by Yip Mei Chu Pandora. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 207-233). / Abstract also in Chinese. / STATEMENT --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vii / ABBREVIATIONS --- p.xiii / LIST OF FIGURES AND TABLES --- p.xvii / Chapter CHAPTER ONE ... --- GENERAL INTRODUCTION / Chapter 1.1 --- the blood cells formation - hematopoiesis --- p.1 / Chapter 1.1.1 --- Hierarchy of hematopoiesis --- p.2 / Chapter 1.1.2 --- Malfunction in the process of hematopoiesis - hematologic neoplasia - Leukemia --- p.6 / Chapter 1.1.2.1 --- Classification of leukemia --- p.7 / Chapter 1.1.2.2 --- Differentiation therapy ´ؤ a new hope in the treatment of leukemia --- p.9 / Chapter 1.2 --- Understanding the pathogenesis of leukemia --- p.12 / Chapter 1.2.1 --- General regulation of hematopoiesis --- p.12 / Chapter 1.2.2 --- Regulation of the differentiation of myeloid lineage --- p.15 / Chapter 1.2.2.1 --- Regulation of myeloid cell differentiation by hematopoietic regulatory protein --- p.16 / Chapter 1.2.2.2 --- Signal transduction pathways in myeloid cell differentiation --- p.20 / Chapter 1.2.2.3 --- Gene regulation of myeloid cell differentiation --- p.22 / Chapter 1.2.2.3.1 --- Transcription factors --- p.23 / Chapter 1.2.2.3.2 --- Myeloid specific genes --- p.31 / Chapter 1.2.2.3.3 --- Protooncogenes and tumor suppressor genes --- p.37 / Chapter 1.2.2.3.4 --- Homeobox genes --- p.42 / Chapter 1.2.2.3.5 --- Cell cycle control in myeloid growth and differentiation --- p.47 / Chapter 1.3 --- Induction of differentiation in myeloid leukemia cell --- p.48 / Chapter 1.3.1 --- Induced myeloid leukemia cell differentiation --- p.48 / Chapter 1.3.2 --- Inducers of myeloid cell differentiation --- p.52 / Chapter 1.3.3 --- Chemical inducers ´ؤ Flavonoids --- p.57 / Chapter 1.3.4 --- Murine myeloid leukemia cell ´ؤ WEHI-3B JCS --- p.60 / Chapter 1.4 --- Aim of study --- p.53 / Chapter CHAPTER TWO ... --- ISOLATION OF GENES THAT ARE DIFFERENTIALLY EXPRESSED DURING BIOCHANIN A INDUCED WEHI-3B (JCS) MYELOID LEUKEMIA CELL DIFFERENTIATION / Chapter 2.1 --- Introduction --- p.65 / Chapter 2.1.1 --- Strategy for searching differentially expressed genes - RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP- PCR) --- p.65 / Chapter 2.1.2 --- Reamplification of PCR products by Touchdown PCR --- p.67 / Chapter 2.1.3 --- Methods for eliminating false positives : Dot blot hybridization screening --- p.68 / Chapter 2.2 --- Materials --- p.70 / Chapter 2.2.1 --- "Cell line, Bacterial strain and Vector" --- p.70 / Chapter 2.2.2 --- Chemicals --- p.70 / Chapter 2.2.3 --- Reagents and nucleic acids --- p.71 / Chapter 2.2.4 --- Kits --- p.72 / Chapter 2.2.5 --- Solutions --- p.72 / Chapter 2.2.6 --- Equipments --- p.73 / Chapter 2.3 --- Methods --- p.74 / Chapter 2.3.1 --- Induction of murine myeloid leukemia cell line -WEHI-3B (JCS) cells by biochanin-A --- p.74 / Chapter 2.3.2 --- Isolation of total RNA by guanidium thiocyanate cesium chloride ultracentrifugation --- p.74 / Chapter 2.3.3 --- RNA fingerprinting by arbitrarily primed PCR --- p.75 / Chapter 2.3.3.1 --- Synthesis of first strand cDNA --- p.75 / Chapter 2.3.3.2 --- Normalization of RNA samples --- p.75 / Chapter 2.3.3.3 --- RAP-PCR --- p.76 / Chapter 2.3.3.4 --- Reamplification of differentially amplified fragment --- p.77 / Chapter 2.3.4 --- First round dot blot hybridization screening --- p.78 / Chapter 2.3.4.1 --- Dot blot --- p.78 / Chapter 2.3.4.2 --- Preparation of cDNA probe --- p.79 / Chapter 2.3.4.3 --- 32P-labelling of cDNA probe --- p.79 / Chapter 2.3.4.4 --- Removal of unincorporated probe by NICK´ёØ column --- p.80 / Chapter 2.3.4.5 --- Estimation of 32P labelling efficiency by scintillation counting --- p.80 / Chapter 2.3.4.6 --- Prehybridization and hybridization --- p.81 / Chapter 2.3.4.7 --- Quantitation of hybridization signal by scanning densitometry --- p.81 / Chapter 2.3.5 --- Second round dot blot hybridization screening --- p.81 / Chapter 2.3.5.1 --- Subcloning of differentially amplified fragments --- p.82 / Chapter 2.3.5.1.1 --- Preparation of vector DNA --- p.82 / Chapter 2.3.5.1.2 --- Synthesis of blunt end PCR product --- p.84 / Chapter 2.3.5.1.3 --- Blunt end ligation --- p.34 / Chapter 2.3.5.1.4 --- Transformation --- p.85 / Chapter 2.3.5.1.5 --- Selection and confirmation by polymerase chain reaction --- p.85 / Chapter 2.3.5.2 --- Dot blot hybridization screening --- p.85 / Chapter 2.4 --- Results --- p.87 / Chapter 2.4.1 --- Spectrophotometric analysis of total RNA --- p.87 / Chapter 2.4.2 --- Normalization of RNA samples --- p.88 / Chapter 2.4.3 --- RNA fingerprinting by arbitrarily primed PCR --- p.39 / Chapter 2.4.4 --- Reamplification of isolated RAP-PCR products --- p.91 / Chapter 2.4.5 --- First round of dot blot hybridization screening --- p.92 / Chapter 2.4.6 --- Subcloning of differentially amplified fragments --- p.100 / Chapter 2.4.7 --- Second round of dot blot hybridization screening --- p.102 / Chapter 2.4.8 --- Comparison of the first and second round of dot blot hybridization screening --- p.106 / Chapter 2.5 --- Discussion --- p.108 / Chapter 2.5.1 --- RNA fingerprinting by arbitrarily primed PCR --- p.108 / Chapter 2.5.2 --- Limitation of RAP-PCR --- p.110 / Chapter 2.5.3 --- Two rounds of dot blot hybridization