Includes bibliographical references (leaves 161-184). / This study shows that HIV-specific cervical T cells can be isolated by cytobrushing and in vitro polyclonal expansion is a useful approach to increase the number of T cells available from mucosal sites. Dynal beads (1:1) in the presence of IL-2, IL-7 and IL-15 resulted in the best yields of cervical T cells while anti-CD3 in the presence of IL-2 best conserved the ex vivo T cell profile. Expanded T cell lines, irrespective of expansion method used, generally maintain their cytokine response profile to HIV anti- gens. This study shows that HIV Gag-specific blood and cervical T cells were largely mono-functional with polyfunctional T cells being detected in women with high blood CD4 count and low plasma viral load. This study confirms that HIV-specific Gag T cell responses detected in the polyclonal expanded female genital tract T cells are associated with those measured in blood during HIV infection.

Studies on Mycobacterium tuberculosis (MTB) antigens are important to improve immunodiagnostics and vaccine efficacy. A novel genome based strategy for antigen discovery is to relate what is highly expressed by bacilli in vivo or in vitro, to what is recognized by human T cells as antigens. As hypoxia is a relevant stimulus that MTB encounters in vivo, whole genome based transcriptional profiles of M. tuberculosis subject to prolonged hypoxia (described as the Enduring hypoxic response (EHR) were analyzed, to guide the discovery of novel potential anitgens, by a combined bioinformatic and empirical approach and to determine evidence of infection stage specific recognition.

The development of an effective HIV-1 vaccine remains a global priority. Neutralizing antibodies (nAbs), which block infection by cell-free virus, are likely to be an important response for vaccines to elicit. However, evidence from the RV144 vaccine trial and non-human primate vaccine studies suggest antibody-dependent cellular cytotoxicity (ADCC) responses, which target virus-infected cells, may also be protective. This thesis uses deep sequencing, together with immune assays, to characterise HIV-1 Envelope evolution associated with both nAb and ADCC responses in early infection, and investigates broadly neutralizing and non-neutralizing monoclonal antibody ADCC activity against subtype C viruses. Recent advances in deep sequencing approaches, coupled with the primer ID method which barcodes each viral genome, enabled us to generate thousands of viral sequences to accurately track viral population dynamics in early infection. In all participants investigated, there was a significant drop in the relative frequency of wildtype (WT) virus following nAb responses. However, in three of the seven participants, when controlling for changes in viral load (VL) over time, we observed that the WT load (frequency of the WT residue x total VL) remained relatively stable despite an effective nAb response. Instead, there was an outgrowth of the escaped virus with a concomitant increase in viral loads. We found that nAbs were inefficient at blocking cell-cell transmission of early WT and escape viruses, identifying this as one mechanism by which viruses may persist despite the presence of nAbs. These results suggest that other antibody effector functions such as ADCC, which target infected cells, may be important to elicit in a protective HIV-1 vaccines. If ADCC responses are important in controlling viral populations, one would expect to find evidence of viral escape from these responses. In all nine participants investigated, we found ADCC responses emerged prior to nAb responses, and in three individuals we observed sequence changes prior to detectable nAbs. To evaluate if these changes were due to ADCC pressure on the virus, we introduced select mutations into infectious molecular clones encoding the cognate early/acute envelope (Env-IMCs). In one participant, the mutation introduced conferred resistance to both nAb and ADCC responses, while in two participants, mutations were identified which resulted in resistance to ADCC but had no effect on neutralization, suggesting escape from ADCC. Longitudinal analysis in one of these participants, which targeted the CD4- binding site, revealed three distinct escape pathways, of which two conferred resistance to ADCC, and confirmed that ADCC responses can directly drive viral evolution in vivo. Finally, we investigated the ADCC activity of eleven anti-HIV-1 monoclonal antibodies (mAbs), including seven broadly neutralizing antibodies (bnAbs) and four non-neutralizing antibodies (nnAbs), against a panel of nine acute subtype C Env-IMCs. We found bnAbs had low to moderate ADCC breadth (11-66%). In contrast, while the two V2 nnAbs we tested were narrow and weak, the two nnAbs targeting CD4-induced epitopes (A32 and C11) mediated the broadest (78-100%) and most potent (0.06-0.81 μg/mL) ADCC against this panel. In addition, a nonlinear relationship was found between ADCC activity and strength of mAb binding to the infected cell surface (rs = -0.5309, p=0.0001). In conclusion, in contrast to studies which evaluated limited number of sequences, utilizing deep sequencing approaches, we found that the WT load remained relatively stable following early nAb pressure, albeit at lower relative frequency to the escape variant. Evasion of antibody responses through cell-cell transmission may contribute to the persistence of WT virus, providing further motivation for the importance of antibody effector functions that target infected cells in a protective HIV-1 vaccine. For the first time, we provide evidence of ADCC-mediated immune pressure in early infection, showing that these responses can exert selective pressure on HIV-1. However, the limited number of sequence changes relative to those observed following nAb pressure suggests that this response does not put as much selective pressure on the virus as nAbs. Lastly, the moderate breadth of bnAb ADCC activity provides evidence that there are common epitopes on free virions and on the surface of infected cells. This indicates bnAbs with potent and broad ADCC should be identified to include in antibody-based treatment and cure strategies, which aim to eliminate infected cells. Altogether, these data suggest that while eliciting nAbs should be the primary goal of HIV-1 vaccine design, ADCC-mediating antibodies may also play an important role.

