Choi Wai-wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 120-138). / Abstracts in English and Chinese. / Statement --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / Abstract (Chinese Version) --- p.v / Table of Contents --- p.vii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- "Hemoglobin ´ؤ Structures, Types and Functions" --- p.1 / Chapter 1.1.1 --- Structures of Hemoglobin --- p.1 / Chapter 1.1.2 --- Types of Hemoglobin --- p.2 / Chapter 1.1.3 --- Functions of Hemoglobin --- p.3 / Chapter 1.2 --- Human Globin Genes and Their Regulation --- p.5 / Chapter 1.2.1 --- Organization of the Human Globin Genes --- p.5 / Chapter 1.2.2 --- Regulation of Globin Gene Expression --- p.6 / Chapter 1.2.2.1 --- The Locus Control Region (LCR) --- p.6 / Chapter 1.2.2.2 --- Cis-Regulatory Elements --- p.7 / Chapter 1.2.2.2.1 --- Promoters --- p.7 / Chapter 1.2.2.2.2 --- Enhancers --- p.7 / Chapter 1.2.2.2.3 --- Silencers --- p.8 / Chapter 1.2.2.3 --- Trans-Acting Factors --- p.8 / Chapter 1.2.2.3.1 --- GATA Family --- p.9 / Chapter 1.2.2.3.2 --- Kruppel-like Factors --- p.9 / Chapter 1.2.2.3.3 --- Nuclear Factor-Erythroid (NF-E) --- p.9 / Chapter 1.2.2.4 --- Chromatin Remodelling --- p.10 / Chapter 1.2.2.5 --- Intergenic Sequences --- p.11 / Chapter 1.3 --- Mechanisms of Hemoglobin Switching --- p.12 / Chapter 1.3.1 --- Autonomous Silencing --- p.12 / Chapter 1.3.2 --- LCR and Globin Gene Interaction --- p.12 / Chapter 1.4 --- Hemoglobinopathies --- p.14 / Chapter 1.4.1 --- α -thalassemia --- p.14 / Chapter 1.4.2 --- β -thalassemia --- p.14 / Chapter 1.4.3 --- Sickle Cell Anemia --- p.16 / Chapter 1.5 --- Therapies for β-thalassemia --- p.16 / Chapter 1.5.1 --- Blood Transfusion --- p.16 / Chapter 1.5.2 --- Bone Marrow Transplantation --- p.17 / Chapter 1.5.3. --- Gene Therapy --- p.17 / Chapter 1.6 --- Gene Switch Therapy --- p.18 / Chapter "1.6,1" --- Pharmacological Induction of HbF --- p.18 / Chapter 1.6.1.1 --- Hydroxyurea --- p.19 / Chapter 1.6.1.2 --- Butyrate --- p.20 / Chapter 1.6.1.3 --- Summary --- p.21 / Chapter 1.7 --- Objectives --- p.22 / Chapter Chapter 2 --- Induction of HbF by LC978 in K562 / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Materials --- p.26 / Chapter 2.2.1 --- Chemicals and Reagents --- p.26 / Chapter 2.2.2 --- Kits --- p.27 / Chapter 2.2.3 --- Buffers and Solutions --- p.27 / Chapter 2.2.4 --- Primers --- p.30 / Chapter 2.2.5 --- Equipment and Other Consumables --- p.30 / Chapter 2.2.6 --- Maintenance of K562 --- p.31 / Chapter 2.2.7 --- Handling and Treatment of utilities for RNA isolation --- p.31 / Chapter 2.3 --- Methods --- p.32 / Chapter 2.3.1 --- Dose-response and time-response study of LC978 in K562 by TMB assay --- p.32 / Chapter 2.3.2 --- Detection of γ -Globin Gene Expression in LC978-induced K562 by RT-PCR --- p.33 / Chapter 2.3.3 --- Fetal Hemoglobin Analysis by Human Fetal Hemoglobin (HbF) ELISA Quantitation Kit --- p.36 / Chapter 2.3.4 --- Statistical Analysis --- p.38 / Chapter 2.4 --- Results --- p.39 / Chapter 2.4.1 --- Dose-response and time-response study of LC978 in K562 by TMB assay --- p.39 / Chapter 2.4.2 --- Detection of γ -Globin Gene Expression in LC978-induced K562 by RT-PCR --- p.45 / Chapter 2.4.3 --- Fetal Hemoglobin Analysis by Human Fetal Hemoglobin (HbF) ELISA Quantitation Kit --- p.48 / Chapter 2.5 --- Discussions --- p.51 / Chapter Chapter 3 --- Signal Transduction Pathways Modulated by LC978 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials --- p.57 / Chapter 3.2.1 --- Chemicals and Reagents --- p.57 / Chapter 3.2.2 --- Kits --- p.57 / Chapter 3.2.3 --- Buffers and Solutions --- p.58 / Chapter 3.2.4 --- Primers --- p.59 / Chapter 3.2.5 --- Equipment and Other Consumables --- p.60 / Chapter 3.2.6 --- Maintenance of K562 --- p.60 / Chapter 3.2.7 --- Handling and Treatment of utilities for RNA isolation --- p.60 / Chapter 3.3 --- Methods --- p.61 / Chapter 3.3.1 --- Identification of Signaling Pathways by Microarray --- p.61 / Chapter 3.3.2 --- Real-time RT-PCR --- p.65 / Chapter 3.4 --- Results --- p.67 / Chapter 3.4.1 --- Identification of Signaling Pathways by Microarray --- p.67 / Chapter 3.4.2 --- Real-time RT-PCR --- p.74 / Chapter 3.5 --- Discussions --- p.80 / Chapter Chapter 4 --- MAPK pathways and HbF induction by LC978 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.2 --- Materials --- p.87 / Chapter 4.2.1 --- Chemicals and Reagents --- p.87 / Chapter 4.2.2 --- Kits --- p.88 / Chapter 4.2.3 --- Buffers and Solutions --- p.88 / Chapter 4.2.4 --- Equipment and Other Consumables --- p.90 / Chapter 4.2.5 --- Maintenance of K562 --- p.90 / Chapter 4.3 --- Methods --- p.91 / Chapter 4.3.1 --- "Roles of three MAPKs ´ؤ ERK, JNK and p38 in LC978-mediated γ -globin gene induction in K562 using CASE´ёØ Kits" --- p.91 / Chapter 4.3.2 --- Effect of p38 inhibitor SB203580 on HbF induction --- p.94 / Chapter 4.3.3 --- Statistical Analysis --- p.97 / Chapter 4.4 --- Results --- p.98 / Chapter 4.4.1 --- "Roles of three MAPKs - ERK, JNK and p38 in LC978-mediated γ -globin gene induction in K562 using CASETM Kits" --- p.98 / Chapter 4.4.2 --- Effect of p38 inhibitor SB203580 on HbF induction --- p.106 / Chapter 4.5 --- Discussions --- p.110 / Chapter Chapter 5 --- Summary and Prospects / Appendix / References

Made available in DSpace on 2016-01-26T12:51:33Z (GMT). No. of bitstreams: 1 joicematosbiselli_tese.pdf: 4248277 bytes, checksum: 46a8e90eea3be6a03251e5b3d7d8b0e5 (MD5) Previous issue date: 2011-11-28 / Fundação de Amparo a Pesquisa do Estado de São Paulo / Trisomy 21 is the genetic basis of Down syndrome (DS), the most common human chromosomal disorder. DS phenotype may include several dysmorphic features, intellectual disability, immunological alteration, congenital heart disease, high risk for specific types of leukemia and neurological alterations. There are basically two hypotheses to explain how the presence of three copies of chromosome 21 results in DS phenotype. The gene dosage effect hypothesis states that over-expression in about 50% of a specific gene or a group of genes located on chromosome 21 present in triplicate in DS individuals is directly responsible for DS features. The second hypothesis suggests the existence of secondary effects of trissomic genes that affect multiple metabolic pathways, resulting in cellular dysfunction. Recent studies show that trisomy 21 results in the over-expression of microRNAs, small molecules of noncoding RNA involved in post-transcriptional gene regulation, which could result in low expression of specific proteins and contribute to DS phenotype. Objective: To identify differentially expressed microRNAs in peripheral blood mononuclear cells of DS and non-DS children and to identify biological processes relevant to DS pathogenesis associated with predicted gene targets of microRNAs differentially expressed in DS children. Casuistic and Methods: Six children with free trisomy 21 and six control children were included in the study. Mature microRNAs were quantified using TaqMan® Low Density Arrays (Applied Biosystems), which enable the quantification of 754 mature microRNAs by real time quantitative polymerase chain reaction (qPCR) using fluorescent probes. The target prediction was performed using the software TargetScanHuman v. 5.2. Information about gene targets was obtained using the software Bioprocess, a database that obtains data from the National Center for Abstract xvi Biotechnology Information (NCBI). Results: Of the 490 mature microRNAs expressed in this cell type, 49 are low-expressed in DS group. The microRNAs located in chromosome 21 did not present differential expression between the