Overcoming the emergence of drug resistance in HIV is a major challenge to the scientific community. We use the established computational method of classical molecular dynamics to investigate the molecular basis of resistance in HIV-1 protease to the inhibitor saquinavir, using the wildtype and the G48V, L90M and G48V7L90M mutant HIV-1 proteases throughout this thesis. Firstly we reveal insights into a G48V mutation-assisted lateral drug escape mechanism from the protease active site. Such a mechanism allows drug escape without the full opening of the flaps of the protease. Furthermore, the mechanism is facilitated by differential drug-protease interactions, induced by mutations that take advantage of the conformational flexibility of the inhibitor. Secondly, we investigate the thermodynamic basis of binding of this set of mutants, using established 'approximate' free energy methods. The absolute and relative free energies of saquinavir binding to this set of proteases are successfully determined using our simulation and free energy analysis protocol and exhibit excellent correlation with experiment. This study is thus a template for an extended study on a larger range of HIV-1 protease-drug combinations. We describe a tool, the 'Binding Affinity Calculator', which has been designed to automate this protocol and which can be routinely applied, using high performance computing and grid technology, to meet the intensive computational demands of such an investigation. The free energy of binding of the NC-pl natural substrate cleaved by the protease is also deter mined. The enhanced flexibility of the substrate over the drug precludes the guarantee of a converged free energy result, even from the 10 ns duration of each simulation. However, qualitative insight into the thermodynamic basis of binding is gleaned as well as the effect of these mutations on the catalytic efficiency of the protease. Furthermore, we combine drug and substrate binding free energies to develop a metric for evaluating the approximate enzymatic fitness of a given mutant protease, computable directly from molecular simulation.

T lymphocyte numbers in the human body are kept constant by homeostatic mechanisms balancing cell gain and loss. These mechanisms eventually fail in HIV infection, which is characterized by progressive immune deficiency attributable to a slow but relentless depletion of CD4+ T cells, the main viral targets. HIV infection is also associated with increased T cell turnover and a state of generalized immune activation. One of the fundamental questions in the field of HIV research is the relation between CD4+ T cell depletion and immune activation. It has been suggested that the virus has a direct effect by killing CD4+ T cells and increased T cell turnover reflects a homeostatic response to CD4+ T cell depletion. Alternatively, chronic immune activation may lead to enhanced turnover of T cells by ongoing proliferation- differentiation and cell death. In both cases, AIDS is the result of an exhaustion of the regenerative capacity of the immune system. To address these questions we examined the consequences of activated CD4+ T cell killing in a virus-free mouse model. Immunodeficiency viruses are highly selective for activated/memory and regulatory CD4+ T cells due to restricted expression of CCR5, the co-receptor for HIV and SIV, or CD 134 (OX40, TNFRSF4), the cellular receptor for FIV. Activated CD4+ T cells were depleted by conditional reactivation of diphtheria toxin gene mediated by Tnfrsf4-driven Cre recombinase expression. Conditional ablation of activated CD4+ T cells resulted in accelerated turnover, with only a minimal apparent effect on their numbers, and was associated with a reduction in CD4:CD8 ratio and development of CD4+ T cell immune deficiency, resembling HIV infection. Importantly, activated CD4+ T cell killing also resulted in generalized immune activation, including lymph node enlargement, B cell expansion, elevated serum levels of proinflammatory cytokines and increased turnover and activation of CD8+ T cells, characteristic of HIV infection. CD8+ T cell activation correlated with lack of regulatory CD4+ T cell function and was prevented upon regulatory CD4+ T cell reconstitution. We therefore propose a causal link between memory and regulatory T cell depletion and immune deficiency and immune activation, respectively.

From 2002 international and national policies transformed the availability of antiretroviral medicines for people living with HIV/AIDS in Africa. By the end of 2008 an estimated 42 percent of people requiring such treatment were accessing medication and anti-retroviral treatment (ART) programmes had been rolled out in many countries (UNAIDS and WHO 2009). However, this expansion was implemented unevenly across and within different countries raising questions about the gap between policy intention and execution. This thesis addresses this gap. It examines the processes of implementation, comparing rapid ART roll-out in Zambia with South Africa where implementation was initially much slower. It draws on both top-down and bottom-up perspectives to better understand factors hindering and enabling implementation. The focus is on actors and their networks and how they were able to exert power on the implementation of policy. It is a qualitative study that relies on document review and over 150 interviews conducted with actors in the policy processes in both countries, during field work in 2007 and 2008. Findings confirmed the importance of communication, resources and structures in determining implementation, but the comparative analysis indicated their influence varied considerably according to context. The findings also offer new insights into how contrasting networks of actors affected implementation. A broad range of actors made it possible to roll-out ART in South Africa despite a hostile policy environment, by drawing on diverse sets of skills and ties that dated back to the anti-apartheid struggle. In Zambia the network which most influenced ART roll-out was an epistemic community of clinicians, which, by forging alliances with PEPFAR implementing agencies was able to rapidly scale up access to treatment in spite of health systems constraints. The economic capital of donors allowed them to shape policy and blur boundaries between state and non-state actors in Zambia while social capital of networks was important in South Africa. Findings suggest that focusing on the sources of power of networks in implementation enriches the understanding of health policy processes.

