Proceedings and Report

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2 Proceedings and Report 76

3 Italian Proteomics Association 6 th Annual National Conference Rettorato dell Università degli Studi (via Verdi, 8) & Dipartimento di Biologia Animale e dell Uomo (via Accademia Albertina, 13) Turin, 21 st - 24 th June, 2011

4 ItPA Executive Board Scientific Committee Urbani Andrea (President) Bini Luca (Vice President) Roncada Paola (Treasurer) Modesti Alessandra (Secretary) Castagnola Massimo Fasano Mauro Organising Committee Pessione Enrica (President) Cavaletto Maria Fattori Paolo Giunta Carlo Nebbia Carlo Novelli Franco Timperio Anna Maria Pessione Enrica

5 The Organization and Scientific Committes acknowledge the support of: ABSciex Agilent Technologies Biorad Bruker Dionex GE Healthcare Thermo Scientific Waters

6 WORKSHOP PROGRAMME

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8 CONGRESS PROGRAMME

9 !"#$%!&!"'()!*+,-!".##! Conference opening Conference Sessions Poster sessions (Aula Magna - Rettorato Università di Torino)!"#$!%&'"($)$*$!"#$+,($-.$/$0,&"1,$!"/%0!*+,-!".##! Last Conference Session (Aula Magna DBAU Dipartimento di Biologia Animale e dell Uomo - Università di Torino)!"#$233#'%4"#$256%&71#($-8$/$0,&"1,$

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11 LECTURES

12 L1 PROTEINS PRODUCED BY PROBIOTIC BACTERIA AS MEDIATORS OF HOST-BACTERIA INTERACTIONS. A FOCUS ON BIFIDOBACTERIA Abelardo Margolles IPLA - CSIC, Ctra. Infiesto s/n Villaviciosa, Asturias, Spain Bifidobacteria are commensal microorganisms of the human gastrointestinal tract which are largely being used in functional foods. Some strains are able to exert health-promoting effects, being considered as probiotics. The scarcity of genetic tools has hindered the development of functional genomic studies in bifidobacteria, like the identification of molecular mechanisms underlying their survival under different environmental challenges. However, some of these experimental obstacles have been successfully overcome with the use of proteomics. The aim of this presentation is to summarize some of the work carried out in our group during the last years, mainly focused on the study of the response of bifidobacteria to gastrointestinal conditions. Bile salts and acid ph represent a key challenge for bifidobacterial survival in the gastrointestinal environment and may, thus, determine their functionality at this location. However, the precise mechanisms employed by bifidobacteria to counteract their effects are poorly understood. The comprehension of these mechanisms may lead to better knowledge of host-microbiota interactions, and to a rational application of probiotic bifidobacteria. We have used proteomics and functional genomics techniques for a deeper understanding of the molecular mechanisms involved in bifidobacteria response to gastrointestinal stress. Acid response involves a number of changes jointly dedicated to mitigate the impact of protons in the cell physiology, mainly by controlling the intracellular ph through different mechanisms. On the other hand, exposure to bile in bifidobacteria produces deep changes in the carbohydrate metabolism, as well as changes in fatty acid biosynthesis and an increase in the amount of molecular chaperones and DNA protection proteins. Remarkably, bile also triggers the synthesis of several membrane proteins involved in the bile resistance phenotype of bifidobacteria. Finally, It is worth mentioning the role of some extracellular proteins from bifidobacteria able to act as intestinal adhesins. We have characterized a moonlighting protein which is an aggregation factor and a mucus binding protein, likely contributing to the colonization capacity of bifidobacteria in the human intestine. L2 PROTEOMICS DRIVEN TRANSLATIONAL RESEARCH IN PANCREATIC CANCER Francesco Novelli Centro Ricerche Medicina Sperimentale (CERMS) Azienda Universitaria Ospedaliera, San Giovanni Battista, Torino; Dipartimento Medicina e Oncologia Sperimentale, Università di Torino, Torino, Italy Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. It is the fourth leading cause of cancer death with a 2% 5-years survival rate. This poor prognosis is due, in part, to a lack of early diagnosis, indeed biomarkers for the early detection of PDAC are lacking. By using a proteomic approach, defined as SERological Proteome Analysis (SERPA) we have defined a number of PDACassociated antigens that are specifically recognized by autoantibodies present in PDAC patients sera. These autoantibodies recognized with high frequency a number of metabolic enzymes and cytoskeletal proteins that are mostly up-regulated in PDAC. The protein recognized with the highest frequency by autoantibodies in PDAC sera was the glycolytic enzyme -enolase (ENOA). Accumulating evidences have revealed that ENOA is a multifunctional protein involved in many physiological and pathophysiological conditions. It is expressed on the surface of many prokaryotic and eukaryotic cells, where it acts as a plasminogen-binding receptor. This binding leads to activation of plasminogen to plasmin and it is critical in tumour cells spreading and tissue invasion. We have found that PDAC cell lines display ENOA on cell surface, where it could be critical in the invasion of PDAC cells through extracellular matrix. We found that treatment of PDAC cells with a mouse monoclonal antibody against human ENOA inhibited plasminogen-dependent migration both in vitro and in vivo. Microarray, Western blot and immunohistochemistry studies have indicated that ENOA is overexpressed in PDAC tissues. ENOA is able to elicit T cell proliferation, activation and cytotoxic specific T lymphocytes (CTL) differentiation both in vitro and in vivo. We have used genetically engineered mice (GEM) that spontaneously develop PDAC very similar to human to analyze the effect of DNA vaccination to human ENOA. Preliminary results indicated that DNA vaccination to ENOA elicits an integrated humoral and cellular immune response that significantly extends survival of GEM. Finally, six ENOA isoforms from pancreatic cell lines were identified by two-dimensional electrophoresis Western blot and mass spectrometry, and used as targets to screen the IgG reactivity of 120 PDAC patients, 40 healthy subjects, 50 non-pdac tumor patients, 46 chronic pancreatitis and 12 autoimmune diseases patients. Antibodies to two phosphorylated isoforms of ENOA (ENOA1,2) were observed in 62% of PDAC sera. Patients with antibodies to ENOA1,2 displayed better disease control upon therapy and longer survival. Our aim is to identify by mass spectrometry analysis the phosphorylated residues in ENOA1,2 isoforms and afterwards to find the epitopes of phosphorylated ENOA that are recognized by PDAC autoantibodies. This information will allow a short-term development of an assay based on ENOA phosphorylated peptides for rapid diagnosis of PDAC. As whole these data demonstrate that SERPA is a powerful strategy to identify tumor associated antigens, like ENOA, that are suitable for both therapeutic and diagnostic (theragnostic) purposes in PDAC.

