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 Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale Toscana TITOLO PROGETTO Seven Tesla MR imaging as preclinical biomarker in populations at risk for Parkinson disease Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Istituto Superiore di Sanita' TITOLO PROGETTO Seroprevalence of Hepatitis E virus infection in Italian blood donors: a survey at national and regional level Area Expertise Population Sciences and Epidemiology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Finanziamento Assegnato euro Methodological, epidemiological, ,00 socio-economic, organizational, managerial emerging public health issues related to the above reported areas CODICE RF Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO NSD1 PHD fingers as potential targets for Acute Myeloid Leukemia Area Expertise Biological Chemistry and Macromolecular Biophysics TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00

2 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale IZSPLV TITOLO PROGETTO APRON: Assay for PRiONs. DEVELOPMENT OF AN ASSAY DETECTING PRIONS IN ANIMALS AND HUMANS AFFECTED WITH PRION DISORDERS IN A PRECLINICAL AND CLINICAL STAGE Area Expertise Veterinary TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Food safety and animal safety Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Piemonte TITOLO PROGETTO Pancreatic cancer therapy based on combination of DNA vaccination and PI3Kgammainhibition Area Expertise Immunology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale ISTITUTO TUMORI MILANO TITOLO PROGETTO MICRORNA IN PAPILLARY THYROID CARCINOMA: PATHWAYS INVOLVED AND POSSIBLE PROGNOSTIC AND THERAPEUTIC IMPACT Area Expertise TIPOLOGIA_RICERCA Oncology 1 - Basic Translational Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale IZSUM TITOLO PROGETTO BIOLOGICAL APPROACH TO DECONTAMINATION OF BIVALVE MOLLUSCS FROM V.PARAHAEMOLYTICUS AND SALMONELLA BY BACTERIOVORAX SPECIES. Area Expertise Veterinary TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Food safety and animal safety Finanziamento Assegnato euro ,00

3 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO Defining molecular targets that govern the capacity of platelets to support hepatitis B virus (HBV)-associated liver immunopathology Area Expertise Immunology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Toscana TITOLO PROGETTO An integrated approach to unravel the genetic causes and molecular pathogenesis of epileptogenic focal cortical dysplasia. Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale HUMANITAS TITOLO PROGETTO Anti-Cholinergic receptors antibodies, cardiovascular autonomic profile and dysautonomia symptoms relationships in Pure Autonomic Failure, Amyotrophic Lateral Sclerosis and Postural Orthostatic Tachycardia Syndrome: evidence for a pathophysiology based th Area Expertise Cardiovascular and Respiratory Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale SAN MATTEO TITOLO PROGETTO Cardiac amyloidosis: molecular mechanism and innovative therapies for a challenging aging related cardiomyopathy Area Expertise Biological Chemistry and Macromolecular Biophysics TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

4 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale ISTITUTO TUMORI MILANO TITOLO PROGETTO SELECTIVE AXILLARY LYMPH NODE DISSECTION VS COMPLETE AXILLARY DISSECTION: A RANDOMISED CLINICAL TRIAL TO ASSESS THE PREVENTION OF LYMPHEDEMA IN BREAST CANCER TREATMENT Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO Rab39b mouse models: pre-clinical studies by using AMPA receptors as possible therapeutic targets to unravel the role of RAB39B in Intellectual Disability and Autism Spectrum Disorder Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale HUMANITAS TITOLO PROGETTO The long pentraxin PTX3 in cancer: a novel player in cancer-related inflammation Area Expertise Immunology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale MONZINO TITOLO PROGETTO Prenylcysteine Lyase as a potential novel player in atherosclerosis Area Expertise Vascular and Hematology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

5 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale Istituto Superiore di Sanita' TITOLO PROGETTO Calcitonin oligomers as a model to study the amyloid neurotoxicity: the focal role played by lipid rafts in the prevention and cure. Area Expertise Biological Chemistry and Macromolecular Biophysics TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale MONDINO TITOLO PROGETTO Inhibition of endocannabinoids-hydrolyzing enzymes as a new therapeutic target for migrainetreatment: studies in animal models and preliminary evaluation in humans Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale AUXOLOGICO TITOLO PROGETTO ALS and FTD: changing paradigms in the genotype-phenotype correlation due to C9orf72 and other gene characterization Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Piemonte TITOLO PROGETTO Cervical cancer screening in younger women. Area Expertise Healthcare Delivery and Methodologies TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00

6 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale MONDINO TITOLO PROGETTO Idiopathic Normal Pressure Hydrocephalus (inph), parkinsonism and dementia: improving the accuracy of diagnosis and the patient care to reverse the symptomatology Neurodegeneration, Phenotypes and Outcome Measures Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Emilia-Romagna TITOLO PROGETTO Multicenter phase II clinical study of adoptive immunotherapy with alloreactive NK cells as consolidation strategy for elderly acute myeloid leukemia patients Area Expertise Oncology 2 - Translational Clinical TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Sicilia TITOLO PROGETTO Role of environment-gene interaction in etiology and promotion of pituitary tumours. Area Expertise Endocrinology, Metabolism, Nutrition and Reproductive Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO Cell-type and subunit specific alterations of NMDA receptors in striatum at early stages of Parkinson's disease Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00

7 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale MARIO NEGRI TITOLO PROGETTO Development of a nanoparticle-based pharmacological approach for amyotrophic lateral sclerosis Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale FATEBENEFRATELLI TITOLO PROGETTO Neuroplasticity and Alzheimer's disease: integrated approach to identify biological and neurophysiological markers. Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale CARLO BESTA TITOLO PROGETTO Subthalamic nucleus deep brain stimulation in Tourette syndrome Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale CASA SOLLIEVO SOFFERENZA TITOLO PROGETTO Are genetic, inflammatory and metabolomic markers of coronary heart disease related to kidney function decline in patients with type 2 diabetes? Area Expertise Population Sciences and Epidemiology 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 Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale MAGGIORE TITOLO PROGETTO Effect of surgical or conservative approach in patients with adrenal incidentalomas on cardiovascular, metabolic, neuropsychological and bone manifestations: respective roles of cortisol secretion and glucocorticoid sensitivity. Area Expertise Endocrinology, Metabolism, Nutrition and Reproductive Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Veneto TITOLO PROGETTO The genetics of sudden cardiac death in athletes and implication for risk prevention Area Expertise Cardiovascular and Respiratory Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Toscana TITOLO PROGETTO LEFT VENTRICULAR HYPERTROPHY IN AORTIC VALVE DISEASE AND HYPERTROPHIC CARDIOMYOPATHY: GENETIC BASIS, BIOPHYSICAL CORRELATES AND VIRAL THERAPY MODELS Area Expertise Cardiovascular and Respiratory Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO Inhibition of Glycolysis by 2-Deoxy-D-Glucose as a Therapeutic Strategy for Polycystic Kidney Disease Area Expertise Digestive, Kidney and Urological Systems TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

9 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale I.E.O. TITOLO PROGETTO Role of the p21-mediated DNA-Damage Response in the immune-surveillance of leukemia stem cells Area Expertise TIPOLOGIA_RICERCA Oncology 1 - Basic Translational Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale I.E.O. TITOLO PROGETTO Clinical utility of a next generation sequencing-based "oncochip" for therapeutic decision in metastatic breast cancer Area Expertise Oncology 2 - Translational Clinical TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale I.E.O. TITOLO PROGETTO Identification of new therapeutical targets of cancer stem cells: study of the mechanistics of mitotic spindle alignment to cortical polarity in mammary stem cell asymmetric divisions Area Expertise Biological Chemistry and Macromolecular Biophysics TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO Role of serotonin in modulating L-DOPA-induced dyskinesia Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00

10 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale Piemonte TITOLO PROGETTO Improving therapeutic appropriateness of Multiple Sclerosis treatments using biological approaches to personalize therapy and save pharmaceutical spending Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale OSPEDALE BAMBINO GESU' TITOLO PROGETTO Role of extracellular vesicles in bone tumour pathogenesis: implications for therapy Area Expertise Musculoskeletal, Oral and Skin Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Innovative biotechnologies Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Abruzzo TITOLO PROGETTO ASSESSMENT OF THE PATHOBIOLOGICAL ROLE, DIAGNOSTIC AND PREDICTIVE VALUE OF THE NOVEL CYTOKINE IL-30 IN PROSTATE CANCER Area Expertise TIPOLOGIA_RICERCA Oncology 1 - Basic Translational Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Istituto Superiore di Sanita' TITOLO PROGETTO Connecting DNA repair and metabolic alterations of obesity in a search for predictive biomarkers Area Expertise Endocrinology, Metabolism, Nutrition and Reproductive Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00

11 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale ISTITUTO TUMORI MILANO TITOLO PROGETTO Molecular characterization of pulmonary sarcomatoid carcinoma, a deadly subtype of lung cancer, for patient management Area Expertise Genes, Genomes and Genetics TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Veneto TITOLO PROGETTO NETosis in atherothrombotic disorders Area Expertise Vascular and Hematology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Emilia-Romagna TITOLO PROGETTO Validation of a novel non-invasive technique based on real time ultrasound B-mode to assess the jugular venous pulse Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale STELLA MARIS TITOLO PROGETTO CareToy: a smart System for early home-based intervention in infants at high risk for Cerebral Palsy Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00

12 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale Emilia-Romagna TITOLO PROGETTO Axillary Reverse Mapping (ARM).Try to identify and spare lymphatic pathways in breast cancer surgery to prevent breast cancer related lymphedema (BCRL) Area Expertise Surgical Sciences, Biomedical Imaging, and Bioengineering TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Veneto TITOLO PROGETTO Development and implant of the photovoltaic artificial retina in the pig with photoreceptor degeneration: towards the human Phase-1 experimentation Area Expertise Bioengineering Sciences and Technologies TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Innovative biotechnologies Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO A novel structural and functional MRI-derived index of axonal myelination: application to MS and correlation with neurophysiology Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale I.E.O. TITOLO PROGETTO Assessment of the therapeutic value of targeting cancer stem cells in Numb-deficient breast cancer. Area Expertise TIPOLOGIA_RICERCA Oncology 1 - Basic Translational Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00

13 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale MAGGIORE TITOLO PROGETTO Early atherosclerosis in patients with steatosis and with chronic hepatitis C: role of visceral adiposity, procoagulant imbalance and endothelial dysfunction in vascular damage Area Expertise Endocrinology, Metabolism, Nutrition and Reproductive Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale DON GNOCCHI TITOLO PROGETTO Mother-child immunogenetic interactions in pregnancy and risk of Autism Spectrum Disorders Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO The prognostic value of collateral flow in acute ischemic stroke secondary to occlusion of intracranial arteries treated by endovascular therapy. Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Piemonte TITOLO PROGETTO EARLY STUDY. MULTICENTER, RANDOMIZED, CLINICAL TRIAL ON THE COMPARISON BETWEENEARLY SURGERY AND CONVENTIONAL THERAPY IN INFECTIVE ENDOCARDITIS. Area Expertise Cardiovascular and Respiratory Sciences TIPOLOGIA_RICERCA Clinico-assistenziale 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 Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale SAN RAFFAELE MILANO TITOLO PROGETTO Identification of biological and imaging events that predict the progression of Takayasu arteritis vascular lesions Area Expertise Immunology TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale MAGGIORE TITOLO PROGETTO Vitamin D Supplementation on Assisted Reproduction Technology (ART) outcomes: a randomized clinical controlled trial and an investigation of the involved biological mechanisms Area Expertise Endocrinology, Metabolism, Nutrition and Reproductive Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Environmental diseases, safety of the work place and occupational diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Toscana TITOLO PROGETTO Novel approaches for screening of humoral primary immunodeficiencies in adult patients Area Expertise Immunology TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Innovative biotechnologies Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Abruzzo TITOLO PROGETTO Effects of multimodal training on cognition, biomarkers, rs-fmri and brain structural integrity in MCI patients Area Expertise Brain Disorders and Clinical Neuroscience 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 Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale Emilia-Romagna TITOLO PROGETTO Olfaction as an index of conversion from MCI to AD: a longitudinal functional connectivity study Area Expertise Integrative, Functional, and Cognitive Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale I.N.R.C.A. TITOLO PROGETTO Report-AGE Project: clinical and biological predictors of physical function decline in older patients with chronic cardiovascular diseases Area Expertise Biology of Development and Aging TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Metabolic and cardiovascular diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale OSPEDALE BAMBINO GESU' TITOLO PROGETTO Targetting the interleukin-6 receptor as a novel therapeutic approach in Duchenne muscular dystrophy to modulate muscle inflammation and regeneration Area Expertise Musculoskeletal, Oral and Skin Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale OSPEDALE BAMBINO GESU' TITOLO PROGETTO Distinct developmental programs of cord blood and bone marrow haematopoietic stem cells reconstitute different B-cell compartments in transplanted patients leading to transplant-specific immune impairment. Cellular and molecular characterization of transi Area Expertise Immunology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00

16 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale IZSPLV TITOLO PROGETTO No more emergencies in DNA species identification: development and harmonization of molecular and next generation methods to determine and quantify origin species in food and to discriminate frauds from unintentional contamination. Area Expertise Veterinary TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Food safety and animal safety Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Veneto TITOLO PROGETTO Targeting mitochondrial calcium handling in core myopathies: an integrative approach towards novel therapeutic strategies Area Expertise Musculoskeletal, Oral and Skin Sciences TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale SANTA LUCIA TITOLO PROGETTO Identifying preclinical phenotypic and biological markers of Alzheimer's Disease in healthy elderly at risk subjects Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale IZSPLV TITOLO PROGETTO A new immunotherapy option for oral melanoma and other canine malignant tumors using a DNA vaccine coding for human chondroitin sulphate proteoglycan 4 (CSPG4), integrated with the validation of candidate gene useful for therapeutic opportunities and prog Area Expertise Veterinary TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00

17 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale S.CAMILLO TITOLO PROGETTO Modulation of visuospatial awareness during multi-tasking in right hemisphere stroke patients: towards novel behavioral and neurofunctional predictors of impairment and recovery of the attentional networks Area Expertise Integrative, Functional, and Cognitive Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Emilia-Romagna TITOLO PROGETTO A NOVEL INTEGRATED GENOMIC AND IMMUNOLOGICAL STRATEGY FOR THE THERAPY OF CHRONIC HEPATITIS B Area Expertise Immunology TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale MAGGIORE TITOLO PROGETTO Effects of maternal nutrition during pregnancy on fetal epigenetic profile: set up of a research infrastructure. Area Expertise Healthcare Delivery and Methodologies TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Innovative biotechnologies Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale Umbria TITOLO PROGETTO Vitamin E, mirna and inflammation: a tunable network in Alzheimer's disease. Area Expertise Brain Disorders and Clinical Neuroscience TIPOLOGIA_RICERCA Biomedica AREA_STRATEGICA Neurologic diseases Finanziamento Assegnato euro ,00

18 ABSTRACT DEI PROGETTI FINANZIATI NEL2015 CON IL BANDO RICERCA FINALIZZATA - FINANZIAMENTO 2013 NELLA SEZIONE Progetti Ordinari INDICE PROGETTI CODICE RF Destinario Istituzionale SAN GALLICANO TITOLO PROGETTO Clinical and Experimental Pathway to Vitiligo Management Area Expertise Musculoskeletal, Oral and Skin Sciences TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Infectious and immunological diseases Finanziamento Assegnato euro ,00 CODICE RF Destinario Istituzionale ISTITUTO TUMORI MILANO TITOLO PROGETTO Towards precision medicine for patients with biliary tract cancers: tailored treatments based on the mutational profile detected on tumor lesions and monitored in liquid biopsies (circulating tumor cells, CTC, and circulating tumor DNA, ctdna) Area Expertise Oncology 2 - Translational Clinical TIPOLOGIA_RICERCA Clinico-assistenziale AREA_STRATEGICA Oncology Finanziamento Assegnato euro ,00 SEGUONO ABSTRACT PROGETTI FINANZIATI

19 Seven Tesla MR imaging as preclinical biomarker in populations at risk for Parkinson disease BANDO 2013 Progetti Ordinari RF RF Cosottini Mirco 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: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Parkinson s disease and other movement disorders (Huntington s, Dystonias, Ataxias). Magnetic resonance imaging Ultra high field 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/Lavoratory Role in the project 1 Toscana Azienda Ospedaliero Universitaria Pisana UO Neuroradiologia 2 Toscana Azienda Ospedaliero Universitaria Pisana UO Neurologia 3 Azienda Ospedaliero Universitaria San Giovanni di Dio e Ruggi d'aragona Azienda Ospedaliero Universitaria San Giovanni di Dio e Ruggi d'aragona UO Neurologia Coordinator MR acquisition Patient recruitment Genetic analysis Patient recruitment Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Ceravolo Roberto Toscana Patient recruitment 04/04/ Pellecchia Maria Teresa Azienda Ospedaliero Universitaria San Giovanni di Dio e Ruggi d'aragona Genetic analysis Patient recruitment 03/06/ /07/ / 5

20 Seven Tesla MR imaging as preclinical biomarker in populations at risk for Parkinson disease BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale Cosottini Mirco Toscana Background and Significance Parkinson s disease (PD) has a long premotor period during which pathological features evolve but do not reach the threshold for presenting with motor symptoms. In this period a clinical diagnosis is not permitted and a preclinical biomarker would be advisable. Identification of subjects with increased risk to evolve to PD would allow a better understanding of disease progression, an early diagnosis even before symptom onset and a prompt treatment. More importantly, neuroprotective therapies might be most successful if given in this preclinical phase. People with the higher risk to evolve to PD are subjects carrying gene mutations for monogenic forms of PD and subjects affected by conditions associated with an increased risk of PD such as hyposmia and Rem Sleep Behaviour Disorder (RBD). Ultra-high field (UHF) MRI is a research tool that improves the image contrast and resolution, and has been tested in different neurological disorders. In PD, targeted UHF-MRI of the midbrain allows to reveal the loss of the normal structure of the substantia nigra (SN). In particular the intermediate component of the SN (compacta ventralis) and the nigrosome1 formation are not recognizable in PD patients allowing a diagnostic accuracy near to 100 % also in patients with short disease duration. Application of UHF 7-Tesla MRI to premotor PD stage could provide a marker of SN degeneration and within the population at risk, could help to identify subjects with the higher possibility to develop Specific aims Aim 1: Aim 2: Aim 3: To identify the changes of the SN anatomy in patients with genetic form of PD (Park1, Park2, Park8) and specific mutation carriers as a preclinical biomarker of disease: the main questions that will be addressed concern the SN changes in different forms of parkinsonisms (monogenic parkinsonism versus typical sporadic PD) and the timeline of the appearance of such SN alterations (monogenic parkinsonism versus gene mutation carriers). To identify the changes of the SN anatomy in subjects at risk of developing PD (RBD or hyposmia): the question addressed will concern which is the percentage of subjects with abnormal nigral aspect in a group of subjects at increased risk for PD. To evaluate longitudinally the clinical course of subjects at risk either genetic or sporadic: the question addressed concerns the potential predictive value of SN anatomy changes in the follow-up and the evaluation of risk of conversion to a clinical PD. Hypothesis: Since the dopamine loss within the SN occurs before the clinical onset of the disease and the inner structure of the SN is detectable at 7T, we hypothesize that target imaging of the SN might represent a radiological specific sign in subjects with monogenic PD forms and a biomarker of increased risk of conversion from preclinical phase to the disease in subjects at risk. Preliminary data: We described in previous works the UHF-7T MR capability to identify the inner structure of the SN and their changes in the early phase (also Hoehn&Yahr stage I) of PD with respect to healthy controls (Cosottini et al, Radiology 2014). We also demonstrated that 7T MR is well tolerated both in healthy subjects and patients with PD (Cosottini et al, Eur Radiol 2014). Preliminary unpublished data have suggested that the nigrosome changes are present also in atypical parkinsonisms as well as in two cases of parkinsonisms associated respectively with Park8 and Park2 mutation. 10/07/ / 5

21 Seven Tesla MR imaging as preclinical biomarker in populations at risk for Parkinson disease BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale Cosottini Mirco Toscana Materials and Methods We will enroll the following study population: 25 patients with monogenic PD (Park1, Park2, Park8); 25 asymptomatic subjects carriers of a gene mutation involved in PD; 40 patients with hyposmia; 40 patients with REM Sleep Behaviour Disorder. All subjects will be clinically evaluated before the 7T MRI scan, to obtain a disease staging in monogenic PD patients, and to exclude mild parkinsonian signs in the population at risk. MR examinations will be performed by using a 7T scanner GE 950 MRI (GE Healthcare Medical Systems, Milwaukee, WI, USA) equipped with a 2ch-tx/32ch-rx head coil (Nova Medical, Wilmington, MA USA). All subjects will undergo a high resolution three-dimensional Susceptibility-Weighted Angiography (SWAN). The SWAN acquisition time is 4 02, the in plane resolution is 312 µm. After the scan, all subjects will be clinically evaluated at every four months to uncover the appearance of any motor signs and non motor symptoms in the population at risk. Impact and Translational Implications Identification of patients with high risk to evolve to PD would allow early diagnosis or even a preclinical detection. Since putative neuroprotective or disease modifying therapies might be more effective in the preclinical phase than when the critical threshold from preclinical to clinical manifestation has been passed, the identification of a biomarker of risk of PD is desirable. A MRI based biomarker would be an objective, not invasive new tool for early PD diagnosis and treatment planning. 10/07/ / 5

22 RF Seroprevalence of Hepatitis E virus infection in Italian blood donors: a survey at national and regional level Pisani Giulio Biomedical/Biomedica Istituto Superiore di Sanita' 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 Infectious Diseases, Reproductive Health, Asthma and Pulmonary Conditions - IRAP Project Keyword 1: Project Keyword 2: Project Keyword 3: The transmission and distribution of infectious, reproductive, asthma/allergy, and non-malignant pulmonary conditions in human populations in relation to person, place, time, environmental exposures, animal/insect vectors and personal characteristics or behaviors. Hepatitis E virus Blood safety 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/Lavoratory Role in the project 1 Istituto Superiore di Sanita' National Center for Immunobiologicals Research and Evaluation, Biological Unit 2 Istituto Superiore di Sanita' Department of Infectious, Parasitic and Immuno-mediated diseases, Viral Hepatitis Unit Coordinator of the project Coordinator of laboratory investigation and result evaluation 3 Istituto Superiore di Sanita' National Blood Center Coordinator of the Italian Blood Transfusion Centres Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Bruni Roberto Istituto Superiore di Sanita' Coordinator of laboratory investigation, result evaluation and study design 26/06/ Pupella Simonetta Istituto Superiore di Sanita' Coordinator of the Italian Blood Transfusion Centres and study design 3 equestre michele Istituto Superiore di Sanita' Next Generation Sequencing and molecular analysis and result evaluation 4 Pezzotti Patrizio Istituto Superiore di Sanità Study design, statistical analysis and result evaluation 22/04/ /08/ /12/ /07/ / 5

23 RF Seroprevalence of Hepatitis E virus infection in Italian blood donors: a survey at national and regional level Pisani Giulio Biomedical/Biomedica Istituto Superiore di Sanita' Background and Significance Hepatitis E virus (HEV) can cause sporadic as well as epidemic acute hepatitis. In Europe, all autochthonous HEV infections are caused by HEV genotype 3 (gt-3) and are linked to the consumption of contaminated food. In general, HEV gt-3 infection remains asymptomatic or occurs as mild self-limited acute hepatitis. During HEV infection, the first antibody to appear is IgM at week 4, followed by IgG at week 5. Usually IgM positivity persists for 2-3 months while viremia appears at week 2 and normally lasts for 2 to 3 weeks but it can last up to 112 days. Recently, seroprevalence studies among blood donors were performed in several places in Europe and they evidenced a large heterogeneity with values of IgG positivity varying from around 5% to more than 50%. Several studies also evidenced presence of HEV IgM and HEV RNA in some sera suggesting that HEV infection in blood recipient is possible. Transfusiontransmitted risk for HEV infections may be relevant for individuals who are immunocompromised or are unable to achieve viral clearance. These patients are at risk of developing chronic HEV infection with increased mortality. Specific aims Aim 1: Aim 2: Aim 3: To estimate the IgG seroprevalence of HEV in Italian blood donors at national and regional level. Up to now there are no nationwide data about HEV prevalence in Italy. Only few and old prevalence data based on studies performed at local or regional level using first generations of ELISA kit are available. To evaluate anamnestic information from blood donors enrolled in the study, in order to identify behavioural risk factors possibly related to HEV transmission. To define the proportion of imported (due to gt-1) and autochthonous (due to gt-3) cases as well as to compare HEV strains from different Italian regions, and evaluate the zoonotic origin of autochthonous infections by comparison of human HEV sequences with sequences from animal viruses available in databases (HEV from pig and wild boar). Hypothesis: Based on recent studies and our preliminary data (see below), it is hypothesized that HEV infection in Italy may be more common than previously believed, especially in some local settings, raising the possibility of transfusion transmission risk. In order to estimate the Italian HEV prevalence, the IgG, IgM and HEV RNA detection in serum of blood donors will be evaluated. The study will involve at least 100 blood unit centers and at least 10,000 serum samples. The high number of blood unit centers is needed to take into account the likely high heterogeneity in the prevalence. In fact, we expect that, assuming that the intraclass correlation be 0.1 among donors of the same center, the estimate of the prevalence will have a 95% confidence intervals of around ±5%. Preliminary data: In a recent survey on a small blood donor population from a region of central Italy, a HEV IgG seroprevalence of 48% ( 95% CI: 43-54) was observed. Multiple binomial regression showed that eating raw dried pork liver sausage was associated with past and present infection in this region (APRR=2.11, 95% CI: 1-3). Among IgG positive samples, 2 samples (1.3%) tested positive for IgM, and one of them was also HEV-RNA positive by Real-Time PCR. Sequence analysis of a viral genome fragment amplified from this latter sample showed that the infecting virus was gt-3. A parallel blood donor survey from another region of central Italy showed quite lower IgG prevalence (9%), suggesting high heterogeneity among Italian regions. These findings prompt for a nationwide large scale study. 10/07/ / 5

24 RF Seroprevalence of Hepatitis E virus infection in Italian blood donors: a survey at national and regional level Pisani Giulio Biomedical/Biomedica Istituto Superiore di Sanita' Materials and Methods The study will consider a stratified two-stage sampling design using the blood unit (cluster) as the first stage of sampling and the donors as the second stage of sampling. Clusters will be sampled with weights proportional to the volume of blood donations performed in the last year. The sampling selection will be based on stratification by region and by randomization for the blood unit centers within each region. Voluntary blood donors within each included center will be selected for the study covering all Italian regions and after taking written consent. Serum samples will be tested for anti-hev IgG by ELISA kits. Positive samples will be tested for anti-hev IgM, presumptive for acute infection. IgM positive samples will be tested by Real-Time PCR for HEV RNA detection. The HEV genome of positive samples will be characterized by a next generation sequencing approach (Illumina MiSeq platform). All statistical analyses will be based on formulae based in the two-sampling design. Impact and Translational Implications 1) Evaluating the prevalence of HEV infection among blood donors at national and regional level and to establish the influence of age, sex, geographical origin and specific risk factors 2) These data can be used by the National Blood Center and regulatory authorities for planning future preventive interventions even at the level of different regions of the country.

25 NSD1 PHD fingers as potential targets for Acute Myeloid Leukemia BANDO 2013 Progetti Ordinari RF RF Musco Giovanna Biomedical/Biomedica Ospedale San Raffaele - Milano LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Biological Chemistry and Macromolecular Biophysics Macromolecular Structure and Function C - MSFC Project Keyword 1: Project Keyword 2: Project Keyword 3: Protein-protein and protein-nucleic acid interactions, small molecule interactions with proteins and nucleic acids, and mechanisms of allostery. structure-function epigenetics Project Request: Animals: X Humans: Clinical trial: The project has already been presented: X Project code reference: RF I declare that the object/s of this application is/are under patent copyright Operative Units INSTITUTION Department/Division/Lavoratory Role in the project 1 Ospedale San Raffaele - Milano 2 Ospedale San Raffaele - Milano Genetics and Cell Biology\ Biomolecular PI NMR Unit Functional genomics of cancer/division of Molecular Oncology Head of the unit in charge of AIM2 3 IFOM - ISTITUTO FIRC DI ONCOLOGIA MOLECOLARE Proteomica funzionale Head of the unit in charge of AIM3 Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Tonon Giovanni Ospedale San Raffaele - Milano Head of the unit in charge of AIM2 24/02/ Bachi Angela IFOM - ISTITUTO FIRC DI ONCOLOGIA MOLECOLARE Head of the unit in charge of AIM3 09/04/ /07/ / 6

26 NSD1 PHD fingers as potential targets for Acute Myeloid Leukemia BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Musco Giovanna Ospedale San Raffaele - Milano Background and Significance The Nuclear receptor-binding SET domain protein 1 (NSD1), an histone methyltransferases, is fused to the nucleoporin NUP98 in up to 16% paediatric cases of human cytogenetically-normal acute myeloid leukemia (AML). Patients presenting this translocation have a dramatic prognosis, with a median survival of few months, among the worst in leukemias, and no effective treatment available. Among the protein domains present in NSD1, a plant homeo-domain finger (PHDv) juxtaposed to a variant PHD finger (C5HCH) is crucial for the inappropriate activation of HOX genes and is required for oncogenesis. Consistent with its putative role as chromatin reader, this region binds H3K4me3 and H3K9me3 in vitro but also provides a hub for the interaction with other proteins critical for NSD1 oncogenic activity such as the transcriptional repressor NIZP1. Other methyltransferases frequently translocated in cancer, such as MLL, rely on interacting proteins as for example DOT1L and BRD4 to deregulate transcription and to unleash the cancer phenotype. Remarkably, highly-promising compounds targeting these interacting partners have recently demonstrated remarkable impact in preclinical studies. PHD domains are critical for oncogenesis. Therefore, the validation of inhibitors targeting the NSD1 PHD domain and the identification of PHD interacting proteins endowed with oncogenic potential are critical steps toward the discovery of more effective therapies for the treatment of these leukemic patients. Specific aims Aim 1: Following the reviewers' insights, we propose the following integrated aims: Structural characterization of PHDv-C5HCH domain as interaction hub for multiple binding events. We will: a) determine the structure of PHDv-C5HCH domain and characterize its interaction with modified histone H3 tails to investigate possible combinatorial reading of multiple marks involved in short-range histone crosstalk. b) screen (in silico and in vitro) for PHDv-C5HCH inhibitors. c) characterize PHDv-C5HCH/NIZP1-C2HR interaction. Aim 2: In vitro and in vivo functional assessment of pharmacological and genetic inactivation of NSD1 PHDv-C5HCH domain. a) explore the effects of the putative PHD inhibitors on NUP98-NSD1-mediated transcriptional activity, histone binding and modifications, progenitor immortalization and oncogenicity, in vitro and in vivo, using myeloid Linbone marrow progenitors. b) explore whether aminoacids involved in the binding of PHDv-C5HCH (predicted by Aim1) impact on NUP98- NSD1 activity. c) assess NUP98-NSD1 interactors oncogenic potential (see AIM3) targeting with shrna the most promising candidates in progenitor cells infected with NUP98-NSD1. Aim 3: Characterization of NSD1 interactome We will investigate the role of the PHD fingers in the recruitment of chromatin interaction partners using a SILACbased quantitative proteomic approach. a) screening of the interaction partners of NSD1 and NUP98-NSD1 in progenitor cell lines (see AIM2) b) assessment the impact of selected inhibitors (from SA1,SA2) on NUP98-NSD1 interactome Hypothesis: Dysregulation of chromatin-binding PHD fingers is emerging as powerful driving force in hematological malignancies. Combining published data and our preliminary observations, we have identified a limited set of drugs putatively binding to NSD1 PHDV-C5HCH. We propose a stringent approach that stems from a structural appraisal of NSD1, followed by biophysical, biochemical and cell biology testing, to thoroughly 10/07/ / 6

27 NSD1 PHD fingers as potential targets for Acute Myeloid Leukemia BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Musco Giovanna Ospedale San Raffaele - Milano explore the potential of these compounds as inhibitors of the NSD1 PHD finger oncogenicity. Based on the successful identification of compounds targeting crucial partners of rearranged proteins in other leukemias, we also propose a comprehensive study to identify and validate NUP98-NSD1 interactors, to identify druggable partners crucial for NUP98-NSD1 oncogenicity Preliminary data: 1. PHDv-C5HCH (1H/13C/15N) assignment is completed. 2. GST-pulldown experiments with unfractionated whole histones and binding assays with Histone Peptide Array suggest binding of PHDv-C5HCH to H3K4me3 and H3K9me3 3. Published GST-pull down assays suggest PHDv-C5HCH/NIZP1-C2HR interactions. C2HR-peptide is available in soluble form for biophysical interaction studies. 4. We have developed a SILAC-based quantitative proteomics protocol to study chromatin bound interactome. 5. Potential ligands identified through a virtual pre-screening 6. Effective isolation, infection and growth of myeloid Lin bone marrow progenitors from Balb/c mice Materials and Methods AIM1 Multidimensional solution NMR spectroscopy, ITC, fluorescence, analytical ultracentrifugation. Virtual Screening with databases of FDA approved molecules (NIH Clinical Collection). Validation of best hits (15) by NMR. AIM2 Retroviral infection of haematopoietic progenitor cells derived from Balb/c mouse bone marrow with mutated NUP98- NSD1. Assessment of mutations and drugs impact on binding to histone tail modifications and on HOX9 expression, in vitro and in in vivo adoptive transfer experiments AIM3 Overexpression of flagged NSD1 and NUP98-NSD1 in SILAC labelled fibroblasts. Enrichment for chromatin-bound interactors with optimized mchip protocol followed by anti-flag immunoprecipitation and MS analysis of purified interactors. SILAC based relative quantitation to discriminate between interactors and background and to evaluate interactors strength. Validation experiments in haematopoietic progenitors. IP experiments with PHD inhibitors to identify PHDv-C5HCH interactions. Impact and Translational Implications There are no effective therapies to treat leukemic patients presenting with NUP98-NSD1 translocations. The elucidation of the structural features, the functional impact, and the oncogenic interactors of the PHD domain will provide an essential springboard for the molecular understanding of the pathogenesis of this disease. Moreover, the therapeutic targeting of a PHD histone reader could unleash a new paradigm in drug development and anticancer agents 10/07/ / 6

28 APRON: Assay for PRiONs. DEVELOPMENT OF AN ASSAY DETECTING PRIONS IN ANIMALS AND HUMANS AFFECTED WITH PRION DISORDERS IN A PRECLINICAL AND CLINICAL STAGE RF Casalone Cristina Biomedical/Biomedica Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle 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 Animal Health - ANHE Project Keyword 1: Project Keyword 2: Project Keyword 3: Study and development of new diagnostic protocols and tools that are easy to use, rapid, reliable, and with a contained cost, to improve surveillance systems for infectious diseases and pathologies of interest to veterinary medicine Innovation in methods or technologies for detection and quantitation of prions in vivo Exosome 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/Lavoratory Role in the project 1 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'aosta Dept. of Neuroscience 2 Istituto Superiore di Sanità Dept. of Cell Biology and Neurosciences 3 Azienda Ospedaliera Universitaria Integrata di Verona Dept. of Neurological and Movement Sciences - Coordinator of the project; - collection of animal tissues and fluids. - RT-QuIC analysis - extraction of exosome - development of an in vivo diagnostic test - collaboration on collection of human fluids - collaboration on RT-QuIC analysis - collaboration on development of an in vivo diagnostic test - collaboration on collection of animal and human tissues and body fluids - collaboration on RT-QuiC analysis - collaboration on development of an in vivo diagnostic test 10/07/ / 6

29 APRON: Assay for PRiONs. DEVELOPMENT OF AN ASSAY DETECTING PRIONS IN ANIMALS AND HUMANS AFFECTED WITH PRION DISORDERS IN A PRECLINICAL AND CLINICAL STAGE RF Casalone Cristina Biomedical/Biomedica Investigators, Institution and Role in the Project Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Pocchiari Maurizio Istituto Superiore di Sanità Research collaborator 1 (C1) Coordinator of OU2 research activities 2 Zanusso Gianluigi Azienda Ospedaliera Universitaria Integrata di Verona Research collaborator 2 (C2) Coordinator of OU3 research activities 25/06/ /09/ Mazza Maria Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'aosta Research collaborator 3 (C3) of OU1 25/07/ Cardone Franco Istituto Superiore di Sanità Research collaborator 4 (C4) of OU2 09/01/1969 Background and Significance Transmissible Spongiform Encephalopathies (TSEs) or prion diseases are rare and invariably fatal neurodegenerative disorders of man and animals characterized by the abnormal deposition of the disease associated prion protein (PrPSc), which results from a conformational change of the normal cellular form (PrPC). PrPSc is detected in a variety of tissues and body fluids, including blood, urine, saliva, and nasal fluids, but no validated diagnostic test is yet available on these easily accessible samples in clinical or preclinical prion-infected hosts. Low amounts of PrPSc are difficult to detect with current biochemical or immunocytochemical assays and recent PrPSc amplification technologies, such as RT-QuIC or PMCA, have not yet been validated in early clinical or preclinical phases of disease. Moreover, in body fluids, these assays might not correctly working because of the potential association of PrPSc with other proteins or soluble components. We recently showed that the RT-QuIC assay is able to detect PrPSc in the olfactory epithelium taken from Creutzfeldt-Jakob disease (CJD) patients by nasal brushing and that plasma exosomes, small vesicles of endocytic origin, carry both infectivity and PrPSc but less contaminants, thus representing a better substrate for PrPSc amplification in body fluids. We propose to investigate whether the RT-QuIC assay would be able to measure PrPSc in easily accessible samples taken at early or preclinical phases of animals and man with TSEs. Specific aims Aim 1: Aim 2: Aim 3: To improve the detection of PrPSc by Real Time-Quacking Induced Conversion (RT-QuIC) on peripheral target tissues and biological fluids of pre-clinical and clinical animals naturally or experimentally infected with prions. To improve the extraction protocol of PrPSc derived-exosomes from biological fluids of animals affected with natural or experimental TSE as well as humans with prion disorders. To develop a non-invasive diagnostic method able to detect PrPSc on body fluids derived-exosomes obtained from animals or humans affected with prion disorders. Hypothesis: Our working hypothesis is that using more sensitive techniques, able to detect small amount of PrPSc, it is possible to obtain subclinical/preclinical and clinical data on prion infection. It has long been argued that the incubation period of a prion diseases could exceed the natural life-span of the host, but that during this clinically silent period, neuropathological and biochemical changes as well as accumulation of PrPSc in peripheral tissues are occurring (Hill et al, 2000). The novel RT-QuIC assay detects PrPSc in brain samples (Peden et al, 2011) and CSF (Atarashi et al, 2011) from symptomatic CJD patients with an improved specificity. According to our preliminary data, obtained in animal and human samples, the research group wishes to identify the most easily accessible peripheral tissue or body fluid for improving the sensitive and 10/07/ / 6

30 APRON: Assay for PRiONs. DEVELOPMENT OF AN ASSAY DETECTING PRIONS IN ANIMALS AND HUMANS AFFECTED WITH PRION DISORDERS IN A PRECLINICAL AND CLINICAL STAGE RF Casalone Cristina Biomedical/Biomedica Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle specificity of PrPSc detection in samples taken in living subjects. Finally, it has been shown that both PrPC and PrPSc are detectable in association with exosomes generated in vitro in animal cell lines (Fevrier et al, 2004; Vella et al, 2007; Alais et al, 2008). Moreover, it has been confirmed that PrPC is associated with exosomes isolated from human plasma (Ritchie et al, 2013). These findings are consistent with the potential use of exosomes in biological fluids for the in vivo diagnosis of TSEs in animal and humans. Preliminary data: PI preliminary work has shown that the RT-QuIC can sensitively detect different sub-types of human PrPSc, goat/bovine BSE, BASE and sheep scrapie. Moreover, in a recent study (Orrù et al. NEJM, in press), C2 observed positive RT-QuIC assays seeded with nasal brushings from sporadic CJD patients, giving >97% sensitivity and 100% specificity for CJD detection. Therefore, nasal brushing coupled with RT-QuIC assay markedly strengthen intravital diagnosis of definite CJD. As regard body fluid derived-exosomes, the group of the PI implemented a sensitive protocol for plasma exosome isolation by using a new precipitation method, which was recently developed in collaboration with Prof. G. Camussi (Univ. Of Turin). By using this novel assay it was possible to extract significant amount of exosomes from a small volume of sheep plasma (1ml) and to detect PrPC in plasma of both healthy and scrapie infected sheep by Western Blotting (WB). Materials and Methods Peripheral tissues (muscles, tonsils, tongue, oral mucosa) and fluids (blood, urine, CSF) samples will be collected from animal experimentally (bovine, goat) or naturally (bovine, goat and sheep) infected with prions (BSE, BASE and scrapie) and human CJD patients. In the first phase, we will identify the target peripheral sample where the detection of PrPSc by RT-QuIC is more feasible. Exosomes will be extracted from body fluids improving the precipitation method used in our preliminary data. In the extracted exosomes, PrPSC will be detected by WB and RT-QuIC. Samples collected will be analysed to assess the sensitivity and specificity of the different diagnostic techniques. Impact and Translational Implications Animal and human public health issues are the major issues of this project, aiming on preventing animal-to-animal, animalto-human and inter-humans prions infection. Available in vivo assays for prions detection in peripheral tissues or body fluids of prion infected animals in a preclinical or clinical stage have an incomplete sensitivity. Therefore, it is demanding to develop a highly sensitive and specific test for minimizing the risk of TSEs transmission in animals and humans. 10/07/ / 6

31 Pancreatic cancer therapy based on combination of DNA vaccination and PI3Kgamma inhibition RF Novelli Francesco Biomedical/Biomedica Piemonte 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. Pancreatic cancer PI3K signaling pathway 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/Lavoratory Role in the project 1 Piemonte Centro Ricerche Medicina Sperimentale, AOU Città della Salute e della Scienza di Torino 2 Università di Torino Dipartimento di Biotecnologie Molecolari e Scienze per la Salute 3 Università di Torino Dipartimento di Biotecnologie Molecolari e Scienze per la Salute Investigators, Institution and Role in the Project Principal investigator Generation of PI3K-gamma knockout mice, characterization of PI3Kgamma inhibitors In vivo imaging and monitoring of tumor growth in pancreatric cancer mouse models Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Ghigo Alessandra Università di Torino Generation of PI3K-gamma knockout mice, characterization of PI3Kgamma inhibitors, Coordinator 2 CONTI LAURA Università di Torino In vivo imaging of tumor growth in pancreatric cancer mouse models, Coordinator 3 Cappello Paola Centro Ricerche Medicina Sperimentale, AOU Città della Salute e della Scienza di Torino Coordinator of vaccination studies, evaluation of immunological paramenters 06/05/ /05/ /01/ /07/ / 6

32 Pancreatic cancer therapy based on combination of DNA vaccination and PI3Kgamma inhibition RF Novelli Francesco Biomedical/Biomedica Piemonte Background and Significance Pancreatic ductal adenocarcinoma (PDA) is still an urgent medical need with little advances in therapy and patient survival. Phospho-inolsitol-3-kinase gamma (PI3Kgamma) is a signal transduction enzyme emerging as a key player in inflammatory reactions supporting tumor growth. Preliminary data obtained in our laboratory have indicated that PDA cells grew less in PI3Kgamma-deficient mice, and this correlated with a decreased number of myeloid cells with a suppressive phenotype. We have identified the glycolytic enzyme alpha-enolase (ENO1) and the cytoskeleton regulatory molecule Ezrin (EZR) as PDAassociated antigens able to induce both humoral and cellular responses in PDA. ENO1-DNA vaccination prolonged survival of Genetically Engineered Mice (GEM) that spontaneously develop PDA. The DNA vaccine elicited high levels of anti-eno1 IgG that trigger complement-dependent cytotoxicity (CDC), recruited CD3 cells into the tumor area, and decreased the number of cells with suppressive activity, namely myeloid-derived suppressive cells (MDSC). However, when the percentage of MDSC was restored to that of the controls, progression was no longer inhibited, and death ensued. In this proposal we intend to test the apparent paradox indicating that a combination of the anti-inflammatory effect of PI3Kgamma blockade with specific vaccination can improve efficacy for the so far unsatisfactory immunotherapy. Specific aims Aim 1: Aim 2: Aim 3: To define the mechanisms underlying PI3Kgamma role in the desmoplastic reaction and MDSC recruitment and activation, and validate its targeting as a novel therapeutic strategy in pancreatic cancer. To validate a new pancreatic cancer immunotherapy based on ENO1 and EZR vaccination and genetic or pharmacological PI3Kgamma inhibition in GEM and in mice orthotopically injected with syngeneic PDA cells. To characterize the immune responses and the antitumor mechanisms elicited by DNA vaccination in the absence of PI3Kgamma. Hypothesis: PDA is the fourth leading cause of cancer mortality in developed countries, with one of the poorest prognoses among all cancers. Although 10-15% of patients are candidates for gross total surgical resection, recurrence is frequent and the overall 5-year survival rate is around 4%. Until recently, chemotherapeutic treatment has provided minimal survival benefit for PDA patients, and there is an ongoing need to develop new therapeutic strategies to treat PDA. PDA is replete with inflammatory cells and associated stroma, which are thought to prevent effective therapeutic responses in humans. Previous studies have shown that myeloid cells promote PDA desmoplasia, growth and metastasis. Pharmacological or genetic blockade of PI3Kgamma suppressed tumor inflammation, desmoplasia, growth and metastasis in orthotopic and spontaneous mouse models of PDA. ENO1 and EZR possess interesting antigenic properties as they induce specific antibodies, along with helper and cytotoxic T cell responses in PDA patients. In this proposal, we will verify the hypothesis that the targeting of MDSC, via PI3Kgamma inhibition, synergizes with ENO1-and EZR-DNA vaccination by inducing a strong and sustained immune response against PDA. Preliminary data: GEM carry pancreas specific Kras (KC) or Kras and p53 (KPC) mutations were crossed with PI3Kgamma knockout mice (KC-PI3Kgamma-/-; KPC-PI3Kgamma-/-). Survival was followed with the Kaplan-Meier analysis and log rank test. In both GEM, the absence of PI3Kgamma significantly delayed tumor progression and increased median survival. To evaluate the stromal-specific role of PI3Kgamma, we orthotopically injected K8484 PDA cells into syngeneic wild type and PI3Kgamma KO mice. After 3 weeks, mice were sacrificed and pancreases were excised for weighing and analysis of myeloid cell infiltration. It was observed that most PI3Kgamma KO mice displayed significantly smaller tumors and a lower percentage of CD11b+/GR1hi cells, usually defined as myeloid suppressor cells. Even macrophage type 1 (M1) cell percentage was increased, though not statistically significant, as well as CD4+ and CD8+ T cells in KO mice compared to WT mice. 09/07/ / 6

33 Pancreatic cancer therapy based on combination of DNA vaccination and PI3Kgamma inhibition RF Novelli Francesco Biomedical/Biomedica Piemonte Importantly, PDA cells in PI3Kgamma-/- mice exhibited reduced tumor desmoplasia, as demonstrated by pricosirius red staining for collagen content. Western blotting analisys showed that CD11b+ myeloid cells but not tumor cells express PI3Kgamma. Materials and Methods KC, KC-PI3Kgamma-/- or KC mice treated with PI3Kgamma inhibitor TG will be vaccinated, with empty, EZR or ENO1-expressing plasmid followed by electroporation starting at 4 weeks of age, every 3 weeks, for three times. WT and PI3Kgamma-/- mice will be also EZR or ENO1-vaccinated before the orthotopical injection of syngeneic K8484 PDA cells. The overall survival will be evaluated. Tumor development will be monitored by in vivo molecular imaging. Some mice/groups will be sacrificed to analyze the pancreas by histochemical analysis and fibrotic reaction by collagen staining. Blood MDSC will be checked by flow-cytometry and their migration into the tumor area will be checked histochemically after pancreas excision. The ability of sera of vaccinated GEM to induce complement-dependent cytotoxicity against PDA cells by IgG to ENO1 or EZR will be evaluated. In vaccinated mice, lymphocyte activation and immunosuppressive markers as well cytokine production will be evaluated. Impact and Translational Implications In this proposal we intend to test the combination of the DNA vaccination and the PI3Kgamma inhibitor to enhance the antitumor response in pancreatic cancer. Of note, our proposed approaches can be easily translated into clinical practice as many of PI3K inhibitors and the pvax plasmid used for the DNA vaccination are approved by the FDA. This project will thus impact on the current knowledge of pancreatic cancer progression and on the therapy of this aggressive and untreatable tumor. 09/07/ / 6

34 RF MICRORNA IN PAPILLARY THYROID CARCINOMA: PATHWAYS INVOLVED AND POSSIBLE PROGNOSTIC AND THERAPEUTIC IMPACT Borrello Maria Grazia Biomedical/Biomedica Fondazione Istituto Nazionale per lo studio e la cura dei tumori - 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: Project Keyword 3: Pathways regulated by oncogenes and tumor suppressors that affect tumor cell phenotype and behavior, such as survival, proliferation, and death Thyroid carcinoma mirna 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/Lavoratory Role in the project 1 Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Dep of Experimental Oncology and Molecular Medicine -Molecular Mechanisms Unit Leader Institution- Design of experiments and data analysis of the whole project. Realization of functional experiments for the identified relevant mirnas. Contributing to the thyroid carcinoma case lists. PI: coordination of the whole project, design and supervision of functional experiments 2 Fondazione IRCCS Ca' Granda - Milano Endocrinology Unit and Dep of Clinical Sciences and Community Health-University of Milan Contributing to the thyroid carcinoma case lists including selection and follow-up of the patients and histological revision and identification of genetic lesions of the tumors 10/07/ / 6

35 RF MICRORNA IN PAPILLARY THYROID CARCINOMA: PATHWAYS INVOLVED AND POSSIBLE PROGNOSTIC AND THERAPEUTIC IMPACT Borrello Maria Grazia Biomedical/Biomedica Investigators, Institution and Role in the Project Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Fugazzola Laura Fondazione IRCCS Ca' Granda - Contributing to the thyroid carcinoma case Milano lists by selection and follow-up of the patients with differential iodine intake ability, and by supervising histological revision and identification of known genetic lesions in the tumors 11/06/ Rizzetti Maria Grazia Fondazione IRCCS Istituto Nazionale dei Tumori- Dep of Experimental Oncology and Molecular Medicine-Molecular Mechanisms Unit/Staff Technician Technical support for experiments in the whole project requiring molecular and cell biology expertise 04/03/1960 Background and Significance Thyroid cancer (TC) is the most prevalent malignancy of the endocrine system. Its incidence has more than doubled in the past decade. Papillary thyroid carcinoma (PTC) is the prevalent histotype (approximately 80% of thyroid carcinomas). Driving tumor initiating events have been identified in about 70% of PTCs. They include BRAFV600E mutation (30-40%), RET (20-30%) and TRK rearrangements and RAS mutations. However, the mechanisms underlying development and progression of PTC have not been fully elucidated. PTC is associated with good prognosis in the majority of cases; nevertheless a fraction of patients progress to metastatic disease and displays a poor prognosis. Indeed most thyroid cancer can be treated effectively by total thyroidectomy followed by adjuvant radioactive iodine (RAI) therapy. However, the ten years survival of patients with metastatic thyroid cancer refractory to RAI therapy is only 10% (against 60% for patients with metastases positive for 131I uptake). In the last years several studies have shown aberrant mirna expression profiles in TC and studied their role. However, the role of cancer deregulated mirnas in the web of neoplastic signal transduction in thyroid remains to be fully elucidated. Furthermore, the involvement of other non coding RNAs as the long intergenic noncoding RNA (lincrna), whose deregulation is involved in several cancer types, has been only recently addressed in thyroid carcinoma. Specific aims Aim 1: Aim 2: Aim 3: To study the biological effects of already identified mirnas, suggested being involved in PTC cancerogenesis by our integrated analysis of PTC cancer modeling and clinical case list expression profiles, which will be challenged as possible therapeutic tools in PTC. To identify differential expression profiles (mirna, mrna and lincrna) of 131I-refractory vs 131I-avid PTC to find a signature of refractoriness useful for patients' management To select a list of mirnas possibly involved in 131I refractoriness, which will be evaluated through functional analyses, including the ability to affect iodine uptake in PTC cells, as a base for novel therapeutic approaches Hypothesis: - Our hypothesis is that obtaining integrated expression profiles of genes and mirna, and combining results from clinical tumors and in vitro cell models of PTC may lead to identify novel players in PTC cancerogenesis - Furthermore, we hypothesize that mirnas deregulation might be one of the molecular mechanisms 10/07/ / 6

36 RF MICRORNA IN PAPILLARY THYROID CARCINOMA: PATHWAYS INVOLVED AND POSSIBLE PROGNOSTIC AND THERAPEUTIC IMPACT Borrello Maria Grazia Biomedical/Biomedica Fondazione Istituto Nazionale per lo studio e la cura dei tumori - leading to 131I refractoriness in a subset of PTCs. Indeed mirnas, by simultaneously orchestrating the expression of different genes, have been implicated in the differentiation process in cancer cells, and might be able to affect iodine uptake in PTC cells. Preliminary data: We have previously defined the expression profiles of genes and mirnas in a case list of PTC surgical samples and in in vitro PTC models based on RET/PTC1 oncogene. We identified commonly deregulated mirnas: up-regulated mir-222, already known to be aberrantly expressed in PTC; and down-regulated mirnas, including mir-199a-3p, that we have identified as a novel oncosuppressor mirna in PTC cells (E. Minna et al Oncotarget, 5: , 2014), mir-214-3p and mir-451a, to be challenged for their involvement in thyroid cancerogenesis. To support our results, we interrogated the public dataset "The Cancer Genome Atlas" (TCGA) for the expression of mir- 199a-3p, mir-214-3p, and mir-451a in thyroid samples, including 203 PTCs and 44 normal thyroids: although showing heterogeneous expression levels, the selected mirnas are significantly underexpressed in PTCs compared with normal thyroids. By comparing mirna profiles of the few 131Irefractory vs 131I-avid PTC cases within this series, mirnas showing a different, though not statistically significant, expression have been identified, prompting us to get more insights by using a new and larger PTC case list that we have selected for this purpose. Materials and Methods - RNA extraction from frozen surgical samples through mirneasy kit (Qiagen) following manufacturer's instructions - Agilent platform for microarray profiling of mirna by chips designed on mirbase v19 and of gene expression, including lincrnas (SurePrint G3 Human GE Microarray). -Data pre-processing and statistical analysis to identifying deregulated mirnas and coding and non-coding RNAs with R/Bioconductor.-Spearman correlation analysis between mirna and mrna expression data, integrated with computational target genes prediction from 6 different algorithms, to select anti-correlated predicted target genes -Gene Ontology and Ingenuity Pathway Analyses of target genes -RealTime-RT-PCR analyses (TaqMan technology, Applied Biosystem) -Functional analyses in PTC cell lines: transfection of mirna-mimic -inhibitors, biochemical and biological assays (proliferation, apoptosis, migration, motility, invasion, in vivo growth in xenografted nude mice) -Analysis of known genetic lesions Impact and Translational Implications The identification of novel players in thyroid carcinogenesis including mirnas, genes and pathways might be useful as preclinical tools/targets to be exploited to hampering neoplastic "program" of PTC, and possibly 131I-resistance. Moreover, the identification of a specific molecular signature of 131I -refractory PTC will help in the risk stratification and in the decision of the best therapeutic option, as the prognosis is not easily predictable at the presentation. 10/07/ / 6

37 RF BIOLOGICAL APPROACH TO DECONTAMINATION OF BIVALVE MOLLUSCS FROM V.PARAHAEMOLYTICUS AND SALMONELLA BY BACTERIOVORAX SPECIES. Ottaviani Donatella Biomedical/Biomedica Istituto Zooprofilattico Sperimentale dell'umbria e delle Marche 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: Development of innovative methods aimed at ensuring high level of safety of processed foods of animal origin. Project Keyword 3: 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/Lavoratory Role in the project 1 Istituto Zooprofilattico Sperimentale dell'umbria e delle Marche Food Control Laboratory; National Reference Laboratory (NRL) for Bacteriological Contamination of Molluscs Institution of Principal Investigator (PI) and Operative Unit (OU) 1. Provide prey strains; selection of Bacteriovorax species; decontamination experiments; Bacteriovorax molecular identification; enumeration of target pathogens and Bacteriovorax; propagation, aliquoting and storage of Bacteriovorax; overall coordination; elaboration of Standardized Operative Procedures (SOPs) and final report; results dissemination 2 Università degli Studi di Teramo Facoltà di Medicina Veterinaria Department of Comparative Biomedical Sciences Institution of OU 2 Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Tiscar Pietro Giorgio Università degli Studi di Teramo Facoltà di Medicina Veterinaria Head of OU2. Selection of Bacteriovorax species; decontamination experiments; Bacteriovorax identification by transmission electron microscopy; contribute to Standarized Operative Procedures (SOPs) and final report; contribute to results dissemination 25/03/ /07/ / 6