In response to a major gap in evidence regarding treatment outcomes among asylum-based refugees. the primary objective of the thesis was to investigate adherence to highly active antiretroviral therapy (HAART) and virological outcomes among refugees and to compare these outcomes with local host communities in one urban, Southeast Asia setting (Sungai Buloh, Kuala Lumpur, Malaysia) and one remote sub-Saharan refugee camp (Kakuma. Kenya) setting. Given limited resources for expanding treatment, questions have been raised as to whether refugees can achieve sufficient levels of adherence and viral suppression to justify sustaining and expanding access. Data sources included a structured questionnaire with self-reported adherence measures, a pharmacy-based prescription refill measure, HIV viral loads, and indepth interviews. Analyses made use of quantitative and qualitative approaches. The thesis begins by presenting the rationale, aims, research questions, and a description of preparatory work. Paper One presents the results of a systematic review of the literature on adherence to HAART and treatment outcomes among conflict-affected and forcibly displaced populations. finding only 17 reports, five of which included less than < 100 clients, adherence estimates in the range of 87-99.5%, and good treatment outcomes. Papers Two and Three present the quantitative findings from both settings, finding no differences in outcomes between refugees and the host community in either setting, but a large difference between the settings. In Malaysia, 83% of clients on HAART for 2:25 weeks were suppressed while only II % were suppressed in Kenya. Female sex, longer time from HIV diagnosis to HAART start, and optimal adherence pharmacy refill schedule were protective in the Malaysian setting while temporary migration for 2: I month (in the previous year) and 2: I hour average transit time to clinic were independent risk factors. Larger household sizes were protective in the Kenyan setting. Paper Four offers an account of patient experiences based on the qualitative findings from both settings, and suggests that systemic barriers and resilient strategies were prevalent in both settings; however. intensive systemic barriers appeared to overwhelm personal resilience in the camp setting. Paper Five positions the work in the context of previous and future research and makes recommendations for programs and policy. The thesis concludes by suggesting that. just as good treatment outcomes were shown to be achievable in a range of forcibly displaced groups. asylum-based refugees were also capable of treatment success and maintain outcomes similar to those of the host communities. There is a clear public health and humanitarian interest in guaranteeing access to ART, promoting optimal adherence. and sustaining viral suppression in all who are in need of treatment. When problems in achieving and sustaining viral suppression occurred, they were not typically due to previous forced displacement, or refugee status itself. Overall, refugees ought to have equal access to HIV treatment based on the principles of fairness, human rights, and individual and population-based public health benefits. Since HIV-positive individuals on HAART with good adherence will rarely transmit HIV to their sexual partners, it is in the enlightened self-interest of host country governments to support HIV programs that serve HIV -positive refugees and host clients equally.

