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Urn U Ottawa urn u Ottawa L’Univcrsilc canadicnnc Canada’s university Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FACULTE DES ETUDES SUPERIEURES 1 ^ = 1 FACULTY OF GRADUATE AND ET POSTOCTORALES U Ottawa POSDOCTORAL STUDIES L’Universitd eanadierme Canada’s university Turaya Naas X u t e u r MLX t h e s e 7 X u t h o r o f T h e s is Ph.D. (Microbiology_______ and Immunology) Department of Biochemistry, Microbiology and Immunology........ Characterization of Structural and Functional Liver Changes in a Transgenic Mouse Model Expressing Genotype 1 a Hepatitis C Virus Core and Envelope Proteins 1 and 2 TITRE DE LA THESE / TITLE OF THESIS Francisco Diaz-Mitoma DIRECTEURpTRECTRi^ cohiRECtWR (cobiREcTRicE) dela ThI ' ^ EXAMINATEURS (EXAMINATRICES) DE LA TH^SE I THESIS EXAMINERS M. Krajden (teleconference) K. Wright S. Li S. Sad Gary W. Slater Le Doyen de la Facuite des itudes supirieures et postdoctoraies / Dean of the Faculty of Graduate and Postdoctoral Studies Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Characterization of Structural and Functional Liver Changes in a Transgenic Mouse Model Expressing Genotype la Hepatitis C Virus Core and Envelope Proteins 1 and 2 By Turaya Naas A thesis Submitted to the Faculty of Graduate Studies in partial fulfillments of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology Department of Biochemistry, Microbiology, and Immunology Faculty of Medicine University of Ottawa © Turaya Naas, Ottawa, Canada, 2007 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Library and Bibliotheque et Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-32415-8 Our file Notre reference ISBN: 978-0-494-32415-8 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. i * i Canada Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis affecting over 170 million people worldwide. The mechanisms behind the pathogenesis of chronic HCV infection are not well understood. The aim of this thesis is to elucidate how the HCV structural proteins affect the dynamic structural and functional properties of hepatocytes and measure the extra- hepatic manifestations induced by these viral proteins. To pursue this goal, a transgenic mouse model was established by expressing Core, El and E2 proteins downstream of a CMV promoter. HCV RNA was detected in transgenic mouse model tissues, such as liver, kidney, spleen, and heart. Immunofluorescence analysis revealed the expression of core, El and E2 proteins predominantly in hepatocytes. Histological analysis of liver cells demonstrated steatosis in transgenic mice older than 3 months, which progressed with mouse age. Electron microscopy analysis revealed alterations in mitochondria, and in the endoplasmic reticulum. The animals became more prone to liver and lymphoid tumor development and hepatocellular carcinoma. It is likely that the HCV structural proteins mediate some of the histological alterations in hepatocytes by interfering with liver biological processes. To study that, we compare differential gene expression patterns in the liver of HCV transgenic and non-transgenic mice using complementary DNA (cDNA) microarrays and confirmed the expression of altered genes by real-time RT-PCR. 15 600 genes were analyzed and 394 genes were identified to be differentially expressed at a statistically significant level, while 196 genes were identified as up-regulated and 198 genes were identified as down-regulated. The expression of HCV structural proteins in transgenic mouse livers induces alterations in the expression of genes involved in many important biological processes such as lipid metabolism and transport, antigen processing and presentation, protein biosynthesis, i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. carcinogenesis, etc. Also, some of the differentially expressed genes were associated with important cell signaling pathways such as Mitogen activated protein kinase (MAPK) pathway and Wnt oncogenic pathway. The third study aims to elucidate the role of immune- mediated cell damage in hepatitis C infection using HCV Transgenic mice. Adoptive transfer of carboxyfluorescein diacetate suuccinimidyl ester (CFSE) labelled splenocytes from HCV immunized mice into HCV transgenic mice was performed. After the adoptive transfer, we observed that there was a significant decrease in the percentage of CFSE-labeled CD4+ and CD8+ T cells in the blood of transgenic mice receiving transfers from immunized donors. Moreover, the percentage of CFSE-labeled CD4+ and CD8+ T cells were significantly higher in the spleen of transgenic and non-transgenic mice when they received transfer from non­ immunized mice. Interestingly, transgenic livers of mice received transfers from immunized mice had significantly high percentage of CFSE-labeled T cells than non-transgenic livers receiving non-immunized transfers. This suggests that the T cells from HCV immunized mice recognized HCV antigens in the liver. Taken together, the research results indicate that our transgenic mouse model is a suitable model to study hepatitis C pathogenesis. ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION I dedicate this thesis to my husband, for his endless support and encouragement during the course of my studies, to my daughter to inspire her, to my family for their never-ending encouragement, and to my father’s immortal soul. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I would like to acknowledge many people who have helped me with unrelenting support and encouragement in so many different ways during my studies. First, I would like to give great thanks to my supervisor Dr. Francisco Diaz-Mitoma for his continual guidance and support through my journey of studies. He always had time to answer my questions, discuss my results and guide me on the right path when I ran into obstacles. I sincerely thank the members of my thesis advisor committee: Dr. Earl Brown and Dr. Craig Lee for their useful clarifications and guidance. I would like to thank Dr. Katrina Gee for reading the thesis and for her helpful comments. I would like also to thank Dr. Marko Kryworuchko and Dr. Ashok Kumar for their helpful suggestions. I would also like to recognize our collaborators: Dr. Susantha Gomis and Dr. Lome Babiuk at Pathology department, University of Saskatoon and Dr. Rudy Mueller at Pathology department, University of Ottawa, for helping in the interpretation of pathological results; Dr. Rashmy Kothary for his assistance in developing the transgenic model, Dr. Fazel Family and Dr. Sieu Phan at National Research Council Canada for helping in analyzing the microarray data. I would also like to thank the technicians in the pathology lab at University of Ottawa and at CHEO for allowing me using their equipment and teaching me different staining techniques. From start to finish of this project, I have made many friends in the lab and I would like to give them a huge thank-you and great respect. I would like to give great thanks to my mentor, Dr. Masoud Ghorbani, for starting me on the project and being patient while teaching me the various techniques. I would like to thank my friends, Catalina Soare and Nicole Scherling, for their endless help in doing experiments and taking a good care of me. I would also like to thank the rest of my lab mates, Khaled Abdulkader, Abdulkarim Alhetheel, Ali Azizi, Ikuri Alvarez-Maya, Sasmita
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