38 RF BIOLOGICAL APPROACH TO DECONTAMINATION OF BIVALVE MOLLUSCS FROM V.PARAHAEMOLYTICUS AND SALMONELLA BY BACTERIOVORAX SPECIES. Ottaviani Donatella Biomedical/Biomedica Istituto Zooprofilattico Sperimentale dell'umbria e delle Marche Background and Significance Bivalves represent a typical seafood responsible of many seafood-poisoning cases worldwide. In Italy recent reports of high prevalence of toxigenic strains in mussels and cases of V.parahaemolyticus gastroenteritis associated with local mussels as source of infection confirm the relevance of this problem. Bivalves exceeding the limits of faecal indicators are depurated or relayed in less contaminated areas before commercialization but these conventional approaches lack in reducing vibrios and Salmonella. New physical and chemical post-harvest processes recently developed to control V.parahaemolyticus and Salmonella are expensive and often kill bivalves. There is a need to investigate new and effective decontamination systems,including the biological ones. Bacteriovorax species (spp), a group of not pathogenic bacteria present in aquatic environments, are parasitic bacteria with the ability to penetrate, reproduce and kill other gram-negative bacteria, including Salmonella and vibrios. It has been demonstrated their role as natural modulators of pathogen bacteria in seawater and oysters. Moreover, recently reported results on their application for elimination of V.parahaemolyticus in oysters are very encouraging. Specific aims Aim 1: To test at the laboratory scale the applicability of Bacteriovorax spp for decontamination of bivalves from V.parahaemolyticus and Salmonella. Aim 2: To obtain Standardized Operative Procedures (SOPs) for the use of Bacteriovorax spp in the decontamination of bivalve molluscs Aim 3: To certificate Bacteriovorax spp useful to this scope Hypothesis: To the light of the recent scientific evidences detailed in the Background and Significance section, we believe that Bacteriovorax spp could be successfully used for biological decontamination of bivalves from V. parahaemolyticus and Salmonella. Different from physical and chemical processing techniques focusing only on post-harvest treatment, Bacteriovorax spp could control the level of pathogens both in growth periods of bivalves and/or at the depuration stage. Preliminary data: In the year 2006 research teams of PI and head of OU2 isolated marine Bacteriovorax from indigenous samples of water and bivalves able to parasitize "in vitro" gram-negative pathogenic bacteria such as vibrios, Salmonella and E.coli (Marozzi..Ottaviani...Tiscar, 2007). In the year 2007 the research team of PI has documented in mussels collected from Adriatic sea, a prevalence of toxigenic V.parahaemolyticus higher than reported in other European and extra-european countries (Ottaviani et al., 2010a). In the years the research team of PI reported in Central Italy 3 cases of acute gastroenteritis due to V.parahaemolyticus, pandemic and not, with local mussels as the most probable source of infection (Ottaviani et al., 2010b ). In the years the research team of PI reported an high prevalence of toxigenic V.parahaemolyticus from all Italian marine coastal waters by an extensive investigation on indigenous bivalves and environmental samples (Ottaviani et al., 2012a). In September 2010 the research team of PI reported in north Italy a sporadic case of gastroenteritis and a family group outbreak both due to nontoxigenic V.parahaemolyticus, with local mussels as the most probable source of infection (Ottaviani et al., 09/07/ / 6

39 RF BIOLOGICAL APPROACH TO DECONTAMINATION OF BIVALVE MOLLUSCS FROM V.PARAHAEMOLYTICUS AND SALMONELLA BY BACTERIOVORAX SPECIES. Ottaviani Donatella Biomedical/Biomedica Istituto Zooprofilattico Sperimentale dell'umbria e delle Marche 2012b). To support our hypothesis, the results on the appplication of Bacteriovorax for elimination of V.parahaemolyticus in oysters by bioaccumulation experiments are very encouraging (Li et al, 2011). Recently it has been also reported the role of marine Bacteriovorax, as natural modulators of pathogens in seawater and oysters (Richards et al, 2012). Li H et al Int J Food Microbiol doi: /j.ijfoodmicro Marozzi S, Ottaviani D,Tiscar PG et al 13th Int Conf European Association of Fish Pathologists Ottaviani D, Leoni F et al Environ Microbiol Reports a Ottaviani D, Leoni F et al Diagn Microbiol Infect Dis b Ottaviani D, Leoni F et al Environ Microbiol (2012a) doi: /j x Ottaviani D, Leoni F et al J Clin Microbiol 50, b Richards GP et al Appl Environ Microbiol Materials and Methods We will evaluate the lytic ability of Bacteriovorax previously isolated and commercial by standard double layer agar plating technique. V.parahaemolyticus and Salmonella strains from NRL collection will be used as preys. Bacteriovorax identification will be performed by sequencing analysis of 16S rdna and transmission electron microscopy. Bacteriovorax will be propagated in coculture with non pathogenic strains or at prey independent status. Decontaminaton experiments will be conducted in tanks under controlled conditions, with mussels contaminated with appropriate mix of target pathogens and Bacteriovorax. Enumeration at different times of target pathogens and Bacteriovorax will be performed by standardized MPN-PCR and double layer agar plating methods, respectively. Experiments will be carried out in triplicate and each experiment repeated three times. Data analysis of variance and least significant differences calculated at the 5% significance level. Impact and Translational Implications The public impact of vibrios and Salmonella in bivalves is relevant and current post-harvest decontamination processes, often expensive and harmful to molluscs, are not always effective. This approach could represent the starting point for the development of low-cost alternative methods, without adverse effects on bivalves. Bacteriovorax and SOPs could be provided to researchers, for further investigations and widely validation of this approach, also at the industrial scale. 09/07/ / 6

40 RF Defining molecular targets that govern the capacity of platelets to support hepatitis B virus (HBV)-associated liver immunopathology Guidotti 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: Immunology Immunity and Host Defense - IHD Project Keyword 1: Project Keyword 2: Project Keyword 3: host-microbe interactions: innate and acquired host immune responses to specific pathogenic organisms including viruses, bacteria, fungi and parasites; host responses to commensal microbes; influence of host factors, including genetic predisposition or resistance to infection. Hepatitis B virus, liver, platelets, effector CD8 T cells Intravital and confocal microscopy 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/Lavoratory Role in the project 1 Ospedale San Raffaele - Milano Immunology, Transplantation and Infectious Diseases Ospedale San Raffaele (OSR) will function as the sole center where experiments are performed and data collected. Relevant to our proposal, OSR provides state-of-the-art resources that include a BSL-3-contained intravital imaging facility, a flow cytometry facility and a Bioimaging center for light and confocal microscopy. Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 08/07/ / 6

41 RF Defining molecular targets that govern the capacity of platelets to support hepatitis B virus (HBV)-associated liver immunopathology Guidotti Luca Biomedical/Biomedica Ospedale San Raffaele - Milano Background and Significance By killing infected cells and by producing cytokines capable of purging HBV from viable hepatocytes, effector CD8 T cells eliminate the virus during self-limited, acute hepatitis (1). Failure to induce these events results in viral persistence. Accordingly, chronic hepatitis is characterized by a functionally inefficient CD8 T cell response that does not eliminate HBV from the liver but maintains a lifelong, low-level chronic necroinflammatory process that ultimately contributes to the development of cirrhosis and hepatocellular carcinoma (HCC) (1). We used proprietary mouse models of acute and chronic HBV infection to i) show that platelets (PLT) promote liver disease by facilitating the hepatic accumulation of pathogenic HBV-specific CD8 T cells (2), and ii) demonstrate that the combined administration of aspirin and clopidogrel (broad inhibitors of PLT function) limits immune-mediated liver damage and prevents the onset of cirrhosis and HCC (3). Undoubtedly, the clinical translation of these findings will benefit from the use of drugs that specifically target the capacity of PLT to support liver immunopathology without affecting hemostasis. Key to this outcome is the identification of molecules mediating the intrahepatic interactions between PLT and liver sinusoidal endothelial cells (LSEC) and between PLT and effector CD8 T cells. This proposal plans to address these issues by taking advantage of animal models and imaging technologies explicitly created for this purpose. Specific aims Aim 1: Aim 2: Aim 3: Identify platelet surface molecules supporting HBV-associated liver immunopathology. Define whether the functional inhibition of targets identified in Aim 1 diminishes the severity of immune-mediated acute or chronic liver injury. None Hypothesis: The hypothesis driving this proposal is that specific platelet surface molecules (including CD44, P-selectin, PECAM-1, a2ß1, glycoprotein (GP)Ib-a, GPVI, GPIIb-IIIa or CD40L) regulate the ability of circulating PLT to adhere to LSEC and, secondarily, interact with pathogenic CD8 T cells. We also hypothesize that the identification of such molecules coupled with their functional inhibition in vivo may represent a significant progress towards the development of new therapeutic strategies for chronic HBV infection and its complications (cirrhosis and HCC). Preliminary data: The mechanisms by which PLT get recruited into the liver and facilitate the hepatic accumulation of HBV-specific CD8 T cells are still poorly understood. In the last few years we set up complex preclinical platforms specifically designed to address these issues. Briefly, we crossed our HBV replication-competent mice with mice whose endogenous PLT lack GPIb-alpha but transgenically express the human counterpart (mgpib-a-null; hgpib-a Tg). These animals can be quantitatively depleted of endogenous PLT (via the injection of in-house developed Abs to hgpib-alpha) and rapidly reconstituted with PLT deriving either from wild type mice (wt) or from genetically modified mice carrying specific defects (in doing so, the transfused mouse PLT cannot be recognized/depleted by circulating Abs to hgpib-a). Coupling this system with single- or multi-photon liver intravital microscopy (IVM) and 3D confocal microscopy (CF) we showed that: i) wt PLT frequently adhere to inflamed or non-inflamed LSEC, forming small and transient aggregates that involve up to PLT (Fig. 1), and ii) PLT aggregates represent a preferential surface onto which circulating effector CD8 T cells arrest their run within the hepatic microcirculation (Fig. 2). Of note, following the PLT-dependent initial arrest, effector CD8 T cells start to slowly crawl within liver sinusoids in search of hepatocellular antigens (Ags); hepatocellular Ag recognition is mediated by protrusions that intravascular T cells extend through the sinusoidal fenestrae (Manuscript in preparation and Figs. 3-4). Further, preliminary experiments utilizing CD44-null PLT to reconstitute 08/07/ / 6

42 RF Defining molecular targets that govern the capacity of platelets to support hepatitis B virus (HBV)-associated liver immunopathology Guidotti Luca Biomedical/Biomedica Ospedale San Raffaele - Milano PLT-depleted animals indicate that platelet CD44 is important to promote PLT-LSEC interacting events ultimately leading to the hepatic accumulation of pathogenic CD8 T cells (Fig. 5). Similar experimental approaches will be used to screen additional molecular candidates. Well-established cellular, molecular, histopathological and biochemical techniques will evaluate the impact that specific inhibitors (small molecules, peptides, Fab fragments or intact Abs) may have on liver disease severity in our models of acute or chronic hepatitis B. Materials and Methods Recipient mice include two transgenic lineages: HBV replication-competent mice and HBsAg-expressing mice experimentally manipulated to develop immune-mediated chronic hepatitis. The first lineage will be also bred against mgpib-a-null/hgpib-a Tg mice. Platelet donor mice include mice lacking CD44, P-selectin, PECAM-1, a2ß1, GPIb-a, GPVI, GPIIb-IIIa or CD40L bred or not against ß-actin-CFP, ß-actin-eGFP or ß-actin-DsRed. CD8 T cell donor mice include HBcAg- or HBsAg-specific TCR transgenic mice bred or not against the fluorescent lineages abovementioned. IVM will be performed on an epifluorescence microscope (Zeiss) equipped with a Colibri system and on a multiphoton microscope (LaVision Biotech) pumped by 2 Ti:Sa laser and 1 OPO. CF will be performed on a Leica SP8 microscope. Offline analysis of IVM data will be performed on workstations equipped with Imaris and MatLab softwares. FACS analyses on intrahepatic leukocytes and virological assays will be performed as described previously. Impact and Translational Implications Termination of chronic HBV infection by available antiviral therapies has been associated with reduced incidence of cirrhosis and HCC. Sadly, most patients do not respond to these therapies with sustained virus elimination. As such, a huge risk remains in store for the ~400 million people chronically infected with HBV worldwide. Undeniably, preclinical approaches aimed at identifying new strategies limiting liver immunopathology and preventing cirrhosis/hcc development will meet a pressing need. 08/07/ / 6

43 RF An integrated approach to unravel the genetic causes and molecular pathogenesis of epileptogenic focal cortical dysplasia. Guerrini Renzo 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: Brain Disorders and Clinical Neuroscience Developmental Brain Disorders - DBD Project Keyword 1: Project Keyword 2: Project Keyword 3: Genetic, metabolic and morphological abnormalities: Developmental abnormalities of brain structure, volume, and ventricular space; congenital CSF abnormalities [hydrocephalus]; developmental aspects of inborn errors of metabolism, storage diseases, and neurotransmitter/receptor function; genetic basis of metabolic and morphological abnormalities; Identification and characterization of genetic mechanisms and development of animal models and therapeutic strategies specifically relevant to disorders of the developing brain. Focal Cortical Dysplasia (FCD) Pathogenetic mechanisms elucidation 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/Lavoratory Role in the project 1 Toscana Pediatric Neurology Unit and Laboratories, Department of Neuroscience, A. Meyer Hospital, Florence 2 Toscana, Pathology Laboratory, A. Meyer Hospital, Florence 3 Toscana, European Laboratory for Non-Linear Spectroscopy (LENS) Pathology Laboratory, A. Meyer Hospital, Florence European Laboratory for Non-Linear Spectroscopy (LENS) Patients enrollment and study. Biological resources collection. Cell sorting. Array CGH analysis. PI3K/AKT/mTOR pathway analysis. Whole exome sequencing analysis. Histological analysis and conventional immunohistochemistry of dysplastic brain specimens. High-resolution 3D imaging of dysplastic brain specimens. 09/07/ / 6

44 RF An integrated approach to unravel the genetic causes and molecular pathogenesis of epileptogenic focal cortical dysplasia. Guerrini Renzo Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Toscana Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 BUCCOLIERO ANNA MARIA Toscana, Pathology Laboratory, A. Meyer Hospital, Florence 2 Costantini Irene Toscana, European Laboratory for Non-Linear Spectroscopy (LENS) 3 Conti Valerio Toscana, Pediatric Neurology Unit and Laboratories, Department of Neuroscience, A. Meyer Hospital, Florence. 4 Barba Carmen Toscana, Pediatric Neurology Unit and Laboratories, Department of Neuroscience, A. Meyer Hospital, Florence. Histological and immunohistochemical analysis of dysplastic brain specimens. High-resolution 3D imaging of dysplastic brain specimens. Targeted resequencing of genes belonging to the PI3K/AKT/mTOR pathway. Whole exome sequencing. Cell sorting. Immunohistochemistry. Clinical and neuropsychological evaluation of patients. Collection of biological specimens. 30/06/ /04/ /04/ /06/1971 Background and Significance Focal cortical dysplasia (FCD) is the most frequent proven finding in pediatric patients undergoing epilepsy surgery and is classified in three different subtypes. Type I FCD is characterized by abnormal cortical layering, Type II FCD includes, in addition to cortical dyslamination, specific cytological anomalies (dysmorphic neurons in Type IIa FCD and dysmorphic neurons and balloon cells in Type IIb FCD) and Type III FCD occurs in combination with other brain lesions (i.e. tumors). In spite of the overwhelming literature on FCD, our knowledge about its molecular pathogenesis is limited. Recently, it has been demonstrated that hyperactivation of the PI3K/AKT/mTOR pathway plays an important role in hemimegalencephaly, a malformation sharing several histopathological features with FCD. An involvement of the PI3K/AKT/mTOR pathway has also been hypothesized in Type IIb FCD since an altered expression or subcellular localization of proteins belonging to this pathway has been demonstrated in balloon cells. We aim to unravel the molecular basis of Type I and Type II FCD, to identify new specific immunohistological markers for these epileptogenic focal brain malformations and to characterize the tridimensional structure of dysplastic tissue. Project results will improve our knowledge about the causes, the pathogenesis and the structural anomalies underlying FCD and its functional consequences. Our study is also expected to identify new potential targets for innovative treatments. Specific aims Aim 1: Unraveling genetic causes of FCD. We will analyze DNA extracted from paired blood/brain specimens of 45 patients with histologically confirmed FCD to explore the presence of pathogenetic CNVs (through array CGH analysis) or point mutations in genes belonging to the PI3K/AKT/mTOR pathway (through targeted resequencing of 42 candidate genes). In samples in whom array CGH and targeted resequencing will result negative we will perform whole exome sequencing. To elucidate whether dysmorphic neurons and balloon cells observed in Type IIa and Type IIb FCD carry specific molecular defects, we will use the above approaches in homogeneous subpopulations of cells obtained via cell sorting. Aim 2: Identification of novel specific immunohistological markers in type II FCD. We aim to increase current knowledge 09/07/ / 6

45 RF An integrated approach to unravel the genetic causes and molecular pathogenesis of epileptogenic focal cortical dysplasia. Guerrini Renzo Clinical health care research/clinicoassistenziale Toscana about the immunological signature of dysplastic neurons and balloon cells in Type IIa and Type IIb FCD and to set up sorting procedures for these cells. We will perform immunophenotyping of dysplastic neurons and balloon cells using antibodies against cell surface markers, specific PI3K/AKT/mTOR pathway proteins and proteins whose involvement in FCD pathogenesis will emerge in the framework of the study. Aim 3: Characterization of the tridimensional structure of dysplastic tissue. We will explore micro-organization anomalies in dysplastic brain samples by characterizing the tridimensional structure of blocks of dysplastic tissue after clarification protocols. We will focus on connections among dysmorphic cells and the surrounding "healthy environment" and on morphology, geometry and density of synaptic junctions. Hypothesis: The distinction among different FCD subtypes is currently based on histopathological examination. Although an involvement of the PI3K/AKT/mTOR pathway has been proposed in Type IIb FCD, genetic validation of this theory is missing. We hypothesize that both Type I and Type II FCD have a genetic basis and that the PI3K/AKT/mTOR pathway plays a pivotal role in the pathogenesis of both FCD subtypes. The experimental plan we propose will allow to validate our hypothesis and will provide novel molecular and cytological markers to improve differential diagnosis of FCD. The demonstration of the involvement of the PI3K/AKT/mTOR pathway in FCD pathogenesis might also lead to develop therapeutic strategies different from surgery, since drugs targeting this pathway (i.e. rapamycin) appear to be effective in the treatment of other pathologies with a similar neuropathological substrate, such as tuberous sclerosis. Preliminary data: We have already collected a cohort of 30 patients surgically treated for drug resistant epilepsy and a histopathological diagnosis of FCD. Array CGH analysis, already performed in 16, revealed in one patient with Type Ib FCD and additional brain anomalies a duplication in 1q21.1-q44. This region includes AKT3, a gene belonging to the PI3K/AKT/mTOR pathway. Immunohistochemistry has demonstrated a hyperactivation of this pathway in the dysplastic tissue of the patient. Materials and Methods We will extract genomic DNA from blood, whole FCD specimens and dysplastic neurons and balloon cells. To obtain homogeneous subpopulations of dysplastic neurons and balloon cells, we will dissociate brain samples (MACS Tissue Dissociation Kit, Miltenyi Biotech) and sort cells on the basis of their size, shape and surface markers using a FACSAria instrument (BD biosciences). We will perform array CGH analysis using the SurePrint G3 Human CGH Microarray Kit 1M (Agilent). For targeted resequencing of PI3K/AKT/mTOR pathway genes and whole exome sequencing, we will use haloplex panels (Agilent) and a GAIIx (Illumina). We will validate filtered variants by direct sequencing. To obtain immunological signature of cells, we will use antibodies against surface markers (e.g., CD133), and PI3K/AKT/mTOR pathway proteins (e.g., RPS6). After tissue clarification (CLARITY protocol), we will characterize the tridimensional structure of the dysplastic tissue using immunohistochemical approaches. Impact and Translational Implications The definition of novel molecular and cytological biomarkers for FCD, as well as the identification of molecular pathways involved in FCD pathogenesis, will provide new insights to better characterize FCD subtypes and to better predict clinical outcome. Data generated within the project will help to understand the mechanisms of drug resistant epilepsy in these patients, providing new targets for innovative pharmacological therapies that may complement or even substitute surgical treatment. 09/07/ / 6

46 Anti-Cholinergic receptors antibodies, cardiovascular autonomic profile and dysautonomia symptoms relationships in Pure Autonomic Failure, Amyotrophic Lateral Sclerosis and Postural Orthostatic Tachycardia Syndrome: evidence for a pathophysiology based therapeutic new strategy. RF Furlan Raffaello Clinical health care research/clinicoassistenziale Istituto Clinico Humanitas LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic 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: Neural control of circulation and respiration Anti-Cholinergic receptors antibodies Power Spectrum Analysis 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/Lavoratory Role in the project 1 Istituto Clinico Humanitas Medicine/Clinica medica/ Syncope Unit Enrolment of PAF and POTS patients. Assessment of cardiovascular autonomic profile both at rest and during a gravitational stimulus (75 head-up tilt). Clinical follow-up. 2 Fondazione Istituto Neurologico "C.Besta", IRCCS 3 Fondazione Salvatore Maugeri IRCCS, Milano Neurology IV/ Neuroimmunology Laboratory Neurorehabilitation Assessement of anti-achr antibodies plasma titers Enrolment, clinical evaluation and follow-up of SLA and PAF patients. 07/07/ / 6

47 Anti-Cholinergic receptors antibodies, cardiovascular autonomic profile and dysautonomia symptoms relationships in Pure Autonomic Failure, Amyotrophic Lateral Sclerosis and Postural Orthostatic Tachycardia Syndrome: evidence for a pathophysiology based therapeutic new strategy. RF Furlan Raffaello Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Istituto Clinico Humanitas Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Andreetta Francesca Fondazione Istituto Neurologico "C.Besta", IRCCS The core laboratory of the Neuroimmunology Unit will provide expertise and facilities for assessing plasma anti-achr antibodies at baseline, before and after selective immunoabsorption and during follow-up 10/10/ MORA GABRIELE Fondazione Salvatore Maugeri IRCCS, Milano Enrolment, clinical evaluation and followup of SLA and PAF patients from outpaitent clinic and neurorehabilitation ward 26/03/ Marchi Andrea Istituto Clinico Humanitas/bioengineer Power spectrum analysis of RR interval and blood pressure variability at baseline, before and after selective immunoabsorption and during clinical follow-up. 09/12/1987 Background and Significance Pure Autonomic Failure (PAF) is a rare neurodegenerative disorder characterized by altered ganglionic functioning and dysautonomia. Failure in sympathetic noradrenergic activity results in orthostatic hypotension and syncope. Deficient cholinergic parasympathetic activity induces constipation, urinary retention, decreased salivation and lacrimation. Notably, up to 40% of PAF patients are characterized by the presence of plasma anti-alfa3 and anti-alfa7 ganglionic cholinergic receptors (anti-achr) antibodies (Abs) which, by disrupting ganglionic transmission, might account for the altered autonomic functioning. Previous single case reports showed that removal of anti-achr Abs by plasmapheresis techniques acutely improved orthostatic tolerance and dysautonomia symptoms in PAF but no clinical medium/long term follow-up was available. There is no data about the relationship among anti-achr Abs plasma titer, dysautonomia symptoms and cardiovascular autonomic profile and about their respective time course after anti-achr removal by plasma immunoabsorption procedure. It is unknown whether more frequent neurodegenerative diseases characterized by concomitant dysautonomia symptoms, such as Amyotrophic Lateral Sclerosis (ALS) and Postural Orthostatic Tachycardia Syndrome (POTS) may share analogous anti-achr profile. This is clinically relevant because Abs removal by immunoabsorption techniques may represent a new effective etiologic therapy for dysautonomia symptoms in these diseases. Specific aims Aim 1: To evaluate the prevalence of plasma anti-achr Abs in patients affected by PAF, ALS and POTS. Aim 2: To address the time course and relationships of anti-achr Abs plasma titers, cardiovascular autonomic profile and dysautonomia symptoms before and after anti-achr Abs plasma removal by selective immunoabsorption technique in PAF, ALS and POTS patients. The core laboratory of the Neuroimmunology Unit of Istituto Neurologico Besta will provide expertise and facilities for assessing plasma anti-achr Abs. The clinical laboratory of Clinica Medica Syncope Unit (Humanitas) will provide the know-how and facilities for patients neural autonomic assessment. 07/07/ / 6

48 Anti-Cholinergic receptors antibodies, cardiovascular autonomic profile and dysautonomia symptoms relationships in Pure Autonomic Failure, Amyotrophic Lateral Sclerosis and Postural Orthostatic Tachycardia Syndrome: evidence for a pathophysiology based therapeutic new strategy. RF Furlan Raffaello Clinical health care research/clinicoassistenziale Istituto Clinico Humanitas Aim 3: To set up a new therapeutic strategy based on selective immunoabsorption technique sessions combined with drug immunosuppression (Prednisone) in patients with anti-achr Abs. Hypothesis: - Changes in anti-achr Abs plasma titer may be mirrored by modifications in the cardiovascular autonomic profile, as assessed by spectrum analysis of RR and arterial pressure variability, and be related to fluctuations in the magnitude of dysautonomia symptoms in PAF, ALS and POTS patients. - Plasma anti-achr Abs might underlie dysautonomia symptoms associated to neurodegenerative disorders such as ASL and POTS - Plasma anti-achr Abs removal by selective immunoabsorption techniques may improve ganglionic transmission leading to clinical amelioration and represent a new effective etiologic therapy for these diseases. Preliminary data: We have assessed anti-achr Abs plasma levels, hemodynamics, spectral indices of cardiac and vascular autonomic modulation and global dysautonomia symptoms by a modified visual analogue scale (VAS) score in a single male affected by PAF, before (March 2010, Baseline, B), after 5 immunoabsorption sessions performed every other month (January 2011, T1) and in December 2011 (T2), after an additional immunoabsorption session (May 2011). Plasma anti-achr Abs were: B 6.0, T and T nmol/l. Orthostatic systolic pressure reduction was: B -55, T1-8 and T2-30 mmhg. Orthostatic norepinephrine increase was: B +18, T and T2 +51 pg/ml. Spectral index of cardiac sympathetic modulation (LFRR) change during orthostasis was: B -9.6, T1 +31 and T2 + 2 n.u. Spectral index of vascular sympathetic modulation (LFSAP) change during orthostasis was: B -9.6, T1 +31 and T2 + 2 mmhg2. Overall symptom score was: B 6.8±0.9, T1 0.2±0.2 and T2 2.0±0.8 (0-10= no symptom-maximum symptom intensity, respectively). Thus, a decline in anti-achr Abs was associated with less orthostatic hypotension and more appropriate cardiac and vascular sympathetic activation upon standing. Intensity of dysautonomia symptoms lessened concomitantly with plasma anti-achr Abs reduction and the overall quality of life improved. Few case reports have documented the presence of plasma anti-achr Abs in POTS. Previous studies have identified variations in cholinergic receptor subunits 3 and 4 in ALS but no plasma Abs were searched for. 07/07/ / 6

49 Anti-Cholinergic receptors antibodies, cardiovascular autonomic profile and dysautonomia symptoms relationships in Pure Autonomic Failure, Amyotrophic Lateral Sclerosis and Postural Orthostatic Tachycardia Syndrome: evidence for a pathophysiology based therapeutic new strategy. RF Furlan Raffaello Clinical health care research/clinicoassistenziale Istituto Clinico Humanitas Materials and Methods a. 25 PAF, 25 ALS and 25 POTS patients with dysautonomia symptoms will be enrolled from S. Maugeri neurorehabilitation and Humanitas Syncope Unit outpatient clinics and wards. Plasma ganglionic anti-achr Abs will be assessed by immunoprecipitation radioassay by complexing epibatidine to a Triton X-100-solubilized ganglionic AChR antigen. Baseline autonomic profile will be assessed by spectrum analysis of RR and blood pressure variability indices (LFRR, LF/HF, LFSAP) and by plasma catecholamine, at rest and during 75 head-up tilt. Dysautonomia symptoms will be evaluated by the Composite Autonomic Scoring Scale (CASS). Anti-AChR positive patients will undergo immunoabsorption sessions once a week, up to the achievement of ganglionic anti-achr plasma level lower than 25% of baseline. Immunosuppressive therapy (prednisone 0.25 mg/kg/day) will follow. b. In all subjects, anti-achr Abs, autonomic profile and dysautonomia symptoms, will be addressed as in a, every 4 months up to 3 years. Impact and Translational Implications The evidence of a relationship among the decline of anti AChR Abs after immunoabsorption sessions, the changes of cardiovascular autonomic profile inducing a more appropriate hemodynamic response during standing and the improvement of dysautonomia symptoms may lead to a new effective therapeutic strategy to be used both in PAF and in other more frequent diseases such as ALS and POTS. This approach may ultimately reduce pharmaceutical and rehabilitative costs for the health system. 07/07/ / 6

50 RF Cardiac amyloidosis: molecular mechanism and innovative therapies for a challenging aging related cardiomyopathy MERLINI GIAMPAOLO Biomedical/Biomedica Fondazione Policlinico San Matteo LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Biological Chemistry and Macromolecular Biophysics Macromolecular Structure and Function B - MSFB Project Keyword 1: Project Keyword 2: Project Keyword 3: Properties of proteins: structural dynamics of proteins, folding and misfolding processes; engineering proteins to enhance function, computer-aided drug design, allostery and cooperativity in enzyme mechanism and control, chaperones, thermodynamic and ele Systemic amyloidosis Amyloid cardiomyopathy 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/Lavoratory Role in the project 1 Fondazione Policlinico San Matteo 2 Università degli Studi di Pavia 3 IRCCS - Istituto di Ricerche Farmacologiche Mario Negri- Milano Laboratori Sperimentali di Ricerca in Biotecnologie Department of Molecular Medicine Department of Molecular Biochemistry and Pharmacology Principal investigator Collaborator Collaborator Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Bellotti Vittorio Università degli Studi di Pavia Researcher collaborator - Leader Unit Università di Pavia 2 DIOMEDE LUISA IRCCS - Istituto di Ricerche Farmacologiche Mario Negri- Milano Researcher collaborator - Leader Unit Istituto Mario Negri 05/02/ /03/ /07/ / 5

51 RF Cardiac amyloidosis: molecular mechanism and innovative therapies for a challenging aging related cardiomyopathy MERLINI GIAMPAOLO Biomedical/Biomedica Fondazione Policlinico San Matteo Background and Significance Amyloid fibril formation, associated with a wide spectrum of serious diseases, is the prototypic pathologic process of protein folding disorder and is emerging as a frequent disease in the elderly. In systemic amyloidosis the mechanisms governing the induction, timing, anatomical distribution, rates of progression, clearance and the pathological effect of amyloid deposition are all poorly understood. The amyloid deposition in the heart dictates the prognosis and the elucidation of mechanism of heart targeting and toxicity is essential for designing and validating new effective treatments. More than 90% of heart amyloidoses are caused by the deposition of amyloidogenic immunoglobulin light chains (LC) or transthyretin (TTR). Extensive work suggests that some structural properties of these proteins are intrinsically responsible for their cardiotropism/cardiotoxicity. A restricted repertoire of LC genotypes is most likely associated to heart amyloidosis, and for TTR the role of protein fragmentation on heart fibrils deposition was recently highlighted. Through a multidisciplinary approach the structure, state of association, dynamic of structural conversion and damaging potential of cardiotropic/cardiotoxic TTR and LC assemblies will be clarified. This will offer the possibility to design new drugs and investigate the pharmacological efficacy of inhibitors of protein aggregation, stabilizers of the native state and modulators of protein expression and toxicity. Specific aims Aim 1: Aim 2: Aim 3: Identification of the LC and TTR molecular mechanisms (different molecular assemblies or direct toxicity associated with the release of reactive oxygen species) underlying cardiotoxicity in vitro and in vivo models. Evaluation of the pharmacological efficacy of inhibitors of protein aggregation, stabilizer of protein native state and protein expression modulators and scavengers. Characterization of molecular signatures of amyloid-related toxicity in ex vivo specimens. Hypothesis: There is strong clinical and pathological evidence that pathophysiology of heart amyloidosis associated to LC and TTR is crucially dictated by the co-presence of both the soluble precursor as well as the insoluble amyloid fibrils. We hypothesize that the contact between the native globular amyloidogenic proteins and the extracellular matrix can prime the amyloid transition. Once the amyloid fibrils are formed, the extracellular matrix component, embedded with amyloid, strongly promotes the oligomerization of the soluble precursor, its amyloid conversion and possibly its cell toxicity. We also hypothesize that unfolding forces, generated by the combination of shear forces of physiologic fluid and contact of protein with hydrophobic biologic surfaces, are sufficient to prime conformational changes causing the oligomerization, amyloidogenesis and toxic gain of function. The assessment of in vitro and in vivo models recapitulating this process would represent an invaluable tool for drug discovery and validation. Preliminary data: Our team has a long-term tradition in investigating molecular mechanisms of amyloid diseases spanning from the characterization of the structural basis of amyloid conversion to the pathologic and clinical scrutiny of the mechanisms of tissue damage by the amyloid process. We have discovered that certain monoclonal LC display a direct cardiotoxic effect and we have recently established an in vivo model recapitulating crucial aspects of toxicity. Regarding TTR, we are investigating a new pathway of amyloidogenesis mediated by a selective proteolytic cleavage that seems particularly relevant for elucidating the molecular basis of cardiotropism and cardiotoxicity. For both these globular amyloidogenic proteins -and for another prototypic protein i.e. ß2-microglobulin- we can now measure the unfolding forces generated by the physiologic shear stress and monitor their effect on protein unfolding, conformational changes and aggregation. This unique know-how represents the best possible condition for addressing the crucial and challenging issue of identifying the toxic species and the basis of selective tissue targeting. 10/07/ / 5

52 RF Cardiac amyloidosis: molecular mechanism and innovative therapies for a challenging aging related cardiomyopathy MERLINI GIAMPAOLO Biomedical/Biomedica Fondazione Policlinico San Matteo Materials and Methods LC and TTR from patient biopsies or as recombinant proteins will be used. The in vitro conformational changes occurring along the amyloid conversion of these proteins will be monitored. The viability of cardiomyocytes and the contractility of adult ventricular cardiomyocytes will be evaluated after treatment with different TTR and LC assemblies. The in vivo toxic effect of different protein conformers will be studied using C. elegans pharynx as heart model. New transgenic C. elegans strains expressing human cardiotoxic LC will be generated as well. Through immunohistochemistry and electron microscopy we will scrutinize in heart biopsies the molecular assemblies responsible for cell damage in LC and TTR amyloidosis. These observations will be correlated with the outcome of the in vivo studies. Overall, this strategy will enable us to evaluate the potential pharmacological efficacy of a large variety of molecules through an integrated in vitro-in vivo experimental approach. Impact and Translational Implications Amyloid cardiac involvement is the leading cause of death in systemic amyloidosis and no effective therapy is available. The understanding of the molecular mechanisms and interactions involved in cardiotropism and cardiotoxicity of LC and TTR will pave the way for a more rational approach in the development of novel targeted therapy.

53 SELECTIVE AXILLARY LYMPH NODE DISSECTION VS COMPLETE AXILLARY DISSECTION: A RANDOMISED CLINICAL TRIAL TO ASSESS THE PREVENTION OF LYMPHEDEMA IN BREAST CANCER TREATMENT RF Gennaro Massimiliano Clinical health care research/clinicoassistenziale 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 - 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 Axillary reverse mapping Breast cancer related lymphedema 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/Lavoratory Role in the project 1 Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano General Surgery 3 - Breast Unit enrollment, surgical treatment, clinical evaluation during follow up. 2 Fondazione IRCCS Istituto Nazionale Tumori - Milano 3 Fondazione IRCCS Istituto Nazionale Tumori - Milano Palliative Care, Pain Therapy and Rehabilitation Nuclear Medicine physical assessment and physical therapy imaging and functional diagnosis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Caraceni Augusto Tommaso Fondazione IRCCS Istituto Nazionale Tumori - Milano 2 MACCAURO MARCO Fondazione IRCCS Istituto Nazionale Tumori - Milano 3 Agresti Roberto Fondazione IRCCS Istituto Nazionale Tumori - Milano coordination physical assessment and follow up preoperative setting and diagnosis of side effects 08/08/ /08/1969 accrual, surgical treatment, follow up 22/05/ /07/ / 6

54 SELECTIVE AXILLARY LYMPH NODE DISSECTION VS COMPLETE AXILLARY DISSECTION: A RANDOMISED CLINICAL TRIAL TO ASSESS THE PREVENTION OF LYMPHEDEMA IN BREAST CANCER TREATMENT RF Gennaro Massimiliano Clinical health care research/clinicoassistenziale Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Background and Significance Axillary lymph node dissection (ALND) is still the treatment of choice for breast cancer patients with nodal involvement. Breast cancer-related lymphedema (BCRL) is the most dreaded complication of ALND and may be a lifelong problem. BCRL may occur in up to 65% in those undergoing AD. BCRL prevalence is increasing as improvements in breast cancer detection and treatment have increased patient survival. The sequelae of BCRL include poor quality of life, poor body image, interference with social functioning and job performance, and increasing health care costs; hence the interest in BCRL prevention. It has recently been suggested that axillary reverse mapping (ARM) can identify and spare the lymphatics from the arm and thus reduce the incidence of BCRL. A recent pilot study at the National Cancer Institute of Milan, evaluated, using radioisotope and lymphoscintigraphy for ARM, the feasibility of selective axillary dissection (SAD) with the purpose of preserving lymphatic drainage of the limb. SAD was feasible in 75% of patients considered. Moreover it was found that 9% had BCRL after SAD compared to 33% after ALND. None of the patients undergoing SAD subsequently developed axillary lymph node disease during a median follow up of 16 months. This initial experience - that SAD is effective in a high proportion of patients - suggested the utility of the present randomised study. Specific aims Aim 1: Efficacy to compare the occurrence of BCRL after SAD and after ALND. For this purpose patients enrolled will receive a physical assessment one month after surgery, with evaluation at 6 and 12 months after surgery that includes oncological assessment, physical assessment, lymphoscintigraphy, and self-evaluation questionnaire to assess the presence BCRL (see also AIM 2). Aim 2: Safety to assess the potential for residual disease in the axilla after SAD by evaluating the status, in the ALND arm, of nodes that are spared and assess the occurrence of axillary relapses during follow-up due to incomplete resection of axillary lymph nodes. Aim 3: Costs To compare costs in the ALND and SAD arms including: lymphoscintigraphy, time required for surgery, and treatments required for lymphedema during follow up. Hypothesis: Several groups have reported injecting blue dye into the arm to map identify and thus allow sparing of the arm's lymphatic drainage in breast cancer patients undergoing ALND. The hypotheses are that the risk of involvement of the ARM nodes is low and that preserving these nodes will prevent BCRL. Thus, SAD may result in less morbidity than ALND, but little is known about its efficacy in reducing BCRL after preservation of ARM nodes, and doubts exist as to the safety of such node-sparing surgery. A randomized clinical trial should give definitive answers regarding the efficacy of SAD in preventing BCRL and the safety of this new surgical approach. Preliminary data: Our previous study assessed the feasibility of node-sparing surgery using radioisotope and lymphoscintigraphy. We found that SAD was feasible in 75% of the 60 patients enrolled. The rate of 11/07/ / 6

55 SELECTIVE AXILLARY LYMPH NODE DISSECTION VS COMPLETE AXILLARY DISSECTION: A RANDOMISED CLINICAL TRIAL TO ASSESS THE PREVENTION OF LYMPHEDEMA IN BREAST CANCER TREATMENT RF Gennaro Massimiliano Clinical health care research/clinicoassistenziale Fondazione Istituto Nazionale per lo studio e la cura dei tumori - BCRL after SAD was less than one third of that found after ALND, Furthermore during follow-up, none of the patients developed axillary node disease after primary treatment. These results encourage development of the technique. It is hard to say, however, whether the success of SAD, which spares hot nodes along axillary vessels, depends entirely on the surgeon's skill (which improves with experience), or whether it also depends on a particular anatomical pattern that allows this type of surgery. Materials and Methods The proposed study is a 2-arm (1:1 allocation) prospective randomised trial. The control arm consist of patients undergoing ALND; the study group of patients undergoing SAD. Patients with breast cancer candidates for axillary dissection, irrespective of the type of breast surgery or adjuvant treatment, are potentially eligible. The primary endpoint is the difference between the risk of developing BCRL in the ALND and SAD arms. With a power of 90% and a type 1 error of 0.05, it is necessary to enrol 79 patients per group to test an assumed difference between the risks of The risk of lymphedema in the ALND group is assumed to be 0.30 under the null hypothesis, and is equal to 0.10 under the alternate hypothesis. We estimate 24 months for enrolment and 12 months for the completion of follow up after the last patient has been recruited. The study requires the written informed consent of patients. Impact and Translational Implications BCRL is a chronic complication that may lead to lifelong impairment. Its importance is increasing as breast cancer survival continues to improve. If our hypotheses that SAD is effective and safe are proven correct, SAD may still optimally stage breast cancer but with less morbidity than conventional ALND. It is also likely that reducing BCRL will reduce the costs of supportive care, especially among working-age women, as well as improving their quality of life. 11/07/ / 6

56 RF Rab39b mouse models: pre-clinical studies by using AMPA receptors as possible therapeutic targets to unravel the role of RAB39B in Intellectual Disability and Autism Spectrum Disorder D'Adamo Patrizia 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. Identification and characterization of genetic mechanisms and development of animal models and therapeutic strategies specifically relevant to disorders of the developing brain. 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/Lavoratory Role in the project 1 Ospedale San Raffaele - Milano 2 Ospedale San Raffaele - Milano Division of Neuroscience/Laboratory of Molecular Genetics of Mental Retardation Department of Oncology/Nuclear Medicine/Preclinical PET facility PI collaborator Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Moresco Rosa Maria Ospedale San Raffaele - Milano collaborator 26/12/ /07/ / 6

57 RF Rab39b mouse models: pre-clinical studies by using AMPA receptors as possible therapeutic targets to unravel the role of RAB39B in Intellectual Disability and Autism Spectrum Disorder D'Adamo Patrizia Biomedical/Biomedica Ospedale San Raffaele - Milano Background and Significance Loss and gain of function mutations in the human RAB39B gene are associated with intellectual disability (ID) and autism spectrum disorder (ASD) (Giannadrea et al. AJHG 2010; Vissers et al. Nat Genet 2010; Vanmarsenille et al. Hum Mut 2013). Our current research focuses on the understanding of the etiopathogenesis of ID and ASD by studying the molecular mechanism subtending these diseases that they often appear clinically associated (Srivastava et al. Neurosc & Biobehav Rev 2014). RAB39B is a member of RAB family controlling intracellular vesicular trafficking in a compartment specificmanner. Preliminary results identified the role of RAB39B in controlling the trafficking from the endoplasmic reticulum (ER) to the Golgi complex and, by that the surface expression of the GluA2 -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit. RAB39B ablation enriches AMPAR of Ca2+-permeable forms, altering post-synaptic activity and loss in synaptic plasticity. The significance of the project is to attempt pharmacological correction of the biochemical and behavioral phenotype on Rab39b mouse models with currently a new available AMPAR drugs. Results of the project will thus shed light on novel mechanisms of neuronal activity and also identify a suitable new strategy to improve the quality of life of patients with ID/ASD. Specific aims Aim 1: Aim 2: Aim 3: Molecular and behavioral analysis of Rab39b mouse models. Based on the results obtained on Rab39b KD mice (see preliminary results), biochemical, anatomical and behavioural analysis will be performed in the new Rab39b mouse models. Analyze brain functional and molecular imaging of Rab39b mouse models. Brain functional and molecular imaging will be defined in our mouse models by radiotracers, to obtain a functional map and to identify possible alterations that might be important for clinical evaluation. Rescue the neurological/behavioral phenotype by pharmacological means. Based on preliminary results, ampakines and newly designed AMPAR modulators will be used to explore the possibility to rescue the Rab39b phenotype. Additional molecules will be used based on Aim 2 results. Hypothesis: The hypothesis to be tested is that loss of RAB39B hinders proper representation of AMPAR and by that causes a loss in synaptic plasticity underlining the cognitive dysfunction in RAB39B-related disorders. Published evidences support the predicted RAB39B/AMPAR/ID-ASD causative link by showing that the regulation of AMPAR trafficking has a direct impact on cognitive abilities and that truncation or deletion of AMPAR subunits are responsible for ID and ASD. Thus, by the study of RAB39B neuronal function we will shed light on a more general phenomenon of receptor trafficking, composition and availability to be tagged in human cognitive disorder as ID/ASD. Preliminary data: Mutations in the RAB39B gene are associated with ID/ASD, but the impact of RAB39B loss of function on synaptic activity is unexplained. In a preliminary study, we showed that RAB39B controls the secretory trafficking and the surface expression of GluA2-AMPAR subunit. The role of AMPARs in synaptic transmission varies depending on the composition of subunits GluA1/A2/A3 they incorporate. RAB39B ablation skews AMPAR composition toward non GluA2-containing Ca2+permeable forms and thereby alters synaptic activity, specifically in hippocampal neurons. We posit that the resultant loss in synaptic plasticity underlies cognitive dysfunction in RAB39B-related disorders. To start test our hypothesis in mouse models we have recently generated a Rab39b knock down (KD) mouse that is viable, fertile with no gross defects in brain morphology in post-natal development. Preliminary results indicate that RAB39B down regulation hinders normal expression of the GluA2-AMPAR subunit in the hippocampus from 30 to 60 days of post-natal development (P) and an increase of the number of dendritic spines. These events appear to be linked to defects in 02/07/ / 6

58 RF Rab39b mouse models: pre-clinical studies by using AMPA receptors as possible therapeutic targets to unravel the role of RAB39B in Intellectual Disability and Autism Spectrum Disorder D'Adamo Patrizia Biomedical/Biomedica Ospedale San Raffaele - Milano social behaviours. As defective AMPAR expression and altered spine numbers have been previously linked to deficit in synaptic pruning, and defect in synaptic pruning has been linked to abnormal social behaviours, often observed in ID/ASD mouse models, we speculate a central role for RAB39B in the pathogenesis of ID/ASD via the control of AMPAR. Materials and Methods We generated Rab39b mouse models (KD, Tg and KO). Biochemical and molecular analysis of AMPAR distribution in Rab39b Tg/KO brains will be done by western blot and immunofluorescence in hippocampal culture. Spine morphology by Golgi staining will be performed. The behavioral analysis will be performed in the behavioral facility directed by the PI, that is equipped with different mazes to assess exploration and emotion (dark/light, open field, emergence and novelty test), learning and memory (radial maze, water maze, fear conditioning, modular test conditioning chamber), social behavior (3- chambers test). Brain functional and molecular imagining will be performed in the laboratory of Preclinical PET imaging directed by the collaborator, using pharmaceutical tracers developed by Radiochemistry lab of the Nuclear Medicine Department. AMPAR modulators will be available thanks to a current collaboration between the PI and F.Hoffmann- LaRoche (CH). Impact and Translational Implications ID/ASD are major social problems worldwide. The study of RAB GTPases has opened new windows on the intracellular trafficking dynamics that control interconnected events responsible for the development and the plasticity of cognitive functions. We defined a new model to explain aspects of ID and ASD suitable for pharmaceutical intervention. Results of the research will have an impact on clinicians facilitating future clinical translation of the identified therapy. 02/07/ / 6

59 The long pentraxin PTX3 in cancer: a novel player in cancer-related inflammation BANDO 2013 Progetti Ordinari RF RF Garlanda Cecilia 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 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. cancer related inflammation, complement and gene instability oncosuppressor and epigenetic modifications 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/Lavoratory Role in the project 1 Istituto Clinico Humanitas Department of Research on Immunity and Inflammation, Laboratory of Experimental Immunopathology ICH will perform the study using in vivo models of carcinogenesis and taking advantage of PTX3- deficient mice, will characterize the molecular mechanisms underlying increased cancer-related inflammation associated to PTX3 deficiency and perform epigenetic studies in human cancer samples to define the role of PTX3 as an oncosuppressor gene. CG will lead the project and coordinate the activity of PhD students. CG has a long lasting experience on PTX3, PTX3- deficient mice, cancer models and cancer. Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 03/07/ / 6

60 The long pentraxin PTX3 in cancer: a novel player in cancer-related inflammation BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Garlanda Cecilia Istituto Clinico Humanitas Background and Significance The link between inflammation and cancer has been a matter of interest since the early twentieth century, because epidemiological studies have revealed that selected inflammatory diseases increase the risk of cancer and because inflammation-associated processes are present also in tumors that are not epidemiologically related to inflammation. Cancer related inflammation (CRI), which consists in leukocyte recruitment, cytokine and chemokine production, tissue remodeling and angiogenesis, contributes to proliferation and survival of malignant cells, angiogenesis, metastasis, subversion of adaptive immunity, and promotes genetic instability, thus affecting different stages of tumor development. CRI is now considered a hallmark of cancer (Carcinogenesis2009,Cell2011). The long pentraxin PTX3 is a soluble pattern recognition molecule produced at sites of inflammation by immune and stromal cells, including macrophages, neutrophils, endothelial cells and fibroblasts. The physiological functions attributed to PTX3 include microbe opsonization, modulation of Complement activation and leukocyte recruitment, regulation of angiogenesis and tissue remodeling (AnnRevImmunol 2005,2010). Gene targeting of PTX3 has revealed that it plays non-redundant roles in resistance to selected pathogens and in modulating inflammatory responses in tissue damage (Nature 2002,JExpMed 2007,Immunity 2014). However, whether PTX3 plays a functional role in cancer-related inflammation has not been investigated. Specific aims Aim 1: Aim 2: Aim 3: The general objective of this proposal is to gain insight into the role of the humoral arm of innate immunity in CRI using the long pentraxin PTX3 as a paradigm. 1) Characterization of the susceptibility of Ptx3-/- mice to chemically-induced carcinogenesis, using models of mesenchymal and epithelial tumors, including 3-MCA-induced fibrosarcoma and skin carcinogenesis induced by DMBA/TPA, and colon carcinogenesis induced by AOM/DSS or AOM alone. 2) Characterization of CRI in Ptx3-/- mice and molecular and cellular mechanisms underlying the oncosuppressive activity of PTX3, by a) defining the source of PTX3 in the tumor, the role of PTX3 derived from the hematopoietic and stromal compartments, analysing leukocyte infiltration and macrophage polarization, angiogenesis, cytokine and chemokine production, Complement deposition, inflammation-induced DNA damage and Tp53 and Kras gene instability; b) addressing the role of PTX3 in regulating CRI, Complement and leukocyte recruitment by analysing the relevance of PTX3 in regulating P-selectin-dependent leukocyte recruitment and factor H-dependent Complement deposition, the role of inos and ROS in inducing DNA damage and of macrophage recruitment and polarization. 3) Characterization of the relevance of PTX3 in human cancer, by investigating whether the human PTX3 gene is silenced in cancers of mesenchymal and epithelial origin and by which epigenetic modifications. We will address a) PTX3 gene methylation in human cancer samples from ICH biobank and cell lines; b) the effect of a demethylating agent (5-AZA-dC) in cancer cells on PTX3 expression, PTX3 promoter and CpG island methylation, histone modifications associated to transcriptional activation and repression, and the binding of transcription factors involved in PTX3 expression (NFkB and AP1) to the promoter. Hypothesis: PTX3 plays regulatory roles in inflammatory responses associated to tissue damage by regulating Complement activation and leukocyte recruitment. Preliminary data show that PTX3-deficiency in the mouse is associated to increased susceptibility to tumor incidence and growth, and increased CRI. In human, PTX3 epigenetic modifications leading to PTX3 silencing have been described in cancer. Based on these data, we hypothesize that PTX3 acts as a regulator of CRI, acting as an oncosuppressor gene. Preliminary data: 1) In pilot experiments, PTX3 was induced during 3-MCA carcinogenesis. 3-MCA-treated Ptx3-/- mice were significantly more susceptible to fibrosarcoma incidence in comparison of Ptx3+/+ (80% vs 40%) and PTX3-/- sarcomas grew faster than wild type tumors. 03/07/ / 6

61 The long pentraxin PTX3 in cancer: a novel player in cancer-related inflammation BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Garlanda Cecilia Istituto Clinico Humanitas 2) 3-MCA fibrosarcomas from PTX3-/- mice were infiltrated by a higher number of macrophages, increased angiogenesis and higher Complement C3 deposition. 3) In silico available data indicate that PTX3 gene is methylated in human cancer. Pilot experiments in colerectal cancer samples confirm increased methylation of PTX3 promoter region and CpG islands in tumor tissue comapred to the normal counterpart. Materials and Methods We will use PTX3-deficient mice, chimeric mice and other available original reagents (anti-ptx3 antibodies, recombinant PTX3, domains); P-selectin-, C3-, PTX3/C3- and PTX3/P-selectin deficient mice; murine models of chemically induced carcinogenesis (3-MCA-induced fibrosarcoma, skin carcinogenesis by DMBA/TPA, and colon carcinogenesis by AOM/DSS or AOM alone). CRI (cytokines, chemokines, leukocytes, angiogenesis), complement activation (C3, C5-9, C4BP and factor H deposition, C5a levels), DNA damage (DNA-damage response markers gamma-h2ax and 53BP1) and gene (Tp53, KRas) instability will be analysed by ELISA, immunohistochemistry, confocal microscopy, FACS analysis, Q-PCR, gene sequencing. inos and ROS inhibitors will be used to address the role of reactive species in increased tumorigenesis and DNA damage. Epigenetic modifications will be analysed in human cancer samples and cell lines by methylation analysis and ChIP. Ethical approvals are available. Impact and Translational Implications This study would unravel the role as oncosuppressor of a humoral component of innate immunity, acting by regulating CRI through modulation of complement and leukocyte recruitment; the potential pathogenetic role of unleashed complement in cancer; the relevance in human cancer of epigenetic modifications leading to PTX3 silencing. Translational implications include: pharmacological targeting of a novel oncosuppressive molecule and of CRI (Complement); use of PTX3 as marker of cancer progression. 03/07/ / 6

62 Prenylcysteine Lyase as a potential novel player in atherosclerosis BANDO 2013 Progetti Ordinari RF RF Banfi Cristina Biomedical/Biomedica Centro Cardiologico S.P.A. Fondazione Monzino 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: Lipoprotein oxidation and metabolism; structure and function of apolipoproteins, lipid-metabolizing enzymes and receptors; cholesterol transport and reverse transport; scavenger receptors and ABC transporters; apoproteins B, E and A-1; gene expression and regulation. Inflammation and lipids Functional proteomics and gene expression 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/Lavoratory Role in the project 1 Centro Cardiologico S.P.A. Fondazione Monzino Unit of Proteomics 2 University of Milan Department of Pharmacological and Biomolecular Sciences 3 University of Naples Federico II Department of Chemical Sciences Coordinator, proteomic and biochemical analysis Studies on animal models of atherosclerosis Functional proteomics Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Bellosta Stefano University of Milan Studies on animal models of atherosclerosis 22/08/ PUCCI PIETRO University of Naples Federico II Functional proteomics 09/02/ Tremoli Elena Centro Cardiologico Monzino IRCCS, Scientific Director Scientific coordinator and responsible for the samples and data management of the IMPROVE Study 18/06/ /07/ / 6

63 Prenylcysteine Lyase as a potential novel player in atherosclerosis BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Banfi Cristina Centro Cardiologico S.P.A. Fondazione Monzino Background and Significance Genetic, clinical and pharmacological studies implicate elevated plasma levels of low density lipoproteins (LDL) in the pathogenesis of atherosclerotic vascular disease, the leading cause of death in industrialized societies. However, at any level of plasma cholesterol, there is a wide variation in the incidence of coronary artery disease. It is evident that, besides cholesterol, many factors might affect the lipoprotein-arterial wall interaction, thus exacerbating the atherogenic process. Among these factors, lipoprotein modifications, mainly oxidation, are crucial for the uptake of LDL by cells in the first stages of atherogenesis. However, the nature of these modifications and the determinants, either lipoprotein intrinsic or extrinsic factors, remain unclear. By proteomics, we discovered that Prenylcysteine Lyase (PCL1), an enzyme involved in the degradation of prenylated proteins, is associated with atherogenic lipoproteins and, in the presence of its substrate, can generate hydrogen peroxide (Banfi C, Proteomics 2009;9: ), which in turn can induce lipoprotein oxidation in vitro (Fig 1). Overall, these observations stress the importance of understanding both PCL1 biology and mechanism(s) of action, which are still unknown, and might point out an important biological role for this enzyme in the development of atherosclerosis. Specific aims Aim 1: Aim 2: Aim 3: To identify gene expression regulators, biological functions, and protein interactors of PCL1 To define mechanisms underlying the effects of PCL1 ablation on atherogenesis in animal models To establish if PCL1 might represent a novel risk factor for atherosclerosis and its progression and/or the incidence of cardiovascular events in human Hypothesis: We hypothesise that PCL1, a new lipoprotein-associated pro-oxidant enzyme, represents a novel risk factor for atherosclerosis. To address this issue we will analyse, in vitro and in vivo, PCL1 from gene regulation to its role in the modification of lipoproteins in terms of biological functions and its involvement in murine atherogenesis. The potential association of PCL1 with subclinical atherosclerosis and vascular events in patients at high risk of CVD will be also addressed Preliminary data: AIM1 Candidate regulators for PCL1 were identified from databases of ChIP coupled with tiling arrays or next-generation sequencing. The PCL1 locus has quite short (<10 kbps) insulator-delimited intergenic regions, poorly conserved and covered by repetitive sequence elements, suggesting that, as other hepatic genes, most of the sequence elements crucial for its transcriptional regulation are in the promoter region paving the way for further analysis. Hepatocytes strongly express PCL1 (The Human Protein Atlas) and HepG2 human hepatocytes model secretes it along with nascent lipoproteins (Fig 2). We overexpressed myc-ddk-tagged wildtype PCL1 in HepG2 cells to identify its binding partners; and we silenced PCL1 (Fig 3, 4) to analyze its influence on the cell proteome/secretome and on protein/lipid content of secreted lipoproteins (Fig 5). Proseek Multiplex assay of cell culture media revealed a significant decrease of selected proteins (4 out of 92 analytes) involved in inflammation following PCL1 silencing. AIM2 We found that mouse lipoproteins are carriers of PCL1, whose content declined from VLDL to LDL reflecting the findings on human lipoproteins. To explore PCL1 potential role in atherosclerosis, we generated PCL1/apoE double knockout mice and fed them with a western-type diet for 4 weeks. PCL1 deletion resulted in a reduction of plasma total cholesterol and triglycerides, lesion size, and neutrophil content (Fig 6, 7). These changes were accompanied by a significant reduction of 10/07/ / 6

64 Prenylcysteine Lyase as a potential novel player in atherosclerosis BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Banfi Cristina Centro Cardiologico S.P.A. Fondazione Monzino triglycerides content in the liver, without signs of hepatic dysfunction (Fig 8). AIM3 The PCL1 measurement by ELISA (detection range pg/ml; LOD 7 pg/ml) shows that it is associated solely with lipoproteins, since it is undetectable in lipoprotein-depleted plasma. In a pilot study, plasma PCL1 median level was 83 pg/ml and 152 pg/ml in healthy subjects (n=15) and in agematched patients with stable angina (n=7, angiographycally documented stenosis >75% in at least 1 major coronary artery), respectively. Data and samples of the IMPROVE study are available Materials and Methods AIM1 Functional proteomic approaches, DNA affinity capture and immunoprecipitation, to identify PCL1 gene expression factors and PCL1 protein partners. Transcriptomic analysis to ascertain the effects of PCL1 gene silencing in hepatocytes HepG2 model AIM2 Analysis of atherogenic profile and phenotype characterisation (by "omics" approaches) of PCL1-KO mice; lipid/protein composition and oxidability of mouse lipoproteins; and proinflammatory, proadhesive, procoagulant status of in vitro cultured vascular cells exposed to lipoproteins isolated from wild type and PCL1-KO mice AIM3 Association of PCL1 (protein and gene mutations) with cardiovascular events will be assessed in a nested case-control study drawing samples from the biologic bank of the IMPROVE study (European Commission, QLG1-CT ) designed to investigate the association between subclinical atherosclerosis, its progression and the onset of vascular events in 3711 subjects with >3 cardiovascular risk factors Impact and Translational Implications Defining the role and functions of PCL1 is relevant to understand its role in lipid oxidation and hopefully in atherosclerosis. Whether PCL1 is confirmed as a novel risk factor for atherosclerosis, then we can develop a new diagnostic tool to test its level in blood, allowing people to find "hardening of blood vessels" earlier. Furthermore, we anticipate that this study could lead to the development of new drugs that inhibit PCL1 activity and block the atherogenic modification of lipoproteins 10/07/ / 6

