Introducción: La pandemia por SARS-Cov-2 ha tenido un impacto negativo en múltiples aspectos de la vida humana, tanto en lo físico, psicológico, económico, social y cultural. Las afecciones de la calidad de vida están asociadas al confinamiento y la libertad de salir, de pasar tiempo con sus amigos o familiares, o de realizar actividades; por lo que se ven privados de la mayor parte de su interacción social. El objetivo de este presente artículo es brindar información sobre cómo afecta el confinamiento y aislamiento social por la pandemia a la calidad de vida y salud mental.

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A distinct feature of contemporary politics is the involvement of international forces in a state's domestic politics, and vice versa. Despite the plethora of literature on the international system and comparative state politics, relatively little addresses the interpenetration of international and domestic systems in intermestic issues. This thesis explores intermestic processes. It argues that states' domestic policies are increasingly formed in an intermestic context and such intermesticity has brought states a dilemma between maintaining effective domestic control and achieving integration into the global economy. This thesis examines China's SARS policy formation in 2003 as a case study. How did the internal health problem come to be addressed in an intermestic context in a country noted for its tight domestic control and long-term aversion to foreign intervention? The question is approached through a textual analysis of the story of China's SARS policy development. This study also identifies the patterns of international influence on China's domestic politics, particularly in the SARS crisis. I interpret the intermestic dynamic as a learning process through which China has chosen to embrace international institutions in its pursuit of national interests in a globalized world. / Master of Arts

Im sechsten Semester des Medizinstudiums an der Julius-Maximilians-Universität Würzburg findet das verpflichtende Praktikum „Impfkurs“ statt. Im Rahmen dieses Kurses wurde vom Sommersemester 2020 bis zum Sommersemester 2021 ein standardisierter online Fragebogen erhoben, der unter anderem demographische Daten sowie Expositionsmöglichkeiten gegenüber SARS-CoV-2 im privaten, beruflichen und universitären Umfeld erfragte. Zusätzlich wurde im gleichen Zeitraum der SARS-CoV-2 Serostatus der Medizinstudierenden erhoben und ausgewertet und dieser mit den Daten des Fragebogens zusammengeführt. Dafür wurden Blutproben entnommen, welche im Labor des Instituts für Virologie der Universität Würzburg mittels Western Blot auf IgG/IgM/IgA Antikörper gegen SARS-CoV-2 untersucht wurden. / In the sixth semester of medical studies at the Julius-Maximilians-Universität Würzburg, the compulsory internship “vaccination course” takes place. As part of this course, a standardized online questionnaire was collected from the summer semester 2020 to the summer semester 2021, which, among other things, collected demographic data and exposure to SARS-CoV-2 in the private, professional and university environment. In addition, the SARS-CoV-2 serostatus of the medical students was collected and evaluated during the same period and merged with the data from the questionnaire. For this purpose, blood samples were taken, which were tested for IgG/IgM/IgA antibodies against SARS-CoV-2 by Western blot.

