Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 102-120).

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

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

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 89-99).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 3 uppsatser.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 161-183). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Sept. 16, 2008). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

Chuck Chi-pang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 138-160). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiii / Abbreviations --- p.xiv / Acknowledgement --- p.xviii / Introduction / Chapter 1. --- Background / Chapter 1.1 --- SARS / Chapter 1.1.1 --- Outbreak and Influence --- p.1 / Chapter 1.1.2 --- Clinical Features --- p.4 / Chapter 1.2 --- SARS-CoV / Chapter 1.2.1 --- Genomic Organization --- p.5 / Chapter 1.2.2 --- Morphology --- p.7 / Chapter 1.2.3 --- Phylogenetic Analysis --- p.9 / Chapter 1.3 --- S Glycoprotein / Chapter 1.3.1 --- Functional Roles --- p.11 / Chapter 1.3.2 --- Structure and Functional Domains --- p.12 / Chapter 1.3.3 --- Interacting Partners --- p.15 / Chapter 1.3.4 --- Viral Entry Mechanism --- p.17 / Chapter 1.4 --- Aim of Study / Chapter 1.4.1 --- Mismatch of SARS-CoV Tissue Tropism and Tissue Distribution of ACE2 --- p.20 / Chapter 1.4.2 --- Presence of Other Interacting Partner(s) --- p.22 / Chapter 1.4.3 --- Significance of the Study Materials and Methods --- p.22 / Chapter 2. --- Plasmid Construction / Chapter 2.1 --- Fragment Design / Chapter 2.1.1 --- Functional Domain Analysis --- p.23 / Chapter 2.1.2 --- Secondary Structure and Burial Region Predictions --- p.24 / Chapter 2.2 --- Vector Amplification / Chapter 2.2.1 --- E. coli Strain DH5a Competent Cell Preparation --- p.30 / Chapter 2.2.2 --- Transformation of E. coli --- p.30 / Chapter 2.2.3 --- Small-scale Vector Amplification --- p.31 / Chapter 2.3 --- Cloning of DNA Fragments into Various Vectors / Chapter 2.3.1 --- Primer Design --- p.32 / Chapter 2.3.2 --- DNA Amplification --- p.35 / Chapter 2.3.3 --- DNA Purification --- p.35 / Chapter 2.3.4 --- "Restriction Enzyme Digestion, Ligation and Transformation" --- p.36 / Chapter 2.3.5 --- Colony PCR --- p.37 / Chapter 2.4 --- DNA Sequence Analysis / Chapter 2.4.1 --- Primer Design --- p.35 / Chapter 2.4.2 --- DNA Amplification and Purification for DNA Sequence Analysis --- p.39 / Chapter 2.4.3 --- Sequence Detection and Result Analysis --- p.40 / Chapter 3. --- "Protein Expression, Purification and Analysis" / Chapter 3.1 --- Protein Expression in E. coli / Chapter 3.1.1 --- Molecular Weight and pI Predictions --- p.41 / Chapter 3.1.2 --- Glycerol Stock Preparation --- p.41 / Chapter 3.1.3 --- Protein Expression Induction --- p.41 / Chapter 3.1.4 --- Protein Extraction --- p.42 / Chapter 3.1.5 --- Affinity Chromatography --- p.42 / Chapter 3.1.6 --- Removal of GroEL --- p.43 / Chapter 3.1.7 --- Protein Solubilization and Refolding --- p.44 / Chapter 3.2 --- Protein Expression in P. pastoris / Chapter 3.2.1 --- Large-scale Plasmid Amplification --- p.46 / Chapter 3.2.2 --- Restriction Enzyme Digestion and Ethanol Precipitation --- p.47 / Chapter 3.2.3 --- Preparation of KM71H Competent Cells --- p.47 / Chapter 3.2.4 --- Electroporation --- p.48 / Chapter 3.2.5 --- Colony PCR --- p.48 / Chapter 