65 RF Calcitonin oligomers as a model to study the amyloid neurotoxicity: the focal role played by lipid rafts in the prevention and cure. Diociaiuti Marco 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: Biological Chemistry and Macromolecular Biophysics Biochemistry and Biophysics of Membranes - BBM Project Keyword 1: Project Keyword 2: Project Keyword 3: Membrane architecture: lipid-protein interactions, membrane protein folding, assembly, structure, and dynamics. Amyloid oligomers: lipid rafts interaction, Neuraminidase effects Neurotoxicity: intracellular Ca2+ unbalance, synaptic plasticity 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/Lavoratory Role in the project 1 Istituto Superiore di Sanita' Tecnologie e Salute Dipartimento del Farmaco 2 Istituto Superiore di Sanita' Centro Nazionale Malattie Rare Biologia Cellulare e Neuroscienze Amyloid oligomers preparation, purification, striuctural caracterization and interaction with model membranes. Experiments on in vitro and ex vivo models to unravel and hinder the molecular mechanisms of amyloid toxicity. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 FRANK CLAUDIO Istituto Superiore di Sanita' Measurement of Ca2+ influx and currents on mice hippocampal primary neuronal cultures. Evaluation of the protective effects of neuraminidase. 05/03/ Gaudiano Maria Cristina Amyloid oligomers preparation and purification. 3 D'ARCANGELO GIOVANNA Università Tor Vergata/ Dip. Electrophysiological experiments to Medicina dei Sistemi/Ricercatore perform functional studies on scos Confermato neurotoxic effects and neuraminidase protection. 4 matteucci andrea Istituto Superiore di Sanità/Dip. Biologia Cellulare e Neuroscienze/Ricercatore Immunohistochemical analysis of brain tissues: the scos neurotoxic effects and the neuraminidase protection. 17/11/ /11/ /11/ /07/ / 6

66 RF Calcitonin oligomers as a model to study the amyloid neurotoxicity: the focal role played by lipid rafts in the prevention and cure. Diociaiuti Marco Biomedical/Biomedica Istituto Superiore di Sanita' Background and Significance A number of degenerative disorders are pathogenetically linked to a process of misfolding and aggregation of specific proteins, the large family of the so-called amyloid proteins. They follow a common aggregation process, leading to characteristic cytotoxic properties that generally proceeds through several states, including the formation of dimers, oligomers and protofibrils, before finally assuming an insoluble fibrillar conformation, characterized by beta-structures. It is now generally agreed that the cytotoxic species are the soluble, oligomeric aggregates, while the insoluble fibrils seem to be relatively non-harmful. Among diseases caused by protein misfolding, neurodegenerative diseases are particularly frequent. They include high prevalence pathologic conditions, such as Alzheimer's (AD) and Parkinson's diseases, as well as rare diseases, such as prion protein diseases and the large family of polyglutamine diseases. The CNS seems therefore a particular suitable environment for diseases caused by cytotoxic, misfolded proteins. Calcitonin is a polypeptidic hormone secreted by the parafollicular C cells of the thyroid gland and is involved in calcium homeostasis. Salmon calcitonin (sct) belongs to the amyloid family (Gaudiano et al., BBA :134) and is characterized by the slowest aggregation rate. This peculiarity renders sct a useful tool to study the amyloid aggregation since the early stages. Specific aims Aim 1: Aim 2: Aim 3: SCO preparation and characterisation. The main goal is to clarify if a peculiar sco structure and/or lipid composition (lipid rafts) triggers the membrane damage. We will first proceed to prepare, using Size Exclusion Cromatography, samples rich in sct Oligomers (scos) formed in the aggregation pathway from the monomer to the mature fibre. To investigate the sco molecular structure, samples will be studied by the combined application of Circular Dichroism spectroscopy and Transmission Electron Microscopy. Finally, to unravel the molecular mechanisms driving the interaction of scos with cellular membranes, synthetic model membranes (liposomed and Langmiur monolayers) mimicking the actual membranes, will be biophysically studied. SCO biological effects. The main goal is to go deep inside the neurotoxic mechanisms of scos. To this aim Ca2+ currents in mice hippocampal cell cultures and synaptic transmission and synaptic plasticity in mice hippocampal slices will be explored. Moreover, apoptosis, neuritic and synaptic damage and glial activation will be investigated in several areas of CNS. Focus on new therapeutic targets. The main goal is to prevent the sco induced neurotoxicity by disrupting the "lipid rafts" components in primary neuronal cultures and brain slices from different regions. This will be done by treating neurons with neuraminidase or ciclodextrin, drugs that affect the molecular structure of GM1 and cholesterol. Hypothesis: Several findings suggest that a common pathologic pathway exists in the case of amyloid-associated diseases, independently on the peptide primary sequence (Glabe, Neurobiol. Aging :570). According to this intriguing hypothesis, amyloid protein toxicity is due to the formation of Ca2+-permeable channels in the plasma membrane, leading to an unbalanced Ca2+ influx and consequent cell death. The structure of the toxic aggregate is common to all amyloid proteins and the damage is due to ion channel formation mediated by the membrane lipid composition. GM1 and cholesterol, the typical components of the "lipid rafts", seem to play a pivotal role in the channel formation. Preliminary data: We have demonstrated that the use of the slowly aggregating sct can be a useful tool for the study of the amyloid structure and toxicity (Diociaiuti et al., Int. J. Mol. Sci :9277). We have also shown in a biophysical study that scos form Ca2+-permeable pores in liposomes, highly reminiscent of the ion channels formed by other amyloid proteins, such as Abeta. sco insertion in synthetic model membrane occurs only when they contain ganglioside GM1 and cholesterol, typical components of the "lipid rafts" (Diociaiuti et al., Biophys. J :2275). This focus the attention on the role-played by the "lipid rafts" in the interaction between plasma membrane and amyloid 10/07/ / 6

67 RF Calcitonin oligomers as a model to study the amyloid neurotoxicity: the focal role played by lipid rafts in the prevention and cure. Diociaiuti Marco Biomedical/Biomedica Istituto Superiore di Sanita' proteins. Recently, we confirmed on primary neurons that scos induce strong damage, that can be inhibited by GM1 and cholesterol depletion (Malchiodi et al., BBA-DIS :406). Materials and Methods ENERGY FILTERED-TRANSMISSION ELECTRON MICROSCOPY. We will investigate at high resolution (about 1 nm) the sco structure and morphology. CIRCULAR DICHROISM. This spectroscopy will give detailed information on the protein secondary structure. MODEL MEMBRANES/ LANGMUIR THROUGH/BREWSTER ANGLE MICROSCOPY. The interaction between scos and synthetic model membranes will be studied preparing both liposomes and planar lipid Langmuir films. IN VITRO STUDIES. Male Balb/C mice will be used. Intracellular calcium concentration and currents will be measured as previously described on primary hippocampal cultures (Frank et al., EJP 1996, FEBS 2004). Electrophysiological experiments will be performed on mice slices as previously reported (D'Arcangelo et al., Br. Res. 2011) EX VIVO STUDIES. In order to evaluate sco neurotoxicity, sections obtained from in vitro experiments will be processed (Matteucci et al., Exp Eye Res 2014). Impact and Translational Implications We have recently stressed, both in model membranes and hippocampal neurons, the focal role-played by GM1 and cholesterol, the typical "lipid rafts" components, in the amyloid oligomer neurotoxicity. We have shown that the depletion in GM1 and cholesterol protects against neurotoxicty. Data outcoming from this study will design novel therapeutic approaches, based on "lipid raft" modification, in the cure and prevention of amyloid neurodegenerative diseases. 10/07/ / 6

68 RF Inhibition of endocannabinoids-hydrolyzing enzymes as a new therapeutic target for migraine treatment: studies in animal models and preliminary evaluation in humans Tassorelli Cristina Biomedical/Biomedica Fondazione Istituto Neurologico Casimiro Mondino 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 migraine endocannabinoid system 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/Lavoratory Role in the project 1 Fondazione Istituto Neurologico Casimiro Mondino Headache Science Centre, Laboratory of Neurophysiology of the Autonomic Integrative Systems Basic part of the study (use and optimization of animal models, in vivo and ex vivo, investigations in animal models of migraine, collection and centralization of data); mrna quantification; collection of plasma from healthy subjects and high frequency migraine sufferers;neurophysiological testing in humans 2 Istituto Italiano di Tecnologia, Genova Dept. of Drug Discovery and Development Endocannabinoids assay; Identification and supply of the inhibitors of endocannabinoid hydrolases 08/07/ / 7

69 RF Inhibition of endocannabinoids-hydrolyzing enzymes as a new therapeutic target for migraine treatment: studies in animal models and preliminary evaluation in humans Tassorelli Cristina Biomedical/Biomedica Investigators, Institution and Role in the Project Fondazione Istituto Neurologico Casimiro Mondino Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Reggiani angelo mario Istituto Italiano di Tecnologia, Genova 2 UO2 coordinator;identification and supply of the inhibitors of endocannabinoid hydrolases 21/10/ Greco Rosaria Fondazione C. Mondino, Laboratory Laboratory of Neurophysiology of the Autonomic Integrative Systems, PhD/ Biologist 4 Bandiera Tiziano Istituto Italiano di Tecnologia, Dept. of Drug Discovery and Development, senior researcher Lab. coordinator; behavioral evaluations of animal model of trigeminal hyperalgesia, immunohistochemical evaluation, mrna quantification UO2 collaborator; Endocannabinoids assays 24/10/ /12/1957 Background and Significance Migraine is an episodic, neurological disorder ranked among the most common, disabling neurological disorders by WHO. Migraine affects 17% of females and 8% of males in the European Population. In 3 % of the general population migraine is present in its chronic form (> 15 days of headache/month), with a very high burden. The available treatments for migraine prophylaxis are quite unsatisfactory both in terms of effectiveness and tolerability. The lack of understanding of the mechanisms behind the development of migraine has been a major limitation to the development of more effective treatments. Hence the need for more specific treatments developed according to their capability to interfere with the circuitries and the processes involved in migraine attacks. Experimental evidence has shown an antinociceptive action of endocannabinoids system, a major player in the determinism of migraine Attacks. Furthermore a reduced endocannabinoid activity has been reported by clinical studies in migraineurs. Therefore, we hypothesize that potentiation of endocannabinoid activity, i.e. via the inhibition of the catabolizing pathway, may represent a new potential therapeutic target for migraine pain. The evaluation in a validated animal model of migraine of the effects of modulating the endocannabinoid system function will hopefully provide new interesting insights into mechanisms involved in the migraine pain and prompt new, possibly more effective druggable targets for this pathology. Specific aims Aim 1: Aim 2: Aim 3: to investigate the role of anandamide and the impact of the inhibition of its degradation (by means of inhibitors of fatty acid amide idrolase., FAAH) in preclinical rodent models of migraine and trigeminal hyperalgesia to evaluate the role of endocannabinoids in healthy subjects and migraine sufferers to screen potential leads for drug development in the migraine pain Hypothesis: Hypothesis: Our hypothesis is that the endocannabinoid system is deeply involved in the pathogenesis of migraine. Therefore, the increase in endocannabinoid levels, obtained via the inhibition of the anandamide - hydrolyzing enzymes, may represent a potential therapeutic option for this condition. 08/07/ / 7

70 RF Inhibition of endocannabinoids-hydrolyzing enzymes as a new therapeutic target for migraine treatment: studies in animal models and preliminary evaluation in humans Tassorelli Cristina Biomedical/Biomedica Fondazione Istituto Neurologico Casimiro Mondino This hypothesis will be tested using the animal/human model of systemic nitroglycerin (NTG) administration, which has proven to be a reliable model of migraine over the years (Buzzi and Tassorelli, 2010). More specifically, in the rodent we will evaluate the effect of FAAH inhibition upon: a) NTG-induced neuronal activation (Fos expression) in the CNS b) Endocannabinoid content at the peripheral and cerebral levels c) Face-rubbing response at the formalin orofacial test, pro-inflammatory cytokines, CGRP, Substance P, nnos expression in brain areas. Then, we will optimize additional FAAH inhibitors (bioavailability and activity) using the preclinical models. In humans we will evaluate endocannabinoid levels and pain thresholds after NTG administration. Preliminary data: Systemic NTG administration in the rat activates cerebral nuclei involved in nociception, as well as in neuroendocrine and autonomic functions (Tassorelli et al., 1995). These changes are likely relevant for migraine pain, since NTG provokes spontaneous-like migraine attacks in migraineurs (Sances et al., 2004). The underlying mechanisms are not known, but it is likely that NTG -induced vasodilation and increased availability of nitric oxide at the trigeminovascular level (Buzzi and Tassorelli, 2010), and possibly also at central sites, may induce a condition of sensitization. This hypothesis was confirmed by the demonstration that NTG administration in rat induces hyperalgesia after plantar (Greco et al., 2014) and orofacial (data unpublished) formalin test. Previously, we have found that NTG-induced hyperalgesia is associated with increased activity of FAAH and increased density of cannabinoid binding sites in specific cerebral areas (Greco et al., 2010). Additionally, pre-treatment with anandamide (AEA), significantly reduced NTG-induced neuronal activation in nucleus trigeminalis caudalis (NTC) (Greco et al., 2011). On the basis of these findings, recently we reported that both URB597 and URB937 (FAAH inhibitors) counteracted NTG-induced hyperalgesia at the Formalin test in the rat. In addition, URB937 significantly reduces NTG-induced neuronal activation in the NTC and in the locus coeruleus (LC). These results may be related to the effect of AEA in the trigeminovascular system, which might counteract NTG-induced hyperalgesia. 08/07/ / 7

71 RF Inhibition of endocannabinoids-hydrolyzing enzymes as a new therapeutic target for migraine treatment: studies in animal models and preliminary evaluation in humans Tassorelli Cristina Biomedical/Biomedica Fondazione Istituto Neurologico Casimiro Mondino Materials and Methods In rats The effect of FAAH inhibition will be evaluated in male rats pre-treated with (NTG, 10 mg/kg, i.p.) o vehicle. We will investigate: - Fos expression on brain slices (immunohistochemistry, IHC); - Endocannabinoids levels in the meninges, trigeminal ganglion, medulla and mesencephalon (liquid chromatography-mass spectrometry, LC-MS/MS) - Face-rubbing response at the formalin orofacial test, levels of pro-inflammatory cytokine expression in specific brain areas (RT-PCR) and CGRP, Substance P and nnos in the NTC (IHC). In humans (healthy subjects and high frequency migraineurs) we will evaluate, before and after NTG administration: - pain processing at the central level by means of the temporal summation threshold of the lower limb nociceptive withdrawal reflex (Sandrini et al., 2005) and the related pain sensation; - plasma endocannabinoid levels by LC-MS/MS (Gaetani et al., 2008). Impact and Translational Implications Current pharmacotherapy for migraine pain includes medications that are not particularly effective, nor well tolerated. The successful completion of the proposed study will elucidate the role of the endocannabinoid anandamide in migraine, thus offering new insights on the mechanisms underlying this common disorder. Furthermore, our studies will provide essential preclinical tests of the potential utility of anandamide-degradation inhibitors (FAAH inhibitors) in migraine. 08/07/ / 7

72 RF ALS and FTD: changing paradigms in the genotype-phenotype correlation due to C9orf72 and other gene characterization Silani Vincenzo Clinical health care research/clinicoassistenziale Istituto Auxologico Italiano 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. Frontotemporal Dementia (FTD) Genetics 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/Lavoratory Role in the project 1 Istituto Auxologico Italiano Neurology and Laboratory of Neuroscience Project coordination, ALS and FTD patients' recruitment, clinical definition and staging, MR, biomarker collection-biobank, C9orf72 screening and Southern blot, C9orf72 methylation, C9RAN Elisa in CSF, fibroblast cultures. 2 Fondazione IRCCS Istituto Neurologico Carlo Besta Laboratory for Molecular Diagnosis of ALS and FTD patients' recruitment, clinical Hereditary Neurodegenerative Diseases definition, and genetic characterization (NGS). 3 Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico Laboratory of Genetics and Biochemistry - Neurology Unit ALS and FTD patients's recruitment, production of ipscs and generation of differentiated cells. In vitro model characterization. 10/07/ / 6

73 RF ALS and FTD: changing paradigms in the genotype-phenotype correlation due to C9orf72 and other gene characterization Silani Vincenzo Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Istituto Auxologico Italiano Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Gellera Cinzia Fondazione IRCCS Istituto Neurologico Carlo Besta PI of UO2, Lab. Coordinator for molecular studies, design of NGS customized exome panel for genetic characterization of ALS and FTD patients. Biobank coordinator. 2 Comi Giacomo Pietro Fondazione IRCCS Ca' Granda - PI of UO3, Lab. Coordinator for cellular Ospedale Maggiore Policlinico studies, generation of ipsc from ALS and FTD patients, differentiation of ipsc into cortical neurons, motor neurons and astrocytes. Definition and characterization of a stem cell derived ALS-FTD in vitro model-ipsc Biobank coordinator. 03/11/ /11/1958 Background and Significance Amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTD) share common genetic, pathologic, and clinical characteristics, and are now considered to be part of the same disease continuum. C9orf72 repeat expansions (RE) are the major genetic cause of ALS and FTD, accounting for 25-50% of familial and 5-10% of sporadic cases worldwide (DeJesus- Hernandez et al. 2011). Given the compelling evidence of somatic mosaicism and clinical heterogeneity (van Blitterswijk et al., 2012; Ticozzi et al., 2014; Akimoto et al., 2014), novel approaches, such as methylation studies (Liu et al., 2014) and assessment of poly(gp) c9ran proteins in the CSF (Su et al., submitted) are needed to draw a comprehensive genotypephenotype correlation in c9orf72+ patients. Furthermore, the design and application of a Next Generation Sequencing (NGS) genetic panel for target resequencing of known and candidate ALS- and FTD-associated genes represents a powerful and effective tool both in clinical and research setting for a better characterization of c9orf72- patients. Skin fibroblast-derived ipscs from patients carrying c9orf72 RE and/or other causative mutations can be used to study the pathogenesis of ALS and FTD and to identify novel therapeutic targets. A correct genotype-phenotype correlation in ALS and FTD is critical in the clinical settings, having a direct impact on therapeutic strategies, on patients' stratification for clinical trials, and on redesigning new assistance models. Specific aims Aim 1: Aim 2: Aim 3: Selection of a cohort of 200 ALS and 100 FTD patients for deep clinical phenotyping (electrophysiological, neuroradiological, neuropsychological-ecas evaluations). Genotype-phenotype correlation in c9orf72+ patients through: 1) determination of c9orf72 status by repeat-primed PCR; 2) RE size assessment by Southern Blot; 3) assessment of c9orf72 methylation; 4) measurement of c9ran protein in the CSF by ELISA. 1) to apply a customized NGS panel for studying a large number of genes causing ALS/FTD overlapping phenotypes (81 genes) in a number of FALS of unknown genetic cause (n=48) and in a large cohort of SALS/FTD patients (>300), in order to identify novel causative genetic variants or possible concurrent genetic determinants for elective diagnostic and therapeutical approaches; 2) Creation of a customized database reporting patients results at genetic level. The database will be publicized. Production of ipscs from fibroblasts of ALS and FTD patients from all Units and differentiation in relevant cell types. IPSCs will be differentiated into cortical and motor neuron and examined by genomic, proteomic, phenotypical and functional analyses. We will investigate autonomous and non autonomous disease features, 10/07/ / 6

74 RF ALS and FTD: changing paradigms in the genotype-phenotype correlation due to C9orf72 and other gene characterization Silani Vincenzo Clinical health care research/clinicoassistenziale Istituto Auxologico Italiano using co-culture with ipsc derived astrocytes. Cells will be available for analysis in other aims generating an Italian ALS/FTD ipsc library. Hypothesis: A comprehensive genotype-phenotype correlation and the identification of novel biomarkers are necessary steps for the stratification of the ALS and FTD patients in homogeneous subgroups. Characterization of C9orf72+ individuals is crucial, given their high prevalence. The use of NGS tools allows the identification of pathogenic mutations in other genes. Development of ipsc lines and ips-derived neurons from these individuals will in turn help broaden the understanding of the pathogenesis of both diseases. Preliminary data: Proponent Institutions are referral Centers for ALS and FTD, recognized in the European ALS Consortium (ENCALS), World Federation of Neurology, and European Academy of Neurology (EAN). They contributed to the European Guidelines for the Diagnosis and Treatment of ALS (Andersen et al., 2005, 2012) and FTD (SINdem). They have screened for causative mutations a large cohort of ALS and FTD patients. Protocols for c9orf72 RE identification/size determination (Ratti et al., 2012; Akimoto et al., 2014), and ELISA assay for identification of poly(gp) c9ran proteins in the CSF (Su et al., submitted) have been optimized. The Laboratories have extensive experience in NGS technologies and have designed a NGS genetic panel for target resequencing of candidate genes. The Laboratories have the capability to characterize skin fibroblast-derived ipscs and to use them as a tool to define disease mechanisms and design new therapeutical strategies (Corti et al., 2012; Nizzardo et al., 2014). Materials and Methods A series of 200 ALS and 100 FTD patients are characterized by ENG/EMG, MUNIX, TMS/MEPs, neuropsychological (ECAS), and MRI-3T evaluations. Genetic screening of C9ORF72 includes fragment-length analysis, RP-PCR, Southern Blot, bisulfite/pcr with TaqMan assay for mrna expression (methylation). CSF C9RAN proteins are tested by ELISA immunoassay. The NGS panel is set up by TruSeq Illumina technology and MiSeq apparatus, data analyzed using Illumina MiSeq Reporter, Illumina Variant Studio and CLC Genomics Workbench softwares. Filtering passing variants are validated by Sanger and evaluated by in silico programs. The HGMD, NCBI-dbSNP, and Exome Variant Server databases are used for SNPs and MAF analysis. We will generate ipscs from ALS/FTD patients using non viral method (Corti, 2012). Cortical neuron, motor neuron, and astrocytes will be obtained from ipscs by applying a multistage differentiation protocol developed for human pluripotent stem cells (Shy, 2012; Krencick, 2011). 10/07/ / 6

75 RF ALS and FTD: changing paradigms in the genotype-phenotype correlation due to C9orf72 and other gene characterization Silani Vincenzo Clinical health care research/clinicoassistenziale Istituto Auxologico Italiano Impact and Translational Implications Both ALS and FTD are incurable diseases with tremendous burden on the NHS. The rapid development of efficacious therapies is an urgent need. By elucidating the genetic basis and pathomechanisms of the two diseases, the project aims to provide novel biomarkers to reduce diagnostic delays and costs due to inadequate pharmacological and nonpharmacological interventions. The clinical and biological stratification of ALS and FTD patients will ultimately provide novel therapeutic targets to exploit.

76 Cervical cancer screening in younger women. BANDO 2013 Progetti Ordinari RF RF Ronco Guglielmo Clinical health care research/clinicoassistenziale Piemonte LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology 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. Cervical cancer screening Human Papillomavirus 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/Lavoratory Role in the project 1 Piemonte AOU Città della Salute e della Scienza di Torino. SC Epidemiologia dei Tumori. CPO Piemonte 2 Piemonte AOU Città della Salute e della Scienza, SC Anatomia ed Istocitopatologia diagnostica e di Screening - Centro Unico Screening Cervico- Vaginale - SGAS Centro Unificato screening Coordination of the study Study Design Data analysis RCT management in Piemonte Molecular analyses Collaboration to study design and management 3 Toscana Istituto per lo Studio e la Prevenzione Oncologica (ISPO) - SC Laboratorio Prevenzione Oncologica RCT management in Toscana Molecular analyses Collaboration to study design and management Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 GILLIO TOS ANNA Piemonte Molecular analyses 14/02/ Carozzi Francesca Maria Toscana Study Coordinator in Toscana molecular analyses 11/09/ /07/ / 6

77 Cervical cancer screening in younger women. BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale Ronco Guglielmo Piemonte Background and Significance HPV-based screening has shown greater efficacy than cytology based screening in preventing cervical cancer and longer low-risk duration after a negative test but is less specific, therefore requiring triage of HPV positive women. Below the age of 30 years cervical cancer is rare and HPV infections are frequent and mainly transient, resulting in many unneeded colposcopies. Some data also suggests larger over-diagnosis of regressive CIN3 and especially CIN2 at younger age. CIN treatment is associated with pregnancy complications. Therefore, HPV-based screening is not recommended at this age but cytology-based screening is recommended. Relevant over-diagnosis is however certainly present also for cytology-detected CIN2 and 3 (just 1/3 of CIN3 progress to invasion in 30 years), particularly at younger age. Based on a Health Technology Assessment report (Ronco et al. Epidemiol Prev 2012) the Italian Ministry of Health delivered a recommendation to regions for starting HPV-based screening at age Using different screening tests at different ages is difficult to explain to women and entails difficulties in the organisation. Specific aims Aim 1: Evaluating, in comparison to current practice, for women below age 30, a screening strategy using HPV testing just to select those who need screening up to 30 years. Evaluating the safety of such strategy by comparing, between the two arms: a) detection rate of histologically determined CIN2+ at baseline. Non-inferiority in experimental arm would shows safety and b) detection rate of histologically determined CIN2+at 30 years. Non-superiority in experimental arm would confirm it. Power: for aims 1a and 1b >80% for a true 20% increase, assuming 6 per 1000 DR in conventional arm (CA). Aim 2: Evaluating the reduced disadvantages of such a strategy by comparing: c) cumulative referral to colposcopy 25 to 30 yrs (including screening at 28 if done). d) cumulative detection of CIN2+ 25 to 30 yrs. Reduction in the experimental arm shows reduced overdiagnosis. Aim 3: -- Power: for aim 2c >90% for a 20% reduction assuming 9% cumulative referral in CA. For aim 2d 90% for 30% reduction assuming 1.5% cumulative detection in CA. Hypothesis: The incidence of invasive cervical cancers within 5.5 years after a negative HPV test is much lower than the incidence of invasive cancers within 3.5 years (see preliminary data). Thus, recalling HPV negative women after 5 years is safe. Over-diagnosis with HPV mainly depends on lesions detected by HPV testing but negative for cytology. Therefore, using the HPV test just to select those women aged 25 years who need screening up to age 30 should not cause overdiagnosis. Moreover, this approach should reduce the number of women who are screened by cytology at 25 and 28 years. Thus, we expect that it will reduce the number of colposcopies and could reduce the over-diagnosis compared with the conventional approach. Preliminary data: An Italian multicentre randomised controlled trial (RCT) on HPV-based screening (NTCC) involving almost 100,000 women showed greater efficacy of HPV testing but registered greater cumulative detection of CIN2+ in the experimental arm, with a large difference in younger women, suggesting possible greater overdiagnosis with HPV testing (Ronco et al. Lancet Oncol 2010). A pooled analysis of the follow-up of 4 European RCTs that reached at least the second screening round (Ronco et al. Lancet 2014), showed that HPV-based screening reduced the incidence of invasive cervical cancer by 60-70% compared to cytology. A clear effect was present from age 30 years. The cumulative risk 10/07/ / 6

78 Cervical cancer screening in younger women. BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale Ronco Guglielmo Piemonte of invasive cancer within 5.5 years after a negative HPV test was about half the cumulative incidence within 3.5 years after a negative cytology. After a cluster-randomized pilot project conducted in Torino, which showed increased participation to HPV-based than to cytology-based screening, the entire region is converting to HPV-based screening. Our institution obtained financial support by the EU (within the FP7-funded COHEAR project, WP3) for a multi-national study on the optimal approach to screening of young women vaccinated for HPV. Materials and Methods Randomised controlled trial. Some women aged 25 years attending for cervical screening in organised programmes will be randomly assigned, with 1:1 ratio, to: - conventional arm. All women will be tested by cytology at age 25 and 28 years and referred to colposcopy if cytology abnormal according to routine protocols - experimental arm. All women tested for DNA of oncogenic HPV types at 25 years. Only HPV positive women will be tested for reflex cytology. They will be referred to colposcopy just on the basis of the cytology result, as in the conventional arm. Only women HPV positive at 25 years will be invited for cytology at 28 years and will be managed as in the conventional arm. Women HPV negative at 25 years will not be re-screened until age 30. At age 30 women from both arms will be invited for HPV-based screening according to standard Italian protocols with HPV (including referral to colposcopy of women who persist HPV positive for 1 year, independently of cytology). Impact and Translational Implications Results, if favourable, can be directly applied to the practice of cervical screening so to minimise undesired effects, discomfort and anxiety for women and costs for the SSN while keeping protection. Using the same primary screening tests at all ages is of great organisational advantage. Results of the present study and of the study supported by EU on the best approach to screening young vaccinated women can easily be combined, resulting in strong positive interaction. 10/07/ / 6

79 Idiopathic Normal Pressure Hydrocephalus (inph), parkinsonism and dementia: improving the accuracy of diagnosis and the patient care to reverse the symptomatology Neurodegeneration, Phenotypes and Outcome Measures RF Pacchetti Claudio Clinical health care research/clinicoassistenziale Fondazione Istituto Neurologico Casimiro Mondino 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: Parkinson s disease and other movement disorders (Huntington s, Dystonias, Ataxias). Dopaminergic degeneration, DAT SCAN, diagnostic accuracy, dopaminergic treatment phenotypes, shunt surgery, outcome measures, longitudinal study, 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/Lavoratory Role in the project 1 Fondazione Istituto Neurologico Casimiro Mondino UOS Parkinson e Disordini del Movimento expertise in clinical diagnosis and care of patients with movement disorders and dementia; expertise in clinical rating scales and cognitive evaluations; qualitative research experts, assistance with data analyses & interpretation, dissemination of findings & study design 2 Azienda Ospedaliero Universitaria Pisana 3 Istituto Superiore Sanità (ISS) UOC Neurologia expertise in assessing brain imaging DAT SCAN, MRI and CT scan; recruiting patients; clinical evaluation with rating scales; assistance to study design Centro Nazionale di Epidemiologia, methodological competence in clinical Sorveglianza e Promozione della Salute epidemiology and statistics analysis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Bonuccelli Ubaldo Azienda Ospedaliero Universitaria Pisana recruiting patients; clinical evaluation with rating scales; assistance to study design 29/11/ D'Errigo Paola Istituto Superiore Sanità (ISS) data collection and statistical analysis; methodology of study design 28/06/ /07/ / 6

80 Idiopathic Normal Pressure Hydrocephalus (inph), parkinsonism and dementia: improving the accuracy of diagnosis and the patient care to reverse the symptomatology Neurodegeneration, Phenotypes and Outcome Measures RF Pacchetti Claudio Clinical health care research/clinicoassistenziale Fondazione Istituto Neurologico Casimiro Mondino Background and Significance Idiopathic normal pressure hydrocephalus (inph) is a complex syndrome of ventriculomegaly, cognitive decline, gait impairment and incontinence that occurs in an elderly. Six studies on the prevalence of inph have been published between 1985 and Prevalence ranged from 0.8% in those aged years and 5.9% in those aged 80 years. Comorbidity and phenotypes are important factors in the prognosis and for the post-operative outcome of shunt surgery for inph. In this regard, inph can be associated with more complex clinical pictures similar to those of a neurodegenerative parkinsonism, typical as PD or atypical as PSP or MSA, or dementia like LBD, FTD and AD. In many cases of inph there is evidence of loss of presynaptic dopamine transporter (DAT) after brain scintigraphy DATSCAN, as in parkinsonism. If the loss of DAT is an inevitable event in the natural history of a inph or otherwise identify a specific subtype of the disease, is currently not known. The identification of an underlying neurodegenerative process and comorbidities and phenotypes should thus be a central part of the clinical management of inph. Improving the diagnostic accuracy should ameliorate the patient care. Is expected that an early ventriculoperitoneal shunt (VPS) should reverse neurological symptomatology. This improving process of care will reduce the costs for the SSN. Specific aims Aim 1: Aim 2: improve the understanding of pathophysiology of inph to test the hypothesis of an underlying neurodegenerative process using DATSCAN imaging outline the prevalence of inph clinical presentations, their DATSCAN profiles and their different dopaminergic response to pharmacological treatment Possible inph subtypes: (1) "pure" movement disorders in absence or in presence of mild cognitive impairment: (i) isolated parkinsonism of gait, (ii) typical parkinsonism (PD), (iii) atypical parkinsonism PSP-like, MSA-like or other, (2) parkinsonism combined with dementia subtypes: (i) LBD-like, FTD-like and AD-like Aim 3: improving the diagnostic accuracy and the care of patients providing an early surgical shunt ("early diagnoses for early treatment"). Hypothesis: inph might be caused by a neurodegenerative process; there is anedoctical evidence of loss of presynaptic dopamine transporter (DAT) after brain scintigraphy DATSCAN, as well as in parkinsonism. The diagnostic effort to distinguish between typical and atypical inph clinical presentation is important for improving the accuracy and for predicting the response to pharmacological and surgical treatment. Preliminary data: In these last 18 months, we observed in our referral centers of Parkinson's and Movement Disorders (Pavia, Pisa), 40 subjects (25 males 15 females) with inph and parkinsonian syndrome (unpublished data). The average age of the patients was 74 years (SD 6.75). The clinical phenotype was characterized by isolated gait disorder as lower body parkinsonism (23 cases); by a typical parkinsonian syndrome resembling PD (5 cases); from a PSP-like (5 cases) and MSA-like parkinsonism (3 cases); a parkinsonian syndrome associated with dementia LBD-like (2 cases) and FTD-like (2 cases). We found a positive DATSCAN in 30 cases (the lost of DAT was asymmetric in 18 and symmetric in 22). In all cases of "extended" parkinsonism an a neurodegeneration was 10/07/ / 6

81 Idiopathic Normal Pressure Hydrocephalus (inph), parkinsonism and dementia: improving the accuracy of diagnosis and the patient care to reverse the symptomatology Neurodegeneration, Phenotypes and Outcome Measures RF Pacchetti Claudio Clinical health care research/clinicoassistenziale Fondazione Istituto Neurologico Casimiro Mondino confirmed by DAT SCAN ; in 10 cases with isolated lower limbs parkinsonism DAT SCAN was negative. Extensive cognitive impairment was found in different degree in all cases and overt dementia in 4 of them. Twenty inph patients were undergone to ventriculoperitoneal shunt (VPS). 14/20 were positive to DAT SCAN. In all patients VPS improved gait disorders at 12 months; after 24 months, patients with PD phenotype worsened. In these latter unlike before, levodopa was later efficacious. There is only one study with a few cases that demonstrates the existence of a postsynaptic damage in the inph (reduction of D2 receptors in the presence of presynaptic activity preserved), carried out by means of SPECT with presynaptic markers (ligands of the dopamine transporter) and postsynaptic (receptor ligands D2). The DAT SCAN -[123I] FP-CIT-SPECT (using a presynaptic ligand, the 123Ioflupane)- represents the main and most widely used diagnostic tool in detecting PD and more generally the degenerative parkinsonism. Materials and Methods Retrospective and longitudinal study; Subjects: those with brain MRI or TC scan suggestive of inph regardless of the clinical picture presented; They will be enrolled from Radiology, Neurology, Neurosurgery and Geriatric Department. Instrumental Methods: RMN a) ventricles symmetrically dilated, out of proportion to sulcal enlargement b)free communication between the ventricular system and subarachnoid space or CT scan a) Evans index> 0.3 b) DATSCAN: patients with inph will undergo DAT SCAN Clinical evaluation: a detailed history, neurologic examination, neuropsychological screening and "ad hoc" questionnaire will be applied for the identification of comorbidities and phenotypes. All patients DAT SCAN positive will be treated with levodopa for 3 months to evaluate their motor response inph subjects treated with VPS or less will be subject to clinical monitoring at 6, 12 and 18 months Those treated with VPS repeated DAT SCAN after 12 months Impact and Translational Implications Many elderly having dementia and parkinsonism possess imaging features of inph. Highlightening the neurodegenerative process underlying inph and its multifaceted clinical presentations, will be useful for improving clinical management and the outcome measures. Improve the diagnostic accuracy will hasten the patient care. This early management could allow to reverse the neurological symptomology. Enhancing the process of care will reduce the costs for the SSN. 10/07/ / 6

82 RF Multicenter phase II clinical study of adoptive immunotherapy with alloreactive NK cells as consolidation strategy for elderly acute myeloid leukemia patients Curti Antonio Clinical health care research/clinicoassistenziale 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 acute myeloid leukemia natural killer cells 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/Lavoratory Role in the project 1 Emilia-Romagna U.O. Ematologia, Dipartimento di Ematologia, Oncologia e Medicina di laboratorio 2 Azienda ospedalierouniversitaria, IRCCS San Martino-IST, Genova 3 Azienda ospedaliera di Perugia, Os. Santa Maria della Misericordia U.O Ematologia U.O. Ematologia, Dipartimento di Medicina, Divisione di Ematologia e Immunologia clinica Principal investigator Co-investigator Co-Investigator Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Lemoli Roberto Massimo Azienda ospedalierouniversitaria, IRCCS San Martino-IST, Genova 2 Velardi Andrea Azienda ospedaliera di Perugia, Os. Santa Maria della Misericordia Full Professor in Hematology, dedicated in the project to patients enrollment and clinical care Ordinary Professor in Hematology, dedicated in the project to patients enrollment and clinical care, biological studies 29/05/ /02/ /07/ / 6

83 RF Multicenter phase II clinical study of adoptive immunotherapy with alloreactive NK cells as consolidation strategy for elderly acute myeloid leukemia patients Curti Antonio Clinical health care research/clinicoassistenziale Emilia-Romagna Background and Significance Acute myeloid leukemia (AML) treatment in adult patients is based on intensive chemotherapy. Complete remission (CR) rate after chemotherapy ranges from 60 to 85%, but the overall survival (OS) is 40% in patients younger than 60 years, and falls to 10% in those older than 60 years. The main reason for such dismal clinical outcome is that many patients, who achieve CR after chemotherapy, still harbor a minimal residual disease (MRD), which eventually leads to relapse and progression, being resistant to further pharmacological treatments. MRD treatment is allogeneic stem cell transplantation (SCT), but such approach has several limitations, especially for over 60-years old patients. Therefore, new strategies addressing relapse prevention, especially in patients not eligible for allogeneic SCT, are highly warranted. Natural killer (NK) cells play a role in the immune control of tumors. They express activating and inhibitory receptors which recognize MHC class I alleles, termed "Killer cell Immunoglobulin-like Receptors" (KIRs). Data from haploidentical T-cell depleted SCT show that alloreactive KIR-L mismatched NK cells are major anti-leukemia effectors, resulting in the protection of AML patients from relapse. Based on these data, we and others have conducted pilot studies of adoptive immunotherapy with NK cells outside the transplant setting. These studies clearly demonstrate the safety and feasibility of such approach with some promising clinical responses. Specific aims Aim 1: Aim 2: Aim 3: To assess the relapse-free survival of AML patients, not eligible for SCT, who undergo adoptive immunotherapy with alloreactive NK cells after the achievement of CR with induction/consolidation chemotherapy. To assess the OS of AML patients infused with alloreactive NK cells. To assess the predictive impact of donor NK cell repertoire, i.e. the frequency of donor alloreactive NK cell clones, on the clinical outcome of AML patients infused with alloreactive NK cells Hypothesis: Based on published reports and our preliminary data (see below), we hypothesize that alloreactive NK cell infusion, which has been already demonstrated to exert significant anti-leukemia effect, may be of clinical benefit in reducing disease recurrence for elderly AML patients in CR. Preliminary data: Our group published the results of a monocentric phase I study of adoptive immunotherapy with highly purified haploidentical NK cells in adult AML patients with relapsing/refractory disease. No specific NK cell-related toxicity was observed (Curti et al, Blood, 2011). Donor-versus-recipient alloreactive NK cells were demonstrated in vivo by the detection of donor-derived NK clones and adoptively transferred NK cells were alloreactive against recipient's leukemic cells. Subsequently, we extended our approach to a cohort of elderly AML patients, who achieved CR after induction/consolidation chemotherapy, as part of consolidation program (NCT ; manuscript in preparation). Seventeen AML patients, in first morphological CR (3 patients showed molecular disease) (median age 64 years, range 53-73) received highly purified CD56+CD3- NK cells from haploidentical KIR-L mismatched donors after fludarabine/cyclophosphamide immunosuppressive chemotherapy, followed by interleukin-(il) 2. Myelosuppression was moderate although 1 patient died due to overwhelming bacterial pneumonia and was censored for clinical follow-up. With a median follow-up of 22.5 months (range, 6-68 months), 9/16 evaluable patients (56%) are alive disease-free, whereas 7/16 (44%) relapsed with a median time to relapse of 9 months (range, 3-51 months). Among relapsed patients, 2 individuals showed a very prolonged CR phase of 24 and 51 months, respectively, in absence of any concomitant anti-leukemia treatment. Three patients, treated with molecular disease, achieved molecular CR lasting 9 and 4 months in 2 cases and 8+ months in the third patient. Adoptively transferred NK cells were alloreactive against recipient's cells, including 08/07/ / 6

84 RF Multicenter phase II clinical study of adoptive immunotherapy with alloreactive NK cells as consolidation strategy for elderly acute myeloid leukemia patients Curti Antonio Clinical health care research/clinicoassistenziale Emilia-Romagna leukemia. More importantly, higher frequencies of NK alloreactive clones in the donors statistically correlated with better clinical outcome. Materials and Methods This is a multicentric, prospective, phase IIb study. Adult AML patients in CR after standard chemotherapy, except those with low-risk disease in molecular CR, not candidates to allogeneic SCT, are eligible. Using a genetic randomization through a 'donor' vs 'no donor' approach, patients will undergo NK cell infusion or followed-up without treatment. Donorrecipients pairs will be selected on the basis of known KIR ligands. NK cells will be obtained from a haploidentical KIR-L mismatched donor. Immunomagnetic purification of NK cells will consist in depletion of CD3+ T cells followed by positive selection of CD56+ NK cells (CliniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany). Treatment before NK cell infusion will include fludarabine 125 mg/m2 and cyclophosphamide 4g/m2. IL-2 will be administered after NK cell infusion. Correlative biological studies will be performed. Accordingly to sample size analysis, 40 patients will be assigned to each arm in a time frame of 30 months. Impact and Translational Implications The incidence of AML increases with age. In patients older than 60 years, clinical outcome is very poor due to the high incidence of relapse also in patients who achieve CR after chemotherapy. Indeed, these patients, who are mostly not eligible for allogeneic SCT, lack a post-cr consolidation programme. In this context, the use of the proposed NK-cell based consolidation strategy may increase the number of patients who maintain CR and thus favourably impact on OS of elderly patients with AML. 08/07/ / 6

85 Role of environment-gene interaction in etiology and promotion of pituitary tumours. BANDO 2013 Progetti Ordinari RF RF Cannavò Salvatore Biomedical/Biomedica Sicilia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology 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: Regulation of cell growth and differentiation by steroid/polypeptide hormones and growth factors. Xenobiotic and pituitary tumorigenesis Gene-environment interaction 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/Lavoratory Role in the project 1 Sicilia UOC DI ENDOCRINOLOGIA, AOU POLICLINICO GAETANO MARTINO, MESSINA IDEATION OF THE PROJECT, COORDINATION, MOLECULAR STUDIES 2 UNIVERSITY OF MESSINA DEPARTMENT OF NEUROSCIENCES COLLECTION OF TISSUE SAMPLES,, STORAGE, GENTYPE - TUMOUR CORRELATIONS, EPIGENETICS AND 3 IRCCS CENTRO NEUROLESI BONINO DIREZIONE SCIENTIFICA Investigators, Institution and Role in the Project IMAGING, ANALYSIS OF RESULTS, GENOTYPE - CLINICAL CHARACTERISTICS CORRELATIONS Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Esposito Felice UNIVERSITY OF MESSINA COLLECTION OF TISSUE SAMPLES 14/03/ Pisani Laura Rosa IRCCS CENTRO NEUROLESI BONINO PULEJO IMAGING, ANALYSIS OF RESULTS, GENOTYPE - CLINICAL CHARACTERISTICS CORRELATIONS 3 Angileri Filippo Flavio UNIVERSITY OF MESSINA TISSUE SAMPLE STORAGE AND GENOTYPE - TUMOUR CHARACTERISTICS CORRELATION 4 Aguennouz M'Hamed UNIVERSITY OF MESSINA EPIGENOMICS AND PROTEOMICS STUDIES 03/09/ /10/ /07/ /07/ / 5

86 Role of environment-gene interaction in etiology and promotion of pituitary tumours. BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Cannavò Salvatore Sicilia Background and Significance Acromegaly is a disease due to pituitary tumours, frequently invasive and aggressive. We reported that prevalence is increased in an area of the province of Messina, identified as a high-risk zone for environmental crisis (HRZ) by the Ministero dell Ambiente of the Italian Government, in which Agenzia Regionale per la Protezione dell Ambiente proven high atmospheric concentrations of non methanic hydrocarbons (NMHC) and of volatile organic compounds (VOC). NMHC and VOC interact with endocrine system, acting as hormone agonists or antagonists through Estrogen Receptor or Aryl Hydrocarbon Receptor (AHR) pathways. Recently, we showed high frequency of a AHR gene variant in acromegalic patients, associated with pituitary tumour invasiveness and some secondary neoplasms. Moreover, we reported increased IL-22 concentrations in patients with pituitary macroadenomas, and hyper-expressed IL-22 receptors in these tumours. This pro-inflammatory cytokine, regulated by AHR activation in Th17 cells, induces tumour progression and metastasis in animal models. A grant of Department of Scientific Research of Italian Government (PRIN ) supports our preliminary studies concerning the role of xenobiotics in pituitary tumorigenesis. Significance of project is to study genetic predisposition to develop pituitary tumours in HRZ, and of mechanisms through which xenobiotics induce tumorigenesis and pharmacological resistance, by genetic, proteomic and metabolomic approachs Specific aims Aim 1: Aim 2: Aim 3: to evaluate frequency of homozygous and heterozygous variants of AHR and other genes encoding proteins involved in xenobiotic detoxification s mechanisms, in patients with acromegaly to investigate epigenetic effects (methylation, deacethylation) of xenobiotic exposition on genes involved in AHR pathway in tumour and normal pituitary tissues of acromegalic patients and animal models to study mrna and protein (hormones, enzymes) expression related to AHR pathway dysregulation in pituitary tumour tissue of acromegalic patients, by a proteomic approach Hypothesis: Dysregulated xenobiotics detoxification could represent a predisposing factor for development, progression or aggressivity of growth hormone secreting pituitary tumours especially in areas characterized by increased pollutants concentration. In addition, it could affect tumour sensitivity to somatostatin-analogs and/or dopamine agonists, decreasing effectiveness of medical treatment, prolonging duration of active disease and increasing finally mortality risk in acromegalic patients Preliminary data: We recently demonstrated that the prevalence of acromegaly is dramatically increased in a high-risk zone for environmental crisis of province of Messina, in comparison with a wide surrounding area, in which disease prevalence is however related to industrialization (1). Increased risk of developing pituitary tumours has been demonstrated also by other authors in the Seveso area, where an industrial incident caused diossine pollution in 1977 (2). Study in vitro and in animals showed that several endocrine disruptors induce abnormal hormone secretion and tumour progression in the pituitary gland, especially through the involvement of Aryl Hydrocarbon Receptor pathway (3-5). The frequency of a heterozygous or homozygous variant of AHR gene, which causes potential dysregulation of hydrocarbons or digoxin detoxification in human and animal pituitary tumour tissue, is increased in patients with acromegaly, and the occurrence of this variant is associated with increased invasiveness of pituitary tumours and occurrence of secondary tumours in acromegalic patients (2). Moreover, in the immune system, xenobiotic activation of AHR pathway induces IL-22 secretion by Th17 cells. A role is postulated in breast and colon tumorigenesis. We showed that serum IL-22 concentration is increased in patients with PRL-secreting and non functioning pituitary tumours, and that IL-22 receptors are hyper-expressed in these tumours in comparison with normal pituitary tissue 04/07/ / 5

87 Role of environment-gene interaction in etiology and promotion of pituitary tumours. BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Cannavò Salvatore Sicilia Materials and Methods An archival series of >200 lymphocitic DNA of acromegalics from different Italian areas, including HRZs, is available. Clinical and biochemical patients' characteristics are recorded in a dedicated database. AHR and AHR Interacting Protein (AIP) has been genotyped in all cases. DHPLC screening analysis of genes encoding for other proteins of AHR pathway will detect variants characterized by DNA sequencing methods. Functional significance of variants will be evaluated by in silico methods. Epigenetic and proteomic studies will be performed in tumour and normal pituitary tissue from patients undergone neurosurgery for pituitary tumours. Epigenetic studies will evaluate DNA methylation and deacethylation and mirna expression of AHR pathway genes. Proteomic studies will be based on bidimentional electrophoresis and spectrometry MALDI-TOF analysis. Results of epigenetic and proteomic studies and patients characteristics will be related to xenobiotics exposition Impact and Translational Implications We will investigate the role of variants of genes of AHR pathway to develop pituitary tumors causing acromegaly and in treatment effectiveness, in subjects exposed to xenobiotics. Acromegaly is considered a rare disease due to an estimated prevalence of about 125 cases per million of inhabitants, but a not negligible number of patients is diagnosed even years after the onset of the disorder or post-mortem. According with recent investigations the true prevalence could be 5-6 times higher

88 RF Cell-type and subunit specific alterations of NMDA receptors in striatum at early stages of Parkinson's disease Calabresi Paolo 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: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Parkinson s disease and other movement disorders (Huntington s, Dystonias, Ataxias). Project Keyword 2: 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/Lavoratory Role in the project 1 Fondazione Santa Lucia Laboratory of Neurophysiology Operative Unit 2 Hospital at the University of Perugia Department of Medicine, Neurological clinic, Lab of Experimental neurology Operative Unit 3 University of Milan Department of Pharmacological and biomolecular sciences Operative Unit Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Di Filippo Massimiliano Hospital at the University of Perugia Unit coordinator 15/01/ Gardoni Fabrizio University of Milan Unit coordinator 25/12/ /07/ / 6

89 RF Cell-type and subunit specific alterations of NMDA receptors in striatum at early stages of Parkinson's disease Calabresi Paolo Biomedical/Biomedica Fondazione Santa Lucia Background and Significance Parkinson's disease (PD) is characterized by massive degeneration of dopamine (DA) neurons of the substantia nigra, dramatic motor and cognitive alterations, and presence of nigral Lewy bodies. Although the exact threshold of DA denervation producing early motor and cognitive symptoms has never been accurately established in PD patients, current hypothesis indicates that loss of ~70% of nigrostriatal DA neurons is necessary for detecting clinical signs. Moreover, the synaptic striatal mechanisms underlying behavioral and motor effects in the early phases of the disease are still mostly unknown. Current knowledge indicates that the strength of glutamatergic signals from the cortex to the striatum is regulated during progression of the disease. Striatal synaptic plasticity is critically controlled by nigral projections influencing endogenous striatal DA levels. Accordingly, characterizing the early synaptic dysfunctions in striatal medium spiny neurons (MSNs) and cholinergic interneurons (ChIs), and their influence on motor behaviour represent a key step for a full comprehension of PD pathogenesis and for setting-up early pharmacological intervention targeting glutamatergic synapse. Even if modifications of NMDA glutamate receptor (NMDAR) function in striatal synapses represent a key element in PD pathogenesis, the specific role of molecular and functional alterations of NMDAR regulatory subunits at MSNs and ChIs at early stages of PD remains to be addressed. Specific aims Aim 1: Aim 2: A molecular, electrophysiological and behavioural approach will be used to investigate and identify molecular mechanisms responsible for striatal cell-type and subunit specific alterations of NMDA receptors at early stages of PD. We will characterize the relevance of these molecular alterations in striatum on motor abnormalities at the early stage of PD. In particular, we will aim at identifying similarities and/or differences in the mechanisms affecting NMDAR subunits function in a model of early PD to identify new possible pharmacological target for initial stages of the disease. Aim 3:. Hypothesis: At the early stages of PD striatal neurons present cell-type and subunit specific alterations of NMDAR functions. Targeting the altered NMDAR subunit composition with specific pharmacological tools and unravelling molecular pathways regulating the expression and synaptic function of NMDAR subunits in MSNs and ChIs will be of pivotal importance for defining novel therapeutic approaches for the treatment of PD at the early stage of the disease. Preliminary data: We found that an increase of synaptic GluN2A- in MSNs and GluN2D-containing NMDARs in ChIs, represents early molecular alterations that could play a crucial role in PD pathogenesis. We demonstrated that alterations of NMDAR GluN2A/GluN2B ratio at MSNs synapses at different stages of DA denervation correlate with motor behaviour abnormalities observed in rat models of PD (Mellone and Gardoni, 2013). We confirmed the increased GluN2A/GluN2B ratio at synapses in the striatum of MPTP-parkinsonian monkeys as well as in tissue from PD patients. GluN2A and GluN2B subunits interact with scaffolding proteins; however, the mechanisms responsible for the specific anchoring of GluN2A-containing NMDARs at synapses remain unknown. We identified Rabphilin3A (Rph3A) as a new molecule interacting with GluN2A regulating its localization at synapses. Notably, a role for Rph3A in PD has been suggested (Chung et al., 2009). Accordingly, we found increased postsynaptic levels of Rph3A in 6-OHDA-lesioned parkinsonian rats. GluN2D-containing NMDARs, enriched in ChIs (Bloomfield et al., 2007), contribute to striatal NMDAR-dependent control of neurotransmitter release (Zhang et al., 2014). Accordingly, we found 08/07/ / 6

90 RF Cell-type and subunit specific alterations of NMDA receptors in striatum at early stages of Parkinson's disease Calabresi Paolo Biomedical/Biomedica Fondazione Santa Lucia increased expression of GluN2D in two early PD models, rats injected in striatum with AAV-alphasynuclein and partial 6-OHDA-lesioned rats. Finally, we investigated the contribution of GluN2D-containing NMDARs in the striatal LTP induction performing patch-clamp recordings of both ChIs and MSNs in slice preparations. We found that in ChIs the evoked excitatory postsynaptic current (EPSC) was dependent on the GluN2D-containing NMDARs since the selective GluN2D antagonist QNZ-46 was able to reduce the EPSC amplitude and to block LTP. Conversely, in MSNs QNZ-46 failed to affect both the EPSC amplitude and LTP suggesting that the GluN2D might play a role in ChIs but not in MSNs physiology. Materials and Methods A molecular, electrophysiological and behavioural approach will be used. Parkinsonian rats will be obtained by unilateral nigral injection of 6-OHDA or AAV-alpha-synuclein. Motor tests (stepping, cylinder) and active avoidance test will be used for behavioural analysis. MSNs and ChIs will be studied in striatal slices by patch-clamp recordings. Membrane properties, NMDAR-mediated EPSCs and synaptic plasticity will be analysed using conventional striatal electrical stimulation and an optogenetic approach. WB and co-immunoprecipitation assays will be used to measure NMDAR subunits and interaction with binding proteins and post-translational modifications; a biochemical approach will be used to purify striatal PSD. Confocal imaging will be used to evaluate GluN2 subunits in MSNs and ChIs. Freshly synthesised human alpha-synuclein will be used for acute treatments. Molecular studies will be validated in post-mortem brains of PD patients. Impact and Translational Implications PD exacts an enormous emotional toll affecting all aspects of life of people leaving with the pathology; the burden for patients, families and society is huge its cost being estimated at 11 billion euro annually. Thus, detecting novel targets characterizing the early stages of the disease and establishing innovative, cell-specific pharmacological tools for interventions on PD patients would have dramatic impact for precocious PD treatment opening to new perspectives for PD treatment. 08/07/ / 6

91 RF Development of a nanoparticle-based pharmacological approach for amyotrophic lateral sclerosis Bigini Paolo Biomedical/Biomedica Istituto di Ricerche Farmacologiche Mario Negri 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. Pharmacology Nanomedicine 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/Lavoratory Role in the project 1 Istituto di Ricerche Farmacologiche Mario Negri 2 Università degli Studi di Padova Biochemistry and Molecular The Principal Investigator (UO1 Leader) will be Pharmacology/Biochemistry and protein involved in the biological studies (in vitro studies, chemistry pharmacokinetics, in vivo studies). In addition he will coordinate the whole project in tight collaboration with the other operative units and the key personnel. Pharmaceutical Sciences The Researcher Collaborator (UO2 Leader) will be involved in the preparation and optimization of nanodrugs. 08/07/ / 7

