Autoimmune Addison’s disease (AAD) is a rare and highly heritable endocrinopathy. It is a complex genetic disease, meaning that it is due to a combination of interacting environmental and genetic factors. To date, the majority of the substantial genetic component to AAD aetiology remains undefined. In this study, a combination of hypothesis-driven (candidate gene) and discovery-driven (genome-wide) approaches have been used to search for novel genetic determinants of AAD. PCR-based approaches were undertaken to study the potential role of the CYP21A1P pseudogene in AAD. CYP21A1P is highly homologous to the CYP21A2 gene which encodes 21-hydroxylase, the primary autoantigen in AAD. In individuals with AAD, CYP21A1P is more likely to be absent from the genomic DNA sequence than in controls. qPCR and in situ hybridisation have been successfully combined to identify CYP21A1P transcripts in thymic, and fetal adrenal, tissue. These data perhaps indicate a role for CYP21A1P in induction of immune tolerance, with its loss being associated with autoimmunity against the steroidogenic apparatus. Taking a broader candidate gene approach, the largest association analysis in AAD to date, of twenty candidate genes in six European AAD cohorts, suggests a role for NF-κB1, IL23A and GATA3 variants in susceptibility to AAD in individual European cohorts, and a role for STAT4 more universally in AAD. SNP array technology has been used to conduct the first genome-wide linkage and association analysis in AAD. The linkage study, including 23 families, has linked regions on chromosomes 6, 7, 9 and 18 to disease. A genome-wide association analysis, comparing the 50 familial AAD cases to the Wellcome Trust 1958 UK Birth Cohort control group, revealed clusters of associated SNPs on chromosomes 2 and 6. iii This body of work has illustrated some of the challenges in investigating a rare, complex genetic disorder, and how international collaboration can help to resolve some of these issues. In the course of this work, in addition to identifying a number of novel genetic determinants to AAD, exciting preliminary results have been generated which will need to be followed up. It is hoped that once these preliminary findings are replicated and further investigated, they will contribute significantly to an increase in our understanding of the pathogenesis of AAD, with the long-term aim of identifying novel means of treating the disease, altering its natural history or even preventing it.

This thesis involves three sections exploring disclosure amongst adolescents with HIV. Part one is a literature review examining the current literature around disclosure for young people with HIV and highlighting the factors that both facilitate and inhibit disclosure. Part two is an empirical paper outlining a qualitative study interviewing young people with perinatally acquired HIV on their attitudes and experiences towards disclosure. Part three is a critical appraisal of the research experience, commenting upon some of the challenges of conducting the research, methodological issues and learning points. All three sections should influence further research and have implications for clinical work in suggesting ways in which young people with HIV can be better supported around disclosure.

No description available.

Antiretroviral therapy (ART) is effective in reducing HIV morbidity and mortality as well as improving patients' quality of life. However, because of several hurdles, resource-poor countries have provided treatment to only a few people in need. Thailand is unusual in having opted to offer universal coverage for therapy. This thesis aims to understand the process by which ART reached the Thai Government agenda, and to explore the lessons learned from the design and implementation of the publicly-organised treatment programme. This study suggests that Thailand's ART programme was influenced by the networks and learning of several actors which evolved over time. During a period of policy continuity between 1992 and 2000, the policy process developed within a relatively closed subsystem dominated by health officials in the Disease Control Department and HIV experts. The cost of antiretrovirals was the major factor restraining treatment coverage. The dramatic shift in ART service towards universal access took place in 2001, as a consequence of drug price reduction and political transformation that allowed participation of new Health Minister, health financing reformists, and an alliance of non-governmental organisations (NGOs). Apparently, local and external treatment experiences inspired these actors to pursue similar paths in Thailand. The rapid policy formulation process was facilitated by common interests, shared experience, previously established collaboration, as well as awareness of interdependency among members of the Ministry of Public Health's Technical and Administration Panels. Learning about the intricacy in ART administration, especially from existing programmes and research studies in the country, played a crucial role in devising treatment expansion plans. The individual expertise of clinical specialists, researchers, health officials, NGOs and PL WHA helped to accelerate lesson drawing from policy feedback, anticipating future obstacles and selecting appropriate policy options. At the sub-national level, the process by which the universal ART policy was translated into action involved another set of actors, comprising hospital administrators, health professionals, officials in the Health Ministry's Regional and Provincial Offices, local NGOs and PLWHA groups. A key feature of policy in this phase was that the front-line workforce struggled to carry out the tasks prescribed by national policy makers. The discrepancy between the programme's expectation and actual therapy delivered in two study provinces was significant, - 2 - resulting from insufficient number of experienced health personnel, increased workload as an effect of parallel reforms in the health and public administration systems, and stigma attached to HIV. To counter these impediments, treatment execution networks of government staff and civic groups were instigated. Collective learning among service providers, supporters and clients had an important role in ART scaling up. Different coping strategies were implemented in study hospitals, aiming to balance the contradictory goals of achieving the allocated targets while maintaining treatment quality. This thesis demonstrates that to understand policy development in such a complex circumstance governments cannot unilaterally deal with particular problems. Employing a policy network concept to address the partnership between state and non-state actors is not only useful but essential as the policy environment has expanded beyond merely state actions, to depend, to some extent, on non-state actors. Moreover, the integration of policy learning model into policy analysis framework can provide insights into the increasingly dynamic interactions between actors, context and processes of public policy in focus.

