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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327107 |
| Date | January 2010 |
| Contributors | Wu, Chi Chung., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, viii, [4], 96 leaves : ill. (chiefly col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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