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דוקטור לפילוסופיה Doctor of Philosophy עבודת גמר )תזה( לתואר Thesis for the degree דוקטור לפילוסופיה Doctor of Philosophy מוגשת למועצה המדעית של Submitted to the Scientific Council of the מכון ויצמן למדע Weizmann Institute of Science רחובות, ישראל Rehovot, Israel מאת By גיורא וולפרט Volpert Giora תאור תפקידו של סרמיד סינטאז 2 בבריאות ובמחלות Delineating the role of ceramide synthase 2 in health and disease מנחה: :Advisor פרופ' טוני פוטרמן Prof. Tony Futerman ניסן התשע"ז April 2017 Acknowledgements As I am approaching the end of my Ph.D., I would like to thank all those who made this journey professional, educational and fun as it was. First I would like to thank my advisor Prof. Tony Futerman for introducing me to the intriguing world of sphingolipids. For knowing when to let me do science my way and when to give me a “kick in the rear-end”. For the guidance throughout endless meetings, brain storming and Skype calls. For the model of organization which I am striving to adopt, and for the opportunity to be part of an enthusiastic, supporting and open group of people that made this period very rewarding and satisfying. I would like to specially thank to Dr. Andy Klein for all of our philosophical talks about scientific and non-scientific matters, for being the perfect collaborator to produce our movies and for just being a friend when I needed it. I want to express my deep appreciation to Dr. Yael Pewzner-Jung and Tammar Joseph for the willingness to help anyone at any time, for the unconditional assistance, technical support and wise advice. I would like to thank Dr. Elad Laviad, Dr. Rotem Tidhar, Dr. Oshrit Ben-David, Dr. Natalia Santos Frreira, Iris D Zelnik, Eden Rosenfeld-Gur, Dr. Einat Vitner (Vintage), Dr. Hila Zigdon (HilaZ), Chen Yaacobi (Cheng), Shani Blumenreich (Sheng), Ayelet Vardi (Ajelet), Lital Sahar (Oligo) and Inbal Polin (Polish). Thank you for being a great research partners, co- workers and friends. To all other students in the present and the past, Dr. Modi Ali, Dr. Marton Magyeri, Dr. Ashish Saroha, Dr. Tamar Farfel-Becker, Dr. Woo-Jae Park, Dr. Soo Min Cho and Jiyoon Kim (Leah) for endless lunch breaks, discussions and research assistance. To our best collaborator, Dr. Shifra Ben-Dor, for giving me the opportunity to work with you and learn from you how one should work professionally, creatively and quickly. To my mother and late father who raised me to be curious and hard working. To my grand mothers and to my brother who try to understand what exactly I am doing in the lab. Thank you to my two lovely daughters Daphne and Abigail for letting me know what’s really important in life. And last but not least to my wife and life companion Yael (Yuli, KofT) Hollender-Volpert. Thank you for your love, support, encouragement and understanding. Thank you for making me a better person, I could not have finished this degree without you. This dissertation is dedicated to you. 2 Table of Contents 1. Abbreviations used............................................................................................................... 4 2. Abstract and main findings ................................................................................................. 5 3. Introduction .......................................................................................................................... 8 3.1. The lipid composition of eukaryotic membranes .................................................................... 8 3.2. SL biosynthesis pathway ......................................................................................................... 9 3.3. Ceramide ............................................................................................................................... 11 3.4. Ceramide as a structural unit of complex sphingolipids ....................................................... 11 3.5. The (Dihydro)ceramide synthases (CerS) family .................................................................. 12 3.6. CerS2 ..................................................................................................................................... 13 3.7. CerS transgenic mice ............................................................................................................. 14 3.8. Endocytosis – more than one way to get inside a cell ........................................................... 15 3.9. CerS in health and disease ..................................................................................................... 17 4. Research objectives ............................................................................................................ 18 5. Materials and methods ...................................................................................................... 19 5.1. Mouse models ....................................................................................................................... 19 5.2. Astrocyte culture ................................................................................................................... 19 5.3. Immunohistochemistry .......................................................................................................... 19 5.4. RNA extraction and polymerase chain reaction .................................................................... 20 5.5. CerS assays ............................................................................................................................ 21 5.6. Lipid analysis ........................................................................................................................ 21 5.7. Proliferation and cell death ................................................................................................... 21 5.8. Western blotting .................................................................................................................... 21 5.9. Uptake of fluorescently-labeled ligands ................................................................................ 22 5.10. Electron microscopy ............................................................................................................ 22 5.11. Transcription factor binding site analysis ........................................................................... 23 5.12. Chromatin immune precipitation......................................................................................... 23 5.13. Reactive oxygen species ...................................................................................................... 23 5.14. Mitochondrial complex IV activity ..................................................................................... 23 5.15. Confocal microscopy ........................................................................................................... 23 5.16. BM chimeras ....................................................................................................................... 24 5.17. SL turnover .......................................................................................................................... 24 6. Results ................................................................................................................................. 25 6.1. Characterization of primary cultured astrocytes from a CerS2 null mouse .......................... 25 6.2. Analyzing the effect of altered SL composition on endocytic pathways .............................. 28 6.3. Characterization of CerS1/CerS2 double knockout mice ...................................................... 36 6.4. Exploring the hyper-sensitivity of CerS2 null mice to DSS induced colitis ......................... 39 6.5. The involvement of CerS dimerization in glioblastoma........................................................ 41 6.6. Other collaborative projects .................................................................................................. 42 7. Discussion............................................................................................................................ 44 8. List of publications from PhD work ................................................................................. 50 9. References ........................................................................................................................... 51 10. Declaration........................................................................................................................ 62 3 1. Abbreviations used ACBP Acyl-CoA binding protein BM Bone marrow CCP/CCV Clathrin coated pit/clathrin coated vesicle CerS Ceramide synthase CHC Clathrin heavy chain ChIP Chromatin immune-precipitation CME Clathrin-mediated endocytosis DSS Dextran sulfate sodium ER Endoplasmic reticulum FASN Fatty acid synthase GBM Glioblastoma GFAP Glial fibrillary acidic protein GLTP Glycolipid transfer protein Hsc70 Heat shock cognate 70 HSF1 Heat shock factor 1 KO/ dKO Knock out/double knock out LacCer Lactosylceramide LCB Long chain base LDL Low density lipoprotein NAC N-acetyl cysteine PAH Pulmonary arterial hypertension PBS Phosphate buffer PM Plasma membrane ROS Reactive oxygen species Sa/SaP/S1P Sphinganine /sphinganine-1-phosphate/sphingosine-1-phosphate Sp1 Specificity protein 1 SLs/ VLC-SLs Sphingolipids /very long chain sphingolipids SM/ SMase Sphingomyelin/sphingomyelinase Tf Transferrin TNFR1 Tumor necrosis factor receptor 1 4 2. Abstract and main findings Ceramides with defined acyl chain lengths are involved in a number of human diseases such like multiple sclerosis and several types of cancer, yet
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