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Characterization of human antiquitin: structural and functional analyses.

Wong, Chun Pong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 130-146). / Abstract also in Chinese. / Thesis Assessment Committee --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / 摘要 --- p.iv / Abstract --- p.vi / List of Abbreviations --- p.viii / List of Figures --- p.xii / List of Tables --- p.xiv / Content --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Classification of aldehyde dehydrogenase --- p.1 / Chapter 1.2 --- Structure and catalytic mechanism of aldehyde dehydrogenase --- p.4 / Chapter 1.3 --- Multiple functions of aldehyde dehydrogenase --- p.11 / Chapter 1.4 --- Background of antiquitin --- p.13 / Chapter 1.5 --- Aim of study --- p.24 / Chapter Chapter 2 --- Structural Analysis of Human Antiquitin --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.30 / Chapter 2.2.1 --- Subcloning and expression of human antiquitin and its mutants --- p.30 / Chapter 2.2.2 --- Purification of human antiquitin and its mutants --- p.31 / Chapter 2.2.3 --- Kinetic properties of human antiquitin and its mutants --- p.32 / Chapter 2.2.4 --- Inhibitor studies of human antiquitin --- p.33 / Chapter 2.2.5 --- X-ray crystallography of human antiquitin ternary complex --- p.34 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- "Subcloning, expression and purification of human antiquitin and its mutants" --- p.36 / Chapter 2.3.2 --- Kinetic properties of human antiquitin and its mutants --- p.41 / Chapter 2.3.3 --- Inhibitor studies of human antiquitin --- p.44 / Chapter 2.3.4 --- X-ray crystallography of human antiquitin ternary complex --- p.47 / Chapter 2.4 --- Discussion --- p.56 / Chapter 2.4.1 --- Substrate specificity of recombinant human antiquitin --- p.56 / Chapter 2.4.2 --- Pyridoxine-dependent seizures and mutations in human antiquitin gene --- p.63 / Chapter 2.4.3 --- X-ray crystallography of human antiquitin ternary complex --- p.76 / Chapter Chapter 3 --- Functional Analysis of Human Antiquitin --- p.79 / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Materials and Methods --- p.83 / Chapter 3.2.1 --- Cell culture --- p.83 / Chapter 3.2.2 --- Transfection of HEK293 cells with siRNA --- p.83 / Chapter 3.2.3 --- Total protein extraction --- p.84 / Chapter 3.2.4 --- Total RNA extraction --- p.85 / Chapter 3.2.5 --- Real-time PCR assay --- p.86 / Chapter 3.2.6 --- Stress responsiveness of transfected HEK293 cells --- p.87 / Chapter 3.2.7 --- Cell growth analysis of transfected HEK293 cells --- p.87 / Chapter 3.2.8 --- Cell cycle profile analysis of transfected HEK293 cells --- p.88 / Chapter 3.2.9 --- Programmed cell death analysis of transfected HEK293 cells --- p.89 / Chapter 3.2.10 --- Confocal immunofluorescence microscopic analysis of transfected HEK293 cells --- p.89 / Chapter 3.2.11 --- Subcellular fractionation of transfected HEK293 cells --- p.90 / Chapter 3.2.12 --- Western blot analysis of transfected HEK293 cells --- p.90 / Chapter 3.3 --- Results --- p.93 / Chapter 3.3.1 --- Condition optimization for siRNA transfection in HEK293 cells --- p.93 / Chapter 3.3.2 --- Knock down of human antiquitin at protein and mRNA levels in HEK293 cells --- p.93 / Chapter 3.3.3 --- Stress responsiveness of transfected HEK293 cells --- p.99 / Chapter 3.3.4 --- Cell growth in transfected HEK293 cells --- p.102 / Chapter 3.3.5 --- Cell cycle profile analysis of transfected HEK293 cells --- p.107 / Chapter 3.3.6 --- Western blot analysis of cell cycle regulatory proteins of transfected HEK293 cells --- p.107 / Chapter 3.3.7 --- Programmed cell death analysis of transfected HEK293 cells --- p.111 / Chapter 3.3.8 --- Confocal immunofluorescence microscopic analysis of transfected HEK293 cells --- p.113 / Chapter 3.3.9 --- Subcellular fractionation of transfected HEK293 cells --- p.116 / Chapter 3.4 --- Discussion --- p.118 / Chapter 3.4.1 --- Lack of response of human antiquitin towards hyperosmotic stress --- p.118 / Chapter 3.4.2 --- Involvement of human antiquitin in cell growth --- p.119 / Chapter 3.4.3 --- Subcellular localization of human antiquitin --- p.124 / Chapter 3.4.4 --- Study of physiological function of human antiquitin using siRNA technique --- p.125 / Chapter Chapter 4 --- Future Prospects --- p.128 / References --- p.130

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326706
Date January 2009
ContributorsWong, Chun Pong., Chinese University of Hong Kong Graduate School. Division of Life Sciences.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xviii, 146 leaves : ill. (some col.) ; 30 cm.
RightsUse 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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