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Subcellular localization-function relationship study in human antiquitin.

Chan, Chi Lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 103-127). / Abstracts in English and 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.xi / List of Tables --- p.xiii / Table of Content --- p.xiv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Classification of the aldehyde dehydrogenase superfamily --- p.1 / Chapter 1.2 --- Structures and catalytic mechanism of ALDH --- p.4 / Chapter 1.3 --- Multiple functions of ALDH --- p.8 / Chapter 1.4 --- Antiquitin - background and recent discoveries --- p.12 / Chapter 1.5 --- Aim of study --- p.19 / Chapter Chapter 2 --- Mitochondrial and Cytosolic Localizations of ALDH7A1 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- Cell culture --- p.26 / Chapter 2.2.2 --- Subcellular fractionation --- p.26 / Chapter 2.2.3 --- Western blot analysis --- p.27 / Chapter 2.2.4 --- Flow cytometric analysis of mitochondria in WRL68 cells --- p.28 / Chapter 2.2.5 --- Transient transfection of various EGFP constructs --- p.29 / Chapter 2.2.6 --- Immunofluorescence staining --- p.31 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Presence of ALDH7A1 in cytosol and mitochondria in WRL68 cells --- p.33 / Chapter 2.3.2 --- Mitochondrial-targeting N-terminal sequence in ALDH7A1 --- p.34 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- In silico and in vitro subcellular localization studies on ALDH7A1 --- p.40 / Chapter 2.4.2 --- Significance of mitochondrial and cytosolic localizations of ALDH7A1 --- p.45 / Chapter 2.4.3 --- Comparison of animal ALDH7A and plant ALDH7B enzymes --- p.48 / Chapter Chapter 3 --- "ALDH7A1: A Potential Regulator for Cell Growth, Cell Cycle and a Potential Biomarker for Cancer (Stem) Cells" / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods --- p.55 / Chapter 3.2.1 --- Cell synchronization --- p.55 / Chapter 3.2.2 --- Semi-quantitative determination of DNA amount in synchronized cells --- p.55 / Chapter 3.2.3 --- Total protein extraction --- p.55 / Chapter 3.2.4 --- Western blot analysis --- p.57 / Chapter 3.2.5 --- Immunofluorescence staining --- p.57 / Chapter 3.2.6 --- Expression and purification of ALDH7A1 and its mutant --- p.57 / Chapter 3.2.7 --- Kinetic analysis of ALDH7A1 and its mutant --- p.58 / Chapter 3.2.8 --- Generation of native ALDH7 A1 and mutant for transfection --- p.58 / Chapter 3.2.9 --- Generation of stable cell line transfectants --- p.59 / Chapter 3.2.10 --- 2D cell culture and ultra-low attachment cell culture --- p.59 / Chapter 3.2.11 --- Collection of total cell lysates --- p.60 / Chapter 3.2.12 --- Western blot analysis --- p.60 / Chapter 3.2.13 --- Growth analysis --- p.61 / Chapter 3.2.14 --- Aldefluor assay --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Expression level of ALDH7A1 at different phases of the cell cycle --- p.62 / Chapter 3.3.2 --- Subcellular distribution of ALDH7A1 in synchronized cells --- p.64 / Chapter 3.3.3 --- Changes in the expression level of key cell cycle regulators and the growth rate after ALDH7A1 knockdown --- p.68 / Chapter 3.3.4 --- Absence of catalytic activity in the purified ALDH7A1 mutant C302S --- p.68 / Chapter 3.3.5 --- Over-expression of ALDH7A1 variants in HEK293 cells --- p.73 / Chapter 3.3.6 --- Growth rates of cells overexpressing different ALDH7A1 variants --- p.73 / Chapter 3.3.7 --- Expression level of ALDH7A1 in various 2D cell types and stem-like cells --- p.76 / Chapter 3.3.8 --- Aldefluor assay on cells over-expressing different ALDH7A1 variants --- p.79 / Chapter 3.4 --- Discussion --- p.82 / Chapter 3.4.1 --- Nuclear localization of ALDH7A1 --- p.82 / Chapter 3.4.2 --- Potential role of ALDH7A1 in cell cycle --- p.86 / Chapter 3.4.3 --- Non-catalytic role of ALDH in cell growth and development --- p.86 / Chapter 3.4.4 --- Relationship between ultra-low attachment culture and stem-like cells --- p.89 / Chapter 3.4.5 --- Up-regulation of ALDHs in cancer and CSCs and the evaluation of applicability of Aldefluor assay in CSC isolation --- p.93 / Chapter 3.4.6 --- Comparison on ALDH7A1 expression level in primary and stem-like cells --- p.98 / Chapter Chapter 4 --- Future Prospects / References --- p.103

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327385
Date January 2011
ContributorsChan, Chi Lung., 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, xvi, 127 leaves : ill. (chiefly 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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