92 RF Development of a nanoparticle-based pharmacological approach for amyotrophic lateral sclerosis Bigini Paolo Biomedical/Biomedica Investigators, Institution and Role in the Project Istituto di Ricerche Farmacologiche Mario Negri Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Morpurgo Margherita Università degli Studi di Padova Researcher Collaborator. Dr Morpurgo will be in charge of the synthesis, functionalization, characterization of ANANAS. She will also coordinate the experiments to optimize the loading and the controlled release of the MM /10/ Bonetto Valentina Istituto di Ricerche Farmacologiche Mario Negri Researcher Collaborator. Dr Valentina Bonetto has a large experience in the field of cyclophilin A and in the studies of preclinical models of ALS. In the present project she will collaborate with the PI to evaluate the functional role of the nanotherapy both by in vitro and in vivo analyses. 13/04/1969 Background and Significance Cyclophilin A (CypA) is a multifunctional protein with peptidyl-prolyl cis-trans isomerase (PPIase) activity which is pharmacologically inhibited by the immunosuppressive drug cyclosporin A (CsA). In pathological conditions CypA is aberrantly secreted extracellularly, where it has a proinflammatory cytokine-like behavior and induces MMP-9 by interacting with its EMMPRIN receptor. We collected several experimental evidences indicating that inhibiting specifically extracellular CypA (ecypa) is a promising therapeutic target for amyotrophic lateral sclerosis (ALS). In particular, we found that a potent CypA inhibitor MM-218, a non-immunosuppressive cell-impermeable CsA analog, both reduces the death of motor neurons in an in vitro paradigm of ALS and increases the survival of the transgenic SOD1G93A mice. Since MM-218 does not cross the blood-brain-barrier (BBB), it was directly administered into lateral ventricles. This way of administration is not easily translated into clinical practice therefore alternative approaches have to be investigated. The present study aims to develop an innovative nanocarrier to boost the delivery of the specific ecypa inhibitor MM-218 to the central nervous system. The nanocarrier Avidin-Nucleic-Nano-ASsemblies (ANANAS), recently developed by our team, is highly biocompatible, biodegradable and particularly convenient for target delivery. The success of this project will provide the basis for a prompt translation to clinical application. Specific aims Aim 1: Aim 2: Aim 3: Development of an ANANAS-based nanocarrier with enhanced BBB penetration. To do that ANANAS functionalization with an apolipoprotein E-derived peptide and anti-trasferrin receptor monoclonal antibodies will be investigated and the best formulation will be chosen for efficacy studies. Development of a nanodrug for ALS. Nanoparticles loaded with the CypA inhibitor MM-218, modified to allow nanoparticle loading and controlled release, will be tested preliminarily in vitro and then in vivo for efficacy, biodistribution and pharmacokinetics. An important outcome measure in vitro and in vivo will be the level of MMP- 9 before and after treatment. In vivo pharmacological study of the best candidate nanodrug in the SOD1G93A transgenic mice. The nanodrug and relative controls will be administered to transgenic mice starting at the onset of symptoms. Doses and treatment regimen will be set considering the previuos pre-clinical trial with MM-218, the 08/07/ / 7

93 RF Development of a nanoparticle-based pharmacological approach for amyotrophic lateral sclerosis Bigini Paolo Biomedical/Biomedica Istituto di Ricerche Farmacologiche Mario Negri pharmacokinetics/biodistribution of the nanodrug in vivo, and the pharmacological activity in vitro. The effect of the treatment will be evaluated by assessing disease progression, survival and histopathological alterations in the spinal cord of the animals. Hypothesis: Under pathological conditions, CypA has a proinflammatory cytokine-like behavior and increases MMP-9 through its EMMPRIN receptor. We hypothesized that MM-218, by inhibiting specifically extracellular CypA, could block toxic downstream molecules such as MMP-9 and its associated pathways, and therefore represent an efficient therapeutic approach for ALS. Intracerebroventricular administration of MM-218 in SOD1G93A mice led to a significant increase of life expectancy. This result strongly supports our working hypothesis and paves the way for the development of a nanocarrier-dependent systemic administration of MM-218 to enhance its blood brain barrier passage and allow repeated administration in a safer and easier way targeted to future translational application from mice to patients. Preliminary data: 1) ALS models: We performed efficacy studies with MM-218 in an in vitro model of ALS, astrocytespinal neuron co-cultures expressing SOD1G93A derived from the SOD1G93A mice, and found that MM-218 rescued motor neuron death at subnanomolar concentrations and is able to downregulate MMP-9. We then performed a preclinical trail with SOD1G93A mice treated with MM-218 by continuous infusion into lateral ventricles and found that MM-218 at 10 microm (in the minipump) significantly increased the mean survival duration after the treatment by 18%. 2) Nanoparticles: The bio-distribution, pharmacokinetics, immunogenicity and stability of ANANAS has been recently investigated in healthy mice. The main outcomes emerging from this study are: a) circulating nanoparticles remain for a long time in the bloodstream; b) they efficiently migrate to organ parenchyma; c) they do not induce immunological reaction after repeated administrations. 08/07/ / 7

94 RF Development of a nanoparticle-based pharmacological approach for amyotrophic lateral sclerosis Bigini Paolo Biomedical/Biomedica Istituto di Ricerche Farmacologiche Mario Negri Materials and Methods 1. Synthesis of biotin-linked MM-218 analogs 2. In vitro assay for PPIase activity of the analogs 3. Synthesis of biotinylated ApoE peptide and biotinylation of anti-trasferrin receptor antibodies 4. Assembly of ANANAS with the BBB transport mediators 5. Uptake studies of CNS-ANANAS formulations in hcmec/d3 cells and in vivo. 6. Conjugation of the best CNS-ANANAS formulation with the MM-218 analogs 7. In vitro MM218 release kinetics from ANANAS in physiological buffers 8. In vivo NP-biodistribution by Fluorescent Molecular Tomography 9. Longitudinal ex-vivo experiments 10. In vitro pharmacological test of biotin-mm-218 analogs tethered or not to CNS-ANANAS 11. Toxicity of the nanodrugs in vitro 12. Analysis of the MMP-9 levels in tissues of mice after administration of the nanodrug 13. Motor neuron survival assay 14. Preparation of astrocyte-spinal neuron co-cultures and treatments 15. MMP-9 analysis 16. Mouse treatments and behavioural trial 17. Histopathology Impact and Translational Implications Despite the considerable number of clinical trials for ALS no treatment has so far been identified, partly because of the lack of efficient methods to deliver drugs into CNS. We aim to develop innovative nanoparticles loaded with MM-218, a drug shown to be effective in a mouse model of ALS. This project if successful will provide the rationale for a pilot clinical trial in ALS patients. Nanocarriers with enhanced BBB penetration would be of interest also for other neurodegenerative diseases. 08/07/ / 7

95 RF Neuroplasticity and Alzheimer's disease: integrated approach to identify biological and neurophysiological markers. Miniussi Carlo Biomedical/Biomedica Centro San Giovanni di Dio Fatebenefratelli 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. Alzheimer's disease Neurophysiological biomarkers 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/Lavoratory Role in the project 1 Centro San Giovanni di Dio Fatebenefratelli 2 Centro San Giovanni di Dio Fatebenefratelli 3 Catholic University School of Medicine Cognitive Neuroscience Section Genetic Laboratory Institute of Human Physiology Neurophysiology on human subjects: non invasive brain stimulation, neuromodulation and TMS-EEG recording Transcriptomic analysis (RNA-seq, real time PCR) in human and animal tissues. Biological profile/phenotype correlations Study of cortical excitability, neuroplasticity and behavior in animal models subjected to tes Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Gennarelli Massimo Centro San Giovanni di Dio Fatebenefratelli 2 Grassi Claudio Catholic University School of Medicine 3 Fracassi Claudia IRCCS Centro San Giovanni di Dio Fatebenefratelli; Research assistant 4 Podda Maria Vittoria Catholic University School of Medicine, Institute of Human Physiology, Assistant Professor Principal Investigator of Unit 2 27/02/1960 Principal Investigator of Unit 3 17/12/1956 Neurophysiological recording and data analyses in humans Electrophysiological experiments in animal models 02/10/ /05/ /07/ / 6

96 RF Neuroplasticity and Alzheimer's disease: integrated approach to identify biological and neurophysiological markers. Miniussi Carlo Biomedical/Biomedica Centro San Giovanni di Dio Fatebenefratelli Background and Significance A growing body of evidence suggests that synaptic dysfunction plays a critical role in the pathophysiology of Alzheimer's disease (AD) (Morrison & Baxter, 2012), the reason is that synaptic failure is an early event in the pathogenesis and that it might be already detectable in subjects in a prodromal state of AD. These changes underlie cognitive impairments expressed, first, as a slowing down and then as an inability to encode new information, progressing afterward to a complete impairment of other cognitive functions. This dynamic process starts from an initial presymptomatic and possibly reversibly, functionally responsive stage of down-regulation of synaptic functions; moving to stages irreversibly associated with degeneration (Arendt, 2009). Many researchers believe that future treatments to slow or stop the progression of AD and preserve brain function will be most effective when administered during the preclinical stages of the disease. Therefore, there is growing interest in the identification of individuals in the earliest symptomatic, as well as presymptomatic, stages of disease, because in this population new therapies may have the greatest chance of success. Through a translational approach including genetic, molecular and neurophysiological analyses, we expect to identify novel biomarkers associated with altered neuroplasticity, which are potentially useful for early diagnosis and prognosis assessment of AD. Specific aims Aim 1: Aim 2: Aim 3: To determine how transcranial electrical and magnetic stimulation (tes, TMS), that probes the reactivity of the brain and its functions a) modifies cognitive performance and b) affects connectivity and excitability of a cortical network in patients at different stages of AD. To characterize the effects of tes in a mouse model subjected to stimulation protocols similar to those applied to humans by: a) investigating the tes-induced changes in cortical excitability and neuroplasticity; b) identifying the underlying molecular mechanisms; c) evaluating behavioral correlates of tes-induced plasticity modulation. To verify the effectiveness of pre-selected putative biomarkers of cognitive impairment by evaluating their expression levels in brain and in blood of the animal model in conjunction with neurophysiological data. To identify new potential peripheral biomarkers correlated with neuroplasticity by studying the whole transcriptomic profile in brain and blood of the animal model (with RNA-seq analysis) and subsequently check if the same correlations also occur in humans. Hypothesis: The premise is that by modifying cortical activity through tes we will be able to test the neuroplastic capability of the cortical networks in subjects genetically characterized, thus unveiling any subthreshold functional alterations of the system. Studies on animal models will parallel patient studies, where we aspect to confirm the key role of some genes associated with neuroplasticity and AD progression, and show how in the animal model such expression levels are reflected in blood tissues. Subsequently, we will identify the same variations that occur in humans by corroborating the match between transcription profiles and neuroplasticity information. Such information will reveal potential new biomarkers that can be tested in human leukocyte as a proxy of the impairment of plasticity. Preliminary data: OU1 demonstrated that is possible to investigate cortical plasticity changes induced by tes combining TMS-EEG. These data show increased cortical excitability after anodal tes and a decreased excitability after cathodal tes. Moreover OU1 has also shown how TMS-EEG can provide dynamic measures of the response of the brain convergence and how graph theory models will provide an excellent method of mapping effective cortical connectivity in a network and its relationship with cognitive functioning. OU2 has already stratified a group of persons affected by AD and MCI according with genetic and neurobiological biomarkers. The classification is consistent with the rationale behind the project which is the subdivision of the human subjects in high and low risk for AD development. OU3 showed that synaptic plasticity, assessed by LTP protocols in brain slices of mice exposed to 10/07/ / 6

97 RF Neuroplasticity and Alzheimer's disease: integrated approach to identify biological and neurophysiological markers. Miniussi Carlo Biomedical/Biomedica Centro San Giovanni di Dio Fatebenefratelli anodal tes, was increased vs. reduced in cathodal tes. Enhanced plasticity was associated with increased expression of genes critical for performances in behavioral tests. Materials and Methods We will study will 3 groups of humans (AD, MCI, healthy controls: total 120) stratified in 2 groups (high risk and low risk for AD) on the basis of an algorithm integrating genetic and peripheral biomarkers data (CSF, plasma, serum, PBMC). Measures of behavioral performance and cortical plasticity will be collected before, during and after tes. TMS-EEG will be used to evaluate cortical reactivity and connectivity. Graph theoretical analysis based on EEG recordings will be used to investigate whether and how tes-induced changes modulate effective connectivity. OU3 will perform parallel experiments in animal models by using tes, behavioral protocols, basic neurophysiological and molecular approaches. Mice (3xTg-AD) will be tested at different ages to match conditions in humans. We will compare the whole transcriptome in brain and blood cells on mice presenting extreme phenotypes in term of neurophysiological data. Identified new biomarkers will be then tested in human leukocyte. Impact and Translational Implications Study of cortical plasticity in response to tes and correlation of the changes observed in groups of patients at different stages of AD with biomarkers identified in animal models and subsequently validated in humans will provide us with crucial novel information for early diagnosis and prognosis of AD. The future horizons of this proposal also include the application of these methods to patients with aetiologies different from AD (e.g., stroke) also for plasticity induction treatments. 10/07/ / 6

98 Subthalamic nucleus deep brain stimulation in Tourette syndrome BANDO 2013 Progetti Ordinari RF RF Albanese Alberto 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: Parkinson s disease and other movement disorders (Huntington s, Dystonias, Ataxias). Tourette syndrome Subthalamic nucleus 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/Lavoratory Role in the project 1 Fondazione Istituto Neurologico Carlo Besta 2 Fondazione IRCCS Ca' Granda Neurologia I Centro Clinico per la Neurostimolazione, le Neurotecnologie ed i Disordini del Movimento Selection of patients, motor cognitive and psychiatric evaluations. Implantation of STN electrodes. Local fields potentials recording and acquisition. Setup of experimenta behavioral tests. Technological support. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Priori Alberto Fondazione IRCCS Ca' Granda Electrophysiological recordings 19/11/ Romito Luigi Michele Patient selection and evaluation 11/07/ /07/ / 6

99 Subthalamic nucleus deep brain stimulation in Tourette syndrome BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale Albanese Alberto Fondazione Istituto Neurologico Carlo Besta Background and Significance Tourette syndrome (TS) is characterized by the chronic presence of tics since childhood/adolescence not necessarily associated to echolalia or coprolalia [1]. Most TS patients fulfill diagnostic criteria for comorbid psychiatric disorders, including obsessive compulsive disorder (OCD), attention deficit and hyperactivity disorder (ADHD), affective disorders and impulse control disorders [2, 3]. It is reckoned that a meaningful proportion of TS patients do not have adequate benefit with drug treatment and the majority of patients have side effects of variable severity [4]. Deep brain stimulation (DBS) trials have been performed in patients with severe TS [5]: most studies published to date are case reports or case series reporting varying selection criteria, stimulation targets and assessment protocols. The most commonly used brain targets have been the centromedian-parafascicular nuclear complex (CM-Pf) [6-8], the anterior capsule in proximity of the nucleus accumbens [9], the nucleus accumbens itself [10] and the globus pallidus internus (GPi) [11, 12]. There is no consensus on the ideal target for treating TS with DBS, because the targets so far chosen have provided inconsistent outcomes on tics and behavioral features [13, 14]. This research project aims at evaluating the safety and efficacy of subthalamic nucleus (STN) DBS on motor and behavioral features of TS patients, by considering homogenous patient subgroups [15]. Specific aims Aim 1: Aim 2: Aim 3: To evaluate efficacy and safety of STN DBS on tics and behavioral features in patients with TS To correlate outcome with the physiological status of STN (recording local fields potentials) and activation of different brain regions (metabolic PET scans) To define the most appropriate stimulation settings for STN DBS in TS Hypothesis: Control of motor and behavioral dysfunctions depends on a network activity rather than on single anatomical structures. Treating with DBS a motor and behavioral disorder, such as TS, requires finding the target with a strategic location along basal ganglia networks. Previous targets have proven not to control appropriately the dual presentation of TS. In addition to the well-known role of STN in motor control, recent data have shown that the STN is characterized by a strategic role in controlling behavioral phenomena and particularly obsessive-compulsive behavior [16]. We hypothesize that patients with severe TS may have motor and behavioral benefit by STN DBS. We plan to study two subgroups of TS patients, those with pure motor phenomenology and another group with prevalent obsessive-compulsive phenomenology. Preliminary data: We have recently performed STN DBS in two patients with severe medication-refractory motor TS, who were followed for 9 and two months respectively (unpublished observation). In both patients, we observed a significant improvement of the Yale Tic Global Severity Scale (YTGSS) [32], by 78% and 81%, respectively. No adverse events were recorded. STN DBS is a well-established procedure in our center, commonly performed on patient with PD. In this case, both TS patients received chronic bilateral STN stimulation, similarly to PD patients. Observation of these pivotal treatment is still ongoing. Previous recordings of LFPs from the STN in patients with PD suggest that this brain structure may have a role in impulsivity and decision making processes [33]. Following the analysis of data obtained from neurophysiological recordings in nine patients with severe TS [34, 35] who underwent Vo/CM-Pf or nucleus accumbens [36] DBS, we found specific oscillatory patterns of LFPs that may support the idea that pathological activity in the entire cortico-basal ganglia thalamic network could determine the main features of TS. 11/07/ / 6

100 Subthalamic nucleus deep brain stimulation in Tourette syndrome BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale Albanese Alberto Fondazione Istituto Neurologico Carlo Besta Materials and Methods This study will be organized as a randomized, double-blind, parallel-arms, research project involving 10 adult patients with a diagnosis of TS based on current criteria [1] and a severity of YTGSS > 35/50 at baseline. Patient selection will consider the more recent recommendations [7, 8]. Candidates for STN DBS will be evaluated by a multidisciplinary team. The patients will be characterized and evaluated before and after surgery at regular intervals. The project will be carried out at the Carlo Besta Institute. Three days after surgery, LFPs will be recorded from STN while patients perform motor and cognitive tasks in two clinical conditions: STN DBS turned off or on. Primary outcome measure will be variations of the score of the YTGSS [37] and neuropsychological rating scales, including the Y-BOCS [38]. A PET study will be performed before and 3 months after switching on STN DBS to correlate the changes of TS features with variations in activation of specific brain regions. Impact and Translational Implications The effects of STN DBS on TS phenomenology is still unknown. We will investigate the role of STN functional modulation by DBS on TS phenomenology, and collect new information on the pathophysiology of TS. This study may have impact on the current indications for DBS in TS patients, broadening the spectrum of therapeutic options. Study of LFPs will highlight the behavioral role of STN and may lead to identify electrophysiological signatures to develop adaptive DBS techniques. 11/07/ / 6

101 RF Are genetic, inflammatory and metabolomic markers of coronary heart disease related to kidney function decline in patients with type 2 diabetes? Menzaghi Claudia Clinical health care research/clinicoassistenziale Ospedale Casa Sollievo della Sofferenza LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular diseases Project Classification IRG: Project Classification SS: Population Sciences and Epidemiology Kidney, Nutrition, Obesity and Diabetes - KNOD Project Keyword 1: Project Keyword 2: Project Keyword 3: Elucidation of the determinants of kidney diseases/conditions, obesity, diabetes, gastro-intestinal conditions, environmental and nutritional influences on health outcomes in human populations in relation to time, place, and personal characteristics. The focus is 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. Genetic and inflammatory markers Metabolomics 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/Lavoratory Role in the project 1 Ospedale Casa Sollievo della Sofferenza 2 Azienda Ospedaliero Universitaria Pisana Research Unit of Diabetes and Endocrine Diseases Department of Clinical and Experimental Medicine Section of Diabetes and Metabolic Diseases Coordinator - Principal investigator Collaborator 3 Consorzio C.A.R.S.O Metabolomics Unit Collaborator Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 PENNO GIUSEPPE Azienda Ospedaliero Universitaria Pisana Patients' data collection and analyses 25/04/ Piccinonna Sara Consorzio C.A.R.S.O Metabolomics study 08/04/ Fontana Andrea Ospedale Casa sollievo della Sofferenza Statistical analyses 10/03/ /07/ / 6

102 RF Are genetic, inflammatory and metabolomic markers of coronary heart disease related to kidney function decline in patients with type 2 diabetes? Menzaghi Claudia Clinical health care research/clinicoassistenziale Ospedale Casa Sollievo della Sofferenza Background and Significance Chronic kidney disease (CKD), mainly indicated by a reduced glomerular filtration rate (GFR) is the leading cause of premature death in patients and type 2 diabetes (T2D). A better understanding of pathogenic mechanisms responsible for GFR decline in such patients is, therefore, urgently needed. In patients with T2D, reduced GFR share a common pathogenic soil with coronary heart disease (CHD), including the deleterious effect played by hyperglycemia, obesity, hypertension, dyslipidemia, low grade inflammation and insulin resistance. Given that the two conditions are under a genetic control, it is reasonable hypothesize they share also a common genetic background. Similarly, based on the same pathogenic and epidemiological background also inflammatory molecules might be common pathogenic risk factors shared by CHD and low GFR. Likewise, serum metabolites may reflect common underlying abnormalities between these two traits, and could serve as novel biomarkers of reduced kidney function. Unfortunately, such hypotheses have never been investigated. Filling up this gap of knowledge, may help identify specific subgroups of patients with T2D at risk for early progression of renal function decline. Specific aims Aim 1: Aim 2: Aim 3: To investigate in Italian patients with T2D whether established genetic and inflammatory markers of CHD are associated with reduced GFR in i) a case-control study of 3,190 patients (egfr considered as dichotomous trait ; i.e.>=or<60ml/min/1.73m2) and ii) a prospective study of 880 independent patients in which rate of GFR decline over time (GFR slope i.e. the relative decline of GFR from baseline to the subsequent GFR measurements) is followed up for 4.5 years. To investigate in the prospective study, above described; whether established serum metabolites linked to CHD are associated with reduced GFR decline. To investigate, prospectively, whether a combined genetic, inflammatory and metabolite multimarker approach, as derived by results obtained from aim 1 and 2, increases in patients with T2D the overall predictability of GFR decline as provided by traditional risk factors. Hypothesis: Our hypothesis states that, genetic and/or inflammatory and/or metabolite markers of CHD, alone or in combination, are also prime movers of GFR decline in T2D and that, they may help setting up a multimarker model for better predicting renal dysfunction in T2D so to refine risk stratification and improve prevention efforts in such patients. Preliminary data: Recruitment of study individuals of all samples utilized for both case-control and prospective studies has been already completed; blood and serum have been also collected and stored at -80C for subsequent analyses and measurements. DNA has been already extracted according to standard procedures and is available for all subjects. Case-control studies: we have established a collaborative research program aimed at investigating the genetics and the pathophysiology of kidney disease among patients with T2D. Our collaborative network provides access to a total of 3,190 patients with T2D. Low GFR status (i.e. GFR<60 ml/min/m2; 638 cases and 2552 controls) has been already defined (Table 1). Prospective Study: this study comprises 880 patients with T2D consecutively recruited at the Endocrine Unit of IRCCS Casa Sollievo della Sofferenza in San Giovanni Rotondo (Italy) from November 2009 through September 2011 (Table 2). The end-point is the rate of GFR decline over time, expressed as ml/min/1.73 m2 per year. This cohort has been already followed-up for 3 years and will be followedup for additional 18 months. Data so far available indicate a GFR decline of 3.2±2.0 ml/min/1.73 m2 per year, a rate similar to that previously reported elsewhere. In patients from SGR 1 sample, elevated serum adiponectin and resistin are independently associated with low GFR (Table 3). 11/07/ / 6

103 RF Are genetic, inflammatory and metabolomic markers of coronary heart disease related to kidney function decline in patients with type 2 diabetes? Menzaghi Claudia Clinical health care research/clinicoassistenziale Ospedale Casa Sollievo della Sofferenza Materials and Methods SNPs of interest will be typed by allelic discrimination assay. Serum adiponectin, resistin, haptoglobin, IL-6, IL-17, IL-18, MCP-1, MIF, TNF-alfa, ICAM-1 and VCAM-1, will be evaluated by multiplex ELISA. 15 selected metabolites will be detected and quantified by Nuclear Magnetic Resonance. Exposures will be: a) Genotype risk score (SNPs combination); b) Inflammatory multimarker score (inflammatory markers combination) and c) Metabolite score (metabolites combination). Outcomes will be in case-control study egfr considered as dichotomous trait; in prospective study the relative GFR decline. The discriminatory power of the prediction models for CKD, both without and with information given by scores will be tested using Survival C-index. Additional predictive Reclassification improvement offered by scores will be assessed by the Net Reclassification Index. Impact and Translational Implications Investigating whether genetic and/or inflammatory and/or metabolomic markers of CHD, alone or in combination, are also prime movers of GFR decline in T2D represents a significant improvement of our understanding of the pathogenic pathway linking CHD and kidney dysfunction in T2D. Setting up a multimarker model for better predicting renal dysfunction in these patients would pave the way for better prevention and treatment strategies, thus eventually reducing diabetes related morbidity rates. 11/07/ / 6

104 Effect of surgical or conservative approach in patients with adrenal incidentalomas on cardiovascular, metabolic, neuropsychological and bone manifestations: respective roles of cortisol secretion and glucocorticoid sensitivity. RF Chiodini Iacopo Clinical health care research/clinicoassistenziale Fondazione Ca'Granda - Ospedale Maggiore Policlinico 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 and Clinical Endocrinology and Reproduction - ICER Project Keyword 1: Project Keyword 2: Project Keyword 3: Growth, development, and disorders of endocrine organs: hypothalamic, pituitary, thyroid, and adrenal physiology, pharmacology, toxicology, and pathophysiology; mechanisms of puberty; endocrine-organ neoplasia; endocrine autoimmunity and immunobiology. subclinical hypercortisolism adrenal incidentalomas 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/Lavoratory Role in the project 1 Fondazione Ca'Granda - Ospedale Maggiore Policlinico 2 "Casa Sollievo della Sofferenza" IRCCS, San Giovanni Rotondo (FG) 3 Policlinico San Donato IRCCS, San Donato Milanese Unit of Endocrinology and Metabolic Disases. Unit of Endocrinology and Diabetology Dpt. of Medical and Surgical Sciences Coordinating Unit. Patients recruitment, conservative and postsurgical follow up. Patients recruitment, conservative and postsurgical follow up. Patients recruitment, conservative and postsurgical follow up. Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Scillitani Alfredo "Casa Sollievo della Sofferenza" IRCCS, San Giovanni Rotondo (FG) 2 corbetta sabrina luigia Policlinico San Donato IRCCS, San Donato Milanese Patients recruitment, conservative and postsurgical follow up. Data analysis and interpretation. Patients recruitment, conservative and postsurgical follow up. Data analysis and interpretation. 01/04/ /02/ /07/ / 6

105 Effect of surgical or conservative approach in patients with adrenal incidentalomas on cardiovascular, metabolic, neuropsychological and bone manifestations: respective roles of cortisol secretion and glucocorticoid sensitivity. RF Chiodini Iacopo Clinical health care research/clinicoassistenziale Fondazione Ca'Granda - Ospedale Maggiore Policlinico Background and Significance Subclinical hypercortisolism (SH) is a status of hypercortisolism without signs of overt glucocorticoid (GC) excess and it is thought to be present in up to the 30% of patients with incidentally discovered adrenal masses (adrenal incidentalomas, AI). Since in patients older than 60 years of age the AI prevalence is about 7%, the prevalence of SH is estimated to be 0.8-2%. Although asymptomatic, SH has been demonstrated to be associated with an increased risk of diabetes, hypertension, major cardiovascular events and osteoporotic fractures and the recovery from SH has been suggested to be beneficial. However, to date, a prospective randomized study assessing in AI patients the overall beneficial effects of the surgical approach and the criteria predictive of the effect of the recovery from SH is lacking. Therefore, the diagnosis itself of SH is still debated and in many AI patients SH remains uncertain and the best therapeutic approach unknown. These uncertainties are also due to the fact that the GC sensitivity varies among individuals. Indeed, the individual GC sensitivity depends on the polymorphisms of GC receptor (GR) and on the different 11-hydroxysteroid-dehydrogenase type 2 enzyme activity, that, catalyzing the shift from active cortisol to the inactive cortisone, regulates the peripheral tissue exposure to active GC. Therefore, the combined evaluation of cortisol secretion and sensitivity could consent to address the approach of choice in the single AI patient. Specific aims Aim 1: Aim 2: Aim 3: To assess the variation of blood pressure control, lipids and glucose metabolism, vascular damage, bone mineral density (BMD), incidence of falls, clinical and morphometric vertebral fractures, body composition, coagulation parameters, neuropsychological aspects and quality of life (QoL) in AI patients with uncertain SH after the surgical removal of the adrenal mass or after a conservative treatment. To assess in AI patients and uncertain hypercortisolism the effect of the surgical and conservative approach on cardiovascular, metabolic and bone manifestations, neuropsychological aspects and quality of life (QoL), in relation to the degree of cortisol secretion and sensitivity. To establish the best clinical-biochemical criteria for diagnosing SH, on the basis of the changes of the outcomes after the surgical or conservative approach, and therefore, for addressing the treatment of choice in the individual patient with AI Hypothesis: In AI subjects with uncertain SH the combined evaluation of the clinical features together with the parameters of cortisol secretion and sensitivity will consent to decide which patient has the greatest probability to ameliorate after surgery. Preliminary data: In AI patients, the SH of adrenal origin is ascertained if cortisol after a dexamethasone suppression test (DST) is >5.0 µg/dl and ACTH <5 pg/ml. In these subjects salivary and/or serum midnight cortisol levels (MSalC and MSeC, respectively) and/or 24hours urinary free cortisol (UFC) levels are often increased. In this condition the surgical treatment is suggested, since it is of benefit in the majority of subjects. In AI patients with cortisol after DST <1.8 µg/dl and normal ACTH, MSalC, MSeC and UFC, SH is excluded. In this condition surgery is performed only if the adrenal mass is >5 cm or if it has radiological features not suggestive of a benign lesion. However, in the majority of AI patients the diagnosis of SH is uncertain, as cortisol levels after DST are between 1.8 and 5 µg/dl and often associated with increased MSalC and/or MSeC and/or UFC and reduced ACTH levels. In these subjects, who are possibly at risk of the chronic consequences of SH, the best approach (i.e surgical treatment or conservative approach with medical therapy of hypertension and/or diabetes and/or osteoporosis) is not known. A previous not randomized study of 07/07/ / 6

106 Effect of surgical or conservative approach in patients with adrenal incidentalomas on cardiovascular, metabolic, neuropsychological and bone manifestations: respective roles of cortisol secretion and glucocorticoid sensitivity. RF Chiodini Iacopo Clinical health care research/clinicoassistenziale Fondazione Ca'Granda - Ospedale Maggiore Policlinico our Group suggested that, in AI subjects with uncertain SH and with hypertension and/or osteoporosis and/or diabetes mellitus, the surgical treatment may be advantageous, while the conservative approach deleterious. Recently, in a multicenter observational study with a long-term follow up, we demonstrated that SH patients are at higher risk of major cardiovascular events. Finally, in a study assessing in AI patients the role of the BclI and N363S polymorphisms of GR, we showed that the presence of vertebral fragility fractures and hypertension was associated with the presence of a GC sensitizing haplotype. Materials and Methods Ninety AI subjects (40-70 years) with single benign AI and uncertain SH will be randomized to surgery or conservative follow up. The sample size is based on a bone mineral density (BMD) variation of 2.5%/year. At baseline and 6, 12 and 24 months (of follow up or after surgery) we will assess UFC, urinary free cortisone (UFCo), cortisol after DST, ACTH and MSalC levels, UFC/UFCo ratio, body weight, blood pressure, lipid and coagulation indexes, glycated hemoglobin, the N363S, ER22/23EK and Bcl1 GR variants, several neuropsychological aspects and QoL, the carotid intima-media thickness, vertebral morphometry and BMD (these latter only at baseline and 24 months). Serum ACTH levels will be assessed by IRMA, cortisol immunofuorimetrically, MSalC, UFC and UFCo by liquid chromatography tandem mass spectrometry, GR polymorphisms by polymerase chain reaction, BMD by Dual X-rayabsorptiometry, vertebral morphometry by X-ray and the neuropsychological aspects and QoL by appropriate questionnaires Impact and Translational Implications In AI patients the SH diagnosis requires an expensive workup and the best approach (surgical or conservative) remains undefined in many patients. This study could help to define an algorithm for addressing the approach of choice in the individual AI patient. The individualized therapeutic approach in AI patents will consent to reduce the economic and social costs of the SH diagnosis and those related to SH consequences, while avoiding useless surgical operations. 07/07/ / 6

107 The genetics of sudden cardiac death in athletes and implication for risk prevention BANDO 2013 Progetti Ordinari RF RF THIENE GAETANO Clinical health care research/clinicoassistenziale Veneto 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: Experimental models, clinical studies and studies on mechanisms of disease states including exercise physiology as related to cardiac and pulmonary metabolism, oxygen, contractility, and respiratory function and regulation Sudden cardiac death Genetics 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/Lavoratory Role in the project 1 Veneto UOC Patologia Cardiovascolare, Azienda Ospedaliera Padova Coordination, sudden cardiac death Registry, autopsy, genetic investigation 2 Azienda ULSS 16 Padova Centro Medicina dello Sport ed Attività 1st level preparticipation screening of competitive motorie, Dipartimento Socio-Sanitario ai atheletes, clinical database Colli 3 Azienda Ospedaliera/Università di Padova UOC Cardiologia, Elettrofisiologia e Centro Genetico-Clinico delle Cardiomiopatie aritmiche 2nd and 3rd level clinical, invasive and non invasive screening 09/07/ / 6

108 The genetics of sudden cardiac death in athletes and implication for risk prevention BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project THIENE GAETANO Veneto Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Schiavon Maurizio Azienda ULSS 16 Padova 1st level preparticipation screening of competitive atheletes, clinical database 28/09/ Domenico Corrado Azienda Ospedaliera/Università di Padova 2nd and 3rd level clinical, invasive and non invasive screening 05/08/ Basso Cristina Azienda Ospedaliera/Università di Padova Registry sudden cardiac death, autopsy 18/09/ PILICHOU KALLIOPI Azienda Ospedaliera/Università di Padova Genetic investigation 26/01/1976 Background and Significance Sudden cardiac death (SCD) in young athletes is a devastating event for the family, the sports medicine team and the local community. The long-lasting Italian experience revealed that preparticipation screening including electrocardiogram (ECG) definitively improves the sensitivity in detecting underlying heart diseases and substantially reduces the risk of SCD in the athletic field (primary prevention). However, clinical heterogeneity and the lack of gold-standard clinical criteria and diagnostic tools led to the failure of timely recognition of asymptomatic subjects. Inherited cardiomyopathies (CMs) account for up to 40% of SCD in the young, and include arrhythmogenic (AC), hypertrophic (HCM) and non structural (ion channel) diseases. Up-to-date more than 120 genes and multiple allelic variants have been related to heritable CMs enabling the transition from discovery through translation with clinical genetic tests. The goal of our multidisciplinary investigation is to achieve further decline of SCD by enabling the early detection of inherited concealed cardiovascular entities, through the combination of traditional non invasive and invasive screening tools (including cardiac magnetic resonance -CMR), with the most updated deep sequencing techniques which offer great advantages in terms of number of patients and genes screened. Specific aims Aim 1: Aim 2: Aim 3: To evaluate the cardiovascular risk in a population of young athletes, through a coordinate approach of three levels Unit to gather data on epidemiological changes in the recent years of SCD during sport activity in competitive and non competitive athletes in North East of Italy. Implementation of DNA and tissue banking and of central databases and registries is a main target. To establish the most appropriate strategy to screen athletes for underlying cardiovascular diseases and whether and in which context new imaging techniques, such as CMR and genetic screening, should be carried out. To determine the prevalence and pathogenicity of genetic variants in the general athletic population and the extent to which they contribute to the phenotype (modifiers). Hypothesis: The synergic employment of traditional screening tools and of the most updated deep sequencing techniques in young people performing sport activity could further reduce the burden of SCD in athletes, by increasing early identification in the pre-symptomatic stage. Preliminary data: Previous studies carried out in the Veneto region have established that the incidence of SCD in the 09/07/ / 6

109 The genetics of sudden cardiac death in athletes and implication for risk prevention BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale THIENE GAETANO Veneto young is 0.8/ per year and of 2.3/ per year in the athletes, the latter progressively decreasing with the increase awareness of diseases at risk and their consequent early identification[corrado et al, 2003]. SCD in the young is mostly arrhythmic and the spectrum of underlying heart diseases is wide: atherosclerosis, AC [Thiene et al, 1988], HCM [Basso et al, 2000 and 2009], myocarditis [Basso et al, 2001], congenital coronary anomalies [Basso et al, 2000], ventricular preexcitation [Basso et al, 2001], Marfan syndrome or bicuspid aortic valve with aortic dissection [Basso et al, 1995]. As far as the athlete, AC and HCM are particular at risk of SCD during effort [Corrado et al, 1998; Corrado et al, 2006; Basso et al, 2008]. Recently, a left ventricular variant of AC has been reported in competitive athletes dying suddenly and preliminary data indicate the limitation of current diagnostic tools including ECG, with the exception of cardiac magnetic resonance (CMR) [Perazzolo Marra et al, 2012]. Our group identified 6 AC-causing genes: cardiac ryanodine receptor type 2 [Tiso et al, 2001], desmoplakin [Rampazzo et al, 2002], transforming growth factor beta3 [Beffagna et al, 2005], desmoglein-2 [Pilichou et al, 2006], desmocollin-2 [Beffagna et al, 2007] and at-catenin [van Hengel, Calore et al, 2012]. Compound/digenic heterozygosity, which is present in 16% of AC-causing desmosomal gene mutation carriers, is a powerful risk factor for lifetime major arrhythmic events and SCD [Rigato et al, 2013]. Overall, about 30-40% of cardiovascular diseases, either structural or non, at risk of SCD in the young and the athlete are genetically determined, thus underlying the potential role of genetic screening for early diagnosis. Materials and Methods A) Study population: - consecutive young athletes who undergo preparticipation screening at the Center for Sports Medicine of Padova, Italy - probands with inherited CM and first degree family members referred to Cardiology Division, Padova, Italy - young people dying during sport activity B) Methods: - clinical screening. First level: physical examination, 12-lead ECG, stress test ECG; second level: echocardiogram, SAECG, Holter, CMR; third level:angiography, biopsy, electrical mapping; - registry of SCD/aborted SCD occurring during sport activity; - postmortem investigation, including molecular autopsy - genetic screening: mutational analysis on a next generation sequencing platform (MiSeq-Illumina). The bioinformatic pipeline will examine more than 120 CM-related genes taking into account different clinical and pathological features. Genetic variants will be properly classified with respect to their actual pathogenicity and association or not with heritable CMs. 09/07/ / 6

110 The genetics of sudden cardiac death in athletes and implication for risk prevention BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale THIENE GAETANO Veneto Impact and Translational Implications The major impact on the Regional and National Health system will be the identification of new diagnostic markers of diseases at risk of SCD. The significance of CMR data will be assessed before its implementation as a cardiac imaging technique at pre-participation screening. The collection of all genetic variants in athletes will enable the generation of an inherited CMs genetic database which is fundamental for interpretation of results and genetic counseling.

111 LEFT VENTRICULAR HYPERTROPHY IN AORTIC VALVE DISEASE AND HYPERTROPHIC CARDIOMYOPATHY: GENETIC BASIS, BIOPHYSICAL CORRELATES AND VIRAL THERAPY MODELS RF Olivotto Iacopo 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 Cardiac Contractility, Hypertrophy, and Failure - CCHF Project Keyword 1: Project Keyword 2: Project Keyword 3: Genetic cardiomyopathies; genotype-phenotype correlation; genomic and proteomic approaches to cardiac hypertrophy and failure. next generation sequencing RNAi-based therapies 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/Lavoratory Role in the project 1 Toscana Heart and Vessel Department and DAI Laboratory 4,Genetic Diagnostics, Azienda Ospedaliera Universitaria Careggi, Florence 2 Fondazione Don Gnocchi, IRCCS Florence 3 Azienda Ospedaliera Universitaria Senese Cardiovascular Rehabilitation Unit Molecular Medicine Unit Cardiac surgery, perioperative assessment,collection of surgical samples.genetic studies. Biophysical characterization of isolated myofibrils and preclinical pharmacological studies. Post-operative assessment and follow-up. Allele silencing by RNAi technique and viral delivery studies Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Zipoli Renato Fondazione Don Gnocchi, IRCCS Florence 2 Sorrentino Vincenzo Azienda Ospedaliera Universitaria Senese 3 Girolami Francesca DAI Laboratory 4,Genetic Diagnostics, Azienda Ospedaliera Universitaria Careggi, Florence Echocardiographic studies. Post-operative assessment and follow-up. Allele silencing by RNAi technique and viral delivery studies 10/04/ /02/1956 Next generation sequencing studies 14/05/ /07/ / 6

112 LEFT VENTRICULAR HYPERTROPHY IN AORTIC VALVE DISEASE AND HYPERTROPHIC CARDIOMYOPATHY: GENETIC BASIS, BIOPHYSICAL CORRELATES AND VIRAL THERAPY MODELS RF Olivotto Iacopo Clinical health care research/clinicoassistenziale Toscana Background and Significance Left ventricular hypertrophy (LVH), whether genetically driven (as in hypertrophic cardiomyopathy - HCM) or secondary (as in aortic stenosis) is associated with adverse outcome irrespective of the etiology. In both models, the degree of hypertrophic response is very heterogeneous, suggesting the impact of a variety of genetic and environmental modifiers. To date, the genetic determinants of LVH are largely unresolved. The potential for LVH regression following pharmacological interventions and surgery, may help unravel the determinants of LVH itself, and may be relevant to other, even more common conditions such as hypertension. To date, however, studies assessing the effects of pharmacological and surgical therapy on regression of LVH are few and based on little translational background. Furthermore, the enormous potential offered by genetic studies using next generation sequencing techniques has been scarcely exploited in this context. In a translational perspective, the present project was conceived to combine a spectrum of pre-clinical and clinical expertise in order to identify the genetic predictors and functional correlates of LVH development and regression in patients with aortic valve disease and HCM, and to use such information to explore novel pharmacological approaches. Furthermore, the use of RNAi techniques and viral delivery in pre-clinical models of HCM will be explored as a viable therapeutic option for genetically-driven LVH. Specific aims Aim 1: Aim 2: Aim 3: To identify clinical, echocardiographic and genetic variables associated with inappropriately high hypertrophic response in patients with aortic valve disease, and their relationship with short- and mid-term outcome following surgical intervention. To assess LVH regression following surgical myectomy in HCM. To identify, by next generation sequencing, the prevalence of genetic mutations affecting sarcomere genes such as MYBPC3, MHY7 and TNNT2, potentially capable of triggering inappropriate hypertrophy, as well as that of genetic modifiers (such as polymorphisms of RAAS-related genes) in determining the degree of hypertrophic response to afterload mismatch. To characterize the biophysical properties of isolated cardiac preparations (intact trabeculae, single cardiomyocytes and single myofibrils) from surgical samples of the same patients enrolled for Aim 1; to investigate their response in vitro to therapeutic concentrations of pharmacological agents potentially applicable to clinical management (such as ranolazine, sildenafil, telmisartan, azilsartan and spironolactone), by determining how they affect myocardial contraction-relaxation cycle, intracellular Ca2+ fluxes and the molecular markers of hypertrophic response. To assess the use of the RNAi technique and viral delivery in TNNT2-mutant mice (an established transgenic model of HCM) and in human and murine cells harbouring HCM-associated mutations, as a novel therapeutic tool for the treatment of dominant inherited cardiomiopathies. Hypothesis: A comprehensive genetic and biophysical assessment of the hypertrophic response, and its in vitro response to novel pharmacological treatment, may prove valuable for the management of patients withaortic valve disease and HCM, and promote therapeutic advances in the field. Furthermore, the recently discovered RNAi technology has proven to be highly effective in selective allele silencing both in vitro and in vivo: the use of the RNAi technique and viral delivery represents a promising therapeutic tool for treatment of dominant inherited cardiomiopathies. Preliminary data: Preliminary data identifying modifier genes and pathways of LVH have been produced using next generation sequencing techniques. We have established a model for the molecular and electrophysiological characterization of isolated cardiomyocytes from patients with genetic LVH, and have recently described the effects of ranolazine on electrical remodeling and calcium homeostasis 10/07/ / 6

113 LEFT VENTRICULAR HYPERTROPHY IN AORTIC VALVE DISEASE AND HYPERTROPHIC CARDIOMYOPATHY: GENETIC BASIS, BIOPHYSICAL CORRELATES AND VIRAL THERAPY MODELS RF Olivotto Iacopo Clinical health care research/clinicoassistenziale Toscana in such model, with very encouraging results (Coppini et al, Circulation, 2013). Early data on the efficiency of sequences aimed at targeting specific genes have been obtained in cultured cells and in vivo murine models. Validation of the ability of these sequences to reduce a specific allele have been obtained by Real Time PCR and protein expression analysis. Materials and Methods In patients referred for aortic valve surgery and HCM patients undergoing surgical myectomy echocardiographic assessment of will LVH be performed pre-and 6 months post-operatively by echo and MRI. Targeted whole exome sequencing by Next Generation Sequencing Analysis will be performed followed by bioinformatics analysis. Patch-clamp, intracellular Ca2+ and force measurements will be performed on cardiomyocytes isolated from the surgical specimens, to evaluate the electro-mechanical alterations occurring in LVH and their reversal by pharmacological interventions. Selection of RNAi sequences will be performed on the basis of their ability to specifically target the mutant alleles.the adenoassociated virus vector AAV-9 will represent our first choice for viral delivery to cells and TNNT2-mutant mice. Alternative vectors will also be considered on the basis of the results obtained Impact and Translational Implications Understanding the molecular basis, modulators and pathophysiologic targets of LVH and its related conditions may open the way to novel treatments impacting the lives of patients with orphan disease, such as HCM, as well as common conditions such as aortic stenosis or hypertension. In genetically driven LVH, the ability of RNAi to attenuate the expression of specific alleles in preclinical studies may lead to a novel, convincing therapeutic strategy based on allele silencing. 10/07/ / 6

114 RF Inhibition of Glycolysis by 2-Deoxy-D-Glucose as a Therapeutic Strategy for Polycystic Kidney Disease magistroni riccardo 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: Digestive, Kidney and Urological Systems Cellular and Molecular Biology of the Kidney - CMBK Project Keyword 1: Project Keyword 2: Project Keyword 3: Identification and characterization of genes that cause kidney diseases in humans and animal models. Including the pathophysiology and cellular and molecular consequences of genetic disorders (including polycystic kidney disease and disorders of tubular function). glucose metabolism 2-Deoxy-D-Glucose Project Request: Animals: X Humans: X Clinical trial: X 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 50% San Raffaele Scientific Institute and 50% Fondazione Telethon. Patent number: US Patent Application n. 61/667,615 filed on July 3, 2012; published online on January 9, 2014 INSTITUTION Department/Division/Lavoratory Role in the project 1 Ospedale San Raffaele - Milano 2 Azienda Ospedaliero Universitaria Policlinico di Modena 3 Ospedale di Montichiari A.O. Spedali Civili di Brescia Division of Genetics and Cell Biology/Division of Nephrology and Dialysis animal model preclinical investigation/ recruiting patients in clinical trial Dipartimento di Medicina e Specialità recruiting patients in clinical trial Mediche/Divisione di Nefrologia Dialisi e Trapianto Unità Operativa di Nefrologia e Dialisi recruiting patients in clinical trial Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 leonelli marco Azienda Ospedaliero Universitaria Policlinico di Modena 2 Capistrano Mariano Ospedale di Montichiari A.O. Spedali Civili di Brescia 3 Boletta Alessandra Ospedale San Raffaele - Milano/Division of Genetics and Cell Biology/ Investigator recruiting patients in clinical trial 04/05/1958 recruiting patients in clinical trial 19/05/1969 animal model preclinical investigation 23/02/ /07/ / 6

115 RF Inhibition of Glycolysis by 2-Deoxy-D-Glucose as a Therapeutic Strategy for Polycystic Kidney Disease magistroni riccardo Biomedical/Biomedica Ospedale San Raffaele - Milano Background and Significance Polycystic kidney disease (PKD) is a life-threatening genetic disease affecting an estimated 12.5 million people worldwide. The hallmark of ADPKD is bilateral renal cyst formation. Mutations in two genes have been associated with the disease: PKD1, accounting for 85% of cases and PKD2 for the remaining 15%. Currently there is no western countries approved drug treatment for this condition. In a recent study published by a member of this Team, Dr. Boletta, a novel potential therapeutic approach has been identified. The group has shown that a profound metabolic alteration at the level of glucose metabolism can be found in ADPKD cellular and animal model systems as well as in the epithelia lining the cysts of patients (Rowe et al, Nat Med, 2013). Importantly, interfering with glycolysis using a glucose analogue, 2-deoxy-D-Glucose (2-DG) effectively slowed down disease progression in two distinct orthologous mouse models of ADPKD (Ksp-Cre:Pkd1flox/- and Pkd1v/v mice, respectively). More recently, the group of Dr. Boletta has generated additional preliminary data (see below) suggesting that administration of 2-DG in murine models more closely resembling the disease results in a very significant delayed disease progression. These data provide a strong rationale for the initiation of clinical studies aimed at testing safety and efficacy of the use of 2- DG in ADPKD patients. We propose here to initiate such studies while we continue refining the studies on the animal models. Specific aims Aim 1: Aim 2: Aim 3: defining the maximum tolerated dosage of 2-DG oral administration in patients affected by ADPKD with normal renal function and progressive disease. Given the promising results obtained in animal models we will test the safety profile of 2-DG in patients affected by ADPKD. We will use increasing doses of 2-DG (2 mg/kg to 88 mg/kg) to establish the best tolerated dose, following the scheme of previous clinical trials performed on neoplastic patients. defining the safety profile of 2-DG in patients affected by ADPKD. During the escalation of 2-DG dosage we will record any adverse event to evaluate the safety profile of the drug. In particular we will monitor the `hypoglycaemic-like manifestations' and the alteration of electric cardiac activity (QTc elongation). refining the optimal dosage and timing for the efficacy of 2-DG in slowing disease progression in animal models of ADPKD. In this aim, we will build on our recent findings and try to optimize the use of 2-DG in preclinical animal models. More specifically, we will use a very long-term mouse model of PKD treated in the presence of low doses of 2-DG that might be compatible with the tolerated doses in humans (see above). Hypothesis: Based on the above data we have hypothesized that therapeutic interventions interfering with glucose metabolism in PKD might represent a novel strategy to retard cyst expansion. In this proposal we hypothesize that low doses of 2-DG will be very well tolerated in humans and at the same time they will still be effective in retarding disease progression in slowly progressive PKD mice models (inactivated at P40 and followed for 6 months). Preliminary data: We previously showed that 2-DG (500mg/kg) in very aggressive models of ADPKD for only two days resulted in an effective disease improvement (Rowe et al, Nat Med, 2013). Previous studies showed that early inactivation of PKD1 (before P13) induces an aggressive PKD model, whereas late inactivation (after P13) results in a milder PKD model. We have found that two-weeks treatment with 100mg/kg of 2-DG effectively reduced disease progression in mice in which inactivation was induced 09/07/ / 6

116 RF Inhibition of Glycolysis by 2-Deoxy-D-Glucose as a Therapeutic Strategy for Polycystic Kidney Disease magistroni riccardo Biomedical/Biomedica Ospedale San Raffaele - Milano at P12, as monitored by kidney weight and Blood Urea Nitrogen (BUN). Furthermore, a slowly progressive disease caused by Pkd1 inactivation at P25 treated for two months with 100mg/kg of 2- DG resulted in a significant reduction in Total Kidney Volume (TKV, monitored by MRI) and improvement of BUN. 2-DG has been studied in rodents with long-term administration showing safety. We do not have personal data on ADPKD patients, however 2-DG has been tested in cancer patients in previous clinical studies (Raez, et al.,cancer Chemother Pharmacol, 2013 ; Stein, et al.,prostate: 2010) and was reported to have a very well tolerated profile with minimal side effects. Thus, we believe that altogether these evidences provide a strong rationale to design clinical trials with 2-DG to assess its safety and efficacy in ADPKD patients. Materials and Methods Clinical Study - A small number of patients with ADPKD, having normal well preserved kidney function and large kidneys will be treated and clinically followed. - Once verified the safety profile in such patients a second group of patients with progressive disease will be recruited and treated. - Dose titration will start with the low dose, eg 2 mg/kg/day, to be gradually increased over 6 weeks. Administration of 2-DG will be by oral route. - Clinical evaluation, blood samples and pharmacokinetics data will be collected. Animal study - Pkd1flox/flox:Tamoxifen Cre mice will be injected a single dose of Tamoxifen (10mg/kg) on postnatal day P40 - The volume of the kidneys as well as the size of the cysts will be evaluated using a 7 Tesla MRI available in our mice facility monthly. - 2-DG dissolved in NaCl will be daily administered by intradermal injections at the dosage of mg/kg - Blood will be withdrawn once a month and several parameters including BUN analyzed. Impact and Translational Implications Renal failure in ADPKD is caused by the progressive renal cyst expansion, which only form in a minority of nephrons. Reducing the rate of cyst expansion is considered a good strategy to preserve the function of the kidney in ADPKD. We have identified a novel therapeutic strategy, which targets the energy demand of the diseased cells and appears very effective in animal models. Thus, 2-DG might be the long-sought-after well tolerated molecules needed for a chronic treatment in ADPKD. 09/07/ / 6

117 RF Role of the p21-mediated DNA-Damage Response in the immune-surveillance of leukemia stem cells PELICCI PIER GIUSEPPE Biomedical/Biomedica Istituto Europeo di Oncologia 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 Acute myeloid leukemia (AML) p21 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/Lavoratory Role in the project 1 Istituto Europeo di Oncologia Department of Experimental Oncology D.I. Applicant Institution Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 11/07/ / 6

118 RF Role of the p21-mediated DNA-Damage Response in the immune-surveillance of leukemia stem cells PELICCI PIER GIUSEPPE Biomedical/Biomedica Istituto Europeo di Oncologia Background and Significance Survival of cancer cell involves inhibition of cell death and escape from clearance by immune responses. According to the "Cancer Stem Cell" hypothesis, tumor growth is maintained by rare cells, the CSCs, endowed with unlimited self-renewal properties. Recent findings show that their growth may be severely influenced by the host immunocompetence. How CSCs evade immune-surveillance, however, is not known. Indirect evidence from our group suggests that the p21-mediated DNA-damage (DD) response might be implicated in the regulation of survival and immune-surveillance of normal and cancer SCs: 1) Adult SCs have evolved a unique DD-response that leads to their immediate expansion and limits self-renewal. This response depends on the activation of the cell cycle inhibitor p21, cell cycle entry, symmetric self-renewing divisions, DD-repair and maintenance of self-renewal. However, DDrepair is never complete, leading to progressive DD accrual and diminishing self-renewal; 2) constitutive-activation of p21 in leukemias, as a consequence of oncogene expression, activates DD-repair, limits DD accumulation and maintains unlimited self-renewal potential of leukemia SCs. Notably, leukemogenesis does not proceed in the absence of p21 expression; 3) p21-null leukemia SCs activate a T-cell mediated immune response against leukemic cells. Transfer of the primed T-cell population into immunodeficient mice blocks in vivo leukemia expansion, suggesting a direct role for p21 in immune evasion. Specific aims Aim 1: Characterization of the immune properties of APLs. We will test if p21 expression in APL blasts regulates immunomodulatory ligands that activate or repress, respectively, a protective or cytotoxic T-cell population, by performing RNAseq analyses of WT vs. p21-/- primary APLs or vs. the same blasts knocked down for p21. Candidates will be validated in vivo functionally, by shrna or overexpression approaches. Aim 2: Characterization of the effector T-cell and of the cellular mechanisms of APL-blast clearance. We will: i) characterize the T cell population responsible for the clearance of p21-/- APLs; ii) analyze cytokine production by effector T-cells; iii) analyze the presence of antigen-specific T-cells in p21-/- or WT leukemic mice; iv) determine whether the clearance of APLs by T-cells requires the contribution of innate immune cells. Aim 3: Investigation of the role of DD in leukemic SC (LSC) immune-clearance. We will: a) determine whether increased DD mimics loss of p21 in APL blasts; b) determine whether DNA-damaged HSCs elicit a T-cell specific immuneresponse; c) characterize p21-/- APL blasts that are tolerated by the anti-apl immune-response. Hypothesis: We hypothesize that the extended self-renewal of LSCs results from avoidance of immunological clearance, and that this is due to high levels of DD and p21 activation. Preliminary data: We showed that the extended self-renewal of LSCs is due to DD accrual and constitutive activation of p21 following oncogene expression (PML-RAR or AML1-ETO). The p21-dependent response involves reversible cell-cycle arrest, DNA repair and maintenance of self-renewal. P21 is indispensable for initiation of AML1-ETO-induced leukemogenesis and to maintain the LSC pool in PML-RAR leukemias (which is markedly reduced in p21-/- APLs and residual LSCs hyperproliferate, accumulate massive DD and functionally exhaust). PML-RAR-p21-/- leukemias are not transplantable in syngenic mice, yet reacquire the ability to initiate leukemogenesis when transplanted into immunodeficient (RAG1-/-) or syngeneic mice after g-irradiation. The growing leukemias are indistinguishable from primary WT APLs and can be re-transplanted into immunodeficient, but not syngeneic mice, suggesting that the expansion of p21-/- LSCs is controlled by the host immune-system. Interestingly, non-irradiated syngeneic mice transplanted with primary p21-/- APLs do not show signs of leukemia during their lifetime, however, APL blasts are still detectable in the bone marrow, suggesting that they represent a residual leukemic population that is tolerated by the host immune system. Further pilot experiments indicated that p21-/- LSCs are 11/07/ / 6

119 RF Role of the p21-mediated DNA-Damage Response in the immune-surveillance of leukemia stem cells PELICCI PIER GIUSEPPE Biomedical/Biomedica Istituto Europeo di Oncologia cleared by T-cells since p21-/- APLs: i) initiate leukemias when transplanted into NOD/SCID or RAG1-/- mice (which lack B, T and T-NK cells), but not in JHT mice (lacking only B-cells), and ii) do not grow in RAG1-/- mice when the recipients are injected with a T cell population primed with p21-/- blasts, but grow with non-primed T cells or T cells primed with WT blasts. Materials and Methods As tumor model, we will use knock-in mice expressing PML-RAR from the endogenous myeloid-specific cathepsin G gene promoter (PRKI mice) in a WT and p21null background. Normal and leukemic SCs and progenitors will be FACS-sorted from mouse bone marrow mononuclear cells. We will assess self-renewal properties in vivo by serial transplantation or SC counting insc primary-transplants, repopulating activities by Competitive Repopulation, engraftment by evaluation of Ly5.1/2 chimerism, extent of DD by quantitative H2AX staining, proliferation by Ki67 staining, and DD response by staining against activated ATM, ATR, p53. T-cells (primed and non-primed) will be recovered from mouse spleens. RNAseq data-sets of WT or p21-/- primary APLs will be analyzed for expression of immunomodulatory ligands, chemokines, cytokines and phagocytosis signals and candidates validated by an in vitro killing assay based on the coculture of APL blasts and WT or primed T-cells. Impact and Translational Implications We do not know if p21 extends LSC self-renewal by activating DD repair, restricting the cell-cycle, or regulating their survival. Likely, it is by a combination of cell intrinsic and cell extrinsic mechanisms. Here we aim to investigate the role of p21 in APL immune clearance and the immune cells involved. Understanding how CSCs evade immune detection is of huge therapeutic importance as re-establishing an immune response to CSCs could allow detection and eradication of tumors at an early stage. 11/07/ / 6

