanità National Center for Epidemiology, Surveillance and Health Promotion 3 AO Città della Salute e della Scienza di Torino Unità di Epidemiologia Clinica e Valutativa coordination of project activities, study design, data management, clinical advice, finalization of the research products, drafting of scientific papers study design, data management, epidemiological and statistical analysis, screening of scientific publications to update scientific knowledge on the topic of the project, drafting of scientific papers Data management, Health care cost evaluation 10/07/ / 6

296 GR The PRIORITY Study - PRedictIng long term Outcomes after Isolated coronary artery bypass surgery BARILI FABIO Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Piemonte Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Lacorte Eleonora Istituto Superiore di Sanità collaboration to the study design of the project, screening of scientific publications to update scientific knowledge on the topic of the project; drafting of scientific papers 27/07/ evangelista andrea AO Città della Salute e della Scienza di Torino 3 Sorge Chiara Regional Health Authority of Lazio- Department of Epidemiology Lazio Regional Health Service Data management, Health care cost evaluation; collaboration in drafting of scientific papers record linkage of clinical data with Hospital Discharge Records and Tax Registry, statistical analysis 11/08/ /08/1978 Background and Significance Coronary heart disease (CHD) is one of major diseases worldwide. A 16% increase in its prevalence is expected within the next 20 years, with a devastating impact in terms of clinical aspects and allocation of resources. Coronary artery bypass grafting (CABG) is the most effective treatment for CHD. More than CABG are performed yearly in Italy. CABG provides the best perioperative results, but little information is available on long-term outcomes and costs. A large part of Italian resources are constantly diverted to funding healthcare, mainly due to the increasing burden of chronic diseases, such as CHD. Policy proposals and demonstration projects are exploring ways to improve care and reduce costs. Predicting perioperative mortality is no longer considered adequate nor sufficient for quality assessment, thus higher attention is being focused on more specific qualitative and economical parameters, such as long-term outcomes and re-hospitalization rates. This is a significant study as it will provide a new preoperative tool to predict long-term outcomes after CABG, thus guiding the clinician in identifying indications/contraindications to surgery and in tailoring the best surgical option on each single patient. It will also allow a better allocation of resources, as it will provide policy makers with a wider understanding of the characteristics of re-hospitalized patients, and the risk factors that contribute to re-hospitalization due to very frequent medical conditions. Specific aims Aim 1: Aim 2: Aim 3: To create a risk score to predict mid- and long-term outcomes after isolated CABG surgery based on patients' preoperative clinical characteristics and surgical procedure options. To build a risk function predicting mid- and long-term re-hospitalization of patients treated with isolated CABG surgery using administrative data properly supplemented by few selected patients' preoperative clinical characteristics. To analyze costs related to the long-term management of patients treated with CABG, considering patients' preoperative characteristics and surgical procedure options. Hypothesis: Individual preoperative characteristics can predict long-term outcomes in patients treated with CABG. Data from literature report that individual preoperative characteristics predicting 30-day mortality after isolated CABG surgery are also significant predictors of long-term outcomes. The Italian CABG Project collected data on 34,310 isolated CABGs providing a short-term risk function. We hypothesize that preoperative clinical data collected in the Italian CABG study can be employed to build a new risk score able to predict 10/07/ / 6

297 GR The PRIORITY Study - PRedictIng long term Outcomes after Isolated coronary artery bypass surgery BARILI FABIO Clinical health care research/clinicoassistenziale Piemonte outcomes at up to 10-year, specific for the Italian population and also tailored on specific subgroups of patients and surgical options. Literature shows that a previous history of AMI, stroke, diabetes, renal failure, etc. can predict rehospitalization 2-year after CABG surgery. Hospital Discharge Records (HDR) routinely collected by the Italian NHS provide information on patients comorbidities which are useful to trace patients' previous clinical history. Our hypothesis is that information from administrative data, supplemented by some preoperative data, if needed, can be used to develop a risk function to predict mid- and long-term re-hospitalization and health-related costs of patients treated with isolated CABG surgery. Preliminary data: The Italian CABG and CABG-2 Projects collected clinical data on all adults who underwent an isolated CABG intervention in Italian cardiac surgery centers (N=41,746). The linkage between a subset of these data and all regional HDR databases was about 95%. The Italian CABG Project developed a risk function to predict 30-day mortality which fits Italian data way better than other American and European pre-set risk scores. Our data show a 4.8% and 8.9% mortality rates at 1 and 3 years respectively, different from the 6.2% and 11.2% rates at the same intervals in US population. In a previous study we found that information from the National HDR database is accurate in reporting patients' clinical history. Our data show that about 31% of patients are re-hospitalized at least once within the first year, the average total LOS is 13.4 days, and about 20% of patients has a LOS >=20 days. A preliminary analysis showed that the use of off-pump circulation significantly reduces long-term rehospitalization for percutaneous coronary intervention, with the subsequent reduction of costs due to hospitalization and adjunctive treatments. Materials and Methods Cohort study including all patients enrolled in the Italian CABG Project ( ) and the Italian CABG-2 Project ( ), who underwent an isolated CABG intervention (IT-CABG cohort). AIM 1. STUDY DESIGN: Prospective cohort study. DATA SOURCE: IT-CABG database for demographic and preoperative clinical characteristics; HDR database linked with the Tax Register (HDR-TR) for FU information. END POINTS: Mortality and MACCE within 1, 3, 5 and 10 years from the intervention. STATISTICAL ANALISYS: Parametric and semi-parametric time-to-event models. AIMS 2-3. STUDY DESIGN: Retrospective cohort study. DATA SOURCE: the HDR database for the clinical history of patients from the IT-CABG cohort; IT-CABG database for additional preoperative characteristics; HDR-TR for FU information; Regional DRG fees for re-hospitalization costs. END POINTS: Readmissions within 1, 2 and 3 years. STATISTICAL ANALISYS: time-to-event model for recurrent event; Regression Models for censored medical costs. 10/07/ / 6