13 L3 L4 AN OMICS APPROACH TO HUMAN LONGEVITY WITHIN A SYSTEMS BIOLOGY PERSPECTIVE Claudio Franceschi Dipartimento di Patologia Sperimentale dell Università di Bologna (Italy) PUSHING THE LIMITS OF PROTEOMICS TECHNIQUES TO UNDERSTAND STRESS AND TOLERANCE IN AN ALLOPOLYPLOID CROP Sebastien Carpentier K.U.Leuven / Research Foundation Flanders (FWO) Faculty of Bioscience Engineering Kasteelpark Arenberg 13 bus Leuven, Belgium Polyploidy and allopolyploidy have played an important role in the evolution of many plants and crops and it generates a great deal of protein polymorphism. Protein polymorphism is a driving force behind crop improvement and the analysis of protein isoforms is important in understanding crop biodiversity and stress tolerance. The combination of two-dimensional electrophoresis (2DE) and 2D DIGE with de novo MS/ MS sequence determination is still the workhorse for proteomics in nonmodel plants (Carpentier et al., 2008) and is an excellent tool to quantify and analyze the different protein isoforms and to identify variety specific proteins and amino acid polymorphisms (Carpentier et al., 2011). However, proteins with multiple membrane spanning domains a class of proteins extremely important in stress reactions- are hardly detected on classical 2-DE gels (Vertommen et al., 2010). Therefore we have also explored the possibility to apply a peptide based shot gun approach and pushed the limits (Vertommen et al., 2011). A peptide based approach in non-model plants demands special precautions to prevent false positive identification of proteins and the reconstruction of peptides into proteins is a big challenge. Therefore, we developed a workflow that included (i) optimization of protein extraction and the peptide separation (2DLC), (ii) the use of MSe and DDA for protein quantification and identification, (iii) performing de novo sequencing to allow a sequence homology search, (iv) visualization of identified peptide-protein associations using Cytoscape to remove redundancy and wrongly assigned peptides and to visualize the protein inference problem. References Carpentier, S., Panis, B., Vertommen, A., Swennen, R., Sergeant, K., Renaut, J., Laukens, K., Witters, E., Samyn, B., Devreese, B., Proteome analysis of non-model plants: a challenging but powerful approach. Mass Spectrometry Reviews, vol. 27, pp Carpentier, S. C., Panis, B., Renaut, J., Samyn, B., Vertommen, A., Vanhove, A.-C., Swennen, R., Sergeant, K., The use of 2Delectrophoresis and de novo sequencing to characterize inter- and intracultivar protein polymorphisms in an allopolyploid crop. Phytochemistry In Press. Vertommen, A., Panis, B., Swennen, R., Carpentier, S., Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants. Planta, vol. 231, pp Vertommen, A., Moller, A. L., Cordewener, J. H., Swennen, R., Panis, B., Finnie, C., America, A. H., Carpentier, S. C., A workflow for peptidebased proteomics in a poorly sequenced plant: A case study on the plasma membrane proteome of banana. J Proteomics In Press.

