<p>The HIV-1 virus encodes a protease essential for the processing of polyprotein precursors into mature viral proteins. This enzyme is a primary target for drug design against AIDS. Concurrent effects of inhibitors targeted to defined regions of the extended active site were investigated using Yonetani-Theorell kinetics to understand its complex specificity requirements. Kinetic data revealed that the simultaneous presence of two specific inhibitors may increase their binding affinity for the enzyme. A 100-fold enhancement in binding affinity was observed in certain instances. Results from this work showed a correlation between inhibitor synergism and substrate specificity thus implicating subsite interactions in enzyme catalysis.</p> <p>To facilitate the analysis of enzyme-inhibitor interactions an improved graphical method, the combination plot, was developed as an alternative to the Yonetani-Theorell plot. The method generates a single straight line rather than a family of lines which is the traditional approach in such kinetic studies. The slope of the plot, 1/α, quantitatively measures the extent and nature of interaction between two inhibitors on their target enzyme. The approach was easily extended to analyze, for the first time, the interaction between three competitive inhibitors on an enzyme. The combination approach potentially has broad applications for kinetic analysis.</p> <p>Combination plots were used in the discovery of gem-dialkyl succinic acid derivatives as a new class of unusually potent reversible inhibitors of carboxypeptidases A and B. 2-Ethyl-2-methylsuccinic acid binds to carboxypeptidases A and B with dissociation constants of 1.1 X 10ˉ⁷ M and 3.4 X 10ˉ⁶ M respectively. The low dissociation constants of the inhibitors for the zinc proteases can be attributed primarily to the gem-dialkyl groups which presumably make very important hydrophobic contacts within the active site. The inhibitors may also act as zinc ligands while possessing sufficient affinity for the carboxyl-recognition site in the enzymes.</p> / Doctor of Philosophy (PhD)

This thesis describes the development of serum and whole blood immunodiagnostic assays for Maedi-Visna virus (MVV). All previously described recombinant MVV ELISA assays utilised either the core p25 or transmembrane (TM) proteins alone, or combined, but as individual proteins. The p25 and TM genes of MVV were cloned individually into the pGEX-2T expression vector. Both proteins were expressed as a combined fusion protein in frame with glutathione S-transferase (GST). The purified recombinant antigens (GST-TM and GST-TM-p25) were used to develop a MVV ELISA. Sera from 46 positive and 46 negative sheep were tested using the GST-TM and GST-TM-p25 ELISAs and a commercial p25 EIA kit. A two-graph receiver operating characteristic (TG-ROC) analysis program was used to interpret the data. The GST-TM-p25 ELISA was more sensitive than the commercial assay which is based on the p25 antigen alone and more specific than the GST-TM ELISA. The GST-TM-p25 ELISA showed a sensitivity and specificity of 100%. The human AIDS lentivirus transmembrane (TM) glycoprotein portion of the envelope viral protein has been identified as the antigen most consistently recognised by antibodies. There is suggestive evidence that the same applies to MVV as the GST-TM fusion protein, expressed in E. coli, has comparable sensitivity to the GST-TM-p25 fusion protein, but lacks specificity. However, due to the hydrophobic nature of the MVV TM protein, purification of the expressed fusion protein required lengthy purification protocols. This was despite the fact that only a truncated version of the TM protein was expressed. This prompted investigating an alternative expression system that could possibly circumvent the above mentioned problems. The yeast Pichia pastoris is known to be suitable for the high-level expression of heterologous proteins which are secreted into the culture supernatant. These features made P. pastoris an attractive host for the expression of the hydrophobic TM protein of MVV. However, limited success was achieved as only low expression levels were obtained and detection and quantification was only accomplished by means of ELISA. Evaluation of the diagnostic performance of the P. pastoris expressed MVV TM-polypeptide was performed using a panel of 36 confirmed negative and positive sera, and evaluated using a TG-ROC analysis programme, which yielded an equal Se and Sp of 83%. The use of a novel rapid immunoassay system, which allows the detection of circulating antibodies in whole blood, has been investigated for use as a MVV diagnostic assay. The central feature of this immunoassay lies in a monoclonal antibody against a glycophorin epitope present on all sheep erythrocytes. A Fab'-peptide conjugate was constructed by coupling a synthetic peptide, corresponding to a sequence from MVV TM protein, to the hinge region of the Fab' fragment of the antisheep erythrocyte antibody. Within the limited number of 10 seronegative and 10 seropositive samples the autologous red blood cell agglutination assay had a sensitivity of 90% and a specificity of 80%. Despite the limitations and difficulties encountered, the use of such rapid whole blood immunodiagnostic assays for MVV holds promise. / Thesis (Ph.D.)-University of Natal, Durban, 1997.

Thesis equivalent (M.P.H.)--University of Michigan, 1947.