298 GR The PRIORITY Study - PRedictIng long term Outcomes after Isolated coronary artery bypass surgery BARILI FABIO Clinical health care research/clinicoassistenziale Piemonte Impact and Translational Implications A new risk score predicting long-term outcomes after CABG in the whole study group and in specific subsets of patients can become a valid tool to support clinicians in choosing appropriate therapeutic strategies for CHD management. The risk function predicting re-hospitalization after CABG surgery can be used by regional and national health authorities to identify sub-group of patients at higher risk of re-hospitalization, to plan prevention priorities and to optimize the allocation of resources

299 GR The predictive values of brain connectivity for the diagnosis of disorder of consciousness using a multimodal magnetic resonance imaging protocol Marino Silvia Clinical health care research/clinicoassistenziale Centro Neurolesi Bonino Pulejo LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Brain Disorders and Clinical Neuroscience Acute Neural Injury and Epilepsy - ANIE Project Keyword 1: Project Keyword 2: Project Keyword 3: Neuropathological, neuroimaging, electrophysiological, and functional studies to monitor the onset, progression and treatment of brain and spinal cord disease and injury; therapeutic approaches and clinical studies; cerebral blood flow and metabolism in the context of clinical neuroimaging. Disorders of Consciousness functional magnetic resonance Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Neurobioimaging Laboratory Project Coordinator, Patient recruitment, neuroimaging expert, neurological examination, MRI examination, data analysis 2 IRCCS Fondazione Santa Lucia - Roma Radiology Department Neuroimaging expert- Patient recruitment, Neuropsychological patient evaluation- MRI examination and analysis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Falletta Caravasso Chiara IRCCS Fondazione Santa Lucia - Roma Neuroimaging expert- Patients recruitment, Neuropsychological patient evaluation- MRI examination and analysis 01/10/ /07/ / 6

300 GR The predictive values of brain connectivity for the diagnosis of disorder of consciousness using a multimodal magnetic resonance imaging protocol Marino Silvia Clinical health care research/clinicoassistenziale Centro Neurolesi Bonino Pulejo Background and Significance Predicting the recovery of consciousness in patients with severe brain injuries is still very challenging with all the ensuing ethical, social consequences. More difficult is to predict how many vegetative state (VS) patients recover to minimally conscious state (MCS). Functional magnetic resonance imaging (fmri) is a noninvasive technique of choice for the study of acquired brain injury (ABI). In the past decades, fmri has been adopted to identify regions specialized in cognitive and/or motor tasks, but more recently the interest has shifted toward a novel fmri approach, i.e. the functional connectivity MRI (fcmri), especially in the resting state condition. Recent studies, have demonstrated resting-state abnormalities in cortical midline structures in VS/unresponsive wakefulness syndrome and MCS patients, even if, the functional implications of these resting-state abnormalities remain unclear. To date, no fmri longitudinal studies were performed in patients with disorder of consciousness (DOC). These findings may have implications for efficacy therapies, and should be taken into account in trials of interventions aimed at improving responsiveness in person with DOC. In addition, these results may have important implications for the clinical utility of resting-state measures as predictive markers of outcome, and for understanding the neural basis of the consciousness and awareness of internally-oriented self-relatedness (Schneider, 2008). Specific aims Aim 1: Aim 2: Aim 3: To apply an advanced multimodal MRI protocol in which structural and functional parameters are combined. These parameters can be correlated with the clinical evaluation of the sabi patients using standardized scales with the aim to find a possible set of markers of consciousness and predict recovery from VS to MCS using a longitudinal design. To investigate the Resting State Network (RSN) spatial topology with smri and fmri, in presence of lesions due to sabi. We aim to apply an approach analysis which estimate automatically, not from prior fmri seeds, a set of functional connectivity cores and related networks. This will allow to characterize the structure of the involved RSNs, to understand the influence of lesions involving the main hubs networks on the resulting functional disconnection, and to overcome some of the difficulties given by the anatomical abnormalities due to the brain injury Active task-requirement and a proper control condition (internally-oriented self-relatedness) is necessary to test for proper neural processing in the resting-state network, and the associated neuronal recruitment and modulation.this is particularly important, given recent reports that some DOC patients exhibit residual cognitive capacity. Hypothesis: We will prospectively assess the potential value of resting fmri to evaluate the functional connectivity of the whole brain, rather than just locally at the site of the structural damage with regard to the consciousness related brain regions and the resting state networks. Specifically, we hypothesize (i) that in DOC patients the features and plasticity of patterns of network connections in the midline brain regions might be closely associated to the level of consciousness and (ii) that the abnormal brain responses while processing internally-oriented self-relatedness tasks may be related to impairments not only in the spatial domain but also with temporal abnormalities in frequency and temporal content changes and spatial reorganization. Thus to provide a complete description of these phenomena we propose a multimodal approach in which structural and functional brain connectivity are combined with the clinical evaluation. Preliminary data: In a fmri study (Marino S, 2009, 2010) it has been shown that some VS patients evolved to MCS within six months, highlighting the prognostic value of fmri. However, an active task requirement and a proper control condition (non-self-referential) is necessary to test for proper neural processing in the resting-state network. This is important, given recent reports that some DOC patients exhibit 10/07/ / 6