groups. Bioinformatics analysis showed that genes involved in several relevant biological process to DS, including apoptosis, reactive oxygen species metabolism, mitochondrial metabolism, immune system, cell aging, cycle and division and control of gene expression, are predicted targets of microRNAs differentially expressed in DS children. Conclusion: DS children present low expression of microRNAs not located on chromosome 21 in peripheral blood mononuclear cells, as compared to children without DS. Biological processes relevant to DS pathogenesis are associated with predicted gene targets of microRNAs differentially expressed in DS children. / A trissomia do cromossomo 21 é a base genética da síndrome de Down (SD), a cromossomopatia humana mais frequente. O fenótipo da SD pode incluir várias características dismórficas, deficiência intelectual, alterações imunológicas, cardiopatias congênitas, risco aumentado para leucemias específicas, alterações neurológicas, entre outras. Existem basicamente duas hipóteses que tentam explicar como a presença de três cópias do cromossomo 21 resulta no fenótipo Down. De acordo com a hipótese do efeito da dosagem gênica , a expressão elevada em cerca de 50% de um gene específico ou de um grupo de genes do cromossomo 21 presente em triplicata em indivíduos com SD seria diretamente responsável pela manifestação de características da síndrome. A segunda hipótese sugere a existência de efeitos secundários de genes trissômicos, que afetariam múltiplas vias metabólicas, resultando em disfunção celular. Estudos recentes mostram que a trissomia do cromossomo 21 resulta na expressão elevada de microRNAs, pequenas moléculas de RNAs nãocodificantes envolvidos na regulação gênica pós-transcricional, o que pode levar à redução da expressão de proteínas específicas e contribuir para o fenótipo da SD. Objetivo: Identificar microRNAs diferencialmente expressos em células mononucleares do sangue periférico de crianças com SD em relação a crianças sem a síndrome e identificar processos biológicos relevantes para a patogênese da SD associados a genes-alvo preditos de microRNAs diferencialmente expressos em crianças com SD. Casuística e Métodos: Foram incluídas no estudo seis crianças com trissomia livre do cromossomo 21 e seis crianças sem a síndrome. A quantificação de microRNAs maduros foi realizada utilizando-se TaqMan® Low Density Arrays (Applied Biosystems), que possibilita a investigação da expressão de 754 microRNAs maduros Resumo xiv pelo método de reação em cadeia da polimerase quantitativa (PCRq) fluorescente em tempo real. A predição de genes-alvo dos microRNAs foi realizada utilizando-se o programa TargetScanHuman v. 5.2. Para obtenção de informações sobre os genes-alvo preditos foi utilizada a ferramenta Bioprocess, um banco de dados alimentado com informações do National Center for Biotechnology Information (NCBI). Resultados: Dos 490 microRNAs maduros expressos no tipo celular investigado, 49 apresentaram expressão reduzida no grupo de crianças com SD. Os microRNAs localizados no cromossomo 21 não apresentaram expressão diferencial entre os grupos. A análise de Bionformática revelou que genes envolvidos em diversos processos biológicos relevantes para a SD, tais como apoptose, metabolismo de espécies reativas de oxigênio, metabolismo mitocondrial, sistema imunológico, envelhecimento, ciclo e divisão celular e controle da expressão gênica, são alvos preditos de microRNAs diferencialmente expressos em crianças com SD. Conclusão: Crianças com SD apresentam expressão reduzida de microRNAs não localizados no cromossomo 21 em células mononucleares do sangue periférico, em relação a crianças sem a síndrome. Processos biológicos relevantes para a patogênese da SD estão associados a genes-alvo preditos de microRNAs diferencialmente expressos em crianças com SD.

