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

Wong Wei-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 221-242). / Abstracts in English and Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / Abstract in Chinese --- p.iv / Abstract in English --- p.vi / List of abbreviations --- p.viii / List of figures --- p.xi / List of tables --- p.xv / Content: --- p.xvi / General introduction --- p.1 / Aldehyde dehydrogenase superfamily --- p.3 / Background of antiquitin --- p.5 / Plant antiqutins (ALDH7B) --- p.5 / Animal antiquitins (ALDH7A) --- p.8 / Human antiquitin information on NCBI --- p.14 / Rationale of studying the inducibility of annquitin and overexpression of it in HEK293 and HepG2 cells --- p.16 / Flowchart 1 Procedure of antiquitin expression studies in the HEK293 and HepG2 cells under stress --- p.19 / Flowchart 2 Procedure to study antiquitin expression in the HEK293 and HepG2 cells after in silico promoter search --- p.20 / Flowchart 3 Procedure to study antiquitin overexpressed HEK293 and HepG2 cells --- p.21 / Chapter Chapter 1 --- Inducibility of antiquitin in the HEK293 and HepG2 cells under hyperosmotic stress / Chapter 1.1 --- Introduction --- p.22 / Chapter 1.1.1 --- Cellular response to hyperosmotic stress --- p.22 / Chapter 1.1.2 --- Methods to study the responses of cells under hyperosmotic stress --- p.24 / Chapter 1.2 --- Materials --- p.26 / Chapter 1.2.1 --- Cell culture media --- p.26 / Chapter 1.2.2 --- Buffers for RNA use --- p.26 / Chapter 1.2.3 --- Buffers for DNA use --- p.27 / Chapter 1.2.4 --- Other chemicals --- p.27 / Chapter 1.3 --- Methods --- p.28 / Chapter 1.3.1 --- Culture of HEK293 and HepG2 cells --- p.28 / Chapter 1.3.2 --- Hyperosmotic stress on HEK293 and HepG2 cells --- p.29 / Chapter 1.3.3 --- MTT assay --- p.29 / Chapter 1.3.4 --- Total RNA extraction --- p.30 / Chapter 1.3.5 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.30 / Chapter 1.3.6 --- Polymerase chain reaction (PCR) --- p.31 / Chapter 1.3.7 --- Quantification of PCR products --- p.31 / Chapter 1.3.8 --- Statistical analysis --- p.33 / Chapter 1.4 --- Results --- p.34 / Chapter 1.4.1 --- Viability of HEK293 and HepG2 cells under hyperosmotic stress --- p.34 / Chapter 1.4.2 --- Validation of RNA quality --- p.34 / Chapter 1.4.3 --- Validation and determination of PCR conditions --- p.40 / Chapter 1.4.4 --- Inducibility of antiquitin in HEK293 cells under hyperosmotic stress / Chapter 1.4.5 --- Inducibility of antiquitin in HepG2 cells under hyperosmotic stress --- p.43 / Chapter 1.4.6 --- Inducibility of aldose reductase under hyperosmotic stress --- p.43 / Chapter Chapter 2 --- "In silico studies of human antiquitin promoter, genomics sequences and open reading frame" --- p.54 / Chapter 2.1 --- Introduction --- p.54 / Chapter 2.1.1 --- Eukaryotic promoters --- p.55 / Chapter 2.1.2 --- Key events in transcriptional initiation --- p.55 / Chapter 2.1.3 --- Alternative splicing of mRNA --- p.57 / Chapter 2.1.4 --- Bipartite nuclear localization signal (NLS) --- p.57 / Chapter 2.2 --- Methods --- p.60 / Chapter 2.2.1 --- Putative promoter studies of human antiquitin --- p.60 / Chapter 2.2.2 --- Putative promoter studies of Arabidopsis thaliana antiquitin --- p.60 / Chapter 2.2.3 --- Analysis for the alternative splicing of human antiquitin mRNA --- p.60 / Chapter 2.2.4 --- Analysis for the nuclear localization signal (NLS) of human antiquitin amino acid sequence --- p.61 / Chapter 2.2.5 --- Nucleotide / amino acid sequence analyses --- p.61 / Chapter 2.3 --- Results --- p.62 / Chapter 2.3.1 --- Computer search for the