301 GR The predictive values of brain connectivity for the diagnosis of disorder of consciousness using a multimodal magnetic resonance imaging protocol Marino Silvia Clinical health care research/clinicoassistenziale Centro Neurolesi Bonino Pulejo retained cognitive capacity, such as initiating mental. We validated the proposed methodology on a group of healthy subjects and the results are encouraging identifying some functionally relevant regions such as the Posterior Cingulate Cortex and Supplementary Motor Area involving with the Default Mode Network and Motor Networks respectively (De Pasquale, 2013). Materials and Methods Twenty VS and 20 MCS patients will be enrolled, according to Coma Recovery Scale- Revised. After a preliminary neurological assessment, in order to ascertain the eligibility of the subjects and their grouping in VS and MSC, they will be undergone to resting fmri on the admission to the rehabilitation center (T0), after 12 (T1) and 24 months (T2). We will conduct an active self-referential processing paradigm, as previously reported (Schneider F, 2008). We will first validate the paradigm in healthy participants to identify brain regions associated with self-referential processing, and several control and validation analyses will be conducted to confirm active task participation in DOC patients. This will allow us to test abnormal signal changes for DOC patients compared to healthy participants. Next, we will examine the correlation between the neural activity changes and the level of consciousness, focusing on the regions exhibiting abnormal activity during the selfreferential task. Impact and Translational Implications Patients in VS and MCS present problems with misdiagnosis (into 40% of the cases) (Schnakers, 2009), prognosis, treatment, and everyday management, with resulting important socio-economic impact. The study of brain resting activity, by using fmri, could improve the differential diagnosis, the outcome and could add information about how many patients from VS to MCS in the middle and long term following evaluation. 10/07/ / 6

302 GR Targeting the lysosome to modulate mtorc1 function and treat obesity-associated metabolic disorders settembre Carmine Biomedical/Biomedica Ospedale San Raffaele - Milano LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Endocrinology, Metabolism, Nutrition and Reproductive Sciences Cellular Aspects of Diabetes and Obesity - CADO Project Keyword 1: Project Keyword 2: Project Keyword 3: Genetics of obesity and diabetes; analysis of the functional consequences of specific genetic alterations concerning obesity and/or diabetes lysosome mtorc1 Project Request: Animals: X Humans: Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Division of genetics and cell biology P.I. unit. The project will be entirely carried out by the unit headed by Dr. Settembre. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Forrester Alison Post-Doc. Development of Aim 1 10/07/ Bartolomeo Rosa Fellow. Development of Aim 2 08/08/ /07/ / 6

