ΑΙΤΗΣΗ ΓΙΑ ΟΙΚΟΝΟΜΙΚΗ ΕΝΙΣΧΥΣΗ ΕΡΕΥΝΗΤΙΚΟΥ ΕΡΓΟΥ ΠΡΟΓΡΑΜΜΑ ΒΑΣΙΚΗΣ ΕΡΕΥΝΑΣ «Κ. ΚΑΡΑΘΕΟΔΩΡΗ» Προκήρυξη 2010

ΕΛΛΗΝΙΚΗ ΔΗΜΟΚΡΑΤΙΑ ΠΑΝΕΠΙΣΤΗΜΙΟ ΠΑΤΡΩΝ ΕΠΙΤΡΟΠΗ ΕΡΕΥΝΩΝ Προς την ΕΠΙΤΡΟΠΗ ΕΡΕΥΝΩΝ ΠΑΝ/ΜΙΟΥ ΠΑΤΡΩΝ ΑΙΤΗΣΗ ΓΙΑ ΟΙΚΟΝΟΜΙΚΗ ΕΝΙΣΧΥΣΗ ΕΡΕΥΝΗΤΙΚΟΥ ΕΡΓΟΥ ΠΡΟΓΡΑΜΜΑ ΒΑΣΙΚΗΣ ΕΡΕΥΝΑΣ «Κ. ΚΑΡΑΘΕΟΔΩΡΗ» Προκήρυξη 2010 Τµήµα : Ιατρικής Ονοµατεπώνυµο Υπεύθυνου Έργου: Διονύσιος Παπαχρήστου Ιδιότητα: Παθολογοανατόµος, Επίκουρος Καθηγητής Ανατοµίας-Ιστολογίας-Εµβρυολογίας, Τµήµα Ιατρικής, Πανεπιστήµιο Πατρών Ονοµατεπώνυµο και ιδιότητα συνεργατών: Κυριάκος Κυπαραίος, Αναπληρωτής Καθηγητής Φαρµακολογίας, Τµήµα Ιατρικής, Παν. Πατρών. Ελένη Παπαδάκη-Πέτρου, Αναπληρώτρια Καθηγήτρια Ανατοµίας-Ιστολογίας- Εµβρυολογίας, Τµήµα Ιατρικής, Παν. Πατρών. Τίτλος του έργου: Metabolic pathways implicated in the pathogenesis and development of obesity-induced osteoarthritis Νέο Έργο Προτεινόµενη Ηµεροµηνία Έναρξης 1.12.2010 Συνολική Δαπάνη του Έργου 33.000 Ευρώ Ενίσχυση από άλλες πηγές (αν υπάρχουν) - Αιτούµενη Συνολική Χρηµατοδότηση 33.000 Ευρώ 1

Ηµεροµηνία 13/04/2010 Ο Επιστηµονικός Υπεύθυνος Διονύσιος Παπαχρήστου Επικ. Καθηγητής Ανατοµίας-Ιστολογίας-Εµβρυολογίας 2

Α) ΤΙΤΛΟΣ (TITLE): Metabolic pathways implicated in the pathogenesis and development of obesity-induced osteoarthritis Β) ΣΤΟΧΟΣ (AIMS OF THE PROJECT): 1. To identify cellular genes and pathways affected by apoe expression in mice, which may be involved in the pathogenesis of obesity-induced OA. 2. To examine histologically articular (hyaline) cartilage and chondroblastic cells from human patients suffering from OA and to assess the protein and gene expression levels of apoe. 3. To assess whether mouse ApoE and human ApoE2, ApoE3 and ApoE4 expression in apoe deficient (apoe -/- ) chondrocytes may induce genetic markers associated with OA. Γ) ΠΕΡΙΛΗΨΗ (ABSTRACT): Osteoarthritis (OA) is the most common joint disease in the USA and Western Europe that affects primarily elderly and adults and is responsible for significant activity limitations and eventually, in some cases, disability. However, the molecular mechanisms that are involved in its pathogenesis are still unclear. One of the major causes of OA is obesity. The past few years a lot of studies have focused on the functional, metabolic link between adipose tissue and cartilage in the development of OA. It has been recently shown that apolipoprotein E (apoe) over-expression promotes diet-induced obesity in mice. In addition, in vitro studies have demonstrated that apoe gene deficiency enhances the reduction of bone in mice fed high-fat diet, while lack of ApoE leads to increased bone formation in mice fed chaw diet. Triggered by these findings, in the present proposal the applicant will use in vivo, biochemichal and molecular methodologies in order to investigate the role of ApoE in the development and progression of OA in humans. Key words: osteoarthritis, obesity, Apolipoprotein E, metabolic syndrom 3

Δ) ΑΝΤΙΚΕΙΜΕΝΟ (SUBJECT OF THE PROJECT): 1) ΣΗΜΕΡΙΝΗ ΓΝΩΣΗ ΣΤΟ ΘΕΜΑ (CURRENT KNOWLEDGE): Background and the problem Osteoarthritis (OA) is a complex disease defined by the Subcommittee on Osteoarthritis of the American College of Rheumatology Diagnostic and Therapeutic Criteria Committee as "A heterogeneous group of conditions that lead to joint symptoms and signs which are associated with defective integrity of articular cartilage, in addition to related changes in the underlying bone at the joint margins" (1). Clinically, OA is characterized by joint pain, tenderness, limitation of movement, crepitus, occasional effusion, and variable degrees of local inflammation. It can occur in any joint but is most common in the hip, knee and the joints of the hand, foot, and spine. From a histopathological stand point OA is characterized by clustering of chondrocytes, fibrillation and fissuring of the articular cartilage, and in more advanced forms, loss of articular cartilage, subchondral bone sclerosis and cyst formation. OA is the most common joint disease in the USA and Western Europe. Indeed, it is estimated that 1 out of 10 people suffers from OA, meaning that worldwide 9.6% of men and 18.9% of women >60 years of age have symptomatic disease. It is the sixth leading cause of years of living with disability at the global level, accounting for 3% of the total global years of living with disability (2). It is believed that by 2030 more that 25 million people, 9.3% of the adults in USA will have activity limitations due to arthritis. It is well-documented that one of the largest and perhaps most preventable risk factors for the development of OA is obesity (3). It is also well-documented that obesity has reached epidemic proportions globally, with more than 1 billion adults overweight - at least 300 million of them clinically obese - and is a major contributor to the global burden of chronic disease and disability. It was initially believed that obesity-related OA was primarily the net result of increased mechanical loading upon the articular joints. However, more resent data have demonstrated that non-mechanical, metabolic factors have also central role in the development of OA in obese people. Therefore, it is not surprising that the last few years a large number of research groups have focused on the study of the molecular mechanisms that govern the relationship between adipose tissue and cartilage. 4

