Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 207-251).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / "December 1999." Typescript. Vita. Includes bibliographical references (leaves 98-104). Also available on the Internet.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 98-104). Also available on the Internet.

Ho Hei Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 189-199). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemiology of the Severe Acute Respiratory Syndrome (SARS) Outbreak --- p.2 / Chapter 1.2 --- The SARS Coronavirus --- p.3 / Chapter 1.2.1 --- Genome organization --- p.7 / Chapter 1.2.2 --- Structural proteins --- p.9 / Chapter 1.2.3 --- Non-structural proteins --- p.11 / Chapter 1.3 --- Introduction to SARS-CoV nsp10 Protein --- p.14 / Chapter 1.4 --- Introduction to SARS-CoV RNA-dependent RNA Polymerase (RdRp) Protein --- p.17 / Chapter 1.5 --- Objectives of the Present Study --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of Glutathione S-Transferase (GST) Fusion/Green Fluorescence Protein (GFP) N1 and C1 Fusion nsplO --- p.26 / Chapter 2.1.1 --- Primer design --- p.26 / Chapter 2.1.2 --- Gene amplification by PCR --- p.28 / Chapter 2.1.3 --- Purification of PCR product --- p.30 / Chapter 2.1.4 --- Enzyme restriction --- p.31 / Chapter 2.1.5 --- Ligation --- p.33 / Chapter 2.1.6 --- Transformation --- p.34 / Chapter 2.1.6.1 --- Preparation of competent cell DH5α --- p.34 / Chapter 2.1.7 --- Mini scale plasmid preparation --- p.36 / Chapter 2.2 --- Subcellular Localization Study --- p.39 / Chapter 2.2.1 --- Midi scale plasmid preparation --- p.39 / Chapter 2.2.2 --- Transfection of GFP recombinant plasmids --- p.41 / Chapter 2.2.2.1 --- Cell culture of Vero E6 cell line --- p.41 / Chapter 2.2.2.2 --- Lipofectamine based transfection --- p.41 / Chapter 2.2.3 --- Fluorescent microscopic visualization --- p.42 / Chapter 2.2.4 --- Western blotting for GFP fusion protein expression --- p.43 / Chapter 2.2.4.1 --- Protein extraction --- p.43 / Chapter 2.2.4.2 --- Protein quantification --- p.44 / Chapter 2.2.3.4 --- SDS-PAGE analysis --- p.45 / Chapter 2.3 --- "Expression of GFP-nsp10 in Vero E6 cells, SARS-CoV Infected Vero E6 Cells and Convalescent Patients' Serum" --- p.47 / Chapter 2.3.1 --- Cell-based immunostaining of VeroE6 cells and SARS-CoV infected Vero E6 cells --- p.47 / Chapter 2.3.1.1 --- Immobilization of Vero E6 cells and SARS-CoV infected Vero E6 cells --- p.47 / Chapter 2.3.1.2 --- Preparation of monoclonal antibodies against SARS-CoV nsp10 --- p.48 / Chapter 2.3.1.3 --- Immunostaining of SARS-CoV nsp10 in Vero E6 cells and SARS-CoV VeroE6 cells --- p.48 / Chapter 2.3.1.4 --- Fluorescent microscopic visualization --- p.49 / Chapter 2.3.2 --- Detection of SARS-CoV nsplO expression in SARS-CoV infected convalescent patients' serum --- p.50 / Chapter 2.3.2.1 --- Western blotting of SARS-CoV nsp10 by SARS-CoV infected convalescent patients' serum --- p.50 / Chapter 2.4 --- Expression of GST fusion SARS-CoV nsp10 in E.coli --- p.51 / Chapter 2.4.1 --- Preparation of competent cells --- p.51 / Chapter 2.4.2 --- Small scale expression --- p.51 / Chapter 2.4.3 --- Large