303 GR Targeting the lysosome to modulate mtorc1 function and treat obesity-associated metabolic disorders settembre Carmine Biomedical/Biomedica Ospedale San Raffaele - Milano Background and Significance Obesity represents a major risk factor in the development of metabolic syndrome and cancer. However, the mechanisms by which lipid accumulation exert such a detrimental effect remain obscure. The mechanistic Target of Rapamycin Complex 1 (mtorc1) is an intracellular kinase that senses and integrates a variety of environmental cues to regulate many major cellular processes. mtorc1 deregulation is implicated in the pathogenesis of several pathological conditions, including cancer, type 2 diabetes, and aging. Recent studies in cell and mouse models showed that lipid overload, such as in the context of obesity, causes an aberrant increase in mtorc1 activity. However, despite the importance of this observation, the mechanism through which lipids induces aberrant mtorc1 activation is still unknown. Lysosomes are organelles devoted to the degradation of specific substrates, including lipids that are continuously delivered to lysosomes by autophagy. However, an excess of lipids alters the composition of the lysosomal membrane and lead to lysosomal dysfunction. Recent studies have revealed that in order to be active mtorc1 has to be located on the lysosomal surface and that mtorc1 activity is dynamically regulated in response to lysosomal nutrient content. We and others recently demonstrated that lysosomal dysfunction led to altered mtorc1 activity. However, the physiological role of this regulatory mechanism and its implication in human pathology is still unknown. Specific aims Aim 1: Aim 2: Aim 3: Manipulation of lysosomal function as a therapeutic approach to rescue the defects of mtorc1 signaling. The recent discovery of a lysosomal gene network and of its master regulator TFEB provides experimental evidence that lysosomal function can be globally controlled. We propose to stimulate lysosomal function through TFEB overexpression in order to enhances lysosomal lipid degradation and rescues lysosomal accumulation of lipids in the liver of mice fed with high fat diet (HFD). Given the central role of the lysosome in the regulation of mtorc1 function, we expect that normalizing lysosomal function will rescue the activity of mtorc1, hence exerting a positive effect on cellular metabolism. We intend to investigate if the lysosome can be pharmacologically targeted to rescue mtorc1 hyperactivation. We want to identify compounds that are able to displace mtorc1 from the lysosomal surface. The machinery that associates mtorc1 to the surface of the lysosome is composed of several proteins and perturbing the function of each of the component of the complex or their association lead to mtorc1 inactivation. These compounds can be considered a novel class of selective mtorc1 inhibitors. Our 5 best hits will be tested in mice fed with HFD, in which mtorc1 is hyperactive. -none- Hypothesis: We hypothesize that changes in the lysosomal function caused by excess of lipids affect the capacity of the lysosome to modulate mtorc1 activity in response to cellular nutrient. This condition may lead to aberrant mtorc1 activation that alter cellular metabolism and promotes de novo lipid biogenesis. This scenario can lead to a vicious cycle that alters cellular metabolism and predispose to cancer development. We intend 1) to demonstrate that enhancing lysosomal function is a way to restore mtorc1 function and normal metabolism in cells and in organs subjected to lipid overload and 2) to identify a novel class of selective mtorc1 inhibitors that target the association of mtorc1 with the lysosome, hence inhibiting mtorc1 function. Preliminary data: This grant proposal is supported by strong preliminary data that we have generated while studying lysosomal regulation by TFEB and mtorc1.(associated to aim 1) We tested the metabolic effects 11/07/ / 6

304 GR Targeting the lysosome to modulate mtorc1 function and treat obesity-associated metabolic disorders settembre Carmine Biomedical/Biomedica Ospedale San Raffaele - Milano of TFEB gain of function in the liver of mice fed with a high-fat diet. We generated mice overexpressing TFEB in the liver which showed increased expression of lysosomal genes and display livers with markedly reduced lipid content compared to HFD controls, suggesting that modulating lysosomal function is a way to ameliorate lipid metabolism.(associated to aim 2) We have set up three different assays to screen library of compounds using an high content microscopy facility:1) Quantification of mtorc1 localization with lysosome. 2) Quantification of level of S6 phosphorylation, as measure of mtorc1 activity. 3) GFP-TFEB nuclear translocation, which is regulated by mtorc1-mediated phosphorylation. Materials and Methods Transgenic mice in which overexpression of TFEB-FLAG can be induced upon tamoxifen treatment will be fed with lipid enriched diet (8 weeks) in order to develop obesity, liver steatosis, insulin sensitivity defects and hyperactivation of mtorc1. Metabolic, histological, biochemical, and molecular biology studies will be performed in Tamoxifen treated vs untreated animals. Primary hepatocytes in which TFEB can be pharmacologically activated will be cultured in the presence of fatty acids and/or in the presence of cholesterol. The activity and the lysosomal localization of mtorc1 will be evaluated by western blotting and immunofluorescence, respectively, in Tamoxifen treated vs untreated cells. Transcriptome analysis will be also performed. In the second aim, images will be acquired with the high content system Operetta (Perkin Elmer) and analyzed by creating a script using Harmony software. Commercial library (1200 FDA approved drugs) will be screened. Impact and Translational Implications The World Health Organization (WHO) predicts that obesity may become soon the principal public health concern. This work will identify lysosome as a a novel therapeutic target for the treatment of metabolic dysfunctions associated to obesity. mtorc1 is often dysregulated in multiple diseases, by identifying drugs targeting mtorc1 association to lysosome we intend to discover a new class of therapeutics designed to rebalance cellular nutrient signaling and metabolic functions. 11/07/ / 6