Nevertheless, the molecular links between obesity and OA has only recently started been investigated. State-of-the-art In this proposal, the applicant will investigate the link between ApoE a crucial component of the lipid and lipoprotein transport system, obesity, and osteoarthritis. Lipoproteins, apolipoproteins, and lipoprotein modifying enzymes constitute the major proteins involved in the maintenance of lipid and lipoprotein homeostasis in the blood. Genetic alterations in these proteins may affect the type, concentration, and properties of the lipoprotein particles in the circulation, and may result to dyslipidemia and atherosclerosis (4). A large volume of studies has documented the strong and positive association between body mass index (BMI) and OA in weight bearing joints, namely hip, knee and foot. Interestingly, BMI is also associated with the development of OA in non-weightbearing joints of hand. In addition, several prospective studies have shown that obesity (BMI>30kgr/m 2 ) increases the relative risk for the development of knee OA by 2-10 fold (5, 6). These clinical data support the hypothesis that the mechanisms that trigger the obesity-related OA are related not only to increased mechanical loading, but also to nonmechanical biochemical/molecular alterations. By contrast to earlier beliefs, white adipose tissue is not merely a fat store but a dynamic player that generates more than 50 cytokines controlling body homeostasis. These cytokines, called adipokines, exert their action via endocrine, paracrine and autocrine cross-talks in a wide variety of physiological and pathological conditions, including inflammation (7). Relatively recent molecular and biochemical studies have highlighted the pivotal role of adipokines in OA pathobiology. More specifically, it has been shown that leptin is crucially implicated in the pathogenesis of OA, acting both locally and systemically (8). The aforementioned data suggest that OA should be considered as a metabolic disease in which factors related to lipid metabolism participate in the induction of morphological and biochemical alteration of joint articular (hyaline) cartilage. Clearly, the need to understand the biological mechanisms that govern the pathogenesis and progression of OA and to develop novel therapeutic strategies to effectively treat this common disease is becoming increasingly pressing. 5

Recent results from the Laboratory of Dr. Kypreos, one of our collaborators, suggest that the chylomicron pathway is central in the development of diet-induced obesity. Specifically Dr. Kypreos and his colleagues found that apoe3 overexpression in mice promotes diet-induced obesity, hepatic steatosis, insulin resistance and glucose intolerance. In contrast, they found that apoe-deficient mice are resistant to the development of these conditions (4, 9). Thus, following a lipid-rich meal, chylomicrons are secreted by the intestine into the circulation. Partial lipolysis of chylomicrons by LpL converts chylomicrons into chylomicron remnants which in the presence of excess ApoE3, are taken up by the liver and the adipose tissue via the LDL-receptor or the LRP or both, resulting in hepatic steatosis, obesity, glucose intolerance and insulin resistance. In the absence of ApoE however, chylomicron remnants cannot bind to the LDL receptor that is present both in the liver and fat, and accumulate in the plasma of the apoe-/- mice. Thus, lack of apoe prevents diet-induced obesity and results in hypercholesterolemia. Driven by the recent observation that the chylomicron transport system has a pivotal role in the development of obesity and that obesity is strongly associated with OA pathogenesis, we were tempted to assume that apoe is also involved in the pathobiology of obesity-induced OA. 2) ΑΝΑΠΤΥΞΗ ΜΕΘΟΔΟΛΟΓΙΑΣ (RESEARCH METHODOLOGY): AIM 1: To identify cellular genes and pathways affected by apoe expression in mice, which may be involved in the pathogenesis of obesity-induced OA. In the present proposal we will extract total RNA from cartilage of apoe-deficient (apoe -/- ) or apoe3 knock-in mice using Qiagen RNeasy kit. The RNA will then be subjected to T7-based RNA amplification using the Ambion MessageAmp II arna Kit. Microarray analysis will be applied to identify genes and signalling pathways that are triggered when these mice are fed western-type diet. For microarray analyses Affymetrix mouse genome 430. 20 GeneChips (containing over 39.000 genes in a single assay) and expressed sequence tags (ESTs) will be used. The knock-out and transgenic animal models proposed in this application, their source, and the pathways they are involved into are listed in Table 1. All animal models are bred at least 9 times on the C57BL/6 background to ensure that any effects on obesity are not due to differences in the genetic background of the mice. Mice will be 6

housed individually and all groups will be allowed free access to food and water, and kept on a 12-hour light cycle. All experiments will be performed under the guidelines of the Protocol for the Protection and Welfare of the animals and the supervision of the Institutional Animal Committee of The University of Patras. We expect that the studies described in this Aim will provide novel in vivo insights in the development of obesity and its associated OA. Furthermore, these studies may reveal new therapeutic targets for the treatment of these conditions. AIM2: To examine histologically hyaline cartilage and chondroblastic cells from humans suffering from OA and to assess the protein and gene expression levels of apoe. In this aim, fresh-frozen and formalin-fixed paraffin-embedded tissues (FFPET) from articular cartilage from femoral heads, femoral condyles and tibial plateaus of patients with primary OA who have undergone hip or knee replacement surgery at the Department of Orthopaedics of the University Hospital of Patras, Greece will be retrieved from Tissue Banks or Archives of the University Hospital of Patras. For this aim we plan to use at least 100 tissue samples from different patients. Each case will be re-examined histologically by the applicant who is an experienced bone pathologist and the OA changes will be graded according to the Mancin Score (MS) according to the following assumption: MS 1-4: mild OA, MS: 5-9 moderate OA and MS: 10-14 severe OA (11). ApoE cellular levels will be examined by both immunohistochemistry (IHC) and Westen blotting. Conventional IHC will be performed on 5 micrometer sections from formalin-fixed, paraffin-embedded tissues (FFPET) from archival material, as previously described (12). For western blot analysis fresh and/or fresh-frozen tissues will be used. Briefly, 40µg of protein (as determined by Bradford Assay) in 20µl of lysate will be separated using 10% PAGE. Separated proteins will be transferred onto nitrocellulose membrane. Following a blocking step with 5% low-fat milk powder, membranes will be exposed to anti-apoe antibody at 4 o C overnight. IR fluorescence signal will be scanned on a LiCor Odyssey scanner. The expression and loacalization of ApoE in cartilge will also be evaluated by confocal microscopy (Leica TCS SP5 II) Fresh-frozen and FFPE tissues will be used for RNA extraction and qrt-pcr analysis (quantitative real-time PCR using SyBr Green detection on a Roche LightCycler 2.0 7

