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

Chan, King Lun Michel. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 111-121). / Abstracts in English and Chinese. / THESIS ASSESSMENT COMMITTEE --- p.i / ACKNOWLEDGEMENTS --- p.ii / 摘要 --- p.iv / ABSTRACT --- p.v / LIST OF ABBREVIATIONS --- p.xi / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Aldehyde Dehydrogenase Superfamily / Chapter 1.1.1 --- Classification and Substrate Specificities of Aldehyde Dehydrogenases --- p.1 / Chapter 1.1.2 --- Multiple Functions of Aldehyde Dehydrogenases and their Roles in Metabolism --- p.4 / Chapter 1.1.3 --- Structural Organization of Aldehyde Dehydrogenases in view of their Catalytic Mechanism --- p.7 / Chapter 1.2 --- Antiquitin / Chapter 1.2.1 --- Discovery and Plant Antiquitins --- p.15 / Chapter 1.2.2 --- Animal Antiquitins --- p.17 / Chapter 1.2.3 --- Human Antiquitin Gene Mutations and Pyridoxine-dependent Seizures --- p.19 / Chapter 1.2.4 --- Previous Findings on Seabream Antiquitin --- p.20 / Chapter 1.3 --- Aims of Study --- p.22 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials / Chapter 2.1.1 --- "Subcloning, Expression and Purification of Human Antiquitin" --- p.24 / Chapter 2.1.2 --- Characterization of Human Antiquitin --- p.25 / Chapter 2.1.3 --- "Crystallization of Human Antiquitin, Diffraction Data Collection and Structure Determination" --- p.26 / Chapter 2.1.4 --- Mutational Studies of Human Antiquitin --- p.26 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- "Subcloning, Expression and Purification of Human Antiquitin" / Chapter 2.2.1.1 --- Subcloning of the Full-length Human Antiquitin cDNA --- p.27 / Chapter 2.2.1.2 --- Bacterial Expression of Recombinant Human Antiquitin --- p.29 / Chapter 2.2.1.3 --- Purification of Human Antiquitin --- p.30 / Chapter 2.2.2 --- Characterization of Human Antiquitin / Chapter 2.2.2.1 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.31 / Chapter 2.2.2.2 --- Size-exclusion Chromatography - Multi-angle Light Scattering --- p.32 / Chapter 2.2.2.3 --- Isoelectric Focusing --- p.33 / Chapter 2.2.2.4 --- pH-rate Profile --- p.33 / Chapter 2.2.2.5 --- Stability Studies --- p.34 / Chapter 2.2.2.6 --- Substrate Specificity Study --- p.35 / Chapter 2.2.3 --- "Crystallization of Human Antiquitin, Diffraction Data Collection and Structure Determination" / Chapter 2.2.3.1 --- Crystallization of Human Antiquitin --- p.36 / Chapter 2.2.3.2 --- Diffraction Data Collection and Model Building --- p.37 / Chapter 2.2.4 --- Mutational Studies of Human Antiquitin / Chapter 2.2.4.1 --- Preparation of Mutant Plasmids --- p.39 / Chapter 2.2.4.2 --- "Expression, Purification and Kinetics Studies of Mutants" --- p.39 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- "Subcloning, Expression and Purification of Human Antiquitin" --- p.43 / Chapter 3.2 --- Characterization of Human Antiquitin --- p.49 / Chapter 3.3 --- "Crystallization of Human Antiquitin, Diffraction Data Collection and Structure Determination" / Chapter 3.3.1 --- "Crystallization, Data Collection and Refinement" --- p.59 / Chapter 3.3.2 --- Human Antiquitin Structure --- p.63 / Chapter 3.4 --- Mutational Studies of Human Antiquitin --- p.75 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- Characterization and Substrate Specificity of Recombinant Human Antiquitin --- p.83 / Chapter 4.2 --- Crystallization and Crystal Structure of Human Antiquitin / Chapter 4.2.1 --- Crystallization --- p.86 / Chapter 4.2.2 --- Overall structure --- p.86 / Chapter 4.2.3 --- Cofactor binding --- p.88 / Chapter 4.2.4 --- Substrate Binding and Catalysis --- p.91 / Chapter 4.3 --- Mutations in Human Antiquitin Gene and their Relationship with Pyridoxine-dependent Seizures --- p.95 / Chapter 4.4 --- Comparison of Antiquitin Gene and Other Aldehyde Dehydrogenases --- p.104 / Chapter CHAPTER 5 --- FUTURE PROSPECTS --- p.106 / LIST OF REFERENCES --- p.111 / APPENDIX --- p.122