putative cis-acting elements on human antiquitin promoter --- p.62 / Chapter 2.3.2 --- Comparison of cis-acting elements found on human antiquitin promoter with those on Arabidopsis thaliana antiquitin promoter --- p.62 / Chapter 2.3.3 --- Possibilities of alternative splicing isoforms of human antiquitin / Chapter 2.3.4 --- Possibilities of bipartite nuclear localization signals on human antiquitin protein --- p.83 / Chapter Chapter 3 --- Overexpression of antiquitin in HEK293 and HepG2 cells and their characterization / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- Cell cycle of a human somatic cell --- p.88 / Chapter 3.1.2 --- Detection of changes in the transcriptome --- p.90 / Chapter 3.1.3 --- Human genome U133 Plus 2.0 array --- p.95 / Chapter 3.1.4 --- Detection of changes in the proteome --- p.96 / Chapter 3.1.5 --- MALDI-TOF MS --- p.97 / Chapter 3.2 --- Materials --- p.99 / Chapter 3.2.1 --- Solutions for cell culture use --- p.99 / Chapter 3.2.2 --- Solutions for cloning --- p.99 / Chapter 3.2.3 --- Buffers for cell cycle analysis --- p.99 / Chapter 3.2.4 --- Buffers for two-dimensional (2D) electrophoresis --- p.100 / Chapter 3.2.5 --- Solutions for silver staining --- p.101 / Chapter 3.2.6 --- Solutions for Coomassie blue protein staining --- p.102 / Chapter 3.2.7 --- Solutions for Western blotting --- p.102 / Chapter 3.2.8 --- Solutions for mass spectrometry --- p.103 / Chapter 3.3 --- Methods --- p.104 / Chapter 3.3.1 --- Hypoosmotic stress --- p.104 / Chapter 3.3.2 --- Heat shock --- p.104 / Chapter 3.3.3 --- Oxidative stress treatment / Chapter 3.3.4 --- Chemical hypoxia --- p.104 / Chapter 3.3.5 --- Treatment of forskolin --- p.106 / Chapter 3.3.6 --- Culture of SHSY5Y cells and its differentiation --- p.106 / Chapter 3.3.7 --- Cloning of pBUDCE4.1/ATQ --- p.106 / Chapter 3.3.8 --- PCR product purification --- p.107 / Chapter 3.3.9 --- Preparation of pEGFP.N1 vector for co-transfection --- p.109 / Chapter 3.3.10 --- Transfection of HEK293 and HepG2 cells --- p.109 / Chapter 3.3.11 --- Assays to characterize transient transfected HEK293 and HepG2 cells --- p.110 / Chapter 3.3.11.1 --- Transfection efficiency monitoring --- p.110 / Chapter 3.3.11.2 --- Cell cycle analysis --- p.112 / Chapter 3.3.11.3 --- Cell doubling time measurement --- p.112 / Chapter 3.3.11.4 --- Stress responsiveness --- p.113 / Chapter 3.3.11.5 --- Oligonucleotide array analysis --- p.113 / Chapter 3.3.11.5.1 --- Total RNA extraction --- p.113 / Chapter 3.3.11.5.2 --- Oligonucleotide array preparations --- p.113 / Chapter 3.3.11.5.3 --- Data analysis --- p.114 / Chapter 3.3.11.6 --- Two-dimensional (2D) electrophoresis --- p.115 / Chapter 3.3.11.6.1 --- Total protein extraction --- p.115 / Chapter 3.3.11.6.2 --- Protein quantification --- p.115 / Chapter 3.3.11.6.3 --- First dimension electrophoresis: isoelectric focusing (IEF) --- p.115 / Chapter 3.3.11.6.4 --- Second dimension electrophoresis: SDS- --- p.116 / Chapter 3.3.11.6.5 --- Silver staining --- p.116 / Chapter 3.3.11.6.6 --- Spots detection --- p.117 / Chapter 3.3.11.7 --- Preparations of samples for MALDI-TOF MS --- p.117 / Chapter 3.3.11.7.1 --- Silver de-staining --- p.117 / Chapter 3.3.11.7.2 --- In-gel tryptic digestion --- p.118 / Chapter 3.3.11.7.3 --- Peptide extraction --- p.118 / Chapter 3.3.11.7.4 --- ZipTip® samples desalting and concentrating --- p.119 / Chapter 3.3.11.7.5 --- MALDI-TOF MS --- p.119 / Chapter 3.3.11.8 --- Western blotting --- p.119 / Chapter 3.3.11.8.1 --- Antibodies probing --- p.120 / Chapter 3.3.11.8.2 --- Enhanced chemiluminescence's (ECL) assay --- p.121 / Chapter 3.4 --- Results --- p.122 / Chapter 3.4.1 --- Inducibility of antiquitin in HEK293 cells under xenobiotic stimulus --- p.122 / Chapter 3.4.2 --- Inducibility of antiquitin in HEK293 and HepG2 cells under chemical hypoxia --- p.122 / Chapter 3.4.3 --- Inducibility of antiquitin in HEK293 and HepG2 cells under hypoosmotic stress --- p.122 / Chapter 3.4.4 --- Inducibility of antiquitin in HEK293 and HepG2 cells under heat shock --- p.122 / Chapter 3.4.5 --- Inducibility of antiquitin in HEK293 and HepG2 cells under forskolin challenge --- p.128 / Chapter 3.4.6 --- Expression of antiquitin in differentiating SHSY5Y cells by retinoic acid and N2 supplement --- p.128 / Chapter 3.4.7 --- Overexpression of antiquitin in HEK293 and HepG2 cells --- p.128 / Chapter 3.4.8 --- Viability of transfected HEK293 and HepG2 cells under hyperosmotic stress --- p.136 / Chapter 3.4.9 --- Cell doubling times of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.10 --- Cell cycle analysis of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.11 --- "Western blot analysis of cyclin D, cyclin A and cyclin B of transfected HEK293 and HepG2 cells" --- p.148 / Chapter 3.4.12 --- RNA quality control tests for oligonucleotide array analysis --- p.148 / Chapter 3.4.13 --- Oligonucleotide array analysis on transfected HEK293 and HepG2 cells --- p.155 / Chapter 3.4.14 --- Two-dimensional electrophoresis of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.15 --- MALDI-TOF MS of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.16 --- Genes and proteins upregulnted in the antiquitin transfected HEK293 and HepG2 cells --- p.190 / Discussion --- p.197 / Reference --- p.221 / Appendix Materials used in the project --- p.243

It is generally believed that ribosome-inactivating proteins (RIPs) are transported to their intracellular targets to express their toxicity. However, studies on the uptake, intracellular transport and apoptotic mechanism of type I RIPS and the cell-binding B chain of type II RIPS are rare. This study is to investigate some problems in these aspects of RIP toxicity. / RCA caused a cell loss at the minimal dose of 50 nM at 24 hr. The main type of cell death was apoptosis, which peaked at 12 hr. The apoptosis proceeded through an extrinsic pathway that involved the activation of caspase-8, but not caspase-9. / RTA caused cell loss at the minimal dose of 50 nM at 24 hr. The main type of cell death was apoptosis, which peaked at 12 hr. RTA was internalised via a clathrin-dependent RME. Like the TCS transport, RTA was not found in the Golgi apparatus. The apoptosis proceeded via the extrinsic pathway that involved the activation of caspase-8 and caspase-3. However, on live rabbits, RTA caused necrotic skin damage. / RTB caused cell loss at the minimal dose of 100 nM at 24 hr. The main type of cell death was initially necrosis, but later became apoptosis, which peaked at 12 hr. / TCS caused a decrease in cell number at the minimum effective dose of 800 nM at 24 hr post administration. The main type of cell death was apoptosis, which peaked at 12 hr. / These results show that (1) the cell-binding B chain is not a precondition for RIP toxicity, because TCS and RTA are also toxic to cells; (2) RTB itself is toxic; (3) without the binding of the B chain to cell surface, the entry and intracellular transport of type I RIPS differ from those of the type II; and (4) both RIPs and single B chain can induce apoptosis. Additionally, the results from live rabbits and cultured cells show that in vivo and in vitro toxicity may involve different cell death mechanisms. RTB-treated NIH 3T3 cells may serve as a model for the switch of cell death from necrosis to apoptosis. (Abstract shortened by UMI.) / We studied trichosanthin (TCS) and ricin A chain (RTA), which are type I RIPS, ricinus communis agglutinin (RCA), which is a type II RIP, and ricin B chain (RTB), which is the cell-binding chain of ricin and RCA. / Sha Ou. / "August 2005." / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3547. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 185-217). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Mak, Keng Hou. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 77-91). / Abstracts in English and Chinese. / Thesis Committee --- p.2 / Declaration --- p.3 / Table of Contents --- p.4 / List of Abbreviations --- p.6 / List of Figures --- p.8 / Abstract --- p.10 / Chapter Chapter 1 --- Introduction --- p.12 / Chapter 1.1 --- Background --- p.12 / Chapter 1.1.1 --- Overview of apoptosis --- p.12 / Chapter 1.1.2 --- Synopsis of the apoptotic pathway --- p.13 / Chapter 1.1.3 --- Defining apoptosis --- p.14 / Chapter 1.1.4 --- Interaction between pro- and anti-apoptotic factors determines cell fate --- p.14 / Chapter 1.1.5 --- DNA fragmentation during the execution phase --- p.15 / Chapter 1.1.6 --- Current understanding of the point of commitment in apoptosis --- p.16 / Chapter 1.1.7 --- Previous studies and hypotheses related to the reversibility of late-state apoptosis --- p.16 / Chapter 1.1.8 --- Unanswered questions --- p.19 / Chapter 1.2 --- "Hypothesis and objectives, Study models and Significance" --- p.19 / Chapter 1.2.1 --- Hypothesis and objectives --- p.19 / Chapter 1.2.2 --- Study models --- p.20 / Chapter 1.2.3 --- Significance --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter Chapter 3 --- Results --- p.30 / Chapter 3.1 --- Dying cells reversed execution stage of apoptosis after removal of apoptotic stimuli --- p.30 / Chapter 3.2 --- Dying cells reversed apoptosis after DNA damage --- p.37 / Chapter 3.3 --- Genetic alterations and transformation occurred after reversal of apoptosis --- p.43 / Chapter 3.4 --- Investigating molecular mechanism driving reversal of apoptosis --- p.50 / Chapter 3.4.1 --- Preparation and characterization of samples for microarray --- p.50 / Chapter 3.4.2 --- Gene ontology enrichment analysis of the expression profile during reversal of apoptosis --- p.52 / Chapter 3.4.3 --- Interfering stress response or anti-apoptotic factors during the reversal of apoptosis drove cells to terminal death --- p.56 / Chapter Chapter 4 --- Discussion --- p.62 / Chapter 4.1 --- "Reversal of apoptosis in ""normal cells"" was observed" --- p.62 / Chapter 4.2 --- Cells surviving apoptosis had their genomes damaged and altered --- p.63 / Chapter 4.3 --- Transformation occurred after reversal of apoptosis --- p.65 / Chapter 4.4 --- Investigating molecular mechanism driving reversal of apoptosis --- p.65 / Chapter 4.5 --- Summary --- p.68 / Chapter Chapter 5 --- Perspectives --- p.70 / Chapter 5.1 --- Could reversal of apoptosis be evolutionarily advantageous? --- p.70 / Chapter 5.2 --- "Reversal of apoptosis as an ""individualistic"" behavior against organismal integrity" --- p.71 / Chapter 5.3 --- Proposed studies --- p.72 / Chapter 5.3.1 --- Other apoptotic targets that may leave persistent effects --- p.72 / Chapter 5.3.2 --- Post- caspase activation regulation of apoptosis --- p.74 / Chapter 5.3.3 --- Identifying correlation between reversal of apoptosis and cancer --- p.74 / Chapter 5.3.4 --- Single cell methods and cell tracking system for further studies --- p.75 / Chapter 5.3.5 --- Notes on studying reversal of apoptosis in relation to phagocytosis --- p.76 / References --- p.77

