Pannexin 1 (Panx1) is a metabolite channel enriched in the brain and known to localize to the cell surface, where it is involved in a variety of neuronal processes including cell proliferation and differentiation. The mechanisms through which Panx1 is trafficked or stabilized at the surface, however, are not fully understood. The proximal Panx1 C-terminus (Panx1CT), upstream of a caspase-cleavage site has been demonstrated to be required for Panx1 cell-surface expression. We discovered a previously unreported putative leucine-rich repeat (LRR) motif within the proximal Panx1CT. I investigated the involvement of this putative LRR motif on Panx1 localization and oligomerization. Deletion of the putative LRR motif or uniquely the highly conserved segment of the putative LRR motif resulted in a significant loss of Panx1 cell surface expression. Finally, ectopic expression of Panx1-EGFP in HEK293T cells increased cell proliferation, which was not recapitulated by a Panx1 deletion mutant lacking the putative LRR motif. Overall the findings presented in this thesis provide new insights into the molecular determinants of Panx1 trafficking and oligomerization. / Graduate / 2020-02-14

Chan Wan Lui Wendy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 158-162). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Abbreviations --- p.ix / List of Figures --- p.xii / List of Tables --- p.xvi / Table of Contents --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.3 / Chapter 2.1 --- Malaria --- p.3 / Chapter 2.2 --- History of malaria --- p.4 / Chapter 2.3 --- Malaria parasites --- p.4 / Chapter 2.4 --- Life cycle --- p.5 / Chapter 2.5 --- Potential use of malaria vaccine --- p.6 / Chapter 2.6 --- Merozoite surface protein 1 (MSP1) --- p.7 / Chapter 2.7 --- Potential use of MSPl --- p.8 / Chapter 2.8 --- Significance of MSPl C-terminal fragments --- p.9 / Chapter 2.8.1 --- Significance of MSP142 --- p.9 / Chapter 2.8.2 --- Significance of MSP119 --- p.11 / Chapter 2.9 --- Production of MSPl C-terminal fragments --- p.12 / Chapter 2.10 --- Plants as bioreactors --- p.12 / Chapter 2.11 --- Expression of MSPl C-terminal fragments in transgenic plants --- p.14 / Chapter 2.12 --- Phaseolin and its sorting signal --- p.19 / Chapter 2.13 --- Protein targeting signals --- p.20 / Chapter Chapter 3 --- Material and methods --- p.23 / Chapter 3.1 --- Introduction --- p.23 / Chapter 3.2 --- Chemical and enzymes --- p.23 / Chapter 3.3 --- Cloning --- p.24 / Chapter 3.3.1 --- MSP142 and MSP119 constructs --- p.24 / Chapter 3.3.2 --- Protein targeting fusion constructs --- p.24 / Chapter 3.3.3 --- GUS fusion Constructs --- p.30 / Chapter (a) --- Particle bombardment --- p.30 / Chapter (b) --- GUS fusion constructs for plant transformation --- p.32 / Chapter (c) --- Modified GUS fusion constructs --- p.38 / Chapter 3.4 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.39 / Chapter 3.4.1 --- Cloning of pSUNl --- p.40 / Chapter 3.4.2 --- Primer sequence --- p.45 / Chapter 3.5 --- Bacterial strains --- p.46 / Chapter 3.6 --- Particle bombardment --- p.46 / Chapter 3.6.1 --- Plant materials --- p.46 / Chapter 3.6.2 --- Microcarrier preparation and coating DNA onto microcarrier --- p.46 / Chapter 3.6.3 --- GUS assay --- p.48 / Chapter 3.7 --- Transgenic expression in Arabidopsis thaliana --- p.49 / Chapter 3.7.1 --- Plant materials --- p.49 / Chapter 3.7.2 --- Agrobacterium transformation --- p.49 / Chapter 3.7.3 --- Vacuum infiltration Arabidopsis transformation --- p.49 / Chapter 3.7.4 --- Selection of successful transformants --- p.50 / Chapter 3.7.5 --- Selection for homozygous plants --- p.51 / Chapter 3.8 --- Transgenic expression in tobacco --- p.51 / Chapter 3.8.1 --- Plant materials --- p.51 / Chapter 3.8.2 --- Agrobacterium transformation --- p.52 / Chapter 3.8.2.1 --- Preparation of Agrobacterium tumefaciens LBA4401 competent cells --- p.52 / Chapter 3.8.3 --- Leaf discs method for tobacco transformation --- p.53 / Chapter 3.8.4 --- GUS staining --- p.54 / Chapter 3.9 --- DNA analysis --- p.55 / Chapter 3.9.1 --- Genomic DNA extraction --- p.55 / Chapter 3.9.2 --- Genomic PCR --- p.55 / Chapter 3.9.3 --- Southern blot --- p.55 / Chapter 3.10 --- RNA analysis --- p.56 / Chapter 3.10.1 --- RNA extraction --- p.56 / Chapter 3.10.2 --- Northern blot --- p.56 / Chapter 3.11 --- Protein analysis --- p.57 / Chapter 3.11.1 --- Protein extraction --- p.57 / Chapter 3.11.2 --- Western blot --- p.58 / Chapter 3.11.3 --- Western blot analysis --- p.58 / Chapter Chapter 4 --- Results --- p.60 / Chapter 4.1 --- Transient assay of gene expression of MSP142 and MSPl19 --- p.60 / Chapter 4.1.1 --- Construction of the GUS fusion constructs --- p.60 / Chapter 4.1.2 --- Particle Bombardment --- p.63 / Chapter 4.2 --- Transgenic analysis of MSP142 and MSPl19 expression --- p.70 / Chapter 4.2.1 --- MSPl42 and MSPl19 constructs and transformation --- p.70 / Chapter 4.2.2 --- Selection of transgenic plants --- p.71 / Chapter 4.2.3 --- Southern analysis --- p.75 / Chapter 4.2.4 --- Northern analysis --- p.77 / Chapter 4.2.5 --- Western analysis --- p.79 / Chapter 4.3 --- Expression of the protein-targeting and GUS fused modified MSP1 constructs --- p.81 / Chapter 4.3.1 --- Construction of the fusion constructs --- p.81 / Chapter (A) --- Protein-targeting constructs --- p.81 / Chapter (B) --- GUS fusion constructs --- p.90 / Chapter B1. --- Constructs for transient assay --- p.90 / Chapter B2. --- Modification of GUS sequence --- p.96 / Chapter B3. --- Constructs for tobacco transformation --- p.100 / Chapter 4.4 --- Transient assay of GUS fused MP42 and MP19 constructs by particle Bombardment --- p.107 / Chapter 4.4.1 --- The GUS fusion constructs --- p.107 / Chapter 4.4.2 --- Modification of GUS --- p.112 / Chapter 4.5 --- Generation of transgenic tobacco --- p.116 / Chapter 4.6 --- Southern analysis --- p.120 / Chapter 4.7 --- Northern analysis --- p.126 / Chapter (A) --- Protein-targeting constructs --- p.126 / Chapter (B) --- GUS fusion constructs --- p.130 / Chapter 4.8 --- Western analysis --- p.133 / Chapter (A) --- Protein-targeting constructs --- p.133 / Chapter (B) --- GUS fusion constructs --- p.139 / Chapter Chapter 5 --- Discussion --- p.146 / Chapter Chapter 6 --- Conclusion --- p.157 / References --- p.158

by Lau On Sun. / Thesis submitted in: November 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 117-127). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Abbreviations --- p.viii / Table of Contents --- p.x / List of Figures --- p.xiii / List of Tables --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Malaria --- p.3 / Chapter 2.1.1 --- Global situation --- p.3 / Chapter 2.1.2 --- Malaria parasite and its life cycle --- p.4 / Chapter 2.1.3 --- Need for a malarial vaccine --- p.5 / Chapter 2.2 --- Merozoite surface protein 1 and its fragments - the advanced malaria vaccine candidate --- p.7 / Chapter 2.2.1 --- Basic research on MSP1 --- p.7 / Chapter 2.2.2 --- Vaccine research on MSP1 --- p.8 / Chapter 2.3 --- Transgenic plants as recombinant protein production systems --- p.11 / Chapter 2.3.1 --- Characteristics --- p.11 / Chapter 2.3.2 --- Plant-based vaccine --- p.13 / Chapter 2.4 --- Expression of MSP 1 C-terminal fragments in transgenic plants --- p.15 / Chapter 2.4.1 --- Previous studies --- p.15 / Chapter 2.4.2 --- Plant-optimized MSP142 cDNA --- p.18 / Chapter 2.5 --- Phaseolin: its promoter and vacuolar-sorting signal --- p.20 / Chapter 2.6 --- Sorting of soluble protein to vacuoles in plants --- p.22 / Chapter 2.7 --- Winged bean lysine-rich protein and translational fusion strategy --- p.24 / Chapter 2.8 --- Hypotheses and aims of study --- p.26 / Chapter Chapter 3: --- Materials and Methods --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Chemicals --- p.28 / Chapter 3.3 --- Bacterial strains --- p.28 / Chapter 3.4 --- Chimeric gene construction --- p.29 / Chapter 3.4.1 --- Construction of the lysine-rich protein fusion constructs --- p.33 / Chapter 3.4.2 --- Construction of the phaseolin-targeting constructs --- p.37 / Chapter 3.4.3 --- Confirmation of sequence fidelity of chimeric genes --- p.42 / Chapter 3.4.4 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.42 / Chapter 3.5 --- Transgenic expression in Arabidopsis and tobacco --- p.44 / Chapter 3.5.1 --- Plant materials --- p.44 / Chapter 3.5.2 --- Agrobacterium transformation --- p.44 / Chapter 3.5.3 --- Arabidopsis transformation and selection --- p.45 / Chapter 3.5.4 --- Tobacco Transformation and Selection --- p.47 / Chapter 3.5.5 --- Genomic DNA isolation --- p.49 / Chapter 3.5.6 --- Southern blot analysis --- p.49 / Chapter 3.5.7 --- Total silique RNA isolation --- p.50 / Chapter 3.5.8 --- Northern blot analysis --- p.50 / Chapter 3.5.9 --- Protein extraction and SDS-PAGE --- p.51 / Chapter 3.5.10 --- Western blot analysis --- p.52 / Chapter 3.5.11 --- Enterokinase digestion of recombinant LRP fusion protein --- p.53 / Chapter 3.5.12 --- Deglycosylation studies of recombinant MSP142-AFVY --- p.54 / Chapter 3.6 --- Confocal immunoflorescence studies of MSPl42-AFVY in tobacco --- p.55 / Chapter 3.6.1 --- Preparation of sections --- p.55 / Chapter 3.6.2 --- Labeling of fluorescence probes --- p.55 / Chapter 3.6.3 --- Image