120 RF Clinical utility of a next generation sequencing-based "oncochip" for therapeutic decision in metastatic breast cancer CURIGLIANO GIUSEPPE 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: 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. next generation sequencing targeted 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/Lavoratory Role in the project 1 Istituto Europeo di Oncologia 2 Istituto Europeo di Oncologia Division of Early Drug Development PI - Applicant Institution Department of Experimental Oncology Operative Unit 2 Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Belloni Elena Carla Istituto Europeo di Oncologia Principal Investigator - Operative Unit 2 09/07/ Bernard Loris Istituto Europeo di Oncologia Collaborator for the sequencing data processing and analysis 19/02/ /07/ / 6

121 RF Clinical utility of a next generation sequencing-based "oncochip" for therapeutic decision in metastatic breast cancer CURIGLIANO GIUSEPPE Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia Background and Significance Many promises of genomics-driven personalised medicine remain unfulfilled to date. In the recent SAFIR02 study on genomics-based therapy allocation in breast cancer, only 13% of patients could be matched with appropriate therapy, and only 3% received any clinical benefit. Most studies so far have relied mainly on copy number variation (CNV), but still too little is known on the clinical impact of the majority of point mutations, which are the preferred mode of oncogene activation but are more complex to study than CNVs. In breast cancer, a handful of bona fide cancer-promoting mutated genes ("drivers", e.g. tp53 or PIK3CA) coexist with a disproportionately higher number of genes that mutate often but are unlikely to participate in the natural history of the disease ("passengers"). Driver genes often mutate at low frequency in certain tumours, but, when present, can efficiently be targeted therapeutically. In addition, the germline genomic make-up influences pharmacological parameters of classical and investigational drugs. With our recently published algorithm for driver-passenger discrimination (DOTSfinder), we have designed a panel of cancer relevant mutations, with 201 putative cancer drivers and 89 pharmacogenomics-relevant specific variants (IEO Oncochip). Here, we wish to test whether extensive genomic information combining copy number measurement and IEO Oncochiptargeted sequencing is useful in guiding clinical decision making for metastatic breast cancer patients. Specific aims Aim 1: Aim 2: Compare progression-free-survival in a 2-arm phase II trial, randomising patients to either receiving genomicsdriven therapy modification (experimental arm) or standard maintenance therapy (control). Compare overall survival, response rates (RECIST v1.1), safety of the strategy as a whole and of the individual targeted agents. To correlate molecular mechanisms in patients with the efficacy endpoints Aim 3:. Hypothesis: Test whether outcome of metastatic breast cancer patients is improved by a strategy of therapy adaptation driven by an NGS-based panel of cancer mutations, the IEO Oncochip. Preliminary data: A comprehensive cancer gene panel with maximised information of clinical utility requires to discriminate between driver and passenger mutations among the plethora of mutations that typically characterise tumour genomes. We recently published a new algorithm (DOTSfinder) that, by weighing much more strongly than its predecessors the functional impact of mutations, identifies putative drivers with better performance and easier implementation. To build the IEO Oncochip, DOTSfinder was run against several TCGA datasets, obtaining tissuespecific drivers. To these, we added putative drivers identified by Vogelstein et al. and 89 variants with high level of evidence for cancer-specific pharmacogenomics interaction extracted from the PharmGKB database. The final panel of 201 genes and 89 variants covers a total of approximately 1050 Kilobases. To in silico simulate the performance of the oncochip in a fictitious clinical cohort, we tested DOTSfinder against the TCGA provisional BRCA database. Among genes mutated with frequency >1% in TCGA breast cancer, Oncochip genes included all well known cancer drivers (p53, PIKRCA, MAP kinases, epigenetic modifiers and transcription factors) but depleted for genes encoding large extracellular proteins of dubious relevance (mucins, titin, SYNEs). Putative drivers were however also scattered among genes with low mutation frequency, with 85% Oncochip genes mutated in at least one case. About 25% of Oncochip genes have a known antineoplastic drug interaction (DGidb), as opposed to <5% of passengers. Thus, a rare driver gene mutation with a higher than average likelihood of being or becoming "druggable", can be identified more easily with a relatively non tissue-specific gene panel like the Oncochip. 10/07/ / 6

122 RF Clinical utility of a next generation sequencing-based "oncochip" for therapeutic decision in metastatic breast cancer CURIGLIANO GIUSEPPE Clinical health care research/clinicoassistenziale Istituto Europeo di Oncologia Materials and Methods Design: prospective phase II randomised trial, comparing treatment administered according to the identified molecular anomaly of the tumour vs without considering the tumour genome analysis. Population: metastatic breast cancer patients with no more than 3 lines of previous chemotherapy and progressing to previous anti-her2 if HER2+ disease. Procedure: following screening, bioptic material from metastases will be analysed for copy number variation and Oncochip mutations. In the experimental arm, treatment recommendations will be made by a multidisciplinary panel, matching treatment to available targeted drugs from currently available investigational drugs. Current panel includes drugs targeting PI3K, mtor, FGFR, AKT, EGFR1-3, MEK, VEGFR, RET and PARP; this panel is expanding. If no targeted drugs can be specifically identified, conventional therapy will be administered considering pharmacogenomic information from the Oncochip. Impact and Translational Implications This will be one of the first studies directly evaluating a combined sequencing/copy number analysis strategy as a tool for clinical decision making. It will analyse multiple genomic parameters of proposed but unconfirmed clinical validity at the same time, establishing the role of next generation genomics in clinical practice. 10/07/ / 6

123 RF Identification of new therapeutical targets of cancer stem cells: study of the mechanistics of mitotic spindle alignment to cortical polarity in mammary stem cell asymmetric divisions MAPELLI MARINA Biomedical/Biomedica Istituto Europeo di Oncologia LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Biological Chemistry and Macromolecular Biophysics Macromolecular Structure and Function C - MSFC Project Keyword 1: Project Keyword 2: Project Keyword 3: Protein interaction networks and signal transduction. asymmetric cell division mitotic spindle orientation 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/Lavoratory Role in the project 1 Istituto Europeo di Oncologia Department of Experimental Oncology D.I. Applicant Institution Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date Background and Significance The impairment of pathways controlling asymmetric cell division (ACDs) of adult stem cells favors the development of human cancer. Recently, the mitotic spindle orientation machinery has emerged as a key player of ACDs, whose deregulation correlates with tumor spreading. Converging evidence supports the notion that during asymmetric divisions specific cortical landmarks instructs the orientation of the mitotic spindle to promote differential placement of daughter cells with respect to the niche, and unequal partitioning of fate determinants. The proposal deals with the structural and functional characterization of mechanisms coupling the mitotic spindle orientation to cellular polarity during asymmetric cell divisions. The main focus of the application is on the working principles of the NuMA/LGN/Gai protein network, and its relation with cortical polarity established by Par proteins, with emphasis on the role of the adaptor Inscuteable and of the junctional protein Afadin. The main feature of the application lies in the combination of structural, biochemical and stem cell biology approaches that will provide a clear view of the interplay between pulling forces acting on astral microtubules and polarity cues instructing the spindle orientation. We believe the availability of detailed molecular information on proteins implicated in stem cell maintenance will eventually pave the way to the development of innovative anti-cancer therapies specifically targeting stem cells. Specific aims 09/07/ / 5

124 RF Identification of new therapeutical targets of cancer stem cells: study of the mechanistics of mitotic spindle alignment to cortical polarity in mammary stem cell asymmetric divisions MAPELLI MARINA Biomedical/Biomedica Istituto Europeo di Oncologia Aim 1: Aim 2: Aim 3: Spindle coupling to polarity involves the recruitment at cortical sites of molecular devices able to establish pulling forces on astral microtubules emanating from the spindle poles. This function is promoted by cortical NuMA that associates with the microtubule motor Dynein and is recruited at the cortex by LGN:Gai complexes. The overall architecture of NuMA:LGN:Gai and the dynamics of its assembly at the cortex still remain elusive. It is known that mitotic kinases such as Aurora-A play fundamental roles in orienting the spindle, possibly via regulatory phosphorylations of NuMA or LGN. Here we propose to test this hypothesis with biochemical approaches and cell biology studies in living cells. These analyses will clarify the molecular mechanisms coordinating the onset of microtubule-pulling forces with mitotic progression. An issue related to how NuMA/LGN/Gai are assembled is how they are recruited and maintained at sites of polarization. Apico-basal polarity is established by the distribution of Par3:Par6:aPKC at the apical cortex, which recruit Inscuteable (Insc). Insc has long been considered the molecular bridge between Par proteins and NuMA/LGN/Gai. Recent data from our and other groups revealed that Insc and NuMA are mutually exclusive partners of LGN, thus challenging the molecular bridge hypothesis. A main line of investigation of this proposal will deal with the formulation of a revised model for the spindle orientation. In particular, we will address 1) how the balance between the Insc-bound and the NuMA-bound LGN pools is set in mitosis, and 2) how Insc interacts with polarity proteins. This analysis will shed light on the fundamental aspect of how the mitotic spindle communicates with cortical polarity, and clarify to which extent it contributes to fate specification in ACDs. An emerging concept in the spindle orientation field is that the actomyosin cortex forming during mitotic rounding plays an active role in transducing external cues that instruct spindle positioning. However the mechanistic link between cortical actin and the spindle orientation machinery is still missing. We have evidence that LGN binds directly to the actin-binding protein Afadin, a junctional protein required for epithelia organization. Here we propose to investigate the function of the Afadin:LGN interaction in symmetric and asymmetric mitoses. These studies will generate evidence to integrate Afadin in the model of spindle orientation in vertebrate epithelia and in breast stem cells. Hypothesis: Described in Aims Preliminary data: For in vitro studies we have purified NuMA:LGN:Gai complexes, and reconstituted Par3:Insc:LGN:Gai assemblies. We have recently obtained a preliminary crystallographic structure of LGN:Insc, and well diffrancting crystals of the LGN:Afadin assembly. To probe our models in living cells, we have raised monoclonal antibodies against NuMA, LGN, Par3, Insc, Afadin and Dlg1, and developed protocols to visualize mitoses in Caco-2 cysts. Materials and Methods To build models of how cortical force generators are coordinated with polarity in asymmetric divisions, we will adopt a multidisciplinary approach. We will characterize the architecture of reconstituted complexes by X-ray crystallography and their thermodynamical properties by biochemical and biophysical methods. To address the role of Aurora-A in spindle placement, we will perform in vitro kinase assays and use chemical inhibition in cells. The activation and cortical anchoring mechanisms of NuMA:LGN:Gai will be studied in three-dimensional organotypic cultures of Caco-2 cells, and in primary breast stem cells imaged at single-cell resolution. We will also employ sphere-forming assays to estimate how defects in spindle orientation impact on breast stem cells proliferative potential. Studies in breast stem cells will be performed in collaboration with Prof. Pier Paolo Di Fiore. 09/07/ / 5

125 RF Identification of new therapeutical targets of cancer stem cells: study of the mechanistics of mitotic spindle alignment to cortical polarity in mammary stem cell asymmetric divisions MAPELLI MARINA Biomedical/Biomedica Istituto Europeo di Oncologia Impact and Translational Implications A new concept in the cancer field is that cancer stem cells may be responsible for relapse and resistance to anticancer therapies. If so, investigating the mechanisms ensuring stem cell asymmetric division is instrumental for the identification of new drug targets for therapeutical intervention. We believe the detailed information delivered by structural and biochemical analyses combined with studies in stem cells might eventually contribute to the identification of novel anticancer therapies.

126 Role of serotonin in modulating L-DOPA-induced dyskinesia BANDO 2013 Progetti Ordinari RF RF Picconi Barbara 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: Brain Disorders and Clinical Neuroscience Clinical Neuroscience and Neurodegeneration - CNN Project Keyword 1: Project Keyword 2: Project Keyword 3: Parkinson s disease and other movement disorders (Huntington s, Dystonias, Ataxias). L-DOPA-induced dyskinetic animal models electrophysiology 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/Lavoratory Role in the project 1 Fondazione Santa Lucia Neurophysiology Principal Investigator 2 Fondazione SDN CEINGE Collaborator 3 Università degli studi di Pisa Department of Biology, Unit of Cellular and Developmental Biology, Collaborator Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 usiello Alessandro Fondazione SDN Responsabile Unità Operativa 19/02/ Pasqualetti Massimo Università degli studi di Pisa Responsabile Unità Operativa 03/09/ /07/ / 6

127 Role of serotonin in modulating L-DOPA-induced dyskinesia BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Picconi Barbara Fondazione Santa Lucia Background and Significance Serotonergic system has been demonstrated to play a crucial role in L-DOPA-induced dyskinesia (LID) (Rylander et al., 2010; Carta and Bezard, 2011; Rylander, 2012). In line, the use of eltoprazine a serotonin 5-HT1A/1B receptor agonist reduces L-DOPA-derived extracellular dopamine (DA) levels (Lindgren et al., 2010), and suppresses LID in rats(carta et al., 2007; Bezard et al., 2013) as well as in MPTP-treated monkeys (Munoz et al., 2008). Besides to the modulatory effect on DA-release under parkinsonian-like condition, we cannot rule out that serotonergic system may also regulate the severity of LID through other mechanisms, including neurotrophic factor release (Rylander et al., 2010). Therefore, based on the complex and still unclear role exerted by serotonergic system on L-DOPA-related motor disturbances, we aim to: i) clarify, beyond serotonin (5-HT) innervation, the selective contribution of 5-HT itself in controlling the onset and the expression of LID, and ii) determine the influence of 5-HT on the anti-dyskinetic properties associated to eltoprazine administration in a mouse model of PD. To accomplish these issues, we will take advantage of a knockout for the rate-limiting enzyme of 5-HT synthesis tryptophan hydroxylase 2 (Tph2). Genetically introduced enhanced green fluorescent protein (egfp) into the Tph2 locus (Tph2::GFP knockin mice) will allow to highlight the serotonergic system independently of 5-HT immunoreactivity (Migliarini et al., 2013). Specific aims Aim 1: Aim 2: Investigate the involvement of 5-HT depletion in induction and severity of L-DOPA-induced dyskinesia (LID). We will use mice genetically depleted of central 5-HT. Control and mutant mice will be sham-operated or lesioned with 6-OHDA, and 6 weeks after surgery they will be treated for 9 days with L-DOPA. Anti-akinetic and dyskinesia will be scored. Subsequently, mice will be evaluated i) for altered striatal synaptic transmission and bidirectional synaptic plasticity, ii) for TH immunostaining and 5-HT circuitry innervations, iii) for striatal Golf/PKA/DARPP32, perk, mtor signaling pathways and BDNF expression. Evaluate the neurobiological correlates associated to the anti-dyskinetic properties of 5-HT1A/1B agonist treatment and the influence of 5-HT on its therapeutic action. To accomplish this task lesioned control and Tph2 KO mice will be treated with L-DOPA + Eltoprazine (5-HT1A/1B agonist). Afterwards, possible antidyskinetic effects will be scored. Subsequently to behavioral studies, we will address in treated animals the effects on synaptic plasticity, signaling transduction and anatomical correlates. Aim 3: - Hypothesis: Neuronal alterations identified in dyskinetic subjects leading to the concept that a combination of maladaptive cellular and molecular changes are needed to develop dyskinetic movements (Picconi et al., 2003; Calabresi et al.; Rylander et al., 2010). Emerging evidence put forward the role of 5-HT transmission in LID (Tanaka et al., 1999; Carta et al., 2007; Eskow et al., 2009). Eltoprazine is the elected candidate with a mixed 5-HT1A/1B agonism profile (Bezard et al., 2013), recently employed in a first explorative study in dyskinetic patients at the University of Lund. Despite its translational relevance, the neurobiological mechanisms underlying the anti-dyskinetic effect of 5-HT1A/1B agonists still remain an unsolved question. To fill this gap, herein we propose to investigate through a multidisciplinary approach ranging from behavior, electrophysiology to signal transduction and neuroanatomical studies the consequences associated to the abilities of eltoprazine to prevent LID in chronically L-DOPA-treated PD mice. Moreover, taking advantage of Tph2 KO mice, which concomitantly display lack of 5-HT synthesis and an intact serotonergic innervation, we also intend to reveal the role of this neurotransmitter on the therapeutic properties of eltoprazine. Preliminary data: Preliminary data indicate that i) eltoprazine does not alter per sè intrinsic electrophysiological properties and behavior of control and parkinsonian animals, ii) BDNF expression protocol by WB 08/07/ / 6

128 Role of serotonin in modulating L-DOPA-induced dyskinesia BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Picconi Barbara Fondazione Santa Lucia has been developed in mice, iii) 1 week treatment with eltoprazine does not alter long term depression in control animals, iv) eltoprazine applied for 1 week in dyskinetic animals significantly reduced LID, v) bidirectional synaptic plasticity recorded from striatal spiny neurons of animals chronically treated with L-DOPA plus Elto appears to be modified by the 5-HT/DA pharmacological manipulation. Materials and Methods Tph2::eGFP knock-in mice. See Migliarini et al OHDA model. Sham-operated controls and 6-OHDA-lesioned control and KO mice will be obtained as previously reported (Errico et al., 2011). Treatments. 6-OHDA lesioned mice will be treated for 9 days with one injection per day of L-DOPA (10mg/kg). The effect of Eltoprazine (Elto) will be examined by treating parkinsonian for 9 days with L-DOPA plus Elto (0.3mg/kg). Dyskinesia will be assessed with a pharmacologically validated mouse model (Lundblad et al., 2004). Electrophysiology. Whole-cell patch-clamp recordings will be utilized to study intrinsic and synaptic properties of striatal neurons in corticostriatal slices in all the experimental groups (Bagetta et al., 2010,2012). WB and immunostaining. Striatal Golf/PKA/DARPP32, perk, mtor signaling pathways and BDNF expression will be performed as in Santini et al., 2010 and Subramaniam et al., In situ hybridization. ISH will be performed for BDNF analysis. Impact and Translational Implications We will clarify the involvement of 5-HT in induction and severity of LID, and iwe will evaluate the neurobiological correlates associated to the anti-dyskinetic properties of eltoprazine, and finally iii) reveal the influence of 5-HT system on 5-HT1A/1B agonists therapeutic action. A better understanding of the complex synaptic and molecular mechanisms underlying 5-HT system modulation occurring at striatal level during LID induction appears to be instrumental towards new pharmacological tools. 08/07/ / 6

129 RF Improving therapeutic appropriateness of Multiple Sclerosis treatments using biological approaches to personalize therapy and save pharmaceutical spending Bertolotto Antonio Clinical health care research/clinicoassistenziale Piemonte 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. personalized medicine saving in pharmaceutical spending 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/Lavoratory Role in the project 1 Piemonte A.O.U. San Luigi Gonzaga, S.C.D.O. Neurologia 2 - CReSM (Centro Riferimento Regionale Sclerosi Multipla) Project coordinator, human samples collection and assays and data analysis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date Caldano Marzia A.O.U. San Luigi Gonzaga, S.C.D.O. Neurologia 2 - CReSM (Centro Riferimento Regionale Sclerosi Multipla)/PharmD Human samples collection, samples analysis, data analysis 20/07/ Spadaro Michela A.O.U. San Luigi Gonzaga, S.C.D.O. Neurologia 2 - CReSM (Centro Riferimento Regionale Sclerosi Multipla)/PhD Human samples collection, samples analysis, data analysis 10/03/ /07/ / 6

130 RF Improving therapeutic appropriateness of Multiple Sclerosis treatments using biological approaches to personalize therapy and save pharmaceutical spending Bertolotto Antonio Clinical health care research/clinicoassistenziale Piemonte Background and Significance The therapy for Multiple Sclerosis (MS) includes first (Interferon beta: IFNb and Glatiramer acetate), and second line treatments (Natalizumab: NAT and Fingolimod) and the anti-cd20 Abs Rituximab (RTX) used off-label. New drugs approved by FDA/EMA will be soon available in Italy (Pegylated IFNb: PEG-IFNb, teriflunomide and Alentuzumab: ALE). Each of these drugs is effective only in a subset of patients, is administered at a fixed dose and at pre-established time without consideration for pharmacokinetic; some can cause serious adverse events. In some patients Anti-Drug Antibodies (ADA) develop and make them non-responders. All drugs are very expensive, ranging from 7000 to /year. As over patients are under treatment in Italy (50% of the MS population) the total expense is about 400 million /year. Our Institution has a long experience in early biological detection of IFNb- and NAT-non-responders and in pharmacoeconomic evaluations. The present project aims to improve the treatment appropriateness by using biological approaches, in particular: - extending the quantification of ADA to PEG-IFNb, RTX and ALE for early detection of non-responders - quantifying blood drug levels and biological activity markers to optimize dose and timing of infusions of NAT and RTX - testing if T-cells adhesion-molecule expression can detect the risk of serious adverse events during NAT treatment Each approach will be analyzed by pharmacoeconomic to achieve the best cost-effectiveness. Specific aims Aim 1: Aim 2: Aim 3: Early detection of non-responders. Detection and titration of Binding/Neutralizing Antibodies (BAbs/NAbs) against NAT and RTX will be performed. Cross reactivity between NAbs against IFNb and PEG-IFNb and between different anti-cd20 monoclonal will be evaluated. The biological activity of PEG-IFNb will be tested by analyzing mrna expression of a specific biomarker. Biological data will be correlated with the responsiveness to the treatment measured by EDSS, MRI and clinical activity. An economic analysis will be performed to evaluate the therapeutic appropriateness. Optimization of dose and time of infusion of NAT and RTX. NAT and RTX responders are defined as patients without clinical activity and without new MRI lesions. NAT and RTX serum concentration in samples stored in our bio-bank will be correlated with responsiveness. The frequency of RTX infusion will be decided according to the number of circulating CD19+ and CD27+ B cells detected by FACS and by a new molecular (RT-PCR) approach set up in our center. This letter method could be used also for the new anti-cd20 drugs. The cost of traditional infusions will be compared with the optimized procedures. To analyze L-selectin expression in T-cells to detect the risk to develop Progressive Multifocal Leukoencephalopthy (PML). In fact, the major adverse event of NAT is PML that involves about 1 out of 1000 patients. The risk of PML limits the use of the highly effective NAT and PML early detection and management are very expensive. Recently, Schwab et al. (Neurology 2013) investigated the influence of NAT on adhesion molecules in T-cells and found a very low expression of L-selectin in patients who will develop PML. Hypothesis: Treatment efficacy depends on drug availability and its biological activity. Dose and timing of infusion can be personalized. Biological marker can stratify the risk of adverse events. Consequently, quantification of drugs level, ADA, biological activity and biomarkers can early identify non-responders patients, improve treatment efficacy and reduce the risk of adverse events. These biological methods can improve allocation of a large amount of pharmaceutical spending. Preliminary data: - CRESM lab is the unique certified Italian lab to evaluate the presence of anti-ifnb and anti-nat Abs since 2002, is able to evaluate the IFNb and RTX biological activity by measuring MxA and CD19 mrna levels respectively, to quantify the NAT and RTX serum concentration - Sera, frozen cells and mrna samples of more than 700 patients at the beginning and during 10/07/ / 6

131 RF Improving therapeutic appropriateness of Multiple Sclerosis treatments using biological approaches to personalize therapy and save pharmaceutical spending Bertolotto Antonio Clinical health care research/clinicoassistenziale Piemonte treatment with IFNb, NAT or RTX are available in the CRESM bio-bank - L-selectin expression was evaluated in 200 MS treated patients - Neurological examination and MRI are performed every 6/12 months in treated patients Materials and Methods patients treated with IFNb (n = 300), NAT (n = 150) and RTX (n = 50) - evaluation of the presence of Neutralizing Antibodies against PEG-IFNb, NAT, anti-cd20 using approved and validated assays and/or commercial kits (CPE, ELISA) - evaluation of drug concentration by ELISA, RIA, HPLC - evaluation of in vivo mrna expression of MxA and CD19 by Real-time PCR - evaluation of CD19, CD27 and L-selectin by FACS - clinical and MRI evaluations - cost-effectiveness analysis Impact and Translational Implications Results of the study might lead to a better management of MS patients: personalized and thus more effective treatments not only allow direct savings in pharmaceutical spending, but also reduce the indirect costs because of slowing of accumulation of disability. This translates in a better allocation of funds for health costs. 10/07/ / 6

132 Role of extracellular vesicles in bone tumour pathogenesis: implications for therapy BANDO 2013 Progetti Ordinari RF RF Muraca Maurizio Biomedical/Biomedica Ospedale pediatrico Bambino Gesù 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 Skeletal Biology Development and Disease - SBDD Project Keyword 1: Project Keyword 2: Project Keyword 3: Studies of molecular pathogenesis and biology of osteosarcoma, in vitro studies and animal models of the effects of primary tumors and metastasis to bone on function. Extracellular vesicles Chemotherapeutics 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/Lavoratory Role in the project 1 Ospedale pediatrico Bambino Gesù Regenerative Medicine 2 University of L'Aquila Department of Biotechnological and Applied Clinical Sciences Coordinator of Project Consultant and Supervisor for in vivo experiments Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Teti Anna Maria University of L'Aquila Consultant and Supervisor for in vivo experiments 2 28/03/ Cappariello Alfredo Ospedale pediatrico Bambino Gesù in vitro and in vivo experiments 25/05/ /07/ / 6

133 Role of extracellular vesicles in bone tumour pathogenesis: implications for therapy BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Muraca Maurizio Ospedale pediatrico Bambino Gesù Background and Significance Extracellular vesicles (EVs) recently emerged as potent means of physiological and pathological cell-cell communication. They are nm sized lipid bilayers, encompassing intracellular and membrane proteins, cytoskeletal components and nucleic acids, dramatically affecting target cells by various means. EVs are also released in biological fluids and are considered new biomarkers of diseases to be exploited for diagnostic purposes. They promise to be a versatile biotechnological tool for smart drug delivery as well. Bone and bone marrow are crowd sites in which cell-cell communication is essential for their physiological processes. This microenvironment is also a fertile soil for cancer dissemination. Bone tumours exist as primary or metastatic lesions that disrupt the balanced activities of resident cells. Crosstalk between tumour and bone cells is crucial for tumour survival and progression and, so far, little is known on EV involvement in bone tumour pathogenesis. Tumour cells are important EV producers and tumour EVs may interfere with the normal communication between the bone/bone marrow cells that largely contribute to the delivery of tumour-promoting factors, such as growth factors, enzymes and mirnas. Bone tumours are generally incurable and currently available treatments are not efficient in prolonging lifespan. Therefore, a deeper understanding of EV biology alongside EV-based biotechnological approaches could help develop effective bone tumour therapies. Specific aims Aim 1: Aim 2: Aim 3: To investigate the involvement of EVs in the transfer of the osteoblast factor Receptor Activator of NF-kB Ligand (RANKL) to target cells and in the activation of osteoclast bone resorption and angiogenesis during bone tumourigenesis. To characterise tumour-induced osteoblast EV RNA and protein profiles to identify molecular pathways and biological processes deregulated by bone malignancies. To employ osteoblast EVs loaded with chemotherapeutics to antagonise bone tumour growth. Hypothesis: We aim at understanding the involvement of EVs in the development of bone malignancies. We hypothesize that EVs contribute to the disruption of the bone cell communication processes, subverting bone turnover and angiogenesis. We will identify molecular pathways involved in EV tumorigenic functions and will exploit their specific tropism to create new methods to target and combat bone tumours. Preliminary data: We have accumulated evidence that EVs are involved in bone biology. We observed that osteoblasts seeded in transwells (pore size 1µm) exchanged fluorescent probes overtime, probably by EVs. EVs were isolated from osteoblast culture media by ultracentrifugation followed by sorting by FACS. Transmission electron microscopy showed that they were intact, irregularly shaped, sized µm and well preserved. FACS analysis showed that 50% of EVs presented on their surface the most potent pro-ostoclastogenic cytokine, RANKL. This EV population labelled with cytoplasmic and membrane fluorescent probes fully integrated with target osteoblasts, suggesting that both membrane-bound and intravesicular components were transferred to target cells. The RNA-specific linker Syto RNA Select revealed the presence of RNA inside the EVs that was transferred to target cells. RANKL-positive EVs injected intraperitoneally in newborn WT CD1 mice delivered fluorescent lipophylic probe especially in bone suggesting potential use for systemic targeted therapy. RANKLpositive EVs also dose-dependently triggered osteoclastogenesis in RANKL KO mice suggesting in vivo biological activity. Osteogenic sarcoma cell line, U2OS and SAOS2, communicated by EVs with osteoblasts, monocytes and endothelial cells transferring fluorescent probes. Osteoblast EVs loaded with the chemotherapeutics doxorubicin (DXR) incubated in vitro with tumour cells induced tumour cell death similar to standard treatment with 1uM of free DXR. HPLC preliminarily revealed that DXR-loaded EVs efficaciously shuttled about 3 ng (10 nm) of DXR to target cells, 11/07/ / 6

134 Role of extracellular vesicles in bone tumour pathogenesis: implications for therapy BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Muraca Maurizio Ospedale pediatrico Bambino Gesù suggesting an enhanced antitumoural potency of DXR when encapsulated in EVs. Materials and Methods The study will be performed in vitro and in vivo. For EVs isolation, primary cells (murine osteoblasts, monocytes) and cell lines (endothelial E9, osteogenic sarcoma U2OS and SAOS2) will be maintained for 6 days in serum-free medium. Supernatants will be ultracentrifuged ( g, 1h, 4 C), and pellets stained with CMFDA/PKH26 probes and sorted by FACS (gate µm). EVs will be immunophenotyped and evaluated by confocal and electron microscopy, FACS, RT-PCR, Western blot, Pathway finder RT-PCR and kinome assays. In vitro, DXR loaded EV anti-tumoural effects will be evaluated by DAPI or TUNEL staining, MTT and Annexin V detection. Mice will be injected intraperitonally with EVs at various densities. For tumour model, intratibial and subcutaneous tumour cell injections will be performed in atymic mice. Survival, cachexia, in situ tumour incidence, incidence of metastases, osteolysis, bone structural parameters, fibrosis, angiogenesis and histological indexes will be analysed. Impact and Translational Implications Primary bone malignancies (1/ ) have poor overall survival rates, and early surgical removal remains the only approach to save patients. Our project could open the avenue to new strategies for smartly targeting bone tumours, thus establishing a solid foundation for the translational significance of our results. Since the life expectancy and patients' quality of life could be very poor, any improvement would be a great scientific and ethical value that could bring real benefits to patients. 11/07/ / 6

135 RF ASSESSMENT OF THE PATHOBIOLOGICAL ROLE, DIAGNOSTIC AND PREDICTIVE VALUE OF THE NOVEL CYTOKINE IL-30 IN PROSTATE CANCER Di Carlo Emma Biomedical/Biomedica Abruzzo 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 Prostate cancer Interleukin-30 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/Lavoratory Role in the project 1 Abruzzo 'G. d'annunzio' University, Department of Medicine and Science of Aging, and CeSI Aging Research Center, Chieti, Italy. 2 Abruzzo 'G. d'annunzio' University, Department of Medicine and Science of Aging, and CeSI Aging Research Center, Chieti, Italy. 3 'Alma Mater Studiorum' University of Bologna, Italy Department of Experimental, Diagnostic and Specialty Medicine, Laboratory of Immunology and Biology of Metastasis. Study conception and design. Analysis of human samples, in vitro experiments, data collection and interpretation. Biostatistical analyses. In vivo experiments in murine models. 10/07/ / 6

136 RF ASSESSMENT OF THE PATHOBIOLOGICAL ROLE, DIAGNOSTIC AND PREDICTIVE VALUE OF THE NOVEL CYTOKINE IL-30 IN PROSTATE CANCER Di Carlo Emma Biomedical/Biomedica Investigators, Institution and Role in the Project Abruzzo Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Manzoli Lamberto Abruzzo He will perform biostatistical analyses and will discuss the results with the PI. 28/10/ Lollini Pier Luigi 'Alma Mater Studiorum' University of Bologna, Italy His Lab will carry out xenograft experiments. He will provide his outstanding experience in the biology of cancers and will discuss with the PI all data obtained in the present project. 03/05/ Sorrentino Carlo 'G. d'annunzio' University, Chieti, Italy. M.D., Pathologist, Resident in Clinical Biochemistry. He will perform cell cultures, microarrays and next-generation sequencing. 05/05/1976 Background and Significance Prostate cancer (PC) is the second most common cause of male cancer-related deaths. Mortality for PC is due to metastatic disease driven by both genetic alterations and multiple signals from the tumor microenvironment. Discrimination of molecular pathways driving tumor growth and progression is crucial to identify novel prognostic markers and targets for treatments. The Interleukin(IL)-27 cytokine subunit p28, known as IL-30, is a 28 kda protein recently recognized as a novel cytokine endowed with its own properties, mainly produced by activated APCs. Its involvement in cancer biology has recently become the subject of intense investigation. It has been shown to both suppress the anti-tumor effects of IL-27 in colon cancer and reduce the survival of colon cancer bearing mice. We have recently provided evidence that IL-30 expression by PC epithelia or Tumor- and Lymph-Node-Infiltrating-Leukocytes correlates with advanced disease grade and stage, and that IL-30 exerts pro-tumor activity in PC cells by stimulating their proliferation, down-modulating the tumor suppressor CMTM3 gene and up-regulating CMKLR1/chemR23 expression driving cancer cells towards chemerin-rich lymph nodes. IL-30 may affect both epithelial and stromal compartments via the IL6Ra(gp80)/gp130 receptor complex, whose expression increases during PC progression. Since it is the new microenvironmental hallmark of PC progression, understanding of its biological functions may have profound clinical implications. Specific aims Aim 1: Aim 2: Two objectives: -To assess the in vitro effects of hril-30 on primary prostate epithelial cells, hpc cell lines, normal and cancerassociated fibroblasts, as well as the biological consequences of IL-30 silencing, by specific sirna, in IL-30 expressing hpc cells. Transcriptome, and possible genetic and epigenetic alterations will be analysed by microarray technology and/or next-generation sequencing (RNA-Seq, DNA-Seq, Methyl-Seq). -To assess the in vivo effects of IL-30 silencing in hpc cells, on PC growth and progression in immunodeficient mice. Since preliminary data reveal that IL-30 regulates pluripotency gene expression in hpc cells, we will assess the biological effects of hril-30: -on prostate Stem Cells (WPE-Stem Cells, ATCC) in vitro, and -in immunodeficient mice s.c. injected with these cells, and locally treated with the cytokine, to investigate whether IL-30 conditioning of prostate Stem Cells/or their niche promotes PC onset and 10/07/ / 6

137 RF ASSESSMENT OF THE PATHOBIOLOGICAL ROLE, DIAGNOSTIC AND PREDICTIVE VALUE OF THE NOVEL CYTOKINE IL-30 IN PROSTATE CANCER Di Carlo Emma Biomedical/Biomedica Abruzzo development. Aim 3: Two objectives: -To assess whether IL-30 expression in prostate tissue and serum at biopsy diagnosis may predict the risk of progression during Active Surveillance. -To assess whether IL-30 expression, alone or in combination with other biomarkers, in prostate tissue and plasma, may predict the risk of disease recurrence or resistance to therapy. We have already collected biological samples and clinico-pathological data from 623 men who underwent NPB, and 1145 PC patients. Hypothesis: IL-30 has displayed pro-tumor activity in hpc, and its expression, in the prostate and draining lymph nodes, by immune or cancer cells, correlates with advanced disease grade and stage. It may thus be supposed that IL-30 expression during carcinogenesis or even before tumor onset, plays a critical role in PC growth and progression and patient behavior. Preliminary data: -PC3 and DU145 cells, expressing both IL6Ra and gp130, were cultured for 24h with or without hril- 30 (100ng/ml). RNA was extracted, and qrt-pcr was performed to assess the stemness gene expression profile. hril-30 significantly up-regulated (Student's T test, p<0.05), in both cell lines, expression of SOX2 (12.5 and 9.2 times), SOX9 (16.4 and 6.8 t.), OCT4A (9.0 and 6.3 t.), SHH (8.6 and 11.0 t.), NOTCH1 (9.1 and 5.5 t.), TAZ (12.7 and 8.0 t.) and YAP1 (7.9 and 5.1 t.). NANOG and BMI1 were significantly (p<0.05) up-regulated only in PC3 cells (8.7 and 11.4, respectively) and KLF4 only in DU145 cells (5.6 t). Data were confirmed at protein level by W.B. -IL-30 level was measured by ELISA in the serum samples from 103 untreated non-metastatic (Stage I-III) PC patients, and n=20 healthy controls matched for age. Serum IL-30 level ranged pg/ml in healthy controls, and pg/ml in PC patients. Tissue microarray and qrt-pcr analyses revealed that PC tissues from patients (25/103) showing the highest IL-30 levels ( pg/ml) were embedded with a prominent IL-30 expressing immune cell infiltrate, whereas in most of the remaining, IL-30 expression was scarce to absent. Materials and Methods Expansion of our BioBank with biological samples and clinic-pathological data from subjects undergoing PNB for suspected PC and RP for PC. Microarray technology and/or next-generation sequencing to detect the biological consequences of: IL-30 treatment of prostatic cell cultures, IL-30 silencing (sirna Technology) in hpc cells. Tumor growth experiments in immunodeficient mice injected with IL-30 silenced hpc cells or with prostate Stem Cells and locally treated with IL-30. Autopsy and histo-pathological analyses. Immunohistochemistry and qrt-pcr of biopsy or surgery samples, and ELISA assay on serum samples, to assess expression of IL-30 and other biomarkers Laser Microdissection of IL-30+ versus IL-30- PC foci with the same Gleason grade, and microarray analyses to assess gene expression alterations associated with IL-30 expression. Biostatistics to correlate IL-30 expression with clinic-pathological data, and assess the prognostic value of the cytokine in prostate tissue and serum. 10/07/ / 6

138 RF ASSESSMENT OF THE PATHOBIOLOGICAL ROLE, DIAGNOSTIC AND PREDICTIVE VALUE OF THE NOVEL CYTOKINE IL-30 IN PROSTATE CANCER Di Carlo Emma Biomedical/Biomedica Abruzzo Impact and Translational Implications PC is a leading cause of cancer death in men. Mortality for PC is related to metastatic disease, thus identification of novel biomarkers of disease progression will improve rational clinical decision making and provide targets for modern preventive or therapeutic approaches to PC. The development of this project a) will provide novel knowledge on the role of IL-30 in cancer biology, and b) may provide an easily detectable novel indicator of PC progression and a target for treatment.

139 RF Connecting DNA repair and metabolic alterations of obesity in a search for predictive biomarkers Dogliotti Eugenia Biomedical/Biomedica Istituto Superiore di Sanita' 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 Clinical and Integrative Diabetes and Obesity - CIDO Project Keyword 1: Project Keyword 2: Project Keyword 3: Prevention and treatment of obesity and diabetes utilizing lifestyle, pharmacologic or surgical interventions. twin study DNA damage and metabolism 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/Lavoratory Role in the project 1 Istituto Superiore di Sanita' Dpt. Environment and Primary Prevention Coordination of the project; "omics" approach: DNA damage signaling phosphoproteomics, metabolomics, lipidomics, inflammatory secretomics; data analysis and interpretation 2 Istituto Superiore di Sanità National Center of Epidemiology/Section of Genetic Epidemiology 3 University of Rome "Tor Vergata"/Policlinico "Tor Vergata" Dpt. of Systems Medicine/Center for the cure of obesity Recruitment of eligible monozygotic twin pairs and quantitative genetic analyses; construction of a database Intervention study and follow-up of obese patients, clinico-physiological characterization, collection of biological samples 10/07/ / 6

140 RF Connecting DNA repair and metabolic alterations of obesity in a search for predictive biomarkers Dogliotti Eugenia 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 Nisticò Lorenza Istituto Superiore di Sanità Recruitment of eligible monozygotic twin pairs and quantitative genetic analyses; construction of a database 2 Sbraccia Paolo University of Rome "Tor Vergata"/Policlinico "Tor Vergata" 3 Di Vito Massimo Istituto Superiore di Sanità/Section of Cellular and Molecular Imaging 4 FERRERI CARLA Consiglio Nazionale delle Ricerche (CNR)/ISOF Bio Free Radicals Intervention study and follow-up of obese patients, clinico-physiological characterization, collection of biological samples Metabolic profiles by 1H-NMR of sera from obese and lean subjects; data analysis and interpretation Membrane lipidomics of erythrocytes by gas chromatography; data analysis and interpretation 25/02/ /09/ /11/ /12/1957 Background and Significance Obesity is a high prevalence multifactorial disease with major comorbidities. The rapid increase of obesity incidence in the population with minimal genetic flux underlines the importance of environmental factors in the pathogenesis of obesity and its metabolic complications. While lifestyle-related obesity risk factors are known, the underlying molecular pathways await clarification. Our working hypothesis is that increased production of metabolic byproducts (e.g. ROS or lipid peroxides) generated by chronic excessive caloric intake, causes persistent DNA damage and chronic DNA damage response (DDR) leading to local and systemic chronic inflammation followed by metabolic derangement. To identify differently regulated molecular pathways in obese versus lean individuals we plan to study 2 different models: a) body mass index (BMI) discordant monozygotic (MZ) twins before and after diet-induced weight loss to determine which pathways are modulated independently of inherited genetic factors, and b) massively obese patients before and after bariatric surgery to verify whether and how these pathways correlate with metabolic dysfunction. To this end we will use a wide range of advanced techniques such as metabolomics, lipidomics, inflammatory secretomics and DDR-targeted phosphoproteomics. Fat biopsies in severely obese patients will allow to address the critical issue of the relevance of the surrogate tissue for pathway analysis. Specific aims Aim 1: Aim 2: Using a population of monozygotic BMI discordant twins to examine the effects of diet-induced weight-loss, independently of inherited genetic factors, on novel targets and metabolic pathways responsive to weight loss. In particular, we plan to explore the role of DNA damage accumulation/ddr activation in modulating inflammation and metabolic status and its response to a weight-loss strategy with a "omics" approach. Using massively obese patients before and after bariatric surgery to verify how these pathways correlate with metabolic dysfunction. The availability of fat biopsies and blood from the same individual will allow investigating the role of blood as surrogate tissue for pathway analysis. Aim 3: Using data from metabolomics, lipidomics, inflammatory secretomics, phosphoproteomics to construct an integrated database with clinical analyses to identify biomarkers of the metabolic status for rationalized therapeutic intervention strategies. 10/07/ / 6

141 RF Connecting DNA repair and metabolic alterations of obesity in a search for predictive biomarkers Dogliotti Eugenia Biomedical/Biomedica Istituto Superiore di Sanita' Hypothesis: We aim to test the hypothesis that persistent DNA damage and chronic activation of systems engaged in DNA repair/ddr modulate metabolic parameters and molecular pathways that play a key role in the pathogenesis of obesity and obesity-related metabolic abnormalities. Changes in these pathways between MZ twins and their modulation by diet shall allow to identify how environment in general and diet in particular can play a role in the onset and progression of obesity and its metabolic complications. We will also verify this hypothesis in a group of severely obese patients undergoing massive weight loss after bariatric surgery. We expect to identify biomarkers to be used for the development of rationalized intervention strategies. Preliminary data: Among 6474 MZ twins of the Italian Twin Registry >18 years, 50 pairs have BMI difference>3 with one twin being <25 kg/m2. A battery of DNA damage/ddr assays (e.g. gamma-h2ax, comet assay, cytome assay, telomere length) was performed in peripheral blood mononuclear cells (PBMC) from 22 MZ twin pairs. The most significant correlation was observed between BMI and micronucleus (MN) frequency. MZ co-twin control study has increased statistical power than conventional case control study due to paired analysis. Preliminary data showed that 20 BMI discordant MZ pairs have 80% power to detect a 20% intra-pair variation of residual gamma-h2ax (alpha=0.05), a test less sensitive than the MN assay. In a pilot study erythrocyte membrane lipidomics showed significant differences between overweight and obese subjects. The sensitivity of a reverse phase protein array (RPPA) using phosphospecific antibodies was evaluated for a DDR player, gamma-h2ax as measured by immunofluorescence. The analysis of the metabolic profile by 1H-NMR of a small set of sera revealed significant differences between lean and obese subjects. The procedures for isolating adipocytes from human adipose tissue biopsies and analysing inflammatory markers have been set up. 10/07/ / 6

142 RF Connecting DNA repair and metabolic alterations of obesity in a search for predictive biomarkers Dogliotti Eugenia Biomedical/Biomedica Istituto Superiore di Sanita' Materials and Methods -At least 30 BMI discordant MZ twin pairs will be recruited from the Italian Twin Registry -A diet-induced weight-loss intervention will be performed on overweight/obese twins -Standard clinical tests will be performed in biological fluids -Serum metabolic profiles will be determined by 1H NMR and erythrocytes membrane lipidomics by gas chromatography -Basal nuclear DNA damage and DDR will be evaluated by the micronucleus assay in PBMC -Activation of relevant DDR proteins in PBMC and adipocytes will be analysed by small-scale phosphoproteomics. -Mitochondria copy number will be determined by QPCR -White adipose tissue from 20 obese patients before and after bariatric surgery, will be collagenase-digested to collect adipocytes that will be morphologically and functionally characterized -Adipocytes conditioned media will be processed for cytokines and chemokines profiling on customized arrays. Parallel analysis will be carried out with supernatants collected from matched PBMC Impact and Translational Implications Our proposal may contribute to identify novel biomarkers of metabolic derangements associated with a chronic positive energy balance resulting in obesity and obesity-related diseases. As metabolic health is central to enhance longevity and functional capabilities, the development of new strategies to prevent metabolic disorders will impact on the quality of life and healthcare budgets. 10/07/ / 6

143 RF Molecular characterization of pulmonary sarcomatoid carcinoma, a deadly subtype of lung cancer, for patient management Pelosi Giuseppe Clinical health care research/clinicoassistenziale 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 - Project Classification IRG: Project Classification SS: Genes, Genomes and Genetics Molecular Genetics - MGA Project Keyword 1: Project Keyword 2: Project Keyword 3: Genomes: Genome stability and modification, gene regulatory networks, gene network programming Pulmonary sarcomatoid carcinoma next generation sequencing 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/Lavoratory Role in the project 1 Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Pathology Unit Responsible for the pathology unit 2 Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Tumor Genomic Unit Responsible for molecular characterization experiments 3 Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Thoracic Surgery Unit Contributor to the collection of surgical specimen Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Sozzi Gabriella Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano 2 pastorino Ugo Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano 3 Gasparini Patrizia Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Responsible of coordinating Tumor Genomic Unit, data analysis and writing report Contributor to the collection of surgical specimen Responsible for molecular characterization experiments, analysis data and writing reports 22/04/ /07/ /07/ /07/ / 6

144 RF Molecular characterization of pulmonary sarcomatoid carcinoma, a deadly subtype of lung cancer, for patient management Pelosi Giuseppe Clinical health care research/clinicoassistenziale Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Background and Significance Genetic alterations suitable for targeted therapy are poorly known issues in pulmonary sarcomatoid carcinoma (PSC), an uncommon (3 cases/100,000 people/year in Italy) and deadly family of non-small cell lung cancer (NSCLC), which includes pleomorphic carcinoma, spindle cell carcinoma, giant cell carcinoma, carcinosarcoma and pulmonary blastoma and whose current treatment is disappointing. These tumors are characterized by stable epithelial-mesenchymal transition resulting in sarcoma/sarcoma-like phenotype switching off during epithelial carcinogenesis of common ancestor lesions. Genetic alterations thus far described in PSC have regarded EGFR and/or KRAS mutations, with more isolated insights into p53, CTNNB1, and c-kit mutations or EGFR and FGFR amplification/polysomy. However, little is known about the prevalence of anaplastic lymphoma kinase (ALK) and c-met genes aberrations, two targetable driver mechanisms recognized in about 5% lung adenocarcinoma, and their reciprocal interactions in PSC. While the treatment with ALK and c-met-inhibitors has been giving promising results in a subset of lung adenocarcinoma, it would be clinically warranted to expand this knowledge to PSC, a strikingly lethal lung cancer subtype. Furthermore, an extended genetic assessment of these tumors by means of next generation sequencing (NGS) analysis could actually offer targetable driver mechanisms for therapeutic interventions in the frame of precision medicine. Specific aims Aim 1: Assessing ALK and c-met alterations in PSC. Performing an extensive characterization of ALK and c-met gene status by fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), biochemical analysis with western blot (WB) for downstream transduction signals and qpcr for the relevant mrna content in a large series of 200 PSC. Aim 2: Genomic landscape of PSC with targeted NGS Investigating our large series of 200 PSC by next generation sequencing (NGS) technology according to the IonTorrent AmpliSeq Comprehensive Cancer Panel encompassing all-exon coverage of 409 cancer-associated genes; Aim 3: Establishing primary tumor cell lines and patient-derived xenografts (PDXs) to confirm the biological relevance of these gene alterations Hypothesis: A multiparametric molecular assessment may be strategically useful for getting predictive information potentially helpful for therapy. The relevance of ALK/c-MET co-amplification to the treatment of PSC has not so far been investigated, but could unveil potential therapy options for designing clinical studies. In this frame of mind, NGS technology is expected to discover new driver mechanisms contributing to targetable therapies in PSC. Preliminary data: We have indicated that ALK gene amplification is a non-random event in about 20% PSC, whereas the role of other genes known to be drivers of cancer development (such as EGFR, KRAS, HER2, BRAF, PIK3CA and CTNNB1) was negligible. Interestingly, all ALK-amplified PSC also showed c- MET amplification, while only few c-met-amplified PSC did not show ALK co-amplification indicating that these two phenomena were closely correlated (p<0.0001) [Pelosi et al, Lung Cancer 2012;77:507; Gasparini et al, Mod Pathol 2014;27 (Suppl2):478A]. Preliminary biochemical assays suggested activation of different downstream signal transduction pathways according to the diverse combinations of cytogenetic alterations: ALK/c-MET co-amplified tumors showed a deeper activation of transduction signals compared to tumors showing isolated c-met amplification or only gene copy 09/07/ / 6

145 RF Molecular characterization of pulmonary sarcomatoid carcinoma, a deadly subtype of lung cancer, for patient management Pelosi Giuseppe Clinical health care research/clinicoassistenziale Fondazione Istituto Nazionale per lo studio e la cura dei tumori - co-gain for ALK and c-met suggesting a critical contribution of this interaction to the development of a subset of PSC. Interestingly, co-amplified ALK/c-MET tumors run a worse clinical course as compared to tumors displaying no specific cytogenetic alterations. We were also able to establish and propagate PSC-bearing PDXs with a successful rate of about 33% and to obtain primary cell lines from PSC specimen. Our expertise in NGS technology, which we are using since two years in our diagnostic routine, will be used to study extensively a large series of PSC for ascertaining nonrandom gene driver mechanisms suitable for personalized medicine. Materials and Methods Due to its rarity, several Institutions, all leading centers for lung cancer, will participate in the project by collecting 200 formalin-fixed, paraffin-embedded or fresh-frozen PSC, on which ALK and c-met gene status will be assessed by FISH, IHC, NGS, mrna and biochemical analysis. Mice carrying PDXs will be treated with ALK and c-met inhibitors to test drug sensitivity upon FISH analysis-based alterations. Statistical analysis will be used as appropriate. Impact and Translational Implications This is a molecular, immunohistochemical and biochemical translational study of ALK and c-met and NGS characterization of PSC aimed at finding a more effective therapy for this highly aggressive tumor. The development of in vitro and in vivo models will allow specific drugs to be safely tested in paired PSC tumor patients in order to translate the results directly to the clinical practice and rationalize the economical efforts of the Italian National Health Service. 09/07/ / 6

146 NETosis in atherothrombotic disorders BANDO 2013 Progetti Ordinari RF RF Pauletto Paolo Biomedical/Biomedica Veneto 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: Atherosclerosis and Inflammation of the Cardiovascular System - AICS Neutrophil extracellular traps venous thromboembolism 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/Lavoratory Role in the project 1 Veneto Department of Medicine, Medicina Unit of principal investigator. Recruitment of Interna I^, Cà Foncello Hospital Treviso, patients and collection/analysis of samples. University of Padova 2 Venetian Institute of Molecular Medicine Laboratory of Experimental Diabetology Investigators, Institution and Role in the Project Performing ex-vivo analysis on NETs formation Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Albiero Mattia Venetian Institute of Molecular Medicine 2 Performing ex-vivo analysis on NETs formation 29/06/ FAGGIN ELISABETTA Department of Medicine, University of Padova Collection and analysis of samples 30/09/ /07/ / 6

147 NETosis in atherothrombotic disorders BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Pauletto Paolo Veneto Background and Significance Atherothrombotic disorders, such as acute coronary syndrome (ACS), cerebrovascular disease (CVD) and venous thromboembolism (VTE) are common diseases associated with significant morbidity and mortality. Thrombus formation within the arteries and the veins is a complex pathophysiological event originating from the interplay between prothrombotic status, vascular damage and inflammatory processes. Albeit in the last two decades the contribution of pro-thrombotic factors, coagulation cascade and platelets activation in the pathogenesis of thrombotic disorders has been deeply investigated, some pathogenetic mechanisms are still elusive. In the last few years, clinical and animal studies highlighted the important contribution of neutrophil activation and neutrophil extracellular traps (NETs) formation in the pathogenesis of vascular thrombosis. Neutrophils stimulated by microbes, proinflammatory agents, reactive oxygen species or activated platelets, release their nuclear material, that forms a web-like extracellular network, and then undergo a new type of cell death, called NETosis. These webs, formed by DNA, histones and neutrophils granule constituents are designated as NETs and are implicated in antimicrobial defence. Recent findings indicate that NETs generation could also play a role in the early phase of thrombogenesis. However, limited data are available in humans on NETs formation and NETosis in the setting of ACS, CVD, and VTE. Specific aims Aim 1: Aim 2: Aim 3: To evaluate markers of NETosis in patients with (1.1) acute arterial thrombotic events (acute coronary syndrome and cerebrovascular disease) and (1.2) acute venous thromboembolism (deep venous thrombosis / pulmonary embolism) To evaluate markers of NETosis in patients with clinical pro-thrombotic conditions: diabetes, cancer, orthopaedic surgery To determine whether serum collected from patient belonging to the above-mentioned groups triggers NETosis in vitro, and which are the molecular mechanisms involved Hypothesis: We hypothesize that NETosis and formation of NETs are actively involved in the pathogenesis of venous and arterial thrombotic events in humans. However, no study investigated so far the role of NETs in acute atherothrombosis. We expect to observe increases in circulating markers of NETosis and NET release in subjects with acute ACS, CVD and VTE events. As we hypothesize that the proinflammatory and prothrombotic milieu typically observed in these patients acts as a trigger for NETosis, similar findings are expected in groups of patients predisposed to atherothrombotic events (such as those with cancer, recent orthopaedic surgery, or diabetes). We predict that serum obtained from patients affected by acute atherothrombotic disorders or the above-mentioned predisponsing conditions will induce more NETs generation as compared to serum from healthy individuals. On the whole, this project will allow obtaining information about the contribution of NETosis and NET release in thrombotic complications of vascular disease. Preliminary data: Our group recently demonstrated that neutrophil count is independently associated with VTE risk and CVD risk factors (Rattazzi et al. 2013). The in vitro methods to assess NETosis and the release of NETs from unstimulated and stimulated (with BMSO or PMA) neutrophils have been set up and standardized at our laboratory. Visualization of neutrophils undergoing NETosis is accomplished through triple immunofluorescence analysis of chromatin-histone complexes, Hoechst and neutrophil elastase. A custom ImageJ plug-in has been set up to detect chromatin decondensation and extrusion from the nucleus. Labelling with Hoechst and elastase also enables the detection of NETs. In vitro methods also allows to study the mechanisms inducing NETosis by modulating critical molecular targets. Using this method, we have been able to demonstrate in vitro that high glucose, but not equimolar mannitol, induces an increase in the release of NETs, casting the basis for the 11/07/ / 6

148 NETosis in atherothrombotic disorders BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Pauletto Paolo Veneto study of NETosis in diabetes. This is being evaluated by determining circulating markers of NETosis (extracellular double-strand DNA, oligo/mononucleosomes, and citrullinated H4 histone) in plasma/serum of diabetic and non diabetic patients. Materials and Methods Patients. For aim 1, we will enrol 40 patients admitted for ACS, 40 subjects with acute VTE, 40 subjects with CVD and 40 healthy controls. For aim 2, we will recruit 20 subjects with diabetes, 20 with solid neoplasm, and 20 with recent hip/knee surgery. Each subject will undergo clinical evaluation and blood sampling to assess: D-dimer, inflammatory cytokines, selectins, cathepsins, vwf, and MPO. NETosis assays. Circulating markers of NETosis will be quantified in serum/plasma of patients and controls by using the following assays: oligo/mononucleosomes (Cell Death Detection ELISA), Neutrophil elastase (ELISA), extracellular doublestrand DNA, and citrullinated H4 histone. Neutrophils will be plated in vitro to evaluate the effects of serum from patients on NETosis and generation of NETs. Modulation of NADPH, mtor, PKC, PAD4 will shed light on the mechanisms of patient serum driven NETosis and release of NETs. The latter will be measured by using a triple immunofluorescence protocol. Impact and Translational Implications The project will clarify the role played by NETs generation in atherothrombotic disorders. In particular, we will investigate the contribution of neutrophils activation to the pathogenesis of ACS, CVD and VTE. We will also study the potential involvement of NETs in clinical pro-thrombotic conditions (such as cancer, diabetes and orthopaedic surgery). Our findings will offer the opportunity to identify novel therapeutic targets and recognize subjects harbouring increased risk of thrombotic events 11/07/ / 6

149 RF Validation of a novel non-invasive technique based on real time ultrasound B-mode to assess the jugular venous pulse Zamboni Paolo Clinical health care research/clinicoassistenziale Emilia-Romagna 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: Evaluation of improvements in technologies underlying medical imaging systems, as well as studies of available medical imaging systems to evaluate novel medical applications. Ultrasound technology Jugular venous pulse 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/Lavoratory Role in the project 1 Emilia-Romagna Azienda Ospedaliero-Universitaria di Project coordinating Unit and responsible for Ferrara - Dipartimento Chirurgico - U.O. ultrasound B-mode technique to assess the Programma di Fisiopatologia Vascolare jugular venous pulse. e Day Surgery 2 Emilia-Romagna - Azienda Ospedaliero-Universitaria di Ferrara 3 Emilia-Romagna - Azienda Ospedaliero-Universitaria di Ferrara Dipartimento Emergenza - U.O. Anestesiologia e Rianimazione Universitaria Dipartimento di Emergenza Urgenza - U.O. Pronto Soccorso e Medicina d' Urgenza Unit responsible for central venous pressure assessment. Unit responsible for diagnostic accuracy of the ultrasound B-mode technique measurements in emergency setting (Aim 2). 10/07/ / 6