Context: Autoimmune polyendocrine syndrome type 1 (APS1) is characterised by multiple autoimmune endocrinopathies and results from mutations in the AIRE (autoimmune regulator) gene. Approximately 80% of patients present with hypoparathyroidism which is suggested to result from autoimmune responses against the parathyroid glands. The calcium-sensing receptor (CaSR), which plays a pivotal role in maintaining calcium homeostasis by sensing blood calcium levels and regulating release of parathyroid hormone, has been identified as a parathyroid autoantibody target in APS1. Aims: The main aims of this study were to characterise APS1 patient anti-CaSR autoantibodies in relation to their prevalence, disease associations, epitopes, specificity, IgG subclass and effects upon CaSR function, and to develop an ELISA to detect CaSR autoantibodies in patient sera. Methodology: Immunoprecipitation; radioligand binding assays; phage-display; ELISA; bioassays; protein expression. Results: Autoantibodies against the CaSR were detected in 16 out of 44 (36%) APS1 patients and in none of 38 healthy control subjects (P = < 0.0001). No statistically significant associations were found between the presence of CaSR autoantibodies and the disease manifestations of APS1 including hypoparathyroidism. The detection of CaSR autoantibodies had a specificity of 83%, and a sensitivity of 39% for the diagnosis of hypoparathyroidism. There were no significant associations between the presence of CaSR antibodies and either sex, age or disease presentation age. However, a significant association between a shorter APS1 duration (< 10 years) and positivity for CaSR autoantibodies was noted (P = 0.019). CaSR autoantibody epitopes were identified between amino acids 41-69, 114-126, 171-195 and 260-340 in the extracellular domain of the receptor. Autoantibodies against CaSR epitopes 41-69, 171-195 and 260-340 were exclusively of the IgG1 subclass. Autoantibody responses against CaSR epitope 114-126 were predominantly of the IgG1 with a minority of the IgG3 subclass. CaSR autoantibodies were analysed for their ability to increase Ca2+-dependent inositol phosphate accumulation in HEK293 cells expressing the CaSR. The results indicated that 4/16 (25%) APS1 patients had anti-CaSR-activating autoantibodies, suggesting that although the majority of APS1 patients do not have CaSR-stimulating autoantibodies, there may be a small minority of patients in whom the hypoparathyroid state is the result of functional suppression of the parathyroid glands. As part of this study, an ELISA was developed using the extracellular domain of the CaSR expressed in Escherichia coli as the antibody-capture antigen. Although this assay was able to detect the presence of autoantibodies in APS1 patient sera, the experimental method still requires further optimisations in order to attain a validated and robust ELISA, as this was not achievable during the present study. Conclusion: The study provides a detailed analysis of the characteristics of CaSR autoantibodies in APS1 patients, although further investigations are required to determine the exact role played by the autoimmune response against the CaSR in the pathogenesis of APS1.

The genetics of complex common diseases are not fully understood, but rare variants with large phenotypic effects contribute to heritability. The objective of this thesis is to identify rare variants of relevance to autoimmune and renal disease, by developing ways of analysing whole genome sequencing (WGS) data and exploring the variants identified. Forward genetic experimental approaches are used, both in mutagenised mice, and in humans with extreme trait forms of the steroid resistant nephrotic syndrome (SRNS) and systemic lupus erythematosus (SLE). This work demonstrates that N–ethyl–N–nitrosourea (ENU) mutations can be distinguished within WGS data, including a hypomorphic mutation in Lamb2 in a strain with the nephrotic syndrome, a murine model for the milder spectrum of human Pierson syndrome. In a B–cell deficient ENU strain the causative mutation in Lyn was isolated by sequencing multiple affected mice and applying an implementation of the Lander–Green algorithm to search for identity by descent. This method for the first time overcomes a rate-limiting step in ENU programmes and offers the potential to accelerate gene discovery, eliminating the need for out–crossing and conventional linkage analysis. Knowledge of the ENU genomic intervals allowed calculation of a mutation frequency, 1.5 mutations per mega base, and modelling of an efficient ENU strategy. Short–lists of candidate variants from 14 unrelated patients with steroid resistant nephrotic syndrome or systemic lupus erythematosus provide a substrate for future experiments, for example a candidate in the Wiskott–Aldrich syndrome gene was excluded using DNA from family members. In conclusion, WGS coupled with identity by descent analysis offers a powerful tool to improve the efficiency of ENU programmes. Rare variant discovery in humans without obvious Mendelian inheritance is more challenging and will require strategies to prioritise variants that combine bioinformatic filters and experimental verification in a high throughput way.

Alien Sex Club is a practice based PhD that addresses current modes of representation of HIV in art, which grows out of the minimalist aesthetic developed during the earlier part of the AIDS crisis by artists such as Felix Gonzalez-Torres. Today however, it is acknowledged that HIV is an interconnected web of problems that should be represented in a holistic way. Rates of HIV transmission are increasing among gay men in the West. HIV is no longer a life threatening illness due to the availability of highly effective antiretroviral drugs. A resulting decrease in the perception of risk, leading to condom fatigue, unprotected sex and recreational drug use may be some of a number of factors that are contributing to the increase in transmissions. Despite changes in the cultural, social and scientific context of HIV, artistic representations of the subject have remained the same as those originated before effective treatment was available. In this PhD, I use art and architecture and draw from contemporary scientific and political approaches in ways that differ from previous modes of representation, to raise new questions about HIV. Alien Sex Club seeks to re-politicise art as an arena for addressing HIV by mobilising a range of visual and aesthetic genres in a curated installation. This take the form of a cruise maze that addresses the complexity of contemporary HIV problems in an academic context. It makes use of spatial design and a maximalist aesthetic to update the representation of HIV. It transposes knowledge about HIV from science, sociology and philosophy into a visual art practice. It uses live art in the form of hospitality and fortune telling in order to question the existing conventions of the art gallery and engage audiences to consider HIV in new ways. It uses autoethnography, a qualitative research method drawn from the social sciences to theorise the methodology of its making. The installation is used as a test site for gathering data of audience responses, which are subjected to textual analysis. In these ways, Alien Sex Club operates as a counter discourse to the prevailing minimalist representations of HIV. The PhD generates knowledge about how to educate audiences on continuing rates of HIV transmission and extends understandings of the nature of the artist as activist.