platform) for the assessment of the levels of apoe mrna expression. Additionally, chondrocytes isolated from cartilage samples of OA patients will be cultured and used for Westen Blot, qrt-pcr and confocal microscopy (Leica) analyses. In addition, using qrt- PCR we will perform geneotype analysis of apoe, which we expect to offer some answers regarding the presence of different apoe isoforms in OA of different grades (13). With this set of experiments we expect to delineate the potential link between apoe expression and OA in humans. AIM 3: To determine how mouse ApoE and human ApoE2, ApoE3 and ApoE4 expression in chondrocytes may lead to the induction of genetic markers associated with OA. Chondrocytes will be released from cartilage of apoe -/- mice by sequential enzymatic digestion, as previously described (14). The isolated chondrocytes will be seeded at high density in tissue culture flasks and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heatinactivated fetal calfserum (FCS) as previously described (15). The cells will be infected with adenoviruses expressing either mouse apoe, or human apoe2, apoe3 and apoe4 as described previously (16). Five, ten or fifteen days post-infection, total RNA will be collected from the cultured cells and qrt-pcr will be used to determine the expression of OA-related genes such as TGFB1, COL1A2, MMP13, ADAM12, ASPN (17, 18). The principal investigator of this proposal and his collaborators [Drs. K.E. Kypreos (University of Patras) and H.C. Blair (University of Pittsburgh)] have extensive experience in almost all of these techniques. As a result no major problems are expected in the implementation of these methodologies. Table 1. Animal models that will be tested in this application, their sources, and the lipoprotein pathway that they are involved. Mouse Model Source Pathway ApoE knock-out Jackson laboratory Chylomicron/VLDL ApoE2 knock-in Taconic farms Chylomicron/VLDL ApoE3 knock-in Taconic farms Chylomicron/VLDL ApoE4 knock-in Taconic farms Chylomicron/VLDL C57BL/6 Dr. Kypreos laboratory Control 8

BIOETHICAL ISSUES The project has been approved by the Biothics Committee of the University of Patras (Approval letter is available upon request) I. Analyses in human specimens Fresh specimens to be analysed will only be obtained from resected diseased tissue for the benefit of the patient as part of their standard care, as determined by their surgeon. This part of the work has been consented by Dr. Elias Panagiotopoulos, Professor at the resident Department of Orthopaedic Surgery, University of Patras, School of Medicine and twill be performed by the applicant (MD and bone pathologist). Patients will be recruited voluntarily, solely on the basis of them requiring surgery for knee or joint replacement. In practice, patients registered for surgery at the University of Patras Regional Hospital will be asked to consider participation. Those patients will be asked to read a Participant Information and Consent Form prior to being admitted into the hospital and to decide whether they agree to be included in the participant list. The person making contact with the patient regarding participation will be the patient s medical carers. The questions asked to potential participating patients will be designed to ensure that the participant understands the reason for collecting the tissue and inform them that their identity and their sample identity will only be known to their surgeon. Only their age and de-identified medical records will be used for research. Furthermore, patients will be informed that the research conducted and publication of the research on their specimen will not identify them and that can opt out of the research specimen donation at any time, even after signing a consent form. Only if requested by the patient, the researcher is prepared to discuss the nature of the research directly with the donating patient. Patients will not be subjected to any supplementary procedure in relation to this project and there is therefore no risk of stress associated with participation in the project. No extra time will be asked of participants specifically for the research project, apart from the time devoted to reading and signing the information and consent form. In any publication and/or presentation, information will be provided in such a way that the participants cannot be identified. Information will be provided as a general description of the functions of ApoE in groups of patients with a similar form of the disease, and not as individual data from a single patient. Thus, there will not be any means to identify patients.both fresh and archival samples used and originally identified by a patient record number will be assigned a new 9

identifier (e.g. OA001, -002 etc). Thus all data relating to patient material will be deidentified and only held by the relevant medical administrators. Password protected electronic data associated with the patient research specimens will be retained in the Department of Anatomy-Histology-Embryology for at least 5 years beyond the date of the end of grant contract. At the end of this period, the data will be erased from the computer hard drive. II. Research on Animals The work proposed here also requires the use of transgenic and knock-out experimental mouse models. All experiments are designed according to the rules and regulations of the Protocol for the Protection and Welfare of the animals, which are strictly enforced by the Institutional Animal Care Committee of The University of Patras. Special consideration has been given to replace, reduce and refine the proposed experiments in order to minimize the use of animals in my studies.the numbers of mice have been calculated carefully and reduced to the minimum required to achieve statistical significance and allow for the statistical interpretation of our results. Specifically, for each of the Aims 1 and 3, we expect to employ 12 mice for each mouse strain listed in Table I. During the course of the experiments, animals will be monitored daily for any symptoms of stress or discomfort. However, since the experiments proposed involve mainly dietary studies on transgenic and knock-out mouse models, we expect that there will be minimal pain or distress associated with our studies.any animals that will be identified to be under pain or distress (sluggish, lethargic, rough fur etc) will be immediately euthanasized. At the end of each experiment, all animals will be euthanasized according to the guidelines of the Protocol for the protection and welfare of animals. The preferred method of euthanasia will be CO2 inhalation followed by cervical dislocation to ensure that mice have been terminated. The entire project will be performed at the University of Patras in Greece and in accordance with the European and Greek law pertinent to the use of experimental animals. The implementation of the applicable rules and laws will be monitored by the Institutional Animal Care Committee. 3) ΣΚΟΠΙΜΟΤΗΤΑ, ΣΗΜΑΣΙΑ ΚΑΙ ΣΥΜΒΟΛΗ (AIM, SIGNIFICANCE AND CONTRIBUTION) The proposed studies are designed to address the contribution of the lipid and 10

lipoprotein transport system in the development of obesity, and osteoarthritis. Obesity and its associated bone and cartilage pathologies constitute growing and serious problems for western societies. As described earlier in this application, osteoarthritis and obesity have reached threatening proportions and affect the quality of life of millions of citizens worldwide (19, 20, 21). The study of the correlation among obesity and osteoarthritis is a relatively new but very interesting and competitive field of biomedical research, which recently has received a lot of attention by the international scientific and medical communities. Obviously, the need to find new therapeutics for the treatment of these conditions is pressing. In this application, I propose to study the functional relationship between the lipid and lipoprotein transport system and the development of OA. This is a novel idea that is based on previous data showing that apolipoprotein E promotes the development of obesity while lack of apoe results in resistance to obesity. It is my expectation that the successful execution of the proposed studies will provide important insights in the mechanisms linking obesity and the lipoprotein transport system with osteoarthritis, and may reveal novel targets for future pharmaceutical interventions for the treatment of these conditions. Taking this project to community level will increases the probability for a successful identification of new drugs for the treatment of OA, thus contributing to the excellence and competitiveness of the University of Patras. Furthermore, it is my deep desire to transfer my knowledge to the faculty, students, and research stuff of the University of Patras Medical School. Below is a brief description of the multistep approach that I plan to follow for this goal: I will engage into teaching musculoskeletal anatomy, histology and molecular biology to undergraduate and graduate medical students. In addition, I will be in charge of providing laboratory training to graduate students and post-doctoral fellows who will be assigned to me for their Master or Ph.D. thesis. At this capacity, I will be able to transfer my knowledge and expertise to the students and researchers of the University. While continuing my ongoing collaborations and exchange of knowledge and expertise with my Greek and other European and American collaborators I will also work towards establishing new research collaborations both with scientists in the host Institute and other European and American Institutes. International collaborations will involve visits of my 11

collaborators to the University of Patras where they will be encouraged to present their work in the form of seminars and interact with the other faculty and students of the University. This will promote the establishment of new collaborative networks and the exchange of methodologies, knowledge, and reagents, among scientists of the host institute and our guests. 4) ΒΙΒΛΙΟΓΡΑΦΙΑ (LITERATURE CITED) 1. Altman R et al. Development of criteriafor the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American RheumatismAssociation. Arthritis & Rheumatism 1986; 29(8):1039-1049 2. Mokdad AH et al. Actual causes if death in the United States. JAMA 2004;291:1238-1245; Kopelman PG. Obesity as a medical problem. Nature 2000;404:635-643 3. Powel A. et al. Obesity: a preventable risk factor for large joint osteoarthritis which may act through biochemical factors. Br J Sports Med 2005;39:4-5 4. Kypreos KE et al. Mechanisms of obesity and related pathologies: role of apolipoprotein E in the development of obesity. FEBS J 2009;276:5720-5727 5. Oliveira SA et al. Body weight, body mass index and incident symptomatic osteoarthritis of the hand, hip and knee. Epidemiology 1999:10;161-166 6. Griffin TM and Guilak F. Why is obesity associated with osteoarthritis? Insights from mouse models of obesity. Biorheology 2008;387-398 7. Gomez R. et al. Adipokines in the skeleton: influence on cartilage function and joint degenerative diseases. J Mol Endocrinol 2009:43;11 18 8. Simopoulou T. et al. Differential expression of leptin and leptin s receptor isoform (Ob-Rb) mrna between advanced and minimally affected osteoarthriticcartilage; effect on cartilage metabolism. Osteoarthritis Cartilage 2007;15:872-883 9. Karagiannides I. et al. Apolipoprotein E predisposes to obesity and related metabolic dysfunctions in mice. FEBS J 2008; 275 (2008) 4796 4809 10. Toussirot TM et al. The Contribution of Adipose Tissue and Adipokines to Inflammation in Joint Diseases. Current Medicinal Chemistry 2007;14:1095-1110 11. Mankin HJ et al. Biochemical and metabolic abnormalities in articular cartilage form osteoarthritic human hips. J Bone Joint Surg 1971;53A:523-537 12. Papachristou DJ et al. Involvement of the p38 MAPK NF-κB signal transduction pathway and COX-2 in the pathobiology of meniscus degeneration in humans. Mol Med 2008;14:160-166 13. Calero O et al. Apolipoprotein E genotyping method by Real Time PCR, a fast and costeffective alternative to the TaqMan and FRET assays. J Neurosc Methods 2009;183:238-240 14. Fahmi H, Di Battista JA, Pelletier JP, Mineau F, Ranger P, Martel-Pelletier J: Peroxisome proliferator-activated receptor gamma activators inhibit interleukin-1beta-induced nitric oxide and matrix metalloproteinase 13 production in human chondrocytes. Arthritis Rheum 2001, 44:595-607 15. Chabane N et al. Human articular chondrocytes express 15-lipoxygenase-1 and -2: potential role in osteoarthritis. Arthritis Res Ther 2009;[E-pub ahead of print] 16. Van Dijk et al. Adenovirus mediated gene transfer. Methods Mol Biol 2001;276:231-247 17. Marshall KW et al. Chondrocyte genomics: implications for disease modification in osteoarthritis. Drug Discov Today 2006;11:825-832 12

18. Swingler TE et al. Degradome expression profiling in human articular cartilage. Arthritis Res Ther. 2009;11(3):R96. [Epub 2009 ahead of print] 19. Zhao L-G et al. Correlation of obesity and osteoporosis: effect of fat mass on the determination of osteoporosis. J Bone Miner Res 2008;23:17 29 20. Kopelman PG. Obesity as a medical problem. Nature 2000;404:635-643 13

Ε. ΧΡΟΝΟΔΙΑΓΡΑΜΜΑ ΕΚΤΕΛΕΣΗΣ ΤΟΥ ΕΡΓΟΥ (MILESTONES OF THE PROJECT) Duration (months): Experimental procedures Collection of fresh-frozen and FFPE tissue samples from patients with osteoarthritis, histologic evaluation and re-evaluation, of the sections Histochemical, immunohistochemical and molecular analyses of the human samples Purchase and feed mice with high-fat diet (Western-type) and collect tissues Perform histological, molecular and microarray assays on tissues and RNA samples, isolated from the cartilage of apoe dificient and apoe knock-in mice Isolate and culture chondrocytes from apoe dificient mice, perform adenovirus-mediated gene transfer experiments and analyses of the infected cells 0 6 12 18 24 30 36 ΣΤ. ΑΝΑΛΥΣΗ ΚΑΙ ΑΙΤΙΟΛΟΓΗΣΗ ΠΡΟΫΠΟΛΟΓΙΣΜΟΥ (BUDGETARY BREAKDOWN) In this application, we request a total of 11000 per year for 3 years. Thus, the total amount requested is 33000. According to the Grant call, 82% of the total will cover the salary of the postgraduate / PhD student. This means that the salary of the student will be 27060 for 3 years ( 9020 per year or 751,6667 per month). The rest of the amount ( 5940 for 3 years / 1980 per year) will be used for consumables. 14

Ζ. ΣΥΝΘΕΣΗ ΕΡΕΥΝΗΤΙΚΗΣ ΟΜΑΔΑΣ ΚΑΙ ΑΠΑΣΧΟΛΗΣΗ ΚΑΘΕ ΜΕΛΟΥΣ (Eπισυνάπτονται πλήρη βιογραφικά στο τέλος της πρότασης) (WORKING GROUP SYNTHESIS AND ROLES IN THE PROJECT) (Full CVs are attched at the end of the proposal) i) Επιστηµονικός υπεύθυνος (Scientific Coordinator of the Project): - Papachristou Dionysios, Anatomic Pathologist, Assistant Professor, Department of Anatomy-Histology-Embryology, Univ. of Patras, School of Medicine. Dr. Papachristou is a bone pathologist with significant experience in bone-related methodologies, trained in high-imapct Institutions of the USA. He will be the suprevisor of the project and will participate in all the phases/experimental procedures. ii) Yποψήφιος Μεταπτυχιακός φοιτητής / Διδάκτορας (Postdoctoral / PhD student): The postdoctoral / PhD studen will be hired in due time iii) Συνεργάτες (Collaborators): - Dr. Kypreos Kyriakos, Associate Professor, Department of Pharmacology, Univ. of Patras, School of Medicine. Dr. Kyriakos Kypreos, is world leader in the study of lipid metabolism and related pathologies, such as metabolic syndrom and atherosclerosis. Dr. Kypreos will provide as the experimental animals needed for the study and will participate in the adenovirus-mediated gene transfer experiments and the analyses of infected cells. - Dr. Helen Papadakis-Petrou, Anatomic Pathologist, Associate Professor and Director, Department of Anatomy-Histology-Embryology, Univ. of Patras, School of Medicine. Dr. Papadakis is an experienced Pathologist, with deep knowledge on the histopathological and molecular analyses of tissue samples. She will participate in the histological, cytological and molecular analyses of human and experimental animal tissues. iv) Συνεργαζόµενα εργαστήρια και κλινικές (Collaborating Laboratories and Clinics) - Dr. Elias Panagiotopoulos, Professor of Orthpeadic Surgery, Univ. Hospital of Patras, Greece and a long-lasting collaborator of Dr. Papachristou, the project supervisor. Dr Panagiotpoulos is an orthopaedic surgeon with extensive experience in the osteoarthritis- 15

