Proteomic analysis of polyglutamine disease in drosophila.

• Other Authors: Lam, Wun.
• Format: Others
• Language: English; Chinese
• Published: 2005
• Subjects: Nervous system > Degeneration > Etiology; Proteomics; Drosophila > Genetics; Neurodegenerative Diseases > etiology; Intranuclear Inclusion Bodies > genetics; Drosophila > genetics; Spinocerebellar Ataxias > genetics
• Online Access: http://library.cuhk.edu.hk/record=b5892385; http://repository.lib.cuhk.edu.hk/en/item/cuhk-325134

Lam Wun. === Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. === Includes bibliographical references (leaves 140-153). === Abstracts in English and Chinese. === ABSTRACT --- p.i === ACKNOWLDGEMENT --- p.iii === TABLE OF CONTENT --- p.iv === ABBREVIATIONS --- p.x === LISTS OF TABLES --- p.xi === LISTS OF FIGURES --- p.xii === Chapter 1. --- INTRODUCTION === Chapter 1.1 --- Neurodegeneration and triplet repeat diseases --- p.1 === Chapter 1.2 --- Polyglutamine diseases --- p.2 === Chapter 1.3 --- Polyglutamine nuclear inclusions --- p.4 === Chapter 1.3.1 --- Kinetics of polyglutamine nuclear inclusion formation --- p.4 === Chapter 1.3.2 --- Roles of protein inclusions in neurodegeneration --- p.7 === Chapter 1.4 --- Polyglutamine pathogenic pathways --- p.8 === Chapter 1.4.1 --- Protein depletion theory --- p.9 === Chapter 1.4.2 --- Induction of apoptotic pathways --- p.13 === Chapter 1.5 --- Previous study on NI proteins --- p.14 === Chapter 1.6 --- Drosophila model for studying polyglutamine diseases --- p.15 === Chapter 1.6.1 --- Drosophila model for studying human diseases --- p.15 === Chapter 1.6.2 --- GAL4/UAS gene expression system --- p.15 === Chapter 1.6.3 --- Drosophila polyglutamine models --- p.17 === Chapter 1.7 --- Objectives of the study --- p.21 === Chapter 2. --- MATERIALS AND METHODS === Chapter 2.1 --- Drosophila genetics --- p.22 === Chapter 2.1.1 --- Drosophila culture --- p.22 === Chapter 2.1.2 --- GAL4/UAS gene expression system --- p.22 === Chapter 2.1.3 --- Eye phenotypic analysis --- p.25 === Chapter 2.1.4 --- Polyglutamine fly models --- p.25 === Chapter 2.1.5 --- Generation and characterization of GFP-polyglutamine transgenic fly models --- p.25 === Chapter 2.2 --- Proteomic identification of nuclear inclusion proteins --- p.26 === Chapter 2.2.1 --- Proteomic identification of NI proteins by SDS-insolubility of NIs --- p.26 === Chapter 2.2.2 --- Proteomic identification of NI proteins by FA-solubility of NIs --- p.27 === Chapter 2.2.2.1 --- Approach overview --- p.27 === Chapter 2.2.2.2 --- Sample preparation for two-dimensional gel electrophoresis --- p.27 === Chapter 2.2.2.3 --- Two-dimensional gel electrophoresis --- p.29 === Chapter 2.2.2.4 --- Polyacrylamide gel staining --- p.31 === Chapter 2.2.2.5 --- Computer analysis of 2D patterns --- p.31 === Chapter 2.2.2.6 --- In-gel trypsin digestion --- p.32 === Chapter 2.2.2.7 --- Mass spectrometric analysis --- p.33 === Chapter 2.2.3 --- Detection of NIs by flow cytometry --- p.34 === Chapter 2.3 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.34 === Chapter 2.3.1 --- Sample preparation for SDS-PAGE --- p.34 === Chapter 2.3.2 --- SDS-PAGE --- p.35 === Chapter 2.4 --- Immunodetection --- p.36 === Chapter 2.4.1 --- Electroblotting --- p.36 === Chapter 2.4.2 --- Western blotting --- p.36 === Chapter 2.4.3 --- Filter trap assay --- p.37 === Chapter 2.5 --- Sav antibody production --- p.38 === Chapter 2.5.1 --- Sav peptide synthesis --- p.38 === Chapter 2.5.2 --- Rabbit immunization --- p.38 === Chapter 2.6 --- Cryosectioning and immunostaining of adult fly heads --- p.39 === Chapter 2.7 --- Alcohol dehydrogenase assay --- p.40 === Chapter 2.8 --- Semi-quantitative reverse transcription- Polymerase Chain Reaction --- p.41 === Chapter 2.8.1 --- Total RNA preparation from fly heads --- p.41 === Chapter 2.8.2 --- Reverse transcription- Polymerase Chain Reaction (RT-PCR) --- p.41 === Chapter 2.9 --- Reagents and buffers --- p.42 === Chapter 3. --- RESULTS === Chapter 3.1 --- Transgenic polyglutamine fly models --- p.48 === Chapter 3.1.1 --- Characteristics of MJD polyglutamine fly model --- p.48 === Chapter 3.1.1.1 --- Overexpression of expanded truncated human MJD proteins in Drosophila