Background: ‘Transactional sex’, is generally defined as a sexual relationship in which money or material goods are exchanged for sex. As this may entail sex with older or multiple partners, it is likely to be associated with young women’s increased vulnerability to HIV or HSV-2 infection. Existing research illustrates that the motivations for young women’s engagement in transactional sex are complex. This PhD study explores the socio-demographic factors that motivate young women to engage in transactional sex and the relationship with HIV or HSV-2 infection. Methods: This thesis uses a combination of quantitative and qualitative methods. The quantitative analysis uses baseline data from HPTN 068, an ongoing randomised controlled trial (RCT) in rural Mpumalanga province, South Africa. Using both descriptive statistics and multivariable logistic regression analysis, I have quantitatively explored the relationship between household socio-economic status and socio-demographics, young women’s self- reported engagement in transactional sex, specific characteristics of her consumption patterns and the relationship between transactional sex and HIV and HSV-2 infection. I have also used qualitative methods to explore the strategies young women deploy for accessing money or gifts, the role that transactional sex plays in meeting these aspirations and the agency she exhibits in these encounters. Results: Multivariable analysis shows that young women from low socio-economic status households who engage in transactional sex have higher odds of consuming a combination of both high cost essential items (such as school fees, transport to school and food and groceries) and entertainment related items (such as beer/alcohol, movie tickets and birth control/condoms) compared to young women who do not engage in transactional sex. In addition, after adjusting for age and household level socio-demographics, young women who report engaging in transactional sex had significantly increased odds of being HIV seropositive. No association with HSV-2 infection was seen. The association between transactional sex and HIV does not appear to be mediated by any of the other dimensions of HIV risk that might ordinarily overlap with transactional sex. Qualitative findings suggest that belongingness, peer acceptance and status were all factors that motivated young women to aspire for items considered needs or wants. In-depth interviews also showed how young women have a deep-seated need to feel financially independent and have aspirations for lucrative job opportunities in the future, which they hope to achieve through education. But, given their current context where economic opportunities are circumscribed, young women use the money received from transactional sex to fill gaps in their needs and wants. Most of them appear to be in “love” with their boyfriends and have romantic notions of what they want from the relationship, but underlying it all very clearly is the need to be provided for by the man, in the form of money. Thus, the balance between the emotional and transactional elements in a relationship is blurred. Young women appear to express considerable agency when it comes to partner choice, but not necessarily after the choice of partner is made; as within the relationship they invariably submit to male authority within unequal gendered power structures. Discussion: Findings suggest that there is not a clear dichotomy between the survival sex of the passive victim and the consumption sex of the active sexual agent; rather, these distinctions are far more nuanced. Engagement in transactional sex seems to be driven by poverty and economic need, but also by aspirations within the context of a globalising emerging economy like South Africa. In addition, these findings also indicate the subtle position that transactional sex or sexual exchange occupies within a continuum of adolescent sexual relationships in South Africa where sexual exchange occurs within the context of loving peer relationships. This potentially weakens the negotiating or bargaining position of young women if they are receiving money or items, thus having implications on HIV prevention and programming.