305 GR Autophagy impairment in patients with chronic HCV infection: impact on steatohepatitis and carcinogenesis. Vescovo Tiziana Biomedical/Biomedica Istituto per le Malattie Infettive Lazzaro Spallanzani LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Infectious and immunological diseases Project Classification IRG: Project Classification SS: Infectious Diseases and Microbiology Virology A and B Study Sections - VIRA and VIRB Project Keyword 1: Project Keyword 2: Project Keyword 3: Virus-host cell interactions: effects on signal transduction; host gene expression; cellular physiology and metabolism; production of interferons, cytokines and chemokines; cytopathology; apoptosis Hepatitis C virus Autophagy and Inflammation Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of Epidemiology and Preclinical Research, Cell Biology and Electron Microscopy Unit 2 National Institute for Infectious Diseases Lazzaro Spallanzani IRCCS Department of Epidemiology and Preclinical Research, Gene Expression and Experimental Hepatology Analysis of the role of autophagy impairment and cholesterol accumulation in triggering inflammation and HCC Investigation of the tumorigenic potential of HCV replicon cells in immune competent mice in relation to autophagy function. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 COZZOLINO ANGELA MARIA National Institute for Infectious Diseases Lazzaro Spallanzani IRCCS Investigation of the tumorigenic potential of HCV replicon cells in immune competent mice in relation to autophagy function. 05/08/ /07/ / 6

306 GR Autophagy impairment in patients with chronic HCV infection: impact on steatohepatitis and carcinogenesis. Vescovo Tiziana Biomedical/Biomedica Istituto per le Malattie Infettive Lazzaro Spallanzani Background and Significance Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. The incidence of HCC is increasing, with new cases and estimated deaths annually. Since the treatment of patients with HCC remains unsatisfactory, improving knowledge of hepatocarcinogenesis may encourage the development of targeted therapies. Chronic infections with Hepatitis B (HBV) or C (HCV) virus are the leading causes of HCC. HCV-induced HCC develops in an environment of inflammation that results from chronic liver damage. Indeed, persistent inflammation induces the progression from steatosis to steatohepatitis which increases the risk to develop HCC, suggesting that lipid accumulation synergistically with inflammation represents a promoting factor of tumorigenesis. However, the molecular mechanisms linking inflammation, steatohepatitis and the onset of HCC are poorly understood. We have demonstrated that in HCV infected cells autophagy is stimulated to counteract the increase of cholesterol levels induced by HCV. Importantly, accumulation of free cholesterol was reported to activate the NLRP3 inflammasome and contribute to chronic inflammation, and cholesterol metabolites were described to be involved in the progression of breast and prostate cancer. Based on these evidences, we propose to investigate whether autophagy defects and cholesterol accumulation mediate the onset of steatohepatitis increasing the risk to develop HCC in HCV patients. Specific aims Aim 1: Aim 2: Aim 3: Study of the effect of autophagy inhibition on cholesterol deposits and the inflammation state of HCV replicon cells. Analysis of the correlation of autophagy levels and cholesterol accumulation with the presence of HCC in patients with chronic HCV infection Investigation of the tumorigenic potential of HCV replicon cells with impaired autophagy and increased cholesterol deposits in immune competent mice. Hypothesis: We aim to clarify whether an impairment of autophagy represents an early event in the multi-step process leading to HCC in HCV patients. Although the development of HCC in HCV patients is deeply associated with chronic inflammation, the processes by which steatohepatitis leads to cancer remain to be elucidated. The identification of specific features of inflammation that correlate with the onset of tumor will be important to monitor patients at higher risk for HCC, diagnose cancer at early stages and develop targeted therapy. We have demonstrated that autophagy is up-regulated in HCV infected cells, and defects in this process result in a strong accumulation of cholesterol induced by HCV (Vescovo 2012). Since accumulation of cholesterol has been described to be a potent inducer of inflammation, we propose to investigate whether autophagy impairment activates inflammation pathways in HCV-infected cells. Subsequently we intend to evaluate whether autophagy defects and cholesterol induced-inflammation response correlate with the development of HCC in HCV patients. Finally, we will assess whether autophagy impairment and cholesterol accumulation leads to increased chronic inflammation and promotes cell transformation of mouse HCV replicon hepatocytes injected in the liver of syngeneic animals. Preliminary data: We have reported that HCV infection triggers a functional autophagic process which plays an important role in counteracting lipid metabolism alterations induced by HCV (Vescovo 2012). We demonstrated that HCV-induced autophagy selectively targets free cholesterol in HCV replicon and HCV-infected cells. Notably inhibition of autophagy leads to a significant increase of free cholesterol induced by HCV replication. Importantly, in liver biopsies of HCV patients an inverse correlation between microvesicular steatosis and autophagy levels was observed indicating a role of autophagy in regulating intracellular lipids levels and in mediating HCV-induced pathogenesis in vivo. Moreover, 11/07/ / 6