Yau, Pak Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 150-167). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / List of abbreviations --- p.vi / List of figures --- p.viii / List of tables --- p.xi / Table of content --- p.xii / Chapter Chapter One: --- General introduction --- p.1 / Chapter 1.1. --- Introduction / Chapter 1.1.1. --- Tumor hypoxia --- p.2 / Chapter 1.1.2. --- Non-coding RNA --- p.6 / Chapter 1.1.3. --- Long non-coding RNAs: regulation and related diseases --- p.7 / Chapter 1.1.3.1. --- aHIF and cancer --- p.11 / Chapter 1.1.4. --- Objective --- p.11 / Chapter Chapter Two: --- Regulation of HIF-lα by aHIF --- p.13 / Chapter 2.1. --- Introduction / Chapter 2.1.1. --- aHIF: a natural antisense long non-coding RNAs --- p.14 / Chapter 2.1.2. --- The relationship between aHIF and HIF-lα --- p.15 / Chapter 2.1.3. --- HIF-lα regulation --- p.19 / Chapter 2.2. --- Materials and Methods / Chapter 2.2.1. --- Cell culture --- p.22 / Chapter 2.2.2. --- Western blot analysis --- p.22 / Chapter 2.2.3. --- RNA isolation and reverse transcription --- p.23 / Chapter 2.2.4. --- Quantitative Real-time PCR --- p.23 / Chapter 2.2.5. --- Plasmids construction --- p.24 / Chapter 2.2.5.1. --- aHIF over-expression clone --- p.24 / Chapter 2.2.5.2. --- Luciferase reporter with HIF-lα 3'UTR --- p.25 / Chapter 2.2.5.3. --- HIF-lα and PTB over-expression vector --- p.25 / Chapter 2.2.5.4. --- PTB knock-down vector --- p.30 / Chapter 2.2.6. --- Stable Clone --- p.30 / Chapter 2.2.7. --- Transfection --- p.31 / Chapter 2.2.8. --- Luciferase reporter assay --- p.31 / Chapter 2.2.9. --- Statistical analysis --- p.32 / Chapter 2.3. --- Results / Chapter 2.3.1. --- Effect of aHIF (FL) on HIF-lα expression --- p.33 / Chapter 2.3.2. --- Effect of aHIF (FL) on HIF-lα 3，UTR --- p.33 / Chapter 2.3.3. --- Effects of aHIF (OL) and aHIF (NOL) on HIF-lα level --- p.37 / Chapter 2.3.4. --- Effects of aHIF (NOL) and aHIF (OL) on HIF-lα 3，UTR --- p.39 / Chapter 2.3.5. --- Effect of aHIF (FL) on HIF-lα 3' UTR in PTBi cells --- p.41 / Chapter 2.3.6. --- Effect of aHIF (NOL) on HIF-lα 3，UTR in PTBi cells --- p.43 / Chapter 2.3.7. --- Effect of aHIF (OL) on HIF-lα 3' UTR in PTBi cells --- p.45 / Chapter 2.4 --- Discussion / Chapter 2.4.1. --- aHIF regulates HIF-la through HIF-la 3' UTR (FL) --- p.47 / Chapter 2.4.2. --- Factors involved in aHIF- HIF-lα interaction --- p.53 / Chapter Chapter Three: --- aHIF regulates drug sensitivity through BNIP3 --- p.58 / Chapter 3.1 --- Introduction / Chapter 3.1.1. --- aHIF and drug sensitivity --- p.59 / Chapter 3.1.2. --- BNIP3: its regulation and functions --- p.61 / Chapter 3.1.3. --- Taxol and its action mechanism --- p.67 / Chapter 3.1.4. --- Objective --- p.69 / Chapter 3.2. --- Materials and Methods / Chapter 3.2.1. --- Cell culture --- p.70 / Chapter 3.2.2. --- Cell viability assay --- p.70 / Chapter 3.2.3. --- Western blot analysis --- p.70 / Chapter 3.2.4. --- Plasmid construction --- p.71 / Chapter 3.2.5. --- Transfection --- p.71 / Chapter 3.2.6. --- Stable clone formation --- p.71 / Chapter 3.2.7. --- Quantitative real-time PCR --- p.71 / Chapter 3.2.8. --- Annexin V binding assay --- p.72 / Chapter 3.2.9. --- DNA fragmentation assay --- p.72 / Chapter 3.2.10. --- Detection of mitochondrial membrane potential by flow cytometry --- p.73 / Chapter 3.2.11. --- Cytochrome c and AIF translocation assay --- p.73 / Chapter 3.2.12. --- Statistical analysis --- p.74 / Chapter 3.3 --- Results / Chapter 3.3.1. --- Effect of aHIF on Taxol and vincristine sensitivity in HepG2 cells --- p.75 / Chapter 3.3.2. --- Effect of HIF-lαi on Taxol and vincristine sensitivity in HepG2 cells --- p.75 / Chapter 3.3.3. --- Effect of aHIF on Taxol-induced apoptosis --- p.78 / Chapter 3.3.4. --- HIF-1α regulation of BNIP3 expression --- p.78 / Chapter 3.3.5. --- Effect of aHIF on BNIP3 expression --- p.81 / Chapter 3.3.6. --- BNIP3 expression in BNIP3i stable transfectant --- p.81 / Chapter 3.3.7. --- The response of BNIP3i cells towards Taxol and vincrisinte --- p.84 / Chapter 3.3.8. --- Effect of BNIP3 on Taxol and vincristine sensitivity in BNIP3i cells --- p.84 / Chapter 3.3.9. --- Taxol- or vincristine- induced apoptosis in BNIP3i cells --- p.87 / Chapter 3.3.10. --- "Effects of aHIF, HIF-lα and BNIP3 on Taxol-induced apoptosis in HepG2 cells" --- p.89 / Chapter 3.3.11. --- Caspases activation in Taxol - or vincristine - induced apoptosis in BNIP3i cells --- p.91 / Chapter 3.3.12. --- Mitochondrial membrane depolarization in Taxol - or vincristine - induced apoptosis in BNIP3i cells --- p.91 / Chapter 3.3.13. --- AIF and cytochrome c expressions in BNIP3i cells --- p.92 / Chapter 3.3.14. --- Effect of aHIF on other chemo- and radio-therapeutics in HepG2 cells --- p.96 / Chapter 3.3.15. --- Effect of HIF-lα on other chemo- and radio-therapeutics in HepG2 cells --- p.96 / Chapter 3.3.16. --- BNIP3i cells became more sensitivity to a number of drugs --- p.99 / Chapter 3.3.17. --- BNIP3i became more resistance to some drugs --- p.99 / Chapter 3.4 --- Discussion / Chapter 3.4.1. --- aHIF affected Taxol sensitivity through BNIP3 --- p.102 / Chapter 3.4.2. --- Mechanism of BNIP3 regulated Taxol or vincristine induced apoptosis --- p.106 / Chapter 3.4.3. --- Possible roles of BNIP3 in response to other therapeutics --- p.110 / Chapter Chapter Four: --- aHIF regulation of tumorigenesis --- p.116 / Chapter 4.1 --- Introduction / Chapter 4.1.1. --- aHIF in cancer biology --- p.117 / Chapter 4.1.2. --- Ras proteins --- p.118 / Chapter 4.1.3. --- K-Ras and cancers --- p.121 / Chapter 4.1.4. --- Regulation of Ras --- p.122 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1. --- Cell culture --- p.124 / Chapter 4.2.2. --- Western blot analysis --- p.124 / Chapter 4.2.3. --- Plasmids construction --- p.124 / Chapter 4.2.4. --- Transfection --- p.124 / Chapter 4.2.5. --- Cell growth assay --- p.124 / Chapter 4.2.6. --- Soft agar assay --- p.125 / Chapter 4.2.6. --- Statistical analysis --- p.125 / Chapter 4.3 --- Results / Chapter 4.3.1. --- Effect of aHIF and HIF-lα on cell proliferation --- p.127 / Chapter 4.3.2. --- Effect of aHIF and HIF-lα on anchorage-independent growth --- p.127 / Chapter 4.3.3. --- Effect of aHIF and HIF -lα on K-Ras expression --- p.130 / Chapter 4.3.4. --- Effect of FTS on cell transfected with aHIF or HIF-lα --- p.130 / Chapter 4.4 --- Discussion / Chapter 4.4.1. --- Role of aHIF in tumorigenesis --- p.133 / Chapter 4.4.2. --- Proposed pathways of aHIF-regulated tumorigenesis --- p.136 / Chapter Chapter Five: --- General discussion and conclusion --- p.140 / Chapter 5.1 --- General discussion --- p.141 / Chapter 5.2 --- Conclusion --- p.146 / Chapter 5.3 --- Future perspectives --- p.147 / Chapter 5.3.1 --- Role ofPTB in aHIF-HIF-lα interaction --- p.147 / Chapter 5.3.2 --- Effect of aHIF (OL) on HIF-lα mRNA 3' UTR --- p.147 / Chapter 5.3.3 --- Effect ofaHIF on AIF --- p.148 / Chapter 5.3.4 --- Confirmation of the involvement of K-Ras --- p.148 / Chapter Chapter Six --- References --- p.150 / Chapter 6.1 --- References --- p.151