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Université Paris Xi Faculté De Médecine Paris-Sud UNIVERSITÉ PARIS XI FACULTÉ DE MÉDECINE PARIS-SUD Année 2010 N°attribué par la bibliothèque THESE Pour obtenir le grade de DOCTEUR DE L’UNIVERSITE PARIS XI Champ disciplinaire : Médecine Ecole Doctorale de rattachement : ED418 Cancérologie, Biologie, Médecine,Santé Présentée et soutenue publiquement par L’Emira Ghida HARFOUCHE Le 12 février 2010 Titre: FIBROBLAST GROWTH FACTOR 2 AND DNA REPAIR INVOLVEMENT IN THE KERATINOCYTE STEM CELL RESPONSE TO IONIZING RADIATION Directrice de thèse: MARTIN Michèle JURY Pr. BOURHIS Jean Président Dr. DOUKY Thierry Rapporteur Pr. LAMARTINE Jérôme Rapporteur Dr. BRETON Lionel Examinateur Dr. MOYAL Elisabeth Examinateur Dr. MARTIN Michèle Directrice de thèse ACKNOWLEDGEMENTS First of all, I would like to thank my thesis advisor, Dr. Michele Martin, for her guidance and her expertise in stem cell radiobiology. I appreciate her enthusiasm and encouragement during the course of the study. I have learned a lot and have grown a lot through all the experiences in her lab, not only as a scientist but also as a person. I would like to acknowledge my thesis committee Dr. Thierry Douky and Pr. Jérôme Lamartine for accepting to be my “rapporteurs”, for evaluating my work, for carefully reading this thesis and giving me helpful instructions and for being part of the jury members, along with Pr. Jean Bourhis, Dr. Lionel Breton, and Dr. Elisabeth Moyal. Their contributions to the judgment of this work and to this thesis as readers are very much appreciated. I would like to thank Dr. Nicolas Fortunel for his encouragement, invaluable advices and discussions that made me leap up one more step as a scientist. He also fortified my interest in skin biology. I would like also to thank Dr. Odile Rigaud and Dr. Sylvie Sauvaigo for teaching me the essentials of the comet assay and for the collaborations we had. I would like also to thank Dr. Serhal and the “Clinique de l’Essonne” for providing us with the human biopsies. I am also grateful to Pierre Vaigot, if not for him and his expertise in flow cytometry, isolating keratinocyte wouldn’t have been possible. I would like to thank Dr. Gilles Lemaitre for his support and constant advices, his permanent positive attitude, and for reading the discussion of my manuscript. I am very fortunate to have met Dr. Claude Gazin who provided me with outstanding encouragement and guidance. He is one of the best scientists I’ve interacted with, and a role model for his altruistic dedication into helping others and finding solutions. He has been inspirational in my development as a scientist and as a person, helping me to see things for what they are and what they can be. Over this last year, I have benefit a great deal from the precious intellectual exchange with him, both on the scientific and cultural levels, and I definitely enriched my “high-standard French vocabulary”! I am also deeply grateful for all the time he dedicated to amend this manuscript despite the short delay, providing me with valuable advices I needed in order to improve it before giving it to the referees. I would like to thank Dr. Walid Rachidi for he has been a coworker and a friend, along with his family. Albeit he is not part of the lab anymore, he accompanied me in my scientific reflections over the last years. Additionally, I would like to thank Dr. Vincent Frouin and Vincent Guillemot for their help in all the statistical aspects. Vincent Guillemot was also a volunteer reader giving me an external point of view. We both shared the same stress of thesis writing and mutual encouragement to get through it. I would like to sincerely thank my friends, Emmanuelle Cadio, Sandra Moratille and Loubna Chadli for helping me with various research techniques and especially helping me keep my sanity all these years! Every day was such an enjoyable time because of them, and I truly appreciate the time we have spent together in and out of the lab. I will cherish all those moments for the rest of my life. (Oh, and I will definitely think of you each time I use words like “utopique”, “idyllique”, “tout bien en tout honneur”!!). Special thanks to Emma for her listening and caring, for believing in me, for her help and patience always and always, and especially during printing and assembling tasks of the thesis. You always helped me cope through thick and thin! I also sincerely thank Elizabeth Jajolet for making the administrative nightmare much easier and especially for being so lovable and continually encouraging me! My fellow lab members, both past and present: Marie-Anne, Amelie, Peggy, Olivier, Sophia, Vincent, Arthur, Johnny, Simon, Sylvain, Lindha, Audrey, Pascal… we shared great moments… each had a contribution at various stages of my research and your help along the way contributed