screening --- p.111 / Chapter CHAPTER THREE... --- CHARACTERIZATION OF THE ISOLATED GENE FRAGMENTS / Chapter 3.1 --- Introduction --- p.113 / Chapter 3.1.1 --- Automated DNA sequencing and analysis --- p.113 / Chapter 3.1.2 --- GenBank and the BLAST homology search --- p.115 / Chapter 3.2 --- Materials --- p.118 / Chapter 3.2.1 --- Selected recombinant plasmids --- p.118 / Chapter 3.2.2 --- Chemicals --- p.118 / Chapter 3.2.3 --- Reagents --- p.118 / Chapter 3.2.4 --- Kits --- p.119 / Chapter 3.2.5 --- Solutions --- p.119 / Chapter 3.2.6 --- Equipment --- p.119 / Chapter 3.3 --- Methods --- p.120 / Chapter 3.3.1 --- Preparation of selected recombinant plasmid DNA --- p.120 / Chapter 3.3.2 --- Restriction digestion of recombinant plasmid DNA --- p.120 / Chapter 3.3.3 --- Automated DNA sequencing --- p.120 / Chapter 3.3.3.1 --- Primer annealing to template --- p.120 / Chapter 3.3.3.2 --- Sequencing reactions --- p.121 / Chapter 3.3.3.3 --- Polyacrylamide gel electrophoresis --- p.121 / Chapter 3.3.3.4 --- Data analysis by ALF manager and DNAsis --- p.122 / Chapter 3.3.4 --- Sequence homology search with databases --- p.122 / Chapter 3.4 --- Results --- p.123 / Chapter 3.4.1 --- Spectrophotometric analysis of selected recombinant plasmid DNAs subcloned with differentially amplified fragments --- p.123 / Chapter 3.4.2 --- Restriction digestion of selected recombinant plasmid DNA --- p.124 / Chapter 3.4.3 --- Sequences of the subcloned differentially amplified fragments --- p.126 / Chapter 3.4.4 --- Sequence analysis of the subcloned differentially amplified fragments --- p.144 / Chapter 3.5 --- Discussion --- p.157 / Chapter 3.5.1 --- Sequence analysis of the isolated gene fragment --- p.157 / Chapter CHAPTER FOUR … --- "EXPRESSION PROFILE OF ISOLATED GENES FRAGMENTS IN MYELOID LEUKEMIA CELL, MOUSE EMBRYO, AND TISSUES" / Chapter 4.1 --- Introduction --- p.162 / Chapter 4.1.1 --- Quantitation of mRNA by Reverse transcription-polymerase chain reaction --- p.162 / Chapter 4.1.2 --- Internal primer design by OLIGO´ёØ ver 34 --- p.167 / Chapter 4.2 --- Materials --- p.168 / Chapter 4.2.1 --- Mice --- p.168 / Chapter 4.2.2 --- Cell lysate --- p.168 / Chapter 4.2.3 --- Total RNAs --- p.168 / Chapter 4.3 --- Methods --- p.169 / Chapter 4.3.1 --- Internal primer design by OLIGO´ёØ ver 34 --- p.169 / Chapter 4.3.2 --- "Isolation of total RNA from biochanin A induced JCS cells, mouse embryos and tissue" --- p.169 / Chapter 4.3.2.1 --- Preparation of cell lysate from mouse embryo and postnatal mouse brain --- p.169 / Chapter 4.3.2.2 --- Isolation of RNA by guanidium thiocyanate cesium chloride method --- p.170 / Chapter 4.3.3 --- Preparation of saggital section of mouse embryo --- p.170 / Chapter 4.3.4 --- Confirmation of differential expression of isolated genes fragments during biochanin A and midazolam induced WEHI 3B (JCS) differentiation and the expression profile in mouse tissues and during mouse embryo development by reverse transcription-polymerase chain reaction --- p.171 / Chapter 4.4 --- Results --- p.173 / Chapter 4.4.1 --- Internal primer design of the sequenced fragments --- p.173 / Chapter 4.4.2 --- Spectrophotometric analysis of total RNA --- p.175 / Chapter 4.4.3 --- Saggital section of mouse embryo --- p.176 / Chapter 4.4.4 --- Normalization of RNA samples --- p.180 / Chapter 4.4.5 --- Analysis of mRNA expression of differentially amplified fragmentsin biochanin A or midazolam induced JCS cells and mouse embryos by RT- PCR --- p.182 / Chapter 4.4.5.1 --- "Genes downregulated at 1 hour, 5 hours and 48 hours after biochanin A induction of JCS cells" --- p.183 / Chapter 4.4.5.2 --- Genes up-regulated at 48 hours after biochanin A induction --- p.183 / Chapter 4.4.5.3 --- Genes constitutively expressed during the course of biochanin A treatment --- p.184 / Chapter 4.4.5.4 --- Genes showing undetectable level of expression in biochanin A induced JCS cells --- p.184 / Chapter 4.4.6 --- Tissue expression of the biochanin A induced-differentially expressed fragments by RT-PCR --- p.188 / Chapter 4.5 --- Discussion --- p.191 / Chapter 4.5.1 --- Expression profiles of isolated differentially amplified fragments --- p.191 / Chapter 4.5.2 --- Comparison of the expression profiles of the isolated gene fragments analyzed by dot blot hybridization screening and RT-PCR --- p.197 / Chapter CHAPTER FIVE ... --- GENERAL DISCUSSION --- p.200 / REFERENCES --- p.207 / APPENDIX --- p.234

The mammalian heart is a remarkable organ in that it must provide for the cardiovascular needs of the organism throughout life, without pausing. Yet, through developmental growth to adulthood and into ageing, the mammalian heart undergoes extensive physiological, morphological and biochemical remodelling. Pivotal to the age-associated alterations in cardiac phenotype is a decline in the proliferative capacity of cardiac myocytes (CMs), which is insufficient to compensate for the basal rate of CM death over time. The terminally differentiated nature of adult CMs also underlies the inability of the heart to repair itself after myocardial damage, such as infarction. As a consequence, existing CMs mount a compensatory hypertrophic response to sustain cardiac output. In parallel, the proliferation rate of resident cardiac fibroblasts, which comprise