collection --- p.56 / Chapter 3.7 --- Bacterial expression of MSP 142 and anti-serum production --- p.57 / Chapter 3.7.1 --- pET expression in E. coli --- p.57 / Chapter 3.7.2 --- Purification of recombinant His-MSPl42 --- p.58 / Chapter 3.7.3 --- Immunization of rabbits --- p.59 / Chapter Chapter 4: --- Results --- p.60 / Chapter 4.1 --- Transgenic analysis of lysine-rich protein fusion constructs --- p.60 / Chapter 4.1.1 --- Construction of the lysine-rich protein fusion constructs --- p.60 / Chapter 4.1.2 --- Selection of transgenic plants --- p.62 / Chapter 4.1.3 --- Southern analysis --- p.65 / Chapter 4.1.4 --- Northern analysis --- p.69 / Chapter 4.1.5 --- Western analysis --- p.71 / Chapter 4.1.6 --- Western analysis with anti-LRP --- p.75 / Chapter 4.1.7 --- Enterokinase digestion of recombinant LRP fusion protein --- p.76 / Chapter 4.2 --- Transgenic analysis of phaseolin vacuolar-sorting signal constructs --- p.80 / Chapter 4.2.1 --- Construction of the phaseolin vacuolar-sorting signal constructs --- p.80 / Chapter 4.2.2 --- Selection of transgenic plants --- p.82 / Chapter 4.2.3 --- Southern analysis --- p.85 / Chapter 4.2.4 --- Northern analysis --- p.89 / Chapter 4.2.5 --- Western analysis --- p.91 / Chapter 4.2.6 --- Deglycosylation studies of recombinant MSPl42-AFVY --- p.96 / Chapter 4.2.7 --- Human serum detection of MSP142-AFVY --- p.100 / Chapter 4.3 --- Confocal immunofluorescence studies of MSP142-AFVY in tobacco --- p.102 / Chapter 4.4 --- Bacterial expression of MSPl42 and anti-serum production --- p.105 / Chapter 4.4.1 --- Expression and purification of recombinant His-MSPl42 in E. coli --- p.105 / Chapter 4.4.2 --- Titer and specificity of the anti-serum --- p.107 / Chapter Chapter 5 --- Discussion --- p.109 / Chapter Chapter 6 --- Conclusion --- p.116 / References --- p.117

by Lo Ka-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 175-197). / Chapter I --- Title page --- p.i / Chapter II --- Thesis committee --- p.ii / Chapter II --- Acknowledgment --- p.iii-iv / Chapter III --- Abstract --- p.v-vi / Chapter IV --- Table of content --- p.vii-xiv / Chapter V --- List of figures --- p.xv-xviii / Chapter VI --- List of tables --- p.xix-xx / Forewords: / Overall objectives --- p.1 / Introduction on the fish used in this research study --- p.2 / Chapter Chapter 1: --- Literature Review on Biomarkers of Stress in Teleosts --- p.5 / Chapter 1.1 --- Definition of stress --- p.7 / Chapter 1.2 --- Classification of stress indicators --- p.8 / Chapter 1.2.1 --- Primary stress indicators --- p.8 / Chapter 1.2.1.1 --- Molecular stress indicators --- p.8 / Chapter 1.2.1.2 --- Hormonal stress indicators --- p.9 / Chapter (I) --- Corticosteroid --- p.9 / Chapter (II) --- Catecholamines --- p.11 / Chapter 1.2.2 --- Secondary stress indicators --- p.12 / Chapter 1.2.2.1 --- Metabolic changes --- p.12 / Chapter (I) --- Glucose metabolism --- p.13 / Chapter (II) --- Lactic acid --- p.14 / Chapter 1.2.2.2 --- Osmoregulatory changes --- p.15 / Chapter 1.2.2.3 --- Haematological changes --- p.16 / Chapter 1.2.2.4 --- Reproductive changes --- p.17 / Chapter 1.2.3 --- Tertiary stress indicators --- p.18 / Chapter 1.2.3.1 --- Histopathological indicators --- p.18 / Chapter 1.2.3.2 --- Ecological indicators --- p.19 / Chapter 1.3 --- Recent trends on the study of biomarkers --- p.20 / Chapter 1.3.1 --- Use of detoxification enzymes for specific indication of toxic pollutants in aquatic environment --- p.20 / Chapter 1.3.1.1 --- Metallothioneins (MTs) --- p.20 / Chapter 1.3.1.2 --- Cytochrome P450 monoxygenase (CYP450) --- p.21 / Chapter 1.3.2 --- Use of HSP 70 as a biomarker in teleost --- p.22 / Chapter 1.4 --- Future perspectives on the study of biomarkers in fish --- p.24 / Chapter Chapter 2: --- Literature Review on Heat Shock Proteins (HSPs) --- p.28 / Chapter 2.1 --- General Characteristics of HSPs --- p.29 / Chapter 2.1.1 --- HSP90 family --- p.30 / Chapter 2.1.2 --- HSP70 family --- p.31 / Chapter 2.1.3 --- HSP60 family (Chaperonin-60) --- p.32 / Chapter 2.1.4 --- Low-molecular weight HSPs (HSP20) --- p.33 / Chapter 2.2 --- Structure of HSP70 encoding gene --- p.33 / Chapter 2.2.1 --- General characteristics of HSP70-encoding gene --- p.33 / Chapter 2.2.2 --- Heat shock transcription factor (HSF) --- p.35 / Chapter 2.2.3 --- Heat shock elements (HSE) --- p.35 / Chapter 2.3 --- Stress-mediated control of HSP70 transcription --- p.36 / Chapter 2.4 --- Characterization of HSP70 expression in teleost --- p.38 / Chapter 2.4.1 --- Tissues-specific expression of HSP70 in teleost --- p.39 / Chapter 2.4.2 --- Inter-relationship of HSP70 expression with