causes eye degeneration --- p.49 === Chapter 3.1.1.2 --- Overexpression of expanded truncated human MJD proteins in Drosophila results in nuclear inclusion formation --- p.49 === Chapter 3.1.1.3 --- Formic acid dissolves fly polyglutamine nuclear inclusions --- p.51 === Chapter 3.1.1.3.1 --- Formic acid dissolves fly polyglutamine NIs as shown by Western blot analysis --- p.51 === Chapter 3.1.1.3.2 --- Formic acid dissolves fly polyglutamine NIs as shown by filter trap assay --- p.53 === Chapter 3.1.2 --- Summary --- p.55 === Chapter 3.2 --- Proteomic identification of nuclear inclusion (NI) proteins --- p.56 === Chapter 3.2.1 --- Proteomic identification of NI proteins by SDS-insolubility of NIs --- p.56 === Chapter 3.2.2 --- Proteomic identification of NI proteins by FA-solubility of NIs --- p.63 === Chapter 3.2.2.1 --- Two-dimensional gels showing differential protein spots as potential NI proteins --- p.63 === Chapter 3.2.2.2 --- NI protein candidates identified by the 2D approach --- p.75 === Chapter 3.2.3 --- Study of polyglutamine NI proteins by flow cytometry analysis --- p.90 === Chapter 3.2.3.1 --- Detection of fly polyglutamine NIs by flow cytometry --- p.90 === Chapter 3.2.3.2 --- Characterization of a new GFP-polyglutamine fly model --- p.92 === Chapter 3.3 --- Characterization of the nuclear inclusion protein candidates --- p.96 === Chapter 3.3.1 --- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) --- p.96 === Chapter 3.3.1.1 --- Confirmation of GAPDH as a NI protein --- p.97 === Chapter 3.3.1.2 --- Discussion --- p.97 === Chapter 3.3.2 --- Receptor of activated protein kinase C (RACK1) --- p.99 === Chapter 3.3.2.1 --- Confirmation of RACK1 as a NI protein --- p.99 === Chapter 3.3.2.1.1 --- Colocalization of RACK1 with NIs --- p.99 === Chapter 3.3.2.1.2 --- Formic Acid extracts RACK1 from NIs --- p.101 === Chapter 3.3.2.2 --- Reduction of soluble RACK1 protein level in polyglutamine fly --- p.101 === Chapter 3.3.2.2.1 --- Soluble RACK1 protein level reduced in polyglutamine fly --- p.101 === Chapter 3.3.2.2.2 --- RACK1 transcript level remains unchanged in polyglutamine fly --- p.103 === Chapter 3.3.2.3 --- Overexpression of RACK 1 partially suppresses polyglutamine degeneration --- p.105 === Chapter 3.3.2.4 --- Discussion --- p.107 === Chapter 3.3.3 --- Warts (Wts) --- p.111 === Chapter 3.3.3.1 --- Overexpression of Wts partially suppresses polyglutamine degeneration --- p.111 === Chapter 3.3.3.2 --- Wts mutant slightly enhances polyglutamine degeneration --- p.113 === Chapter 3.3.3.3 --- Genetic analysis of Warts pathway in polyglutamine pathogenesis --- p.113 === Chapter 3.3.3.3.1 --- Overexpression of Salvador partially suppresses polyglutamine degeneration --- p.116 === Chapter 3.3.3.3.2 --- Hpo mutant slightly enhances polyglutamine degeneration --- p.119 === Chapter 3.3.3.3.3 --- Overexpression of DIAP1 partially suppresses polyglutamine degeneration --- p.119 === Chapter 3.3.3.4 --- Discussion --- p.121 === Chapter 3.3.4 --- Alcohol dehydrogenase (Adh) --- p.122 === Chapter 3.3.4.1 --- Adh activity is reduced in polyglutamine flies --- p.122 === Chapter 3.3.4.2 --- Overexpression of Hsp70 partially restores the reduced Adh activity in polyglutamine flies --- p.122 === Chapter 3.3.4.3 --- Discussion --- p.125 === Chapter 3.3.5 --- Genetic analysis of other NI protein candidates --- p.127 === Chapter 3.3.5.1 --- Overexpression of CG7920 protein partially suppresses polyglutamine degeneration --- p.127 === Chapter 3.3.5.2 --- Pten dsRNA slightly enhances polyglutamine degeneration --- p.129 === Chapter 3.3.6 --- Summary --- p.131 === Chapter 4. --- DISSCUSSION === Chapter 4.1 --- Protein depletion theory --- p.133 === Chapter 4.2 --- Comparison of different approaches for identification of NI proteins --- p.134 === Chapter 4.3 --- Long-term significance --- p.136 === Chapter 4.4 --- Future studies --- p.137 === Chapter 4.4.1 --- Characterization of other NI protein candidates --- p.137 === Chapter 4.4.2 --- Study of NI proteins by an alternative approach --- p.137 === Chapter 4.4.3 --- Study of NI proteins using other polyglutamine fly models --- p.137 === Chapter 5. --- CONCLUSION --- p.139 === Chapter 6. --- REFERENCES --- p.140