Thesis equivalent (M.P.H.)--University of Michigan, 1947.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 75-80).

The overall emphasis in this thesis is the study of the regulation of virus infections by natural killer (NK) cells. In initial analyses, vaccinia virus (VV)-infected cells were found to be more sensitive to NK cell-mediated lysis during a discrete period of time post-infection. This enhanced susceptibility to lysis correlated with enhanced triggering (but not binding) of the effector cells and a concomitant decrease in target cell H-2 class I antigen expression. Furthermore, VV-infected cells became resistant to lysis by allospecific cytotoxic T lymphocytes (CTL) at a time when they were very sensitive to killing by NK cells or VV-specific CTL. This suggested that alterations in class I MHC antigens may affect target cell sensitivity to lysis by NK cells. The hypothesis that viral peptide charging of H-2 class I molecules can modulate target cell sensitivity to NK cell-mediated lysis was tested by treating target cells with synthetic viral peptides corresponding to the natural or minimal immunodominant epitopes defined for virus-specific CTL, and then target cell susceptibility to NK cell-mediated lysis was assessed. None of the 12 synthetic viral peptides used were able to significantly alter target cell lysis by NK cells under any of the conditions tested. In order to determine if H-2 class I molecules were required in the regulation of a virus infection by NK cells in vivo, intact or NK depleted (treated with anti-asialo GM1 antiserum) β2-microglobulin-deficient [β2m (-/-)] mice, which possess a defect in H-2 class I antigen expression, were infected with the prototypic NK-sensitive virus, murine cytomegalovirus (MCMV). In anti-asialo GM1-treated β2m (-/-) mice, as well as in β2m + (H-2 class I normal) control mice also treated with anti-asialo GM1 a significant enhancement in splenic MCMV titers as compared to NK-intact animals, was observed. When thymocyte expression of H-2 class I molecules (H-2Db) in normal mice was analyzed, it was found that following MCMV infection, H-2Db expression was significantly greater than the low level of expression found in uninfected thymocytes. In marked contrast, thymocytes from β2m (-/-) mice did not display any detectable H-2Db before or after infection. These in vivoresults demonstrate that NK cells can regulate a virus infection, at least in the case of MCMV, independent of H-2 class I molecule expression. Thymocytes from uninfected normal mice were found to be very sensitive to NK cell-mediated lysis, whereas those from MCMV-infected animals were completely resistant, presumably due to the protective effects of MCMV-induced interferon (IFN). However, thymocytes from MCMV-infected β2m (-/-) mice were only slightly protected from lysis by NK cells, consistent with the inverse correlation between MHC class I antigen expression and sensitivity to NK cell-mediated lysis. These results provide in vivoevidence suggesting a requirement for MHC class I molecules in IFN-mediated protection from lysis by NK cells. In addition to the analysis of H-2 class I molecules on target cells, the identity of a molecule present on the surface of all NK cells and other cytotoxic effector cells, which is recognized by a monoclonal antibody (mAb) generated in this laboratory designated CZ-1, and can also modulate NK cell triggering, was also of interest. This laboratory has previously reported that this antigen is upregulated on cytotoxic (and other) lymphocytes following a virus infection in vivo, or upon activation in vitro. Using competitive FACS analysis and fibroblasts transfected with various isoforms of CD45, it was found that mAb CZ-1 recognizes a sialic acid-dependent epitope associated with a subpopulation of CD45RB molecules.