seasonal variation and thermotolerance of teleost --- p.40 / Chapter 2.4.3 --- Induction of HSP70 in teleost upon acute thermal stress --- p.41 / Chapter 2.4.4 --- Induction of HSP70 in teleost by non-thermal stressors --- p.43 / Chapter 2.4.4.1 --- Heavy metal-induced HSP70 expression --- p.43 / Chapter 2.4.4.2 --- Handling stress-induced HSP70 expression --- p.43 / Chapter Chapter 3: --- "Induction of HSP70 in blood cells of seabream, Sparus sarba subjected to in vivo and in vitro thermal stress" --- p.48 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Materials and Methods --- p.52 / Chapter 3.2.1 --- Overall experimental design --- p.52 / Chapter 3.2.2 --- Fish --- p.53 / Chapter 3.2.3 --- Blood sampling --- p.53 / Chapter 3.2.4 --- Preparation of blood cells --- p.54 / Chapter 3.2.5 --- Thermal stress regimes --- p.54 / Chapter 3.2.5.1 --- Time couse of HSP70 induction profile in blood cells after in vitro exposure to thermal stress --- p.54 / Chapter 3.2.5.2 --- HSP70 synthesis in blood cells taken from fish subjected to in vivo hyper- thermic stress --- p.55 / Chapter 3.2.5.3 --- Transcriptional inhibitory effect of actinomycin D on the synthesis of HSP70 in blood cells subjected to in vitro thermal stress --- p.55 / Chapter 3.2.6 --- Protein analysis --- p.56 / Chapter 3.2.7 --- Gel electrophoresis --- p.57 / Chapter 3.2.8 --- Immunoblotting (Western blot analysis) --- p.57 / Chapter 3.2.9 --- Autroradiography --- p.58 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- Time course of HSP70 induction profile in blood cells subjected to in vitro thermal treatments --- p.59 / Chapter 3.3.1.1 --- Results of immunoblotting from blood cells of fish acclimated to 26°C --- p.59 / Chapter 3.3.1.2 --- Results of immunoblotting in blood cells from 18°C-acclimated fish --- p.60 / Chapter 3.3.1.3 --- Results of immunoblotting in blood cells from fish acclimated to 20°C --- p.60 / Chapter 3.3.1.4 --- 35S-methionine labelling of de novo protein synthesis in blood cells of fish acclimated to 15 and 20°C --- p.61 / Chapter 3.3.2 --- Blood cell HSP70 levels in 20°C-acclimated fish subjected to in vivo hyperthermic stress --- p.61 / Chapter 3.3.3 --- Transcriptional inhibitory effect of actinomycin D on HSP70 induction in blood cells subjected to in vitro thermal stress --- p.62 / Chapter 3.4 --- Discussions --- p.60 / Chapter 3.4.1 --- Characteristics of HSP70 induction in blood cells of seabream subjected to in vitro temperature stress --- p.72 / Chapter 3.4.1.1 --- Induction profile of HSP70 in blood cells --- p.72 / Chapter 3.4.1.2 --- Time course ofHSP70 induction in blood cells --- p.75 / Chapter 3.4.1.3 --- Effect of acclimation temperature of fish on the induction of HSP70 --- p.76 / Chapter 3.4.2 --- Comparison of HSP70 induction in in vitro and in vivo thermal treatment on blood cells --- p.78 / Chapter 3.4.3 --- "Effect of transcriptional inhibitor, actinomycin D, on the de novo synthesis of HSP70" --- p.80 / Chapter 3.5 --- Conclusions --- p.70 / Chapter Chapter 4: --- "Effects of seasonal variation and transportation stress on level of HSP70, serum glucose and serum Cortisol in seabream, Sparus sarba" --- p.86 / Chapter 4.1 --- Introduction --- p.87 / Chapter 4.2 --- Materials and methods --- p.90 / Chapter 4.2.1 --- Overall experimental design --- p.90 / Chapter 4.2.2 --- Fish and blood sampling --- p.91 / Chapter 4.2.3 --- Preparation of blood samples --- p.92 / Chapter 4.2.4 --- Determination of HSP70 levels in blood cells sampled from seabream upon different seasons --- p.92 / Chapter 4.2.5 --- Immunoblotting analysis --- p.92 / Chapter 4.2.6 --- Enzyme-linked Immnosorbent Assay (ELISA) --- p.93 / Chapter 4.2.7 --- Measurement of serum parameter in seabream --- p.95 / Chapter 4.2.7.1 --- Serum glucose --- p.95 / Chapter 4.2.7.2 --- Serum Cortisol --- p.96 / Chapter 4.3 --- Results --- p.96 / Chapter 4.3.1 --- Determination of HSP70 levels in blood cells sampled from seabream in different seasons --- p.96 / Chapter 4.3.1.1 --- Immunoblotting analysis --- p.96 / Chapter 4.3.1.2 --- Enzyme-linked immunosorbent assay (ELISA) --- p.96 / Chapter 4.3.2 --- Serum analysis of seabream sampled from fish farm in different seasons --- p.98 / Chapter 4.3.2.1 --- Serum glucose --- p.98 / Chapter 4.3.2.2 --- Serum Cortisol --- p.99 / Chapter 4.4 --- Discussions --- p.117 / Chapter 4.4.1 --- Characterization of HSP70 expression in blood cells of seabream --- p.117 / Chapter 4.4.2 --- Dynamicity of HSP70 content and thermo- tolerance of fish in different seasons --- p.118 / Chapter 4.4.3 --- Effect of transportation stress on HSP70 induction in blood cells