Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: With a cure for HIV and AIDS still absent, non-nucleoside reverse transcriptase inhibitors (NNRTIs) play a major role in the current antiretroviral treatments used, which have shown to improve and prolong the lives of HIV patients significantly. However, with rapid mutations of the HI virus, the use of these drugs is becoming limited, thereby highlighting the need for the development of new NNRTIs. Previous work by our research team has led to the development of a cyclopropyl-containing indole-based compound with an inhibition activity (IC50 value) of 0.1 μM, as determined in an in vitro single-cycle, non-replicative phenotypic assay. Therefore, in this project, we focussed on enhancing the intermolecular interactions of our compound to three major areas in the NNRTI binding pocket, namely the Tyr181, the Val179, and the Lys101 binding pockets. Hereby we were able to obtain both improved and lower potencies, with our most active compound having an inhibition activity (IC50 value) of 1 nM. For the interaction to the Tyr181 binding pocket, we were thus unable to synthesise a heterocyclic ring system onto our molecule as opposed to the previously used phenyl ring. Secondly, for the interaction to the Lys101 binding pocket we were able to synthesise a tetrazole ring system and an amide functionality onto the 2-position of the indole. Lastly, in our quest to synthesise the cyclopropyl moiety onto our compound for the interaction in the Val179 binding pocket, we were able to investigate the full inhibition effect of this interaction by synthesising a similar compound with no interaction in this binding pocket. Moreover, we were able to synthesise a new compound with a methoxy moiety for this interaction with an inhibition activity (IC50 value) of 1 nM. With this compound only being submitted for efficacy evaluation as a racemic compound mixture, this opened a new door for research possibilities for our team. / AFRIKAANSE OPSOMMING: In die awesigheid van 'n geneesmiddel vir MIV en VIGS, speel nie-nukleosied omkeerbare transkripsie inhibitore ("NNRTIs")'n groot rol in die huidige antiretrovirale behandeling. Ongelukkig ondergaan die MI virus mutasies, wat dus die gebruik van hierdie antiretrovirale middels beperk. Hierdie beklemtoon dus die noodsaaklikheid vir die ontwikkeling van nuwe "NNRTIs". Vorige werk wat deur ons navorsings groep verrig is, het gelei tot die ontwikkeling van "n siklopropiel bevattende indol verbinding, met "n inhibisie aktiwiteit ("IC50" waarde) van 0.1 μM. Gevolglik, het ons in hierdie projek gefokus om die intermolekulêre interaksies van hierdie verbinding in drie hoof areas in die "NNRTI" bindings ruimte te verbeter, genaamd die Tyr181, die Val179, en die Lys101 bindings ruimtes. Hierdie projek het dus beide verbeterde en ook laer inhibisie aktiwiteits resultate gelewer, waar die mees aktiewe verbinding 'n inhibisie aktiwiteit ("IC50" waarde) van 1 nM behaal het. Vir die interaksie na die Tyr181 bindings ruimte, was ons dus onsuksesvol om 'n heteroaromatiese ring te sintetiseer as plaasvervanger vir die oorspronklike feniel ring. Tweedens, vir die interaksie na die Lys101 bindings ruimte, was ons in staat om 'n tetrazol ring en 'n amied funksionaliteit aan die 2-posisie van die indol te sintetiseer. In ons stryd om die siklopropiel ring aan ons verbinding te sintetiseer vir die interaksie in die Val179 bindings ruimte, was ons in staat om die volledige effek van hierdie interaksie te bepaal deur 'n soortgelykke verbinding te sintetiseer met geen interaksie in die Val179 bindings ruimte nie. Daarenbowe, het ons 'n verbining gesintetiseer met 'n inhibisie aktiwiet ("IC50" waarde) van 1 nM, waarvan die aktiwitiet van slegs die rasemiese mengsel van die verbinding bepaal is. Hierdie vinding het dus 'n nuwe navorsings deur vir ons groep geopen.