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [129]-135).

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

The study of Alzheimer’s Disease (AD) has been hindered by the absence of animal models of late-onset/age-related AD (also termed sporadic AD) (95% of AD cases) since current transgenic mouse models exhibit pathological changes dependent on overexpression of mutant human genes linked to early-onset, familial AD (5% of cases). Oxidative stress is considered to be a causative factor in age-related AD, and we have found that aldehyde dehydrogenase 2 (Aldh2) null mice exhibit not only oxidative stress, but also display many AD-like pathologies. The current study used behavioral analysis to assess whether Aldh2-/- mice also exhibit memory and cognition deficits. Male and female wild type and Aldh2-/- mice were tested monthly beginning at three months of age, using the open field novel object recognition test (a measure of recognition memory), as well as spontaneous alternations in the Y-maze (a measure of spatial working memory). In both tasks, significant decreases in performance occurred in Aldh2-/- mice by 3.5-4 months of age, and this progressively declined over the next three months compared to wild type mice. Sex-related differences in memory impairment were not observed. These results, together with the findings that AD-like pathologies are also present, suggest that Aldh2-/- mice represent a new, oxidative stress-based model of age-related cognitive impairment and AD. This model may prove useful both for assessing AD therapeutics and for gaining better insight into the pathogenesis of AD. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2013-09-26 11:20:00.023

Organic nitrates such as glyceryl trinitrate (GTN) are commonly used to treat myocardial ischemia and congestive heart failure. GTN is proposed to act as a prodrug that requires bioactivation for pharmacological activity. However, continuous administration results in tolerance development, limiting its clinical usefulness. Aldehyde dehydrogenase 2 (ALDH2) has been proposed to be the primary enzyme responsible for GTN bioactivation, and ALDH2 inactivation has been proposed as the sole basis of nitrate tolerance. In the present study, we utilized an in vivo GTN tolerance model to investigate the role of ALDH2 in GTN bioactivation and tolerance. We assessed changes in ALDH2 protein, mRNA and activity levels in rat blood vessels during chronic GTN exposure (0.4 mg/hr for 6, 12, 24 and 48 hr) in relation to changes in vasodilator responses to GTN. A time-dependent decrease in both ALDH2 expression and activity occurred (80% in tolerant veins and 30% in tolerant arteries after 48 hrs exposure to GTN), concomitant with decreased vasodilator responses to GTN. However, after a 24 hr drug-free period following 48 hr GTN exposure, the vasodilator responses to GTN had returned to control values, whereas ALDH2 expression and activity were still markedly depressed. The dissociation between reduced ALDH2 activity and expression, and the duration of the impaired vasodilator responses to GTN in nitrate-tolerant blood vessels, suggest factors other than changes in ALDH2-mediated GTN bioactivation contribute to nitrate tolerance. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-10-03 16:14:49.313

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by Choy Ka-Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 107-125). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / 論文摘要 --- p.II / ABSTRACT --- p.III / ABBREVIATIONS --- p.V / TABLE OF CONTENTS --- p.VI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- PURIFICATION OF GRASS CARP ALDH FROM MITOCHONDRIA --- p.18 / Chapter CHAPTER 3 --- PURIFICATION & CHARACTERIZATION OF GRASS CARP ALDH --- p.49 / Chapter CHAPTER 4 --- CONCLUSION --- p.104 / REFERENCES --- p.107