scale expression of GST-nsp10 in optimized conditions --- p.54 / Chapter 2.5 --- Purification of GST fusion SARS-CoV nsp10 --- p.55 / Chapter 2.5.1 --- Glutathione Sepharose 4B affinity chromatography --- p.55 / Chapter 2.5.2 --- Superdex 75 gel filtration chromatography --- p.56 / Chapter 2.6 --- "CD Measurement, NMR and Crystallization Study of SARS-CoV nsp10" --- p.57 / Chapter 2.6.1 --- CD measurement --- p.57 / Chapter 2.6.2 --- NMR spectroscopy --- p.58 / Chapter 2.6.3 --- Crystallization of nsp10 --- p.58 / Chapter 2.7 --- "Glutathione-S-Sepharose Pull-down assay, 2D Gel Electrophoresis and Mass Spectrometry" --- p.59 / Chapter 2.7.1 --- GST pull-down assay --- p.59 / Chapter 2.7.2 --- Two-dimension gel electrophoresis --- p.59 / Chapter 2.7.2.1 --- First dimensional isoelectric focusing (IEF) --- p.59 / Chapter 2.7.2.2 --- Second dimension SDS-PAGE --- p.60 / Chapter 2.7.2.3 --- Silver staining --- p.61 / Chapter 2.7.3 --- Protein identification by mass spectrometry --- p.63 / Chapter 2.7.3.1 --- Data acquisition --- p.65 / Chapter 2.8 --- Proliferative study of SARS-CoV nsp10 in VeroE6 Cell Line and Mouse Splenocytes --- p.66 / Chapter 2.8.1 --- Assay of mitogenic activity by 3H-thymidine incorporation --- p.66 / Chapter 2.9 --- "Cloning, Expression and Purification of GST fusion SARS-CoV RNA-dependent RNA Polymerase (RdRp) Full- length Protein" --- p.67 / Chapter 2.9.1 --- Construction of GST-RdRp-full length expression plasmid --- p.67 / Chapter 2.9.2 --- Expression and purification of GST-RdRp full-length protein --- p.68 / Chapter 2.10 --- "Cloning, Expression and Purification of GST Fusion SARS-CoV RNA-dependent RNA Polymerase (RdRp) Catalytic Domain" --- p.70 / Chapter 2.10.1 --- Construction of GST-RdRp Catalytic Domain (p64) and MBP-RdRp-p64 expression plasmids --- p.70 / Chapter 2.10.2 --- Expression and purification of GST fusion catalytic domain of SARS-CoV RdRp (GST-p64) --- p.71 / Chapter 2.10.3 --- Expression and purification of MBP fusion catalytic domain of SARS-CoV RdRp --- p.72 / Chapter 2.11 --- "Cloning, Expression and Purification of the His-thioredoxin Fusion N-terminal Domain of SARS-CoV RdRp (pET32h-pl2)" --- p.74 / Chapter 2.11.1 --- Construction of His-thioredoxin fusion N-terminal domain of SARS-CoV RdRp (pET32h-pl2) expression plasmid --- p.74 / Chapter 2.11.2 --- Expression and purification of His- thioredoxin fusion N-terminal domain of SARS-CoV RdRp (pET32h-pl2) --- p.74 / Chapter 2.12 --- Interaction Study of RdRp Catalytic Domain and N-terminal Domain --- p.76 / Chapter 2.13 --- Electrophoretic Mobility Shift Assay of SARS-CoV Genomic RNA Strands with RdRp Full-length sequence --- p.76 / Chapter 2.13.1 --- Preparation of RNA transcripts --- p.76 / Chapter 2.13.2 --- EMSA --- p.77 / Chapter 2.14 --- Non-radiometric and Radiometric RdRp Assays --- p.78 / Chapter 2.14.1 --- Non-radiometric RdRp assay--luciferase coupled enzyme assay --- p.78 / Chapter 2.14.2 --- Radiometric RdRp assay ´ؤ filter-binding enzyme assay --- p.79 / Chapter 2.15 --- Western Blot Analysis for Interaction Study --- p.80 / Chapter Chapter 3 --- Results and Discussion on SARS-CoV nsplO --- p.81 / Chapter 3.1 --- "Cloning, Expression