approximately 60% of total cardiac cells, increases, replacing healthy myocardium with fibrotic scar tissue. Together, CM hypertrophy and fibroblast hyperplasia progressively reduces cardiac function and the ability of the heart to adapt to environmental stressors or damage. Under continued stress or through natural ageing, the heart progresses to a failing state in which cardiac output can no longer meet the demands of the body. The societal impact of ageing-associated decline in cardiac function is great, with heart failure affecting around 8% of over 65s and consuming approximately 2% of the NHS budget. These statistics are set to rise with an ageing population. The substantial phenotypic alterations characteristic of ageing and disease-associated cardiac remodelling requires a wholesale reprogramming of the CM transcriptome. In many biological systems, although yet to be established in adult myocytes, epigenetic mechanisms underlie the transcriptome changes that arise. I hypothesised that alterations in the epigenetic landscape of CMs mediate the transcriptome remodelling that determines the phenotypic transformations that occur in cardiac ageing, hypertrophy and disease. To test this hypothesis, I examined CM-specific changes in DNA cytosine modifications, long non-coding RNA (lncRNA) expression and histone tail lysine methylation marks – epigenetic marks with central roles in transcriptional regulation in many biological systems. I examined how these changes correlate with alterations in the CM transcriptome during disease and ageing. Understanding how alterations in the transcriptome and epigenome contribute to phenotypic changes using whole tissue data is confounded by the heterogeneous nature of the heart, coupled with ageing and disease-associated changes in relative cellular composition. To overcome this, I validated a method to isolate CM nuclei specifically from post-mortem heart tissue. This method also has the advantage that it could be applied to frozen tissue, allowing access to archived material. LncRNAs are functional RNA transcripts longer than 200 bases are emerging as important regulators of gene expression. Common mechanisms of gene expression regulation by lncRNAs include by antisense suppression, as guide/co-factor molecules to direct chromatin modifying components or splicing factors to locations in the genome. Transcriptome profiling in healthy and failing human CMs identified an increase in expression of the lncRNA MALAT-1, which was consistently observed in rodent models of pathology and in ageing. Loss-of-function investigations revealed a potential anti-hypertrophic function for this lncRNA. Specifically, MALAT-1 knock down in vitro in CMs incited spontaneous hypertrophy with features reflecting pathological remodelling in the heart and hypertrophy induced by pro-hypertrophic mediators in vitro. ix In addition, novel uncharacterised transcripts were identified as differentially expressed in cardiovascular disease, including a lncRNA at 4q35.2, which was found significantly downregulated in CMs from human failing hearts. DNA methylation is a stable epigenetic modification and is generally associated with transcriptional repression. It is established by de novo DNA methyltransferases (DNMTs) in early development to determine and maintain differentiated cell states and is ‘copied’ to daughter strands in DNA synthesis by the maintenance DNMT1. Methylcytosine (MeC) can be subject to further processing to hydroxymethylcytosine (hMeC) through a TET protein-mediated oxidation reaction. This serves as a means to actively remove methylation marks as well as hMeC being a novel epigenetic modification in its own right. For the first time, I identified the cardiac myocyte genome as having a high global level of hMeC, comparable with that in neurones. I also discovered an age-associated increase in gene body hMeC that coincided with the loss of proliferative capacity and plasticity of CMs. In parallel, gene body DNA MeC levels decrease in CM ageing. Both these phenomena in gene bodies corresponded with a non-canonical upregulation in expression of genes particularly relevant to cardiac function. This relationship between gene body methylation and transcription rate is strengthened with age in CMs. Recent work in the laboratory had identified the pervasive loss of euchromatic lysine 9 dimethylation on histone 3 (H3K9me2) as a conserved feature of pathological hypertrophy and associated with re-expression of foetal genes. Concurrently, expression and activity of the enzymes responsible for depositing H3K9me2, euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/GLP and EHMT2/G9a) were reduced. Consistently, microRNA-217-induced genetic or pharmacological inactivation of Ehmts was sufficient to promote pathological hypertrophy and foetal gene re-expression, while suppression of this pathway protected from pathological hypertrophy both in vitro and in mice. In summary, I provide new insight into CM-specific epigenetic changes and suggest the epigenome as an important mediator in the loss of plasticity and cardiac health in ageing and disease. Epigenetic mediators and pathways identified as responsible for this remodelling of the CM epigenome suggests opportunities for novel therapy approaches.