to making my stay a real hub for learning. I am very grateful to Nancy Sayar and Jessy Asmar as for they have been wonderful friends since I was 10! There are so many times that a graduate student must fall and learn from fruitless experiments. They always said to me that“it matters not if a person falls as long as they get back on their feet trying to get stronger!” My friends not only lifted me up each and every time in order to reach my ultimate goals, but they also spared me lots of wisdom in life. You will always be part of my life. My thanks go to Maya, Maria, Eliane, Aurelien, Georges, Ahmad, Carole, Roula, Layal, Richard, Gerard, Ali, Mohamad, Ramzi, Kamel… you’ve been exceptional motivators and you constantly supported me. Finally, and most importantly, I would like to thank my parents, Samir and Rima, and my brother Fawzy, for their unconditional love, support and understanding. I cannot thank them enough for their trust and encouragement. Mom and Faw who’ve always been my best friends and dad my best guide. It makes me so happy to make them proud. My deepest thanks will always be theirs, and all the accomplishments in my life will always be dedicated to them… List of abbreviations Adh Adhering aFGF acidic Fibroblast Growth Factor ASC Adult Stem Cell ATM Ataxia Telangiectasia Mutated BAD Bcl-2 Associated Death promoter BCC Basal Cell Carcinoma BER Base Escision Repair bFGF basic Fibroblast Growth Factor BIR Break Induced Replication BMP Bone Morphogenic Protein BrdU BromodeoxyUridine BRET Background Radiation Equivalent Time CFE Colony-Forming Efficiency CGH Comparative Genomic Hybridization ChK Checkpoint Kinase CNV Copy Number Variant CPD Cyclobutane Pyrimidine Dimers Cs137 Cesium 137 DDR DNA Damage Response DNA Deoxyribonucleic acid DP Dermal Papilla DSB Double Strand Break ECM ExtraCellular Matrix EG Embryonic Germ EGF Epithelial Growth Factor EGFR Epithelial Growth Factor Receptor EPU Epidermal Proliferative Unit ESC Embryonic Stem Cells EYA Eyes Absent FACS Fluorescence Activated Cell Sorting FGF Fibroblast Growth Factor FGFR Fibroblast Growth Factor Receptor GRB2 Growth factor receptor-bound protein 2 Gy Gray HF Hair Folicle HP1 Heterochromatin Protein 1 HR Homologous Recombination HSC Hematopoietic Stem Cell HSGAG Heparan Sulfate GlycosAminoGlycans HSPG Heparan Sulfate ProteoGlycans HUVEC Human Umbilical Vein Endothelial Cell IFE InterFollicular Epidermis IFN Interferon IL Interleukin IPA Ingenuity Pathway Analysis iPS induced Pluripotent Stem cell IR Ionizing Radiation Itg Integrin IVF In Vitro Fertilization JNK JunKinase K Keratin KAP1 KRAB domain-associated protein 1 KGF Keratinocyte Growth Factor KSC Keratinocyte Stem Cell LMM Low Molecular Mass LRC Label Retaining Cell MAP2K1 Mitogen-activated protein 2 kinase 1 MAPK1 Mitogen-activated protein kinase 1 MeSC Melanocyte Stem Cell MMR MisMatch Repair MMS Methyl-MethanoSulfonate MRN MRE11-RAD50-NBS1 MSC Mesenchymal Stem Cell mtDNA mitochondrial DNA nDNA nuclear DNA NER Nucleotide Excision Repair NHEJ Non Homologous End Joining P Progenitor PARP Poly(ADP-Ribose) Polymerase PDGF Platelet Derived Growth Factor PI3K Phosphatidyl-Inositol 3-Kinase PP2A Protein phosphatase 2A PTPN11 protein tyrosine phosphatase, non-receptor type 11 Rad Roentgen absorbed dose Rem Roentgen equivalent man RNA RiboNucleic Acid ROS Reactive Oxygen Species RT-PCR Real Time Polymerase Chain Reaction SC Stem Cell SCC Squamous Cell Carcinoma SCNT Somatic Cell Nuclear Transfer SG Sebaceous Gland siRNA small interference RNA SNP Single Nucleotide Polymorphism SP Side Population SSA Single Strand Annealing SSB Single Strand Break Sv Sievert TA Transient Amplifying TGF Transforming Growth Factor TNF Tumour Necrosis Factor Trf-R Transferrin Receptor UI Upper Isthmus UV UltraViolet VEGF Vascular Endothelial Growth Factor XTT 2,3bis[2-methoxy-4-nitro-5-sulfoxyphenyl]-2H-tetrazolium 5-carrboxyanilide inner salt γ-H2AX Histone 2A variant X phosphorylated on serine 139 5-FU 5-FluoroUracil TABLE OF CONTENTS INTRODUCTION……………………………………………………………………………..……1 Chapter 1: Stem cells, the building blocks of the body…………………….………….….4 I- General features on stem cells………………………………………….………….....4 II- Embryonic stem cells………………………...………………………………..…......6 III- Human embryonic germ cells…………….……………………………………….....9 IV- Adult stem cells…...……………………………………………………………......10 V- What are similarities and differences between embryonic and adult stem cells?......17 VI- What are iPS?............................................................................................................18 VII- Stem cells therapy …………………………………………………………………19 Chapter 2: Skin, the protector shield of the body………………………………………..21 I- The skin………………………………………………………………...…………...21 II- Anatomy of the skin………………………………………………………………...22 III- Differentiation
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