by Chan Chuek Kee. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 165-201). / Abstract --- p.i / Table of Content --- p.iv / List of Abbreviations --- p.viii / List of Tables --- p.x / List of Figures --- p.xii / Acknowledgement --- p.xiii / Chapter Chapter 1. --- Introduction / Chapter 1.1. --- Discovery and historical events --- p.1 / Chapter 1.2. --- General characteristics of rotavirus --- p.3 / Chapter 1.2.1. --- Virus morphology --- p.3 / Chapter 1.2.2. --- Physicochemical properties --- p.3 / Chapter 1.2.3. --- Virus stability and inactivation --- p.4 / Chapter 1.2.4. --- Genome structure --- p.4 / Chapter 1.2.5. --- Rotavirus proteins --- p.5 / Chapter 1.2.6. --- Morphogenesis --- p.8 / Chapter 1.3. --- Characteristics of rotavirus infection --- p.9 / Chapter 1.3.1. --- Morbidity and mortality --- p.9 / Chapter 1.3.2. --- Clinical features --- p.11 / Chapter 1.3.3. --- Pathogenesis --- p.13 / Chapter 1.3.4. --- Seasonal variation of rotavirus infection --- p.13 / Chapter 1.3.5. --- Nosocomial rotavirus infection --- p.14 / Chapter 1.3.6. --- Route of transmission --- p.14 / Chapter 1.4. --- Classification and epidemiology of rotaviruses --- p.15 / Chapter 1.4.1. --- Rotavirus groups --- p.15 / Chapter 1.4.2. --- Rotavirus subgroups --- p.16 / Chapter 1.4.3. --- Rotavirus serotypes --- p.17 / Chapter 1.4.4. --- Rotavirus electropherotypes --- p.20 / Chapter 1.4.5. --- "Relationship between subgroup, serotype and electropherotype" --- p.23 / Chapter 1.4.6. --- Mechanisms contributing to strain variations --- p.24 / Chapter 1.5. --- Detection of rotavirus --- p.28 / Chapter 1.5.1. --- Electron microscopy (EM) --- p.28 / Chapter 1.5.2. --- Virus isolation --- p.29 / Chapter 1.5.3. --- Serological methods --- p.30 / Chapter 1.5.4. --- RNA analysis --- p.30 / Chapter 1.5.5. --- Nucleic acid hybridization --- p.31 / Chapter 1.6. --- Prevention and control of rotavirus infection --- p.32 / Chapter 1.6.1. --- Host resistance --- p.32 / Chapter 1.6.2. --- Vaccine development --- p.33 / Chapter 1.6.3. --- Passive immunization --- p.35 / Chapter 1.7. --- Objectives of this study --- p.36 / Chapter Chapter 2. --- Materials and methods / Chapter 2.1. --- Materials --- p.38 / Chapter 2.1.1. --- Reagents for tissue culture work --- p.38 / Chapter 2.1.2. --- Reagents for electropherotyping --- p.39 / Chapter 2.1.3. --- Reagents for ELISA --- p.42 / Chapter 2.1.4. --- Reagents for hybridization assay --- p.43 / Chapter 2.2. --- Methods / Chapter 2.2.1. --- Collection of specimens --- p.46 / Chapter 2.2.2. --- Rotavirus strains --- p.46 / Chapter 2.2.3. --- Monoclonal antibodies --- p.48 / Chapter 2.2.4. --- Detection of rotavirus antigen by ELISA --- p.49 / Chapter 2.2.5. --- Rotavirus electropherotyping --- p.50 / Chapter 2.2.6. --- Serotyping of rotavirus by fluorescent foci neutralization --- p.52 / Chapter 2.2.7. --- Serotyping of rotavirus by oligo- nucleotide probes hybridization --- p.55 / Chapter 2.2.8. --- Rotavirus serotyping by ELISA --- p.59 / Chapter 2.2.9. --- Rotavirus subgrouping by ELISA --- p.61 / Chapter Chapter 3. --- Results / Chapter 3.1. --- Age and sex distribution of the study population --- p.63 / Chapter 3.2. --- Detection of rotavirus by ELISA --- p.63 / Chapter 3.3. --- Seasonal distribution of rotavirus infections --- p.67 / Chapter 3.4. --- Genetic diversity of human rotaviruses --- p.74 / Chapter 3.5. --- Subgroup determination by ELISA --- p.99 / Chapter 3.6. --- Rotavirus serotypes by fluorescent foci neutralization (FFN) --- p.102 / Chapter 3.7. --- Rotavirus serotypes by ELISA --- p.107 / Chapter 3.8. --- Rotavirus serotypes as determined by oligonucleotide probes --- p.110 / Chapter 3.8.1. --- Dot hybridization --- p.110 / Chapter 3.8.2. --- Northern blots of electrophoresed RNAs --- p.118 / Chapter 3.9. --- Temporal distribution of rotavirus serotypes --- p.124 / Chapter 3.10. --- "Association between serotype, subgroups and electropherotypes" --- p.128 / Chapter 3.11. --- Unusual rotavirus strains --- p.135 / Chapter 3.12. --- Nosocomial rotavirus infection --- p.135 / Chapter Chapter 4. --- Discussion / Chapter 4.1. --- Seasonal variation of rotavirus infection --- p.141 / Chapter 4.2. --- Molecular epidemiology of rotavirus infection --- p.143 / Chapter 4.3. --- Subgrouping of human rotavirus strains --- p.146 / Chapter 4.4. --- Serotyping rotaviruses by ELISA --- p.147 / Chapter 4.5. --- Serotyping rotaviruses by oligonucleotide probe hybridization --- p.150 / Chapter 4.6 --- Advantage and disadvantage of different methods for serotyping of rotaviruses --- p.152 / Chapter 4.7. --- Distribution of rotavirus serotypes --- p.153 / Chapter 4.8. --- Association between rotavirus serotype and electropherotype --- p.156 / Chapter 4.9. --- Nosocomial rotavirus infection --- p.158 / Chapter 4.10. --- Unusual rotavirus strains --- p.159 / Chapter 4.11. --- Concluding remark --- p.162 / Chapter 4.12. --- Future studies --- p.164 / References --- p.165

Call number: LD2668 .T4 1965 A22 / Master of Science

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy