La protéine multidomaine Pr55Gag de VIH-1 joue un rôle crucial dans les étapes finales de la réplication virale, notamment lors de la reconnaissance et la sélection de l’ARN génomique ainsi que lors de la production de nouvelles particules virales. Outre son rôle structural, Pr55Gag chaperonne aussi les séquences d’acides nucléiques, une propriété cruciale pour la dimérisation de l’ARN génomique et l’hybridation de l’amorce ARNt à l’ARN génomique. Des partenaires cellulaires comme la protéine ribosomale RPL7 sont supposées être recrutées par Pr55Gag afin d’augmenter son potentiel chaperon. Afin d’étudier le mécanisme d’hybridation des acides nucléiques par Gag et RPL7, nous avons examiné leur effet sur la réaction d’hybridation entre dTAR, la version ADN de l’élément de transactivation virale et sa séquence complémentaire cTAR. Nos résultats révèlent que Gag et RPL7 présentent des mécanismes différents pour promouvoir l’hybridation cTAR/dTAR. Utilisés de concert, RPL7 peut aider Gag à chaperonner des séquences stables de l’ARN génomique que Gag seule pourrait difficilement chaperonner. Ce renforcement par RPL7 de l’activité chaperonne de Gag jouerait un rôle critique dans l’assemblage du virus. / The multidomain Pr55 Gag protein of HIV-1 plays a crucial role during late stages of viral replication, notably for the recognition and selection of genomic RNA as well as for the production of new viral particles. In addition to its structural role, Pr55 Gag also chaperones nucleic acid sequences, a property which is crucial for genomic RNA dimerization and annealing of the primer tRNA to the genomic RNA. Cellular partners like ribosomal protein RPL7 are thought to be recruited by Pr55 Gag to enhance its chaperoning potential. To investigate the nucleic acid annealing mechanism of Gag and RPL7, we examined their effect on the annealing reaction between dTAR, the DNA version of the viral transactivation element and its complementary cTAR sequence taken as relevant model HIV-1 sequences. Our data show that Gag and RPL7 exhibit different mechanisms for promoting the cTAR/dTAR annealing. When used together, RPL7 can help Gag to chaperone stable sequences of the genomic RNA that Gag would hardly be able to chaperone alone. This RPL7-driven boost in Gag chaperone activity is thought to be critical in the viral assembly process.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Gag polyprotein is the precursor of HIV-1 structural proteins, required for correct assembly, budding and maturation of viral particle within HIV-1 life cycle. The process of maturation into an infectious virion is dependent on Gag and GagPol cleavage at nine predefined sites by HIV-1 proteinase. Its disruption is one of the main targets of HIV treatment. HIV-1, however, develops resistance to the proteinase inhibitors by creating mutations in both the proteinase and the substrate. The Gag processing by HIV-1 proteinase is a highly sequential process, that happens in specific order and rate. Previous biochemical studies determined the kinetic data of these processes using oligopeptides representing naturally occuring cleavage sites. This thesis describes the cleavage of the Gag polyprotein itself, which is the natural substrate of HIV-1 proteinase. For this purpose, the full-length Gag polyprotein was recombinantly prepared in bacterial expression system. The cleavage was carried out and its products were analyzed via SDS-PAGE and Western blotting. The substrate specificity of the wild-type and mutant HIV-1 proteinase with respect to the full-length wild-type Gag polyprotein was compared. Substantial differences were observed between the rates of individual steps of cleavage by the wild-type and mutant...