related surgical procedures., who will provide us the fresh tissue obtained from the surgical treatment of patients with oteoarthritis. In addition, he is expected to have significant contibution eveluationg the clinical signficance of our experimental data. - Dr. Harry Blair, Professor of Pathology, Dept. of Medicine, University of Pittsburgh, Pittsburgh, PA, USA. Dr. Blair, is a long-lasting collaborator of Dr. Papachristou s and an expert in the field of bone biology with emphasis in the study the metabolic bone diseases. He will provide us the primary chondrcytes for cells culture and will offer his expertise in the deveolment and application of bone/cartilage related methodologies. - Η. ΚΑΤΑΣΤΑΣΗ ΠΡΟΓΡΑΜΜΑΤΩΝ ΤΗΣ ΤΕΛΕΥΤΑΙΑΣ ΤΡΙΕΤΙΑΣ (GRANTS DURING THE LAST THREE YEARS): 16

Π Ρ Ο Υ Π Ο Λ Ο Γ Ι Σ Μ Ο Σ Δ Α Π Α Ν Ω Ν (BUDGET) 1. ΥΠΟΤΡΟΦΙΕΣ ΜΕΤΑΠΤΥΧΙΑΚΟΙ ΦΟΙΤΗΤΕΣ (SCHOLARSHIPS POSTDOCTORAL STUDENTS) (82 %) ΟΝΟΜΑΤΕΠΩΝΥΜΟ (NAME) ΜΗΝΕΣ ΑΠΑΣΧΟΛΗΣΗΣ ΜΕΣΗ ΜΗΝΙΑΙΑ ΣΥΝΟΛΙΚΗ ΔΑΠΑΝΗ (TOTAL) (DURATION) ΑΜΟΙΒΗ (SALARY PER MONTH) To be hired in due time 36 751,6667 27060 2. ΑΝΑΛΩΣΙΜΑ (CONSUMABLES) ΠΕΡΙΓΡΑΦΗ (DISCRIPTION) ΠΟΣΟ (AMOUNT) PCR reagents 1500 (RNA isoltayion, Taq-Polymerase, primers, probes) Reagants for Micrarray analyses 4000 Animals 440 Cell culture reagents Monoclonal antibodies, 0, available to th PI through Western blotting reagents, Consumables for tissue the Dept. Of Anatomydecalcification, embedding and sectioning Histology-Embryology Σύνολο (Total) 5940 3. ΜΕΤΑΚΙΝΗΣΕΙΣ (TRAVEL EXPENCES) ΠΕΡΙΓΡΑΦΗ (DISCRIPTION) ΠΟΣΟ (AMOUNT) 0 ΣΥΝΟΛΙΚΗ ΔΑΠΑΝΗ ΕΡΓΟΥ (ΤOTAL BUDGET OF THE PROJECT) 33.000 17

Βιογραφικό Επιστηµονικού Υπευθύνου (Curriculum Vitae of the scientific coordinator) Προσωπικά στοιχεία (Personal information) Επώνυµο (Surename) Όνοµα (Given Name) Θέση στο Ίδρυµα (Current Position in the Institution) Τµήµα (Department) Email Τηλέφωνο επικοινωνίας (Telephone) Papachristou Dionysios Assistant Professor Anatomy-Histology-Embryology, School of Medicine, Univ. of Patras papachristoudj@med.upatras.gr, dip13@pitt.edu +30-2610-996121, +30-2610-969188, +30-6972818212 Έτος (Year) Ίδρυµα (Institution) Τίτλος (πτυχίο, διδακτορικό) [Title (Degree, PhD)] 1995 University of Patras, School of Medical Doctor Medicine 2003 University of Patras, School of PhD Medicine (BIE) 2004 University of Pittsburgh, School Research Fellow in Pathology of Medicine, Pittsburgh, PA 2006 University of Patras, School of Specialty of Anatomic Pathology Medicine 2007 University of Pittsburgh, School of Medicine, Pittsburgh, PA Instructor of Pathology 2009 University of Pittsburgh, School Assistant Professor (Adj.) of Pathology 18

of Medicine, Pittsburgh, PA 2009 University of Patras, School of Medicine Assistant Professor Κύρια ερευνητική δραστηριότητα (Main scientific interests and activity) - Bone and soft tissue tumor pathobiology and pathology (Bone 2003, Anticancer Res 2005, Histopathology 2005, Hum Pathol 2006, Int J Biochem Cell Biol 2007, Histopathology 2007, J Foot Ankle Surg 2007, Eur J Cancer 2008, Orthopedics 2008, Cancer Genet Cytogenet 2009, Acta Orthop Belg 2009, Arch Orthop Trauma Surg 2009, ) - Bone biology and metabolic bone disease (Histochem Cell Biol 2005, Cell 2006, FEBS Lett 2007, Mol Med 2008, Bioessays 2009, Arch Oral Biol 2009, Eur J Phys Rehabil Med 2009, Med Sci Monit 2010) - Gastrointestinal system biology, osbesity (J Endocrinol Invest 2005, Gastrointest Endosc 2006, Pancreatology 2006, Am J Gastroenterol 2006, Am J Gastroenterol 2007, JOP 2007, Dig Dis Sci 2010) - Brain Pathology (Brain Pathol 2009) Επιστηµονικές εργασίες τα τελευταία 7 χρόνια (σε διεθνή περιοδικά µε κριτές) [List of publications the last 7 years (in peer reviewed journals)] 1. Papachristou GI, Muddana V, Papachristou DJ, Stello K, Whitcomb DC. Epidermal growth factor serum levels and the 61 G/A polymorphisms in patients with acute pancreatitis. Dig Dis Sci 2010 [Epub ahead of print] 2. Athanassiou V, Papachristou DJ, Panagopoulos A, Saridis A, Scopa C, Megas P. Histological comparison of autograft, allograft-dbm, xenograft, and synthetic grafts in trabecular bone defect: an experimental study. Med Sci Monit 16:BR24-31;2010 3. Mazis N, Papachristou DJ, Zouboulis P, Tyllianakis M, Scopa CD, Megas P. The 19