published_or_final_version / Anatomy / Master / Master of Philosophy

Organic nitrates, such as nitroglycerine (NTG), are widely used for their potent vasodilator capacity in the management of coronary artery disease and heart failure. Unfortunately, their beneficial effect is rapidly lost due to the development of nitrate tolerance, which is translated by an impaired vasorelaxation to NTG and an increased oxidative stress production. Although the mechanisms of the development of nitrate tolerance are still not fully elucidated, much interest has been focused in treating nitrate-receiving patients together with other drugs in order to overcome the development of nitrate tolerance. The Nitric Oxide generating enzyme, eNOS, and the superoxide anion generating enzyme, NAD(P)H oxidase, have been suggested to play a role in the development of nitrate tolerance. The aim of this study was to analyse the underlying mechanism by which ramipril, an ACE inhibitor and rosuvastatin, a new molecule of the statin class, are able to protect against the development of nitrate tolerance in the aortas isolated from rats, wild-type (wt) and eNOS-/- mice. These results show that ramipril as well as rosuvastatin are able to protect against the development of nitrate tolerance in the wt and eNOS-/- mice aortas suggesting that eNOS is not necessary for their protective effect. The aortas from nitrate tolerant rats and mice showed a significant increase in the NAD(P)H oxidase activation compared to the aortas from the control and from the co-treated ramipril+NTG or rosuvastatin+NTG animals. In line with these findings were the results obtained by RT-PCR analysis: the mRNA expression of the different subunits of the NAD(P)H oxidase, such as gp91phox, p22phox, were significantly decreased after rosuvastatin or ramipril treatment in wt and eNOS-/- mice aortas. Apocynin, the NAD(P)H oxidase inhibitor was also able to inhibit the development of nitrate tolerance in the rat and mouse aortas. In conclusion, these results suggest that rosuvastatin and ramipril are able to protect against the development of nitrate tolerance by counteracting the nitrate-induced oxidative stress. The mechanism of protection involves a direct interaction with the NAD(P)H oxidase pathway and seems to be completely independent of the eNOS pathway.

Louis, Tong Kwok-leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 121-130). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGEMENTS --- p.III / LIST OF CONTENTS --- p.IV / LIST OF TABLES --- p.VIII / LIST OF FIGURES --- p.X / ABBREVIATIONS --- p.XII / GENERAL ABBREVIATIONS --- p.XII / VARIOUS NOMENCLATURES OR ABBREVIATIONS FOR SRSVS --- p.XIII / OBJECTIVES OF THE STUDY --- p.XIV / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- HISTORICAL PERSPECTIVE --- p.2 / Chapter 1.2 --- CLINICAL FEATURES OF HUMAN SRSV INFECTION --- p.10 / Chapter 1.3 --- EPIDEMIOLOGY --- p.12 / Chapter 1.4 --- PHYSICAL CHARACTERISTICS OF SRSV --- p.14 / Chapter 1.5 --- STABILITY OF NORWALK VIRUS --- p.15 / Chapter 1.6 --- DIAGNOSTIC TOOLS FOR SRSVS --- p.15 / Chapter 1.7 --- GENOMIC ORGANIZATION OF SRSVS --- p.16 / Chapter 1.8 --- MOLECULAR TECHNOLOGIES --- p.19 / Chapter CHAPTER 2 --- MATERIALS --- p.20 / Chapter 2.1 --- FAECAL SAMPLES FROM 1986 TO 1992 --- p.21 / Chapter 2.2 --- OTHER FAECAL SAMPLES FROM 1995 TO 1998 --- p.21 / Chapter 2.3 --- AGE GROUPS OF ALL THE SAMPLES --- p.21 / Chapter