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Karenia brevis dinoflagellate blooms off the west coast of Florida can create devastating effects on marine communities when they release a neurotoxin known as a brevetoxin. These blooms, informally referred to as red tides, can cause massive fish kills, necessitate closures of shellfish fisheries, and can even leave lingering toxins that impact shelf communities long after the bloom has dissipated. As a result, much effort has been put into studying K. brevis bloom initiation and dynamics. However, how K. brevis blooms impact Florida's fisheries is not fully understood because the relationship between K. brevis cell counts and fish mortality is poorly described. To study this relationship and the ecosystem response to K. brevis blooms, Ecopath with Ecosim (EwE) modeling is used to force K. brevis bloom mortality on the shelf ecosystems by using a recently developed time series that indexes K. brevis bloom severity. This index dynamically drives K. brevis bloom mortality in EwE in a historical reconstruction scenario from 1980 to 2009. Three hypotheses on ecosystem response are explored using Gag grouper as a case study. We postulate a) that K. brevis blooms impose bottom-up and top-down effects on the food web, b) that episodic perturbations by these blooms shape the community structure and c) that fishing pressure exacerbates those effects. Results support the hypothesis that K. brevis blooms pose top-down food web pressures, which is seen by evidence of trophic cascading. Changes in community structure with bloom mortality are also evidenced by changes seen in biodiversity and richness. An exacerbation of those effects as a result of heavy fishing pressure is evident, however, is only seen during severe bloom events. Little to no changes were found in the mortality from K.brevis blooms during blooms of average severity, and less mortality was imposed on the system during blooms of particularly low severity. However, this may be an artefact of the mode of action of K. brevis in EwE. Investigation of bloom effects on Gag showed that natural mortality rates of Gag appear to be largely influenced by mortality incurred during K. brevis blooms relative to the low rate of predation on Gag. Moreover, consumption rates of Gag on its prey were found to increase under a realistic schedule of these blooms. This may be due to a combination of effects, including increased mortality on competitors (making more prey available for Gag) and a lowering of the mean age of the Gag stock, which increases population productivity.

Chemokines are small immune system proteins mediating leukocyte migration and activation, and are important in many aspects of health and diseases. Some chemokines also have the ability to block HIV-1 infection by binding to the HIV-1 co-receptors CCR5 (CC chemokine receptor 5) and CXCR4 (CXC chemokine receptor 4). The first part of this work is to determine the mechanism of action of a human herpesvirus-8 encoded viral chemokine analog vMIP-II (viral macrophage inflammatory protein-II) by characterizing its interactions with endothelial surface glycosaminoglycans (GAGs) and cell surface receptors. Nuclear magnetic resonance (NMR), mutagenesis and molecular-docking were conducted and results show that vMIP-II tightly binds glycosaminoglycans using residues distributed along one face of the protein, such as R18, R46 and R48, and that there is a shift in the GAG binding site between the monomer and dimer form of vMIP-II where the N-terminus is involved in GAG binding for the dimer. This study, for the first time, provides a model that explains the mechanism of how quaternary structure affects chemokine-GAG binding. Mutagenesis and competition binding assays were conducted to study the receptor-binding mechanism of vMIP-II. Preliminary results suggest that vMIP-II uses the same positively charged binding surface comprising R18, K45, R46 and R48 to interact with the negatively charged N-termini of CCR5 and CXCR4. NMR studies on how vMIP-II interacts with N-terminal peptides of CCR5 and CXCR4 is on-going. The second part of this work was to rationally design HIV-1 entry inhibitors based on our knowledge of the mechanisms of chemokine-receptor binding and HIV-1 cell entry. We successfully designed two chimeric HIV entry inhibitors composed of CCR5-targeting RANTES variants (5P12-RANTES and 5P14-RANTES) linked to a gp41 targeting C-peptide, C37. In in vitro assays, chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 showed the highest anti-viral potency yet published with IC50 values as low as 0.001 nM against certain virus strains. On human peripheral blood mononuclear cells, the chimeric inhibitors also exhibited very strong inhibition against R5-tropic and X4-tropic viruses, with IC50 values as low as 0.015 nM and 0.44 nM, respectively. A clear delivery mechanism was observed and characterized. These fully recombinant inhibitors can be easily produced at low cost and are excellent candidates for HIV microbicides.