150 RF Validation of a novel non-invasive technique based on real time ultrasound B-mode to assess the jugular venous pulse Zamboni Paolo Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Emilia-Romagna Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 VERRI MARCO Emilia-Romagna - Azienda Ospedaliero-Universitaria di Ferrara 2 Cisno Caterina Emilia-Romagna - Azienda Ospedaliero-Universitaria di Ferrara 3 De Bonis Pasquale Emilia-Romagna - Azienda Ospedaliero-Universitaria di Ferrara 4 Malagoni Anna Maria Emilia-Romagna - Azienda Ospedaliero-Universitaria di Ferrara Responsible for central venous pressure assessments Responsible for diagnostic accuracy of the ultrasound B-mode technique measurements in emergency setting (Aim 2) Responsible for diagnostic accuracy of the ultrasound B-mode technique measurements in a specialized Unit (Aim 2) 05/12/ /12/ /10/1979 Project manager 07/07/1965 Background and Significance The evaluation of the jugular venous pulse (JVP), defined as the movement of expansion of the jugular veins due to changes in pressure in the right atrium, is traditionally considered the open window to assess central venous blood volume, cardiac preload, presence and type of cardiac dysrhythmias, sepsis and many other emergency pathological conditions, providing an estimate of the true right atrial pressure or central venous pressure (CVP). Physical examination of JVP and direct cannulation of the venous system are both used to measure CVP. However, the classic physical examination with variable inclination of the upper body and the sternal angle as the reference point is not simple to be performed, and its accuracy is no better than 50/60 %, as well as the routine placement of central venous catheters is unfeasible and not without risk. Although several alternative methods to assess CVP either noninvasively or in a minimally invasive approach have been proposed, no one has been currently introduced in routine clinical practice. Relying on an extensive experience in ultrasound (US) technique, our multidisciplinary research team hypothesized to derive the JVP waveform through the US B-mode sequential assessment of the cross sectional area of the internal jugular vein along the cardiac cycle. A preliminary pre-clinic study was performed to develop a proper technique and to assess its accuracy and internal validity. Specific aims Aim 1: Aim 2: To validate in a clinical setting a novel non-invasive technique based on real time ultrasound B-mode to assess the JVP (bed-side post processing analysis). To verify if the proposed method can be transferred from our hemodynamics and fluid dynamics lab to the clinical setting, by using any kind of US equipment widely available in any Hospital in our Country, also by operators without a specific qualification in US technique, after an appropriate training. Aim 3: - Hypothesis: A new method, based on original software for post-processing bed-side analysis of real time ultrasound B- mode, might be able to non-invasively, accurately, rapidly measure the JVP. This might be useful in a variety of multidisciplinary emergency and elective clinical situations. Preliminary data: A preliminary study was carried out in the hemodynamics and fluid dynamics lab of the Programma 10/07/ / 6

151 RF Validation of a novel non-invasive technique based on real time ultrasound B-mode to assess the jugular venous pulse Zamboni Paolo Clinical health care research/clinicoassistenziale Emilia-Romagna di Fisiopatologia Vascolare e Day Surgery of the Azienda Ospedaliero-Universitaria of Ferrara, in collaboration with the Department of Physics of the University of Ferrara, to study the variation of the cross sectional area of the internal jugular vein during the cardiac cycle by means of real time B- mode images sequence. It was also tested if an automatic procedure could reliably substitute a time wasting operator tracing. Thanks to an algorithm and the development of a related original software, the assessment can be performed in post-processing analysis at bed-side. Materials and Methods The study will be prospective and blinded. The new US-JVP method will be compared to the gold standard of direct invasive CVP measurement in patients from an intensive care unit, with operators blinded to the patients' condition and values of the direct measure of CVP. The validation processes will include assessment of the diagnostic power of the US- JVP including evaluation of sensibility, specificity, predictive positive and negative values, accuracy and precision. Repeatability and reliability will be also evaluated. We plan also to evaluate the diagnostic accuracy of the US-JVP measurements in operators not specifically qualified in US technique, before and after an appropriate training. Measurements will be tested and re-tested pre and after the training. The statistical approach and analysis will be followed and carried out by an University Institute of Statistics (subcontract). Impact and Translational Implications In case of positive results, the new US-JVP technique might be proposed in clinical practice for rapid, non-invasive, and cost effective evaluation of CVP by means of equipment widely available in the Hospitals of our Country, and by different operators appropriately trained. Cardiovascular US-JVP assessment could be useful in a variety of multidisciplinary emergency and elective clinical situations. 10/07/ / 6

152 RF CareToy: a smart System for early home-based intervention in infants at high risk for Cerebral Palsy Cioni 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: Therapeutic interventions and brain plasticity: Medical, surgical, pharmacological, and behavioral interventions; plasticity and rehabilitation in the developing brain; clinical studies in children Early intervention Information and communications Technologies 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/Lavoratory Role in the project 1 Fondazione Stella Maris Department of Developmental Neuroscience Design of clinical specification of ICT platform, design of RCT and validation of the study, Enrolment of patients, clinical assessment of infants, designing of rehabilitation packages, MRI acquisitions and data analysis, analysis of the sleep 2 Scuola Superiore Sant'Anna Biorobotic Institute Revision and Designing of ICT platform, mechatronic devises and tele-rehabilitation module, analysis of sensors signals 3 Azienda Ospedaliera Sanitaria Pisana Investigators, Institution and Role in the Project Neonatal Intensive Care Unit Infants' enrolment and assessment Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Dario Paolo Scuola Superiore Sant'Anna Engineer: coordination of designing and building of ICT platform 2 bartalena laura Azienda Ospedaliera Sanitaria Pisana Neonatologist: infant's enrolment and assessment, clinical interpretation of data 27/07/ /07/ Faraguna Ugo Fondazione Stella Maris Sleep studies design and analysis 14/06/ /07/ / 6

153 RF CareToy: a smart System for early home-based intervention in infants at high risk for Cerebral Palsy Cioni Giovanni Clinical health care research/clinicoassistenziale Fondazione Stella Maris Background and Significance Cerebral Palsy (CP) is the largest cause of childhood physical disability. In Italy, every year, there are about 1080 new cases. Long life disabilities associated with CP result in considerable efforts by families and in relevant economic costs for Health Service. The combined use of Prechtl's General Movement Assessment (GMA) and Magnetic Resonance Imaging (MRI) has high sensitivity and specificity in prediction of CP since the first early months of life. Early diagnosis allows beginning Early Intervention (EI) during the critical period of brain plasticity improving neurodevelopmental outcomes and enhancing brain plasticity. Different kind of EI programs have been used in the clinical setting demonstrating promising results but more studies, mainly Randomized Clinical Trials (RCT), are needed. In any case, providing a treatment intensive enough and at affordable costs is very critical, even in developed countries. Biotechnologies and tele-rehabilitation could represent an immediate and promising approach. Recently, the CareToy (CT) Project has developed a new technological smart modular system as a tele-rehabilitation tool for EI in infants. CT allows an intensive, individualized, home-based and family-centered intervention, managed remotely by trained clinical staff. Finally, we propose a novel non-invasive approach to monitor the biological effects of remote EI on brain maturation in CP patients, by studying quantity and quality of motor activity and sleep. Specific aims Aim 1: Aim 2: Aim 3: To develop,on the base of the already existent CareToy (CT) modular system,a new setting of toys and rehabilitation packages suitable for personalize Early Intervention of young infants at high risk for CP To assess,by means of RCT,the effectiveness of CT system to improve the motor development outcome of infants at high risk for CP To identify the aspects of child development (such as visual development, mother-infant interaction, sleep organization) on which CT intervention has the highest impact compared to the standard care(sc) Hypothesis: We have hypothesized that: i) the personalized intensive and active intervention can be obtained by means of biomechatronic devises as the CT system that can combine home-based intervention, personalized treatment, clinical supervision with tele-rehabilitation ii) infant's motor development,even in case of brain lesion,is highly sensitive to intensive stimulation in the first months of life, during the critical period for motor development iii) CT home intervention system is suitable to promote perceptual and cognitive development and also sleep organization Preliminary data: FSM and SSSA are collaborating in the context of an European project ( developing and experimenting the CT System, a smart modular system for infant's rehabilitation at home.the CT platform is mainly composed by biomechatronic toys, a sensorized mat, a vision module, a telerehabilitation module and software for data acquisition, processing and integration. During the CT training infants perform activities to promote reaching, grasping, postural control, visual attention while the CT system captures quantitative data.moreover by tele-rehabilitation, the clinical staff can manage remotely each training, designing individualized exercises, assessing their validity and updating them in order to match individual changes with therapeutic needs. With the collaboration of the Neonatology Unit of Pisa for the recruitment, the CT system has been recently used in a pilot study with preterm infants, born between 28 and 32+6 weeks of gestational age, considered at low risk for neurodevelopmental disorders.the infants aged 2-9 months of corrected age (CA) performed a daily training for 4 weeks (approximately 30 minutes/day).to evaluate the effectiveness of CT training infants have been assessed through neurodevelopmental 11/07/ / 6

154 RF CareToy: a smart System for early home-based intervention in infants at high risk for Cerebral Palsy Cioni Giovanni Clinical health care research/clinicoassistenziale Fondazione Stella Maris tests (Infant Motor Profile, Alberta Infants Motor Scale, Teller Acuity Cards and Bayley III-Cognitive subscale) before (T0) and after the intervention(1 week, T1 and 4 weeks, T2).The preliminary data have showed a good compliance of infants and of their caregiver that have completed the whole training period.the CT system appears a playful and user-friendly environment for the infants and caregiver and very useful for the clinical staff for the remote management of the rehabilitation.moreover, the preliminary clinical results suggest that CT training improve neurodevelopment in this group of infants and that the effects continue in the follow-up Materials and Methods A RCT in infants at high risk for CP on the base of congenital brain lesions detected by MRI will be carried out.infants will be screened during neurological follow up using GMA.After parents' consent, at 3 months of CA infants with abnormal GMA will be randomized in 2 groups, one will perform the CT training while the other will continue SC.During the CT training infants will perform individualized goal-directed activities that will be monitored and modified, according to infants' developmental needs, remotely by clinical staff.it will be around 30 minutes per day and will last 6 weeks.infant Motor Profile will be chosen as primary outcome measure.the time line of assessments will be baseline (in the week preceding the onset of the treatment),t1 and T2(1 and 4 weeks after the end of the treatment) and T3 at 18 months of CA.Moreover, sleep-waking patterns will be assessed by actigraphic recordings,after a concurrent actigraphyic versus sleep EEG validation in the population of interest Impact and Translational Implications This study will contribute to highlight the effects of EI on neurodevelopment of infants at high risk for CP.These effects could represent an important contribution to reduce the costs of health services.ct training could become relatively inexpensive and can expand the accessibility of rehabilitation to a large number of infants.moreover,tele-rehabilitation approach could become a tool i)helpful in the monitoring of intervention efficacy and ii)suitable also for other developmental disabilities 11/07/ / 6

155 RF Axillary Reverse Mapping (ARM).Try to identify and spare lymphatic pathways in breast cancer surgery to prevent breast cancer related lymphedema (BCRL) CARCOFORO PAOLO Clinical health care research/clinicoassistenziale Emilia-Romagna 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 surgery Lymphedema 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/Lavoratory Role in the project 1 Emilia-Romagna Azienda Ospedaliero-Universitaria di Ferrara -Dipartimento Chirurgico - U.O. Clinica Chirurgica 2 Emilia-Romagna -Azienda Ospedaliero-Universitaria di Ferrara 3 Emilia-Romagna -Azienda Ospedaliero-Universitaria di Bologna Azienda Ospedaliero-Universitaria di Ferrara -Dipartimento Neuroscienze e Riabilitazione - U.O. Medicina Riabilitativa Azienda Ospedaliero-Universitaria di Bologna -Dipartimento Emergenza/Urgenza - U.O. Chirurgia generale e dei trapianti Leader Project Co-leader Rehabilitation coordinator Co-Leader enrollment unit 10/07/ / 6

156 RF Axillary Reverse Mapping (ARM).Try to identify and spare lymphatic pathways in breast cancer surgery to prevent breast cancer related lymphedema (BCRL) CARCOFORO PAOLO Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Emilia-Romagna Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 BASAGLIA NINO Emilia-Romagna -Azienda Ospedaliero-Universitaria di Ferrara 2 Taffurelli Mario Emilia-Romagna -Azienda Ospedaliero-Universitaria di Bologna 3 DE TROIA ALESSANDRO Azienda Ospedaliero- Universitaria di Ferrara - Dipartimento Chirurgico - U.O. Clinica Chirurgica 4 BONAZZA SIMONA Azienda Ospedaliero- Universitaria di Ferrara - Dipartimento Chirurgico - U.O. Clinica Chirurgica Co-leader Rehabilitation coordinator 19/11/1948 Co-Leader enrollment unit 31/08/1953 enrollement and methodologist 03/11/1983 enrollement and methodologist 07/07/1983 Background and Significance Breast cancer-related lymphoedema (BCRL) is a debilitating and distressing condition affecting approximately one out five breast cancer survivors. BCRL is a chronic swelling of the upper arm following axillary lymph nodes surgical treatment. It is associated to a significant functional, psychological and social morbidity, with an heavy impact on life quality.several studies reported BCRL incidence between 6.7% and 62.5% for different population cohorts.randomised controlled trials reported that sentinel lymph node biopsy (SLNB) when compared with axillary lymph node dissection (ALND) leads to a significant reduction in postoperative complications. However, the advent of SLNB doesn t solve the problem of BCRL with a concrete chance to develop a lymphoedema after single SLNB around 7%. Health care cost of BCRL rehabilitative treatment is not available in literature yet. Few studies considered incidence, risk factors and treatment costs of BCRL among working-age women after breast cancer treatment, reporting that BCRL population had significantly higher rehabilitative medical costs ($14,877 to $23,167) with twice as much risk to develop BCLR complications, such as lymphangitis or cellulitis when compared to BCRL free population (OR = 2.02, P =.009). Specific aims Aim 1: Aim 2: Aim 3: To evaluate corresponding pathway between SLN breast draining and the ARM node draining for arm and to demonstrate correlation to lymphedema onset when same pathway occurred, in order to prove correlation between ARM node removal and lymphedema onset. The follow-up will be based on instrumental and clinical skills for detection of early signs of BCRL To evaluate possible modifications in arm lymphatic drainage due to SLNB associated with ARM procedure in patients selected for ALND with ARM procedure 3 To assess and estabilish useful large scale methods for further studies the ARM procedure usefulness in reducing lymphedema onset risk both for SLNB and ALND patients. Hypothesis: Axillary reverse mapping (ARM) procedure claims to map and preserve arm lymphatic drainage during ALND and/or during SLNB, reducing BCRL development. ARM is developed as result of assumption that arm s lymphatic pathway isn t involved by metastatic pathway of primary breast cancer. When main drainage lymph node is removed during SLN biopsy, lymphatic drainage disruption onset will be expected 10/07/ / 6

157 RF Axillary Reverse Mapping (ARM).Try to identify and spare lymphatic pathways in breast cancer surgery to prevent breast cancer related lymphedema (BCRL) CARCOFORO PAOLO Clinical health care research/clinicoassistenziale Emilia-Romagna with BCRL risk increase.. When associated to ARM, fluorescence imaging technique is useful for detecting lymphatic drainage from arm and it allows differentiation of fluorescent ARM nodes and/or lymphatics between radioguided detected SLN. Preliminary data: Up to date there are few reports on methods to assess the oncologic safety of preserving ARM nodes during ALND. Studies reported criteria for pathological analysis of ARM nodes, such as intraoperative macroscopic evaluation or intraoperative cytology. Our preliminary experience with ARM included 50 patients, undergoing to ALND for invasive breast cancer.all ARM procedures were radioguided. For 5 patients diagnostic method was improved with ICG (idocyanine green) photodynamic technique. ARM lymph node was identified in 48 patients The targeted limph node was excised. 3 out of 48 limph nodes were positive for metastasis. Due to these preliminary observations, to localize the lymphatics draining of the arm, both with radioactive tracer (radioguided) and ICG (photodynamic guide) techniques should give physicians sensitive and reliable diagnostic method to avoid/reduce BCRL onset risk, without affecting patient survival rate. Materials and Methods Patients enrolled will be subjected to radioguided SLNB associated with identification and preservation of lymphatic drainage of the upper extremity, exploiting the photodynamic technique. If the SLN will coincide with the ARM node identified by the photodynamic technique will be excised for pathological definitive analysis. Patients in group who have SLN positive for metastasis will undergo ALND associated with ARM (radioguided) procedure in order to asses possible alterations in lymphatic drainage pathways of the arm after SLNB procedure.arm lymph node will be isolated from the other axillary lymph nodes and preserved. All patients will sign an informed consent The post-operatory follow up will be conducted in inpatient and outpatient setting (early and delayed surgical complications, early and delayed lymphedema).the power analysis, based on preliminary results, reports that 150 women are required to achieve a power of 0.9 and to detect a significant difference ( <0.05). Impact and Translational Implications Currently, routine clinical, instrumental and sonographical testing have a limited prediction early and certain onset of BCRL, with high level impact on rehabilitation and management of patients affected by primary breast cancer treatment complication. The strength of this project, that make it quickly transferable to the regional health care system is the cost decrease for rehabilitative management of BCRL as main complication of breast cancer treatment. 10/07/ / 6

158 RF Development and implant of the photovoltaic artificial retina in the pig with photoreceptor degeneration: towards the human Phase-1 experimentation Pertile Grazia Biomedical/Biomedica Veneto 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 Biomaterials and Biointerfaces - BMBI Project Keyword 1: Project Keyword 2: Project Keyword 3: New biomaterials and fabrication techniques for tissue engineering, transport and perfusion aspects of tissue engineering, and bioreactors. retinitis pigmentosa artificial retina 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/Lavoratory Role in the project 1 Veneto Oftalmologia, Ospedale Sacro-Cuore Don Calabria, Negrar 2 Istituto Italiano di Tecnologia Department of Neuroscience and Brain Technologies Coordination and Surgical procedure Functional evaluation of the prosthesis 3 Politecnico di Milano Dipartimento di Fisica Engineering of the prosthesis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Ghezzi Diego Istituto Italiano di Tecnologia Functional evaluation of the prosthesis 07/11/ Lanzani Guglielmo Politecnico di Milano Engineering of the prosthesis 17/07/ /07/ / 5

159 RF Development and implant of the photovoltaic artificial retina in the pig with photoreceptor degeneration: towards the human Phase-1 experimentation Pertile Grazia Biomedical/Biomedica Veneto Background and Significance There is currently no cure for genetic degenerative diseases such as Retinitis pigmentosa (RP) leading to a complete loss of photoreceptors, exception made for the epiretinal or subretinal implant of retinal prostheses producing electrical stimulation of the remaining retinal network. However, silicon-based artificial devices were poorly successful so far in effectively restoring sight because of a series of problems including need of power supply, scarce biocompatibility, stiffness, fixed geometries and size of the electrodes, high impedance levels, resistive currents and heat generation. Recently, we documented the suitability of organic photovoltaic polymers for generating an active interface with living cells. We demonstrated that neurons can be grown onto these materials and that such hybrid interface is able, upon light stimulation, to depolarize and induce firing in primary neurons cultured on its surface (Ghezzi et al., Nature Comm. 2011). Subsequently, we demonstrated that the very same organic device was able to restore light sensitivity in explants of blind retinas interfaced with the prosthesis (Ghezzi et al., Nat. Photonics 2013). Our prototype has many advantages with respect to the siliconbased prostheses that are currently on clinical testing, namely higher biocompatibility, no need of power supply, and intimate connection to live tissues thanks to the organic photo-transduction mechanism. Specific aims Aim 1: Engineering of the organic retinal prosthesis for long-term implantation in pig eyes: material selection, design and characterization. We will optimize the interface in terms of organic polymer composition, type, shape, flexibility and porosity of the substrate and protective coatings to improve long-term functioning and large retinal coverage. Aim 2: Aim 3: Surgical implantation of the prostheses and evaluation of long-term tolerability. We will: (i) chemically induce rod/cone degeneration with iodoacetic acid; (ii) functionally analyze lesioned animals to assess the extent of functional impairment four weeks after the lesion; (iii) refine the surgical implant of the optimized device in subretinal position in the degenerate retina of the pig. Physiological testing of the visual function of implanted pigs bearing photoreceptor degeneration. We will: (i) monitor the surgical implant by in vivo imaging and, after the functional tests, by histochemistry; (ii) study the efficiency of the implanted device in rescuing the visual function and acuity by pupillometry, electroretinographic (ERG) and visually-evoked field potentials (VEP) recordings; (iii) evaluate the persistence of the effects over time. The outcome of this objective will be the key milestone to access human experimentation. Hypothesis: The poor success of drug treatments, gene therapies or stem cell transplantations in the cure of retina degeneration encouraged to attempt a restoration of vision using electrical stimulation of the spared retinal network by implanted retinal prostheses. The project aims at transferring our preliminary results obtained in RCS rats to man by optimizing and implanting the organic photovoltaic prosthesis in the eye of the pig with photoreceptor degeneration and study its tolerability and rescue of blindness, as a necessary testing step before approaching the Phase-1 experimentation in man. With respect to silicon retina prostheses, the organic photovoltaic approach is the only one that can achieve a completely autonomous and effective device with similar physiological performances but with the potential of covering large areas of the retina. Preliminary data: We recently implanted the prosthesis in the subretinal space of a rat model of RP (RCS rats). Results demonstrated that the implant is well tolerated for up to 5 months after surgery and yields a full recovery of light-sensitivity as indicated by the restoration of pupillary reflex, electroretinogram, visually evoked field potentials in primary cortex and performance in visually-driven behavioral tests to levels closely similar to control animals. The simplicity of the mechanism of stimulation and the advantages versus silicon-based devices make it a promising solution to be adopted in larger eyes such as the porcine and human eyes. 09/07/ / 5

160 RF Development and implant of the photovoltaic artificial retina in the pig with photoreceptor degeneration: towards the human Phase-1 experimentation Pertile Grazia Biomedical/Biomedica Veneto Materials and Methods We will start with the engineering of the retinal prostheses for the implantation in the porcine/human eye and for covering at least the 50% of the retinal surface. The prosthesis will be implanted in the subretinal space of pigs bearing a chemically induced degeneration of the photoreceptors, by using the surgical procedure called ab-interno approach allowing the unfolding of the prosthesis under the retina. The tolerability of the implant will be followed up to 18 months by in-vivo Optical Coherence Tomography and, at the experimental end, by post-mortem morphological analysis. We will assess the capability of the prosthesis in restoring light sensitivity by monitoring light-evoked pupillary reflexes, electroretinogram (ERG), visual field potentials in the occipital cortex and light-driven behavior in the implanted pigs. All these steps should provide the required validation in order to apply for Phase I clinical trial. Impact and Translational Implications The impact of new and better artificial retina prostheses is extremely high in case of progressive bilateral blindness occurring in the advanced stages of RP. The implantation of such a device in RCS rats already yielded very promising results in terms of long-term tolerability and rescue of light-sensitivity. To come closer to a clinical application in selected RP patients, we propose now to develop the prosthesis and implant it in the human-like eye of the pig.

161 RF A novel structural and functional MRI-derived index of axonal myelination: application to MS and correlation with neurophysiology Cercignani Mara 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 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. g-ratio myelination 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/Lavoratory Role in the project 1 Fondazione Santa Lucia Neuroimaging Laboratory Study conception and design;implementation of MRI acquisition protocols and image analysis pipeline; MRI data collection and analysis Investigators, Institution and Role in the Project 1 Key Personnel Institution/Org./Pos. Role in the project Birth Date 11/07/ / 6

162 RF A novel structural and functional MRI-derived index of axonal myelination: application to MS and correlation with neurophysiology Cercignani Mara Clinical health care research/clinicoassistenziale Fondazione Santa Lucia Background and Significance Multiple sclerosis (MS) is an inflammatory condition affecting the central nervous system (CNS), characterised by inflammation, demyelination and cumulative axonal injury ultimately resulting the accumulation of permanent disability. MS is the most common cause of non-traumatic disability in young adults, with approximately 520,000 sufferers in Europe and 60,000 in Italy. While MRI has become an invaluable tool in the diagnosis of MS, T2 lesion volumes correlate poorly with the clinical status of patients with MS. Quantitative MRI techniques, such as magnetization transfer (MT) and diffusion have been proposed as alternative approaches. Recently it was suggested that the combination of these two techniques can be used to provide measures of the g-ratio, which is the ratio of the inner to the outer diameter of a myelinated axon. The g- ratio is a reliable index of axonal myelination, and, as such, can be related to the physiology and function of an axon. Demyelination, remyelination and axonal degeneration are expected to affect the g-ratio, on turn partially explaining the observed impairment in MS, as well as changes in neurophysiological measures such as evoked potential (EP). Whole-brain g-ratio mapping in humans in vivo has thus the potential to be a sensitive marker of pathology in myelinated axons, and to yield clinically useful information. An optimal g-ratio value, which accounts for speed and fidelity of conduction, was derived to be around 0.7 in the CNS. Specific aims Aim 1: Aim 2: Aim 3: We propose to set up a clinical study named GRACE (G-RAtio mapping and Correlation with Evoked potentials), which will extensively investigate the usefulness of g-ratio mapping across MS phenotypes, and will validate its clinical usefulness by correlating it with visual and somato-sensory EP, routinely performed in MS patients for diagnostic purposes. The primary aim of this project is to implement a clinically feasible MRI protocol designed to measure whole brain g-ratio in patients with MS and to estimate the g-ratio across MS phenotypes. As MS is known to interact with axon myelineation, we expect changes in the g-ratio to occur, and to impact directly on axonal conductivity. We therefore aim to establish the association between the g-ratio in the brain of patients with MS and neurophysiological data obtained from EP (somato-sensory, visual, and motor EP). It is recognised that not all patients with a single episode of inflammation affecting the CNS (Clinically isolated syndrome, CIS) will go on to develop further episodes as define MS. Similarly, predicting the time of progression to secondary progressive form from relapsing-remitting (RRMS) onset is challenging, while a small proportion of patients have a benign course. Currently, we lack reliable biomarkers to predict the disease course and offer an accurate prognosis. A tertiary aim will thus be to evaluate the prognostic value of the g-ratio in patients with CIS and RRMS. Hypothesis: Our main hypothesis is that the g-ratio measured by MRI is more sensitive to MS pathology than other MRI biomarkers, and than MT and diffusion parameters taken in isolation.we expect g-ratio changes to be correlated with neurophysiological measures obtained from EP, and such correlations to be specific to selected brain regions. We also predict that g-ratio changes will evolve with the progression of the disease, and thus we anticipate the g-ratio to increase in the early phases, when demyelination is manily occurring. The complex combination of remyelination and axonal degeneration will be reflected by pseudonormalisation of the g-ratio in more advanced phases. We thus anticipate to observe differences between RR and SP MS patients, and between baseline and follow-up scans in CIS. Preliminary data: We have applied both diffusion and MT to the investigation of MS in several studies, demonstrating the sensitivity of these techniques to tissue damage. Critical for the current research proposal, we have set up a protocol to acquire the MRI data required for g-ratio (quantitative MT and a model of diffusion called NODDI) computation in 40 minutes on a 3T MRI system, indicating the feasibility of this study. Preliminary data were acquired on a group healthy controls, and maps of the g-ratio were 11/07/ / 6

163 RF A novel structural and functional MRI-derived index of axonal myelination: application to MS and correlation with neurophysiology Cercignani Mara Clinical health care research/clinicoassistenziale Fondazione Santa Lucia generated, showing a flat profile, and values within the expected range. Materials and Methods We aim at recruiting 20 patients with relapsing-remitting (RR) MS, and 20 patients with secondary progressive (SP) MS, who will receive a single MRI scan. Twenty patients with clinically-isolated syndrome (CIS) will be also recruited and will recive 2 MRI scans, at baseline, and after 1 year from baseline. A group of 20 healthy participants will also be recruited and receive 2 scans, 1 year a part. All patients with receive motor, visual and somato-sensory EP and MRI on a 3T system. : The g-ratio has been shown to be a function of the fiber volume fraction (FVF) and of the myeline volume fraction (MVF). A method to combine qmt and NODDI towards the estimation of these parameters was presented recently. MVF is linearly related to the macromolecular pool size F, which is computed from quantitative MT. FVF is obtained as FVF=MVF+(1- MVF)(1- viso) vic, where viso and vic are the volume of the isotropic and of the intra-cellular water compartemnts, respectively, both estimated by NODDI. Impact and Translational Implications In the world there are around 2.5 million of people affected by MS, the most common cause of disability in young adults. The absence of any reliable predictor of the individual disease course remains a challenge and a burden to both patients and their caregivers. Our proposal has an excellent potential for providing a new MRI biomarker with increased sensitivity and specificity. Clinicians will have a powerful instrument to help diagnosis, prognosis and patient management, beyond current MRI use 11/07/ / 6

164 RF Assessment of the therapeutic value of targeting cancer stem cells in Numb-deficient breast cancer. Tosoni Daniela Biomedical/Biomedica Istituto Europeo di Oncologia 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: Project Keyword 3: Pathways regulated by oncogenes and tumor suppressors that affect tumor cell phenotype and behavior, such as survival, proliferation, and death Breast cancer, cancer stem cells, epithelial-mesenchymal transition, metastasis Notch, p53 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: For both the patent and the patent application the patent owner is: IFOM Fondazione Istituto FIRC di Oncologia Molecolare Patent number: Patent No. US 7,901,876; Patent Application No. PCT/EP2005/ (Publication n. WO2006/037462) Operative Units INSTITUTION Department/Division/Lavoratory Role in the project 1 Istituto Europeo di Oncologia 2 IFOM Fondazione Istituto FIRC di Oncologia Molecolare Programme of Molecular Medicine - Department of Experimental Oncology Laboratory of Mechanisms of Tumor cells migration D.I. Applicant Institution Operative Unit 2 Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Scita Giorgio IFOM Fondazione Istituto FIRC di Oncologia Molecolare Principal Investigator - Operative Unit 2 09/02/ /07/ / 6

165 RF Assessment of the therapeutic value of targeting cancer stem cells in Numb-deficient breast cancer. Tosoni Daniela Biomedical/Biomedica Istituto Europeo di Oncologia Background and Significance Numb is a potent tumor suppressor in the human mammary gland, able to inhibit the Notch oncogene and concomitantly sustain the tumor suppressor activity of p53 by hindering its Mdm2-mediated degradation (Pece et al., JCB 2004; Colaluca et al., Nature 2008). Indeed, loss of Numb, which is due to its abnormal ubiquitination and proteasomal degradation, is a feature of ~30% of human breast cancers, associated with aggressive disease and poor prognosis (Pece et al., JCB 2004; Colaluca et al., Nature 2008). Our recent unpublished results indicate that the tumor suppressor function of Numb in the mammary gland is linked to its dual role in the homeostasis of the mammary tissue hierarchy: Numb controls asymmetric self-renewing divisions of mammary stem cells (MaSC), while also ensuring proper progenitor maturation and terminal differentiation. Numb dysfunction causes MaSCs to undergo uncontrolled symmetric self-renewing divisions and expansion in number, and induces phenotypic plasticity and stem cell (SC) features at various stages of progenitor maturation. These morphogenetic defects are associated with the appearance of cells with intrinsic SC and epithelial-to-mesenchymal transition (EMT) traits, and enhanced tumorigenicity. Enforced expression of Numb in a mouse model of Numb-KO tumors and in Numb-deficient human breast cancers reduces their cancer SC (CSC) content and inhibits tumor growth, demonstrating that reverting Numb dysfunction is an effective anti-csc strategy. Specific aims Aim 1: Aim 2: Aim 3: 1. To establish whether pharmacological interference with the Notch pathway using gamma-secretase inhibitors (GSIs), which hinder Notch cleavage and activation, and restoration of the p53 pathway with the Mdm2 inhibitor Nutlin, individually or in combination, is an effective strategy to counteract the growth of Numb-deficient tumors by action on the number and tumorigenic potential of CSCs. 2. To address mechanistically whether and how targeted inhibition of Notch and restoration of p53 affects tumorigenesis in Numb-deficient tumors, focusing on the effects of these treatments on the consequences of loss of Numb in the mammary gland, in particular the subversion of asymmetric cell division in the MaSC compartment and the acquisition of SC traits via EMT in the progenitor compartment. 3. To assess whether the EMT-induced acquisition of mesenchymal and SC traits imparts cell motility and invasiveness enhancing the metastatic potential of Numb-deficient tumors, and whether targeting the dysfunction of Notch and p53, alone or in combination, reverts these phenotypes. Hypothesis: We hypothesize that the aggressive behavior and poor prognosis of Numb-deficient breast tumors might reflect their high content of CSCs that display an aggressive phenotype. This view is in keeping with our previous observations that poorly differentiated, highly aggressive breast cancers are enriched in CSCs (Pece et al., Cell 2010). We also hypothesize that EMT related to loss of Numb, besides being associated with maturation defects and the acquisition of SC traits in the progenitor compartment, contributes to the aggressive clinical behavior of Numb-deficient tumors by enhancing the motility/invasiveness of CSCs. On these grounds we predict that pharmacological targeting of the molecular mechanisms responsible for the aggressive phenotype of Numb-deficient CSCs might be an effective therapeutic strategy to cure Numbdeficient breast cancers and curb metastatic spread of the disease. In this scenario, the two Numb-related pathways, Notch and p53, are the candidate circuitries that will be investigated in this proposal. Preliminary data: In a Numb-KO murine model, we have recently found that Numb acts at various levels of the mammary gland hierarchy to counteract the emergence of CSCs: Numb prevents the expansion and acquisition of tumorigenic potential in MaSCs by controlling their asymmetric self-renewing division and proliferative rate; Numb also acts in progenitors to suppress their EMT-associated phenotypic plasticity and to prevent reversion to a SC state. Our data also indicate that Numb ablation in different types of epithelial cells enhances the formation of circular dorsal ruffles (CDR), specialized migratory protrusions associated with increased motility, and facilitates the acquisition of a 10/07/ / 6

166 RF Assessment of the therapeutic value of targeting cancer stem cells in Numb-deficient breast cancer. Tosoni Daniela Biomedical/Biomedica Istituto Europeo di Oncologia mesenchymal mode of invasion in a 3D-matrigel assay. Restoring Numb expression in these models reverts the morphological changes and cellular phenotypes resulting from Numb loss. Materials and Methods We will assess the therapeutic potential of GSI and Nutlin in tumor cells derived from a Numb-KO murine model of breast cancer, and in human primary epithelial cells from Numb-deficient breast tumor biopsies. We will also use mouse and human normal primary mammary epithelial cells, or established cell lines, to study the pharmacological reversion of EMTrelated phenotypes. Using both in vitro and in vivo phenotypic read-outs, we will assess the efficacy of the different drug treatments: i) To inhibit proliferation, motility/invasion and tumor organogenesis in 2D- and 3D-Matrigel assays. ii) To control CSC symmetric/asymmetric self-renewal and life-span using mammosphere-forming and serial propagation assays (Pece et al., Cell 2010). ii) To inhibit the tumorigenic and invasive/metastatic potential of Numb-deficient CSCs in pathogenetically relevant Numbdeficient xenografts coupled with intravital, two-photon imaging (Frittoli et al., 2014, in Press). Impact and Translational Implications Our goal is the pre-clinical assessment of the therapeutic potential of targeting the two Numb-regulated pathways, Notch and p53, as an effective anti-csc therapy in Numb-deficient breast cancers. The use of a pathogenetically relevant Numb- KO murine model and of Numb-deficient human primary tumor cell-derived xenografts will allow direct extrapolation of results to naturally occurring Numb-deficient breast cancers, thus promoting their rapid translation to the clinic. 10/07/ / 6

167 RF Early atherosclerosis in patients with steatosis and with chronic hepatitis C: role of visceral adiposity, procoagulant imbalance and endothelial dysfunction in vascular damage Fracanzani Anna Ludovica Clinical health care research/clinicoassistenziale Fondazione Ca'Granda - Ospedale Maggiore Policlinico 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 Clinical and Integrative Diabetes and Obesity - CIDO Project Keyword 1: Project Keyword 2: Project Keyword 3: Body composition, the mechanisms which regulate it, and the metabolic consequences of distribution patterns of adipose tissue. Steatosis and early atherosclerotic damage Chronic hepatitis C 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/Lavoratory Role in the project 1 Fondazione Ca'Granda - Ospedale Maggiore Policlinico 2 Università degli Studi di Milano 3 Fondazione Ca'Granda- Ospedale Maggiore Policlinico Internal Medicine and Metabolic Diseases Clinical Sciences and Community Health Internal Medicine PI: Design of the study for the identification and implementation of non invasive and not expensive exams for early detection of cardiovascular risk. Oversight of the results and coordination of the different groups. KP: Evaluation of visceral adiposity, microvascular damage, with particular attention in pre diabetic and diabetic status. Data analysis and interpretation. Coordination of laboratory analysis on coagulation imbalance, clinical data interpretation Collection data analysis and interpretation 10/07/ / 6

168 RF Early atherosclerosis in patients with steatosis and with chronic hepatitis C: role of visceral adiposity, procoagulant imbalance and endothelial dysfunction in vascular damage Fracanzani Anna Ludovica Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Fondazione Ca'Granda - Ospedale Maggiore Policlinico Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 tripodi armando Università degli Studi di Milano Coordination of laboratory analysis on coagulation imbalance, clinical data interpretation 2 Airaghi Lorena Fondazione Ca'Granda- Ospedale Maggiore Policlinico collection data of pre diabetic, diabetic and obese patients with steatosis, analysis and interpretation of the results 09/07/ /08/ D'Ambrosio Roberta Fondazione Ca'Granda - Ospedale Maggiore Policlinico 4 Forzenigo Laura Virginia Fondazione Ca'Granda- Ospedale Maggiore Policlinico Coordination of patients with chronic hepatitis C, and clinical data collection, data analysis. 23/11/1980 Data analysis and interpretation of MRI 26/01/1962 Background and Significance Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome which includes obesity, diabetes etc., affects 20-34% of Western countries population and is associated with increased mortality for cardiovascular and liver disease. Chronic hepatitis C (CHC), which presents in 50% steatosis, has been found associated with carotid atherosclerosis. Recently a procoagulant-imbalance has been detected in NAFLD, progressing from simple steatosis to fibrosis and cirrhosis and found associated with higher intima-media thickness (IMT), a well-accepted method of assessing subclinical atherosclerosis. It has been suggested that in endothelial dysfunction, the initial lesion of atherosclerosis, immuno-inflammation plays a key role. Recently ectopic fat accumulation within and around organs such as the heart and the liver have been linked to an increased cardiovascular risk, and it has been reported that epicardial fat correlates with several cardiac comorbidities, including coronary artery disease and left ventricular dysfunction. In addition, it may be possible to identify in patients with NAFLD and with CHC, early cerebral microvascular damage using magnetic resonance spectroscopy (MRS), a non-invasive, ionizing radiation-free analytical technique able to identify and quantify metabolic changes in several brain diseases. Thus these different approaches may help to diagnose and possibly treat early atherosclerosis. Specific aims Aim 1: Aim 2: Aim 3: To evaluate and compare the prevalence and severity of carotid atherosclerosis in a cohort of patients with CHC with different hepatitis virus C genotypes, with and without steatosis, in patients with NAFLD and in controls. To define whether epicardial fat thickness can predict early atherosclerotic vascular damage and whether the amount of epicardial fat thickness correlates with the severity of vascular damage. Results will be evaluated in relation to hepatitis C virus, cardio-metabolic risk factors, endothelial serum markers and liver histology. To investigate the complex interplay between the procoagulants (i.e., thrombin generation drivers) and their naturally occurring anticoagulant counterparts (i.e., thrombin neutralization drivers) operating in vivo in chronic HCV infection and in NAFLD. The results of procoagulants-imbalance will be correlated with endothelium serum markers and early atherosclerotic damage (IMT) and will be defined whether the procoagulant imbalance is able to detect severity of liver disease (fibrosis). To evaluate and quantify brain metabolites, by MRS, to define whether they can identify cerebral micro vessels changes. Results will be correlated with metabolic alterations, with markers of endothelium damage and early carotid atherosclerosis. 10/07/ / 6

169 RF Early atherosclerosis in patients with steatosis and with chronic hepatitis C: role of visceral adiposity, procoagulant imbalance and endothelial dysfunction in vascular damage Fracanzani Anna Ludovica Clinical health care research/clinicoassistenziale Fondazione Ca'Granda - Ospedale Maggiore Policlinico Hypothesis: In this grant proposal we hypothesize that the identification and implementation of non invasive and not expensive exams screening for early detection of cardiovascular risk will reduce inappropriate medical interventions, provide new tools for treatment of early cardiovascular manifestation, decrease disease burden. In addition the possibility to identify specific metabolites expression of microvascular changes by MRS will allow to quantify early cerebral damage in the presence of steatosis. Indeed we think that data from our analysis could help to allow an optimization of both economic and human resources of National Health Care System. Preliminary data: We previously showed that patients with NAFLD have higher prevalence of early atherosclerosis documented by higher values of carotid IMT and more prevalent carotid plaques than controls. Preliminary data in a small series of patients with CHC with or without steatosis and in 125 patients with NAFLD indicate a higher prevalence of carotid atherosclerosis in CHC than in NAFLD (IMT > 0.9 mm, 30% vs 21%, p=0,01, carotid plaques prevalence 37% vs 27%, p=0,02). Procoagulantimbalance, evaluated by high endogenous-thrombin-potential ratio (with/without thrombomodulin) and low Protac-induced-coagulation-inhibition, was recently found by our group in patients with steatosis which increased from simple steatosis to metabolic cirrhosis. The hypercoagulable state was independently associated with higher carotid IMT (O.R. 5.7 (95% C.I ) and severe liver fibrosis (O.R. 6.1 (95% C.I ). A study of brain metabolites, measured by MRS, to define pattern typical of human cerebral vascular disease is ongoing. Materials and Methods We plan to enroll 200 patients with CHC (100 with and 100 without steatosis) and 200 with NAFLD. We calculated the sample size using a non inferiority design, with one-sided test. To evaluate the procoagulant imbalance we calculate a sample size of at least 100 patients with CHC and 100 with NAFLD, (>80% power). At enrolment a) complete biochemical and clinical evaluation, b) ultrasound to evaluate the presence of steatosis, measurements of carotid intima-media thickness (IMT) and detection of plaques, c) transthoracic echocardiogram to measure epicardial fat d) the thrombin generation test in the presence/absence of thrombomodulin and the thrombin generation test in the presence/absence of Protac, e) dosage of endothelial cells dysfunction molecules (intercellular adhesion molecule-1 (ICAM-1), specific molecules (Endocan) by ELISA will be performed. In a subgroup of patients with NAFLD and with CHC quantification of brain metabolites, by MRS will be performed (about 50 cases). Impact and Translational Implications This study should allow to identify patients at risk of developing cardio/cerebral vascular disease and to clear at least in part the mechanisms underpinning this process. This could potentially provide therapeutic tools able to prevent severe comorbidities in patients with NAFLD and CHC. The project will hopefully support the use of screening programs for cardiovascular disease in these patients allowing informed decisions by policy makers. 10/07/ / 6

170 Mother-child immunogenetic interactions in pregnancy and risk of Autism Spectrum Disorders BANDO 2013 Progetti Ordinari RF RF Guerini Franca Rosa Biomedical/Biomedica 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: 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. Immunogenetics Human Leukocyte Antigens 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/Lavoratory Role in the project 1 Fondazione Don Carlo Gnocchi 2 Istituto Neurologico Nazionale IRCCS C. Mondino (Pavia) Molecular Medicne and Biotechnologies Coordinator and Genetic analysis Child Neuropsychiatry Unit 3 University of Sassary Department of Clinical and Experimental Medicine; Pediatic Neuropsychiatry Unit Investigators, Institution and Role in the Project Patients enrolment Patients enrolment Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Chiappedi Matteo Alessio Istituto Neurologico Nazionale IRCCS C. Mondino (Pavia) diagnosis and subjects enrollment 21/09/ Sotgiu Stefano University of Sassary diagnosis and subjects enrollment 15/09/ Bolognesi Elisabetta Fondazione Don Carlo Gnocchi (Milano) Immunologic and genetic investigation 16/03/ /07/ / 6

171 Mother-child immunogenetic interactions in pregnancy and risk of Autism Spectrum Disorders BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Guerini Franca Rosa Fondazione Don Carlo Gnocchi Background and Significance The immune system is suspected to play a role in the impairment of the central nervous system (CNS) that characterizes children with autistic spectrum disorders (ASD)( (Westover et al., 2011 Ashwood et al., 2010; Saresella et al., 2009). Recent evidences indicate a central role for NK cells in the pathogenesis of ASD. Thus, a prevalence of activating Killer immunoglobulin like receptors (KIR)together with their Human leukocyte Antigen cognate (KIR-HLA) complexes was reported in ASD children (Torres et al., 2011) and in their mothers (Guerini et al., 2014), indicating that in children that will develop ASD, NK cells are abnormally activated during fetal development. These data are in agreement with evidences suggesting that immune activation during prenatal life is a risk factor for ASD At the fetal/maternal interface, uterus NK cells that express maternal KIR molecules interact with. classical HLA-C and nonclassical HLA-G molecules on fetal trophoblast; the net effect of these interactions will determine whether NK will or will not be activated. HLA-G, in particular, is responsible for the induction of tolerance during pregnancy; recent results show that this phenomenon is differently modulated by diverse isoforms of HLA-G. Notably, epidemiological data indicate that repeated abortivity, a medical condition associated with immune activation, is common in mothers of ASD children and is predicted by HLA-G isoforms endowed with reduced tolerogenic abilities. Specific aims Aim 1: Aim 2: Aim 3: To define the immunogenetic maternal-fetal KIR-HLA patterns associated with ASD and to investigate whether particular HLA ligands may be specifically involved in ASD development To verify whether isoforms of HLA-G resulting in down-regulation of gene expression and lack of soluble HLA-G in serum are associated with ASD. To address the existence of possible connections between clinical phenotypes and immune genetic patterns in ASD Hypothesis: condition of chronic immune activation and inflammation persisting throughout pregnancy interferes with with physiologic brain development and is associated with the development of ASD. Such immune activation is possibly driven by a milieu favoring NK activation and/or obstacling the phisiological onset of immune tolerance during pregnancy. Notably, epidemiological data indicate that repeated abortivity, a medical condition associated with immune activation, is common in mothers of ASD children. Preliminary data: The observation that autism is likely associated with given alleles encoding within the HLA region was evidenced in our previous studies in two different cohort of ASD patients and their families (Guerini et al., 2009; Guerini et al., 2011). In particular, a 6 Mb region spanning the HLA region ranging from the HLA-DR to the Hemochromatosis (HFE) gene, was analyzed with a particular focus on the alfa and beta blocks microsatellites. Results showed the presence of associations between ASD and a region mapping in the alfa block of the HLA region. This region includes the HLA-G gene; HLA-G, is a ligand of KIR that is involved in the generation of tolerance during pregnancy. Seven different isoforms of HLA-G are generated by an alternative splicing of the primary transcript; most of the HLA-G polymorphisms found in the promoter are located in regulatory regions and, thus, might affect gene expression (Tan et al., 2005, Ober et al., 2006). Notably, a 14-bp sequence in the 3'- UTR associates with significantly reduced levels of both HLA-G mrna (O'Brien et al., 2001; Hviid et al., 2002) and soluble HLA-G (Hylenius et al., 2004). Recent results showed an increased frequency of KIR activating genes and their HLA ligands in ASD, suggesting that the KIR/HLA interactions may be of importance in this condition (Torres et al., 2012). Notably, a strong skewing toward activating KIR genotypes was observed in mothers of ASD children, suggesting that the NK genetic activating milieu seen in ASD patients is a consequence of the KIR distribution in ASD mother, who transmit half of their haplotype to the children (Guerini et al., 2014). 10/07/ / 6

172 Mother-child immunogenetic interactions in pregnancy and risk of Autism Spectrum Disorders BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Guerini Franca Rosa Fondazione Don Carlo Gnocchi Materials and Methods Two distinct cohort of ASD families will be enrolled upon signature of informed consent approved by the Ethical Advisory Board of the Don Gnocchi Foundation. Two hundred ASD children from continental Italy and 150 ASD children from Sardinia, together with their parents and their healthy siblings will be enrolled. DNA will be isolated from peripheral blood or saliva. Genotyping for the 14bp+/14bp- poly morphism in the 3' UTR of the HLA-G gene will be performed by a PCR and run on a 4% agarose gel. KIR polymorphism and HLA- A, B and C alleles will be defined by SSP. HLA-G Exons 2 and 3 will be sequenced, whereas HLA-G* 0106 will be detected by RFLP. HLA-G soluble antigen will be detected in serum of ASD children by ELISA test. Transmission disequilibrium test analysis will be performed to asses specific pattern of HLA transmission to ASD children. Case control analysis will be performed comparing results with published population data. Impact and Translational Implications The definition of prognostic immunogenetic biomarkers of developmental disorders could allow a preventive screening of parents and possibly interventions aiming at reducing immune activation in pregnancy. Moreover a precocious evaluation of neurodevelopment in subjects at risk might also result in precocious rehabilitative interventions which are of crucial importance in children. 10/07/ / 6

173 The prognostic value of collateral flow in acute ischemic stroke secondary to occlusion of intracranial arteries treated by endovascular therapy. RF Stanzione Paolo 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: 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. Acute stroke treatment 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/Lavoratory Role in the project 1 Fondazione Santa Lucia Neuroscience/Stroke Unit Patients selection and recruitment. Neuroradiological assessment. Clinical Follow up. Neurophysiological assessment. Data Analysis.Coordination of the project. 2 Azienda Ospedaliero Universitaria Careggi 3 Spedali Civili di Brescia Università degli Studi di Brescia SOD Neuroradiologia Interventistica U.O. Neuroradiologia Investigators, Institution and Role in the Project Patients selection and recruitment. Neuroradiological assessment. Clinical Follow up. DAta Analysis. Patients selection and recruitment. Neuroradiological assessment. Clinical Follow up. DAta Analysis. Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 salvatore mangiafico Azienda Ospedaliero Universitaria Careggi 2 Gasparotti Roberto Spedali Civili di Brescia Università degli Studi di Brescia Patients selection and recruitment. Neuroradiological assessment. Clinical Follow up. DAta Analysis. Patients selection and recruitment. Neuroradiological assessment. Clinical Follow up. DAta Analysis. 05/09/ /08/ /07/ / 6

174 The prognostic value of collateral flow in acute ischemic stroke secondary to occlusion of intracranial arteries treated by endovascular therapy. RF Stanzione Paolo Clinical health care research/clinicoassistenziale Fondazione Santa Lucia Background and Significance Endovascular therapy has emerged as an alternative strategy in the landscape and management of acute ischemic stroke (AIS). Recently, endovascular devices that allow for mechanical thrombectomy have been developed and are widely used in patients who either are ineligible for IV thrombolysis or in whom persistent large intracranial vessel occlusion (LIVO) is present following IV thrombolysis (IVT). Up to date advanced treatment protocols and techniques have been developed to improve arterial recanalization efficiency. Recent clinical trials have shown that ET is not superior to but as safe as IVT. However, recent results seem to indicate that ET may be a successful therapy especially when applied in patients with LIVO. In this regard a better patients selection for ET is needed to identify which subgroups of AIS could benefit more from this new therapeutic approach. One crucial factor that could determine a favourable outcome after ET may be the collateral flow (CF). CF indicates a physiological mechanism occurring in the first hours of AIS that allows alternative vascular pathways to compensate local critical cerebral blood flow reduction. The developing of a valid CF seems to be one of the main process counteracting rapid cellular death and promoting functional recovery in AIS. Despite its importance, the impact of CF in determining the effect of mechanical thrombectomy in AIS secondary to LIVO has been poorly investigated. Specific aims Aim 1: Aim 2: Aim 3: To evaluate whether CF assessed at admission can be a predictor of good or poor functional outcome in patients with AIS secondary to LIVO treated with mechanical thrombectomy independently from other co-factors (i.e. stroke severity, lesion volume, time from symptom onset etc.) and if CF can widen the time window for ET by sustaining the mechanism of ischemic penumbra. To evaluate if CT angiography (CTA) comparably to the digital subtraction angiography (DSA) may be used to quickly predict a good response to ET in the very early phase of AIS. To evaluate how CF at admission can impact functional recovery in the sub acute phase of stroke as revealed by multimodal assessment of cortical reorganization. Hypothesis: In AIS arterial revascularization to restore perfusion in the ischemic territory remains the principal therapeutic approach. Although the recruitment of cerebral collateral circulation has been explored in chronic hemodynamic insufficiency, relatively little attention has been devoted to the role of pretreatment CF in patients with AIS who are candidates for ET. Recent studies seem to indicate that a relationship may exist between baseline angiographic collaterals and recanalization after ET, independently from the efficiency of reperfusion. Our hypothesis is that compensation of cerebral blood flow reduction induced by CF could maintain the ischemic penumbra for longer time, before irreversible neuronal damage occurred. This in turn should lead to a better response to ET also in those patients presenting with a longer delay from symptom onset. So far preliminary evidence for the role of CF in determining the impact of ET on functional outcome has been obtained by means of DSA. We hypothesize that CTA of cervical and intracranial arteries, a more feasible and fast examination, could provide similar prognostic information as DSA. This would allow a more prompt clinical decision on treating selected subgroups of AIS. Finally we hypothesize that CF assessed at admission could determine a favorable functional cortical reorganization assessed with multimodal neurophysiological, neuroimaging and behavioral assessment, by preserving spared neuronal populations undetectable by standard structural MRI examination. Preliminary data: We have already evaluated the impact of CF on the effects of ET in 45 patients with AIS secondary to LIVO. In particular we retrospectively analyzed pre-treatment brain NCT, CTA of cervical and intracranial arteries and DSA and post-treatment DW-MRI performed within one week of symptoms onset (unpublished data). After dichotomization in good and poor CF we found that good CF was related to higher pre-treatment Alberta Stroke Program Early CT score (ASPECTS) on NCT and SI- 11/07/ / 6

175 The prognostic value of collateral flow in acute ischemic stroke secondary to occlusion of intracranial arteries treated by endovascular therapy. RF Stanzione Paolo Clinical health care research/clinicoassistenziale Fondazione Santa Lucia CTA, shorter thrombus length, more severe symptoms at admission, pronounced clinical improvement at 24 hours, smaller lesion volume and higher post-treatment ASPECTS on DW-MRI, less mortality and favorable outcome at 3 months. Materials and Methods Patients with AIS secondary to LIVO will undergo brain non-contrast CT (NCT), perfusion CT and CTA of cervical and intracranial arteries. CTA will be used to determine a CF grading system whereas CTA-source images (CTA-SI) and perfusion CT will be evaluated for ASPECTS and lesion volume calculation. A clot burden score will used to measure thrombus length and site of occlusion. Patients will undergo DSA, considered the gold standard, to confirm arterial occlusion and to assess CF. They will be treated by ET preceded or not by IVT according to time from symptoms onset. We will assess: i) CF on DSA as an independent predictor of functional outcome; ii) reliability and inter-observer agreement of CF grading system evaluation between DSA and CTA; iii) ASPECTS on CTA-SI as an independent predictor of functional outcome and final infarction size (as assessed by MRI). Functional outcome will be evaluated with multimodal follow up (including MRI) during recovery phase at 1 and 3 months. Impact and Translational Implications AIS represents the second cause of death in developed countries and an increasing economic burden. The only approved therapy for AIS is IVT and a disconcertingly low percentage (< 5%) of patients is treated mostly due to the brief temporal therapeutic window. Moreover the poor response of patients with AIS secondary to LIVO to IVT is well known. This project could provide new perspective on the usefulness of CF in guiding treatment decision-making for patients candidates to ET. 11/07/ / 6

176 EARLY STUDY. MULTICENTER, RANDOMIZED, CLINICAL TRIAL ON THE COMPARISON BETWEEN EARLY SURGERY AND CONVENTIONAL THERAPY IN INFECTIVE ENDOCARDITIS. RF Cecchi Enrico 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: Cardiovascular and Respiratory Sciences Clinical and Integrative Cardiovascular Sciences - CICS Project Keyword 1: Project Keyword 2: Project Keyword 3: Clinical, population, or translational studies of the responses of the cardiovascular system to trauma or surgery: arrhythmias associated with cardiac surgery or cardiopulmonary bypass, cardiac sudden death, resuscitation, stenting, pacemakers;,cardiovascular injury and repair, and myocardial ischemia/reperfusion injury. INFECTIVE ENDOCARDITIS RANDOMIZED TRIAL 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/Lavoratory Role in the project 1 Piemonte Cardiologia Coordinating Center 2 Città della Salute e della Scienza di Torino 3 Città della Salute e della Scienza di Torino Cardiochirurgia SSCVD Epidemiologia Clinica e Valutativa, CPO Piemonte Coordinating Center for Cardiac Surgery On-line database and Statistical Center Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 La Torre Michele William Città della Salute e della Scienza di Torino Contribuited to the study design and coordinates the study for cardiac surgery 29/01/ Castiglione Anna Città della Salute e della Scienza di Torino Contributed to the study design and statistical analysis CRF and database on-line by CPO web site 03/09/ /07/ / 5

177 EARLY STUDY. MULTICENTER, RANDOMIZED, CLINICAL TRIAL ON THE COMPARISON BETWEEN EARLY SURGERY AND CONVENTIONAL THERAPY IN INFECTIVE ENDOCARDITIS. RF Cecchi Enrico Clinical health care research/clinicoassistenziale Piemonte Background and Significance Infective endocarditis (IE) is a disease with a high in-hospital mortality (about 20%) and timely cardiac surgery may be effective in decreasing mortality in IE involving left heart chambers. Guidelines (Habib G; Eur Heart J 2009) have given an indication for emergency or urgent cardiac surgery in all cases with severe acute heart failure, when the infection is not controlled or in presence of large vegetations after an embolic event. In severe heart valve insufficiency without heart failure, cardiac surgery is considered an elective intervention, while in cases with large vegetations >15mm but without other complications, cardiac surgery is indicated on an urgent basis, but clinical recommendation is weak and based on limited evidence: class IIb, level of evidence C. The rationale for early cardiac surgery is based on the hypothesis that a surgical procedure performed in the acute phase is relatively easy and effective in reducing mortality; moreover the early removal of infectious material may prevent the onset of complications related to tissue destruction or embolic events. Unfortunately, with the exception of one small randomized trial (Kang D; N Engl J Med 2012), this hypothesis is largely based on observational studies, suggesting that cardiac surgery is associated with a reduced mortality on the long term, but these studies are at high risk of bias and do not allow to draw reliable conclusions on specific subgroups (Delahaye F; Arch Cardiovasc Dis 2011). Specific aims Aim 1: Aim 2: Aim 3: Because of the lack of good quality evidence supporting early cardiac surgery and the high variability among centres (both in Italy and worldwide), the conduction of a rigorous pragmatic randomized trial comparing an early surgical treatment with a traditional treatment, with a possible delayed surgical intervention, is considered a high priority for improving the outcomes of IE patients. To address this open question we designed a randomized controlled trial with the following specific aims: AIM 1: to evaluate whether an early surgical strategy in selected cases with IE is more effective than conventional therapy to improve 1-year survival without stroke. AIM 2: to compare the cumulative risk of embolic events during admission and in the following year between the two arms. AIM 3: to calculate the costs of the two strategies during 1 year and to perform a cost-effectiveness analysis. Hypothesis: The study hypothesis is that early cardiac surgery compared to elective surgery or medical therapy may decrease 1-year mortality and outcome data in selected patients with IE involving left cardiac chambers. Preliminary data: Unpublished data from the Italian Registry on IE (RIEI) (Cecchi E; J Cardiovasc Med 2008): the RIEI collected data from 680 consecutive patients with definite IE who were admitted to 15 Italian hospitals from August 2006 through August In-hospital mortality was 14%. The IE of the left heart were 556 (82%) and 440 patients (65%) would have been eligible for the present trial. 11/07/ / 5

178 EARLY STUDY. MULTICENTER, RANDOMIZED, CLINICAL TRIAL ON THE COMPARISON BETWEEN EARLY SURGERY AND CONVENTIONAL THERAPY IN INFECTIVE ENDOCARDITIS. RF Cecchi Enrico Clinical health care research/clinicoassistenziale Piemonte Materials and Methods This is a multicenter, randomised open-label trial in IE patients. The study will enroll consecutive cases with definite IE according to Duke criteria through an initial screening. In each participating center, patients will be stratified according to prognostic risk based on the involved valve (native or prosthetic) and the presence of vegetations or valve insufficiency and randomized with a 1:1 ratio. In both arms the medical therapy will be assured according to international guidelines. To demonstrate (with alpha=0.05, beta=0.20) a reduction of mortality without stroke in the experimental arm, with an hazard ratio of 0.50, the study need to enroll 400 patients (with 2 years of accrual an 1 year of follow-up). Two interim analyses will be performed and an independent data monitoring committee will evaluate the need of an early stopping of the study. Impact and Translational Implications This large randomized study has the potential to provide strong evidence in favor (or against) an early surgical treatment of IE patients. In case of positive results, the study will prompt an updating of existing guidelines towards more stringent recommendations and will change the organization of the hospital treatment to assure a timely multidisciplinary prognostic evaluation and an improved survival for these relatively young patients.