Hu Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 95-122). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Table of contents --- p.vi / List of figures --- p.x / List of tables --- p.xi / Abbreviations --- p.vii / Chapter CHAPTER ONE: --- INTRODUCATION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.2 / Chapter 1.1.1. --- Epidemiology of HCC --- p.2 / Chapter 1.1.2. --- The major risk factors --- p.2 / Chapter 1.1.3. --- Management of HCC --- p.3 / Chapter 1.2 --- Mechanisms of multidrug resistance (MDR) in cancer cells --- p.4 / Chapter 1.2.1. --- Major mechanisms in reduced drug accumulation --- p.5 / Chapter 1.2.1.1. --- P-glycoprotein (P-gp) --- p.6 / Chapter 1.2.1.2. --- Multidrug Resistance-associated Protein (MRP) --- p.7 / Chapter 1.2.1.3. --- Other effluxes --- p.8 / Chapter 1.2.2. --- Inhibition of apoptotic signaling pathways --- p.11 / Chapter 1.2.2.1. --- TP53 and multidrug resistance --- p.11 / Chapter 1.2.2.2. --- Anti-oncogene PTEN and drug resistance --- p.13 / Chapter 1.2.2.3. --- Influence of BCL2 family on drug resistance --- p.14 / Chapter 1.3 --- The chemotherapeutic agent of doxorubicin --- p.15 / Chapter 1.4 --- Aims of study --- p.18 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.20 / Chapter 2.1 --- Cell culture --- p.21 / Chapter 2.1.1 --- Cell lines and cell culture --- p.21 / Chapter 2.1.2 --- Subculture --- p.23 / Chapter 2.1.3 --- Cryopreservation --- p.23 / Chapter 2.1.4 --- Recovery of cryopreserved culture --- p.24 / Chapter 2.1.5 --- Cell number counting --- p.24 / Chapter 2.2 --- MTT experiments --- p.26 / Chapter 2.2.1 --- Determination of cell seeding density --- p.26 / Chapter 2.2.2 --- Cytotoxic assay --- p.27 / Chapter 2.3 --- Spectral Karytyping (SKY) --- p.27 / Chapter 2.3.1 --- Pretreatment of chromosome slides for SKY --- p.28 / Chapter 2.3.2 --- Hybridization --- p.28 / Chapter 2.3.3 --- Detection --- p.29 / Chapter 2.4 --- Positional expression profiling --- p.30 / Chapter 2.4.1 --- RNA extraction --- p.32 / Chapter 2.4.2 --- Reverse transcription and cDNA labling --- p.34 / Chapter 2.4.3 --- Probe purification and hybridization --- p.34 / Chapter 2.4.4 --- Image acquisition and data analysis --- p.35 / Chapter 2. 5 --- Quantitative RT-PCR --- p.37 / Chapter 2.5.1 --- RNA extraction --- p.37 / Chapter 2.5.2 --- Primer design --- p.37 / Chapter 2.5.3 --- Reverse transcription --- p.37 / Chapter 2.5.4 --- Quantitative PCR --- p.39 / Chapter 2.6. --- Statistical analysis --- p.40 / Chapter CHAPTER 3 --- RESULTS --- p.43 / Introduction --- p.44 / Chapter 3.1 --- Doxorubicin resistance in HCC cell lines --- p.44 / Chapter 3.2 --- Candidate drug resistance genes --- p.56 / Chapter 3.3 --- The roles of chromosomal instability --- p.58 / Chapter 3.4 --- Candidate resistance genes identified in chromosome 10 --- p.69 / Chapter CHAPTER 4 --- DISCUSSION --- p.75 / Introduction --- p.76 / Chapter 4.1 --- In vitro cell models facilitate drug resistance investigations --- p.11 / Chapter 4.2 --- Aneuploidy and DX resistance --- p.78 / Chapter 4.3 --- The role of known resistance genes on chromosome 10 --- p.79 / Chapter 4.4 --- Identification of novel DX resistance genes on chromosome 10 --- p.80 / Chapter 4.5 --- Common drug resistance genes --- p.83 / Chapter 4.5.1. --- The roles of classical drug resistance --- p.85 / Chapter 4.5.2. --- Inhibition of apoptosis and deregulation of cell cycle --- p.86 / Chapter CHAPTER 5 --- PROPOSED FUTURE STUDIES --- p.90 / Chapter 5.1. --- Validate significant in vitro findings by clinical trials --- p.91 / Chapter 5.2. --- Molecular mechanisms in inactivation of ECHS1 in resistant cells --- p.92 / Chapter 5.3. --- Future utilization of cDNA microarray data --- p.93 / REFERENCES --- p.95 / PUBLICATION --- p.122

Wong Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 73-85). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Abbreviations --- p.viii / Chapter 1 --- Introduction --- p.1 / Overview --- p.1 / Chapter a. --- Steroidogenesis --- p.1 / Chapter i --- Ectopic steroidogenesis --- p.2 / Chapter ii --- Steroidogenesis in the pituitary --- p.5 / Chapter iii --- Proteins and enzymes involved in steroidogenesis and their distribution in brain and other sites --- p.6 / Chapter iv --- Regulation of adrenal steroidogenesis --- p.11 / Chapter b. --- Regulation of pituitary ACTH secretion --- p.13 / Chapter i --- Hypothalamic regulation --- p.13 / Chapter ii --- Paracrine regulation --- p.14 / Chapter iii --- Feedback regulation --- p.15 / Chapter c. --- Aims of the study --- p.15 / Chapter 2 --- Materials and methods --- p.18 / Chapter a. --- Materials --- p.18 / Chapter b. --- In vivo experiments --- p.25 / Chapter i --- Steroidogenic enzyme mRNA expression in rat and mouse pituitary --- p.25 / Chapter ii --- "Effects of hormonal treatments on weights of body, adrenal glands and testis, and steroidogenic enzyme mRNA expression in rats" --- p.25 / Chapter iii --- Effects of adrenalectomy and/or gonadectomy on the expression of steroidogenic enzyme mRNAs in rat pituitary --- p.26 / Chapter c. --- In vitro experiments --- p.26 / Chapter i --- Effects of CRF and forskolin on cAMP production in mouse