Dissertation (M. Appl. Sc.) -- Lincoln University, 2008. / Also available via the World Wide Web.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Mar 10

Thesis (MMedVet (Altil)) - University of Pretoria, 2002. / Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

No description available.

Thesis (M.S.)--Duquesne University, 2009. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 113-138) and index.

Ph.D.

Plasmodium falciparum, the most pestilential of the malaria parasite species, is responsible for ~450,000 direct deaths annually. Clinical disease is a consequence of the blood stage of the parasite’s lifecycle involving a plethora of host-parasite interactions. Key to these interactions are the P. falciparum reticulocyte binding-like homologue (PfRh) proteins responsible for binding erythrocyte receptors and gaining entry to host cells. For example, PfRh4 binds to human complement receptor-1 (CR1) on erythrocytes for sialic-acid-independent invasion. Another protein important for invasion is the PfRh5-interacting protein (PfRipr), an essential member of the PfRh5-associated invasion complex (PAIN-complex) along with CyRPA, the cysteine-rich protective antigen. Loss of function of PfRipr in P. falciparum parasites prevents erythrocyte entry and ablates Ca2+-influx into the erythrocyte; essential events during invasion. This study aimed to biochemically and structurally investigate truncated recombinant versions of PfRh4 and PfRipr. Homology modelling suggested that PfRh4 is rich in alpha-helical secondary structure. The sequence of PfRipr suggested the presence of ten epidermal growth factor-like (EGF) modules, two towards the N-terminus and eight in the C-terminal domain. In this project, monoclonal antibodies made against recombinant PfRh4 were shown, via indirect immunofluorescent assays, to localize to the apical tip of merozoites. Monoclonal antibody 5H12, raised against PfRh4, reduces parasite invasion of erythrocytes by ~75% in growth-inhibition assays with neuraminidase pre-treated erythrocytes. Attempts to produce a stable truncated recombinant PfRh4 protein for structural studies were unsuccessful. An ELISA-based assay using ten alanine-scan mutants suggested the CR1-binding site lies outside of amino acids 283 – 341 of PfRh4. PfRipr truncations, defined by the boundaries of EGF-like repeats predicted based on sequence homology, were produced recombinantly in Escherichia coli and Pichia pastoris. These proteins had a circular dichroism signature suggestive of β-strand-containing proteins with disordered regions. EGF-containing PfRipr truncations did not bind recombinant PfRh5 according to ELISA and size-exclusion chromatography assays. EGFs 1-2, 5-7 and 7-10 of PfRipr did not bind CyRPA via size-exclusion chromatography or NMR. Crystallisation trials performed on EGF modules failed to yield crystals suitable for data collection. A 15N isotopically-labelled sample of EGF5-7 gave good quality HSQC NMR spectra. A suite of three-dimensional NMR spectra collected on a 13C,15N-EGF5-7 sample, at three different temperatures, allowed for >86% of backbone assignments. T1/T2 relaxation analysis and heteronuclear NOE data were suggestive of an elongated, rigid protein undergoing intermolecular self-association. Further evidence for EGF5-7 being an elongated protein was provided via SAXS analysis. Chemical shifts facilitated prediction of secondary structure in EGF 5-7 consistent with an EGF-like fold. Melting studies performed on EGF5-7 showed no evidence of denaturation over the temperature range 20 °C - 95 °C indicating a thermally-stable protein. The addition of Ca2+ to the 15N-EGF5-7 sample caused chemical shift perturbations consistent with high-affinity binding. The discovery of inhibitory monoclonal antibodies recognising a conformational epitope on EGF7 provided evidence of the functional importance of this region within PfRipr. The work described in this thesis provides methods for the industrially-scalable production and biophysical investigations of P. pastoris or E. coli-produced disulfide-rich P. falciparum antigens of interest to vaccinologists.

Human infection by Salmonella Typhi has been occurring for the last 50,000 years and still accounts for ∼ 22million new cases each year worldwide. Through faeco-oral transmission, this human-restricted infection disproportionately affects the most impoverished sections of endemic communities where adequate sanitation infrastructure and effective vaccination approaches are lacking. Development of new control measures to accurately measure the burden of disease and to prevent infection with new vaccine candidates are hindered by an incomplete understanding of host-pathogen interactions and of what constitutes a protective human response after exposure. In this thesis I describe the practical application of a recently developed human challenge model of typhoid infection in assessing new control measures, including the evaluation of the oral single-dose vaccine candidate, M01ZH09. In performing a large, double-blind, placebo-controlled study, I was able to measure the direct protective efficacy (PE) of vaccination with either M01ZH09 or 3-dose Ty21a by performing human challenge with 104CFU Salmonella Typhi, Quailes strain, 28-days later. Using clinical and microbiological definitions to confirm typhoid diagnosis during a 14-day period after ingestion, I found insignificant levels of protection afforded by a single dose of M01ZH09 (12.9%), and a low PE after Ty21a vaccination (35%), demonstrating the stringency of the model and the endpoints used. Many additional insights into pathogen dynamics and host responses were found highlighting several important characteristics of oral vaccination. M01ZH09 was highly immunogenic, and both active vaccines significantly reduced bacterial burden (bacteraemia and stool shedding) while having no effect on symptomatic severity of infection in those diagnosed. M01ZH09 receipt resulted in a significantly longer incubation period, suggesting underlying protective responses were being generated. Further findings included the first objective demonstration of primary bacteraemia occurring after typhoid exposure, and frequent asymptomatic infection or stool shedding in those exposed but remaining well. Overall, these data also demonstrated significant protective effects against challenge by anti-Vi antibody status and age at baseline. Taking these factors into account, M01ZH09 and Ty21a vaccination did convey an overall protective advantage against developing typhoid infection, each reducing the risk of diagnosis by ~two-fold during the challenge period.