179 RF Identification of biological and imaging events that predict the progression of Takayasu arteritis vascular lesions Sabbadini Maria Grazia Clinical health care research/clinicoassistenziale 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: Immunology Hypersensitivity, Autoimmune, and Immune Project Keyword 1: Project Keyword 2: Project Keyword 3: Treatment of immune-mediated diseases: antigen specific and non-specific drug and biologic approaches to tolerance to self or foreign antigens including vaccination, gene therapy, peptide and altered ligand approaches as well as cell-based approaches; development of biomarkers of disease and related activities, and outcome assessments in clinical studies; determinants of response to therapy. large vessel vasculitis biomarkers 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/Lavoratory Role in the project 1 Ospedale San Raffaele - Milano 2 Istituto Superiore di Sanità - Roma Internal Medicine Dipartimento del Farmaco Patient recruitment Analyses of peripheral blood cells 3 Ospedale San Raffaele - Milano Nuclear Medicine Imaging analyses Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Malorni Walter Istituto Superiore di Sanità - Roma 2 Fallanca Federico Ospedale San Raffaele - Milano Collaborator. He will be in charge of autophagy and redox assessment of circulating cells. Collaborator. He will be in charge of imaging studies of vascular inflammation and progression. 04/08/ /04/ /07/ / 6

180 RF Identification of biological and imaging events that predict the progression of Takayasu arteritis vascular lesions Sabbadini Maria Grazia Clinical health care research/clinicoassistenziale Ospedale San Raffaele - Milano Background and Significance Takayasu arteritis (TA) is a chronic relapsing inflammatory disease that comprises patchy granulomatous arteritis of the aorta, the pulmonary artery and their major branches. TA affects young people and is associated with premature mortality and considerable morbidity. Arterial wall remodeling results in stenosis/occlusion or aneurysm formation. TA morbidity and mortality is mainly due to acute cardiovascular complications and to the progression of vascular lesions. Despite its clinical relevance for the clinical outcome, direct information on the inflammatory response within the vessel wall is lacking and the extent of systemic inflammation is used to guide clinical management. Criteria used to assess TA clinical activity have not been validated and do not take into account vascular thickening and/or other radiological variables that could reflect vascular progressive involvement. Moreover criteria for TA activity have a suboptimal sensitivity and specificity and fail to identify patients that undergo vascular progression. Patients treated with biological agents, like tocilizumab, that directly quench the acute phase response in particular require novel tools to assess clinical efficacy. The novel insight on the role of intravascular immunity in guiding vessel wall remodeling and the possibility to study in detail metabolic events and the activation state of circulating cells might offer clues to predict the progression of vascular lesions in TA patients. Specific aims Aim 1: Aim 2: Aim 3: To identify in a prospective clinical study patients with TA that undergo or not vascular progression, using an integrated imaging approach based on PET-CT, magnetic resonance imaging and ultrasonography. To evaluate the ability of cell-associated and soluble biomarkers to correlate with vascular progression on TA patients. We will in particular evaluate the activation state of circulating lymphocytes, monocytes, neutrophils and platelets, their interaction and the presence and characteristics of microparticles derived from leukocytes, platelets and endothelial cells. Sex- and age-matched healthy controls and patients with granulomatosis with polyangiitis (GPA) will serve as controls. Since oxidative stress plays an important role in TA enhancing inflammation and breaking down immune tolerance, and since autophagy, which is defective in a plethora of autoimmune diseases, could contribute to arteritis onset and progression, we will evaluate oxidative imbalance as well as spontaneous and induced autophagy in circulating cells in patients with TA, healthy subjects and control patients with CPA and their association to vascular progression. Hypothesis: The progression of vascular lesions is the main determinant of TA clinical outcome. It reflects poorly characterized immune-mediated events taking place within the vessel wall, which are only partially associated with the extent as systemic inflammation as revealed by acute phase reactants. Recent data suggest that the activation state and metabolic features of circulating blood cells might reflect the maladaptive vessel wall remodeling. Main goal of this study is to verify the ability of clinical, biological and imaging features to predict the development of vessel occlusion or of aneurysms. This will allow to identify the biological pathways involved in the smoldering inflammation that jeopardize vascular integrity in TA, an information critical for more effective therapeutic intervention to be developed, and to define novel criteria to assess TA clinical activity based on vascular involvement, to identify patients that require earlier and more aggressive treatments. Preliminary data: 1. Identification of patients undergoing vascular progression by PET-CT and MRI. 2. Complete depletion of circulating neutrophil granule content occurs in a subset of TA patients and does not correlate with the extent of systemic inflammation. 3. Systemic levels of the soluble pattern recognition receptor PTX3 are higher in TA patients undergoing vascular progression. 4. Autophagy regulation and resistance in the peripheral blood of patients with autoimmune diseases 10/07/ / 6

181 RF Identification of biological and imaging events that predict the progression of Takayasu arteritis vascular lesions Sabbadini Maria Grazia Clinical health care research/clinicoassistenziale Ospedale San Raffaele - Milano is under investigation in our Labs. Materials and Methods This study will rely on the cohort of 51 TA patients at San Raffaele Scientific Institute, Milan. Blood will be retrieved during the periodic visits and patients monitored by PET-CT, MRI and ultrasonography. Data for disease activity (NIH, BVAS, ITAS, DEI-TAK) and the vasculitis damage index will be collected. Ex vivo studies will be carried out on purified mononuclear cells and on neutrophils and platelets using biochemical (western blotting, flow cytometry) or molecular (gene expression profiling for autophagy and cytokine signatures) analyses. Soluble biomarkers will be assessed by ELISA on plasma and serum. Cytometric bead array will be used for cytokines. Microparticles will be retrieved by plasma sequential ultracentrifugation. Sex- and age matched healthy subjects and consecutive patients with GPA will serve as controls. Progressing and relapsing patients will be defined at the end of the follow up period and events correlating/predicting the outcome identified. Impact and Translational Implications The lack of relevant information predicting disease progression/relapse is an urgent unmet need: these patients are those with the worst clinical outcome and require more aggressive and precocious immunosuppressive treatments This need is likely to grow in the next future, given the high costs of biologic drugs. To be able to allocate patients to clusters that might benefit by immunosuppression, disease-modifying drugs, IL6 and TNF pathway blockers would have a major impact on TA management. 10/07/ / 6

182 RF Vitamin D Supplementation on Assisted Reproduction Technology (ART) outcomes: a randomized clinical controlled trial and an investigation of the involved biological mechanisms Somigliana Edgardo Clinical health care research/clinicoassistenziale Fondazione Ca'Granda - Ospedale Maggiore Policlinico LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Environmental diseases, safety of the work place and occupational diseases Project Classification IRG: Project Classification SS: Endocrinology, Metabolism, Nutrition and Reproductive Sciences Integrative and Clinical Endocrinology and Reproduction - ICER Project Keyword 1: Project Keyword 2: Project Keyword 3: Disorders of the female and male reproductive system: pathophysiology, mechanisms, and treatment of polycystic ovary syndrome (PCOS), endometriosis, hypogonadism, precocious puberty, pituitary adenomas, leiomyomas, and uterine fibroids. vitamin D infertility 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/Lavoratory Role in the project 1 Fondazione Ca'Granda - Ospedale Maggiore Policlinico 2 Ospedale San Raffaele IRCCS, Milano UOSD Procreazione Medicalmente Assistita UO Ostetricia e Ginecologia Principal Investigator/Coordinator Collaborator; Embryological procedures, collection of samples. Molecular analysis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Viganò Paola Ospedale San Raffaele IRCCS, Milano collaborator. Embryological procedures, collection of samples. Molecular analysis 19/07/ Ferrari Stefania Fondazione Ca'Granda - Ospedale Maggiore Policlinico 4 PAFFONI ALESSIO Fondazione Ca'Granda - Ospedale Maggiore Policlinico collaborator, collection of samples and molecular analysis. collaborator. Embryological procedures, collection of samples. 11/06/ /01/ /07/ / 6

183 RF Vitamin D Supplementation on Assisted Reproduction Technology (ART) outcomes: a randomized clinical controlled trial and an investigation of the involved biological mechanisms Somigliana Edgardo Clinical health care research/clinicoassistenziale Fondazione Ca'Granda - Ospedale Maggiore Policlinico Background and Significance Vitamin D is derived from the dermatological conversion of pro-vitamin D under the essential effect of solar ultraviolets. Diet participates to the human necessity for only 10%. This extremely particular and unique mean of actually satisfying our needs represents an important concern. Indeed, the radically mutated WOR habits of humans in Western Worlds is not compatible with an adequate exposure to sunlight and as a consequence, vitamin D deficiency is very common (50-90%). Vitamin D plays an important role in human physiology and pathology. The receptor for vitamin D (VDR) regulates 2-3% of the human genome. Accordingly, vitamin D insufficiency has been shown to increase the risk of several diseases including cancers, auto-immune diseases, infections and pregnancy complications. In recent years, there is growing evidence of a role of vitamin D also in reproductive health (Vanni etal, 2014). VDR is expressed in the ovary, the endometrium and the myometrium. Vitamin D insufficiency favors the development of fibroids and endometriosis (Paffoni et al., 2013). Of utmost interest here is that there is evidence in animals and humans that vitamin D may also influence female fertility. Assisted Reproductive Technology (ART) success rate was shown to be higher in women with appropriate reserves of vitamin D (Ruddick et al., 2012). However, to date, there there are no prospective clinical trials testing the effectiveness of vitamin D supplementation in infertile women scheduled for ART Specific aims Aim 1: Aim 2: Aim 3: The study aims at determining the potential benefits of vitamin D supplementation in improving clinical pregnancy rate in women undergoing ART. To investigate whether vitamin D supplementation might improve clinical and embryological parameters such as: number of cancelled cycles, number of available oocytes, number of good quality embryos, units of FSH administered per retrieved oocyte, implantation rate of transferred embryos. To investigate the effect of vitamin D supplementation on oocyte and endometrium quality at a molecular level through a comparative analysis between treated and untreated patients based on the analysis of cumulus cells, follicular and endoemtrial fluids. In particular: Cumuls cells: A real-time PCR analsysis will be performed to assess comaprative expression of genes known to be associated with IVF outcome, i.e. GREM1, PTGS2, HAS2, STAR, COX2, VCAN. Pool of follicular fluids from aspirated follicles (>=18 mm diameter): chemiluminescnt assays to assess Bmp-15, Gdf-9, 25-hydroxyvitamin D [25(OH)D] levels. Endometrial secretions aspirated prior to embryo transfer: a multiplex immunoassay will be performed to describe the cytokine profile of treated and untreated patients. Interleukin(IL)-1ß,, IL-10, IL-12, IL-17, tumor necrosis factor (TNF)-a, eotaxin, dickkopf homologue-1, glicodelin A, macrophage migration inhibitory factor, monocyte chemotactic protein-1, interferon-gamma inducible protein-10, vascular endothelial growth factor, interferongamma will be analyzed given their known involvement in human embryo implantation. Hypothesis: Vitamin D deficiency negatively affects pregnancy rates with an effect mediated through endometrial receptivity and oocyte-embryo quality. Vitamin D supplementation and restoration of vitamin D levels may actually improve the prognosis of infertile women undergoing ART procedures. This effect can be seen as an increase in clinical pregnancy rate per started cycle in supplemented women compared to placebo (from 20% to 30%). Moreover, the molecular profiles of endometrium, follicular fluid and cumulus cells are modified in vitamin D-supplemented women with respect to molecules known to be associated with IVF outcome and implantation process. Preliminary data: In a recent contribution of our group on 334 women undergoing IVF, we have detected an OR of clinical pregnancy of 2.1 (95%CI: ) in women with vitamin D sufficiency (>30 ng/ml) compared to those with vitamin D insufficiency (<30 ng/ml) (Paffoni et al., submitted). 11/07/ / 6

184 RF Vitamin D Supplementation on Assisted Reproduction Technology (ART) outcomes: a randomized clinical controlled trial and an investigation of the involved biological mechanisms Somigliana Edgardo Clinical health care research/clinicoassistenziale Fondazione Ca'Granda - Ospedale Maggiore Policlinico Materials and Methods A randomized double blinded placebo controlled study. Two Italian infertility Units will be involved. Inclusion criteria for IVF women:1) Age years, 2) <3 previous in vitro fertilization (IVF) attempts. Exclusion criteria: 1) Poor responders 2) cycle using frozen embryos or oocytes. P1: Eligible women will be randomized according to a computer-generated randomization list. At the time of randomization, thus 1-4 weeks prior to initiate ovarian hyper-stimulation, women will be given the drug (either 300,000 IU of 25-OH-vitamin D or placebo in a single oral administration). IVF procedures, follow-up and data analysis will be performed according to standard protocols. Recruited women will provide a blood sample at the time of randomization and at oocyte retrieval for 25(OH)D and calcium tests; assessments will be performed after cycle completion. P2: from a subgroup (n=50) of age-matched good-prognosis cases and controls we will collect and analyze samples as indicated in "AIM 3" Impact and Translational Implications The working habits of humans in Western Worlds do not allow an adequate exposure to sunlight and thus hamper the provision of sufficient amount of vitamin D. However, insufficiency can be simply overcome as vitamin D supplementation is an economic, simple and effective treatment. The present study could give the evidence of a simple and inexpensive intervention to improve reproductive health and the cost-effectiveness profile of infertility trearments. 11/07/ / 6

185 Novel approaches for screening of humoral primary immunodeficiencies in adult patients BANDO 2013 Progetti Ordinari RF RF Matucci Andrea Clinical health care research/clinicoassistenziale Toscana LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Innovative biotechnologies Project Classification IRG: Project Classification SS: Immunology Immunity and Host Defense - IHD Project Keyword 1: Project Keyword 2: 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. 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/Lavoratory Role in the project 1 Toscana Biomedicine, IUmmunoallergology Unit, AOU Careggi, Florence Principal investigator 2 Toscana Immunology Unit, AOU Meyer, Florence Co-Investigator Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 moriondo maria Toscana Co-Investigator 13/03/1970 Background and Significance The aim of the present project will be to perform screening of primary immunodeficiencies (PID), in particular Common variable Immunodeficiency (CVID) with 2 differnet methods. The first will be a new patented tandem mass spectrometry (TMS) method, which identify at a negligible cost (< 0.05 per patient) severe PID such as ADA and PNP deficiency, including late-onset phenotypes. For the second, we will develop a multiplex Realtime PCR method including primers/probes for TREC and KREC; the method is expected to identify both severe T cell defect and B cell defect. Sensitivity and specificity of the two methods will be evaluated. Integration of our results with results obtained by other working groups in this research, will allow to evaluate cost effectiveness of the two screening approaches and will provide indicators important for allocating resources, health planning and healthcare utilization. Specific aims 10/07/ / 5

186 Novel approaches for screening of humoral primary immunodeficiencies in adult patients BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale Matucci Andrea Toscana Aim 1: To extend the low cost TMS test performed in Tuscany so that both early-onset and late-onset CVID are identified and to evaluate the cost-effectiveness of the procedure. The test will be extended in different Italian Regions. Actually TMS and not TREC can identify both early and delayed-onset phenotype of PID. Reaching our aim 1, we will prevent, with the earliest diagnosis, damages for the patients and expenses associated with treatment and hospitalization before diagnosis. Aim 2: Aim 3: To include kappa-deleting recombination excision circles (KRECs) into a multiplex Realtime PCR reaction together with TREC and evaluate sensitivity and specificity of that method in identifying PID, particularly CVID in adult patients. Actually one of the main problem of TREC method is that it can identify only immunodeficiencies with T cell defect. Inclusion of KREC together with TREC in a multiplex Realtime PCR will allow to overcome that problem To make diagnosis, who will develop a late-onset phenotype of CVID deficiency. In this way we will be able to study immune deterioration over time, understand the mechanisms which underlie the appearance of immunologic or neurologic symptoms and evaluate the correct timing for starting treatment Hypothesis: CVID meet all criteria considered as necessary to include a disease in a newborn screening program: high mortality and morbidity, the ability to be cured if it is recognized early, an early pre-symptomatic phase, the availability of simple and inexpensive tests for markers of the disease, and defined and available treatments (3). Data published in other western countries demonstrated that newborn screening can be cost-saving (3). A pilot project of newborn screening for ADA and PNP deficiency using TMS, started in 2011 in Tuscany, demonstrated that TMS can identify at birth not only newborns who have the most severe phenotype and will develop early-onset ADA-deficiency, but also those who will develop a delayed-onset SCID. TREC analysis fails in identifying the latter (5). The TMS method and the TREC method are not mutually exclusive, since they identify different patients. TREC actually can identify all severe T cell defect at birth. Adding to TREC analysis the possibility to identify also severe B cell defects would increase sensitivity of the method in the diagnosis of immunodeficiencies and, identifying more patients in the pre-symptomatic period, would improve the cost-effectiveness ratio. Preliminary data: As for TMS, preliminary results show that TMS has a negligible cost in those settings where tandem mass is already used for routine newborn screening (over 20,000,000 children per year in the world). TMS and not TREC can identify delayed-onset ADA deficiency and prevent severe infections and permanent sequelae associated with that phenotype. Association of KREC to TREC increases the number of screened disease so improving cost-effectiveness of the molecular method. However specificity will be confirmed by the present research. 10/07/ / 5

187 Novel approaches for screening of humoral primary immunodeficiencies in adult patients BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale Matucci Andrea Toscana Materials and Methods As for TREC/KREC analysis, DNA will be extracted from a single 3.2 mm punch of the DBS using QIAamp DNA Mini kit (Qiagen) and analyzed by real-time, quantitative PCR for TREC, KREC and ß-actin as previously described (5). Calibration curves will be generated by 10-fold serial dilution of plasmids containing TREC, KREC and ß-actin sequences. DBS will also be collected from PID patients (positive controls) during control visits. Sensitivity and specificity analysis of TREC/ KREC multiplex Realtime PCR, as well as cost-effectiveness analysis will be performed on DBS taken from 30,000 DBS CVID per year. The same DBS obtained for other routine screenings will be used. No extra blood will be required. Statistical analysis will be performed by the researcher of the Research Unit, using the SPSSX statistical package. Impact and Translational Implications The TMS method we propose has proven to have in preliminary results the highest sensitivity and specificity in diagnosis of ADA and PNP deficiency. The cost-effectiveness analysis of the two methods, obtained merging results obtained in different working groups of this research will be a key point for decisions on allocation of healthcare resources.

188 RF Effects of multimodal training on cognition, biomarkers, rs-fmri and brain structural integrity in MCI patients Sensi Stefano Clinical health care research/clinicoassistenziale Abruzzo 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. dementia and "omics" cognitive 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/Lavoratory Role in the project 1 Abruzzo AUSL Pescara. Center for Cognitive Recruitment and rehabilitation strategies for MCI Stimulation for Dementia. San Valentino patient, Neuropsychological evaluations Hospital. DSB SCAFA 2 Center of Excellence on Aging (CeSI) University G. d'annunzio Chieti-Pescara Molecular Neurology, Genomics, and Proteomics Units metabolomics, proteomics, ionomics, genomics 3 AUSL Chieti-Lanciano-Vasto MRI UNIT Chieti Hospital FMRI, MRI Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Ciavardelli Domenico Center of Excellence on Aging (CeSI) University G. d'annunzio Chieti-Pescara Proteomic, Metabolomic, and Ionomic Analysis 10/06/ CAULO MASSIMO AUSL Chieti-Lanciano-Vasto FMRI, MRI 05/04/ Gatta Valentina Center of Excellence on Aging (CeSI) University G. d'annunzio Chieti-Pescara Genotyiping 02/04/ /07/ / 6

189 RF Effects of multimodal training on cognition, biomarkers, rs-fmri and brain structural integrity in MCI patients Sensi Stefano Clinical health care research/clinicoassistenziale Abruzzo Background and Significance Aging is the major risk factor for Alzheimer's disease (AD). Early use of imaging and biological markers can identify at risk subjects decades before the appearance of cognitive decline. It is therefore imperative to act during this extended preclinical phase and promote endogenous responses in brains that still have a great cognitive reserve and optimal levels of neural plasticity. Beside pharmacological intervention, a great deal of interest resides on ways that allow modulation of brain plasticity through cognitive stimulation and /or aerobic exercise. Cognitive enrichment as well as physical activity promote brain plasticity in the elderly, increase neurotrofism and reduce the incidence of AD. Neural correlates of training programs are rarely investigated with multimodal neuroimaging techniques. Furthermore, not much it is known on how training programs on MCI individuals can delay the appearance of dementia later on in life. A key area of improvement in the field is the standardization of training protocols. We are in need of sets of activities that are easily done at home or assisted living places and are universally reproducible. Specific aims Aim 1: We propose the use of highly standardized sets of training activities. All the activities are uploaded in accessible websites and generate an automatic data outputs. The proposal will make use for the aerobic training (AT) of Technogym machines (machines operated by personalized USB keys that are uniques as all the activities are logged, thereby generating a data file containing all the parameters concerning exercise difficulty, lenght, compliance to programs, strength, and metabolic consumption). For cognitive stimulation (CT) we will employ pcbased rehabilitative software. The goal is to provide a standardized and reproducible set of training activities that can be monitored for quantity, quality, and patient compliance. All the data concerning the performed activities will go to a central web site, a cloud, and be available for detailed analysis. In AIM 1, We will evaluate and compare effects of combined activities [Multimodal Training, (MT): Cognitive Training (CT) + Aerobic Training (AT)] on cognitive performances of 90 MCI individuals. MCI subjects (age range years old, y.o.) will be randomly divided in two groups: trainend and control. Training will last for 9 months (T0-T9). The control group, for the same T0-T9 interval, will go on with unchanged daily living routines. Aim 2: Aim 3: Here we will test MT effects on brain functioning and plasticity (rs-fmri and structural MRI) in the trained group and compare then with changes occuring in the control group in the T0-T9 interval. Here we will test MT effects on molecular parameters in the trained group and compare with changes occuring in the control group in the T0-T9 interval. To that aim all subjects will be assayed for plasma BDNF levels, metabolomic, proteomic and ionomic changes occuring before and after the training. Genotyping of a panel of genes associated with susceptibility to late onset AD will be tested along (as we did in Pieramico et al., 2012) with known dopamine receptor related polymorphysms. Hypothesis: The hypothesis is that early intervention when the cognitive reserve and brain integrity are still largely preserved or at least not too compromised can help. We think that a mix of stimulating activities, MT, that are standardized and low cost can translate in reproducible protocols to be implemented in clinical settings (nursing homes, respite care centers, etc). As we want to maximize the effects, the relative weight of AT vs CT in producing beneficial effects is not investigated. The idea is to produce and validate therapeutic protocols where MT is seen as a whole, a non-pharmacological olistic approach. Preliminary data: This project capitalizes from a previous study (Pieramico et al., 2012) showing that Multimodal Training (MT) promotes increased memory performances, modifications in functional connectivity (FC), and brain integrity in healthy elderly individuals. (see: 11/07/ / 6

190 RF Effects of multimodal training on cognition, biomarkers, rs-fmri and brain structural integrity in MCI patients Sensi Stefano Clinical health care research/clinicoassistenziale Abruzzo 3Adoi%2F %2Fjournal.pone ) Materials and Methods 1) Recruitment and Screening: participants will undergo medical, psychiatric, and neurological examination by expert in dementia. Exclusion criteria are history of drug abuse, stroke, untreated hypertension, diabetes, psychiatric or neurodegenerative diseases. Groups will be matched for sex, age and years of education. The Mini Mental State Examination, the phonemic verbal fluency (FAS), Trail Making Test (TMT), Frontal Assessment Battery (FAB), Babcock Story Recall, tests will be performed. 2) T0 and T9: trained MCI individuals will be evaluated at T0 and after 9 months (T9) and compared against their respective control groups for: a) - Neuropsychological evaluations b) Neuroimaging - Rs-fcMRI - Regional volume and cortical thickness using Volumetric T1-weighted MR images. - White matter microstructure/connectivity using DTI b) Genotyping for AD-related or Dopamine-related genes c) Plasma BDNF levels ELISA d) ICP-MS for proteomic, metabolomic, ionomic analysis Impact and Translational Implications We are set to identify and test relatively inexpensive, easily applicable, and cost-effective interventions that are able to prevent and/or delay age-related cognitive decline in MCI subjects. The overall benefit for society and the socio-economical implications of this approach are relevant. The longer an individual remains cognitively efficient, the greater the chances she/he has to maintain satisfactory functional levels in daily living activities and reduce meducalization costs. 11/07/ / 6

191 RF Olfaction as an index of conversion from MCI to AD: a longitudinal functional connectivity study Nichelli Paolo Frigio Biomedical/Biomedica Emilia-Romagna LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Neurologic diseases Project Classification IRG: Project Classification SS: Integrative, Functional, and Cognitive Neuroscience Somatosensory and Chemosensory Systems - SCS Project Keyword 1: Project Keyword 2: Project Keyword 3: Chemosensation: olfaction, taste, vomeronasal and trigeminal chemosensory systems studied with approaches ranging from molecular techniques to human psychophysics. functional connectivity MCI conversion to AD 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/Lavoratory Role in the project 1 Emilia-Romagna AUSL MO/ NOCSAE/Clinical Neurosciences Department/Neurology Unit 2 Emilia-Romagna AUSL MO/ NOCSAE/Mental Health Department/Clinical Psychology Unit PI; project coordantion; clinical evaluation (neurological examination, RMN, lumbar puncture); patients selection neuropsychological evaluation and differential diagnosis 3 University of Modena and Reggio Emilia University of Modena and Reggio Emilia data analysis - Department of Biomedical, Metabolic and Neural Sciences Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Molinari Maria Angela Emilia-Romagna Neuropsychologist; co-investigator; neuropsychological evalutaion and differential diagnosis 2 Benuzzi Francesca University of Modena and Reggio Emilia 02/10/1965 Neuropsychologist; data analysis 28/07/ /07/ / 5

192 RF Olfaction as an index of conversion from MCI to AD: a longitudinal functional connectivity study Nichelli Paolo Frigio Biomedical/Biomedica Emilia-Romagna Background and Significance Deficits of the olfactory system have been reported to be associated with several neurodegenerative diseases such as Alzheimer's disease (AD). Autoptical data showed in AD a specific concentration of lesions (senile plaques, neurofibrillary tangles NFT) in the peripheral and central olfactory cortex, and in layers II and III of the entorhinal cortex. In AD, NFT formation begins in predisposed sites and then spreads to other structures in a predictable sequence according to Braak's stages (transentorhinal, limbic and neocortical). Moreover, NFTs in the olfactory bulb appear in the early Braak's stages, even before spreading to the entorhinal cortex. Recently, a correlation between hippocampal volume and olfactory function has been found thus supporting the hypothesis of a neuroanatomical association between the olfactory system and memory circuits. Within the limbic and the memory system a predominant role is played by the amygdala which is also involved in olfaction supporting the emotional content of odors. Mild Cognitive Impairment (MCI) defines a cognitive (amnestic and non-amnestic) syndrome that denotes a transitional stage between normal aging and dementia. Populations affected by MCI have been shown to be at higher risk than the general population for developing dementia. Recently, it has been demonstrated that patients with MCI and olfactory deficits would have a higher likelihood of conversion to AD. Specific aims Aim 1: Aim 2: Aim 3: First aim of the study is to evaluate whether odor discrimination ability could increase accuracy in identification of patients with high risk of conversion to AD (MCI due to AD). This will be achieved assessing well-know biomarkers (beta-amyloid and tau protein values), conventional neuroradiological signs (hippocampal atrophy) and cognitive deficit (memory as measured by FCSRT and Memory Binding) and the odor identification performances (Smell Identification Test, UPSIT) in a longitudinal study. Second aim of the study is the evaluation of the neuroanatomical correlates of odor discrimination deficit. In a subgroup of patients, resting-state data will be collected to detect early changes in functional connectivity of the olfactory system using the amygdala as seed. Third aim of the project, is the correlation of resting-state functional connectivity data with odor identification as well as memory performances. Hypothesis: The olfactory system is affected since the earlier stages of AD. Data come mainly from autoptical studies but at present, there are no studies correlating the odor discrimination ability and the integrity of the neuroanatomical structures subserving olfaction. We hypothesize that the in vivo evaluation of the clinical (odor discrimination) and functional (resting state functional connectivity) integrity of the olfactory system and their correlation could help in identifying patients with high risk of conversion to AD. Moreover, functional connectivity data, will be used to test the hypothesis that ability to identify and recognize odorants could depend on the involvement of medial temporal lobe structures, which are affected in the early stages of AD and considered responsible for both smell identification and memory processes. Preliminary data: Preliminary evaluation of clinical data of MCI patients of UO di Neurologia showed that at least 60% of patients reported olfactory dysfunction such as iposmia, inability to discriminate odors and reduction of the perception of flavor. 11/07/ / 5

193 RF Olfaction as an index of conversion from MCI to AD: a longitudinal functional connectivity study Nichelli Paolo Frigio Biomedical/Biomedica Emilia-Romagna Materials and Methods A total of 150 amnesic MCI patients will be recruited and evaluated at baseline and after two years. At baseline, a clinical (neurological exam, structural MRI and lumbar puncture) and neuropsychological examination will be performed. Cognitive tests will include measure of memory abilities (FCSRT, Memory Binding, Digit Span F-B, Story Recall), attention, executive function, and language. Odor discrimination will be assessed by means of The Smell Identification Test (UPSIT; Sensonic Inc). In a subgroup of patients, the resting state functional connectivity will be performed over a 10 minute scanning session. A total of 300 dynamic volumes will be acquired (TR= 2000; voxel size: 80x80x80; number of slice: 35). A seed-based analysis will be performed using the amygdala as the seed and SPM8 (Wellcome Trust Centre for Neuroimaging) as software. After two years, patient will undergo a second examination including the cognitive testing and the functional resting-state study. Impact and Translational Implications The study could help in identifying MCI patients with an high risk to convert to AD with the following effects: -a significant increase in the accuracy of identification of MCI due to AD (eligible for pharmacological disease-modified treatment) and MCI not due to AD; - a significant reduction of the health economic impact since only an early pharmacological treatment could modify the progression of AD. 11/07/ / 5

194 RF Report-AGE Project: clinical and biological predictors of physical function decline in older patients with chronic cardiovascular diseases Bustacchini Silvia Clinical health care research/clinicoassistenziale Istituto Nazionale di Riposo e Cura per Anziani LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Metabolic and cardiovascular 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: Modeling of complex regulatory networks such as those affecting cardiovascular function, circadian rhythms, frailty and postural control, and their alteration with age. Cardiovascular diseases Physical performance 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/Lavoratory Role in the project 1 Istituto Nazionale di Riposo e Cura per Anziani Scientific Direction Project management, study design, patients' recruitment, data collection and management, data analyses Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date Bonfigli Anna Rita Istituto Nazionale di Riposo e Cura per Anziani 4 Lisa Rosamaria Bernadete Itala Istituto Nazionale di Riposo e Cura per Anziani Project management, study design, data analyses Patients' recruitment, data collection and management 24/07/ /05/ /07/ / 5

195 RF Report-AGE Project: clinical and biological predictors of physical function decline in older patients with chronic cardiovascular diseases Bustacchini Silvia Clinical health care research/clinicoassistenziale Istituto Nazionale di Riposo e Cura per Anziani Background and Significance The onset of frailty and disability in older patients results from multiple, co-existing causes: it is not only due to the physiological aging, but also to several age-related chronic diseases such as cardiovascular diseases, COPD, diabetes and cancer which do often coexist (Okonkwo OC, Cerebrovasc Dis 2010; Dodd JW, Eur Respir J 2010). In particular, these conditions often occur in older patients with CVD presenting also a multimorbidity status, frequent hospitalizations, and high mortality rates, rendering the clinical evaluation and management decisions complex in this population (Afilalo J, Curr Cardiovasc Risk Rep 2011). In this context, the evaluation of physical performance together with clinical characteristics, and biological markers may be relevant in predicting rates of functional decline, rehospitalization, and death in CVD patients over time (Singh et al, Eur Heart J. 2014; Volpato et al, J Gerontol A Biol Sci Med Sci. 2011). Many studies confirm the role of inflammation in the pathophysiology of chronic diseases, thus, suggesting that it might contribute to the onset and progression of functional decline (Corsonello A et al, Biogerontology 2010). Although this condition is extremely common in older patients, few and relatively small studies have addressed this issue combining the biological, clinical and functional perspective in patients with CVD. Specific aims Aim 1: Aim 2: To identify a network of clinical and biological profiles and their complex relationships in older patients with chronic cardiovascular diseases. To analyse the role of identified profiles to explain physical function decline after one-year follow-up. Aim 3: To integrate the existing electronic health records (EHR) with administrative database. Hypothesis: We hypothesize that, in older patients with chronic cardiovascular diseases, the evaluation of physical performance together with clinical characteristics and biological markers may be relevant in predicting rates of physical functional decline and adverse outcomes such as rehospitalization and mortality. Preliminary data: A preliminary feasibility analysis of the project has been conducted within the INRCA hospital network, setting up a systematic collection of clinical data and biological parameters, funded within the INRCA intramural research program. The database includes to date 3136 patients. Mean age was 84.4 years [range ], women were 56.3 %, with mean length of stay of 9.2 days. Cardiovascular diseases are the most common diagnosis and, in particular, CHF is present in 494 patients (15,8%). The feasibility of biological sample collection and storage, and of lab analysis was confirmed. There is still a fragmentation of the clinical and administrative information among and whitin the hospitals. The effort in reducing this fragmentation can yield a high return on investment. 10/07/ / 5

196 RF Report-AGE Project: clinical and biological predictors of physical function decline in older patients with chronic cardiovascular diseases Bustacchini Silvia Clinical health care research/clinicoassistenziale Istituto Nazionale di Riposo e Cura per Anziani Materials and Methods An observational prospective study design with a one-year follow up will be implemented. Patients with diagnosis of chronic CV diseases aged 65+ will be recruited from INRCA hospitals and clinics. This is a 3-year project (M1-M36). 7 Work Packages (WPs) are planned. WP1. Literature review, experts meeting and development of the research protocol (M1-M6) WP2. Realization of a data set joining existing and newly developed DBs (M3-M8) WP3. Consensus-building and training of staff (M4-M8) WP4. Creation of a geriatric Informed consent form and approval of the ethical committee (M6-M8) WP5. Data Collection (M9- M33), including CGA and clinical, biological and physical performance data WP6. Data analysis (M10-M36), including exploratory analyses of clinical and biological parameters to identify patient profiles by clustering techniques. Predictive models will be used to analyse physical function decline at follow up. WP7. Reporting and dissemination (M34-M36) Impact and Translational Implications Results obtained with the project could be transferred to public health system in terms of better resources planning and allocation for older people with CVD, with specific focus on patient at risk of physical function decline. Indeed, the early identification of high risk profiles may lead to implement different pattern of care, avoiding for example costly hospitalizations and institutionalizations.

197 RF Targetting the interleukin-6 receptor as a novel therapeutic approach in Duchenne muscular dystrophy to modulate muscle inflammation and regeneration de benedetti fabrizio Biomedical/Biomedica Ospedale pediatrico Bambino Gesù 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 Skeletal Muscle Biology and Exercise Physiology - SMEP Project Keyword 1: Project Keyword 2: Project Keyword 3: Skeletal muscle diseases: evaluation of genetics, gene function, and development of vertebrate and invertebrate genetic models; pathophysiology of skeletal muscle disorders and diseases, including the muscular dystrophies, atrophy, myotonia, periodic paralysis, malignant hyperthermia, and inflammatory myopathies; pharmacological interventions and pre-clinical approaches; cell and gene therapies for skeletal muscle diseases. interleukin-6 muscle necrosis and regeneration 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/Lavoratory Role in the project 1 Ospedale pediatrico Bambino Gesù 2 Universita' di Roma La Sapienza 3 Consiglio Nazionale delle Ricerche U.O. Rheumatology Dept Histology and Medical Hembryology patients and animal models, molecular analysis in vivo studies of regeneration markers Institute of Translational Pharmacology cell differentiation studies Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 laura pelosi Universita' di Roma La Sapienza in vivo studies of regeneration markers 08/11/ Bracci Laudiero Luisa Consiglio Nazionale delle Ricerche cell differentiation studies 04/06/ /07/ / 6

198 RF Targetting the interleukin-6 receptor as a novel therapeutic approach in Duchenne muscular dystrophy to modulate muscle inflammation and regeneration de benedetti fabrizio Biomedical/Biomedica Ospedale pediatrico Bambino Gesù Background and Significance Duchenne muscular dystrophy (DMD), caused by dystrophin gene mutations, is characterized by myofiber necrosis and ineffective tissue regeneration that leads to substitution of normal muscle with fibro-adipose tissue. Inflammation plays a crucial role in DMD by favouring muscle necrosis, inhibiting regeneration, promoting atrophy and progression of muscle damage. Indeed, glucocorticoids are the only available treatment that slows DMD progression. Interleukin-6 (IL-6) is involved in the induction and maintenance of chronic inflammation, inducing CD8+ T cell differentiation, M1 macrophage activation, mononuclear cell recruitment. IL-6 seems to affect muscle homeostasis, myonuclear accretion and satellite cell proliferation. However, its role is unclear with high IL-6 having been associated with either muscle repair or muscle wasting. This apparent discrepancy with diverse effects could be due to the peculiar IL-6 receptor system: in the classical cissignalling IL-6 binds to the membrane IL-6 receptor (mil-6r) forming a receptor complex with gp130. While IL-6R expression is restricted to few cell types, gp130 is ubiquitous. In the so-called trans-signalling, IL-6 binds the soluble IL-6R (sil-6r), present at high concentration at inflammatory sites, and activates gp130 expressing cells. It is not known whether an imbalance in cis- and trans-signalling pathways affects satellite cell proliferation/differentiation and abnormal muscle repair in general, or in particular in DMD. Specific aims Aim 1: To evaluate the role of IL-6 in the progression of muscle damage in dystrophin deficient mdx mice and provide the rational for clinical use of anti-il-6 receptor antibodies in DMD. In this aim we will use a monoclonal antibody binding both sil-6r and mil-6r to study the effects of IL-6R system blockade on: - inflammation/necrosis processes (myofiber death, inflammatory infiltrate) - regeneration (muscle stem cell activation, proliferation, and differentiation) - physical performance Aim 2: To provide evidence of the therapeutic efficacy of either mil-6r or sil-6r inhibition on muscle regeneration: given the uncertainties of IL-6 effects on satellite cell activation, proliferation and differentiation, we will investigate in vitro and in vivo the role of IL-6 in muscle repair focusing on the potential different activities of the mil-6r vis a vis the sil-6r. We will assess the different effects of IL-6 binding to the two receptor forms on the differentiation and proliferation of murine myoblasts (C2C12) and human myoblasts (HSkM), using specific neutralizing antibodies or sgp130fc fusion protein. We will assess in mdx mice if differentiation of satellite cells and remodelling of the damaged muscle tissue are differentially affected by specific inhibition of either cis or trans-signalling.this will provide the rational for the choice of the pathway to be inhibited as a possible therapeutic approach in DMD Aim 3: To investigate IL-6 and IL-6 mediated events in biopsies of DMD patient. We will correlate IL-6 expression with expression of markers of muscle necrosis, inflammation and regeneration. Moreover, we plan to evaluate IL-6, mil-6r and gp130 mrna expression levels and correlate them with the amount of infiltrate, expression of MCP-1, VCAM-1, ICAM-1and activation of IL-6 signal transduction in myofibers in order to demonstrate and provide the evidence for a close relation between IL-6 and disease features in DMD Hypothesis: Our hypothesis is that the inflammatory cytokine IL-6 is a key mediator of muscle inflammation, damage and regeneration failure in DMD, and, therefore, a potential therapeutic target in DMD. Based on the our preliminary data, we suggest that IL-6 and, possibly, trans-signalling are directly involved in the 11/07/ / 6

199 RF Targetting the interleukin-6 receptor as a novel therapeutic approach in Duchenne muscular dystrophy to modulate muscle inflammation and regeneration de benedetti fabrizio Biomedical/Biomedica Ospedale pediatrico Bambino Gesù pathogenesis of DMD and neutralization of the IL-6R signalling in vivo may slow disease progression Preliminary data: Our preliminary data show - DMD patients: IL-6 is expressed at high levels in muscles and serum IL-6 levels are elevated - High IL-6 levels promote muscle damage and inhibit regenerationin in mdx mice (in mdx mice crossed with IL6 transgenic) - Blockade of IL-6 activity by a IL-6R antibody binding both mil-6r and sil-6r decreases myofiber necrosis and mononuclear cell infiltration and ameliorates physical performance of mdx mice - IL-6 and mil-6r are highly expressed by myofiber and by muscle satellite cells of mdx mice - C2C12 myoblasts express mil-6r and addition of sil-6r affects the expression of differentiation markers Materials and Methods We will use mdx mice to assess the role of IL-6 in the progression of muscular dystrophy. In mdx mice (4CV non-revertants) and their wild-type controls, we will evaluate fiber type composition by immunhistochemistry and western blot (WB) with specific antibodies direct to MyHC isoforms and regenerative capacity by immunofluorescence and WB analysis of muscle regeneration markers (Pax-7, MyoD and embryonic MyHC). Functional analysis will be performed with voluntary free wheel running and involuntary running on treadmill to evaluate tolerance to exercise. IL-6, sil-6r with or without neutralizing antibodies (specific for both mil-6r and sil-6r) or sgp130fc fusion protein (inhibiting only the sil-6r) will be used in vitro on C2C12 and HSkM cells to evaluate the proliferation and differentiation by WB, qrt-pcr, confocal analysis. Muscle biopsies of DMD patients are already available and the expression of IL-6 and the markers of inflammation and regeneration will be analyzed by qrt-pcr Impact and Translational Implications Proving the role of IL-6 in muscle will establish the viability of IL-6 as a therapeutic target opening a new perspective in the treatment of DMD. The demonstration of the central of IL-6 and the specific receptor pathways involved in muscle degeneration will provide the rational for a treatment targeted directly to IL-6 receptor using neutralizing antibodies 11/07/ / 6

200 Distinct developmental programs of cord blood and bone marrow haematopoietic stem cells reconstitute different B-cell compartments in transplanted patients leading to transplantspecific immune impairment. Cellular and molecular characterization of transitional and memory B cells derived from haematopoietic stem cells of cord blood, bone marrow or peripheral blood isolated from transplanted patients. RF Carsetti Rita Biomedical/Biomedica Ospedale pediatrico Bambino Gesù 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: innate immunity to microorganisms: cells, receptors, cytokines, chemokines, and soluble mediators that provide early protection from injury due to pathogens and their products or responses to commensal organisms. Innate immune cells including but not limited to NK cells, phagocytes, gamma/delta and NK T cells, B-1 cells, dendritic cells, and mast cells. Receptors including but not limited to molecules expressed by these cells engaged in innate immunity, including chemokine and other G-protein coupled receptors, Toll-like receptors, NK cell activation and inhibitory receptors, phagocytic receptors, pattern recognition receptors, Fc receptors, adhesion receptors, co-stimulatory molecules, and cytokine receptors. haematopoietic stem cell transplantation memory B cells 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/Lavoratory Role in the project 1 Ospedale pediatrico Bambino Gesù 2 Ospedale pediatrico Bambino Gesù Immunology and pharmacotherapy Area,Immunology lab. Immunology and pharmacotherapy Area,Immunology lab. PI, project design, samples collection, human and mouse molecular and cellular studies, MIR analysis dentification and molecular analysis of memory B cells, cellular and animal studies, MIR analysis 3 Ospedale pediatrico Bambino Gesù Onco-Haematology Area, Haematology Dept. Patient selection and follow-up, sample collection, clinical management 11/07/ / 6

201 Distinct developmental programs of cord blood and bone marrow haematopoietic stem cells reconstitute different B-cell compartments in transplanted patients leading to transplantspecific immune impairment. Cellular and molecular characterization of transitional and memory B cells derived from haematopoietic stem cells of cord blood, bone marrow or peripheral blood isolated from transplanted patients. RF Carsetti Rita Biomedical/Biomedica Investigators, Institution and Role in the Project Ospedale pediatrico Bambino Gesù Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Piano Mortari Eva Ospedale pediatrico Bambino Gesù 2 brescia letizia pomponia Ospedale pediatrico Bambino Gesù identification and molecular analysis of memory B cells, cellular and animal studies, MIR analysis Patient selection and follow-up, sample collection, clinical management 19/01/ /07/1979 Background and Significance Hematopoiesis occurs in the bone marrow (BM) where stem cells (HSC) differentiate into several lineages and continuously replenish the periphery. B cells also develop in the BM and leave at the transitional B-cell stage to differentiate into maturenaïve B cells and participate to the adaptive immune response. We assume that after birth a unique type of HSC generates all lineages, but before birth different HSC types can be found in the yolk sac, aorto-mesogonadal region and fetal liver. In the adult mouse fetal liver HSC persist in the spleen and generate B-1a B cells throughout life. In human IgM memory B cells, also known as innate memory B cells, are functionally equivalent to B-1a B cells. Of the two types of human memory B cells, the switched embody the more sophisticated adaptive immune response, whereas innate/igm memory B cells represent the first-line defense. Cord blood HSC generate transitional B cells that rapidly differentiate into IgM memory B cells in response to innate signals. Adult HSC also generate transitional B cells, but these do not respond effectively to innate signals. Sources of HSC include: BM from HLA-identical sibling or unrelated HLA-matched (MUD) donor, Umbilical Cord Blood (UCB) or peripheral blood when haploidentical HSC from a mismatched family member are used. We do not know whether HSC of infant type can be found in the bone marrow of sibling and MUD or can be mobilized and reach the peripheral blood in the case of Haplo-HSCT. Specific aims Aim 1: Aim 2: Aim 3: To demonstrate whether IgM memory B cells can be generated from all types of HSC, independently from the source. We propose to compare the reconstitution of the memory pool in children with HSC transplantation from different sources. We will calculate the frequency and numbers of IgM and switched memory B cells developing after transplantation, sort and sequence the two populations in order to determine VH family usage, frequency and distribution of somatic hypemutations (SHM). To prove the origin of IgM memory B cells in a humanized mouse model. We will transplant mice with HSC from different sources and compare the reconstitution 2-3 and 4 months after cell injection. At these times we will analyze BM, spleen, thymus and intestine in order to detect different type of human B cells. We will also analyze other cell lineages, including T cells (CD4, CD8, Treg, alpha beta and gamma delta), NK and NK T cells. To identify the cellular and molecular features that distinguish transitional B cells of cord blood from the adult type. Several microrna are differentially expressed in CD34pos HSC from UCB and BM. In addition microrna regulating both TLR9 expression and function have been identified. We will identify by deep sequencing microrna differentially expressed by transitional B cells derived in vivo (isolated from the peripheral blood of patients) from all HSC sources. We will also compare microrna expression in each type of transitional B cells before and after TLR9 ligation. 11/07/ / 6

202 Distinct developmental programs of cord blood and bone marrow haematopoietic stem cells reconstitute different B-cell compartments in transplanted patients leading to transplantspecific immune impairment. Cellular and molecular characterization of transitional and memory B cells derived from haematopoietic stem cells of cord blood, bone marrow or peripheral blood isolated from transplanted patients. RF Carsetti Rita Biomedical/Biomedica Ospedale pediatrico Bambino Gesù Hypothesis: As in the mouse also in human two types of HSC contribute to generate the immune system. Fetal type HSC, also found in UCB, produce the first-line defense, represented by innate/igm memory B cells. BM HSC generate the adaptive immune system, including its most specialized product, switched memory B cells. Preliminary data: 1.Transitional B cells from UCB or generated in patients transplanted with UCB rapidly differentiate into innate memory B cells in response to CpG. Transitional B cells sorted from BM or generated in patients with MUD transplants do not respond to CpG. 2.Rapid reconstitution of the IgM memory pool after UCBT, delayed in other types of transplants; switched memory B cells are equally generated in all types of transplants. 3.Higher expression of TLR9 in transitional B cells from UCB or from the peripheral blood of patients receiving UCB transplants, lower in transitional B cells from BM or from peripheral blood of patients with MUD transplants. 4. Transitional B cells are the only B cell type in the peripheral blood at day 90 after transplantation. 5. Innate/IgM memory B cells can be generated without GC, T cells or gamma chain cytokines. They can be unmutated and generally have a low frequency of randomly distributed SHM. They preferentially use VH3. Switched memory B cells only derive from the GC, have a high frequency of SHM localized in the antigen-binding site and preferantially use VH1. Materials and Methods -UCB, BM or PBMC will be isolated by density-gradient centrifugation. Transitional B cells will be identified as CD24+CD38+, stem cells as CD34+. -RNA will be isolated using Total RNA Plus Kit. To compare transitional B cells in CB and PBMC of transplanted patients we will use deep sequencing (IGA Techno). We will detect both mrna and microrna. Deep sequencing will also be used to compare memory B cells from patients transplanted with HSC from different sources. -Humanized mice are a promising translational model for studying human immunity. The injection of human stem cells in Rag-gchain -/ mice has allowed in the past to replenish all the cell types of the immune system (B, T and NK cells) without the risk of rejection. We will transplant newborn mice, via intrahepatic injection, with CD34+ HSC from CB, BM or periphery. The animals should be irradiated before transplantation to eliminate residual mouse immune cells and also to promote human cell engraftment and regeneration. 11/07/ / 6

203 Distinct developmental programs of cord blood and bone marrow haematopoietic stem cells reconstitute different B-cell compartments in transplanted patients leading to transplantspecific immune impairment. Cellular and molecular characterization of transitional and memory B cells derived from haematopoietic stem cells of cord blood, bone marrow or peripheral blood isolated from transplanted patients. RF Carsetti Rita Biomedical/Biomedica Ospedale pediatrico Bambino Gesù Impact and Translational Implications The slow reconstitution of the immune system causes high susceptibility to infection after HSCT. Fetal type HSC reconstitute early immune defences whereas BM HSC more effectively replace the adaptive immune system. When only one type of HSC is transplanted, part of immune functions will not be replaced. Knowing the intrinsic reconstitution capacity of each type of HSC will guide the choice of better transplantation strategies and the development of patient-specific therapeutic interventions.

