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Myriam Thesis Final UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE (ED 414) Génétique Moléculaire, Génomique, Microbiologie (GMGM) – UMR 7156 THÈSE présentée par : Myriam Sanjuán Vázquez soutenue le : 29 Janvier 2018 pour obtenir le grade de : Docteur de l’Université de Strasbourg Discipline/ Spécialité : Aspects moléculaires et cellulaires de la biologie Study of proteins implicated in centronuclear myopathies by using the model of yeast Saccharomyces cerevisiae THÈSE dirigée par : Mme FRIANT Sylvie GMGM, Université de Strasbourg RAPPORTEURS : Mme TRONCHERE Hélène Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse M. BITOUN Marc Institut de Myologie, Paris AUTRES MEMBRES DU JURY : M. LESCURE Alain Institut de Biologie Moléculaire et Cellulaire, Strasbourg Acknowledgements I am grateful to have had the opportunity of making and completing this doctoral thesis. It has been a process of both professional and personal growth. In the process, several people have participated in this achievement. I will start by respectfully thanking the members of the jury for accepting to read and evaluate my thesis work, Dr. Alain Lescure, Dr Hélène Tronchère and Dr. Marc Bitoun. I really appreciate your interest. I would like to thank Dr. Sylvie Friant for accepting me in her research team when I was then “just” an erasmus student; later she gave me the opportunity to continue my studies with a doctorate. I am grateful to Dr. Joern Putz, who as erasmus coordinator, put me in contact with Sylvie Friant to my M2S4 internship and who then informed us about the possibility of applying for an IDEX Fellowship at the University of Strasbourg. Thanks really to the University of Strasbourg for its policy of opening up to the reception of new students coming out of France and by the IDEX Fellowship which has made it possible to finance me during these 3 years and 3 months. I have very good memories of these years as student in this University and in this city so beautiful that is Strasbourg. Thanks to the members of the team. Thanks to Bruno Rinaldi, Speedy Gonzales of the team; he has always solutions for all, either for problems in the experiments at the laboratory as in experiments at home. At the beginning, I worked with him to learn the techniques in yeast cells. His good character and generosity has helped me to feel at home in the lab during these 5 years. Thanks to Johan de Craene, who although is not in the team (University of Tours has won a good part) was essential for the successful completion of my master project and my continuation on the team. I appreciated his passion for science, which he transmitted to me and I greatly enjoyed our scientific conversations in which I learned a lot with a great fascination and enthusiasm. Thanks to Séverine Bär also for her support, for your attentive listening and interest in our scientific or not discussions, she is always there to help. Thanks to other PhD students in the department, all of us have gone through the same thing and at the same time, it's been a very valuable support. Thanks to Matthieu Raess for the calm he transmits to me and for the help throughout these years, with him I have learned to take small thing less seriously and look at the positive side of things many times. Thanks to Gaétan Bader for their friendliness and their help always available at the most needed moments, I am still remembering my “mi-parcours” of thesis and my beginnings in the OpenLab project. Thanks to Romuald Loutre by his affable character and interesting conversations. Thanks to Sylvain Debard for his interest and advice in my experiments on oxidative stress. By the way, thanks to Dimitri Bertazzi, who although I saw him once and I do not know at all, I've heard and said his name more times what I can remember in last years; He defended his thesis just until I came to the laboratory and his experiments have allowed that I continue with my own. Thanks to Roberto Silva Rojas, student erasmus at the time, with whom I started the dynamin project Thanks to the rest of the people in the department, each has brought something special: Virgile Jarrige, Damien Jeandard, Anne-Marie Heckel, Hubert Becker, Bruno Senger, Laurence Huck, Frédéric Fischer, Anna Smirnova, Alexandre Smirnov, Nina Entelis, Ivan Tarassov, Benoît Masquida, Denise Stuber, Nathaniel Yakobov, Marine Hemmerle, Daphné Laporte, Yuhei Araiso, Ludovic Enkler, Aline Keilbach, Noemi Bindel. Thank you to the organizers and responsible members of the OpenLAB operation, especially Serge Potier, Laurence Marechal-Drouard, Vincent Leclerc, Stéphane Vincent et Lucile Schneider. It has been a wonderful and very rewarding experience for 2 years. It has allowed me to share the science in a practical way with the students in the high schools through Alsace. Thanks to my family and friends; the great strength that supports me and allows me to move forward. It would have been impossible to do this doctorate without them. Thanks to my grandparents who rest in peace, I keep the memory of my grandfather “calacala” saying “go into the bullring with the courage of a bullfighter, with your head high and ready to take the bull by the horns”; my grandmother “aia” who had better memory than me, and reminded me of my past achievements to encourage me in the present. Thanks to my mother, Mari Carmen, who listens to me, understands me, supports me, and who believes in me more than anybody; that is something so valuable that I never be able to give the favor back. Thanks to my father, Ricardo, who gave me the passion for understanding science and the world that surrounds us. The two are an example of perseverance. Thanks to my sisters Cristina and Paloma, from whom there is so much to learn. Thanks to Mari Cruz Colodrón, who unfortunately will not see this completed work, without her I would have never finished not even the Bachelor's degree in biochemistry; she has been essential in my life and a decisive person so that I have reached where I am now. I. INTRODUCTION .......................................................................................................................... 6 1.1. THE DIFFERENT CENTRONUCLEAR MYOPATHIES ......................................................................... 6 1.1.1. X-linked centronuclear myopathy due to mutations in the MTM1 gene ............................. 9 1.1.2. Centronuclear myopathie due to mutations in the DNM2 gene ....................................... 10 1.1.3. Myopathy due to mutations in the PYROXD1 gene .......................................................... 12 1.1.4. Centronuclear myopathy due to mutations in the BIN1 gene and in the RYR1 gene ....... 12 1.1.5. Current research for treatment for XLCNM myopathies ................................................. 13 1.1.6. Clinical data and health-care for patients suffering of CNM myopathies ....................... 15 1.1.7. The sarcomere structure and the excitation-contraction coupling mechanism of muscles .................................................................................................................................................... 16 1.2. THE THREE STUDIED PROTEINS MTM1, DNM2 AND PYROXD1. .............................................. 20 1.2.1. MTM1 and the myotubularins family of protein ............................................................... 20 1.2.2. The dynamin DNM2, a GTPase protein ........................................................................... 28 1.2.3. The oxidoreductase Pyroxd1 protein ................................................................................ 35 1.3. THE BUDDING YEAST SACCHAROMYCES CEREVISIAE, THE MODEL OF THIS STUDY ...................... 36 1.3.1. Yeast Saccharomyces cerevisiae membrane trafficking ................................................... 38 1.3.2. The yeast vacuole and classification of vacuolar defective yeast mutants ....................... 40 1.4. THE LIPID PHOSPHOINOSITIDES (PI) AS ESSENTIAL ACTORS OF MEMBRANE FUNCTIONS. ........... 43 1.4.1. The phosphoinositide PI3P, a substrat of MTM1 ............................................................. 46 1.4.2. The phosphoinositide PI(3,5)P2 , a substrat of MTM1 ..................................................... 48 1.4.3. The phosphoinositide PI5P, a product of MTM1 phosphatase activity ............................ 52 II. OBJECTIVE OF THE PHD THESIS AND BACKGROUND .............................................. 54 2.1. THE MYOTUBULARIN MTM1 AND ITS PH-GRAM DOMAIN ..................................................... 54 2.2. THE DNM2 DYNAMIN AND YEAST MITOCHONDRIA FUSION ...................................................... 56 2.2.1. Objectives of the hDNM2 study in yeast ........................................................................... 56 2.2.2. Preliminary data on the hDNM2 humanization of yeast cells .......................................... 56 2.3. THE OXYDOREDUCTASE PYROXD1 AND YEAST OXIDATIVE STRESS ....................................... 61 III. RESULTS ................................................................................................................................... 66 3.1. THE MYOTUBULARIN MTM1 AND ITS PHOSPHATASE ACTIVITY IN YEAST ................................ 66 3.1.1. Interactions between the PH/GRAM and the catalytic domain reconstitute an active myotubularin MTM1 phosphoinositide phosphatase .................................................................. 66 3.1.2. Perspectives on the MTM1 study in yeast cells ...............................................................
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