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Lentivector-Mediated Transfer of Bmi-1 and Telomerase in Muscle

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Lentivector-Mediated Transfer of Bmi-1 and Telomerase in Muscle UNIVERSITE DE GENEVE FACULTE DES SCIENCES Département de Zoologie et Biologie Animale Professeur Duri RUNGGER ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE FACULTE DES SCIENCES DE LA VIE Laboratoire de Virologie et Génétique Professeur Didier TRONO ________________________________________________________________________________ Lentivector-Mediated Transfer of Bmi-1 and Telomerase in Muscle Satellite Cells Yields a Duchenne Myoblast Cell Line with Long-Term Genotypic and Phenotypic Stability THESE présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologique par Christophe CUDRE-MAUROUX de Autigny (FR) Thèse N° 3726 Genève Atelier de reproduction d’Uni Mail 1 2006 2 Lentivector-Mediated Transfer of Bmi-1 and Telomerase in Muscle Satellite Cells Yields a Duchenne Myoblast Cell Line with Long-Term Genotypic and Phenotypic Stability 3 Remerciements Je tiens à remercier les diverses personnes qui m'ont aidé pendant cette thèse. Je ne peux les nommer toutes, mais je tiens surtout à exprimer ma profonde gratitude aux personnes suivantes. A mon directeur de thèse, le Prof. Didier Trono. Ses conseils judicieux vont certainement encore résonner dans ma tête pendant longtemps. A mes experts, le Prof. Duri Rungger et le Prof. Joachim Lingner. Je les remercie chaleureusement d’avoir accepté de faire partie de mon jury de thèse. A mes parains de thèse, les Prof. Stylianos Antonarakis et Pierre Spierer. Ils ont été d’une grande aide pendant cette thèse. A tous les membres de mon laboratoire, de mon Département de Génétique et Microbiologie (qui a été renommé Dépatement de Microbiologie et Médecine Moléculaire) et du CMU qui m’ont aidés. Je suis particulièrement reconnaissant envers Sandrine Vianin et Elisabeth Bühlmann qui m’ont aidé dans diverses techniques, et envers le Dr Isabelle Tchou qui m’a particulièrement aidé lors de l’écriture de ma thèse. A tous mes amis aussi qui n’ont parfois rien à voir avec la biologie mais qui m’ont permis de surmonter certains moments difficiles pendant cette thèse. Parmi eux, il y a mes amis du Foyer d’étudiants de Saint-Boniface et parmi eux Antoine Didry-Demarle, mais aussi Daniela Schlaefli et le Dr Blanca Rubi. Enfin, encore un grand merci à ma famille de m’avoir aidé pendant toutes mes études et pendant cette thèse. Je suis très reconnaissant envers mon père René, ma mère Rita et mon frère Philippe qui est d’ailleurs aussi sur le point de terminer un doctorat. 4 CONTENTS RESUME EN FRANCAIS........................................................................................... 8 ABSTRACT.............................................................................................................. 19 ABBREVIATIONS.................................................................................................... 21 CHAPTER 1: INTRODUCTION................................................................................ 24 1.1 GENERAL CONCEPTS OF SENESCENCE AND ONCOGENESIS ................. 25 1.1.1 Senescence and immortalization ..................................................................................... 25 1.1.2 Differences in senescence and oncogenesis between human and mouse cells ............... 25 1.1.3 Are there senescence clocks? .......................................................................................... 28 1.1.4 Pathways toward senescence........................................................................................... 30 1.1.5 The p16INK4a-Rb pathway................................................................................................ 33 1.1.6 The ARF-p53 pathway.................................................................................................... 37 1.1.7 Senescence, quiescence and apoptosis............................................................................ 42 1.1.8 Escape from senescence and crisis.................................................................................. 42 1.1.9 Crisis and genetic instabilities......................................................................................... 46 1.1.10 Somatic mutation theory of carcinogenesis .................................................................. 47 1.1.11 Properties of cancer cells............................................................................................... 48 1.1.12 How many mutations are required to trigger oncogenesis? .......................................... 49 1.1.13 Pathways leading to tumorigenesis ............................................................................... 50 1.1.14 Cancer, stem cells, and cancer stem cells...................................................................... 52 1.1.15 Context counts............................................................................................................... 54 1.1.16 The cellular environment .............................................................................................. 55 1.1.17 Escape from the immune system................................................................................... 58 1.1.18 Immune senescence....................................................................................................... 59 1.1.19 Tests of carcinogenesis.................................................................................................. 60 1.2 GENES USED FOR IMMORTALIZATION......................................................... 61 1.2.1 Telomerase ...................................................................................................................... 62 1.2.2 Alternative lengthening of telomeres (ALT)................................................................... 66 1.2.3 Bmi-1............................................................................................................................... 67 1.2.4 SV40 large T antigen....................................................................................................... 71 1.2.4.1 Large T domains................................................................................................................................... 72 1.2.4.2 TAg, p53 and pRB................................................................................................................................74 1.2.4.3 Other functions of TAg ........................................................................................................................ 75 1.2.4.4 Small t .................................................................................................................................................. 76 1.2.4.5 17kT ..................................................................................................................................................... 78 1.2.4.6 Utility of TAg in immortalization ........................................................................................................ 78 1.2.5 Bcl-2................................................................................................................................ 78 5 1.3 OF THE UTILITY OF CELL LINES .................................................................... 82 1.4 LENTIVIRAL IMMORTALIZATION.................................................................... 84 1.5 WHY TO IMMORTALIZE HUMAN MYOBLASTS? ........................................... 87 1.6 MUSCLES AND SATELLITE CELLS: AN OVERVIEW..................................... 89 1.6.1 Fusion of myoblasts ........................................................................................................ 92 1.6.2 Satellite cell markers and heterogeneity.......................................................................... 95 1.6.3 Factors modulating satellite cell activity......................................................................... 99 1.6.4 Muscle regeneration ...................................................................................................... 101 1.7 MUSCULAR DYSTROPHIES ...........................................................................103 1.7.1 General definition of muscular dystrophies .................................................................. 103 1.7.2. Duchenne Muscular Dystrophy (DMD)....................................................................... 108 1.7.3 Dystrophin..................................................................................................................... 108 1.7.4 Dystrophin deficiency and muscle degeneration .......................................................... 110 1.7.5 Therapeutic strategies for DMD.................................................................................... 111 1.7.6 A dual high capacity hybrid Adeno/AAV vector.......................................................... 115 1.7.7 Models of DMD, and their limitations.......................................................................... 119 CHAPTER 2: FIRST STUDY: LENTIVIRAL IMMORTALIZATION OF MYOBLASTS ...........................................122 2.1 GENERAL INTRODUCTION FOR THE FIRST STUDY....................................123 2.2 RESULTS OF THE FIRST STUDY ...................................................................127 2.3 CONCLUSION OF THE FIRST STUDY............................................................128 CHAPTER 3: SECOND STUDY: COMPLEMENTATION OF DUCHENNE MUSCULAR DYSTROPHY MUSCLE CELLS BY DUAL HIGH-CAPACITY HYBRID VIRAL VECTORS..........................165 3.1 GENERAL INTRODUCTION FOR THE SECOND STUDY...............................166
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