CHAPTER 3 --- METHODS --- p.23 / Chapter 3.1 --- EXTRACTION OF RNA FROM PATIENT STOOL OR VOMIT SAMPLES --- p.24 / Chapter 3.2 --- REVERSE TRANSCRIPTION - POLYMERASE CHAIN REACTION (RT-PCR) FOR SRSVS --- p.25 / Chapter 3.2.1 --- Principle of RT-PCR assay for SRSV --- p.25 / Chapter 3.2.2 --- Reverse transcription (cDNA Synthesis) --- p.26 / Chapter 3.2.3 --- Polymerase chain reaction --- p.26 / Chapter 3.2.4 --- Electrophoresis (in the PCR product room) --- p.31 / Chapter 3.2.5 --- Controls for PCR assay --- p.32 / Chapter 3.2.6 --- Interpretation of SRSV polymerase gene PCR --- p.32 / Chapter 3.3 --- RT-PCR USING INOSINE CONTAINING PRIMERS FOR THE CAPSID REGIONS --- p.34 / Chapter 3.3.1 --- RT-PCR for the capsid region of SRSV genome --- p.34 / Chapter 3.3.2 --- Interpretation of SRSV capsid gene PCR --- p.36 / Chapter 3.4 --- SOLID PHASE IMMUNE ELECTRON MICROSCOPY FOR THE DETECTION OF SRSV --- p.37 / Chapter 3.5 --- OPTIMIZATION OF CONDITIONS FOR SRSV RT-PCR --- p.37 / Chapter 3.5.1 --- Titration of primers --- p.37 / Chapter 3.5.2 --- Titration of MgCl2 --- p.38 / Chapter 3.5.3 --- "Titration ofdNTPs, MgCl2 and Taq polymerase (pH 9.0)" --- p.38 / Chapter 3.6 --- SPECIFICITY OF SRSV RT-PCR --- p.38 / Chapter 3.7 --- PURIFICATION OF PCR PRODUCTS PRIOR TO CLONING …… --- p.38 / Chapter 3.8 --- CLONING OF THE PURIFIED DNA INTO pGEM-T EASY VECTOR --- p.39 / Chapter 3.8.1 --- Introduction --- p.39 / Chapter 3.8.2 --- Sequence of the pGEM-T Easy Vector --- p.42 / Chapter 3.8.3 --- Ligation --- p.44 / Chapter 3.8.4 --- Transformation of competent bacterial cells --- p.44 / Chapter 3.8.5 --- Small-scale preparations of plasmid DNA --- p.45 / Chapter 3.8.6 --- Purification of miniprep using QIAprep Miniprep --- p.45 / Chapter 3.8.7 --- Restriction analysis of small-scale preparations of plasmid DNA --- p.45 / Chapter 3.9 --- CYCLE SEQUENCING OF CLONED SRSV AMPLICONS --- p.46 / Chapter 3.9.1 --- Targets for Sequencing --- p.46 / Chapter 3.9.2 --- Procedures of cycle sequencing --- p.46 / Chapter 3.9.3 --- Gel electrophoresis --- p.48 / Chapter 3.9.4 --- Sequencing conditions --- p.49 / Chapter 3.10 --- SEQUENCE ANALYSIS --- p.49 / Chapter CHAPTER 4 --- REAGENTS AND BUFFERS --- p.51 / Chapter 4.1 --- REAGENTS AND BUFFERS FOR RNA EXTRACTION --- p.52 / Chapter 4.2 --- REAGENTS AND BUFFERS FOR REVERSE TRANSCRIPTION (cDNA SYNTHESIS) --- p.52 / Chapter 4.3 --- REAGENTS AND BUFFERS FOR PCR --- p.53 / Chapter 4.4 --- GEL ELECTROPHORESIS OF PCR PRODUCTS --- p.53 / Chapter 4.5 --- PURIFICATION OF PCR PRODUCTS --- p.54 / Chapter 4.6 --- REAGENTS FOR CLONING THE DNA INSERT INTO pGEM-T EASY VECTOR --- p.54 / Chapter 4.6.1 --- "pGEM-T Easy Vector System (Promega Corporation, USA)" --- p.54 / Chapter 4.6.2 --- Isopropylthio-β-D-galactoside (IPTG) stock solution --- p.54 / Chapter 4.6.3 --- X-Gal --- p.54 / Chapter 4.6.4 --- Luria-Bertani (LB) medium --- p.55 / Chapter 4.6.5 --- LB plates with ampicillin --- p.55 / Chapter 4.6.6 --- LB plates with ampicillin/IPTG/X-Gal --- p.55 / Chapter 4.6.7 --- SOC medium --- p.55 / Chapter 4.6.8 --- Mini-prep purification --- p.56 / Chapter 4.6.9 --- Mini-prep analysis --- p.56 / Chapter 4.6.9.1 --- Lambda DNA-Hind IIIφX-174 DNA-Hae III Digest --- p.56 / Chapter 4.6.9.2 --- Not I --- p.58 / Chapter 4.7 --- REAGENTS AND BUFFERS FOR CYCLE SEQUENCING --- p.58 / Chapter 4.7.1 --- SequiTherm EXCEĹёØ II Long-Rea´dёØ DNA Sequencing Kit-AL´FёØ --- p.58 / Chapter 4.7.2 --- Sequencing primers --- p.59 / Chapter 4.8 --- REAGENTS FOR SEQUENCING GEL CASTING --- p.59 / Chapter CHAPTER 5 --- RESULTS --- p.61 / Chapter 5.1 --- RESULTS OF RT-PCR OPTIMIZATION --- p.62 / Chapter 5.1.1 --- Magnesium chloride and pH of PCR reaction buffer --- p.62 / Chapter 5.1.2 --- Concentration of primers --- p.64 / Chapter 5.1.3 --- "Titration of dNTPs, MgCl2 and Taq polymerase (pH 9.0)" --- p.65 / Chapter 5.2 --- RESULT OF SENSITIVITY TEST --- p.66 / Chapter 5.3 --- RESULTS OF SPECIFICITY TEST --- p.67 / Chapter 5.4 --- RESULTS OF THE PCR USING INOSINE CONTAINING POL PRIMERS --- p.70 / Chapter 5.5 --- RESULTS OF PCR USING INOSINE CONTAINING CAPSID PRIMERS --- p.73 / Chapter 5.6 --- RESULTS OF SOME SAMPLES RETESTED BY SPIEM --- p.75 / Chapter 5.7 --- RESULTS OF SPORADIC OUTBREAKS --- p.77 / Chapter 5.7.1 --- A sporadic outbreak in 1996 --- p.77 / Chapter 5.7.2 --- Sporadic outbreak in a kindergarten in 1997 --- p.79 / Chapter 5.7.3 --- Sporadic outbreak at a hotel in 1998 --- p.79 / Chapter 5.7.4 --- Application of the RT-PCR to contaminated shellfish --- p.80 / Chapter 5.8 --- RESULTS OF MINI PREP ANALYSIS WITH NOT I DIGESTION --- p.85 / Chapter 5.9 --- RESULT OF ELECTROPHEROGRAM OF A SELECTED SPECIMEN FROM THE AUTOMATIC SEQUENCING --- p.86 / Chapter 5.9 --- RESULT OF ELECTROPHEROGRAM OF A SELECTED SPECIMEN FROM THE AUTOMATIC SEQUENCING --- p.86 / Chapter 5.10 --- RESULTS OF ALL TRIMMED DNA SEQUENCES --- p.87 / Chapter CHAPTER 6 --- DISCUSSION --- p.112 / REFERENCES --- p.122 / APPENDIX --- p.131 / APPENDIX I: Electron micrograph of SRSV particles --- p.132 / APPENDIX II: Confirmation for specificity test --- p.133 / APPENDIX III: Sequencing amplicons using capsid primers --- p.135 / APPENDIX IV: Sequencing amplicons (outbreak) using pol primers --- p.136 / APPENDIX V: Electropherogram (direct sequencing) --- p.138 / APPENDIX VI: Other RT-PCR results using pol primers --- p.139 / APPENDIX VII: Results of RT-PCR using capsid primers --- p.149 / APPENDIX VIII: Mini prep analysis --- p.158

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

In most eukaryotes, chromosomes ends are protected by telomeres which are formed by repetitive DNA, specialized binding proteins, and higher order structures. Telomeres become shorter following replication due to the positioning and degradation of terminal RNA primers, as well as resection by nucleases. Extensive telomere shortening over many cell cycles elicits a DNA damage checkpoint that culminates in senescence or, in the absence of tumor suppressor pathways, apoptosis. These effects block the expansion of cells with unstable genomes, but can also precipitate disease in tissues that rely on regeneration for function. In many unicellular eukaryotes and proliferative human cells including cancer cells, telomeres can be maintained by the telomerase reverse transcriptase (TERT) and its associated RNA (TR). The elongation of telomeric DNA by telomerase depends on the telomerase essential N-terminal (TEN) and C terminal reverse transcriptase (RT) domains. We found that human TEN interacted with single-stranded telomeric DNA and restored function, in trans, to an hTERT mutant lacking hTEN. Telomerase required hTEN residues for activity, telomere maintenance, and extension of cellular replicative lifespan. Two inactive hTERT variants bearing mutations in TEN and RT domains, respectively, cooperated to regenerate telomerase activity in