Chan, Wing Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 129-152). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / 摘要 --- p.5 / Table of Contents --- p.7 / List of Abbreviations and symbols --- p.13 / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemics and pandemics of influenza virus --- p.17 / Chapter 1.2 --- Biology of influenza virus --- p.19 / Chapter 1.2.1 --- Types of influenza virus --- p.19 / Chapter 1.2.2 --- Viral structure and viral proteins --- p.20 / Chapter 1.2.3 --- Life cycle of influenza virus --- p.21 / Chapter 1.2.4 --- An ever-changing virus --- p.22 / Chapter 1.3 --- Pathogenesis and immunology of influenza virus --- p.24 / Chapter 1.3.1 --- Diseases and symptoms caused by influenza virus infection --- p.24 / Chapter 1.3.2 --- Production of cytokines during influenza virus infection --- p.25 / Chapter 1.3.3 --- Immune responses in the hosts --- p.27 / Chapter 1.4 --- Non-structural protein 1 (NS1) --- p.28 / Chapter 1.4.1 --- Overview of NS1 --- p.28 / Chapter 1.4.2 --- Roles of NS1 in influenza virus infection --- p.29 / Chapter 1.5 --- Aims of study --- p.33 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.34 / Chapter 2.1.1 --- Chemical reagents --- p.34 / Chapter 2.1.2 --- Buffers --- p.37 / Chapter 2.1.2.1 --- Preparation of buffers --- p.37 / Chapter 2.1.2.2 --- Commonly used buffers --- p.37 / Chapter 2.1.3 --- Strains and plasmids --- p.40 / Chapter 2.1.4 --- Primer list --- p.40 / Chapter 2.2 --- Methods --- p.42 / Chapter 2.2.1 --- Preparation of competent cells --- p.42 / Chapter 2.2.2 --- Molecular cloning --- p.43 / Chapter 2.2.2.1 --- Amplification of the target genes by PCR --- p.43 / Chapter 2.2.2.2 --- Agarose gel electrophoresis --- p.43 / Chapter 2.2.2.3 --- Extraction and purification of DNA from agarose gels --- p.44 / Chapter 2.2.2.4 --- Restriction digestion of DNA --- p.45 / Chapter 2.2.2.5 --- Ligation of digested insert and expression vector --- p.45 / Chapter 2.2.2.6 --- Transformation and plating out transformants --- p.46 / Chapter 2.2.2.7 --- Verification of insert by PCR --- p.46 / Chapter 2.2.2.8 --- Mini-preparation of plasmid DNA --- p.47 / Chapter 2.2.2.9 --- Confirmation of insertion in the miniprep DNA by restriction digestion --- p.48 / Chapter 2.2.2.10 --- Sequencing of the plasmid DNA --- p.48 / Chapter 2.2.3 --- Cell culture --- p.53 / Chapter 2.2.3.1 --- Cultivation of human lung epithelial NCI-H292 cells --- p.53 / Chapter 2.2.3.2 --- Transfection of cell culture --- p.53 / Chapter 2.2.4 --- Western blot analysis --- p.54 / Chapter 2.2.4.1 --- Protein extraction --- p.54 / Chapter 2.2.4.2 --- Determination of protein concentration --- p.54 / Chapter 2.2.4.3 --- Protein Blotting --- p.55 / Chapter 2.2.4.4 --- Membrane blocking and antibody incubations --- p.56 / Chapter 2.2.4.5 --- Detection of proteins --- p.57 / Chapter 2.2.5 --- Total RNA extraction --- p.58 / Chapter 2.2.5.1 --- Preparation of cell culture for total RNA extraction --- p.58 / Chapter 2.2.5.2 --- Spectrophotometric analysis of total RNA --- p.58 / Chapter 2.2.5.3 --- Agarose gel electrophoresis of total RNA --- p.59 / Chapter 2.2.6 --- DNA Microarray --- p.60 / Chapter 2.2.6.1 --- Preparation of biotin-labeled antisense cRNA --- p.60 / Chapter 2.2.6.2 --- "Hybridization, washing and scanning of DNA microarray chips" --- p.60 / Chapter 2.2.6.3 --- Data processing and analysis --- p.61 / Chapter 2.2.7 --- Quantitative real-time PCR (QRT-PCR) --- p.62 / Chapter 2.2.7.1 --- Preparation of cDNA --- p.62 / Chapter 2.2.7.2 --- Analysis of mRNA gene expression by QRT-PCR --- p.62 / Chapter Chapter 3 --- Roles of NS1A and NS1B on cellular gene expression / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Results --- p.67 / Chapter 3.2.1 --- NS1 protein expression in transfected NCI-H292 cells --- p.67 / Chapter 3.2.2 --- Purity and integrity of total RNA extracted --- p.67 / Chapter 3.2.3 --- Microarray data processing and analysis --- p.70 / Chapter 3.2.3.1 --- Genes perturbed by NS1A --- p.88 / Chapter 3.2.3.1.1 --- Effect of NS1A on antiviral gene expression --- p.91 / Chapter 3.2.3.1.2 --- Regulation of JAK-STAT pathway by NS1A --- p.92 / Chapter 3.2.3.2 --- Genes perturbed by NS1B --- p.93 / Chapter 3.2.3.2.1 --- Effects of NS1B on IFN-stimulated gene expression --- p.96 / Chapter 3.2.3.3 --- Genes perturbed by both NS1A and NS1B --- p.96 / Chapter 3.2.4 --- Verification of differentially expressed genes --- p.98 / Chapter 3.3 --- Discussion --- p.100 / Chapter 3.3.1 --- Human lung epithelial cell line as a model --- p.100 / Chapter 3.3.2 --- DNA microarray technology --- p.101 / Chapter 3.3.3 --- Different actions of NS1A and NS1B on host cell gene expression --- p.102 / Chapter 3.3.4 --- Novel roles of NSIA --- p.103 / Chapter 3.3.5 --- Novel role of NSIB --- p.107 / Chapter 3.3.6 --- Implications --- p.108 / Chapter Chapter 4 --- "Roles of NSIA, NS1B and NS1C on cytokine expression" / Chapter 4.1 --- Introduction --- p.109 / Chapter 4.2 --- Results --- p.113 / Chapter 4.2.1 --- NS1 protein expression in transfected NCI-H292 cells --- p.113 / Chapter 4.2.2 --- Purity and integrity of total RNA extracted --- p.113 / Chapter 4.2.3 --- QRT-PCR --- p.116 / Chapter 4.3 --- Discussion --- p.119 / Chapter 4.3.1 --- Human lung epithelial cell line as a model for cytokine study --- p.119 / Chapter 4.3.2 --- Implications of different cytokine patterns induced by different NS1 proteins --- p.120 / Chapter Chapter 5 --- General Discussion and Future Perspectives --- p.125 / References --- p.129
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326293 |
| Date | January 2008 |
| Contributors | Chan, Wing Tung., Chinese University of Hong Kong Graduate School. Division of Molecular Biotechnology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, 153 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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