307 GR Autophagy impairment in patients with chronic HCV infection: impact on steatohepatitis and carcinogenesis. Vescovo Tiziana Biomedical/Biomedica Istituto per le Malattie Infettive Lazzaro Spallanzani we observed that in HCV replicon cells with impaired autophagy and increased cholesterol levels there is an increase of IL-1 beta production suggesting that cholesterol accumulation may actually trigger inflammation in these cells. Based on these preliminary results, we aim to understand whether autophagy defects and cholesterol accumulation triggered-inflammation associates to the presence of steatohepatitis, and promotes the development of HCC. Materials and Methods Autophagy in HCV replicon cells will be inhibited by either genetic or pharmacological approaches. The induction of inflammation in autophagy deficient cells will be examined at gene expression and cytokines level. Liver cancer and noncancer tissues will be collected from HCV patients undergoing surgical resection or liver transplantation. Autophagy will be analysed by western blotting using an anti LC3 antibody. Cholesterol and neutral lipids will be quantified by luminometric assay. HCV MMHD3 cells with impaired autophagy will be injected in the liver of newborn mice and compared to the counterpart with functional autophagy and MMHD3 not carrying a HCV replicon. Inflammation and tumor growth will be verified 1, 3, 6 and 12 months following cells injection. Injected cells will be visualized by immunohistochemistry using specific antibodies against HCV. To assess the role of cholesterol deposits in HCV-induced inflammation, mice will be regularly fed with cholesterol lowering drugs. Impact and Translational Implications Chronic HCV infection and associated steatohepatitis represent risk factors for HCC development, but the molecular mechanisms responsible for HCV-induced tumorigenesis remain largely unknown. The present study aims at elucidating whether chronic inflammation due to impaired lipid-selective autophagy represents a promoting factor for HCC in HCV patients, and whether the pharmacological reduction of intracellular harmful lipid deposits may reduce the probability to develop liver tumors. 11/07/ / 6

308 A new strategy for the detection of residual leukemia cells BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Paganin Maddalena Biomedical/Biomedica Veneto LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Oncology 2 - Translational Clinical Cancer Biomarkers - CBSS Project Keyword 1: Project Keyword 2: Project Keyword 3: The use of specific assays or global molecular profiling to identify novel biomarkers based on DNA, RNA, protein, lipids, or metabolites obtained from tumor tissue or bodily fluids Minimal residual disease Acute lymphoblastic leukemia Project Request: Animals: Humans: X Clinical trial: The project has already been presented: Project code reference: I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Dipartimento di Salute della Donna e del Bambino -SDB- Laboratorio della Clinica di Oncologia-Ematologia Pediatrica, Università degli Studi di Padova Manager Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 11/07/ / 5

309 A new strategy for the detection of residual leukemia cells BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Paganin Maddalena Veneto Background and Significance The prognostic impact of minimal residual disease (MRD) in childhood ALL has been extensively inspected. Trial AIEOP- BFM-ALL include standardized quantitative assessment of MRD for stratification, based on immunoglobulin (Ig) and TCR gene rearrangements as polymerase chain reaction targets. However the present techniques have three main limit: the analysis of junctional regions of Ig/TCR genes is really laborious and complex, moreover the analysis is not feasible for all patients and finally the sensibility/specificity is limited to the detection of around 1 leukemia cells on normal cells. The T-ALL occurs in the 10%-15% of pediatric ALL cases. Recurrent somatic mutations have been identify in different genes (like NOTCH1, FBXW7, PTEN, IL7R) cooperating to the malignant T cell transformation. After the publication of the human genome, a new generation of sequencer have been developed, these technologies are going to improve medical diagnosis (for example new Ig/TCR based MRD assays are in development). In this context, we would like to use the somatic mutations as leukemia specific genetic markers and apply next-generation ultra-deep sequencing technologies to detect the presence of residual ALL cells during the therapy. The development of this novel approach to monitor the response to therapy would allow to be more fast, precise, quantitative, sensitive, compared to the currently used standard technologies; moreover it could be potentially applied to all patients. Specific aims Aim 1: Aim 2: Aim 3: Determinate the sensitivity, precision, and quantitative capacity of a new possible diagnostic method for minimal residual disease detection. We are going to quantify the presence of the somatic mutation by ultra-deep next generation-sequencing applied in bone marrow samples collected during treatment in patients with T-ALL as marker of the residual T-ALL cells. We are going to compared the results to the IG/TCR allele-specific oligonucleotide polymerase chain reaction and multiparameter flow cytometry. Identify new marker for the patients that cannot be followed by the gold-standard MRD assays. We are going to analyze if the level of MRD identify with the next-generation sequencing of somatic mutations is predictive of outcome in the patients without Ig/TCR gene rearrangements who cannot be followed by Real-Time PCR technology during therapy. Pinpoint, through the frequency of somatic mutation, the different clones and sub-clones in the leukemia population at diagnosis to follow it during therapy and correlate the specific different mutation to the response to therapy. Hypothesis: The somatic mutations can be used as genetic marker for each specific leukemia clone in T-ALL patients and the high-throughput ultra deep next generation-sequencing allow the precise detection of the single cancer cell in a bulk of normal cells. Preliminary data: 1. Mutation analysis of T-ALL oncogenes and tumor suppressor genes identified NOTCH1and FBXW7 mutations, resulting in activation of NOTCH1 signaling, in 56% (68/121) and 22% (29/129) respectively of peditric T-ALL patients. In addition, mutations in signaling pathways (PTEN and IL7R) were found in 16% (6/37) and 7% (16/211) of T-cell leukemias. This data demonstrate that in our T-ALL pediatric cohort the mutations are present at an expected frequency and this cohort is representative and available for the subsequent analysis of the project. 2. To examine the specific frequency of the mutated clone detected by Sanger Sequencing in the diagnosis T-ALL bone marrow samples we apply deep-sequencing to the mutated patients for IL7R gene exon 6. We were able to detect the mutation previously identify by Sanger sequencing. We determining the presence of mutated alleles at a frequency that vary from 50% to 0.57% (mutations 11/07/ / 5