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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

Chu Ling Hon Matthew. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 201-223). / Abstracts in English and Chinese. / Declaration --- p.i / Thesis/Assessment Committee --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Acknowledgements --- p.viii / General abbreviations --- p.xi / Abbreviations of chemicals --- p.xv / Table of Contents --- p.xvi / List of Figures --- p.xxiii / List of tables --- p.xxviii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Severe Acute Respiratory Syndrome (SARS) - Three Years in Review --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Clinical presentation --- p.3 / Chapter 1.1.3 --- Diagnostic tests --- p.5 / Chapter 1.2 --- Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) --- p.7 / Chapter 1.2.1 --- SARS - Identification of the etiological agent --- p.7 / Chapter 1.2.2 --- The coronaviruses --- p.9 / Chapter 1.2.3 --- The genome organization of SARS-CoV --- p.11 / Chapter 1.2.4 --- The life cycle of SARS-CoV --- p.13 / Chapter 1.3 --- Spike Glycoprotein (S protein) of SARS-CoV --- p.15 / Chapter 1.3.1 --- SARS-CoV S protein --- p.15 / Chapter 1.3.2 --- S protein-driven infection --- p.17 / Chapter 1.4 --- SARS-CoV S Protein Fusion Core --- p.22 / Chapter 1.4.1 --- Heptad repeat and coiled coil --- p.22 / Chapter 1.4.2 --- The six-helix coiled coil bundle structure --- p.25 / Chapter 1.5 --- 3C-like Protease (3CLpro) of SARS-CoV --- p.28 / Chapter 1.5.1 --- Extensive proteolytic processing of replicase polyproteins --- p.28 / Chapter 1.5.2 --- SARS-CoV 3CLpro --- p.30 / Chapter 1.5.3 --- Substrate Specificity of SARS-CoV 3CLpro --- p.31 / Chapter 1.6 --- SARS Drug Development --- p.32 / Chapter 1.6.1 --- Drug targets of SARS-CoV --- p.32 / Chapter 1.6.2 --- Current anti-SARS drugs --- p.36 / Chapter 1.7 --- Project Objectives --- p.39 / Chapter 1.7.1 --- Characterization of SARS-CoV S protein fusion core --- p.39 / Chapter 1.7.2 --- Characterization of SARS-CoV 3CLpr0 substrate specificity --- p.40 / Chapter 2 --- Materials and Methods --- p.42 / Chapter 2.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.42 / Chapter 2.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.42 / Chapter 2.1.2 --- Recombinant protein approach --- p.43 / Chapter 2.1.2.1 --- Plasmids construction --- p.43 / Chapter 2.1.2.2 --- Protein expression and purification --- p.52 / Chapter 2.1.2.3 --- Amino acid analysis --- p.57 / Chapter 2.1.2.4 --- GST-pulldown experiment --- p.58 / Chapter 2.1.2.5 --- Laser light scattering --- p.61 / Chapter 2.1.2.6 --- Size-exclusion chromatography --- p.62 / Chapter 2.1.2.7 --- Circular dichroism spectroscopy --- p.62 / Chapter 2.1.3 --- Synthetic peptide approach --- p.64 / Chapter 2.1.3.1 --- Peptide synthesis --- p.64 / Chapter 2.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.65 / Chapter 2.1.3.3 --- Size-exclusion high-performance liquid chromato-graphy --- p.66 / Chapter 2.1.3.4 --- Laser light scattering --- p.66 / Chapter 2.1.3.5 --- Circular dichroism spectroscopy --- p.67 / Chapter 2.2 --- Identification of SARS-CoV Entry Inhibitors --- p.70 / Chapter 2.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.70 / Chapter 2.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.74 / Chapter 2.2.3 --- Molecular modeling --- p.75 / Chapter 2.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.79 / Chapter 2.3.1 --- Protein expression and purification --- p.79 / Chapter 2.3.2 --- """Cartridge replacement"" solid-phase peptide synthesis" --- p.80 / Chapter 2.3.3 --- Peptide cleavage assay and mass spectrometric analysis --- p.83 / Chapter 3 --- Results --- p.84 / Chapter 3.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.84 / Chapter 3.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.84 / Chapter 3.1.2 --- Recombinant protein approach --- p.87 / Chapter 3.1.2.1 --- "Plasmids construction of pET-28a-His6-HRl, pGEX-6P-l-HR2 and pGEX-6P-l-2-Helix" --- p.87 / Chapter 3.1.2.2 --- Protein expression and purification --- p.92 / Chapter 3.1.2.3 --- GST-pulldown experiment --- p.101 / Chapter 3.1.2.4 --- Laser light scattering --- p.103 / Chapter 3.1.2.5 --- Size-exclusion chromatography --- p.105 / Chapter 3.1.2.6 --- Circular dichroism spectroscopy --- p.107 / Chapter 3.1.3 --- Synthetic peptide approach --- p.112 / Chapter 3.1.3.1 --- Peptide synthesis --- p.112 / Chapter 3.