of seabream --- p.121 / Chapter 4.4.4 --- Dynamicity of serum glucose level in seabream subjected to seasonal variations --- p.123 / Chapter 4.4.5 --- Effect of transportation stress on the serum glucose level of seabream in different seasons --- p.124 / Chapter 4.4.6 --- Dynamicity of senam Cortisol level in seabream subjected to seasonal variations --- p.125 / Chapter 4.4.7 --- Effect of transportation stress on the serum Cortisol level of seabream in different seasons --- p.126 / Chapter 4.4.8 --- "Comments on the use of HSP70, serum Cortisol and serum glucose as biomarkersin environmental supervision" --- p.126 / Chapter 4.5 --- Conclusions --- p.129 / Chapter Chapter 5: --- "In vitro and in vivo effects of Cortisol, dexamethasone and adrenaline on the induction of HSP70 in seabream, Sparus sarba" --- p.131 / Chapter 5.1 --- Introduction --- p.132 / Chapter 5.2 --- Materials and methods --- p.133 / Chapter 5.2.1 --- Overall experimental design --- p.133 / Chapter 5.2.2 --- Acclimation of fish and regimes of treatment --- p.133 / Chapter 5.2.3 --- Serum Cortisol and adrenaline analysis --- p.135 / Chapter 5.2.4 --- "Protein analysis, gel electrophoresis, immuno- blotting and ELISA analysis" --- p.136 / Chapter 5.3 --- Results --- p.137 / Chapter 5.3.1 --- "HSP70 level in blood cells treated with Cortisol, dexamethasone and adrenaline in vitro" --- p.137 / Chapter 5.3.2 --- "Serum hormones and HSP70 level in tissues of fish injected with Cortisol, adrenaline and dexamethasone invivo" --- p.137 / Chapter 5.3.2.1 --- Serum Cortisol and adrenaline level of fish after hormone injections --- p.137 / Chapter 5.3.2.2 --- "HSP70 level in blood cells, brain and liver tissue of fish after hormone injections" --- p.138 / Chapter (I) --- Level of HSP70 in blood cells of fish after hormone injections --- p.138 / Chapter 5.4 --- Discussions --- p.156 / Chapter 5.4.1 --- In vitro and in vivo study of the hormonal effect on HSP70 level in blood cells of seabream --- p.156 / Chapter 5.4.2 --- Hypothetical mechanism of hormone-receptor mediated HSP70 regulation --- p.158 / Chapter 5.4.3 --- In vivo study of the hormonal effect on HSP70 level in blood cells of seabream --- p.160 / Chapter 5.4.4 --- In vivo study on the hormonal effect of HSP70 synthesis in liver of seabream --- p.163 / Chapter 5.4.5 --- In vivo study on the hormonal effect of HSP70 synthesis in brain tissue of seabream --- p.165 / Chapter 5.4.6 --- HSP70 level in different tissues of fish in relation to the induction and sensitivity against stress --- p.166 / Chapter 5.5 --- Conclusions --- p.169 / Chapter Chapter 6: --- Summary --- p.171 / References --- p.175

Cheung Shu Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 124-137). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.iv / Contents --- p.v / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemiology of Breast Cancer --- p.1 / Chapter 1.1.1 --- Epidemiology of Breast Cancer in Females --- p.1 / Chapter 1.1.2 --- Incidence and Morality of Female Breast Cancer in Hong Kong --- p.2 / Chapter 1.1.3 --- Epidemiology of Breast Cancer in Males --- p.3 / Chapter 1.2 --- Risk Factors for Female Breast Cancer --- p.4 / Chapter 1.2.1 --- Genetic Risk Factors --- p.4 / Chapter 1.2.2 --- Hormonal Risk Factors --- p.6 / Chapter 1.2.2.1 --- Endogenous Hormonal Risk Factors --- p.7 / Chapter 1.2.2.2 --- Exogenous Hormonal Risk Factors --- p.8 / Chapter 1.2.3 --- Other Environmental Risk Factors --- p.9 / Chapter 1.3 --- Oncogenetic Basis of Female Breast Cancer --- p.10 / Chapter 1.4 --- Hormonal Basis of Female Breast Cancer --- p.12 / Chapter 1.4.1 --- Mechanisms of Hormone Action --- p.12 / Chapter 1.4.1.1 --- Estrogen and Progesterone --- p.12 / Chapter 1.4.1.2 --- Prolactin --- p.14 / Chapter 1.4.2 --- Hormonal Regulation of Normal Breast Development --- p.15 / Chapter 1.4.3 --- Hormonal Regulation of Breast Carcinogensis and Its Subsequent Progression --- p.17 / Chapter 1.4.3.1 --- Androgen --- p.17 / Chapter 1.4.3.2 --- Estrogen --- p.18 / Chapter 1.4.3.3 --- Progesterone --- p.20 / Chapter 1.4.3.4 --- Prolactin --- p.22 / Chapter 1.5 --- Animal Models for Breast Cancer --- p.23 / Chapter 1.5.1 --- Mouse Models --- p.24 / Chapter 1.5.2 --- Rat Models --- p.25 / Chapter 1.5.2.1 --- Carcinogen Induced Rat Models --- p.26 / Chapter 1.5.2.2 --- Hormone Induced Rat Models --- p.28 / Chapter 1.5.2.3 --- Spontaneously Developed Rat Models --- p.31 / Chapter 1.6 --- Aims of Study --- p.34 / Tables and Figures --- p.35 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Origin and Supply of Noble Rats --- p.37 / Chapter 2.2 --- Supply of Materials --- p.37 / Chapter 2.3 --- Induction of Mammary Tumors by Singe Dose of Chemical Carcinogens in Female Rats --- p.38 / Chapter 2.3.1 --- Induction by 7，12-Dimethylbenz[a]anthracene in Female Noble Rats --- p.38 / Chapter 2.3.2 --- Induction by N-Methyl-N-Nitrosourea in Female Sprague- Dawley Rats --- p.38 / Chapter 2.4 --- Induction of Mammary Tumors by Long-Term Treatments with Steroid Hormone --- p.39 / Chapter 2.4.1 --- Preparation of Steroid Hormone-filled Silastic® Tubings --- p.39 / Chapter 2.4.2 --- Surgical Implantation of Silastic® Tubings --- p.40 / Chapter 2.4.3 --- Protocols of Hormonal Treatments --- p.40 / Chapter 2.5 --- Collection of Spontaneously Developed Mammary Tumors in Noble Rats --- p.41 / Chapter 2.6 --- Transplantation of Spontaneously Developed Mammary Tumors into Noble Rats --- p.41 / Chapter 2.7 --- Bilateral Ovariectomy of Female Noble Rats bearing Spontaneously Developed Mammary Tumors --- p.42 / Chapter 2.8 --- Measurement of Mammary Tumor Growth --- p.43 / Chapter 2.9 --- Whole Mount Preparation of the Hormone-Treated Mammary Glands in Noble Rats --- p.44 / Chapter 2.10 --- Histological Examination of Mammary Gland and Tumors in Noble Rats --- p.45 / Chapter 2.11 --- Detection of Protein Expression of Hormone Receptors in Normal Mammary Glands and Mammary Tumors of Noble Rats --- p.45 / Chapter 2.11.1 --- Antibodies --- p.45 / Chapter 2.11.2 --- Immunohistochemistry --- p.47 / Chapter 2.11.3 --- "Protein extraction, SDS-PAGE and western blotting analysis" --- p.48 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Gross Appearance of Mammary Tumors --- p.51 / Chapter 3.2 --- Incidence Rate of Mammary Tumors --- p.53 / Chapter 3.2.1 --- Spontaneously Developed Mammary Tumors in Noble Rats --- p.53 / Chapter 3.2.2 --- Hormone Induced Mammary Tumors in Female Noble Rats --- p.53 / Chapter 3.2.3 --- DMBA Induced Mammary Tumors in Female Noble Rats --- p.54 / Chapter 3.2.4 --- NMU Induced Mammary Tumors in Female SD Rats --- p.54 / Chapter 3.3 --- Histology of Normal and Lactating Mammary Glands in Female Noble Rats --- p.54 / Chapter 3.4 --- Histopathology of Mammary Tumors --- p.55 / Chapter 3.4.1 --- Histopathology of Spontaneously Developed Mammary Tumors in Noble Rats --- p.55 / Chapter 3.4.2 --- Histopathology of Hormone Induced Mammary Tumors in Female Noble Rats --- p.59 / Chapter 3.4.3 --- Histopathology of DMBA Induced Mammary Tumors in Female Noble Rats --- p.60 / Chapter 3.4.4 --- Histopathology of NMU Induced Mammary Tumors in Female SD Rat --- p.60 / Chapter 3.5 --- Whole Mount Preparation of Mammary Glands under Hormonal Treatments --- p.61 / Chapter 3.6 --- Effects of Bilateral Ovariectomy on the Growth of Spontaneously Developed Mammary Tumors --- p.61 / Chapter 3.7 --- Transplanability of the Spontaneously Developed Mammary Tumors in Noble Rats --- p.62 / Chapter 3.8 --- Examination of the Malignancy of Mammary Tumors by Immunohistochemical analysis of Epithelial Keratin Expression --- p.62 / Chapter 3.9 --- Immunohistochemical Analysis of Expression and Localization of Hormone Receptor Protein in Normal and Neoplastic Mammary Tissues of Female Noble Rats --- p.63 / Chapter 3.9.1 --- Expression and Localization of Hormone Receptors in Control Tissue --- p.63 / Chapter 3.9.2 --- Expression and Localization of Estrogen Receptor α --- p.64 / Chapter 3.9.3 --- Expression and Localization of Estrogen Receptor β --- p.65 / Chapter 3.9.4 --- Expression and Localization of Progesterone Receptor --- p.65 / Chapter 3.9.5 --- Expression and Localization of Androgen Receptor --- p.66 / Chapter 3.9.6 --- Expression and Localization of Prolactin Receptor --- p.66 / Chapter 3.10 --- Western Blot Analysis of Expression of Hormone Receptor Proteins in Normal and Neoplastic Mammary Tissues of Female Noble Rats - --- p.67 / Chapter 3.10.1 --- Expression of Estrogen Receptor α --- p.67 / Chapter 3.10.2 --- Expression of Estrogen Receptorβ --- p.68 / Chapter 3.10.3 --- Expression of Progesterone Receptor --- p.68 / Chapter 3.10.4 --- Expression of Androgen Receptor --- p.69 / Chapter 3.10.5 --- Expression of Prolactin Receptor --- p.69 / Figures and Tables --- p.71 / Chapter Chapter 4 --- Discussions / Chapter 4.1 --- Comparison of the Incidence Rate of Spontaneously developed Mammary Tumors in Noble Rats with the Previously Reported Incidence Rate --- p.102 / Chapter 4.2 --- Comparison of the Incidence rate of Spontaneously Developed Mammary Tumors in Noble Rats with the Incidence Rate in Other Rat Strains --- p.103 / Chapter 4.3 --- Crucial Factors Influencing the Incidence Rate of Spontaneously Developed Mammary Tumors in Noble