Lin Sze-Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 111-127). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v / Abbreviations --- p.x / Scientific Names --- p.xii / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Gonadotropins --- p.1 / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Function --- p.1 / Chapter 1.1.3 --- Regulation --- p.2 / Chapter 1.1.3.1 --- Gonadotropin-releasing hormone (GnRH) --- p.3 / Chapter 1.1.3.2 --- Dopamine --- p.4 / Chapter 1.1.3.3 --- Sex steroids --- p.5 / Chapter 1.1.3.3.1 --- Functions --- p.5 / Chapter 1.1.3.3.2 --- Working mechanism´ؤEstrogen signaling pathway --- p.7 / Chapter 1.1.3.4 --- Gonadal peptides --- p.9 / Chapter 1.1.3.4.1 --- Functions --- p.9 / Chapter 1.1.3.4.2 --- Working mechanism一Activin signaling pathway --- p.11 / Chapter 1.2 --- Transcriptional regulation of pituitary gonadotropin subunit genes at the promoter level --- p.13 / Chapter 1.2.1 --- Transcriptional regulation of mammalian glycoprotein a subunits --- p.13 / Chapter 1.2.1.1 --- GnRH --- p.14 / Chapter 1.2.1.2 --- Activin --- p.15 / Chapter 1.2.1.3 --- Steroids --- p.15 / Chapter 1.2.2 --- Transcriptional regulation of mammalian FSHβ and LHβ subunits --- p.16 / Chapter 1.2.2.1 --- Regulation of LHβ expression by GnRH --- p.17 / Chapter 1.2.2.1.1 --- Roles of SP-1 binding sites on LHβ promoter --- p.17 / Chapter 1.2.2.1.2 --- Effect of SF-1 on LHp expression --- p.17 / Chapter 1.2.2.1.3 --- Effect of Egr-1 on LHp expression --- p.18 / Chapter 1.2.2.1.4 --- "Synergistic effect ofSP-1, SF-1 and Egr-1 on LHp expression." --- p.18 / Chapter 1.2.2.1.5 --- Effect of Pitx-1 on LHβ expression --- p.19 / Chapter 1.2.2.1.6 --- "Effect of SF-1, Egr-1 and Pitx-1 on LHβ expression of other mammalian counterparts" --- p.19 / Chapter 1.2.2.1.7 --- Effect of other transcription factors on mammalian LHβ expression --- p.19 / Chapter 1.2.2.2 --- Regulation of LHβ expression by steroids and activin --- p.20 / Chapter 1.2.2.3 --- Regulation of FSHβ expression by activin and GnRH --- p.20 / Chapter 1.2.2.4 --- Regulation of FSHβ expression by steroids --- p.21 / Chapter 1.2.2.5 --- Regulation of FSHβ expression by other transcription factors --- p.22 / Chapter 1.2.3 --- Transcriptional regulation of fish FSHβ and LHβ subunits --- p.22 / Chapter 1.3 --- The project objectives and long-term significance --- p.24 / Chapter CHAPTER 2 --- CLONING OF ZEBRAFISH FSHB AND LHB PROMOTERS. --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Chemicals --- p.27 / Chapter 2.2.2 --- Animals --- p.27 / Chapter 2.2.3 --- Isolation of genomic DNA --- p.28 / Chapter 2.2.4 --- Cloning of promoters of zebrafish FSHβ and LHβ from the genomic DNA --- p.28 / Chapter 2.2.5 --- Construction of the reporter plasmids containing zebrafish FSHβ and LHβ promoters --- p.30 / Chapter 2.2.6 --- Cell culture and transient transfection --- p.31 / Chapter 2.2.7 --- SEAP reporter gene assay --- p.32 / Chapter 2.2.8 --- β-galactosidase reporter gene assay --- p.32 / Chapter 2.2.9 --- Data analysis --- p.33 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Cloning of zebrafish FSHβ and LHβ promoters --- p.33 / Chapter 2.3.2 --- Sequence characterization of zebrafish FSHβ and LHβ promoters --- p.34 / Chapter 2.3.3 --- Basal FSHp and LHβ promoter activities in LβT2 cells --- p.35 / Chapter 2.4 --- Discussion --- p.36 / Chapter CHAPTER 3 --- ROLES OF ACTIVIN/SMADS AND ESTROGEN/ERS IN THE REGULATION OF ZEBRAFISH FSHB AND LHB PROMOTER ACTIVITY --- p.51 / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Chemicals --- p.56 / Chapter 3.2.2 --- Animals --- p.56 / Chapter 3.2.3 --- Isolation of total RNA --- p.57 / Chapter 3.2.4 --- Rapid amplification of full-length cDNA (RACE) --- p.57 / Chapter 3.2.5 --- Construction of expression plasmids --- p.57 / Chapter 3.2.6 --- cell culture and transient transfection --- p.59 / Chapter 3.2.7 --- SEAP reporter gene assay --- p.59 / Chapter 3.2.8 --- p-galactosidase reporter gene assay --- p.59 / Chapter 3.2.9 --- Data analysis --- p.59 / Chapter 3.3 --- Results --- p.60 / Chapter 3.3.1 --- Cloning and sequence characterization of zebrafish Smad 4 (zfSmad 4) --- p.60 / Chapter 3.3.2 --- Smads regulate FSHβ transcription in LβT2 cells --- p.61 / Chapter 3.3.3 --- Smads regulate LHβ transcription in LPβT2 cells --- p.61 / Chapter 3.3.4 --- Functionality of the two forms of Smad 4 cloned --- p.62 / Chapter 3.3.5 --- Estrogen and ERs regulate zJFSHβ transcription in LβT2 cells --- p.63 / Chapter 3.3.6 --- Estrogen and ERs regulate zfLHβ transcription in LβT2 cells --- p.63 / Chapter 3.4 --- Discussion --- p.64 / Chapter CHAPTER 4 --- PROMOTER ANALYSIS FOR SMAD RESPONSIVE ELEMENT AND ESTROGEN RESPONSIVE ELEMENT IN ZEBRAFISH FSHB AND LHB PROMOTERS --- p.82 / Chapter 4.1 --- Introduction --- p.83 / Chapter 4.2 --- Materials and Methods --- p.85 / Chapter 4.2.1 --- Chemicals and animals --- p.85 / Chapter 4.2.2 --- Construction of SEAP reporter plasmids containing different lengths of zfFSHβ promoter --- p.85 / Chapter 4.2.3 --- Construction of SEAP reporter plasmids containing different lengths of zfLHβ promoter --- p.85 / Chapter 4.2.4 --- Site-directed mutagenesis --- p.86 / Chapter 4.2.5 --- cell culture and transient transfection --- p.87 / Chapter 4.2.6 --- SEAP reporter gene assay --- p.87 / Chapter 4.2.7 --- P-galactosidase reporter gene assay --- p.87 / Chapter 4.2.8 --- Data analysis --- p.88 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Localization of Smad-responsive element (SRE) on zfFSHβ promoter --- p.88 / Chapter 4.3.2 --- Localization of estrogen-responsive element (ERE) on zfLHβ promoter --- p.89 / Chapter 4.3.3 --- Localization of estrogen-responsive element (ERE) on zfFSHβ promoter --- p.90 / Chapter 4.3.4 --- Confirmation of SRE by site-directed mutagenesis --- p.91 / Chapter 4.3.5 --- Confirmation of ERE by site-directed mutagenesis --- p.92 / Chapter 4.4 --- Discussion --- p.92 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.106 / Chapter 5.1 --- Overview --- p.106 / Chapter 5.2 --- Contribution of the present research --- p.107 / Chapter 5.3 --- Future research direction --- p.108 / REFERENCE: --- p.111

by Cheung Kwok Kuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 111-130). / Chapter Chapter 1 --- General introduction of human ovarian cancer / Chapter 1.1 --- Epidemiology --- p.1 / Chapter 1.2 --- Symptoms and diagnosis --- p.4 / Chapter 1.3 --- Etiology --- p.5 / Chapter 1.3.1 --- Factors associated with decreased risks --- p.6 / Chapter 1.3.2 --- Factors associated with increased risks --- p.8 / Chapter 1.4 --- Classification of ovarian cancer --- p.12 / Chapter 1.5 --- Molecular basis of ovarian cancer --- p.18 / Chapter 1.6 --- Project aim --- p.29 / Chapter Chaper 2 --- "DOC-2, a differentially expressed gene in human ovarian cancer" / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and Methods --- p.35 / Chapter 2.2.1 --- Expression of DOC-2 in human ovarian tissues --- p.35 / Chapter 2.2.1.1 --- Preparation of specimen --- p.35 / Chapter 2.2.1.2 --- Immunohistochemical studies of the expression of DOC-2 protein in human ovarian tissues --- p.35 / Chapter 2.2.1.3 --- Quantitation of immunoreactivity --- p.38 / Chapter 2.2.2 --- Effect of DOC-2 transfection on growth rate of the ovarian cancer cell lineSKOV3 --- p.39 / Chapter 2.2.2.1 --- Cell line --- p.39 / Chapter 2.2.2.2 --- Transfection of DOC-2 to SKOV3 ovarian carcinoma cell line --- p.39 / Chapter 2.2.2.3 --- Growth curve of the transfected ovarian carcinoma cell lines --- p.40 / Chapter 2.2.3 --- In vivo tumorigenicity study --- p.42 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- Expression of DOC-2 in human ovarian tissues --- p.44 / Chapter 2.3.2 --- Effects of DOC-2 transfected gene on the growth rate of the human ovarian cancer cell line SKOV3 --- p.46 / Chapter 2.3.2.1 --- Standard curves for calculating cell density from absorbance --- p.46 / Chapter 2.3.2.2 --- The effect of DOC-2 transfection on the growth rate of the human ovarian cancer cell line SKOV3 --- p.47 / Chapter 2.3.3 --- In vivo tumorigenicity --- p.48 / Chapter 2.4 --- Discussion --- p.50 / Chapter Chapter 3 --- DOC-2 expression in mouse embryonic development / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.60 / Chapter 3.2.1 --- Expression of murine homolog of DOC-2 (p96) during mouse embryonic development --- p.60 / Chapter 3.2.1.1 --- Preparation of paraffin-embedded mouse embryo sections --- p.60 / Chapter 3.2.1.2 --- Preparation of OCT-embedded mouse embryo sections --- p.61 / Chapter 3.2.1.3 --- Immunohistochemistry of murine homolog of DOC-2 (p96) on mouse embryos --- p.61 / Chapter 3.2.2 --- Effect of antibody blocking for DOC-2 protein on the growth of embryonic kidney in vitro --- p.62 / Chapter 3.3 --- Results --- p.64 / Chapter 3.4 --- Discussion --- p.69 / Chapter Chapter 4 --- Apoptosis / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.1.1 --- Current methods for the detection of apoptosis --- p.74 / Chapter 4.1.1.1 --- Agarose gel electrophoresis --- p.75 / Chapter 4.1.1.2 --- Flow cytometric analysis --- p.76 / Chapter 4.1.1.3 --- 3-OH end labelling --- p.77 / Chapter 4.1.1.4 --- Nuclease assay --- p.78 / Chapter 4.1.2 --- Apoptosis in normal physiology and oncogenesis --- p.78 / Chapter 4.1.3 --- p53 and apoptosis --- p.80 / Chapter 4.1.4 --- bcl-2 and apoptosis --- p.83 / Chapter 4.2 --- Materials and Methods --- p.92 / Chapter 4.2.1 --- Expression of p53 and bcl-2 in human ovarian tissues --- p.92 / Chapter 4.2.1.1 --- Preparation of specimens --- p.92 / Chapter 4.2.1.2 --- Immunohistochemical studies of the expression of p53 and bcl-2 proteins in ovarian tissue --- p.92 / Chapter 4.2.2 --- In stiu terminal transferase-mediated dUTP nick and labelling (TUNEL) --- p.94 / Chapter 4.3 --- Results --- p.96 / Chapter 4.3.1 --- Expression of p53 and bcl-2 in human ovarian tissues --- p.96 / Chapter 4.3.2 --- Apoptosis in human ovarian tissues --- p.99 / Chapter 4.4 --- Discussion --- p.101 / Chapter Chapter 5 --- Concluding Remarks --- p.108 / References --- p.111 / Appendix --- p.131 / Figures and legend --- p.138

Cheuk, Wai Ka. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 96-108). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Abbreviations --- p.xii / Chapter CHAPTER 1 --- General introduction / Chapter 1.1 --- Metal Contaminations in the environment --- p.1 / Chapter 1.2 --- Biology of Heavy Metal Ions --- p.3 / Chapter 1.2.1 --- Essential and non-essential metal ions --- p.3 / Chapter 1.2.2 --- Toxicities and origins of heavy metal ions --- p.5 / Chapter 1.3 --- Monitoring Of Heavy Metal Contaminations In Aquatic Environment --- p.9 / Chapter 1.3.1 --- Monitoring in chemical approach --- p.9 / Chapter 1.3.2 --- Monitoring in biological approach: biomarkers --- p.11 / Chapter 1.4 --- Metallothionein (MT) --- p.12 / Chapter 1.4.1 --- Biological functions of MT and its regulation --- p.12 / Chapter 1.4.2 --- MT isoforms --- p.14 / Chapter 1.4.3 --- Mechanisms of MT gene regulation --- p.15 / Chapter 1.4.3.1 --- Zinc pool hypothesis --- p.20 / Chapter 1.4.3.2 --- Protein kinase cascade --- p.21 / Chapter 1.5 --- Metal responsive element (MRE) --- p.22 / Chapter 1.6 --- MRE-Binding Transcription Factor-1 (MTF-1) --- p.30 / Chapter 1.6.1 --- Structure of MTF-1 --- p.30 / Chapter 1.6.2 --- Physiological functions of MTF-1 --- p.32 / Chapter 1.6.3 --- The role of MTF-1 in MT gene regulation --- p.33 / Chapter 1.6.4 --- Regulation of MTF-1 by various heavy metals --- p.34 / Chapter 1.7 --- Zebrafish (Daino reio) --- p.36 / Chapter 1.8 --- Project aim --- p.37 / Chapter CHAPTER 2 --- Materials and Methods / Chapter 2.1 --- Cell Culture --- p.40 / Chapter 2.1.1 --- ZFL cell line --- p.40 / Chapter 2.1.2 --- SJD cell line --- p.41 / Chapter 2.2 --- Alarmar blue̐ưؤ M assay --- p.41 / Chapter 2.3 --- First strand cDNA synthesis --- p.42 / Chapter 2.3.1 --- Metal treatment of the SJD and ZFL cell lines --- p.42 / Chapter 2.3.2 --- Isolation of total RNA --- p.43 / Chapter 2.3.3 --- Quantification of mRNA by spectrophotometer --- p.43 / Chapter 2.3.4 --- Reverse Transcription --- p.44 / Chapter 2.4 --- Quantifications of mRNA levels by using real-time PCR technique --- p.44 / Chapter 2.4.1 --- Primer design --- p.44 / Chapter 2.4.2 --- PCR components and cycling condition --- p.45 / Chapter 2.4.3 --- Determination of relative amount of target gene present in the samples --- p.49 / Chapter 2.5 --- Cloning of zMT-II gene promoter and its transient expression studies --- p.50 / Chapter 2.5.1 --- Purification of genomic DNA --- p.50 / Chapter 2.5.2 --- Preparation of Escherichia coli competent cell --- p.51 / Chapter 2.5.3 --- PCR-Cloning of a 1.4 kb zMT-II gene promoter --- p.51 / Chapter 2.5.4 --- Purification of plasmid DNA --- p.53 / Chapter 2.5.5 --- Transient transfection of plasmid into SJD and ZFL cells --- p.54 / Chapter 2.5.6 --- Heavy metal treatments and measurement of luciferase activities --- p.54 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- Toxicities of various heavy metal ions --- p.56 / Chapter 3.2 --- Relative mRNA fold induction of zMT in SJD and ZFL cell lines --- p.59 / Chapter 3.3 --- The zMT-II gene and its induction by metal ions in zebrafish cell-lines --- p.63 / Chapter 3.4 --- MTF-1 mRNA levels in SJD and ZFL cell lines exposed to heavy metal ions --- p.74 / Chapter CHAPTER 4 --- Discussion / Chapter 4.1 --- Comparison of metal toxicities in the two cell lines studied --- p.78 / Chapter 4.2 --- zMT gene expression study --- p.80 / Chapter 4.2.1 --- zMT mRNA regulation by heavy metal ions in the two cell lines --- p.80 / Chapter 4.2.2 --- The potential use of MT regulation as exposure biomarker --- p.82 / Chapter 4.3 --- Structure of the zMT-II gene promoter region --- p.82 / Chapter 4.4 --- Metal responsiveness of zMT-II promoter --- p.84 / Chapter 4.5 --- Mechanism of MT gene expression and the MTF-1 mRNA inductions in SJD and ZFL cell lines --- p.86 / Chapter 4.6 --- Concluding Remarks --- p.93 / References --- p.96

<p>The <i>Murine Pneumotropic Virus </i>(MPtV), in contrast to the other <i>MurinePolyomavirus</i> (MPyV), appears to be non-tumourigenic in its natural host. Instead, MPtV causes acute pneumonia and can serve as a model in studies of polyomavirus-induced disease. In initial experiments, MPtV large T-antigen (LT) was expressed in a heterologous system. LT was characterized with regard to its metabolic stability and cell immortalizing activity and, after purification, to its specific DNA binding. </p><p>The absence of permissive cell culture system for MPtV has hampered its study. We made attempts to widen the host range of the virus by modifying the regulatory and late regions of the genome. The enhancer substitution mutant (KVm1), having a transcriptional enhancer substituted with a corresponding DNA segment from MPyV, was able to replicate in mouse 3T3 cells and form virus particles that were infectious in mice. However, efficient infection of cells in vitro was not achieved with this mutant virus, possibly due to the absence of virus-specific receptors on the cells. The capsid protein substitution mutants, having capsid protein genes of MPyV, for which receptors are present on a variety of cell types, showed also no cytopathic effect, despite an enhanced viral DNA replication and assembly of virus particles. </p><p>MPtV-DNA extracted