The HIV-1 accessory protein Vpu counteracts the restriction factor tetherin, and induces its cell surface downregulation and ubiquitin-dependent endosomal degradation. We have identified an acidic/dileucine based-sorting determinant, ExxxLV, in the second alpha helix of the Vpu cytoplasmic tail as being required for efficient tetherin antagonism. Mutation of this motif prevents tetherin degradation and downregulation, but not Vpu/tetherin interaction. This effect is similar to Vpu phospho-mutants, which are unable to recruit the SCF-β-TrCP E3 ubiquitin ligase complex. However, ExxxLV mutants are able to bind to β-TrCP and ESCRT-0 component HRS. Tetherin and ExxxLV mutants accumulate at the cell surface and in early endosomes implying that post-binding trafficking of Vpu/tetherin complexes is required to prevent tetherin transit to viral assembly sites on the plasma membrane. Our preliminary data shows that the ExxxLV motif interacts with the trafficking machinery via the clathrin adaptor AP-1 in a manner that is dependent on the tyrosine-based sorting signal in tetherin, although functional redundancy in clathrin trafficking pathways cannot yet rule out other adaptor proteins being involved. A study performed in our laboratory examined the variation of vpu alleles in HIV-1 infection, and identified several variants in which isoleucine residues in the first alpha helix of the cytoplasmic tail were mutated. We show that these isoleucine mutants were defective for inducing tetherin degradation, despite being capable of interacting with tetherin, thus pheno-copying ExxxLV and phospho-mutants. However, unlike both these mutants, isoleucine mutants fail to interact with HRS, which is required for Vpumediated tetherin degradation and antagonism. Vpu ExxxLV, isoleucine and phospho-mutants share localisation defects, in which they accumulate at the plasma membrane and in early endosomes. Interestingly, tetherin antagonism, in all of these mutants, can be rescued by C-terminal addition of a clathrinbinding motif. This implies that these mutants might be defective for the same reason: they fail to interact with the clathrin trafficking machinery. Further experiments that will elucidate this hypothesis are underway.

No description available.

Autoimmune Addison’s disease (AAD) is a debilitating condition and affected patients rely on lifelong steroid replacement. Despite treatment, many patients have increased morbidity and mortality. The disease’s rarity has precluded large scale genomic or cellular studies in humans, resulting in an incomplete picture of the pathophysiology of AAD. This thesis details my research on the pathophysiology and novel therapeutic approaches in AAD. I performed two candidate gene studies on susceptibility alleles that have been implicated in other autoimmune diseases to explore potential causal pathways of these genetic determinants in AAD. The common variant 307*Ser allele of CD226 gene was found to contribute to AAD susceptibility as part of autoimmune polyendocrinopathy type 2. Two genetic variants from a panel of rare and functionally defective variants in the sialic acid acetylesterase (SIAE) gene were identified but they were not significantly associated with AAD. I explored new therapeutic approaches in AAD using a therapeutic regimen of parenteral ACTH1-24. This study demonstrated the presence of residual adrenal function in patients with established AAD, which was remediable to ACTH1-24 therapy. Clinical remission was induced in 2 of 13 study participants. However, some patients developed cutaneous reaction following ACTH1-24 injection, with one failing to sustain clinical remission. I proceeded to investigate the potential immunologic effects induced by ACTH therapy using ELISA and immunoblotting methods. Auto-reactivity against ACTH1-24 and ACTH 1-39 was detected, which could account for the side effects and treatment resistance observed. Finally, I attempted to characterise adrenocortical progenitor cells through cell cultures, immunocytochemistry and flow cytometry. A mesenchymal stem cell-like (MSC) population was isolated from human adrenals for the first time. My studies have advanced knowledge relevant to regenerative medicine approaches to adrenal failure, and the finding of residual adrenal function in some Addison’s disease patient opens an important therapeutic window for this condition.