Rats --- p.104 / Chapter 4.4 --- Comparison of the T+E2 Induced Mammary Tumors with the T+DES Induced Mammary Tumors in Female Noble Rats --- p.105 / Chapter 4.5 --- Comparison of the Incidence Rate & Latency Period of the Hormone Induced Mammary Tumors in Noble Rats with the Previously Reported Data --- p.106 / Chapter 4.6 --- Comparison of the Phenotypic Behaviors in Spontaneously Developed Mammary Tumors with the Hormone Induced Mammary Tumors in Female Noble Rats --- p.107 / Chapter 4.7 --- Comparison of the Behaviors of Carcinogen Induced Mammary Tumors with Spontaneously Developed & Hormone Induced Mammary Tumors in Female Noble Rats --- p.109 / Chapter 4.8 --- "Comparison of Expression Patterns of Hormone Receptor Proteins in Spontaneously Developed, Hormone Induced & Carcinogen Induced Mammary Tumors in Female Noble Rats" --- p.111 / Chapter 4.9 --- "Expressions of ERα & ERβ Proteins in Spontaneously Developed, Hormone Induced and Carcinogen Induced Mammary Tumors in Female Noble Rats" --- p.112 / Chapter 4.10 --- "Expressions of PR Proteins in Spontaneously Developed, Hormone Induced and Carcinogen Induced Mammary Tumors in Female Noble Rats" --- p.115 / Chapter 4.11 --- "Expressions of AR Proteins in Spontaneously Developed, Hormone Induced and Carcinogen Induced Mammary Tumors in Female Noble Rats" --- p.116 / Chapter 4.12 --- "Expressions of PRLR Proteins in Spontaneously Developed, Hormone Induced and Carcinogen Induced Mammary Tumors in Female Noble Rats" --- p.120 / Chapter Chapter 5 --- Conclusions --- p.123 / References --- p.124

Abstract not available

<p>Integrins are receptors presented on most cells. By binding ligand they can generate signalling pathways inside the cell. Those pathways are a linkage to proteins in the cytosol. It is known that tumor cells can survive and proliferate in the absence of a solid support while normal cells need to be bound to ligand. To understand why tumour cells act that way, we first have to know how ligand-binding to integrins affect the cell. This research field includes studies on activation of proteins by integrins and the following protein-protein interactions.</p><p>The part of the research that I did, focused on the activation of PI3K by integrins and the question whether Ras is included in that pathway. I also studied the conformation changes of the integrins and tried to identify factors which regulate these changes.</p><p>Known is that Ras can activate PI3K. But we wanted to know if this is a step in the activation of PI3K by integrins. So if this would be a fact then Ras must be activated by integrins.</p><p>To see if integrins could activate Ras I did a pull down assay. GTP loaded Ras was isolated through its affinity for Raf. Only when Ras is in its activated state then it is GTP loaded, otherwise it is GDP loaded. In the experiment we also compared the β1A and the β1B splice variants. As result we could see that both splice variants probably can activate Ras. By blotting with anti-PI3K antibody we looked if PI3K had bound to Ras but no clear result could be obtained.</p><p>Integrins presented on blood cells are mostly in the inactive state while adherent cells have integrins which are mostly in the active state. PI3K has been shown, for blood cells, to be involved in the conformation regulation of integrins. Possibly, there is a positive circle that for blood cells just has to be switched on. It could be that the integrins in adherent cells are active because the cells are adhesive. By being adhesive, PI3K is activated. PI3K may then activate the integrins, through which the integrins stay in the active state. This circle could be broken at two points: we could inhibit PI3K or we could make the cells un-adhesive. I analysed this in cell attachment assay and by binding of conformation-specific integrin antibodies in FACScan. From the results we could not find any evidence that the whole idea around the positive circle is correct. Surprisingly we saw that the integrin value at the surface decrease if you add PI3K inhibitor. This could be due to distribute recirculation of integrins from the cytoplasm to the cell surface.</p><p>β1- and β3-integrins are both widely spread, but no functional difference could be shown already. Previous results suggest that there is a difference between migrations of those two types. To ensure this suggestion I did a wound assay. Hereby I compared the migration of different cell types, with different integrins on their surface and on different ligands.</p>