14 L5 L6 PLANT GENOMIC INFORMATIONAL CONTENT AT THE START OF THE POSTGENOMIC ERA Francesco Salamini Edmund Mach Fondation, Istituto Agrario di S. Michele all Adige e Parco Tecnologico Padano. After 10 years from the sequencing of the first plant genome, the current state of the field is here reviewed. Plant genomes can be relatively small but, nevertheless, they are the result of ancient and recent genome duplications (WGS); in addition, a common observation is that plant speciation frequently is the result of an allopolyploidy event. The genome status of these organisms is thus characterized by a large number of genes (for example, the recently sequenced apple genome contains more then 57,000 genes, a number deriving mainly from a 50 million years old duplication of almost all chromosomes). The presentation first introduces the major conclusions learned from the sequencing of the apple and grape genomes, as carried out at the Mach Foundation, Istututo Agrario di S. Michele all Adige, Trento. Of relevant interest in these two species are genes controlling disease resistance (365 in grape and 992 in apple) and those responsible for product quality (62 and 110 genes encoding enzymes responsible for fenolics and terpenoids in grape; 1,246 genes related to volatiles, aromatic-compounds, pigments, antioxidant and sorbitol biosynthesis in apple). A summary of relevant results deriving from the two mentioned species, compared to those of other 17 plant genomes today available, is also introduced. Of relevance to the proteomic meeting are two particular issues: the amplification in plants of secondary metabolism-related genes, which supports a large redundancy of some gene families (this creates problems when associating specific genes to the proteins they encode); the second issue concerns RNA and DNA sequence differences at the exonic level, a finding which call for more subtle and effective protein identification methods. nextprot, THE HUMAN PROTEIN KNOWLEDGE PLATFROM IN THE CONTEXT OF THE HUMAN PROTEOME PROJECT. Amos Bairoch Swiss Institute of Bioinformatics and University of Geneva In September 2008, the UniProt/Swiss-Prot group achieved a major milestone: the first complete manual annotation of the full set of human proteins (derived from about 20'000 genes). This corpus of data which is quite rich in information pertinent to modern biomolecular medical research, clearly shows that there is a large gap in our knowledge of human proteins in terms of functional information as well as protein characterization (PTMs, protein/protein interactions, subcellular locations, etc). This gap resides not only in the available experimental information, but also in the way this information has been stored, which is far from being sufficient to help researchers making sense of what all these human proteins do in our bodies! Therefore, in the framework of CALIPHO, a new interdisciplinary group created jointly by the University of Geneva and the SIB, we are developing nextprot, a new humancentric protein knowledge resource, with the aim to help researchers answer pertinent questions. To achieve this goal we need to build up on a corpus of both curated knowledge -originating mainly from the UniProtKB/ Swiss- Prot knowledgebase -and carefully selected and filtered highthroughput data pertinent to human proteins. Such a data gathering and grading effort are complemented by the development of tools that allow such data to be analyzed. nextprot is a common development of the SIB and of GeneBio SA. In January 2011 we came out with the first release of nextprot (available at beta.nextprot.org). We will present its current content and what we are planning to add in the near future. This is especially important in the context of HPP as we plan to develop nextprot as one of the knowledge pillar of this crucial scientific initiative. We hope you will enjoy working with nextprot and will help us making it evolve by telling us of your specific needs.