310 A new strategy for the detection of residual leukemia cells BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Biomedical/Biomedica Paganin Maddalena Veneto at a level lower than 10% could not be detected by Sanger Sequencing). Using high-throughput ultra deep next generation-sequencing we are able to identify with greater accuracy mutations detected by Sanger sequencing, this result demonstrate that we can use this technology to perform the analysis of the minimal residual disease in the bone marrow of the followup during therapy in T-ALL patient, at least with IL7R gene. Materials and Methods Samples: 1. DNA from the bone marrow of 100 T-ALL patients available as the Laboratory of Oncology Haematology Clinic is the AIEOP Bio-Bank center. Methods: 1. Analysis of pathogenetic recurrently mutated genes NOTCH1, FBXW7, PTEN, IL7R with Next-generation amplicon sequencing: 454 chemistry, Universal Tailed Amplicon Library, 454 Junior Platform. 2. Exome sequencing of diagnosis T-ALL with Ion Torrent Platform and division of somatic mutations for frequency to separate the leukemia bulk in clones and sublcones. 3. Ultra-deep amplicon sequencing of pathogenetic mutated gene at day 33 and day 78 of therapy for disease detection and comparison with the data optained with gold-standard techniques (real-time PCR and flow-cytometry). Impact and Translational Implications This project would allow the development of a diagnostic system to analyze the minimal residual disease with increase sensitivity and specificity compared to the gold-standard technique. The development of a somatic-mutation approach to monitor the response to treatment in patient with leukemia could be extensively applied to all type of cancer, as all the cancers can be characterization for somatic mutations usable as genetic markers. 11/07/ / 5

311 Role of Thymic stromal lymphopoietin on immune cell polarisation and cancer development BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Fornasa Giulia Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Immunology Immunology Fellowships and Area - F07 Project Keyword 1: Project Keyword 2: Project Keyword 3: Mechanisms, prevention, and treatment of diseases when the immune system has a major role Thymic Stromal Lymphopoietin Colorectal cancer Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: X I declare that the object/s of this application is/are under patent copyright Patent owner: IEO - Istituto Europeo di Oncologia Patent number: PCT/EP2014/ (pending) Operative Units INSTITUTION Department/Division/Laboratory Role in the project 1 DI CERTIFICATION Department of experimental Oncology DI Applicant Institution 2 Fondazione IRCCS Cà Granda, Ospedale Policlinico di Milano Unità Operativa di Gastroenterologia ed Endoscopia Operative unit 2 Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Caprioli Flavio Andrea Fondazione IRCCS Cà Granda, Ospedale Policlinico di Milano Principal Investigator - Operative Unit 2 11/05/ /07/ / 6