effect of different physical activity levels on muscle fiber size and type distribution of lumbar multifidus. A biopsy study on low back pain patient groups and healthy control subjects. Eur J Phys Rehabil Med. 45:459-67;2009 4. Klironomos G, Bravou V, Papachristou DJ, Gatzounis G, Varakis J, Parassi E, Repanti M, Papadaki H. Loss of Inhibitor of Growth (ING-4) Is Implicated in the Pathogenesis and Progression of Human Astrocytomas. Brain Pathol. 2009 [Epub ahead of print] 5. Papachristou DJ, Papachroni KK, Papavassiliou GA, Pirttiniemi P, Gorgoulis VG, Piperi C, Basdra EKFunctional alterations in mechanical loading of condylar cartilage induces changes in the bony subcondylar region. Arch Oral Biol. 54(11):1035-45;2009 6. Papachristou DJ, Papachroni KK, Basdra EK, Papavassiliou AG.Signaling networks and transcription factors regulating mechanotransduction in bone. Bioessays. 2009 Jul;31(7):794-804 7. Kontogeorgakos VA, Xenakis T, Papachristou D, Korompilias A, Kanellopoulos A, Beris A, Brigman B. Cortical desmoid and the four clinical scenarios. Arch Orthop Trauma Surg. 129:779-785;2009. 8. Papachristou DJ, Palekar A, Surti U, Cieply K, McGough RL, Rao UN. Malignant granular cell tumor of the ulnar nerve with novel cytogenetic and molecular genetic findings. Cancer Genet Cytogenet. 2009 May;191(1):46-50 9. Papachristou DJ, Papadakou E, Basdra EK, Baltopoulos P, Panagiotopoulos E, Papavassiliou AG. Involvement of the p38 MAPK NF-κB signal transduction pathway and COX-2 in the pathobiology of meniscus degeneration in humans. Mol Med 14:160-166;2008 10. Papathanassiou ZG, Megas P, Petsas T, Papachristou DJ, Nilas J, Siablis D. Radiofrequency Ablation of Osteoid Osteomas: An Overview. Orthopedics 31:1118-11122;2008 11. Papachristou DJ, Gkretsi V, Rao UN, Papachristou GI, Papaefthymiou OA, Basdra EK, Wu C, Papavassiliou AG. Expression of integrin-linked kinase and its binding partners in chondrosarcoma: association with prognostic significance. Eur J Cancer. 44:2518-2525;2008 20

12. Papadopoulou A, Papachristou DJ, Pirtiniemi P., Papavassiliou AG, Basdra EK. Load application induces changes in the expression level of SOX9, FGFR3 and VEGF in condylar chondrocytes. FEBS Lett 581:2041-2046;2007 13. Kontogeorgakos VA, Lykissas MG, Mavrodontidis AN, Sioros V, Papachristou D, Batistatou AK, Beris AE. Turret Exostosis of the Big Toe. J Foot Ankle Surg 46:130-132;2007 14. Anoun E, Slivka A, Papachristou DJ, Gleeson F, Whitcomb D, Papachristou GI. Rapid evolution from the first episode of acute pancreatitis to chronic pancreatitis in human subjects. Journal of Pancreas 8:12001-12006;2007 15. Papachristou DJ, Gkretsi V, Tu Y, Shi X, Chen K, Larjava H, Rao UN, Wu C. Increased cytoplasmic level of migfilin is associated with higher grades of human leiomyosarcomas. Histopathology 51: 499-508;2007 16. Papachristou DJ, Papavassiliou AG. Osteosarcoma and chondrosarcoma: new signalling pathways. Int J Biochem Cell Biol 39:857-862;2007 17. Papachristou GI, Gleeson FC, Papachristou DJ, Petersen BT, Baron TH. Endoscopist administrated sedation during ERCP: Impact of chronic narcotic/benzodiazepine use and predictive risk of reversal agent utilization. Am J Gastroenterology 102:738-743;2007 18. Papachristou DJ, Papavassiliou AG. Osteosarcoma and chondrosarcoma: new signaling pathways as targets for novel therapeutic interventions. Int J Biochem Cell Biol. 39:857-862;2007. 19. Papachristou GI, Papachristou DJ, Morinville VD, Slivka A, Whitcomb DC Chronic alcohol consumption is a major risk factor for pancreatic necrosis in acute pancreatitis.. Am J Gastroenterol. 101:2605-2610;2006. 20. Papachristou DJ, Goodman MA, Cieply K, Hunt JL, Rao UN. Comparison of allelic losses in chondroblastoma and primary chondrosarcoma of bone and correlation with fluorescence in situ hybridization analysis. Hum Pathol. 37:890-898;2006. 21. Papachristou GI, Papachristou DJ, Avula H, Slivka A, Whitcomb DC. Pancreatology. Obesity increases the severity of acute pancreatitis: performance of APACHE-O score and correlation with the inflammatory response. 6:279-285;2006. 21

22. Sun L, Peng Y, Sharrow AC, Iqbal J, Zhang Z, Papachristou DJ, Zaidi S, Zhu LL, Yaroslavskiy BB, Zhou H, Zallone A, Sairam MR, Kumar TR, Bo W, Braun J, Cardoso-Landa L, Schaffler MB, Moonga BS, Blair HC, Zaidi M. FSH directly regulates bone mass. Cell. 125:247-60;2006 23. Papachristou D, Pirttiniemi P, Kantomaa T, Agnantis N.J., Basdra EK. The Fra-1, Fra2, JunB and JunD transcription factors are involved in the signal transduction pathway of mechanical loading of condylar chondrocytes. Europ J Orthond 28:20-26;2006 24. Papachristou DJ, Papachristou GI, Papaeuthimiou OA, Agnantis NJ, Basdra EK, Papavassiliou AG. The MAPK-AP-1/Runx-2 signaling axes are implicated in chondrosarcoma pathobiology either independently or via up-regulation of VEGF. Histopathology 47:565-574;2005 25. Papachristou GI, Papachristou DJ, Schoedel K, McGrath K, Slivka A. Systemic silicosis involving the pancreas. Gastrointest Endosc 1:170-172; 2006 26. Papachristou DJ, Pirttiniemi P, Kantomaa T, Papavassiliou AG, Basdra EK. JNK/ERK AP-1/Runx2 induction paves the way to cartilage load-ignited chondroblastic differentiation. Histochem Cell Biol 124:215-223;2005 27. Batistatou A, Papachristou DJ. Food for thought: the sequel! Vegetable cells looking at the pathologist who is looking at the vegetable cells». Int J Surg Pathol 12:49;2004 28. Papachristou DJ, Batistatou A, Paraskevaidis E, Agnantis NJ. Aggressive angiomyxoma of vagina: a case report and review of the literature. Eur J Gynaecol Oncol 25:519-21;2004 29. Stefanou D, Batistatou A., Zioga A, Arkoumani E, Papachristou DJ, Agnantis NJ. Immunohistochemical expression of VEGF and c-kit in cutaneous melanocytic lesions. Int J Surg Pathol 12:133-8; 2004 30. Expression of VEGF and association with microvessel density in benigh prostate hyperplasia and prostate cancer. Stefanou D, Batistatou A, Kamina S, Papachristou DJ, Agnantis NJ. In Vivo 18:155-160;2004 31. D.J. Papachristou, A. Batistatou, N.J. Agnantis. Human Osteosarcomas: an up to date novel pathological and molecular approach. Acta Orthopaedica et Traumatologica Hellenica 55;2004 22