from virus in lung tissue of infected mice had a heterogeneous enhancer segment. A majority of the DNA molecules had a structure differing from the standard-type. A 220 base-pair insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was a prominent variation. Other genome variants showed complete or partial deletions of the insertion and surrounding sequences in the viral enhancer. In relation to the standard-type, all variant genomes showed differences in the activities of transcriptional promoters and the origin DNA replication. Analysis by DNA reassociation showed that a large number of nucleotide sequences related to the 220 base-pair insert in the MPtV genome were present in mouse and human DNA, but not in <i>Escherichia coli</i> DNA. Together, the data suggest that the 220 base-pair insertion is related to a transposable element of a novel type.</p>

<p>In summary there currently exist techniques to discover miRNA however both require many calculations to be performed during the identification limiting their use at a genomic level. Machine learning techniques are currently providing the best results by combining a number of calculated and statistically derived features to identify miRNA candidates, however almost all of these still include computationally intensive secondary-structure calculations. It is the aim of this project to produce a miRNA identification process that minimises and simplifies the number of computational elements required during the identification process.</p>

The Murine Pneumotropic Virus (MPtV), in contrast to the other MurinePolyomavirus (MPyV), appears to be non-tumourigenic in its natural host. Instead, MPtV causes acute pneumonia and can serve as a model in studies of polyomavirus-induced disease. In initial experiments, MPtV large T-antigen (LT) was expressed in a heterologous system. LT was characterized with regard to its metabolic stability and cell immortalizing activity and, after purification, to its specific DNA binding. The absence of permissive cell culture system for MPtV has hampered its study. We made attempts to widen the host range of the virus by modifying the regulatory and late regions of the genome. The enhancer substitution mutant (KVm1), having a transcriptional enhancer substituted with a corresponding DNA segment from MPyV, was able to replicate in mouse 3T3 cells and form virus particles that were infectious in mice. However, efficient infection of cells in vitro was not achieved with this mutant virus, possibly due to the absence of virus-specific receptors on the cells. The capsid protein substitution mutants, having capsid protein genes of MPyV, for which receptors are present on a variety of cell types, showed also no cytopathic effect, despite an enhanced viral DNA replication and assembly of virus particles. MPtV-DNA extracted from virus in lung tissue of infected mice had a heterogeneous enhancer segment. A majority of the DNA molecules had a structure differing from the standard-type. A 220 base-pair insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was a prominent variation. Other genome variants showed complete or partial deletions of the insertion and surrounding sequences in the viral enhancer. In relation to the standard-type, all variant genomes showed differences in the activities of transcriptional promoters and the origin DNA replication. Analysis by DNA reassociation showed that a large number of nucleotide sequences related to the 220 base-pair insert in the MPtV genome were present in mouse and human DNA, but not in Escherichia coli DNA. Together, the data suggest that the 220 base-pair insertion is related to a transposable element of a novel type.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 2, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 107-112). Also available on the Internet.