Dendritic cells (DCs) are prime mediators of innate and adaptive immunity. In humans the DC population comprise only 0.1% of all leukocytes, making their isolation and ex vivo manipulation difficult. Since study of DC activity in vitro requires large numbers of DCs to be readily available, a cell line model, KG-1, was selected. KG-1 cells are a cytokine-responsive human CD34+ myelomonocytic cell line and can be induced to differentiate to a DC phenotype. A range of differentiation agents and protocols were compared, and differentiation efficiency was determined using both morphological features and cell surface marker expression. Expression of CD83, CD11c, CD123, CD86, HLA-DR and DC-SIGN was assessed by immunofluorescence and flow cytometry. KG-1 cells stimulated with 10 ng/ml PMA and 100 ng/ml Ionomycin were found to be the ideal model for obtaining Dendritic Like Cells (DLCs) in vitro. The effect of lactic acid bacteria on KG-1 differentiation was also tested using two immunomodulatory strains, Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052. After 5 days of incubation with R0011 the KG-1 cells expressed DC-specific surface markers CD83, CD86, CD11c, CD123, DC-SIGN and HLA-DR. Lactobacillus rhamnosus R0011 induced a marked rise in CD83 expression with a mean fluorescence intensity of 115.3 after 5 days, suggesting this strain promoted KG-1 differentiation to DLC. Analysis of cytokine by KG-1 DLC indicated that constitutive production of pro-inflammatory cytokines TNF-α and IL-12 was minimal. However IL-10 and TGF-β were detected after TLR-agonist stimulation of R0011-differentiated KG-1s. This study aimed to develop and assess the KG-1 cell model for screening effects of mediators and microbes on DC. / UOIT

Integrins are receptors presented on most cells. By binding ligand they can generate signalling pathways inside the cell. Those pathways are a linkage to proteins in the cytosol. It is known that tumor cells can survive and proliferate in the absence of a solid support while normal cells need to be bound to ligand. To understand why tumour cells act that way, we first have to know how ligand-binding to integrins affect the cell. This research field includes studies on activation of proteins by integrins and the following protein-protein interactions. The part of the research that I did, focused on the activation of PI3K by integrins and the question whether Ras is included in that pathway. I also studied the conformation changes of the integrins and tried to identify factors which regulate these changes. Known is that Ras can activate PI3K. But we wanted to know if this is a step in the activation of PI3K by integrins. So if this would be a fact then Ras must be activated by integrins. To see if integrins could activate Ras I did a pull down assay. GTP loaded Ras was isolated through its affinity for Raf. Only when Ras is in its activated state then it is GTP loaded, otherwise it is GDP loaded. In the experiment we also compared the β1A and the β1B splice variants. As result we could see that both splice variants probably can activate Ras. By blotting with anti-PI3K antibody we looked if PI3K had bound to Ras but no clear result could be obtained. Integrins presented on blood cells are mostly in the inactive state while adherent cells have integrins which are mostly in the active state. PI3K has been shown, for blood cells, to be involved in the conformation regulation of integrins. Possibly, there is a positive circle that for blood cells just has to be switched on. It could be that the integrins in adherent cells are active because the cells are adhesive. By being adhesive, PI3K is activated. PI3K may then activate the integrins, through which the integrins stay in the active state. This circle could be broken at two points: we could inhibit PI3K or we could make the cells un-adhesive. I analysed this in cell attachment assay and by binding of conformation-specific integrin antibodies in FACScan. From the results we could not find any evidence that the whole idea around the positive circle is correct. Surprisingly we saw that the integrin value at the surface decrease if you add PI3K inhibitor. This could be due to distribute recirculation of integrins from the cytoplasm to the cell surface. β1- and β3-integrins are both widely spread, but no functional difference could be shown already. Previous results suggest that there is a difference between migrations of those two types. To ensure this suggestion I did a wound assay. Hereby I compared the migration of different cell types, with different integrins on their surface and on different ligands.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 228-272). Also available on the Internet.