32. Batistatou A, Papachristou DJ. Nevus cells growing within a lymphatic valve. Int J Surg Pathol 12:150;2004 33. Papachristou DJ, A. Batistatou, G.P. Sykiotis, J. Varakis, A.G. Papavassiliou. Activation of the JNK AP-1 signal transduction pathway is associated with the pathogenesis and progression of human osteosarcomas. Bone 32:364-371; 2003 Επιστηµονικές εργασίες στο αντικείµενο της πρότασης τα τελευταία 10 χρόνια (σε διεθνή περιοδικά µε κριτές, συµπεριλαµβανοµένων σχετικών επίσης διπλωµάτων ευρεσιτεχνίας, βιβλίων, βραβείων, κ.ά.) [Scientific publications associated with the project proposal during the last 10 years (publications in peer reviewed journals, patents, books, awards e.t.c,)] 1. Athanassiou V, Papachristou DJ, Panagopoulos A, Saridis A, Scopa C, Megas P. Histological comparison of autograft, allograft-dbm, xenograft, and synthetic grafts in trabecular bone defect: an experimental study. Med Sci Monit 16:BR24-31;2010 2. Papachristou DJ, Papachroni KK, Papavassiliou GA, Pirttiniemi P, Gorgoulis VG, Piperi C, Basdra EKFunctional alterations in mechanical loading of condylar cartilage induces changes in the bony subcondylar region. Arch Oral Biol. 54(11):1035-45;2009 3. Papachristou DJ, Papachroni KK, Basdra EK, Papavassiliou AG.Signaling networks and transcription factors regulating mechanotransduction in bone. Bioessays. 2009 Jul;31(7):794-804 4. Papachristou DJ, Papadakou E, Basdra EK, Baltopoulos P, Panagiotopoulos E, Papavassiliou AG. Involvement of the p38 MAPK NF-κB signal transduction pathway and COX-2 in the pathobiology of meniscus degeneration in humans. Mol Med 14:160-166;2008 5. Papachristou DJ, Gkretsi V, Rao UN, Papachristou GI, Papaefthymiou OA, Basdra EK, Wu C, Papavassiliou AG. Expression of integrin-linked kinase and its binding partners in chondrosarcoma: association with prognostic significance. Eur J Cancer. 44:2518-2525;2008 23

6. Papadopoulou A, Papachristou DJ, Pirtiniemi P., Papavassiliou AG, Basdra EK. Load application induces changes in the expression level of SOX9, FGFR3 and VEGF in condylar chondrocytes. FEBS Lett 581:2041-2046;2007 7. Papachristou DJ, Gkretsi V, Tu Y, Shi X, Chen K, Larjava H, Rao UN, Wu C. Increased cytoplasmic level of migfilin is associated with higher grades of human leiomyosarcomas. Histopathology 51: 499-508;2007 8. Papachristou DJ, Papavassiliou AG. Osteosarcoma and chondrosarcoma: new signalling pathways. Int J Biochem Cell Biol 39:857-862;2007 9. Papachristou DJ, Papavassiliou AG. Osteosarcoma and chondrosarcoma: new signaling pathways as targets for novel therapeutic interventions. Int J Biochem Cell Biol. 39:857-862;2007 10. Papachristou DJ, Goodman MA, Cieply K, Hunt JL, Rao UN. Comparison of allelic losses in chondroblastoma and primary chondrosarcoma of bone and correlation with fluorescence in situ hybridization analysis. Hum Pathol. 37:890-898;2006 11. Papachristou GI, Papachristou DJ, Avula H, Slivka A, Whitcomb DC. Pancreatology. Obesity increases the severity of acute pancreatitis: performance of APACHE-O score and correlation with the inflammatory response. 6:279-285;2006 12. Sun L, Peng Y, Sharrow AC, Iqbal J, Zhang Z, Papachristou DJ, Zaidi S, Zhu LL, Yaroslavskiy BB, Zhou H, Zallone A, Sairam MR, Kumar TR, Bo W, Braun J, Cardoso-Landa L, Schaffler MB, Moonga BS, Blair HC, Zaidi M. FSH directly regulates bone mass. Cell. 125:247-60;2006 13. Papachristou D, Pirttiniemi P, Kantomaa T, Agnantis N.J., Basdra EK. The Fra-1, Fra2, JunB and JunD transcription factors are involved in the signal transduction pathway of mechanical loading of condylar chondrocytes. Europ J Orthond 28:20-26;2006 14. Papachristou DJ, Papachristou GI, Papaeuthimiou OA, Agnantis NJ, Basdra EK, Papavassiliou AG. The MAPK-AP-1/Runx-2 signaling axes are implicated in chondrosarcoma pathobiology either independently or via up-regulation of VEGF. Histopathology 47:565-574;2005 24

15. Papachristou DJ, Pirttiniemi P, Kantomaa T, Papavassiliou AG, Basdra EK. JNK/ERK AP-1/Runx2 induction paves the way to cartilage load-ignited chondroblastic differentiation. Histochem Cell Biol 124:215-223;2005 16. D.J. Papachristou, A. Batistatou, N.J. Agnantis. Human Osteosarcomas: an up to date novel pathological and molecular approach. Acta Orthopaedica et Traumatologica Hellenica 55;2004 17. Papachristou DJ, A. Batistatou, G.P. Sykiotis, J. Varakis, A.G. Papavassiliou. Activation of the JNK AP-1 signal transduction pathway is associated with the pathogenesis and progression of human osteosarcomas. Bone 32:364-371; 2003 Honors/Awards: 1. 2009-today: Financial administrator of the managing team of the Metsnet network of the Univerity of Patras that focuses on the study of the Metabolic Syndrom and related pathologies 2. 2007-2009: Principal Investigator in the research project Molecular, Immunohistochemical and Morphological Predictors of Local and Distant Recurrent Sarcomas and Outcome in Bone and Soft Tissue Sarcomas IRB #: 0612060, University of Pittsburgh, Pittsburgh, PA: University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. 3. May 2008, 1 st Award, Hellenic Society of Pathology 4. October 2006, Vicky Zachariou 1 st Award, EEXOT 5. May 2006, JC Agnantis 1 st Award winner, Bone and Soft Tissue Pathology, European Intercongress of Pathology, International Academy of Pathology 6. April 2004, Sotirios Papastamatis 1 st Award winner, Hellenic Medical Association 7. May 2003, 1 st Award winner, Mediterranean Conference, International Academy of Pathology 25