Includes abstract. / Includes bibliographical references (leaves 110-135). / Recent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix (ECM) components found in the stroma, including type I collagen. Previous in vivo studies in our laboratory have shown that type I collagen mRNA levels are decreased in stage II and III breast tumour tissue compared to adjacent normal tissue.

Includes bibliographical references (leaves 120-133). / Ergothioneine and mycothiol are the two major low molecular weight thiols present in mycobacteria. The generation of mycothiol-deficient mutants has demonstrated its role in protecting M tuberculosis against oxidative and nitrosative stress. To date, no ergothioneine-deficient mutants have been identified and the role of ergothioneine in mycobacteria remains unknown. The work in this thesis was performed with the aim of better understanding the function of ergothioneine in mycobacteria, by studying its biosynthesis and the conditions affecting its production.

The intrinsic resistance of Mycobacterium tuberculosis ( Mtb ) to antibiotics is generally attributed to multiple factors, most significantly the low permeability of the mycobacterial cell wall, the operation of various drug inactivating systems, and the activity of efflux pumps. This study aimed to investigate the role of various components of the "intrinsic resistome" that limit the efficacy of antitubercular agents. The DNA damage response: the SOS response was hypothesized to play a role in antibiotic- mediated cellular death, and that disabling the mycobacterial SOS response, by generating non-cleavable LexA mutants (lexA Ind-), could be used as a tool to validate antibiotic-mediated cell death. To this end, the M. smegmatis (Msm) cleavable LexA was shown to be essential for induced mutagenesis and damage tolerance and that an intact DNA damage repair system is required to respond to antibiotic-mediated DNA damage. In contrast, Mtb cleavable LexA was required for induced mutagenesis but not necessarily damage sensitivity. In addition, the Mtb SOS response does not contribute significantly to remediation of antibiotic-mediated DNA damage. Collectively, these data suggest that DNA repair mechanisms differ between the mycobacterial species and despite effectively inactivating the LexA-dependent DNA repair mechanism(s) in Msm and Mtb, these organisms are able to circumvent this pathway and successfully remediate damaged DNA sustained under various conditions. Furthermore, Mtb auto-bioluminescent reporter strains were generated by introducing the lux operon downstream of the recA or radA promoters. Analysis of a panel of antimicrobials against these strains allowed for the identification of true DNA-damaging agents and the evaluation of the kinetics of the DNA-damage response, in a concentration- and time-dependent manner. Efflux-mediated drug resistance: This study aimed to evaluate the interactions between pairwise combinations of selected antimicrobials and efflux pump inhibitors (EPIs), in vitro and ex vivo, and to identify a novel verapamil (VER)-analogue with improved efficacy against Mtb. Subsequently, a candidate EPI was identified, with equivalent in vitro synergistic effects to VER when used in combination with various antibiotics but with reduced cytotoxic effects, ex vivo, when compared to VER. Mycothiol-mediated protection : It was hypothesized that undetectable levels of mycothiol ( MSH ) in Mtb would potentiate the use of current antibiotics. To investigate the contribution of the cellular antioxidant, MSH, to the mitigation of antimicrobial efficacy, this study aimed to disrupt MSH production by conditionally knocking-down expression of the essential gene, mshC. The mshC knock-down mutants (in all configurations) were not anhydrotetracycline (ATC)-regulatable in liquid or on solid medium, which was subsequently validate d with quantitative gene expression analysis. These data suggest that a tetracycline (Tet)-based conditional expression system may not be applicable to mshC. In conclusion, Mtb has a multitude of inherent mechanisms to subvert the effects of antimicrobial treatment. This study has contributed to the understanding of certain aspects of the intrinsic resistome and in doing so, established tools that can be used in future drug discovery programmes.

