The role of polyglutamine oligomer in pathogenesis of polyglutamine diseases.

• Other Authors: Wu, Chi Chung.
• Format: Others
• Language: English; Chinese
• Published: 2010
• Subjects: Nervous system > Degeneration > Pathophysiology; Oligomers; Neurodegenerative Diseases > physiopathology; Polymers
• Online Access: http://library.cuhk.edu.hk/record=b5894444; http://repository.lib.cuhk.edu.hk/en/item/cuhk-327107

Wu, Chi Chung. === "September 2010." === Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. === Includes bibliographical references (leaves 86-96). === Abstracts in English and Chinese. === Abstract --- p.i === Abstract (Chinese version) --- p.iii === Acknowledgments --- p.iv === List of Abbreviations --- p.v === List of Tables --- p.vii === List of Figures --- p.viii === Chapter 1. --- INTRODUCTION === Chapter 1.1. --- Neurodegenerative disorders 一 a brief overview --- p.1 === Chapter 1.2. --- Polyglutamine diseases --- p.1 === Chapter 1.3. --- Polyglutamine protein conformers and toxicity --- p.5 === Chapter 1.4. --- in vivo modeling of polyglutamine diseases in Drosophila === Chapter 1.4.1. --- GAL4/UAS transgene expression system in Drosophila --- p.13 === Chapter 1.4.2. --- Temporal control of transgene expression systemin Drosophila --- p.15 === Chapter 1.4.3. --- Drosophila as a model to study polyglutamine diseases --- p.16 === Chapter 1.5. --- in vitro polyglutamine diseases models --- p.19 === Chapter 1.6. --- Aim of study --- p.23 === Chapter 2. --- MATERIALS AND METHODS === Chapter 2.1. --- Drosophila culture and manipulation === Chapter 2.1.1. --- Drosophila culture --- p.25 === Chapter 2.1.2. --- Pseudopupil assay of adult retinal degeneration --- p.25 === Chapter 2.2. --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) === Chapter 2.2.1. --- Protein extraction from adult Drosophila heads --- p.26 === Chapter 2.2.2. --- Preparation of SDS-polyacrylamide gel and electrophoresis --- p.27 === Chapter 2.2.3. --- Western blotting --- p.28 === Chapter 2.2.4. --- Immunodetection --- p.29 === Chapter 2.3. --- Solubilization of SDS-insoluble protein --- p.31 === Chapter 2.4. --- Filter retardation assay --- p.31 === Chapter 2.5. --- Immunoprecipitation --- p.32 === Chapter 2.6. --- Nucleocytoplasmic fractionation --- p.33 === Chapter 2.7. --- PCR cloning === Chapter 2.7.1 . --- Drosophila DNA preparation --- p.34 === Chapter 2.7.2. --- Construction of pGEX4T3-MJDflQ27/81 expression plasmid --- p.34 === Chapter 2.8. --- in vitro aggregation assay === Chapter 2.8.1. --- Expression and purification of GST-MJDAQ27/81 protein --- p.36 === Chapter 2.8.2. --- in vitro aggregation --- p.37 === Chapter 2.8.3. --- Native slot-blot --- p.38 === Chapter 2.9. --- Reagents and buffers === Chapter 2.9.1. --- Reagents for Drosophila culture --- p.39 === Chapter 2.9.2. --- Reagents for SDS-PAGE --- p.39 === Chapter 2.9.3. --- Reagents for filter retardation assay --- p.42 === Chapter 2.9.4. --- Reagents for immunoprecipitation --- p.43 === Chapter 2.9.5. --- Reagents for nucleocytoplasmic fractionation --- p.43 === Chapter 2.9.6. --- Reagents for PCR cloning --- p.44 === Chapter 2.9.7. --- Reagents for in vitro aggregation assay --- p.46 === Chapter 3. --- Establishment of a GAL80ts-mediated transgenic Drosophila model of Machado-Joseph Disease (MJD) === Chapter 3.1. --- Introduction --- p.48 === Chapter 3.2. --- Results === Chapter 3.2.1. --- GAL80ts-mediated expression of expanded full-length MJD protein caused progressive neuronal degenerationin Drosophila --- p.49 === Chapter 3.2.2. --- Detection of SDS-insoluble expanded full-length MJD protein and its correlation with neuronal degeneration === Chapter 3.2.2.1. --- Progressive neuronal degeneration is not mediated by progressive accumulation of expanded full-length MJD protein --- p.51 === Chapter 3.2.2.2. --- SDS-soluble expanded full-length MJD protein does not correlate with progressive neuronal degeneration --- p.53 === Chapter 3.2.2.3. --- Progressive accumulation of SDS-insoluble expanded full-length MJD protein correlate with progressive neuronal degeneration --- p.55 === Chapter 3.3. --- Discussion --- p.57 === Chapter 4. --- Detection of conformational changes of expanded full-length MJD protein and its association with neuronal degeneration === Chapter 4.1. --- Introduction --- p.60 === Chapter 4.2. --- Results === Chapter 4.2.1. --- Expanded full-length MJD protein underwent conformational changes from monomer to fibrils and such conformational changes correlated with neuronal degeneration --- p.61 === Chapter 4.2.2. --- Mechanistic studies of how conformational changes of expanded full-length MJD protein triggers neuronal degeneration === Chapter 4.2.2.1. --- Expanded full-length MJD protein gradually accumulated in the nucleus during the course of neurodegeneration --- p.62 === Chapter 4.2.2.2. --- Fibrillar expanded full-length MJD protein caused transcriptional dysregulation of endogenous Hsp70 gene --- p.66 === Chapter 4.2.3. --- Consolidation of the role of fibrillar expanded full-length MJD protein in neuronal degeneration --- p.67 === Chapter 4.3. --- Discussion --- p.72 === Chapter 5. --- Attempts to generate new conformation-specific antibody against recombinant expanded full-length MJD proteins === Chapter 5.1. --- Introduction --- p.75 === Chapter 5.2. --- Results === Chapter 5.2.1. --- Recombinant expanded full-length MJD protein underwent conformational changes during in vitro aggregation --- p.75 === Chapter 5.3. --- Discussion --- p.77 === Chapter 6. --- GENERAL DISCUSSION --- p.81 === Chapter 7. --- CONCLUSION --- p.84 === Chapter 8. --- REFERENCES --- p.86