Access to anti-retroviral drug (ARV) therapy in Nigeria has been a big challenge. Despite the fact that ARVs have been demonstrated to improve quality of life, reduce AIDS prevalence and AIDS deaths, many people in Nigeria still do not have access to ARV therapy. At the time this study was started, the ARV access rate was 16.6%. This Grounded Theory study examines the experiences of HIV positive people accessing ARVs in Abuja, Nigeria. 30 Patients living with HIV/AIDS were interviewed in an iterative manner. The results of the Grounded Theory analysis were triangulated with the documentary analysis of preliminary and secondary literature. As reported by the participants of the study, patients initially found it very difficult to access treatment. Stigma emerged as the main concern of the research participants. Although access to anti retroviral treatment has improved over the years, different forms of stigma still pose important barriers to access in this group of participants. The results of this research suggest that stigma occurs at individual, familial, community, organisational and national levels. The main concern of research participants was resolved mainly through the use of social connections. The contribution to knowledge is the development of the Social Connection Theory. 5 main stages that patients pass through when they attempt to access ARVs were identified in this study. During each of these stages, the patient may experience barriers through stigma or other forms of structural issues such as poverty. They may also move from one stage to another through social connectors who assist them to access ARVs. In the Social Connection Theory, it is argued that in African settings, social connectors play a vital role in influencing the way that patients access antiretroviral treatment. Social connectors are social acquaintances of the patient who help shape their health care seeking decisions. They play a vital role in supporting and linking HIV positive persons to where they can access ARVS. Social connection serves as a useful tool for empowering HIV patients to overcome different obstacles and access treatment. However, these processes do not occur in a structural vacuum. Structural factors such as religion, gender, politics and the economy were also found to shape the way stigma is experienced in Nigeria and how people access HIV treatment. To improve access to ARVs, it is suggested that while taking cognisance of structural forces, multidisciplinary strategies should be developed that integrate social connectors at different critical points in the access continuum.

The type 1 insulin like growth factor receptor (IGF-1R) is a cell surface receptor that mediates proliferation and cell survival. The aim of this project was to explore the contribution of the IGF-1R to the DNA damage response in human prostate cancer cells. Previous work from our group showed IGF-1R to be over expressed in prostate cancer. Furthermore, IGF-1R depletion enhances sensitivity of prostate cancer cells to DNA damaging agents, and IGF-1R depletion or inhibition delays DSB repair. Prior work in melanoma cells indicated that IGF-1R down-regulation enhances radiosensitivity and impairs the function of ATM, a key protein in the response to DNA DSB. This led to the initial hypothesis that IGF-1R signalling can influence ATM function, possibly via the PI3K-AKT pathway. Two putative AKT phosphorylation sites were identified on ATM and initial experiments detected a 350 kDa protein equivalent to the size of ATM, that was phosphorylated on RXRXXS/T motif(s) in response to IGF-1. However, it did not prove possible to identify this immunoreactivity in ATM immunoprecipitates, and AKT inhibition, although radiosensitizing, did not induce features of defective ATM function. Therefore this project was halted and two new projects exploring the role of IGF signalling in DNA repair were undertaken. The first project continues from work published from our laboratory showing that IGF-1R undergoes nuclear translocation in prostate cancer cells. The function of nuclear IGF-1R is unclear, and mass spectrometry (MS) was utilized to identify potential nuclear binding partners. Nuclear extraction and IGF-1R immunoprecipitation methods were modified in order to achieve optimal conditions for subsequent MS analysis. Three independent MS analyses have been performed aiming to identify binding partners of nuclear IGF-1R in cells grown in complete medium, cells that were serum-starved and IGF-1 treated, or exposed to irradiation-induced DNA damage. Candidate binding partners included transcription factors such as Bruton’s tyrosine kinase associated protein 135 (BAP-135) also known as general transcription factor II-I (TF II-I), suggesting possible involvement of nuclear IGF-1R in the regulation of transcription. Initial validation experiments suggested IGF-1 induced complex formation between IGF-1R and TF II-I, and also tyrosine phosphorylation of TF II-I in response to IGF-1, raising the possibility that IGF-1 influences TF II-I function. The second project is based on a high throughput screen completed by another member of our laboratory, that sought to identify DNA repair-related proteins that when depleted increased the sensitivity of prostate cancer cells to IGF-1R inhibition. One of the potential hits is RAD51, a recombinase central to the strand invasion step of homologous recombination (HR). Initial experiments validated RAD51 knockdown by the screen siRNAs, and showed that RAD51 depletion did indeed enhance sensitivity to a novel IGF-1R inhibitor in PTEN wild type prostate cancer cells. Loading of RAD51 onto single stranded DNA requires BRCA2, and in a PTEN wild type colorectal cell line model, lack of BRCA2 was shown to sensitize colorectal cancer cells to IGF-1R inhibition, compared to isogenic cells that expressed functional BRCA2. Finally, experiments also sought to mimic effects of RAD51 depletion using a small molecule CDK1inhibitor, R0-3306, recently shown to impair HR by inhibiting BRCA1 function. In prostate cancer cells, R0-3306 at the determined GI50 value (1 μM) suppressed formation of RAD51 foci, consistent with HR attenuation. Furthermore, CDK1 inhibition phenocopied the effects of RAD51 depletion, sensitizing prostate cancer cells to IGF-1R inhibition with a 2.4 fold reduction in GI50 value and 13 fold reduction in the GI80. These data suggest that the sensitivity of human prostate cancer cells to IGF-1R inhibition can be enhanced by genetic and chemical approaches to suppress HR, and that BRCA mutant cancers may be intrinsically sensitive to IGF-1R inhibition. The ultimate aim of this work is to understand how IGF-1R biology influences DNA damage repair, in order to guide the development of new treatments for prostate cancer.