204 No more emergencies in DNA species identification: development and harmonization of molecular and next generation methods to determine and quantify origin species in food and to discriminate frauds from unintentional contamination. RF Peletto Simone Biomedical/Biomedica Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle 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. Food fraud Species identification 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/Lavoratory Role in the project 1 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'aosta 2 Istituto Zooprofilattico Sperimentale della Lombardia e dell'emilia- Romagna Laboratory of Genetics and Immunobiochemistry Laboratory of Nucleic Acid Technology Applied to Food 3 Istituto Superiore di Sanità Department of Veterinary Public Health and Food Safety Project coordination; Development of DNA quantification methods by real time qpcr; Development of NGS methods for animal and plant DNA; Contamination studies Development of DNA quantification methods by qpcr and droplet Digital qpcr; Contamination studies Stardardization of DNA quantification methods; Organization of inter-laboratory ring trials; Contamination studies Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Bertasi Barbara Istituto Zooprofilattico Sperimentale della Lombardia e dell'emilia-romagna Development of DNA quantification methods by qpcr and droplet Digital qpcr; Contamination studies 2 Gattuso Antonietta Istituto Superiore di Sanità Stardardization of DNA quantification methods; Organization of inter-laboratory ring trials; Contamination studies 14/06/ /12/ /07/ / 6

205 No more emergencies in DNA species identification: development and harmonization of molecular and next generation methods to determine and quantify origin species in food and to discriminate frauds from unintentional contamination. RF Peletto Simone Biomedical/Biomedica Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle Background and Significance The presence of undeclared animal species in food (species substitution fraud) has implications concerning not only commercial (i.e. valuable products replaced with less valuable ones) and ethical (i.e. meat of animals considered pet or prohibited by religion) aspects, but also health aspects, in case the food contains an undeclared ingredient capable of causing allergies/intolerances in the consumer, or substances harmful to health. In 2013, the horsemeat scandal has made headline news across Europe, showing that big gaps can be present in the traceability system, facilitated by the global market. In this context, it appeared how big is the risk of the easy passage from "simple food frauds" to dangerous "food crimes". Large scale frauds can indeed constitute a high threat to public health, because "undeclared" means also "uncontrolled", and massive presence of uncontrolled meat in the market could get along with a high microbiological or chemical risk. For these reasons, to react to the horse meat scandal DG SANCO took strong actions and EU Commission focussed major attention to fighting frauds, establishing a specific Task Force of DG SANCO and planning to introduce fraud control in the Reg. 882/2004. The monitoring plan promptly organized for horse meat showed on one hand that DNA analysis methods can be powerful tools for control but on the other hand the need for a standardized and harmonized diagnostic system completely prepared to face this "new" type of crisis. Specific aims Aim 1: Aim 2: Aim 3: To develop, evaluate and harmonize protocols, based on DNA analysis, to accurate quantify species in meat products. A comprehensive approach for species identification and quantitative determination will be applied, including optimization of extraction methods, selection of a reliable DNA quantification system, assessment of the most suitable molecular targets and reference standards. To develop a Next Generation Sequencing (NGS) method for the identification of unexpected animal and plant DNA present in meat product, in order to provide a complete quali-quantitative picture of the DNA composition in complex mixtures and to unravel the presence of DNA from unconventional and exotic species. To investigate unintentional contamination applying the methods in different scenarios. Different scenarios of unintentional contamination will be re-created. Methods will be standardized in order to meet an appropriate cut-off able to clarify the type of contaminations (fraud or cross-contaminations). Correlation between DNA detection and protein detection will also be studied, to assess implications for public health concerning allergies. Decontamination procedures will be evaluated to advise for Good Manufacturing Practices. Hypothesis: The horse meat scandal showed the extent, the impact and the potential implications for public health of the species substitution fraud. It also highlighted how crucial is to be able to discriminate between frauds and unintentional contaminations, in terms of legal prosecution. Molecular methods based on DNA analysis appear to be the only applicable to complex samples as meat products, but it is necessary to develop methods, also using innovative techniques, of which detection limits and accuracy in quantifying DNA, according to different species and different type of products, is clearly determined, in order to define their field of application in the prospective of control programs for food frauds. Harmonized procedures, guaranteeing comparable results in different laboratories, need to be delivered in order to have a homogeneous surveillance network. Preliminary data: The EU monitoring plan for horse meat demonstrated the effectiveness of qualitative methods, but the decision to establish a cut-off value (1%) to discriminate between fraud and unintentional contamination raised many issues related to quantitative approaches. The laboratories involved in the project have carried out preliminary experiments by applying some quantitative commercial kits 10/07/ / 6

206 No more emergencies in DNA species identification: development and harmonization of molecular and next generation methods to determine and quantify origin species in food and to discriminate frauds from unintentional contamination. RF Peletto Simone Biomedical/Biomedica Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle on field samples (raw meat and processed food) spiked with horse meat at different concentrations. Results revealed difficulties related to sample preparation from complex matrices, differences in DNA yield/quality related to the suggested extraction methods, especially with processed food, and lack of concordance between commercial quantitative tests. This scenario strongly suggests the need to standardize DNA preparation procedures and to set up a reliable quantitative approach, using new genetic targets. Materials and Methods Aim 1: Ad hoc prepared meat products with known species composition and representative of different degrees of processing will be employed. Qualitative and Quantitative molecular methods based on mithocondrial DNA detection, SINE and other genomic targets will be developed, standardized and harmonized, using Real Time qpcr and the innovative droplet Digital PCR. Reproducibility of the methods will be evaluated through ring trials. Aim 2: A NGS method and bioinformatics pipeline will be set up for the determination of expected and unexpected animal or plant DNA. NGS is expected to overcome some limitations of traditional methods because it can also determine non target DNA Aim 3: Different scenarios of unintentional contamination will be re-created either in the laboratory or using the production lines of food companies. DNA and protein cross-over from one species to another will be investigated. Effectiveness of commonly used decontamination procedures will be evalutated. Impact and Translational Implications The project will allow the Ministry of Health to plan efficient monitoring control programs for food frauds and to promptly respond to emergencies or crisis, taking advantage of a network of comparable laboratories and of the development and harmonization of the best suitable tests. The results of the study on cross-contamination and decontamination will be useful for decision makers and the food industry to decide for the implementation of fit for purpose Good Manufacturing Practices. 10/07/ / 6

207 RF Targeting mitochondrial calcium handling in core myopathies: an integrative approach towards novel therapeutic strategies Pegoraro Elena Biomedical/Biomedica Veneto 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 Skeletal Muscle Biology and Exercise Physiology - SMEP Project Keyword 1: Project Keyword 2: Project Keyword 3: Skeletal muscle diseases: evaluation of genetics, gene function, and development of vertebrate and invertebrate genetic models; pathophysiology of skeletal muscle disorders and diseases, including the muscular dystrophies, atrophy, myotonia, periodic paralysis, malignant hyperthermia, and inflammatory myopathies; pharmacological interventions and pre-clinical approaches; cell and gene therapies for skeletal muscle diseases. Mitochondrial Calcium Signaling Core myopathies 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/Lavoratory Role in the project 1 Veneto Department of Neurosciences NPSRR/Clinica Neurologica-Azienda Ospedaliera Padova/Neuromuscular Laboratory, Padova, Italy 2 University of Padova Department of Biomedical Sciences, Padova, Italy Principal investigator Investigator Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Szabadkai Gyorgy University of Padova Responsible of performing functional and morphological inamging and biochemical analysis of cellular models derived from animals and patients 25/03/ /07/ / 6

208 RF Targeting mitochondrial calcium handling in core myopathies: an integrative approach towards novel therapeutic strategies Pegoraro Elena Biomedical/Biomedica Veneto Background and Significance Disruption of cellular Ca2+ homeostasis underlies most muscle pathologies, in particular core myopathies, such as Central Core Disease (CCD) and Multi Minicore Disease (MmD), caused by mutations of the ryanodine receptor (RyR1) Ca2+ release channel. The unifying feature of these debilitating diseases is the loss mitochondria from the myofibers, but we have no understanding of the pathomechanism downstream of RyR1 mutations and thus no effective treatment of the condition has yet been developed. Our preliminary data indicate that defective RyR1 mediated signalling in CCD/MmD leads to maladaptive, enhanced IP3R expression, IP3 mediated Ca2+ release and consequent elevated Ca2+ accumulation in mitochondria, suggesting that Ca2+ mediated mitochondrial dysfunction underlies the disease. Moreover, we have described a human disease caused by mutations in the MICU1 gene, which leads to over-activation of the mitochondrial Ca2+ uptake machinery and triggers a similar pathological phenotype in patient muscle. Here we will test the hypothesis that Ca2+ overload in mitochondria of CCD and MmD muscle disrupts the function of the organelle, leading to its degradation and compromised cellular energy supply, representing a key pathomechanism of the disease. Ultimately we will explore whether genetic or pharmacological inhibition of mitochondrial Ca2+ overload can represent a viable strategy to prevent the formation of central cores and muscular dysfunction in RyR1 mutant animal models. Specific aims Aim 1: Establish cellular, animal and human models of core myopathies. Cell lines, RyR1 receptor mutant knock-in animals (I4895T) and differentiated myotubes from patient biopsies will be maintained. Aim 2: Establish the relationship between deregulation of muscle Ca2+ signalling, mitochondrial function and cell function/fate in core myopathy models. Using the above established models we will measure bioenergetic, morphological, signalling parameters of mitochondria in a series of models carrying different CCD/MmD mutations, to establish phenotype/genotype relationships. Aim 3: Determine the effect of genetic and pharmacological inhibition of mitochondrial Ca2+ uptake on the development of symptoms of core myopathies We will identify and use small molecule inhibitors of mitochondrial Ca2+ uptake and Ca2+ induced dysfunction, as well as overexpress/silence modulators of the Ca2+ uptake machinery (e.g. MICU1) to validate the pharmcological target. Hypothesis: Our central hypothesis is that RyR1 mutations leading to CCD/MmD ultimately cause Ca2+ overload in mitochondria, disrupting the function and structure of the organelle, leading to its degradation and compromised cellular energy supply. We propose that this pathomechanism is a key unifying feature of the disease, thus inhibiting mitochondrial Ca2+ uptake and/or Ca2+ mediated mitochondrial damage will be a novel promising therapeutic strategy to tackle this debilitating disease. Preliminary data: Four sets of preliminary data instigated the principal hypothesis and corroborate the aims of the proposal. 1. We have shown that mitochondrial Ca2+ overload due to loss-of-function mutations of MICU1 leads to human disease including muscle weakness and core pathology. 11/07/ / 6

209 RF Targeting mitochondrial calcium handling in core myopathies: an integrative approach towards novel therapeutic strategies Pegoraro Elena Biomedical/Biomedica Veneto 2. Using cellular models we have shown that loss of function of RyR1 (following its knockdown) significantly enhanced IP3 induced Ca2+ signals. Since it is has been previously well established by us and others that IP3 mediated signalling is tightly linked to mitochondrial Ca2+ uptake, these results strongly corroborate our model of pathogenesis in core myopathies. 3. Forty five muscle biopsies showing extensive areas with reduced mitochondrial activity defining cores or minicores have already been collected for the study. We have identified 20 causative RYR1 and 3 SEPN1 gene mutations. For each muscle biopsy linked extensive clinical and morphological data and DNA samples have been assembled. Moreover, for 34/45 patients cultured myoblast cell lines have been generated. 4. We have developed an assay to screen chemical compounds to identify specific inhibitors of mitochondrial Ca2+ uptake. For this purpose we have validated a high throughput live cell based assay using recombinant targeted aequorin probes to differentiate between modulators of global cellular and specific mitochondrial Ca2+ signals and performed a screen on a library of 1040 compounds (NINDS-II). Materials and Methods The success of this application depends on two principal components of which the PIs are renowned experts. First, the laboratory GS will perform functional and morphological imaging and biochemical analysis of cellular models derived from animals and patients. Second, EP is a well-known clinical neuromuscular expert with ample experience in detailed patient phenotyping, biobanking and gene expression analysis ensuring the availability of well characterized clinical cohorts of patients and samples. Impact and Translational Implications Whilst a large number of mutations have already been identified to cause CCD/MmD, the pathomechanism of the disease phenotype is not understood. We approach the disease from an entirely novel perspective, which if proved will have a massive impact on (i) the basic biological understanding of the pathomechanism, i.e. maladaptation to the lack of RyR function; as well as on (ii) translational potential by developing novel therapeutic strategies to alleviate the phenotype of a crippling disease. 11/07/ / 6

210 RF Identifying preclinical phenotypic and biological markers of Alzheimer's Disease in healthy elderly at risk subjects Caltagirone Carlo 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: Neuroimaging, functional, biochemical, and neuropathological studies to assess the onset, progression, treatment, and development of biomarkers for brain disorders. preclinical dementia at risk subjects 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/Lavoratory Role in the project 1 Fondazione Santa Lucia Clinical and Behavioural Neurology Laboratory Project coordination Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Spalletta Gianfranco Development of functional, cognitive and neuropsychiatric matrix. Neuroimaging acquisition and analysis. Lipidomic analyses 2 Koch Giacomo Neurophysiological data acquisition and analyses 08/08/ /12/ /07/ / 6

211 RF Identifying preclinical phenotypic and biological markers of Alzheimer's Disease in healthy elderly at risk subjects Caltagirone Carlo Clinical health care research/clinicoassistenziale Fondazione Santa Lucia Background and Significance Promising studies in dementia revealed the early use of phenotypic, imaging and biological markers identify at risk subjects decades before the appearance of cognitive decline. The costs of dementia are projected to be out of scale in the next two decades. It is therefore imperative to act during the extended asymptomatic phase and promote endogenous responses in neural systems that still have a great cognitive reserve and optimal levels of plasticity. Here, we test cost-effective tools to identify early progress of subtle cognitive decline when deficits are below the symptomatic threshold but represent a potential warning sign of disease. By unraveling the cognitive, neuropsychiatric, functional, neurophysiological, brain structural, and lipidic changes associated to subtle levels of neurodegeneration in middle age individuals, we will provide a robust tool that can be employed to test the effectiveness of therapeutic intervention when the brain can potentially still gain maximum benefits from therapy. We will also offer molecular insights on the neural bases and allow the identification of biological signatures of these processes. The overall benefit for society and the socio-economical implications of such approach is relevant. By identifying preclinical/symptomatic changes we will favor early intervention. The longer an individual remains cognitively efficient, the greater the chances she/he has to maintain satisfactory functional levels in daily living activities. Specific aims Aim 1: Aim 2: Aim 3: To develop a novel phenotypic, neuropsychological, neuropsychiatric and functional matrix that is sensitive and specific in detecting subtle signs of incipient (2-year time frame) cognitive decline and neurodegeneration. We aimed at discriminating middle-aged normal subjects who at risk of developing Alzheimer's Disease (AD) from middle-aged subjects not at risk. To compare the brain macro (volumetric) and micro (diffusivity) structural and neurophysiological (cortical plasticity and transmission) changes occurring in the 2-year time frame between the two study groups. To assess longitudinal changes in a set of peripheral blood lipids, reflecting cell membrane integrity and sensitive to early neurodegeneration, to improve the prediction of the phenoconversion from normal functioning to early cognitive impairment. Hypothesis: The hypothesis tested here is that, selecting subjects at risk of developing AD, the combination of phenotypic, neuroimaging, neurophysiological and lipidomic evaluations improves the prediction of early signs of dementia development. The idea is that even a modest deterioration in test performance (with scores nevertheless within the normal range) possibly indicates that these individuals are already substantially tapping on their cognitive reserve. This condition should be paralleled by early signs of structural brain and neurophysiological changes and by lipid alterations recently shown to predict MCI-AD conversion in elderly healthy individuals. Preliminary data: We have shown that volume and DTI-derived parameters of specific brain areas are key predictors of working memory (1), declarative memory in aging (2), education-mediated brain reserve (3) and subclinical apathy in healthy individuals (4). Further, we demonstrated that these brain structural neuroimaging parameters may predict healthy aging (5) and progressively change from healthy subjects to MCI and AD patients (6). From a neurophysiological point of view, we recently demonstrated that when tested with noninvasive repetitive transcranial magnetic stimulation (rtms), using theta burst stimulation (TBS) protocols, AD patients typically show a marked impairment of LTP-like cortical plasticity (7,8). Finally, several studies have pointed out that aberrant lipid metabolism is implicated in AD pathophysiology and we have shown significant alterations of plasma fatty acids (9) in MCI and AD subjects when compared to healthy subjects. 10/07/ / 6

212 RF Identifying preclinical phenotypic and biological markers of Alzheimer's Disease in healthy elderly at risk subjects Caltagirone Carlo Clinical health care research/clinicoassistenziale Fondazione Santa Lucia (1) Neuroscience ;171: ; (2) Neurology ;74: ; (3) Hum Brain Mapp :282-9; (4) Hum Brain Mapp : ; (5) Neuroimage :29-36; (6) J Alzheimers Dis : ; (7) Clinical Neurophysiology : ; (8) J Alzheimers Dis :593-9; (9) Alzheimers Dis : Materials and Methods We will include 400 first-degree relatives of AD patients, considered as at risk (AR) of developing AD, and 200 subjects with negative familiar anamnesis for AD (not at risk -NAR). AR and NAR will be stratified as having low, intermediate and high adjunctive risk according to the Mid-Life Dementia Risk Score. All participants will be recruited in the first 9 months of the project and tested immediately after recruitment (T0), and 1 (T1) and 2 (T2) years later. AR will be divided into two groups, with and without cognitive decline, according to an extended and innovative neuropsychological-neuropsychiatric-functional battery sensitive to the earliest detectable pathological changes in cognition. All subjects will undergo volumetric and DTI magnetic resonance imaging as well as paired pulse-repetitive TMS and oscillatory EEG analyses at T0 and T2. Finally lipid profile will be derived from peripheral blood sampling. Impact and Translational Implications AD represents a public health problem due to its growing estimated prevalence in world population. The differentiation of pathogenic courses in presymptomatic phases of AD through the development of research criteria and accurate operational tools would allow clinicians to implement more effective treatment-rehabilitative interventions. The present project encourages valid primary prevention approaches, with an improvement of patients' quality of life and public health cost savings. 10/07/ / 6

213 A new immunotherapy option for oral melanoma and other canine malignant tumors using a DNA vaccine coding for human chondroitin sulphate proteoglycan 4 (CSPG4), integrated with the validation of candidate gene useful for therapeutic opportunities and prognostic evaluations RF Ferrari Angelo Biomedical/Biomedica Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Oncology Project Classification IRG: Project Classification SS: Veterinary Animal Health - ANHE Project Keyword 1: Project Keyword 2: Project Keyword 3: Studies on neoplastic pathologies in animals, including evaluations of those aspects regarding animal health, epidemiological surveillance, and comparative pathology Canine malignancies DNA vaccine 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/Lavoratory Role in the project 1 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'aosta National Reference Center for Veterinary and Comparative Oncology, CEROVEC, GE; Laboratory of Genetics and Immunobiochemistry, TO Coordination. Tumor diagnosis, classification. CSPG4 gene expression analysis by RT qpcr targeted RNAseq of biomarkers by NGS platform 2 University of Torino Dep. of Molecular Biotechnology and Health Sciences, TO; Department of Veterinary Science, Grugliasco (TO) Immunogenicity testing, safety, therapeutic CSPG4 vaccination in dogs with resected oral malignant melanoma 3 Istituto Zooprofilattico Sperimentale della Sardegna Laboratory of Clinical Diagnosis-Animal facilities Immunological assays. Cell subsets identification in flow cytometry 11/07/ / 6

214 A new immunotherapy option for oral melanoma and other canine malignant tumors using a DNA vaccine coding for human chondroitin sulphate proteoglycan 4 (CSPG4), integrated with the validation of candidate gene useful for therapeutic opportunities and prognostic evaluations RF Ferrari Angelo Biomedical/Biomedica Investigators, Institution and Role in the Project Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Cavallo Federica University of Torino -Management of the experimental activities of Unit 2 -Vaccine production, vaccination and monitoring of the induced immune response 2 NICOLUSSI PAOLA Istituto Zooprofilattico Sperimentale della Sardegna 3 MODESTO PAOLA Istituto Zooprofilattico Sperimentale Piemonte, Liguria e Valle d'aosta; Laboratory of Genetics and Immunobiochemistry,Research scientist,dvm PhD-Not permanent position 4 Buracco Paolo Dep. of Veterinary Science, Grugliasco (TO); Full professor -Immunological assays. Cell subsets identification in flow cytometry -CSPG4 gene expression analysis by RT qpcr -Targeted RNAseq of candidate biomarkers by NGS platform in tumors -Provide access to surgically resected malignant melanoma canine patients and to their tumor samples, sera and PBMC; clinical follow-up 13/05/ /04/ /01/ /08/1956 Background and Significance Malignant melanoma (MM) is the sixth most common cancer worldwide. In its early stages, MM is highly curable by surgery, but about one third of patients will experience recurrence and metastasis; once metastasis occurred, MM is often fatal. Inherent immunogenicity and the role played by immunological events in MM natural history have led to develop cancer vaccines for its treatment. Despite several successful studies in murine models, therapeutic efficacy was so far disappointing. More appropriate vaccination targets and pre-clinical models are thus urgently needed. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several features with human disease that could make dog a more relevant preclinical model. Indeed, similarities between human and canine MM make spontaneous canine MM an excellent model for exploring novel immunotherapies. Among the potential antigens for DNA vaccination, chondroitin sulphate proteoglycan 4 (CSPG4) represents an attractive target for MM immunotherapy because of its restricted distribution in normal tissues, its high expression in melanoma stem and differentiated cells, and its role in tumor cell proliferation, migration and invasion. Moreover, the CSPG4 expression in human and canine MM is similar, being 80% and 60% respectively. Being expressed by variety of human tumor types DNA vaccination against CSPG4 could be expanded to different types of disease. Specific aims Aim 1: Aim 2: To test the safety immunogenicity (including cell mediated response) and therapeutic efficacy of CSPG4 vaccination as alternative option for the management of canine MM to assess the power of combinatorial strategies of CSPG4 vaccination with ipilimumab administration. To evaluate the expression of CSPG4 and other candidate biomarkers, considering both frequency and levels of expression in MM, blood and other canine tumor histotypes (i.e. mammary carcinoma, osteosarcoma, squamous 11/07/ / 6

215 A new immunotherapy option for oral melanoma and other canine malignant tumors using a DNA vaccine coding for human chondroitin sulphate proteoglycan 4 (CSPG4), integrated with the validation of candidate gene useful for therapeutic opportunities and prognostic evaluations RF Ferrari Angelo Biomedical/Biomedica Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle cell carcinoma, haemangiopericytoma). Aim 3: To correlate the gene expression of CSPG4 and other candidate markers with clinical outcomes and disease-free survival times. Hypothesis: CSPG4 is an early cell surface marker involved in tumor cell proliferation, migration and invasion and is highly conserved in its structural and functional properties through phylogenetic evolution. CSPG4 expression by both stem and differentiated melanoma cells and its role in tumor progression suggest it may be an ideal vaccination target. Positive results obtained by vaccinating client-owned dogs carrying naturally occurring, surgically resected CSPG4+ MM with a plasmid that codes for human CSPG4 endorse this hypothesis and indicate that xenogeneic vaccination is able to overcome host unresponsiveness to the selfantigen. Gene expression analysis in cancer studies allows the detection and quantification of relative expression level of genes involved in development, progression and tumor chemoresistance. It can be a useful tool for therapeutic opportunities and prognostic evaluations. Our hypothesis is that a correlation might exist between CSPG4 expression levels and the vaccination effectiveness. CSPG4 expression analysis could therefore represent a valuable tool to predict disease-free survival times. RNA-seq is a technology that uses the capabilities of next-generation sequencing to investigate gene expression changes and to potentially identify genes significantly associated with tumor cell proliferation, migration and invasion. Targeted RNA-seq of genes involved in the CSPG4 signaling pathways will be used to measure the abundance of transcripts of interest. This approach will provide both quantitative and qualitative information that may allow to identify novel biomarkers and to get more information about genes involved in cytoskeletal reorganization, survival and chemoresistance and migration. Preliminary data: We have recently shown in a pilot clinical trial in client-owned dogs with surgically resected stage II- III CSPG4+ oral malignant melanoma that intramuscular electroporation (electrovaccination) of a plasmid that codes for human CSPG4 is safe and induce an antibody response against CSPG4. This immune response was associated with survival prolongation when compared to dogs treated with surgery alone. Data from human trials indicate that anti-ctla4 mab (ipilimumab) administration may result in an improved cancer immunotherapy outcome. Preliminary data show that ipilimumab crossreacts with canine CTLA4. Materials and Methods Patients will be enrolled into 2 arms: vaccinated with human CSPG4 with/without Ipilimumab. Sera and PBMC will be collected after each vaccination to characterize humoral/cellular immune responses and the induced action mechanisms. Median survival and disease-free times will be analyzed. Histopathology: H&E staining; tumor classification (WHO); Immunohistochemistry for specific markers of MM and other tumor types. Gene expression analysis: evaluation of the CSPG4 expression by RT qpcr in different canine tumor histotypes and in blood samples. To normalize gene expression data a panel of suitable reference genes will be optimized. Targeted RNAseq, using MiSeq platform, that allows to investigate of user-defined target transcripts. Candidate genes, involved in pathways activated by CSPG4, will be selected. Expression levels will be correlated to clinical outcomes and diseasefree survival time. Flow-cytometry: peripheral blood and lymph nodes cell subsets identification. 11/07/ / 6

216 Modulation of visuospatial awareness during multi-tasking in right hemisphere stroke patients: towards novel behavioral and neurofunctional predictors of impairment and recovery of the attentional networks RF Zorzi Marco 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: Integrative, Functional, and Cognitive Neuroscience Cognitive Neuroscience - COG Project Keyword 1: Project Keyword 2: Project Keyword 3: Attention: influences of attention on information flow within the brain in human and animal studies. Hemispatial neglect; stroke neuroimaging; fmri; EEG; resting state 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/Lavoratory Role in the project 1 Ospedale San Camillo Laboraroty of Neuropsychology management, data collection, data analysis, dissemination Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Pitteri Marco Coordination of neuropsychological testing and rehabilitation 2 gasparoli elisabetta Screening and recruitment of stroke patients 01/03/ /08/ Campostrini Stefania Electrophysiological recordings 14/07/ /07/ / 6

217 Modulation of visuospatial awareness during multi-tasking in right hemisphere stroke patients: towards novel behavioral and neurofunctional predictors of impairment and recovery of the attentional networks RF Zorzi Marco Biomedical/Biomedica Ospedale San Camillo Background and Significance Following right hemisphere damage (RHD), many stroke patients show deficits related to the disruption of the neural mechanisms subserving spatial attention orienting and awareness, the best known being left hemispatial neglect. Neglect dramatically affects both patients and their families, as well as the outcome of rehabilitation, because it is associated with severe impairments in virtually all everyday activities. Spatial awareness for contralesional visual targets in RHD patients can be severely impaired by a concurrent task that requires the deployment of additional attentional resources (a condition that often occurs in everyday cognitive activities). Multi-tasking affects the deployment of spatial attention and modulates the electrophysiogical signatures of early visuo-spatial processing in neurologically intact subjects. In RHD patients multi-tasking can lead to failures in consciously perceiving contralesional visual events, even in patients who show normal performance on standard clinical tests for neglect assessment. Our hypothesis is that behavioural and neurofunctional markers of susceptibility to multi-tasking is a better predictor of patients' performance in everyday life activities as well as of the future outcome of functional recovery than standard neuropsychological assessment. Moreover, in a pilot rehabilitation study, we will investigate the behavioral and neural effects of computer-based training that includes multi-tasking as a core feature. Specific aims Aim 1: Aim 2: Aim 3: Validation of visuospatial monitoring under dual-task as computerized diagnostic tool. Our paradigm uncovers subtle or mild forms of neglect that are undetected by standard paper-and-pencil tests. Validation on a large population of patients will fully assess the diagnostic potential and the correlation with a standardized scale for measuring neglect in everyday life activities. Identify behavioural and neurofunctional markers of susceptibility to multi-tasking and assess their predictivity of future outcomes of functional recovery. EEG and fmri will be used to detect the neural signatures of the modulatory effect of multi-tasking on visuospatial processing. We will also assess whether these effects can be linked to resting state activity, especially in the dorsal attention network and in the posterior visual processing network, as well as to individual patterns of brain connectivity. Assess behavioral and neural changes following computer-based training that includes multi-tasking in a pilot rehabilitation study. Treatment will be based on our recent adaptive videogame that is specifically designed for training attention and executive functions in neurological patients. We will assess changes in both functional and structural connectivity. Hypothesis: 1. Visuo-spatial monitoring performance during multi-tasking is better correlated with standardized measures of neglect in everyday life activities than standard paper-and-pencil neuropsychological assessment scores 2. Behavioral and neurofunctional markers of susceptibility to multi-tasking are predictive of future functional recovery 3. Computerized training of visuospatial attention that includes multi-tasking re-shapes connectivity patterns in the attention networks and is an effective tool for neglect rehabilitation Preliminary data: The computerized task has been extensively piloted in small-scale studies. Visuo-spatial monitoring is performed either in isolation (single task) or together with a concurrent task (dual-task). This topdown manipulation of attentional load affects the detection of contralesional targets. Severe visual extinction or neglect can emerge even in patients who show normal performance on standard clinical tests for neglect assessment (Bonato et al., 2010, 2012, 2013). Our task it is more sensitive than cued-detection (Posner task) in uncovering residual deficits (longitudinal study; Bonato et al., 2012). Electrophysiological results on healthy participants show that multi-tasking affects early components of ERP responses to peripheral targets (see supporting figure). 11/07/ / 6

218 Modulation of visuospatial awareness during multi-tasking in right hemisphere stroke patients: towards novel behavioral and neurofunctional predictors of impairment and recovery of the attentional networks RF Zorzi Marco Biomedical/Biomedica Ospedale San Camillo The rehabilitation study will exploit our adaptive videogame designed for training attention and executive functions in neurological patients (Montani et al. 2014). Playing involves visuospatial orienting, selective attention and multitasking as core features. A short training protocol (<10 hours) induced cognitive enhancement in healthy participants as well as changes in resting state EEG activity (see supporting figure). Materials and Methods We will recruit 60 RHD patients following first stroke. Lesion extension will be quantified by structural MRI. Resting state fmri and DTI data will be collected to assess brain connectivity. All patients, following neuropsychological assessment, will perform the computerized dual-task paradigm. Brain connectivity indices will be correlated with individual differences in the dual-task paradigm as well as with the functional recovery after rehabilitation. Patients who show mild or no signs of neglect on standard paper-and-pencil tests will be included in task-based fmri and EEG studies (i.e., during the dual task paradigm); analyses will focus on BOLD modulation in critical nodes of the attention networks, and on components of stimulus-evoked EEG potentials associated with automatic stimulus processing. All patients with neglect will be included in the videogame-based rehabilitation study. DTI and rfmri data will be collected to assess treatment effects on brain connectivity patterns. Impact and Translational Implications Neglect symptoms are often more severe in everyday life than in paper-and-pencil tests for neglect assessment. Our computerized task is insensitive to compensatory strategies and uncovers subtle or mild forms of neglect that are not detected by standard tests. The discovery of markers that are predictive of functional outcome would inform and optimize the implementation of rehabilitation. Videogame-based training would substantially reduce treatment costs and allow selftraining upon discharge. 11/07/ / 6

219 RF A NOVEL INTEGRATED GENOMIC AND IMMUNOLOGICAL STRATEGY FOR THE THERAPY OF CHRONIC HEPATITIS B Ferrari Carlo Biomedical/Biomedica Emilia-Romagna 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-microbe interactions: innate and acquired host immune responses to specific pathogenic organisms including viruses, bacteria, fungi and parasites; host responses to commensal microbes; influence of host factors, including genetic predisposition or resistance to infection. chronic hepatitis B, genome wide microarray analysis T cell exhaustion, T cell functional reconstitution 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/Lavoratory Role in the project 1 Emilia-Romagna Azienda Ospedaliero-Universitaria di Parma/Unit of Infectious Diseases and Hepatology/ Laboratory of Viral Immunopathology 2 Emilia-Romagna / Università degli Studi di Parma 3 Lazio / Università degli Studi di Roma "La Sapienza" Department of Biochemistry and Molecular Biology Department of Internal Medicine and Medical Specilties Coordination; patient recruiment; transcriptomic analysis; immunological validation; data mining Transcriptomic analysis; data mining Molecular validation; data mining Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Ottonello Simone Emilia-Romagna / Università degli Studi di Parma 2 Levrero Massimo Lazio / Università degli Studi di Roma "La Sapienza" Transcriptomic analysis; data mining 23/07/1951 Molecular validation; data mining 28/08/ /07/ / 6

220 RF A NOVEL INTEGRATED GENOMIC AND IMMUNOLOGICAL STRATEGY FOR THE THERAPY OF CHRONIC HEPATITIS B Ferrari Carlo Biomedical/Biomedica Emilia-Romagna Background and Significance In chronic HBV infection virus-specific T cells are hypo-responsive to antigen stimulation and functionally defective. T cell exhaustion by persistent exposure to high antigen concentrations is believed to be a cause of T cell impairment, which in turn contributes to virus persistence. Reconstitution of an efficient anti-viral T cell response may thus represent a rational strategy to treat chronic HBV patients and to complement the effect of available anti-viral therapies. At present, treatment of chronic HBV infection is mainly based on nucleos(t)ide analogue (NUC) therapy, which must be lifelong in most patients. Therefore, there is an urgent need to define novel strategies to shorten the duration of therapy and to achieve virus eradication. Based on data from other natural and animal models of chronic viral infection, blockade of overexpressed inhibitory pathways has been tested in chronic HBV infection to restore the T cell function, but efficacy has been partial. However, a comprehensive evaluation of the regulatory mechanisms involved in T cell dysfunction has never been done by a systematic transcriptome analysis directly performed on exhasted virus-specific T cells. In this proposal misregulated genes/pathways associated with HBV persistence and T cell exhaustion will be identified and validated through a genome-wide approach with the aim of developing novel strategies for targeted manipulations of the immune response as novel anti-viral therapies for HBV infection Specific aims Aim 1: Aim 2: To target misregulated genes/pathways identified in HBV-specific and total CD8 cells from chronic naive HBV patients, in order to develop novel therapeutic T cell functional reconstitution strategies for chronic HBV infection. To assess to what extent genes/pathways misregulation can be corrected by prolonged viral suppression and inflammation control induced by NUC treatment and to identify persistently misregulated genes/pathways as therapeutic targets for complete control of infection and HBV eradication in NUC treated patients. Aim 3: - Hypothesis: Misregulated genes/pathways identified by transcriptome analysis of virus-specific CD8 cells from chronic HBV patients represent potential targets for antiviral immune response reconstitution, as a therapeutic strategy to treat chronic hepatitis B. Identification and validation of therapeutic targets might be extended and facilitated by transcriptome analysis of more easily obtainable total CD8 cells since they partially mirror virus-specific transcriptional signatures and show also unique molecular features, as a likely expression of chronic immune activation and hyperfunction of negative regulatory networks. Preliminary data: Virus-specific CD8 cells from 4 naive HBeAg- chronic active HBV patients and from 4 subjects who resolved spontaneously an HBV acute infection were sorted and gene profiles associated with virus clearance or persistence were characterized by transcriptome analysis. Comparison of the two groups of patients by moderated t-test allowed to generate a list of 442 differentially expressed genes (DEG) (p<0.01, FC>2), 73 of them being more deeply misregulated (p<10-4, FC ). A number of perturbed pathways/processes were then identified by gene ontology, pathway analysis and GSEA in CD8 cells of chronic patients, including pathways related to electron transport chain, proteasome degradation, cellular response to stress, cellular metabolisms and intracellular signaling. Functional validation of misregulated mitochondrial-related pathways in virus-specific CD8 cells by fluorescent probes in flow cytometry confirmed a marked alteration of the mitochondrial function in chronic HBV patients. Treatment of HBV-specific T cells with mitochondrial-targeted antioxidant drugs able to enhance the mitochondrial function lead to better anti-viral T cell responses. The transcriptome analysis was performed also on total CD8 cells from 5 naive HBeAg- chronic active patients and 4 subjects in the resolution phase of acute infection. Data mining revealed a 11/07/ / 6

221 RF A NOVEL INTEGRATED GENOMIC AND IMMUNOLOGICAL STRATEGY FOR THE THERAPY OF CHRONIC HEPATITIS B Ferrari Carlo Biomedical/Biomedica Emilia-Romagna unique CD8 trascriptional profile associated with chronic persistence, including misregulation of some immunologically relevant pathways related to cytokine signaling and regulation of the immune response which may have an effect on the HBV-specific T cell function. On the other hand, some relevant biological processes related to mitochondria and cellular metabolisms showed a consensual misregulation in HBV-specific and total CD8 cells. Materials and Methods Candidate target genes identified by microarray analysis on HBV-specific and total CD8 T cells sorted from convalescent and chronic patients (naive and NUC-treated) will be validated by real time-pcr, PCR arrays and/or the NanostringTM beads arrays technology on a larger group of patients and confirmed at a protein level by flow cytometry. Available blocking/stimulatory antibodies targeting receptor proteins found to be involved in chronic T cell dysfunction will be employed to restore anti-viral responses. Validation of misregulated pathways in chronic patients will be performed by flow cytometry with specific fluorescent dyes. Next, confirmed dysfunctional pathways will be manipulated by treating virusspecific and total CD8 T cells with stimulator/inhibitor compounds and different classes of epigenetic drugs to reconstitute an efficient antiviral T cell response. T cell function will be tested ex vivo and in vitro by cytokine production, cytotoxicity and proliferation assays. Impact and Translational Implications NUC therapy for chronic hepatitis B imposes high health care costs and discomfort for the patients, because they frequently undergo NUC treatment for life. A genome-wide analysis of virus-specific and total CD8 T cells will provide novel information on the pathogenesis of T cell dysfunction in HBV infection allowing to design novel T cell restoration strategies as possible first line therapies for chronic HBV infection or combined treatments to shorten NUC therapies and eradicate HBV infection. 11/07/ / 6

222 RF Effects of maternal nutrition during pregnancy on fetal epigenetic profile: set up of a research infrastructure. Bosari Silvano Biomedical/Biomedica Fondazione Ca'Granda - Ospedale Maggiore Policlinico LETTER OF INTENT - ABSTRACT New strategies for diagnostic, therapeutic and clinical care in Innovative biotechnologies 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: Behavioral genetics and heritability biobank epigenetics 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/Lavoratory Role in the project 1 Fondazione Ca'Granda - Ospedale Maggiore Policlinico 2 Università degli Studi di Milano Unit of Pathology Department of pathophysiology and transplantation Set up of the maternal/fetal biobank; sample collection; microrna studies; collection and evaluation of clinical data Methylation studies; collection and evaluation of nutritional information Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Tabano Silvia Maria Università degli Studi di Milano Set up and performing methylation studies; Evaluation of the results. 2 3 Alberico Daniela Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico/Dipartimento per la salute della mamma, del neonato e del bambino. 4 lonati caterina Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico Recruitment of pregnant women; dispensation of the nutritional questionnaire; sample collection Analysis of the nutritional questionnaire; performing molecular analyses 03/08/ /07/ /08/ /07/ / 6

223 RF Effects of maternal nutrition during pregnancy on fetal epigenetic profile: set up of a research infrastructure. Bosari Silvano Biomedical/Biomedica Fondazione Ca'Granda - Ospedale Maggiore Policlinico Background and Significance The in utero environment can exert profound effects on fetal health and have lifelong consequences. Nutrition is one environmental condition experienced in early life: its effects are mostly due to the action of food components in modulating histone code, DNA methylation and mirna expression. Although accumulating evidences highlight the importance of maternal nutrition during pregnancy, studies investigating the dietary habits of pregnant women and their correlations with neonatal status are currently lacking. In addition, the methylation and mirna profile of the newborn is still poorly characterized and the effects of maternal diet on epigenetics signatures have not been investigated. The difficulty of obtaining appropriate material for molecular investigation, together with annotated clinical data, hampers the execution of such studies. The creation of a biobank to collect and store maternal/neonatal (M/N) samples, well characterized for maternal nutritional status and diet habits during pregnancy is the starting point to address the above issues. Significance: to set up a biobank of M/N samples, representing the resource for materials for this project and a repository of biological specimens for future studies; to establish the epigenetic signatures using M/N specimens, defining which are most useful for the present and future projects; to determine whether specific epigenetic signatures are associated with maternal nutritional status and dietary habits. Specific aims Aim 1: Aim 2: Aim 3: To create a biobank of maternal, fetal and placental samples. Specific operating procedures will be developed in order to ensure the collection of specimens with high molecular quality suitable for innovative genomic techniques. In particular sample transfer from the operating room to the biobank, tissue collection, storage and clinical data processing will be standardized. All samples stored in the biobank will derive from patients well characterized clinically by: i) complete obstetrical information (including BMI and increasing of weight across pregnancy); ii) nutritional questionnaire, reporting the maternal dietary habits during pregnancy; iii) newborn clinical features (fetal and placental weight, other neonatal measures, APGAR score). To define the epigenetic profile of mothers and offsprings, focusing in particular on candidate molecules involved in fetal growth, by investigating, at birth, DNA methylation and microrna (mirna) expression profile. To reach this issue, a subset of samples stored at the biobank will be analyzed. Samples will be selected based on the maternal Body Mass Index (BMI, kg/m2) and will be stratified in four classes, as follow: 1) BMI<18.5, 2) 18.5<BMI<24.9, 3) 25.0<BMI<29.9, and 4) BMI<30.0. To correlate the epigenetic signatures found in mothers and offsprings with the mother's nutritional status (BMI) and dietary habits. Hypothesis: The expression pattern of genes involved in fetal growth and metabolism can be influenced by the maternal nutritional status and dietary habits during pregnancy, through alterations of DNA methylation and/or mirna expression. The epigenetic modulation of these genes can influence the outcome of pregnancy and the offspring health. Preliminary data: The project includes researchers with different skills, to better address all the aspects of the research. The duration of the project is 3 years. The PI is the director of the Pathology Unit at the Host Institution (HI). He also coordinates a research Unit deeply involved, from several years, in mirna profiling studies by low density array platform, differential gene-expression signatures and bioinformatic analyses. In addtion, the PI group has acquired experience in tissue biobanking. In particular, since 2007 they collected more than 900 lung tumor and normal tissue specimens, thus establishing one of the most important collection of thoracic pathological samples in the region. The other unit leader (ST) has extensive experience in DNA methylation analyses and studied epigenetics features of loci important in fetal growth and development. She also participated to the 10/07/ / 6

224 RF Effects of maternal nutrition during pregnancy on fetal epigenetic profile: set up of a research infrastructure. Bosari Silvano Biomedical/Biomedica Fondazione Ca'Granda - Ospedale Maggiore Policlinico European grant: "Influence of Dietary Fatty Acids on the Pathophysiology of Intrauterine Foetal Growth and Neonatal Development", focusing on the research of placental biomarkers associated to intrauterine development. Finally, the request for the approval of a fetal/maternal biobank and the questionnaire for the dietary habits survey have been recently submitted to the Ethical Committee of the HI. Materials and Methods Samples: Maternal and cord blood, placenta, from 2000 pregnancies will be recruited at Clinica Mangiagalli, belonging to the HI and stored at the biobank. Inclusion criteria: European women, without chronic medical conditions, with single, spontaneous pregnancies, without neonatal malformations and/or genetic anomalies. Samples from 200 pregnancies, equally subgrouped in different classes based on BMI, will be subjected to the epigenetic analyses. Methods: 1) Collection of complete obstetrical/nutritional information of pregnant women. 2) Analysis of the questionnaire on the mothe'rs dietary habits: calculation of intake of calories/day, type of diet, carbohydrates, lipids, proteins %, folate/others supplementation. 3) Epigenetic analyses: - methylation profile evaluated by: methylation SNPs array (Microarray Scanner, Agilent) and massarray EpiTYPER (Sequenom) - mirna exression profile (Life Technologies) A complete laboratory equipment for the epigenetic studies is present at the HI. Impact and Translational Implications Given the increasing evidences about the correlations between maternal nutrition and pregnancy outcome, the comprehension of the molecular mechanisms responsible for its role, finalized to the set up an early detection should be a health priority. Translational implications: M/N samples available in the biobank of HI for research purposes; methylation and mirna profiling: establishment of feasibility and appropriate material; correlation between clinical information and epigenetic features. 10/07/ / 6

225 Vitamin E, mirna and inflammation: a tunable network in Alzheimer's disease. BANDO 2013 Progetti Ordinari RF RF Mecocci Patrizia Biomedical/Biomedica Umbria 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. mirna micronutrients 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/Lavoratory Role in the project 1 Umbria DIP DI MEDICINA, SEZIONE DI GERONTOLOGIA E GERIATRIA 2 IRCCS CENTRO NEUROLESI BONINO 3 UNIVERSITA' STUDI DI PERUGIA DIP DI NEUROLOGIA, DIVISIONE MEDICINA DIP. DI MEDICINA, SEZIONE DI FARMACOLOGIA U.O. N. 1 U.O. N. 2 U.O. N. 3 Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Marra Angela IRCCS CENTRO NEUROLESI BONINO PULEJO 2 Riccardi Carlo UNIVERSITA' STUDI DI PERUGIA RESPONSABILE U.O. N. 2 21/10/1962 RESPONSABILE U.O. N. 3 03/10/ /07/ / 6

226 Vitamin E, mirna and inflammation: a tunable network in Alzheimer's disease. BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Mecocci Patrizia Umbria Background and Significance Alzheimer's disease (AD), the most frequent type of dementia worldwide, represents one of the main causes of severe disability in old age people, with incredibly high impact in human and financial costs. Despite the great efforts of research, our knowledge of the etiopathogenetic mechanisms of AD and, consequently, a pharmacological therapy able to modify the natural course of the disease are still lacking. Although its etiology is not known yet, most experts agree that AD, like other common chronic conditions, probably develops as a result of multiple factors rather than a single cause. So, new studies need to address contribution of different pathways for AD etiopathogenesis. Increasing age, genetic factors, genetic modulation, oxidative stress, inflammation, accumulation of altered proteins, cardiovascular risk factors, diet, have been involved in AD pathogenesis, but their specific role and mechanisms of action are still unclear. Recently microrna (mirna), an abundant class of short non coding RNA, have been discovered as gene modulators. Since, as recently suggested, they control cognitive and immune functions, both altered in AD, and are influenced by micronutrients, such vitamins, their role as a link among different pathogenetic pathways is extremely intriguing. Vitamin E family (tocopherols and tocotrienols) seems also to be involved in AD, as showed in population studies, and -tocopherol has been proposed, with contrasting results, as therapy in AD. Specific aims Aim 1: Aim 2: Aim 3: The first step of this study is the evaluation of several chemokines and cytokines as well as of all eight vitamin E forms (alpha-, beta, gamma- and delta-tocopherol; alpha-, beta, gamma- and delta-tocotrienol) in plasma of subjects with AD and in age-matched controls, by means of multiplex and HPLC-coularray, respectively. On the basis of the observed differences, a group of mirna more strictly related to the inflammatory pattern previously detected (here called inflam-mirna), will be also measured in plasma samples. Correlation analyses will be then performed between each of the eight vitamin E forms and the mirna differently expressed between groups, in order to detect which form of vitamin E, among the eight, may be considered the most influent in modulating the mirna patterns of inflammation. This one will be chosen for the subsequent study in animal model (aim 3). Inflam-miRNA previously detected in plasma samples will be measured in human brain specimens (hippocampal, prefrontal and temporal cortex) of AD and normal subjects, in order to evaluate if there is a similar central/peripheral expression and to confirm a potential pathogenetic role of the detected inflam-mirna in AD. In order to establish the role of vitamin E in modulating the expression of inflam-mirna and of inflammatory proteins, APPswe/PS1dE9 mice will be supplemented or not with the form of vitamin E that showed the most strict relationship with inflam-mirna (see aim 1). After diet supplementation, the inflam-mirna previously detected will be evaluated in brain and in plasma of treated and untreated mice. Hypothesis: A growing number of epidemiological studies has shown that diet can influence the risk for several chronic disease. Whilst this is most clearly established for metabolic diseases, such as diabetes, less is known for neurodegenerative pathologies. Recently attention has been paid on the role of micronutrients and gene expression trough the action of mirna, that are present both in the central nervous system (CNS) and in the immune system. Since in AD a chronic inflammatory status is often detectable in CNS and in peripheral tissues, it seems conceivable to hypothesize that micronutrients modulate mirna and they in turn modulate (neuro)-inflammation. Among micronutrients, alpha-tocopherol has been widely studied in neurodegenerative diseases for its antioxidant activity. It has been tested in AD for prevention and for therapy, with contrasting results. More recently other forms of vitamin E have been studied for their action as modulators of inflammation and gene expression. This new field of research appears promising for the development of new therapies as well as for prevention. Preliminary data: Cross-sectional and longitudinal studies in Italian, Swedish and Finnish populations have shown that 11/07/ / 6

227 Vitamin E, mirna and inflammation: a tunable network in Alzheimer's disease. BANDO 2013 Progetti Ordinari RF RF Biomedical/Biomedica Mecocci Patrizia Umbria plasma levels of the forms of vitamins E are different in AD and cognitively healthy subjects and that different forms can influence the development of AD. Materials and Methods Cross-sectional and longitudinal studies in Italian, Swedish and Finnish populations have shown that plasma levels of the forms of vitamins E are different in AD and cognitively healthy subjects and that different forms can influence the development of AD. Impact and Translational Implications AD therapy remains a complex clinical challenge. Study on inflam-mirna expression and vitamin E plasma levels in AD patients will be useful for AD risk assessment and lead to AD prevention and develop new effective therapies. Costeffectiveness and immediate availability of vitamin E therapy could improve the quality of life of AD patients and alleviate National Health Service costs. In the future, new molecules may be developed to target inflam-mirna for novel efficacious therapies in AD. 11/07/ / 6

228 Clinical and Experimental Pathway to Vitiligo Management BANDO 2013 Progetti Ordinari RF RF LEONE GIOVANNI 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 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: Studies of skin interactions with the environment: photoaging, UV sensitivity reactions; role of skin in transepidermal delivery of drugs; role of skin as a barrier against infectious, mechanical, and other toxic insults vitiligo mitochondrial alterations, metabolic markers 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/Lavoratory Role in the project 1 Istituti fisioterapici ospitalieri Phototherapy Unit - Istituto Dermatologico Santa Maria e San Gallicano patients enrollment and clinical management, coordination 2 stituti fisioterapici ospitalieri - Istituto Dermatologico Santa Maria e San Gallicano Lab Cutaneous Physiopathology & CIRM in vitro tests, management clinical and experimental data, statistical analysis Investigators, Institution and Role in the Project Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Picardo Mauro Michele stituti fisioterapici ospitalieri - Istituto Dermatologico Santa Maria e San Gallicano design in vitro protocols and experimental data management 03/12/ /07/ / 5

229 Clinical and Experimental Pathway to Vitiligo Management BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale LEONE GIOVANNI Istituti fisioterapici ospitalieri - Istituto Dermatologico Santa Maria Background and Significance Vitiligo is an acquired skin disease characterized by the disappearance of pigment cells affecting 0,5-2% of population. Evidence suggests a link between melanocyte-intrinsic abnormalities and an autoimmune-mediated destruction as possible pathogenic mechanism. Vitiligo melanocytes due to an increased ROS generation express a pre-senescent phenotype and after the exposure to pro-oxidants undergo to degeneration and/or death. The subsequent release of DAMPs and unfolded proteins may activate cells of the innate immune system and facilitate the autoimmune process. However, no clear explanations on the biochemical mechanisms underlying oxidative stress have been still produced. The impairment of Electron Transport Chain (ETC) complex I, the expression of malate dehydrogenase and the effects of the macropore inhibitors suggest mitochondria as the site of uncontrolled ROS production. Identify the mechanisms leading to the metabolic defects can provides explanations for cell degeneration and for the induced immune activation, could provide new therapeutic targets. Whether the relationship between metabolic paths and immune deregulation will be defined, a biochemical profile to follow the natural and therapy-modified history of the disease could be determined. The comparison between degenerative process in vitiligo and those observed in neurodegenerative diseases could put vitiligo as a model for studying pathologies based on mitochondrion defects and oxidative-driven autotoxicity. Specific aims Aim 1: Aim 2: Aim 3: To define whether mitochondrial abnormalities occur in vitiligo melanocytes focusing on energetic and specifically glucose-related metabolisms, respiratory chain efficiency, ATP production and activity of factors regulating mitochondrial biogenesis. To clarify whether the metabolic alterations leads to a degenerative process inducing a skin inflammatory reaction activating the innate immunity. The key points will be: a) the biological response to growth factors and subsequent biological effects due to the activation of specific nuclear receptors; b) the generation of peroxidation by-products by a mild oxidative stress and their capability to activate blood derived dendritic cells. To identify parameters of disease status through the correlation between markers of oxidative stress and cell degeneration detectable in serum and skin with the evolution of the manifestations and their response to therapies. Hypothesis: Vitiligo melanocytes present a pre-senescent phenotype probably due to a continuous oxidative stress. Mitochondria, which play a crucial role in mediating the oxidative stress-related aging process, may be the source of ROS generation possibly due to defects in the Krebs cycle and ETC efficiency. The consequential alteration of AMP/ATP ratio interferes with the AMPK expression and therefore with the glucose metabolism. Over expression of AMPK decreases mtor pathway and activate PGC1a altering the network regulating mitochondrial biogenesis, cellular aging and inflammatory status. These phenomena may represent the biochemical basis for the appearance of a senescent phenotype and the increased susceptibility to external stress and the consequent cell degeneration in vitiligo cells. Preliminary data: Early results show that vitiligo melanocyte express a of a pre senescent secretome including the ß- amyloid, and present an activation of the autophagy pathway. A defect in the expression of hexokinases was associated with a decrease of ATP production and an elevated AMPK expression. Higher levels of 7 dehydrocholesterol and of IGFBP3 were detected in melanocyte culture and in the plasma of an initial cohort of patients. Preliminary experiments suggest that 7 dehydrocholesterol activate blood derived dendritic cells. In perilesional skin areas NALP1 and IL1b expression correlate with inflammatory infiltrate and with disease activity. In a limited set of biopsies from non-lesional areas p16 was observed in addition to the overexpression of senescence associated markers. 11/07/ / 5

230 Clinical and Experimental Pathway to Vitiligo Management BANDO 2013 Progetti Ordinari RF RF Clinical health care research/clinicoassistenziale LEONE GIOVANNI Istituti fisioterapici ospitalieri - Istituto Dermatologico Santa Maria Materials and Methods Vitiligo patients (n=51) classified according to VETF criteria will be enrolled. Healthy subjects (n=51) sex and age-matched will be included. Relevant clinical and biochemical data will be registered in dedicated software. Epidermal melanocytes from non-lesional areas of vitiligo patients (n=30) and from healthy subjects (aesthetic surgery, n=30) will be studied after exposition or not to mild pro-oxidant stimuli. Mitochondrial functions will be evaluated by cytomic to detect ETC protein and glucose metabolizing enzymes, by Seahorse analyser to evaluate ETC function and ATP production. Growth factors, cytokines and key signalling proteins will be analysed by ELISA, PCR and WB in cells treated with interfering RNA and/or mitochondrial ETC improving molecules. Biopsies will be analysed by Immunohistochemestry. Plasma parameters will be determined by gas chromatography mass spectrometry or by ELISA assays. Bioinformatics tools, with univariate and multivariate analysis will be employed. Impact and Translational Implications Linking mitochondria impairment to cellular degeneration will give opportunities to define targeted treatments to arrest vitiligo progression. The identification of peripheral biomarkers of metabolic process can contribute to evaluate the evolution of the disease. The cellular functional similarities and the common ontogenesis between melanocytes and neurons will offer possible innovative knowledge and tools to approach other degenerative diseases.

231 Towards precision medicine for patients with biliary tract cancers: tailored treatments based on the mutational profile detected on tumor lesions and monitored in liquid biopsies (circulating tumor cells, CTC, and circulating tumor DNA, ctdna) RF Daidone Maria Grazia Clinical health care research/clinicoassistenziale 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 - 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 biliar tract cancers liquid biopsies 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/Lavoratory Role in the project 1 Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Department of Experimental Oncology and Molecular Medicine, Biomarkers Unit Molecular analyses on tumor lesions, isolation and mutational analysis of CTCs, mutational analysis of ctdna; project coordination 2 Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano Department of Medical Oncology 1 Patient recruitment and follow-up; planning of personalized treatments according to molecular data, 3 Azienda Ospedaliera Universitaria Integrata di Verona Department of Pathology and Diagnostics; ARC-NET Research Center Patient recruitment and follow-up, molecular analyses on tumor lesions 11/07/ / 6

232 Towards precision medicine for patients with biliary tract cancers: tailored treatments based on the mutational profile detected on tumor lesions and monitored in liquid biopsies (circulating tumor cells, CTC, and circulating tumor DNA, ctdna) RF Daidone Maria Grazia Clinical health care research/clinicoassistenziale Investigators, Institution and Role in the Project Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Key Personnel Institution/Org./Pos. Role in the project Birth Date 1 Celio Luigi Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Milano 2 Scarpa Aldo Azienda Ospedaliera Universitaria Integrata di Verona 3 Cappelletti Vera Grazia Fondazione IRCCS Istituto Nazionale per lo studio e la cura dei tumori - Milano/Department of Experimental Oncology/PhD Clinical management of patients and data analysis Mutational analyses, patient recruitment and data analysis Mutational analysis of tumor lesions and CTCs, data interpretation and analysis 18/09/ /06/ /02/ Appierto Valentina Fondazione IRCCS Istituto Nazionale per lo studio e la cura dei tumori - Milano/Department of Experimental Oncology/PhD Mutational analysis of ctdna, data interpretation and analysis 11/11/1968 Background and Significance Biliary tract cancers (BTC) including intraephatic or extraepathic colangiocarcinomas and gallbladder carcinomas are clinically heterogeneous, notwithstanding the common origin from the biliary epithelium. They are genetically distinct entities sharing a very poor prognosis, and no effective targeted agents have been so far approved for BTC. Our group recently demonstrated that specific molecular alterations can be associated to different BTC categories, and drug actionable pathways are evident in 68% of cases, thus making BTC patients suitable for targeted therapies tailored to their genotype. However, an effective implementation of such treatments also requires to consider the spatial/temporal heterogeneity and the plasticity/clonal evolution of cancer cells. Indeed these concerns could be solved by serial monitoring of tumor genotypes, but access to tumor tissue presents limitations since biopsies are invasive, and lesions to be sampled may be completely/partially inaccessible and not fully reflecting tumor biologic heterogeneity. Recent studies demonstrated that circulating tumor cells and DNA (CTC, ctdna), shed from primary and metastatic lesions, can be detected in blood of cancer patients and may allow a non-invasive analysis of tumor genome evolution during treatment. No data are currently available on CTCs and ctdna in BTC, but their potential to act as "liquid biopsies" makes them promising tools to back up targeted therapy studies. Specific aims Aim 1: Aim 2: TO INVESTIGATE THE FEASIBILITY OF A TUMOR GENOTYPE SCREENING PERFORMED ON LIQUID BIOPSIES (by detecting CTCs and ctdna) AND COMPARED TO THE CORRESPONDING TUMOR LESION TO IDENTIFY BTC PATIENTS POTENTIALLY CANDIDATES TO PERSONALIZED THERAPIES.Published results on the prevalence of genetic alterations in BTC provide support to the possibility to successfully detect them also in the blood, as powerful and predictive biomarkers instrumental for precision/personalized medicine TO EVALUATE THE BENEFIT FOR PATIENTS WITH INTRAEPHATIC OR EXTRAEPATHIC COLANGIOCARCINOMAS OF TARGETED THERAPIES TAILORED ON DNA MUTATIONS DETECTED IN THEIR PRIMARY TUMORS. BTC represent an attractive target for molecular treatments, and the possibility to successfully perform mutational analyses for actionable mutations using low amount of DNA even from routinely 11/07/ / 6

233 Towards precision medicine for patients with biliary tract cancers: tailored treatments based on the mutational profile detected on tumor lesions and monitored in liquid biopsies (circulating tumor cells, CTC, and circulating tumor DNA, ctdna) RF Daidone Maria Grazia Clinical health care research/clinicoassistenziale Fondazione Istituto Nazionale per lo studio e la cura dei tumori - processed paraffin tissues is instrumental for planning personalized treatments and providing patients with an early access to innovative drugs Aim 3: TO ASSESS THE RELEVANCE OF CTCs AND ctdna FOR TRACKING CANCER RESPONSE TO TARGETED TREATMENTS AND FOR AN EARLY IDENTIFICATION OF TREATMENT RESISTANCE ASSOCIATED TO THE OCCURRENCE OF NEW MUTATIONS. An effective implementation of personalized treatments requires tools able to monitor tumor response overcoming spatial-temporal differences related to lesion heterogeneity, and liquid biopsies could represent a non-invasive approach potentially amenable to routine use, whose specificity and sensitivity need to be prospectively assessed within clinical trials Hypothesis: 1. BTC represent an attractive target for molecular treatments; it is now possible to identify molecular subsets of tumors that can be explored for specific drug targeting in clinical trials. 2. CTC and ctdna evaluation might represent theranostic tools for i) monitoring response to target treatment based on tumor mutational profile and ii) tracing treatment-induced molecular evolution of the disease. Preliminary data: Adequate BTC patient recruitment is warranted by the accrual rate at the two clical Centers (64 intraephatic or extraepathic colangiocarcinomas were treated and followed-up at INT in 2013). In both Institutions, Biobanks are currently collecting and storing from consented patients the leftover material after diagnosis as FFPE and frozen specimens, and mutational profiles are routinely performed using the Ion AmpliSeq technology and the Ion Torrent Personal Genome Machine at the Pathology Departments. ARC-Net Research Center developed a custom panel specific for the actionable mutations (including TKRs, RAS/RAF/MAPK/ERK, mtor and TGFbeta) detected in 68% of BTC (Simbolo M, Oncotarget 2014). Preliminary experiments confirmed the feasibility to perform mutational analysis at a single-cell-level from CTC pre-enriched blood sample preparations (by using dedicated filters) successively processed by DEPArray (an approach based on single cell movement in di-electrophoretic cages) to allow the isolation of single and 100% purified CTC groups. Materials and Methods DNA from FFPE tumor sections will be extracted using the QIAmp DNA FFPE tissue kit (Qiagen) ng of DNA will be used in mutational analyses through the Ion AmpliSeq technology and the Ion Torrent PGM using the custom panel including genes frequently mutated in BTCs according to our data (Simbolo et al., Oncotarget 5: , 2014). AmpliSeqLibrary Kit 2.0 will be used for preparation of amplicon libraries. Sequencing data will be analyzed with the Torrent Suite Software. Selected tissue mutations will be monitored in plasma by droplet digital PCR. CTCs enriched using ScreenCell CC filters will be enumerated based on cytological criteria and their mutational profile will be assessed after isolation by DEPArray analysis selecting based on specific staining and morphology. Ampli1 WGA Kit will be used for DNA amplification. The case series consisted of 50 consented patients for aim 1 and 60 patients entered tailored personalized treatments approved by IRB and Ethics Committee. 11/07/ / 6

234 Towards precision medicine for patients with biliary tract cancers: tailored treatments based on the mutational profile detected on tumor lesions and monitored in liquid biopsies (circulating tumor cells, CTC, and circulating tumor DNA, ctdna) RF Daidone Maria Grazia Clinical health care research/clinicoassistenziale Fondazione Istituto Nazionale per lo studio e la cura dei tumori - Impact and Translational Implications Advances in BTC genomics here exploited raise hopes of increased precision to plan effective treatments for a tumor type at present characterized by modest therapy approaches, and personalized treatments based on tumor genotype to patients whose access to clinical trials was limited. Liquid biopsy consideration within clinical trials could contribute to accelerate genomic analysis for personalized medicine with measurable benefits to patients and appropriateness tools to National Health System.