3.2.6 --- Protein Expression Induction and Time Course Study --- p.49 / Chapter 3.2.7 --- Deglycosylation --- p.49 / Chapter 3.3 --- Protein Analysis / Chapter 3.3.1 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.50 / Chapter 3.3.2 --- Western Blotting --- p.50 / Chapter 3.3.3 --- Mass Spectrometry --- p.51 / Chapter 3.3.4 --- N-terminal Sequencing --- p.52 / Chapter 3.3.5 --- Size Exclusion Chromatography --- p.52 / Chapter 4. --- Identification of Interacting Partner(s) / Chapter 4.1 --- VeroE6 Preparation / Chapter 4.1.1 --- Cell Culture --- p.53 / Chapter 4.1.2 --- Protein Extraction and Western Blotting --- p.53 / Chapter 4.2 --- Pull-down Assay --- p.54 / Chapter 4.3 --- Two-dimensional Gel Electrophores --- p.is / Chapter 4.3.1 --- Isoelectric Focusing --- p.56 / Chapter 4.3.2 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.56 / Chapter 4.3.3 --- Silver Staining --- p.57 / Chapter 4.4 --- Mass Spectrometry / Chapter 4.4.1 --- Destaining --- p.58 / Chapter 4.4.2 --- In-gel Digestion --- p.58 / Chapter 4.4.3 --- Desalting by Zip-tip --- p.59 / Chapter 4.4.4 --- Loading Sample --- p.59 / Chapter 4.4.5 --- Peptide Mass Detection and Data Analysis --- p.59 / Results / Chapter 5. --- S Protein Expression / Chapter 5.1 --- Plasmid Construction --- p.61 / Chapter 5.2 --- Molecular Weight and pi Predictions --- p.63 / Chapter 5.3 --- Protein Expression and Optimization in E. coli / Chapter 5.3.1 --- "Comparison of Expression Levels, Solubility and Purities of S Protein Fragments" --- p.64 / Chapter 5.3.2 --- "Alteration of the Solubility in Various Cell Strains, Expression Conditions and Lysis Buffers" --- p.68 / Chapter 5.3.3 --- Identification and Remove of the non-target proteins --- p.72 / Chapter 5.3.4 --- Unfolding and Refolding --- p.79 / Chapter 5.4 --- Protein Expression and Optimization in P. pastoris / Chapter 5.4.1 --- "Expression Levels, Solubility and Purities of Various S Protein Fragments" --- p.85 / Chapter 5.4.2 --- Characterization of De-N-glycosylated Recombinant Proteins --- p.89 / Chapter 6. --- Identification of Interacting partners / Chapter 6.1 --- Practicability of Pull-down Assay / Chapter 6.1.1 --- ACE2 Extraction --- p.95 / Chapter 6.1.2 --- Pull-down of ACE2 by the P. pastoris-expressed recombinant RBD --- p.96 / Chapter 6.2 --- Pull-down Assay and Two-dimensional Gel Electrophoresis --- p.97 / Chapter 6.3 --- Identification of Putative Interacting Partners by MALDI-TOF-TOF --- p.107 / Chapter 7. --- Discussion / Chapter 7.1 --- S Protein Expression in E. coli / Chapter 7.1.1 --- Improving Recombinant Protein Expression Level and Solubility --- p.114 / Chapter 7.1.2 --- S Recombinant Protein Bound by GroEL --- p.117 / Chapter 7.2 --- S Protein Expression in P. pastoris / Chapter 7.2.1 --- Advantages of Using P. pastoris --- p.119 / Chapter 7.2.2 --- Variation of S Fragment Expression Levels --- p.120 / Chapter 7.2.3 --- Sizes of S Protein Fragments --- p.123 / Chapter 7.3 --- Identification of Interacting Partners / Chapter 7.3.1 --- Relationship between S Protein and Putative Interacting Partners --- p.124 / Chapter 7.3.2 --- Failure of Finding ACE2 --- p.125 / Chapter 7.3.2 --- Difficulty in the Identification of Protein Spots --- p.126 / Chapter 7.4 --- Conclusion --- p.131 / Chapter 7.5 --- Future Perspective --- p.132 / Chapter 8. --- Appendix --- p.133 / Chapter 9. --- References --- p.138