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PARK15) in Neurons Functional analysis of the parkinsonism-associated protein FBXO7 (PARK15) in neurons Ph.D. Thesis in partial fulfillment of the requirements for the award of the degree "Doctor rerum naturalium" in the Neuroscience Program at the Georg-August-Universität Göttingen Faculty of Biology Submitted by Guergana Ivanova Dontcheva born in Gabrovo, Bulgaria Aachen 2017 Functional analysis of the parkinsonism-associated protein FBXO7 (PARK15) in neurons Ph.D. Thesis in partial fulfillment of the requirements for the award of the degree "Doctor rerum naturalium" in the Neuroscience Program at the Georg-August-Universität Göttingen Faculty of Biology Submitted by Guergana Ivanova Dontcheva born in Gabrovo, Bulgaria Aachen 2017 Members of the Thesis Committee: P.D. Dr. Judith Stegmüller, Reviewer Department of Cellular and Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany Department of Neurology, University Hospital, RWTH Aachen, Germany Prof. Dr. Anastassia Stoykova, Reviewer Department of Molecular Developmental Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Prof. Dr. Nils Brose Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany Date of submission: 03 May, 2017 Date of oral examination: 23 June, 2017 Affidavit I hereby declare that this Ph.D. Thesis entitled "Functional analysis of the parkinsonism-associated protein FBXO7 (PARK15) in neurons" has been written independently with no external sources or aids other than quoted. Guergana Dontcheva April 2017 Aachen, Germany Table of Contents Table of Contents ................................................................................................................................................................. i List of Figures ...................................................................................................................................................................... iii List of Tables........................................................................................................................................................................ iv Abstract................................................................................................................................................................................. 5 1. Introduction.................................................................................................................................................. 6 1.1. Parkinson's Disease..................................................................................................................................... 6 1.2. The Parkinsonian pyramidal syndrome ................................................................................................... 7 1.3. Fbxo7 and FBXO7......................................................................................................................................... 9 1.3.1. FBXO7 functions in cell cycle and proteostasis...................................................................................... 9 1.3.2. Function of FBXO7 in proteasomal stability .........................................................................................12 1.3.3. Functions of FBXO7 in mitophagy: the PINK1, Parkin and FBXO7 interaction ...............................13 1.4. The Cytoskeleton: stability and dynamics.............................................................................................15 1.4.1. The MAP1B family .....................................................................................................................................16 1.4.2. The role of MAP1B LC1 in microtubular transport..............................................................................17 1.4.3. The Mitochondria-UPS relationship in neurodegenerative disorders.............................................18 1.5. Aim of the study ........................................................................................................................................19 2. Materials and Methods ............................................................................................................................20 2.1. Materials .....................................................................................................................................................20 2.1.1. Laboratory Equipment .............................................................................................................................20 2.1.2. Reagents, Chemicals and Kits..................................................................................................................20 2.1.3. Antibodies...................................................................................................................................................21 2.1.4. Enzymes ......................................................................................................................................................22 2.1.5. Solutions, Media and Buffers ..................................................................................................................23 2.2. Methods......................................................................................................................................................25 2.2.1. Molecular cloning......................................................................................................................................25 i. Molecular cloning of the MAP1S LC expression plasmids ................................................................ 25 ii. Bacterial transformation, inoculation and plasmid verification ....................................................... 27 iii. Molecular cloning of vector-based RNA interference oligonucleotides .......................................... 28 2.2.2. Immortalized cell lines maintenance and transfection ......................................................................29 i. HEK 293T cells passaging: ................................................................................................................. 29 ii. HEK 293T cell transfection: ............................................................................................................... 29 iii. SH-SY5Y cells passaging:.................................................................................................................... 29 iv. SH-SY5Y transfection:........................................................................................................................ 30 2.2.3. Primary cortical cell culture generation, maintenance and transfection .......................................30 i. Primary cortical culture generation .................................................................................................. 30 ii. Primary cortical culture transfection ................................................................................................ 31 2.2.4. Immunocytochemistry .............................................................................................................................31 2.2.5. Axonal and total dendritic length analysis............................................................................................31 2.2.6. Biochemical Methods ...............................................................................................................................32 i. Cell lysis ............................................................................................................................................. 32 ii. Protein concentration determination............................................................................................... 32 iii. Co-immunoprecipitation................................................................................................................... 32 iv. Ubiquitination assay/ Interaction strength assay ............................................................................. 33 v. Quantitative mass spectrometry sample preparation ..................................................................... 33 vi. SDS-PAGE and Western Blotting ....................................................................................................... 34 i fl/fl 2.2.7. NEX-Cre;Fbxo7 mouse line generation and genotyping ................................................................35 i. Generation of NEX-Cre;Fbxo7fl/fl transgenic mice ............................................................................ 35 ii. Isolation of genomic DNA ................................................................................................................. 35 iii. Genotyping........................................................................................................................................ 36 2.2.8. Immunohistochemistry ............................................................................................................................37 i. Transcardial perfusion and fixation of mouse brains ....................................................................... 37 ii. Post-fixation paraffin embedding and sectioning............................................................................. 37 iii. Deparaffinization, rehydration and antigen retrie val....................................................................... 38 iv. Immunohistological staining of tissues embedded in paraffin......................................................... 38 v. Nuclear
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