and Purification of SARS-CoV nsp10 in Prokaryotic Expression System" --- p.81 / Chapter 3.1.1 --- Cloning and expression of SARS-CoV nsp 10 --- p.81 / Chapter 3.1.2 --- Purification of GST-nsp10 by GST affinity chromatography --- p.84 / Chapter 3.1.3 --- Purification of nsp10 by size exclusion chromatography --- p.85 / Chapter 3.1.4. --- "Yield, purity and stability of SARS-CoV nsp 10" --- p.88 / Chapter 3.2 --- SARS-CoV nsp10 Sequence Alignment and Protein Structure Prediction --- p.89 / Chapter 3.2.1. --- Sequence alignment of SAR-CoV nsp10 with known viral proteins --- p.91 / Chapter 3.2.2 --- Protein structure prediction - homology modeling --- p.93 / Chapter 3.3 --- Circular Dichroism Analysis of nsp10 --- p.96 / Chapter 3.3.1 --- CD spectrum of SARS-CoV nsp10 --- p.98 / Chapter 3.3.2. --- Effect of divalent metal ions on SARS-CoV nsp10 --- p.99 / Chapter 3.4 --- Nuclear Magnetic Resonance Analysis of nsp10 --- p.101 / Chapter 3.4.1 --- Sample preparation for NMR Experiment --- p.102 / Chapter 3.4.2 --- Protein structure determination by NMR --- p.103 / Chapter 3.5 --- Crystallization of SARS-CoV nsp10 --- p.105 / Chapter 3.5.1 --- Sample preparation of nsp10 for crystallization --- p.105 / Chapter 3.5.2 --- Screening conditions for crystallization --- p.106 / Chapter 3.6 --- "Antigenic, Immunofluorescene and Subcellular Localization Studies on the SARS-CoV nsp10" --- p.110 / Chapter 3.6.1 --- Antigenic and immunofluorescene studies on the SARS-CoV nsp10 --- p.110 / Chapter 3.6.2 --- Subcellular localization of SARS-CoV nsp10 --- p.115 / Chapter 3.7 --- Proliferative Study of nsp10 --- p.120 / Chapter 3.7.1. --- Influence of proliferative effect on the host cell --- p.121 / Chapter 3.8 --- A Proteomics Strategy for Interaction Study of nsp10 --- p.124 / Chapter 3.8.1 --- 2D SDS-PAGE analysis of proteins associating with the nsp10 bait --- p.125 / Chapter 3.8.2 --- Silver staining of proteins associating with the nsp10 bait and their identification by mass spectrometry --- p.127 / Chapter 3.9 --- Discussion on SARS-CoV nsp10 --- p.129 / Chapter Chapter 4 --- Results and Discussion on SARS-CoV RdRp / Chapter 4.1 --- "Cloning, Expression and Purification of SARS-CoV RdRp Full-length, Catalytic Domain and N-terminal Domain" --- p.139 / Chapter 4.2 --- Interaction Study of RdRp Catalytic Domain and its N-terminal Domain --- p.147 / Chapter 4.3 --- Functional Analysis of RNA Binding by the SARS-CoV RdRp --- p.149 / Chapter 4.4 --- Characterization of RdRp by Non-radioactive RdRp Assay ´ؤ Luciferase-coupled Enzyme Assay --- p.152 / Chapter 4.5 --- Characterization of RdRp by Radioactive RdRp Assay ´ؤ 32P Incorporation Assay --- p.157 / Chapter 4.6 --- Discussion on SARS-CoV RdRp --- p.161 / Chapter Chapter 5 --- General Discussion / General Discussion --- p.170 / Appendix --- p.172 / References --- p.189

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

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "August 2001." Typescript. Vita. Includes bibliographical references (leaves 172-183). Also available on the Internet.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2008" Includes bibliographical references.