vitro. hTEN interacted with several regions of hTERT suggesting that dimerization may occur via TEN-TERT interactions. The in vivo defect of certain hTEN mutants may involve an inability to interact with factors that recruit the enzyme to the telomere and/or stimulate activity. Human homologs of the S. cerevisiae recruitment factor Est1 interacted with telomerase in a species-specific manner. The TPR domain of hEST1A interacted with the N-terminus of hTERT. The TPR domain of ScEst1 was required for telomere length maintenance by telomerase, and, paradoxically, also negatively regulated telomere length. In preliminary experiments, hTERT interacted with hPOT1/hTPP1. This interaction may stimulate the elongation of telomeres by telomerase. The DNA and protein interactions described herein expand our knowledge of telomerase and present new targets for the manipulation of telomerase function in human disease.

In most eukaryotes, chromosomes ends are protected by telomeres which are formed by repetitive DNA, specialized binding proteins, and higher order structures. Telomeres become shorter following replication due to the positioning and degradation of terminal RNA primers, as well as resection by nucleases. Extensive telomere shortening over many cell cycles elicits a DNA damage checkpoint that culminates in senescence or, in the absence of tumor suppressor pathways, apoptosis. These effects block the expansion of cells with unstable genomes, but can also precipitate disease in tissues that rely on regeneration for function. In many unicellular eukaryotes and proliferative human cells including cancer cells, telomeres can be maintained by the telomerase reverse transcriptase (TERT) and its associated RNA (TR). The elongation of telomeric DNA by telomerase depends on the telomerase essential N-terminal (TEN) and C terminal reverse transcriptase (RT) domains. We found that human TEN interacted with single-stranded telomeric DNA and restored function, in trans, to an hTERT mutant lacking hTEN. Telomerase required hTEN residues for activity, telomere maintenance, and extension of cellular replicative lifespan. Two inactive hTERT variants bearing mutations in TEN and RT domains, respectively, cooperated to regenerate telomerase activity in vitro. hTEN interacted with several regions of hTERT suggesting that dimerization may occur via TEN-TERT interactions. The in vivo defect of certain hTEN mutants may involve an inability to interact with factors that recruit the enzyme to the telomere and/or stimulate activity. Human homologs of the S. cerevisiae recruitment factor Est1 interacted with telomerase in a species-specific manner. The TPR domain of hEST1A interacted with the N-terminus of hTERT. The TPR domain of ScEst1 was required for telomere length maintenance by telomerase, and, paradoxically, also negatively regulated telomere length. In preliminary experiments, hTERT interacted with hPOT1/hTPP1. This interaction may stimulate the elongation of telomeres by telomerase. The DNA and protein interactions described herein expand our knowledge of telomerase and present new targets for the manipulation of telomerase function in human disease.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains viii, 174 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 42-47).

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains x, 169 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.