and rat pituitary cell-lines --- p.28 / Chapter ii --- Expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.28 / Chapter iii --- Effects of CRF on the expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.29 / Chapter d. --- Statistical analysis --- p.29 / Chapter 3 --- Results --- p.30 / Chapter a. --- In vivo experiments --- p.30 / Chapter i --- Steroidogenic enzyme mRNA expression in rat and mouse pituitary --- p.30 / Chapter ii --- "Effects of hormonal treatments on weights of body, adrenal glands and testis, and steroidogenic enzyme mRNA expression in rats" --- p.38 / Chapter iii --- Effects of adrenalectomy and/or gonadectomy on the expression of steroidogenic enzyme mRNAs in rat pituitary --- p.47 / Chapter b. --- In vitro experiments --- p.51 / Chapter i --- Effects of CRF and forskolin on cAMP production in mouse and rat pituitary cell-lines --- p.51 / Chapter ii --- Expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.55 / Chapter iii --- Effects of CRF on the expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.57 / Chapter 4 --- Discussion --- p.58 / Further studies --- p.71 / References --- p.73 / Appendix 1 --- p.86

Xiao Ping. / "September 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 153-176) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Szeto Yuk Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 257-283). / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.vi / Abstract --- p.ix / Contents --- p.xi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoies --- p.is / Chapter 1.1.1 --- Ontogeny of the hematopoietic system --- p.1 / Chapter 1.1.2 --- Hierarchy of hematopoietic cells --- p.3 / Chapter 1.1.3 --- Characteristics of a functional blood system and the need for regulation --- p.11 / Chapter 1.1.4 --- Interrupted hematopoiesis -- Leukemia --- p.13 / Chapter 1.2 --- Regulation of myeloid cell differentiation / Chapter 1.2.1 --- Regulation of hematopoiesis --- p.16 / Chapter 1.2.2 --- Models of hematopoiesis --- p.18 / Chapter 1.2.3 --- Genes regulation of myeloid cell differentiation and its study --- p.21 / Chapter 1.2.4 --- Genes differentially expressed and involved in myeloid cell differentiation --- p.24 / Chapter 1.3 --- Induced myeloid cell differentiation / Chapter 1.3.1 --- Induced myeloid cell differentiation --- p.46 / Chapter 1.3.2 --- WEHI-3B JCS cells --- p.48 / Chapter 1.3.3 --- Chemical inducers -- Flavonoids and benzodiazepines --- p.51 / Chapter 1.4 --- The aim of study --- p.59 / Chapter Chapter Two --- Cytokine Expression in Biochanin A- and Midazolam-treated JCS cells / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Cytokine and myeloid differentiation --- p.62 / Chapter 2.1.2 --- Phenotypic studies biochanin A- and midazolam-treated JCS cells --- p.65 / Chapter 2.1.3 --- Cytokine regulation at transcriptional level --- p.68 / Chapter 2.1.4 --- Cytokine mRNA phenotyping by a semi-quantitative approach --- p.69 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Cell line --- p.72 / Chapter 2.2.2 --- Chemicals and buffers --- p.72 / Chapter 2.2.3 --- DIG system --- p.73 / Chapter 2.2.4 --- Enzymes and nucleic acids --- p.73 / Chapter 2.2.5 --- Solutions --- p.74 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Isolation of total RNA by guanidinium thiocyanate/cesium chloride isopycnic gradient --- p.75 / Chapter 2.3.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.76 / Chapter 2.3.3 --- Southern blotting --- p.79 / Chapter 2.3.4 --- Cycle titration and dot blotting --- p.79 / Chapter 2.3.5 --- DIG 3' end labeling of probes --- p.81 / Chapter 2.3.6 --- Hybridization and stringency wash --- p.81 / Chapter 2.3.7 --- Chemiluminescent detection --- p.82 / Chapter 2.3.8 --- Quantitation by densitometry --- p.82 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Analysis of total RNA --- p.83 / Chapter 2.4.2 --- mRNA phenotyping --- p.85 / Chapter 2.4.3 --- Summary of mRNA phenotyping results --- p.98 / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- mRNA phenotyping --- p.100 / Chapter 2.5.2 --- Cytokine gene regulation --- p.106 / Chapter 2.5.3 --- mRNA quantitation using the current method --- p.108 / Chapter Chapter Three --- Identification and Isolation of Genes that are Differentially Expressed during Midazolam-induced JCS Cell Differentiation / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Methods for studying differentially expressed genes --- p.110 / Chapter 3.1.2 --- RNA fingerprinting by arbitrarily-primed PCR (RAP-PCR) and differential display (DDRT-PCR) --- p.113 / Chapter 3.1.3 --- Re-amplification of PCR products by touchdown PCR --- p.118 / Chapter 3.1.4 --- Strategies to avoid false positives --- p.119 / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Cell line and bacterial culture --- p.121 / Chapter 3.2.2 --- Chemicals --- p.121 / Chapter 3.2.3 --- Enzymes and nucleic acids --- p.122 / Chapter 3.2.4 --- Kits --- p.122 / Chapter 3.2.5 --- Solutions --- p.122 / Chapter 3.3 --- Methods / Chapter 3.3.1 --- Isolation of total RNA --- p.124 / Chapter 3.3.2 --- First strand cDNA synthesis --- p.124 / Chapter 3.3.3 --- RNA fingerprinting by arbitrarily-primed PCR --- p.124 / Chapter 