15 ABSTRACTS

16 1.O.1 1.O.2 FARM ANIMAL PROTEOMICS: A COST ACTION Paola Roncada* Istituto Sperimentale Italiano Lazzaro Spallanzani, Milano The use of proteomic strategies to investigate animal health and disease has been limited by the lack of international coordination and collaboration. The COST action FAP- Farm Animal Proteomics, FA1002, that is started in November 2010, form a network of the leading European scientists who are focused on farm animal proteomics, but it is also designed to encourage participation of researchers who are at an early stage of utilizing the range of proteomic methods which are becoming available. This network benefit the European economy by providing advanced analytical tools to enhance animal production, health and welfare, as well as in the assessment of food quality and safety related to the protein in food produced from animal origin. The action works through three major workgroups: wg1 proteomics and animal health, that focus on research relevant to animal health covering biomarkers of infectious, parasitic and metabolic diseases,. The WG2, proteomics of food of animal origin- provide communication in fundamental research in proteomics related to food production, quality and food safety. The focus of Working Group 3, Advancing Methodology for Farm Animal Proteomics - is about technical aspects of animal proteomics, from sample collection/ preparation to protein separation, identification, and quantification in production animal body fluids, tissues and post harvest derived products. The action organizes meeting every year and the dissemination of results of the collaboration are published in the website that is the main source of informations. Challenge of the action is also to use EU found to help young scientists to join several laboratory in different EU COST member. ANALYSIS OF THE SECRETOME OF THE GROUP B STREPTOCOCCUS Roberta Galbo a, S. Papasergi b, M. Garibaldi b, I. Pernice a, C. Lo Passo a, F. Mandanici b, L. Romeo b, P. Trieu-Cuot c, G. Teti b and C. Beninati b a Department of Life Sciences, University of Messina, Italy b The Elie Metchnikoff Department, University of Messina, Italy c Unit of Gram Positive Pathogens Biology, Pasteur Institute, Paris, France Group B streptococci (GBS), are important human pathogens, particularly affecting neonates. In recent years, proteomics analysis of surface proteins of group A and B streptococci has greatly contributed to the identification of novel virulence factor and candidate vaccines. In the present study we examined for the first time, by proteomics, the complex of the covr/covs (covr/s) two component system, since inactivation of this system is thought to mimic the physiological status of the GBS infections phase. We identified a total of 37 proteins, about 95% of which were predicted to be secreted or surface-associated by informatics analysis. Fourteen of the 37 identified proteins were secreted by both strains, while 13 were found exclusively in DcovRS supernatants. The latter comprised, by informatics analysis, previously unstudied cell wall associated proteins and/or homologs of putative virulence factors 1,2,3,4 present in other bacterial species. Our data suggest that proteomics analysis of the GBS secretome might be a useful tool for the rapid identification of novel virulence factors. Corresponding author: Paola Roncada, PhD Istituto Sperimentale Italiano Lazzaro Spallanzani Sezione di Proteomica c/o Facoltà di Medicina Veterinaria Via Celoria 10 Milano Italy References: Roberta Galbo Researcher and Aggregate professor of Genetic Dept. of Life Science, University of Messina, Italy Salita Stagno D Alcontres 31, Messina, Italy Tel Fax References: [1] Tiwari R, Qin A, Artiushin S, Timoney JF, Se 18.9, an anti-phagocytic factor H binding protein of S. Equi, Veterinary Microbiology, 2006, 121 (1-2): [2] Zhu H, Liu M, Sumby P, Lei B, The secreted esterase of Group A streptococcus is important for invasive skin infection and dissemination, Infection and Immunity, 77(12): [3] Liu M, Zhu H, Zhang J, Lei B, Active and passive immunizations with the streptococcal esterase Sse protect mice against subcutaneous infection with GAS, Infection and Immunity, 2007, 75(7): [4] Buchanan JT, Simpson AJ, Aziz RK, Liu GY, Kristian SA, Kotb M, Feramisco J, Nizet V, DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps, Current Biology, 2006, 16(4):