Αριθµός ετεροαναφορών και σηµαντικές σ διεθνείς συνεργασίες (Number of hetero-citations and Significant International Collaborations) Number of hetero-citations: 320 Significant International Collaborations - Dr. Harry Blair, Professor of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA - Dr. Uma Rao, Professor of Pathology, Head of the Bone-Soft Tissue Pathology Division, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA - Dr. Geogre Michalopoulos, Professor of Pathology, Head and Chief of the Dept. of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA - Dr. Georgios Papachristou, Assist. Professor of Gastroenterology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA - Dr. Carry Wu, Professor of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA - Dr. Carry Inwards, Professor of Pathology, Head of the Bone Pathology Division, Mayo Clinic, Rochester, MN, USA - Dr. Alexander Payatakes, Assistant Professor of Orthopaedic Surgery, Penn State Hershey Bone and Joint Institute, Hershey, PA, USA Συνολικός αριθµός δηµοσιεύσεων σε περιοδικά (Total number of publications in peer-reviewed journals): 33 26

Papachristou Dionysios, Curriculum Vitae Personal information: Title: (Pathologist) MD, PhD Current position: Assistant Professor of Anatomy-Histology-Embryology, University of Patras Medical School, Patras, Greece; Assistant Professor (Adj.) of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA Surname: Papachristou Given Name: Dionysios Citizenship: Greece Contact Information: University of Patras Medical School, Department of Anatomy-Histology-Embryology, Rio, TK 26500, Greece. Telephone: +30-2610-996121, +30-2610-992391 FAX: +30-2610-969178 e-mail: papachristoudj@med.upatras.gr, dip13@pitt.edu Education: 1. M.D., University of Patras, Greece, Greece: 1988-1995 2. Ph.D., University of Patras School of Medicine, Patras, Greece, 1999-2003: Biochemistry. Thesis Advisor: Dr Athanassios G. Papavassiliou. THESIS TITLE: Regulation of the proto-oncogenes c-jun and c-fos (transcription factor AP-1) in human osteosarcomas (my PhD study was supported by the PENED Program #99ΕΔ293. 3. Pathology Specialty: University Hospital of Patras, Greece; University of Pittsburgh Medical Center (UPMC), Pittsburgh, PA, USA: 2001-2006 Employment History / Experience: 1) September 18, 2009-present: Assistant Professor in Anatomy-Histology-Embryology: University of Patras Medical School, Rio, Patras, Greece. 2) September 2, 2009 present: Assistant Professor (Adj.): Department of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, U.S.A. 3) September 1, 2008-September 1, 2009: Assistant Professor: Department of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, U.S.A. 4) September 1, 2007-August 31, 2008: Instructor: Department of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, U.S.A. 5) May 2006-July 2006: Visiting Clinician, Department of Pathology, Mayo Clinic, Rochester, MN, U.S.A. 65) September 2003-June 2005. Research Fellow, Department of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, U.S.A. Areas of Expertise: Histology, molecular anatomic pathology, bone and soft tissue pathology, bone biology with emphasis to mechanotranduction, metabolic and inflammatory bone diseases, pathobiology of obesity-related bone diseases, surgical pathology Honors/Awards: 1. May 2008, 1 st Award, Hellenic Society of Pathology 2. October 2006, Vicky Zachariou 1 st Award, EEXOT 27

3. May 2006, JC Agnantis 1 st Award winner, Bone and Soft Tissue Pathology, European Intercongress of Pathology, International Academy of Pathology 4. April 2004, Sotirios Papastamatis 1 st Award winner, Hellenic Medical Association 5. May 2003, 1 st Award winner, Mediterranean Conference, International Academy of Pathology Societies-Memberships: 1. United States and Canadian Academy of Pathology (USCAP) 2. European Society of Pathology (ESP) 3. European Calcified Tissue Society (ECTS) 4. American Society of Bone and Mineral Research (ASBMR) 5. International Bone and Mineral Society (IBMS) 6. European Association for Cancer Research (EACR) 7. Hellenic Medical Society Research Grants: 2010-2014: Marie Curie Reintegration Grants (IRG), Call : FP7-PEOPLE-RG-2009, #256402-«apoeostearthritis» (100.000 Euros) 2007-2010: Molecular, Immunohistochemical, Morphological analyses and Predictors of Local and Distant Recurrent and Outcome in Bone and Soft Tissue Sarcoma IRB #: 0612060, University of Pittsburgh, Pittsburgh, PA, USA, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA List of publications 34. Papachristou GI, Muddana V, Papachristou DJ, Stello K, Whitcomb DC. Epidermal growth factor serum levels and the 61 G/A polymorphisms in patients with acute pancreatitis. Dig Dis Sci 2010 [Epub ahead of print] 35. Athanassiou V, Papachristou DJ, Panagopoulos A, Saridis A, Scopa C, Megas P. Histological comparison of autograft, allograft-dbm, xenograft, and synthetic grafts in trabecular bone defect: an experimental study. Med Sci Monit 16:BR24-31;2010 36. Mazis N, Papachristou DJ, Zouboulis P, Tyllianakis M, Scopa CD, Megas P. The effect of different physical activity levels on muscle fiber size and type distribution of lumbar multifidus. A biopsy study on low back pain patient groups and healthy control subjects. Eur J Phys Rehabil Med. 45:459-67;2009 37. Klironomos G, Bravou V, Papachristou DJ, Gatzounis G, Varakis J, Parassi E, Repanti M, Papadaki H. Loss of Inhibitor of Growth (ING-4) Is Implicated in the Pathogenesis and Progression of Human Astrocytomas. Brain Pathol. 2009 [Epub ahead of print] 38. Papachristou DJ, Papachroni KK, Papavassiliou GA, Pirttiniemi P, Gorgoulis VG, Piperi C, Basdra EKFunctional alterations in mechanical loading of condylar cartilage induces changes in the bony subcondylar region. Arch Oral Biol. 54(11):1035-45;2009 39. Papachristou DJ, Papachroni KK, Basdra EK, Papavassiliou AG.Signaling networks and transcription factors regulating mechanotransduction in bone. Bioessays. 2009 28