3.3.4 --- First round cDNA probe screening --- p.126 / Chapter 3.3.5 --- Subcloning of differentially amplified fragments --- p.129 / Chapter 3.3.6 --- Second round cDNA probe screening --- p.133 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Spectrophotometric analysis of total RNA --- p.134 / Chapter 3.4.2 --- Normalization of samples --- p.135 / Chapter 3.4.3 --- RNA fingerprinting of arbitrarily-primed PCR --- p.136 / Chapter 3.4.4 --- Re-amplification of PCR products --- p.138 / Chapter 3.4.5 --- First round cDNA probe screening --- p.139 / Chapter 3.4.6 --- Subcloning of the differentially amplified fragments --- p.143 / Chapter 3.4.7 --- Second round cDNA probe screening --- p.145 / Chapter 3.4.8 --- A comparison of the first and second screening --- p.149 / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- Towards the steps to isolate differentially expressed genes --- p.151 / Chapter 3.5.2 --- Expression profiles predicted at different stage of the procedures --- p.156 / Chapter 3.5.3 --- Representation of the total mRNA in the cell --- p.158 / Chapter 3.3.4 --- Comparison of the original and modified protocol of RAP-PCR --- p.159 / Chapter 3.3.5 --- Advantages of the modified protocol and further refinements --- p.163 / Chapter Chapter Four --- Characterization of the Putative Differentially Expressed Genesin Midazolam-induced JCS cells / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- DNA sequencing --- p.165 / Chapter 4.1.2 --- Automated DNA sequencing and analysis --- p.168 / Chapter 4.1.3 --- Genbank and BLAST homology search --- p.171 / Chapter 4.1.4 --- Internal primer design for RT-PCR --- p.174 / Chapter 4.1.5 --- Genes involved in both myeloid cell differentiation and embryonic development --- p.177 / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Selected recombinant plasmids --- p.180 / Chapter 4.4.2 --- Total RNAs --- p.180 / Chapter 4.2.3 --- Chemicals --- p.180 / Chapter 4.2.4 --- Enzymes and nucleic acids --- p.181 / Chapter 4.2.5 --- Kits --- p.181 / Chapter 4.2.6 --- Solutions --- p.181 / Chapter 4.3 --- Methods / Chapter 4.3.1 --- Preparation of selected recombinant plasmid DNA --- p.182 / Chapter 4.3.2 --- Sequencing --- p.182 / Chapter 4.3.3 --- Data analysis and assessment by ALF manager and DNAsis --- p.184 / Chapter 4.3.4 --- Sequence search by BLASTN program --- p.185 / Chapter 4.3.5 --- Primer design by Oligo´ёØ ver. 34 --- p.186 / Chapter 4.3.6 --- Differential expression confirmed by RT-PCR --- p.186 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Analysis of selected recombinant plasmid DNA --- p.187 / Chapter 4.4.2 --- Sequencing results --- p.191 / Chapter 4.4.3 --- BLASTN search results --- p.212 / Chapter 4.4.4 --- Primer design of the sequenced fragments --- p.222 / Chapter 4.4.5 --- "Expression profile of the isolated genes in midazolam-, biochanin A- induced JCS cells and mouse embryos" --- p.223 / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Sequence analysis of the isolated gene fragments --- p.233 / Chapter 4.5.2 --- Expression profiles of the isolated genes --- p.236 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Studies on leukemic cell differentiation / Chapter 5.1.1 --- Differentiation pathways revealed by different inducers --- p.241 / Chapter 5.1.2 --- Lineage preference during differentiation --- p.243 / Chapter 5.2 --- Differentiation program triggered by midazolam / Chapter 5.2.1 --- Signaling pathways initiated by biochanin A and midazolam --- p.245 / Chapter 5.2.2 --- Differentially expressed genes during midazolam-induced differentiation --- p.247 / Chapter 5.2.3 --- Expression patterns of the isolated differentially expressed genesin midazolam and biochanin A-induced JCS cells --- p.248 / Chapter 5.2.4 --- Myeloid genes in embryonic development --- p.250 / Chapter 5.3 --- Future studies of the isolated fragments --- p.252 / Chapter 5.4 --- Conclusion --- p.256 / Reference --- p.257 / Append --- p.ix / Chapter A1. --- Ambiguity codes for sequencing --- p.i / Chapter A2. --- Myeloid cell lines --- p.ii / Chapter A3. --- Details of manufacturer's products --- p.iii / Chapter A4. --- List of machine and equipment --- p.v

Cheng Chin Hung. / Thesis submitted in: December 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 138-145). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.i / ACKNOWLEDGMENT --- p.iv / ABBREVIATIONS --- p.v / ABSTRACT --- p.vi / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1 --- Endogenous Antisense RNAs --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Class --- p.2 / Chapter 1.3 --- Natural Antisense RNAs in Prokaryotes and Viruses --- p.3 / Chapter 1.4 --- Endogenous Antisense RNAs in Eukaryotes --- p.8 / Chapter 1.4.1 --- Distribution --- p.8 / Chapter 1.4.2 --- Conserved Pattern of Antisense Transcription --- p.10 / Chapter 1.5 --- Potential Functions of Antisense RNAs --- p.10 / Chapter 1.5.1 --- Template for Translation --- p.11 / Chapter 1.5.2 --- Regulation of Sense Gene Expression --- p.12 / Chapter 1.5.2.1 --- Nucleus --- p.13 / Chapter 1.5.2.1.1 --- Transcriptional Regulation --- p.13 / Chapter 1.5.2.1.2 --- Post-transcriptional Nuclear Regulation --- p.14 / Chapter 1.5.2.2 --- Cytoplasm --- p.16 / Chapter 1.5.2.2.1 --- Messenger Stability --- p.16 / Chapter 1.5.2.2.2 --- Translation --- p.17 / Chapter 1.6 --- Possible Mechanism of Antisense-mediated Regulation --- p.18 / Chapter 1.6.1 --- Two Possible Mechanisms --- p.18 / Chapter 1.7 --- Novel Endogenous Antisense RNA Against Cation Channel --- p.23 / Chapter 2 --- CNG1 Cation Channel --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Classification and Distribution of CNG Channels --- p.25 / Chapter 2.3 --- Structure of CNG Channels Gene Gamily --- p.27 / Chapter 2.4 --- Interactions Between CNG Channels and Ca2+ --- p.29 / Chapter 2.5 --- Distribution of CNG Channels in the Central Nervous System --- p.30 / Chapter 2.6 --- CNG Channels Function in CNS --- p.31 / Chapter 3 --- Aim of Study --- p.33 / Chapter Chapter Two: --- Materials and Methods --- p.35 / Chapter 4 --- Materials --- p.35 / Chapter 4.1 --- Library --- p.35 / Chapter 4.2 --- Multiple Tissue Blots --- p.35 / Chapter 4.3 --- Paraffin Sections --- p.35 / Chapter 5 --- Library Screening of Human Brain cDNA Library --- p.37 / Chapter 5.1 --- Amplification of Human Brain cDNA Library Stock --- p.38 / Chapter 5.2 --- Primary Screening --- p.38 / Chapter 5.3 --- Hybridization --- p.39 / Chapter 5.4 --- Secondary Screening --- p.40 / Chapter 5.5 --- Tertiary Screening --- p.40 / Chapter 6 --- Clones confirmation by Manual Sequencing --- p.41 / Chapter 6.1 --- Plasmid DNA Preparation --- p.41 / Chapter 6.2 --- DNA Sequencing --- p.41 / Chapter 6.3 --- Primer Walking Strategy --- p.44 / Chapter 7 --- Probe Preparation for Northern Blot and In-Situ Hybridization --- p.45 / Chapter 7.1 --- Probe for Anti-CNGl --- p.45 / Chapter 7.1.1 --- Enzyme Digestion --- p.45 / Chapter 7.1.2 --- Self-ligation --- p.47 / Chapter 7.1.3 --- Transformation --- p.47 / Chapter 7.1.4 --- Insert Confirmation --- p.48 / Chapter 7.1.5 --- Second Round Modification of cDNA Clone --- p.48 / Chapter 7.2 --- Probe for Sense CNG1 Gene --- p.49 / Chapter 7.2.1 --- RT-PCR Amplification from Cultured human Epithelial Cell Line ECV304 --- p.49 / Chapter 7.2.2 --- Automatic Sequencing --- p.49 / Chapter 7.2.3 --- Cloning of PCR Product --- p.50 / Chapter 7.2.4 --- Transformation --- p.50 / Chapter 7.2.5 --- Clone Confirmation --- p.50 / Chapter 8 --- Northern Hybridization --- p.51 / Chapter 8.1 --- Probe Linealization --- p.51 / Chapter 8.2 --- Labeling of Riboprobe with Radioisotope 32P --- p.53 / Chapter 8.3 --- Prehybridization and Hybridization with Radiolabeled RNA Probes --- p.54 / Chapter 9 --- In Situ Hybridization --- p.56 / Chapter 9.1 --- Preparation of Anti-CNGl Probe --- p.56 / Chapter 9.2 --- Preparation of Sense CNG1 Probe --- p.59 / Chapter 9.3 --- Testing of DIG-labeled RNA Probe --- p.61 / Chapter 9.4 --- Pre treatment --- p.61 / Chapter 9.5 --- "Prehybridization, Hybridization and Posthybridization" --- p.62 / Chapter 9.6 --- Colorimetric Detection of DIG Label --- p.63 / Chapter Chapter Three: --- Results --- p.64 / Chapter 10 --- Isolation and Sequence Analysis of cDNA Clones --- p.64 / Chapter 11 --- Northern Blot Analysis of anti-CNGl RNA in Human Brain Multiple Tissues --- p.72 / Chapter 11.1 --- Human Brain Blot IV --- p.72 / Chapter 11.2 --- Human Brain Blot II --- p.75 / Chapter 11.3 --- Human Multiple Tissues Blot --- p.77 / Chapter 12 --- In Situ Hybridization Analysis of anti-CNGl RNA Expression in Human Embryonic and Adult Brain Regions --- p.80 / Chapter 12.1 --- Expression of Anti-CNGl RNA in Human Embryonic Brain Regions… --- p.80 / Chapter 12.1.1 --- Hippocampus --- p.80 / Chapter 12.1.2 --- Frontal Cortex --- p.84 / Chapter 12.1.3 --- Visual Cortex --- p.88 / Chapter 12.2 --- Expression of Anti-CNGl RNA in Human Adult Brain Regions --- p.91 / Chapter 12.2.1 --- Occipital Cortex --- p.91 / Chapter 12.2.2 --- Frontal Cortex --- p.95 / Chapter 12.2.3 --- Hippocampus --- p.99 / Chapter 13 --- Expression of Sense CNG1 mRNA in Human Embryonic and Adult Brain Regions --- p.102 / Chapter 13.1 --- Expression of Sense CNG1 mRNA in Human Embryonic Brain Regions --- p.102 / Chapter 13.1.1 --- Frontal Cortex --- p.102 / Chapter 13.1.2 --- Visual Cortex --- p.107 / Chapter 13.1.3 --- Parahippocampus --- p.111 / Chapter 13.2 --- Expression of CNG1 mRNA in Human Adult Brain Region --- p.113 / Chapter 13.2.1 --- Frontal Cortex --- p.113 / Chapter Chapter Four: --- Discussion --- p.117 / Chapter 14.1 --- Cloning of Endogenous Anti-CNGl Transcript --- p.117 / Chapter 14.2 --- Neuron-specific Coexpression of Anti-CNGl and CNG1 Transcriptsin Central Nervous System --- p.124 / Chapter 15 --- Implications --- p.128 / Chapter 15.1 --- Endogenous Anti-CNGl Down-regulate Expression of CNG1 Channel --- p.128 / Chapter 15.2 --- Coordinated Co-expression of Sense and Antisense CNG1 Transcripts --- p.129 / Chapter 15.3 --- CNG1 Channel Functions in Human Nervous System --- p.130 / Chapter 15.3.1 --- CNG1 Channel Provides a Novel Ca2+ Entry Mode --- p.130 / Chapter 15.3.2 --- Activation of CNG1 Channel Through G-protein-linked Receptors --- p.130 / Chapter 15.3.3 --- Activation of CNG1 Channel Through Nitric Oxide --- p.131 / Chapter 15.3.4 --- Synaptic Plasticity and CNG1 channel --- p.131 / Chapter 15.3.5 --- A Role of CNG1 Channel in Development --- p.135 / Chapter 16 --- Conclusion --- p.136 / Chapter 17 --- Future Studies --- p.137 / References --- p.138