312 Role of Thymic stromal lymphopoietin on immune cell polarisation and cancer development BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Clinical health care research/clinicoassistenziale Fornasa Giulia Istituto Europeo di Oncologia Background and Significance Inflammation impacts every step of cancer development, initiation, growth and metastatic progression. Inflammation can have opposite effects on tumors. Chronic inflammation can increase the risk of tumor development and promote progression, but acute inflammation can also influence the host immune response to tumors. This balance is mainly defined by the different inflammatory mediators involved, representing a wide therapeutic opportunity. Thymic stromal lymphopoietin (TSLP) is a cytokine primarily expressed by epithelial cells, keratinocytes and stromal cells, mostly in the lung, skin and gut. TSLP is involved in several physiological and pathological immune activities. In the gut, TSLP exerts anti-inflammatory activities acting on intestinal DCs. On the contrary, it plays a pathogenic role in several disorders like atopic dermatitis and asthma driving allergic Th2 responses. Also in cancer TSLP shows this dichotomy, overexpression of TSLP is associated with the progression of breast, pancreatic and lung cancer, but it has been shown that high levels of TSLP released by barrier-defective skin result in resistance to tumorigenesis. In humans, two isoforms of TSLP exist, the long (159 AA) and the short isoform (63 AA) with two independent promoters. Long TSLP is widely studied in the literature but little is known concerning the short TSLP. Our data suggest that the two TSLPs have opposite activities, with long and short TSLP being pro and anti-inflammatory, respectively. Specific aims Aim 1: Aim 2: Aim 3: Analysis of TSLP isoform expression in colorectal cancer (CRC) tissues. Several studies report that TSLP overexpression in the tumor microenvironment contributes to tumor growth following Th2 differentiation. But they do not deal with the two different transcript variants, the long and the short TSLP. Our aim is to study the differential expression of the two isoforms in tumor samples in order to discern the contribution of each one in tumor development. Considering the importance of TSLP in the gut we will analyze tissue samples from CRC patients, both neoplastic mucosa and healthy tissue. We will evaluate if the differential expression of the short/long TSLP correlates with a different polarization of the T cell infiltrate and thus with a pro- or antitumorigenic environment. Analysis of functional effects and mechanisms of action of TSLP isoforms. We are planning to conduct functional studies in vitro to evaluate the biological effects of the two isoforms and, in particular, evaluate their capability of favoring one or another T cell subset generation. From the literature we know that long isoform favors the development of allergic Th2 cells and short TSLP has only been described as an anti-microbial protein. We plan to identify all the cellular targets and the signaling pathways of the short isoform. Therapeutic potential of short TSLP. Several diseases have been associated with the over expression of TSLP. In all of the cases the deleterious effect of TSLP could be attributed to the long isoform. Our data suggest that short TSLP has anti-inflammatory properties and, being a very small protein, is a very good candidate as a drug. We will test short TSLP anti-tumorigenic activity in vivo in two murine models of colon cancer, the APC(Min/+) mice, that develop spontaneously polyps in the intestine and the AOM-DSS protocol, a chemically-induced model of colitisassociated colon carcinogenesis. Hypothesis: Until now the two TSLP isoforms have been almost completely ignored by the literature. We found that short TSLP has anti-inflammatory properties and is the only isoform expressed in healthy tissues. We hypothesize that expression of TSLP isoforms is modulated during inflammation and this has an impact on the polarization of the immune response and cancer development. Preliminary data: We demonstrated that short TSLP is the only isoform expressed in healthy tissue and exerts antiinflammatory activities by affecting the capacity of peripheral blood cells to produce inflammatory cytokines. Moreover, short TSLP ameliorates experimental colitis and prevents endotoxin shock in vivo. The long, instead, is expressed only during inflammation. TSLP isoform ratio is altered during 11/07/ / 6

313 Role of Thymic stromal lymphopoietin on immune cell polarisation and cancer development BANDO 2013 PROGETTI GIOVANI RICERCATORI GR Clinical health care research/clinicoassistenziale Fornasa Giulia Istituto Europeo di Oncologia several inflammatory disorders, including celiac disease and ulcerative colitis. Preliminary data on CRC patients show a significant reduction of short TSLP in tumor tissue compared to the healthy counterpart, contrarily to other types of cancer where it has been shown that total TSLP is greatly upregulated. Materials and Methods CRC biopsies will be collected in our institute and in the Policlinico di Milano (Dr. Flavio Caprioli). We have the possibility to analyze the neoplastic mucosa and the non-cancer `healthy tissue from the same patient. Tumor samples will be snap frozen for RNA and protein extraction or fixed for paraffin embedding. In parallel, T cell infiltrate will be isolated and the polarization evaluated by intracellular staining. We already generated the tools to distinguish the two isoforms, specific primers for the qpcr and an antibody specific for long TSLP. Functional studies will be conducted on human primary cells isolated from healthy peripheral blood, focusing on DC and T cell activation and polarization. Specific signaling pathways will be analyzed on TSLP-conditioned cells with a custom phospho-kinase array kit. The in vivo models of intestinal cancer will be conducted in the animal facility of the IEO campus; both models have already been successfully setup in the host laboratory Impact and Translational Implications The role of TSLP in cancer is not clear, so far studies in epithelial cancers had assigned to TSLP both oncogenic and tumor-suppressive effects. This dichotomy may be due to the existence of two isoforms of TSLP that have opposite functions. This aspect should be taken into account in the development of therapeutic agents. Acting on the balance between short/long TSLP we have the possibility to regulate both the inflammatory potential of long and the tolerogenic/homeostatic role of short TSLP. 11/07/ / 6