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.116 / Chapter 3.1.3.3 --- Size-exclusion high-performance liquid chromatography --- p.117 / Chapter 3.1.3.4 --- Laser light scattering --- p.122 / Chapter 3.1.3.5 --- Circular dichroism spectroscopy --- p.124 / Chapter 3.2 --- Identification of SARS-CoV Entry Inhibitors --- p.129 / Chapter 3.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.129 / Chapter 3.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.131 / Chapter 3.2.3 --- Molecular modeling --- p.135 / Chapter 3.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.141 / Chapter 3.3.1 --- Protein expression and purification --- p.141 / Chapter 3.3.2 --- Substrate specificity preference of SARS-CoV 3CLpr0 --- p.142 / Chapter 3.3.3 --- "Primary and secondary screening using the ""cartridge replacement strategy""" --- p.142 / Chapter 4 --- Discussion --- p.149 / Chapter 4.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.149 / Chapter 4.1.1 --- Design of recombinant proteins and synthetic peptides of HR regions --- p.149 / Chapter 4.1.2 --- Recombinant protein approach --- p.151 / Chapter 4.1.3 --- Synthetic peptide approach --- p.153 / Chapter 4.1.4 --- Summary of the present and previous studies in the SARS-CoV S protein fusion core --- p.157 / Chapter 4.2 --- Identification of SARS-CoV Entry Inhibitors --- p.167 / Chapter 4.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.167 / Chapter 4.2.2 --- Identification of peptide inhibitors --- p.168 / Chapter 4.2.3 --- Identification of small molecule inhibitors --- p.172 / Chapter 4.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.183 / Chapter 4.3.1 --- A comprehensive overview of the substrate specificity of SARS-CoV 3CLpro --- p.184 / Chapter 4.3.2 --- The development of the rapid and high-throughput screening strategy for protease substrate specificity --- p.188 / Appendix --- p.191 / Chapter I. --- Nucleotide Sequence of S protein of SARS-CoV --- p.191 / Chapter II. --- Protein Sequence of S protein of SARS-CoV --- p.194 / Chapter III. --- Protein Sequence of 3CLpro of SARS-CoV --- p.195 / Chapter IV. --- Vector maps --- p.196 / Chapter 1. --- Vector map and MCS of pET-28a --- p.196 / Chapter 2. --- Vector map and MCS of pGEX-6P-l --- p.197 / Chapter V. --- Electrophoresis markers --- p.198 / Chapter 1. --- GeneRuler´ёØ 1 kb DNA Ladder --- p.198 / Chapter 2. --- GeneRuler´ёØ 100bp DNA Ladder --- p.198 / Chapter 3. --- High-range Rainbow Molecular Weight Markers --- p.199 / Chapter 4. --- Low-range Rainbow Molecular Weight Markers --- p.199 / Chapter VI. --- SDS-PAGE gel preparation protocol --- p.200 / References --- p.201

Chong Lin-Tat. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 76-78). / Abstracts in English and Chinese. / Chapter Chapter 1 --- introduction / Chapter 1.1 --- Severe acute respiratory syndrome Coronavirus (SARS CoV) --- p.13 / Figure 1.1 Genome organization and putative functional ORFs of SARS CoV --- p.14 / Chapter 1.2 --- SARS main protease / Chapter 1.2.1 --- Three dimensional structure --- p.15 / Figure 1.2 Ribbon illustration of the SARS-coronavirus main protease --- p.17 / Figure 1.3 Surface representations of P1 and P2 substrate-binding pocket of main protease --- p.18 / Chapter 1.2.2 --- Substrate specificities --- p.19 / Table 1.1. Eleven predicted cleavage sites of SARS CoV main protease --- p.21 / Chapter 1.3 --- Protein-based FRET assay system --- p.22 / Figure 1.4. The principle of fluorescent resonance energy transfer (FRET) --- p.24 / Chapter 1.4 --- Objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General Techniques / Chapter 2.1.1 --- Preparation and transformation of competent E. coli DH5a and23 BL21 (DE3)pLysS --- p.26 / Chapter 2.1.2 --- Minipreparation of plasmid DNA (Invitrogen) --- p.27 / Chapter 2.1.3 --- Spectrophotometric quantitation DNA --- p.28 / Chapter 2.1.4 --- Agarose gel electrophoresis / Chapter 2.1.5 --- Purification of DNA from agarose gel (Invitrogen) / Chapter 2.1.6 --- Restriction digestion of DNA fragments --- p.29 / Chapter 2.1.7 --- Ligation of DNA fragments into vector / Table 2.1. Standard recipe of ligation reaction --- p.30 / Chapter 2.1.8 --- SDS-PAGE electrophoresis --- p.31 / Table 2.2. Standard recipe of separating gel for SDS-PAGE --- p.32 / Table 2.3. Standard recipe of stacking gel for SDS-PAGE --- p.33 / Chapter 2.2 --- Sub-cloning and site-directed mutagenesis / Chapter 2.2.1 --- Sub-cloning of SARS Co V main protease --- p.34 / Chapter 2.2.2 --- Sub-cloning of Substrate / Chapter 2.2.3 --- Site-directed mutagenesis of substrate variant --- p.35 / Table 2.4 Primer sequence for generating substrate variants --- p.36 / Table 2.5. Standard recipe of Polymerase Chain Reaction (PCR) --- p.40 / Table 2.6. Polymerase Chain Reaction (PCR) profile --- p.41 / Chapter 2.3 --- Sample preparation / Chapter 2.3.1 --- Expression of recombinant proteins --- p.42 / SARS CoV main protease / Substrate and substrate variants / Chapter 2.3.2 --- Purification of recombinant proteins / SARS CoV main protease / Substrate and substrate variants / Chapter 2.4 --- Protein-based FRET kinetic analysis --- p.45 / Chapter 2.5 --- A model for substrate-enzyme binding by docking simulation --- p.46 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Preparation of SARS CoV main protease and substrate / Chapter 3.1.1 --- Expression and purification of SARS main protease --- p.48 / Figure 3.1. Purification profile of SARS CoV main protease --- p.49 / Chapter 3.1.2 --- Expression and purification of substrate and substrate variants --- p.50 / Figure 3.2. Purification profile of substrate and substrate variants --- p.51 / Chapter 3.2 --- A novel protein-based FRET assay system was established / Chapter 3.2.1 --- "With the cleavage of active main protease, absorbance at 528nm dropped while signal at 485nm were slightly increased" --- p.52 / Figure 3.3. Absorbance at 528nm dropped and 485nm increased with the substrate hydrolysis --- p.53 / Chapter 3.2.2 --- FRET efficiency ratio (528/485) decreased over time --- p.54 / Figure 3.4. FRET efficiency ratio (528/485) decreased over time --- p.55 / Chapter 3.2.3 --- Comparable kcat/Km value of SARS CoV main protease was obtained --- p.56 / Figure 3.5. Catalytic parameter (kcat/ Km) was determined from the slope of straight Line --- p.57 / Chapter 3.3 --- Main protease activity towards substrate variants at different substrate-binding sites (S2'-S2) --- p.58 / Table 3.1. Kinetic parameterrs of 76 substrate variants in descending order --- p.59 / Chapter 3.3.1 --- S2'substrate-binding site --- p.60 / Chapter 3.3.2 --- S1' substrate-b inding site / Chapter 3.3.3 --- S1 substrate-binding site / Chapter 3.3.4 --- S2 substrate-binding site / Figure 3.6. Kinetic analysis of some typical substrate variants against main protease --- p.62 / Figure 3.7. SDS-PAGE analysis of some typical substrate variants against main protease --- p.63 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Quantitative and high-throughput analysis by protein-based FRET assay system --- p.64 / Chapter 4.2 --- Substrate specificities of SARS CoV main protease at S2'-S2 subsites / Chapter 4.2.1 --- β-strand conformation was preferred at S2，subsite / Chapter 4.2.2 --- Residues with small aliphatic side chain were preferred at S1 ´ة subsite --- p.65 / Chapter 4.2.3 --- "Glutamine at S1 subsite was absolutely conserved, but alternatives were disclosed" --- p.66 / Figure 4.1. Glutamine was not absolutely conserved in S1 subsite --- p.67 / Chapter 4.2.4 --- Hydrophilic residues were tolerated at S2 subsite --- p.68 / Figure 4.2. Hydrophilic residues were tolerated at S2 subsite --- p.70 / Table 4.1. Summary of types of residues preferred at individual subsites --- p.71 / Chapter 4.3 --- Predicted conformation of substrate towards SARS CoV main protease at S2' and S1' subsites --- p.72 / Figure 4.3. Small residues were preferred at S1´ة subsite and Val at S2' subsite was more favoured than the native one --- p.73 / Chapter Chapter 5 --- Summary --- p.74 / Chapter Chapter 6 --- Future work --- p.75 / References --- p.76

