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

Indiana University-Purdue University Indianapolis (IUPUI) / The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.