HIV-infected patients present with a range of pathologies that have been associated with mitochondrial toxicity, and more specifically, induced through exposure to the main class of antiretrovirals used in anti-HIV therapy (HAART), nucleoside analogue reverse transcriptase inhibitors, or NRTIs. It has recently been found that such patients have an excess of mitochondrial DNA mutations when exposed to NRTIs; however, the underlying pathophysiological process of this remains undetermined. To elaborate upon this further, a range of molecular approaches were developed to study the behaviour of mitochondrial DNA mutations, both large scale deletions and single point mutations, in both tissue samples and in vitro. High throughput, ultra-deep DNA sequencing technology was also utilised to characterise mitochondrial DNA mutation burden in detail. Here I present data from physiological samples of HIV-infected individuals receiving NRTIs indicating an increased level of point mutation heteroplasmy and the level of the common mitochondrial deletion, compared to HIV-infected individuals who are NRTI treatment naïve. The mechanisms of this mutation level increase are elucidated through in vitro studies indicating that deletions accumulate through a size dependent mechanism during exposure and a point mutation level increase is mediated through a bottleneck mechanism. The possibility of NRTI de novo mutagenesis is refuted through the use of next generation sequencing technologies; the data also further supports a bottleneck mechanism of increased point mutation level. I also refute the idea that compounds associated with mitochondrial biogenesis can reduce mitochondrial depletion during NRTI exposure and that there appears to be no genetic predisposition to pathologies in sub-Saharan African individuals. I therefore conclude that there is an accelerated clonal expansion of pre-existing mitochondrial DNA mutations through NRTI exposure, which is mediated by a size dependent replication bias for deletions, and a molecular bottleneck effect which accelerates drift for point mutations. These data suggest an acceleration of normal cellular ageing through mitochondrial mechanisms in HIV-infected individuals.

Bloodstream infection (BSI) caused by nontyphoidal serotypes of Salmonella is one of the most important causes of morbidity and mortality in sub-Saharan Africa (SSA). Invasive nontyphoidal Salmonella disease (iNTS) is especially associated with HIV in adults and HIV, malaria and malnutrition in children in this setting. HIV-infected subjects are at particular risk of recurrent disease, both from recrudescence from a sanctuary site and re-infection. Whole- genome sequencing has revealed a novel sequence type (ST) of S. Typhimurium, ST313, is particularly associated with iNTS in SSA and that ST313 display the genomic signature of differential host adaptation. Little is known about African strains of S. Enteritidis. The Malawi Liverpool Wellcome Trust Clinical Research Programme (MLW) has conducted BSI surveillance in adult and paediatric medical patients in Blantyre, Malawi since 1998, enabling long term trends in iNTS to be described. Paediatric iNTS disease has been placed in the context of malaria, malnutrition and seasonality using structural equation modelling. An observational cohort was recruited to describe changes in adult BSI cases following the roll-out of ART in Blantyre. A longitudinal cohort study with enhanced microbiological surveillance was undertaken to investigate the site of persistence of NTS in HIV infected adults and the effect of recurrence upon the emergence of antimicrobial resistance. Lastly whole-genome sequencing was undertaken to compare invasive, African strains of S. Enteritidis with a reference isolate and to construct a global phylogeny of this serotype. There have been three epidemics of invasive Salmonella disease in Blantyre, all of which were preceded by the emergence of a multidrug resistant (MDR) strain. These were caused initially by S. Enteritidis which was then followed by S. Typhimurium, but most recently an epidemic of MDR S. Typhi has started. NTS has declined from epidemic levels, but remains an important cause of BSI in Blantyre and has been demonstrated to acquire cephalosporin iv and fluoroquinolone resistance just once through an IncHI2 plasmid. The decline in paediatric malaria is complex and multi-factorial. It cannot be attributed solely to malaria control interventions as has been the case in other settings. All causes of BSI are falling in adults and this is likely to be attributable in part to the hugely successful HIV treatment programme in Blantyre. The rapid initiation of ART appears to rapidly confer protection against recurrence of iNTS and even though more adult patients are surviving into convalescence, in this cohort, no further emergence of extended resistance was seen. Just as there is a distinct African clade of S. Typhimurium, a distinct clade of S. Enteritidis has emerged in SSA, which also possess the genomic signature of differential host adaptation, a novel prophage repertoire and a novel, MDR plasmid. In Blantyre, iNTS disease has declined from its epidemic peaks, but remains an important cause of BSI. The epidemiology of iNTS is more complex in Blantyre than other settings, but it seems likely that improved in-patient care and measures to control the HIV-pandemic impact on survival and recurrence. Two clades of NTS have emerged from host promiscuous serotypes, both of which have genomic degradation in genes governing potential host range and there is a critical need to understand the environmental niches and transmission pathways of these differentially adapted, invasive clades.