HIV-1 genetic diversity is a major obstacle for developing an effective vaccine. My hypothesis is that HIV-1 genetic diversity can be characterized and that cross-clade immunogens can be predicted at the population level. I systematically investigated positive selection (PS) pressures on HIV-1 Env and Gag proteins based on the analysis of the sequences collected from the Los Alamos Sequence Database. I identified PS sites, investigated PS patterns, correlated PS with the known functional sites of the two proteins, calculated frequencies of HLA alleles targeting CTL epitopes, and compared PS patterns among major subtypes. The results showed that PS pressure was widely dispersed across the entire regions of both HIV-1 Env and Gag proteins, suggesting the conserved regions are under host immune response pressure. The neutralizing antibody, non-neutralizing antibody, and CTL responses were found to be the major forces driving genetic diversity of HIV-1 env and gag genes at population level. However, PS pressures on both Env and Gag proteins remain stable over time, suggesting genetic diversity of HIV-1 driven by host immune responses changed very little over the last 29 years. Furthermore, the results also demonstrated that up to 70% PS sites were shared among the major HIV-1 clades, implying the existence of cross-clade immunogenicity. A number of potential cross-clades immunogens were predicted to elicit CTL or neutralizing antibody responses from Env and Gag proteins. I also detected a significant correlation between HLA allele frequencies and host CTL responses elicited by Accessory/Regulator’s proteins at population level. Moreover, I detected an association between the frequency of HLA-B7 supertype and the number of identified optimal CTL epitopes. The results suggest HLA class I allele frequencies in a population influence the evolution of HIV-1. I also systematically evaluated the utility of ultra-deep pyrosequencing to characterize genetic diversity of HIV-1 gag genes within quasispecies. The results showed that ultra-deep pyrosequencing of amplified HIV genes is a better method than the traditional Sanger-clone-based method in the comprehensive characterization of genetic diversity of HIV-1 quasispecies, especially in detecting low frequency variations. In conclusion, my thesis provides important information for rational design of an effective HIV-1 vaccine.

HIV-1 genetic diversity is a major obstacle for developing an effective vaccine. My hypothesis is that HIV-1 genetic diversity can be characterized and that cross-clade immunogens can be predicted at the population level. I systematically investigated positive selection (PS) pressures on HIV-1 Env and Gag proteins based on the analysis of the sequences collected from the Los Alamos Sequence Database. I identified PS sites, investigated PS patterns, correlated PS with the known functional sites of the two proteins, calculated frequencies of HLA alleles targeting CTL epitopes, and compared PS patterns among major subtypes. The results showed that PS pressure was widely dispersed across the entire regions of both HIV-1 Env and Gag proteins, suggesting the conserved regions are under host immune response pressure. The neutralizing antibody, non-neutralizing antibody, and CTL responses were found to be the major forces driving genetic diversity of HIV-1 env and gag genes at population level. However, PS pressures on both Env and Gag proteins remain stable over time, suggesting genetic diversity of HIV-1 driven by host immune responses changed very little over the last 29 years. Furthermore, the results also demonstrated that up to 70% PS sites were shared among the major HIV-1 clades, implying the existence of cross-clade immunogenicity. A number of potential cross-clades immunogens were predicted to elicit CTL or neutralizing antibody responses from Env and Gag proteins. I also detected a significant correlation between HLA allele frequencies and host CTL responses elicited by Accessory/Regulator’s proteins at population level. Moreover, I detected an association between the frequency of HLA-B7 supertype and the number of identified optimal CTL epitopes. The results suggest HLA class I allele frequencies in a population influence the evolution of HIV-1. I also systematically evaluated the utility of ultra-deep pyrosequencing to characterize genetic diversity of HIV-1 gag genes within quasispecies. The results showed that ultra-deep pyrosequencing of amplified HIV genes is a better method than the traditional Sanger-clone-based method in the comprehensive characterization of genetic diversity of HIV-1 quasispecies, especially in detecting low frequency variations. In conclusion, my thesis provides important information for rational design of an effective HIV-1 vaccine.

Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are important, natural polysaccharides which occur in biological (connective) tissues and have various biotechnological and medical applications. Additionally, there is increasing evidence that chemically (over)sulfated GAGs possess promising properties and are useful as implant coatings. Unfortunately, a detailed characterization of these GAGs is challenging: although mass spectrometry (MS) is one of the most powerful tools to elucidate the structures of (poly)saccharides, MS is not applicable to high mass polysaccharides, but characteristic oligosaccharides are needed. These oligosaccharides are normally generated by enzymatic digestion. However, chemically modified (particularly sulfated) GAGs are extremely refractive to enzymatic digestion. This study focuses on the investigation of the digestibility of GAGs with different degrees of sulfation by bovine testicular hyaluronidase (BTH). It will be shown by using an adapted spectrophotometric assay that all investigated GAGs can be basically digested if the reaction conditions are carefully adjusted. However, the oligosaccharide yield correlates reciprocally with the number of sulfate residues per polymer repeating unit. Finally, matrix-laser desorption and ionization (MALDI) MS will be used to study the released oligosaccharides and their sulfation patterns.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references.