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Functional Role of Ubiquitin Proteasome System in Idiopathic Inflammatory Myopathies

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Functional Role of Ubiquitin Proteasome System in Idiopathic Inflammatory Myopathies Functional role of ubiquitin proteasome system in idiopathic inflammatory myopathies Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by SALYAN BHATTARAI from Nepal Berlin, 2016 This work was performed from October 2013 to August 2016 under the supervision of Prof. Dr. Simone Spuler and close instruction of PD Dr. med. Eugen Feist. Major part of this work were achieved at the Research Laboratory of Rheumatology in the Department of Rheumatology and Clinical Immunology, Charité Universitätsmedizin Berlin and the cell culture experiments were accomplished at the laboratory of Prof. Simone Spuler in Experimental and Clinical Research Center, Berlin. The work was funded by German Research Foundation grant DFG GRK1631 (MyoGrad). Supervisor and 1st Reviewer: Prof. Dr. med. Simone Spuler Institute of Chemistry and Biochemistry Department of Biology, Chemistry and Pharmacy Freie Universität Berlin and Muscle research unit Experimental and Clinical Research Center (ECRC) Charité Universitätsmedizin Berlin 2nd Reviewer PD Dr. med. Eugen Feist Department of Rheumatology and Clinical Immunology Charité Universitätsmedizin Berlin Date of Defense: 20th March, 2017 Acknowledgements My first sincere thanks goes to Prof. Dr. Simone Spuler and Dr. Eugen Feist for allowing me to complete my doctoral thesis under their close supervision and instruction. I am very grateful for their kind support and outstanding scientific guidance. I also thank Dr. Khetam Ghannam and Dr. Lorena Gamboa Martinez from Dr. Fiest’s laboratory for being great colleague, creating good scientific environment in the laboratory and helping me for uncountable times from the first day of my Ph.D. In addition, I really enjoyed discussing science with Dr. Ghannam which helped me to move forward with the project. I would like to thank Prof. Olivier Benveniste from La Pitié-Salpêtriére Hospital, UPMC, for the fruitful discussion and support when I encountered problems. I want to deeply thank Prof. Werner Stenzel from Department of Neuropathology, Charite Universitätsmedizin Berlin for his time, effort and valuable critical comments on the results, particularly on histology. Thank you for providing valuable patients materials for the project, without which the project could not have been success. My acknowledgement also goes to Dr. Sabine Krause from Friedrich-Baur Institute, Munich for giving us the human samples and Prof. Hermen S. Overkleeft for providing immunoproteasome inhibitors. From Prof. Spuler’s laboratory, I would like to thank Stephanie Meyer-Liesener, Dr. Andreas Marg and Adrienne Rothe for their excellent assistance on cell culture and immunofluorescence and for providing me the material when required. I would also like to thank entire team of MyoGrad for providing me an excellent platform to conduct my Ph.D. degree. Most of all, I would like to thank my family for their love, care and support for every moment of my life. Contents List of Figures .............................................................................................................................. i List of Supplementary Figures ............................................................................................... ii List of Abbreviation .................................................................................................................. iii 1. Abstract ................................................................................................................................. 1 1.1. English ........................................................................................................................................ 1 1.2. Deutsch ....................................................................................................................................... 3 2. Introduction .......................................................................................................................... 5 2.1. Skeletal muscle ......................................................................................................................... 5 2.2. Idiopathic inflammatory myopathies .................................................................................. 6 2.2.1. Polymyositis (PM) ............................................................................................................ 7 2.2.2. Sporadic inclusion body myositis (IBM) .................................................................... 7 2.2.3. Immune mediated necrotizing myopathy (IMNM) .................................................... 9 2.2.4. Dermatomyositis (DM) .................................................................................................. 10 2.3. Pathologic mechanism of IIMs ........................................................................................... 11 2.3.1. PM and sIBM pathogenesis ......................................................................................... 11 2.3.2. IMNM pathogenesis ....................................................................................................... 13 2.3.3. DM pathogenesis............................................................................................................ 13 2.4. The ubiquitin proteasome system (UPS) ......................................................................... 15 2.4.1. Proteasome degradation pathway ............................................................................. 15 2.4.2. The Proteasome complex ............................................................................................ 16 2.4.3. Immunoproteasome ...................................................................................................... 17 2.4.4. Function of immunoproteasome ................................................................................ 19 3. Background ........................................................................................................................ 24 4. Aim ........................................................................................................................................ 27 5. Materials and methods .................................................................................................... 28 5.1. Antibodies ................................................................................................................................ 28 5.2. Buffers and solution .............................................................................................................. 29 5.2.1. Western blot .................................................................................................................... 29 5.2.2. For Proteasome activity ............................................................................................... 30 5.2.3. Flow cytometry (FACS) buffer .................................................................................... 31 5.3. Primers sequence .................................................................................................................. 31 5.4. Patients and samples ............................................................................................................ 32 5.5. Human Primary myoblast culture ...................................................................................... 32 5.6. Cytokines and inhibitor treatment to cells ...................................................................... 33 5.7. Immunohistochemistry (tissue) ......................................................................................... 34 5.8. Immunofluorescence (Cells) ............................................................................................... 34 5.9. Western Blot ............................................................................................................................ 35 5.10. Oxyblot .................................................................................................................................. 36 5.11. Proteasome activity measurements .............................................................................. 36 5.12. Probe based competition assay in primary myoblast .............................................. 37 5.13. RNA isolation, reverse transcription and real time quantitative polymerase chain reaction (RT-PCR) ................................................................................................................... 38 5.14. Transfection of cells with β5i/LMP7 shRNA ................................................................ 38 5.15. Flow cytometry ................................................................................................................... 39 5.16. Statistics ............................................................................................................................... 39 6. Results ................................................................................................................................. 41 6.1. General clinical analysis ...................................................................................................... 41 6.2. Proteasome subunits expression in muscle biopsies of IIMs ................................... 42 6.3. Proteasome CT-L and C-L activities in IIMs muscle biopsies .................................... 43 6.4. Subcellular localization of proteasome subunits expression in IIMs muscle biopsies ...............................................................................................................................................
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