In part ONE of this thesis, including the most up to date information on SARS virology, disease transmission, pathogenesis and laboratory diagnosis will be summarized and presented, including the results of many studies in which I have participated (these references will be underlined as they appear in text). This of course summarizes knowledge that is now known in 2006 but was largely unknown during the initial outbreak. In part TWO, six original clinical studies performed at PWH will be presented: study (1) describes the clinical manifestations and severity of SARS, and its potential to cause major hospital outbreaks; (2) demonstrates the importance of epidemiological linkage in diagnosing SARS; (3) reports the clinical outcomes of a stepwise treatment protocol, which includes the use of corticosteroid therapy as an immunomodulant; (4) demonstrates that corticosteroid therapy can retard viral clearance, and should be used judiciously; (5) demonstrates that a more robust humoral response is associated with severe SARS, thus indicating that passive immunity treatment strategies seem only suitable either during early illness or as prophylaxis; and (6) shows that SARS has few early discriminating laboratory features compared to other causes of community-acquired pneumonia, thus a high index of suspicion is needed to recognize this infection in the absence of worldwide transmission. A thorough review of the relevant published material will be included in the discussion section of each study. / Severe Acute Respiratory Syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus. It caused a global outbreak in 2003, resulting in more than 8000 infections, 700 deaths, and major social and economic disruption. In the initial phase of the SARS outbreak, the medical profession had no knowledge regarding the responsible pathogen, nor the clinical manifestations of SARS and the course of illness. There was no reliable diagnostic tool and no known effective therapy. But for the first time in medical history, we witnessed the rapid accumulation of knowledge on a disease as it evolved, which in turn assisted its management and control. / Since conducting randomized-controlled trials during the 2003 crisis was almost impossible, most of the presented studies are either descriptive or case-controlled in design. However, these studies have laid foundations for recent and future research into the clinical diagnosis and management of SARS. Moreover, the construction of the SARS clinical database has contributed to the work of other investigators, which has resulted in over thirty-six publications. It is my hope that these research endeavors can contribute to the understanding of this emerging, deadly disease. / Lee Lai Shun, Nelson. / "April 2006." / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0205. / Thesis (M.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 264-292). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

A finales de 2020 se aprobaron las vacunas desarrolladas en el mundo occidental contra el virus SARS-CoV-2, que ya están siendo inoculadas, conjuntamente con vacunas chinas y rusas. Mientras tanto, estamos en una segunda oleada de la enfermedad y el nuevo coronavirus se ha ido transformando para permitirse una mejor propagación, alojamiento y replicación en el ser humano. La enfermedad se manifiesta ahora con nueva sintomatología, mayor contagio, inclemencia y variación en el número de fallecimientos. La infección de la gestante por coronavirus se está presentando con severidad y consecuencias materno perinatales. Ya se inició la vacunación en gestantes y madres lactantes, previa conversación con su ginecólogo sobre los riesgos y beneficios. Este artículo ofrece un breve rel los acontecimientos que tuvieron lugar durante la transición de 2020 a 2021. / In late 2020, vaccines developed in the Western world against the SARS-CoV-2 virus were approved and are currently being inoculated, together with Chinese and Russian vaccines. In the meantime, we are in a second wave of the disease and the new coronavirus has been transforming to allow for better propagation, harboring and replication in humans. The disease now manifests itself with new symptoms, greater contagiousness, severity and variation in the number of deaths. Coronavirus infection of pregnant women is occurring with harshness and maternal and perinatal consequences. Vaccination has been initiated in pregnant women and nursing mothers, after discussion with their gynecologists about risks and benefits. This article provides a b ok place during the transition from 2020 to 2021.