Yuen Chi Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 84-106). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xii / Symbols and Abbreviations --- p.xv / Scientific names --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Pituitary --- p.1 / Chapter 1.2 --- Gonadotropins (GTHs) --- p.3 / Chapter 1.2.1 --- Structure --- p.3 / Chapter 1.2.2 --- Function --- p.5 / Chapter 1.2.3 --- Regulation --- p.7 / Chapter 1.2.3.1 --- Neuroendocrine hypothalamic regulators --- p.9 / Chapter 1.2.3.1.1 --- Gonadotropin-releasing hormone (GnRH) --- p.9 / Chapter 1.2.3.1.2 --- Dopamine (DA) --- p.11 / Chapter 1.2.3.2 --- Endocrine regulators from the gonads --- p.12 / Chapter 1.2.3.2.1 --- Gonadal steroids (T and E2) --- p.12 / Chapter 1.2.3.2.2 --- Negative steroid effect on pituitary GTH regulation --- p.12 / Chapter 1.2.3.2.3 --- Positive steroid effect on pituitary GTH regulation --- p.13 / Chapter 1.2.3.3 --- Paracrine regulators from within the pituitary --- p.14 / Chapter 1.3 --- Activin --- p.14 / Chapter 1.3.1 --- Structure --- p.14 / Chapter 1.3.2 --- Function --- p.16 / Chapter 1.4 --- Follistatin (FS) --- p.17 / Chapter 1.4.1 --- Structure --- p.17 / Chapter 1.4.2 --- Function --- p.19 / Chapter 1.5 --- Temporal Variations in the GTH Expressional and Releasing Profile and Sex Steroid Level in the Goldfish --- p.19 / Chapter 1.5.1 --- Hormone changes during annual cycle --- p.20 / Chapter 1.5.2 --- Hormone changes during ovulatory cycle --- p.21 / Chapter 1.6 --- Objectives of the Present Study --- p.23 / Chapter Chapter 2 --- "Effects of Dopamine on the Expression of Gonadotropin (GTH) Subunits in the Dispersed Pituitary Cells of the Goldfish, Carassius auratus" --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Materials --- p.27 / Chapter 2.2.2 --- Primary culture of dispersed goldfish pituitary cells --- p.28 / Chapter 2.2.3 --- mRNA analysis --- p.29 / Chapter 2.2.4 --- Data analysis --- p.30 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Effects of DA on GTH-Iβ and GTH-IIβ expression --- p.30 / Chapter 2.3.2 --- Effects of DA D1 and D2 agonists on GTH-Iβ expression --- p.33 / Chapter 2.3.3 --- Effects of DA D1 and D2 antagonists on DA- inhibited GTH-Iβ expression --- p.33 / Chapter 2.3.4 --- Effects of α-adrenergic agonists on GTH-Iβ expression --- p.33 / Chapter 2.4 --- Discussion --- p.37 / Chapter Chapter 3 --- Seasonal Variation of Activin-regulated Goldfish Pituitary GTH-Ip and GTH-IIβ Expression and Evidence for the Involvement of Gonadal Steroids --- p.40 / Chapter 3.1 --- Introduction --- p.40 / Chapter 3.2 --- Materials and Methods --- p.42 / Chapter 3.2.1 --- Materials --- p.42 / Chapter 3.2.2 --- Gonadectomy of the goldfish --- p.42 / Chapter 3.2.3 --- Primary culture of dispersed pituitary cells --- p.43 / Chapter 3.2.4 --- mRNA analysis --- p.43 / Chapter 3.2.5 --- Data analysis --- p.44 / Chapter 3.3 --- Results --- p.44 / Chapter 3.3.1 --- Effects of goldfish activin B on the expression of GTH-Iβ and GTH-IIβ --- p.44 / Chapter 3.3.2 --- Seasonal variation of activin-regulated expression of GTH-Iβ and GTH-IIβ --- p.45 / Chapter 3.3.3 --- Effects of gonadectomy on basal and activin- regulated expression of GTH-Iβ and GTH-IIβ --- p.45 / Chapter 3.3.4 --- Effects of sex steroids on basal and activin- regulated expression of GTH-Iβ and GTH-IIβ in vitro --- p.50 / Chapter 3.4 --- Discussion --- p.57 / Chapter Chapter 4 --- Evidence for the Autocrine/Paracrine Regulation of Gonadotropin Expression by Activin in the Goldfish Pituitary --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Materials and Methods --- p.63 / Chapter 4.2.1 --- RNA isolation --- p.63 / Chapter 4.2.2 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.63 / Chapter 4.2.3 --- Primary culture of goldfish pituitary cells --- p.63 / Chapter 4.2.4 --- Slot-blot analysis --- p.64 / Chapter 4.2.5 --- Data analysis --- p.64 / Chapter 4.3 --- Results --- p.65 / Chapter 4.3.1 --- Expression of activin βB subunit and activin type IEB receptor in the goldfish pituitary --- p.65 / Chapter 4.3.2 --- Effects of human activin A on goldfish GTH-Iβ and GTH-IIβ expression --- p.65 / Chapter 4.3.3 --- Effects of follistatin on basal and activin- regulated GTH-Iβ and GTH-IIβ expression --- p.69 / Chapter 4.4 --- Discussion --- p.69 / Chapter Chapter 5 --- General Discussion --- p.77 / Chapter 5.1 --- Overview --- p.77 / Chapter 5.2 --- Contribution of the Present Study --- p.78 / Chapter 5.2.1 --- Dopamine as a potential neuroendocrine regulator in the differential regulation of GTH-Iβ and GTH-IIβ expression --- p.78 / Chapter 5.2.2 --- Seasonal variation of the effects of activin on GTH-Iβ and GTH-IIβ expression --- p.79 / Chapter 5.2.3 --- Autocrine/paracrine regulation of GTH expression by activin --- p.79 / Chapter 5.3 --- Future Prospects --- p.81 / References --- p.84

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

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 53-58).