Angiotensin-I converting enzyme (ACE) is an important drug target in the treatment of heart disease due to its role in the regulation of blood pressure. ACE contains two domains, the N- and C-domains, both of which are catalytically active and heavily glycosylated. Glycosylation is one of the most important forms of post-translational modification, having a wide range of functions including protein folding, modulation of the immune response, and providing targeting signals. Glycosylation is required for the expression of active ACE and structural studies of ACE have been fraught with severe difficulties because of surface N-glycosylation of the protein. This problem has been addressed to a large extent with respect to the C-domain, where the role of glycosylation has been extensively characterised and a minimally glycosylated form was able to crystallise reproducibly. As yet, little is known about the degree and importance of N-linked glycosylation on the N-domain. The generation of minimally glycosylated N-domain, however, requires a greater understanding of the relative importance of the individual N-linked glycosylation sites.

Includes abstract. / Includes bibliographical references. / This study investigated the frequency and timing of cytotoxic T-lympthocyte (CTL) escape and its pathogenic consequences on HIV-1 subtype C disease progression.

The Fragile X-related disorders arise from an unusual mutation in the X-linked FMR1 gene. The mutation involves expansion, or an increase in the number of repeats, in a CGG•CCG repeat tract located in its 5' untranslated region. FMR1 alleles carrying 55-200 repeats are called Premutation (PM) alleles, and cause Fragile X associated tremor/ataxia syndrome (FXTAS) and Fragile X-associated primary ovarian insufficiency (FXPOI). FMR1 alleles having more than 200 repeats are referred to as full mutation (FM) alleles and cause Fragile X syndrome (FXS). These different alleles arise by intergenerational expansion of the repeat tract from smaller unstable alleles by a mechanism that is unknown. We have shown that in addition to germ line expansion, somatic expansion also occurs in a human cell line in vivo and in a FX PM mouse model. In the mouse model, we found that the extent of somatic instability is dependent on age, gender and tissue. Specifically, organs such as brain, liver and gonads are susceptible to expand more than heart and kidney and expansion is much more frequent in males than in females. No differences were found between male and female mice in the levels of the DNA repair proteins that had already been implicated in repeat expansion in model systems of other disorders thought to arise via a similar mechanism. Neither were there any differences between males and females in the amounts of proteins produced from X-linked DNA repair genes. We also showed that estrogen did not protect against expansion. However, we found that PM alleles expanded exclusively when they were located on the active X chromosome. Thus some of the differences between males and xii females in the level of somatic expansion might be due to the fact that females undergo X inactivation and thus have the PM allele on the inactive X chromosome in half (~50%) of their cells. It also indicates that transcription and/or an open chromatin configuration is required for expansion in the FX PM mouse.

Includes abstract. Includes bibliographical references (leaves 79-93).