17 1.O.3 SOIL PROTEOMICS: INTERFERENCE OF SOIL SOLID PHASES ASSESSED BY MODEL STUDIES Laura Giagnoni 1, Andrea Migliaccio 1, Loretta Landi 1, Daniel van der Lelie 2, Safyh Taghavi 2, Paolo Nannipieri 1, Giancarlo Renella 1 1 Department of Plant, Soil and Environmental Sciences, University of Florence, Florence, Italy 2 RTI International, Center for Agricultural and Environmental Biotechnology, 3040 Cornwallis Road Research, Triangle Park, NC 27709, USA Soil proteomics is a very promising approach for a deep understanding of the ecological and biological functions expressed by the highly diverse soil microbial communities, responsible for the soil functionality. Previous soil studies demonstrate that a critical point for implementing soil proteomics is the possibility to determine the location of the proteins extracted from soil, as most of the soil constituents (e.g. sand, clay hydrooxides, humic substances) can stabilize proteins actively or passively released by the soil microorganisms, making difficult to distinguish between newly expressed and soil-stabilized proteins. To better understand the potentials of soil proteomics and specifically address the effects of various soil solid phases on direct protein extraction from soil, we have used a model study, based on the use of the Gram-negative soil-borne bacterium Cupriavidus metallidurans CH34, with known genome and proteome, inoculated into microcosms containing quartz sand, clays, humic acids and an artificial soil. The bacterial proteome was analyzed by 2-DE and mass spectrometry approach. Based on the previous results which showed that the presence of the high reactive clay montmorillonite gave the largest interference in the bacterial proteome analysis as compared to low reactive clay kaolinite, we also inoculated C. metallidurans CH34 in artificial soils with a different kaolinite:montmorillonite ratios. The results from this study showed that the presence of high reactive clays in soils may negatively influence the potential of recognize the proteomic signature of bacteria in soil microbial communities, likely due to irreversible proteins adsorption. We conclude that more model studies are needed to improve the protein extraction and purification from soils. Corresponding author: Dr. Laura Giagnoni, Department of Plant, Soil and Environmental Sciences, Piazzale delle Cascine, Firenze (FI), Italy, tel O.1 IN VITRO VITAMIN K TREATMENT IS NOT CAPABLE TO RESQUE UNEFFICIENT MGP-CARBOXYLATION IN PXE FIBROBLASTS Giulia Annovi, Federica Boraldi, Daniela Quaglino Dept. Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy Abnormal soft connective tissue mineralization has been related to mutations in the MGP (Matrix Gla Protein) gene or as a consequence of an insufficient vitamin K-dependent gamma-carboxylation of specific Glaresidues on the MGP protein. Interestingly, low levels of vitamin K and reduction of carboxylated-matrix Gla Protein (Gla-MGP) have been demonstrated in the plasma of patients affected by pseudoxanthoma elasticum (PXE), a rare genetic disorder associated to mutations in the ABCC6 gene, and characterized by the progressive mineralization of elastic fibers. Consistently, we have recently reported that fibroblasts, isolated from PXE patients and cultured in vitro, have an altered protein profile exhibiting an insufficient vitamin-k-dependent carboxylation of MGP, a down-regulation of protein disulfide isomerase (PDI) and an upregulation of calumenin (CALU), two proteins of the endoplasmic reticulum that are involved in vitamin K cycle, by assuring the appropriate reduced environment, necessary for regenerating the vitamin in its active form, and by inhibiting the gamma-carboxylase activity, respectively. It has been therefore suggested that vitamin K supplementation might be capable to restore MGP carboxylation in PXE and to inhibit and/or delay the pathologic occurrence of the calcification process. However, very recent studies in the PXE animal model (abcc6-/- mice) failed to demonstrate the efficacy of vitamin K supplementation in counteracting the mineralization of soft connective tissues, thus rising several questions on the ability of cells to utilize the vitamin and to efficiently activate MGP restoring the appropriate environment. Aim of the present study was to investigate, in vitro, the effects of vitamin K1 and K2 supplementation on control as well as on PXE fibroblasts, used as a useful in vitro model to monitor the ability of the vitamin to counteract abnormal MGP carboxylation. Data from the present study indicate that both control and PXE fibroblasts are able to utilize vitamin K1 and K2 and to significantly increase protein carboxylation, however neither vitamin K1 or vitamin K2 supplementations are capable to inhibit the mineralization process and to positively modulate MGP, its expression being down-regulated at high doses of vitamin K2. It can be suggested that the role of vitamin K1 and especially of vitamin K2 on the calcification process is mediated by changes in the carboxylation as well as in the expression of MGP, and that other factors, possibly involving phosphate deposition, should be taken into consideration for an effective inhibition of the mineralization process in peripheral tissues. Prof. Daniela Quaglino Dipartimento Scienze Biomediche Via Campi 287, Modena, Italy Te /5446