Antiretroviral therapy (ART) suppresses HIV viremia, but it does not eliminate the stable cellular reservoir of integrated viral DNA that is generated early during infection. As a consequence, low levels of HIV are detected in patients with consistent viral suppression (HIV-1 RNA < 50 copies/ml) in CD4 cells in different body compartments including plasma, peripheral cells and CD4 T-cell subsets. We first explored the levels of HIV-1 RNA below the cut-off of clinical assay (HIV-1 RNA load < 50 copies/ml, herein referred to as 'residual viremia') in a group of patients who switched from Atripla to Eviplera due to toxicity. Furthermore, we studied HIV-1 DNA and RNA loads in peripheral blood of patients with stable virological suppression (HIV-1 RNA < 50 copies/ml) while being on first line-ART with 1 NNRTI and 2 NRTIs. The measured virological markers included the residual HIV-1 RNA load in plasma quantified by an ultrasensitive assay; total and integrated HIV-1 DNA in both peripheral blood mononuclear cells (PBMC) and the CD4 T-cell subsets using quantitative PCR methods; 2-LTR circular HIV-1 DNA in PBMC using droplet digital PCR assay. Furthermore, a panel of cellular and immunological markers of immune activation, as measured by flow cytometry and ELISA assay respectively, were available for the analysis. Additionally we explored alternative methods to quantify and to study HIV-1 persistence. In patients that switched from Atripla to Eviplera we observed that overall the patients showed maintained virologic suppression until the end of the observation; however, 4 patients experienced virologic rebound with HIV-1 RNA > 50 copies/ml and this was predicted by a progressive increase in residual HIV-1 RNA levels over time. Patients on suppressive ART were studied with a cross sectional approach and we observed detection of residual HIV-1 RNA and 2-LTR circular DNA in a proportion of patients and total HIV-1 DNA in PBMC the whole study group. Additionally, we found that residual HIV-1 RNA levels and total HIV-1 DNA were associated with sCD27 and CD8+HLA-DR/DP/DQ+ T-cells, respectively. Following setting-up of the Alu-gag qPCR assay, we measured integrated HIV-1 DNA in PBMC and we confirmed the association with CD8+HLA-DR/DP/DQ+ T-cells. In the context of assay development, we proposed the Alu-5LTR assay as an alternative to the Alu-gag assay for integrated HIV-1 DNA quantification and preliminary results showed increased sensitivity. Furthermore, we identified and characterized the tools needed to set up an in vitro system to study direct and cell-to-cell HIV-1 infection. Results from my thesis showed the importance of residual HIV-1 RNA monitoring to detect virus rebound. Moreover, patients on suppressive ART for a long period showed HIV detection in plasma and in cells which was associated with high levels of immune activation, suggesting a role for the host immune environment in HIV persistence. In the context of assay developments for more sensitive quantification of integrated HIV DNA reservoir, our proposed Alu-5LTR assay as described in this thesis may be a potential alternative.