18 2.O.2 SEQUENTIAL APPLICATION OF URINARY PEPTIDE CLUSTERS OBTAINED AFTER WEAK-CATION EXCHANGE MAGNETIC BEAD PURIFICATION TO DETECT RENAL CELL CARCINOMA Erica Gianazza 1,2, Yuri E.M. van der Burgt 2, Italo Zoppis 3, Massimiliano Borsani 3, Giancarlo Mauri 3, Clizia Chinello 1, Valeria Squeo 1, Giancarlo Albo 4, Stefano Signorini 5, André M. Deelder 2, Marzia Galli Kienle 1 and Fulvio Magni 1,* 1 Dept. of Experimental Medicine, University of Milano- Bicocca, Monza, Italy. 2 Dept. of Parasitology, Biomolecular Mass Spectrometry Unit, LUMC, Leiden, The Netherlands. 3 Dept. of Informatics, Systems and Communication, University of Milano-Bicocca, Milano, Italy. 4 Dept. of Specialistic Surgical Sciences, Urology Unit, University of Milano, Ospedale Maggiore Policlinico Foundation, Milano, Italy. 5 Desio Hospital, University of Milano-Bicocca, Milano, Italy. Renal Cell Carcinoma (RCC) is the most common kidney malignancy. Since RCC is highly resistant to radio- and chemotherapies patients have a poor prognosis. Therefore, biomarkers for early diagnosis are urgently needed. Previously we have identified various peptides in serum and urine of RCC patients that discriminate from healthy individuals with very high diagnostic potential. In this study we have extended the investigation to patients with a non-rcc tumor using a combination of weak cation exchange magnetic bead (WCX-Mb) purification of urine and matrixassisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Urinary protein profiles of 85 controls (Ctrl), 79 clear cell RCC (RCC) and 23 non-rcc patients (non-rcc) were obtained and statistically evaluated aiming for detection of potential biomarkers. Multiple peptides were observed with intensity / area differences large enough to distinguish controls from patients. Two clusters were build based on a Support Vector Machine (SVM) algorithm containing three and five m/z-values, respectively. The SVM clusters allow the discrimination between the three classes through a sequential decision process. We achieved a significant rate of predicted values (i.e., precision ratios greater than 80%) with good specificity and sensitivity (i.e., recall ratios greater than 77%) for both clusters by considering their discrimination task. Moreover the application of a Genetic Algorithm (GA) resulted in similar classification performance. In conclusion, the results indicate that peptides extracted using WCX display a diagnostic capability to discriminate RCC patients from controls. Moreover the m/z-values included in the clusters may be used to detect non-rcc tumors. The present work has been supported by grant FIRB n. RBRN07BMCT of the Italian Ministry of Research and by grant IIT Project SEED: IPG- CHIP. Prof. Fulvio Magni Dept. of Experimental Medicine, University of Milano-Bicocca Via Cadore 48, Monza, Italy Phone: Fax: O.3 MODULATION OF URINARY PEPTIDOME IN HUMANS EXPOSED TO HIGH ALTITUDE HYPOXIA Veronica Mainini A*, Erica Gianazza A*, Clizia Chinello A, Grzegorz Bilo B, Miriam Revera B, Andrea Giuliano B, Gianluca Caldara B, Carolina Lombardi B, Alberto Piperno C, Fulvio Magni A, Gianfranco Parati B, on behalf of HIGHCARE investigators A Department of Experimental Medicine, University of Milano-Bicocca, Monza, Italy B Department of Cardiology, S. Luca Hospital, IRCCS, Istituto Auxologico Italiano, Milan, Italy C Department of Clinical Medicine and Prevention, University of Milano-Bicocca, Monza, Italy *equally contributing author. The exposure of healthy subjects to high altitude represents a good model to explore the pathophysiology of diseases related to tissue hypoxia and is a good model to evaluate pharmacological approaches potentially useful as a therapy for chronic diseases related to hypoxia. The aim of our study was to explore the urinary peptidome to detect alterations induced by the exposure of subjects to different altitudes (sea level, 3500 and 5400 mt a.s.l.) and/or to pharmacological treatment. Urines were collected from 47 subjects, randomly and blindly assigned to Placebo (n=24) or Telmisartan treatment (n =23). Samples were purified by the use of hydrophobic interaction magnetic beads, then analysed by MALDI-TOF MS. Results showed that the urinary peptidome is not affected by the administration of Telmisartan, neither at sea level nor at altitude. Conversely, 6 peptides were demonstrated to be differentially expressed in association with the exposure to altitude. In detail, we assessed that two of these peptides were down regulated at high altitude, suggesting molecules involved in the early phase of acclimatization. Other two peptides, instead, were demonstrated to be up-regulated at 5400 mt, suggesting a later modulation process. Finally, two peptides were downregulated at 3500 mt compared to sea level, but were comparable at sea level and at 5400 mt. These results suggest a potential late and compensatory adaptation to hypobaric hypoxia. This is the first study describing modulation of the urinary peptidome in response to the exposure to hypobaric hypoxia and could provide useful information on acclimatization processes. The present work has been supported by grant FIRB n. RBRN07BMCT of the Italian Ministry of Research. Veronica Mainini, PhD Biomedical Technologies Department of Experimental Medicine, University of Milano-Bicocca, Via Cadore 48, Monza (MB), Italy address: Phone: Fax:

19 3.O.1 3.O.2 EFFECTS OF AGEING IN MUSCLE TISSUE: THE CONTRIBUTION OF PROTEOMICS Daniele Capitanio1, Michele Vasso1,2, Cecilia Gelfi1,2 1 Department of Sciences and Biomedical Technologies, University of Milan, Segrate (MI), Italy. 2 Institute of Bioimaging and Molecular Physiology, National Research Council, Segrate (MI), Italy. Ageing induces a progressive functional decline affecting the entire organism, therefore, the loss of muscle mass and function (sarcopenia) contributes significantly to a loss of functional autonomy. In muscles, the age-related degenerative changes produce alteration of morphology associated to muscle fiber atrophy, loss of satellite cells, and remodelling of neuronal structures. Morphological changes induced by ageing indicate a decrement of muscle fiber size, a change in fiber type distribution and appearance of mitochondrial aggregates with an increment of lipofuscin. This functional and morphological changes are also observed in nerves, in which ageing induces biochemical, morphological and functional variations both in myelin and in axons. Beside that, mitochondrial metabolism plays a pivotal role in aging, as its alteration influences the muscle function. In particular, the two morphologically distinct mitochondrial sub-fractions, named sub-sarcolemmal (SS) and intermyofibrillar (IMF) according to their diverse cellular localization, play different roles in muscle tissue. The modulation of the respiration rate, proteins and lipids composition, and the diverse biochemical (e.g. protein import) properties, contributes to muscle cell adaptation during ageing. The SS sub-fraction is more influenced by muscle changes and is more affected by ageing. Proteomics has been utilized to monitor, at the molecular level, the decline of muscle tissue, nerves innervating it and mitochondrial machinery Corresponding author: Cecilia Gelfi Associated Professor Università degli Studi di Milano Via F.lli Cervi, Segrate (MI) Tel Fax PROTEOMIC INVESTIGATIONS ON DIFFERENTIAL POST-TRANSLATIONAL MODIFICATIONS OF TRANSTHYRETIN IN MULTIPLE SCLEROSIS Damiana Pieragostino a,b, Piero Del Boccio a,b, Maria Di Ioia c, Sonia Bucci a,b, Luisa Pieroni d,e, Simona D Aguanno d,e, Viviana Greco d,e, Diego Centonze d, Giovanna De Luca a,c, Carmine Di Ilio a,b, Alessandra Lugaresi a,c, Paolo Sacchetta a,b, Andrea Urbani a,d,e acentro Studi sull Invecchiamento (Ce.S.I.), Fondazione Università G. d Annunzio, Chieti, Italy bdip. di Scienze Biomediche, Università G. d Annunzio di Chieti e Pescara, Italy cdip. di Neuroscienze ed Imaging, Università G. d Annunzio di Chieti e Pescara, Italy dcentro Europeo Ricerca sul Cervello (CERC), IRCCS- Fondazione S. Lucia, Roma edip. di Medicina Interna, Università di Roma Tor Vergata, Roma, Italy Multiple Sclerosis (MS) is a multifactorial disease involving central nervous system that result in global health issues. Thus, the need of understanding the molecular machinery involved in this pathology to develop more effective pharmacological therapeutics. Cerebrospinal Fluid (CSF) is a helpful biological fluid to study MS as it collects molecules released from various cell types as a result of the pathology. By studying CSF of MS patients through proteomic approaches we described two non canonical oxidative post-traslactional modifications on CSF Transthyretin (TTR), a 55 kda omotetrameric protein involved in Thyroid Hormone (TH) transport into central nervous system (up to 80% in man). These isoforms, reconductable to Thio-cysteine and Sulpho-cysteine adducts, discriminate MS patients vs subjects with other neurological diseases. These non-canonical isoforms were not highlighted in serum of MS patients, showing a specific phenomenon of CNS. Western Blot analysis of TTR, in non-denaturing conditions, showed a correlation between the modified TTR and an atypical TTR dimer in CSF samples. These results could be very interesting for to better understand the role of TTR in TH transport in MS disease, since TH physiologically helps the oligodendrocytes precursor to turn into re-myelinating oligodendrocytes regulating fundamental cell activities, including cell cycle. Hence TTR redox imbalance might eventually modify TTR transport capacity in multiple sclerosis. This work is supported by FISM Research Fellowship Cod.2010/B/14. Damiana Pieragostino, Ph.D. Department of Biomedical Sciences "G. d'annunzio" University Via dei Vestini 31, Chieti Pescara, Italy phone: Fax: Mobile:

20 3.O.3 4.O.1 PROTEOMIC PROFILING OF BRAIN STEROID 5 ALPHA REDUCTASES IN SLEEP-DEPRIVATED RATS. Alessio Soggiu*1, Marco Bortolato2, Paola Devoto1, Cristian Piras3, Luigi Bonizzi4, Paola Roncada5 1 Section of Neurophysiology and Neurochemistry, Department of Neurosciences B.B. Brodie, University of Cagliari, (Cagliari), Italy 2 Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles 3 Department of Zootechnical Science, University of Sassari, Italy 4 DIPAV, Facoltà di Medicina Veterinaria, Università degli Studi di Milano 5 Istituto Sperimentale L. Spallanzani, Milano, Italy. Steroid 5α reductase (S5αR) is rate-limiting enzyme of one of two major metabolic pathways in brain steroidogenesis. Recent evidence indicates that neuroactive steroids may involved in pathogenesis of schizophrenia spectrum disorders. Moreover, S5αR inhibition has been shown to induce therapeutic effects in animal models of schizophrenia and in several disorders associated to dopaminergic hyperreactivity (1). In rodents, sleep deprivation (SD) is known to induce a series of behavioural patterns, including sensory-motor gating deficit, which might be reflective of psychosis and mania and are countered by antipsychotics (2). The aim of this study was to evaluate, the impact of SD on expression levels of S5αR isozymes in rat brain. After 72 h of SD, rats were sacrificed and brain areas dissected out. Quantitative 1D and 2D western blotting (WB) with antibodies were performed on four brain areas of both control group and SD group (n = 8 each): medial prefrontal cortex (mpfc), nucleus accumbens (nacc), ippocampus (IPPO) and amigdala (AMG). 1D and 2D WB revealed that the expression of S5αR-1 and S5αR-2 was significantly augmented in SD animals in comparison to controls (p < 0.05) in mpfc and nacc area. In IPPO and AMG expression of S5αR-1 and S5αR-2 was unchanged in SD animals in comparison to controls. Regarding therapeutic effect of S5αR inhibition on gating deficit, data suggest that S5αR increase might cause altered balancing in neurosteroid levels, and it could be responsible of behavioral disruption observed in SD animals. SIALYLATION DEFINING CELL INVASIVENESS IN BREAST CANCER Giuseppe Palmisano1, Rikke Leth-Larsen2, Martin Larsen1 1Department of Biochemistry and Molecular Biology, The University of Southern Denmark, Odense, Denmark; 2Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark Background: breast cancer is the leading female cancer in Europe and USA with the metastasis as the main reason for mortality. Aberrant sialylation on the cell surface has been correlated with poor prognosis and metastatic spread of several human malignancies. Due to that, understanding the sialobiology of tumor cells (oncosialobiology) with the identification of tumor-specific sialylated glycoprotein alterations will add important knowledge to breast tumor therapy. Methods: human breast tumour cells were transplanted into mice and cell line was selected for enhanced growth rate, faster metastasis using GFP probe and animal survival rate. The selected isogenic non-metastatic (NM2C5) and metastatic (M4A4) cell lines were grown in SILAC medium. After cell lysis and membrane protein extraction, hydrazide chemistry and a new method based on the combination of TiO2-HILIC chromatography were applied to enrich and quantitatively map the N-linked glycoproteome using MS-based detection. The quantitative glycan analysis was performed using an array of SA-specific lectins and GPCµLC-MS/MS in negative ion mode, using a LTQ-Orbitrap mass analyser. Results: Oncosialobiology is a new point of view of investigating the link between sialylation and metastasis using MS-based techniques and the SA-containing oligosaccharides represent a molecular target for cancer therapy. Moreover we have investigated the potential pitfalls in large scale N-linked glycoproteomic analyses providing new solution to avoid them. *Corresponding author. Giuseppe Palmisano; Institution: Department of Biochemistry and Molecular biology, University of Southern Denmark. Address: Campusvej 55, DK-5230, Odense M, Denmark. Ph: *Corresponding author Alessio Soggiu, PhD Department of Neurosciences B.B. Brodie, Section of Neurophysiology and Neurochemistry University of Cagliari, Monserrato (CA) Phone: Fax: References 1) Paba S, Frau R, Godar SC, Devoto P, Marrosu F, Bortolato M (2011) Curr Pharm Des. 17:151. 2) Frau R, Orrù M, Puligheddu M, Gessa GL, Mereu G, Marrosu F, Bortolato M (2008) Int J Neuropsychopharmacol. 11:947. Acknowledgements This work was supported by a fellowship financed by RAS (Regione Autonoma della Sardegna, PO Sardegna FSE L.R. 7/2007 Promozione della ricerca scientifica e dell innovazione tecnologica in Sardegna )

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