The Saccharomyces cerevisiae Rmi1 protein is a component of the highly conserved Sgs1-Top3-Rmi1 complex, which is required for the maintenance of genome stability. The rmi1Δ deletion mutant has proven difficult to study because it exhibits very poor growth, and rapidly accumulates second site suppressor mutations. Furthermore, deletion of the putative HJ resolvase genes, MUS81-MUS81 or SLX1-SLX4 in rmi1Δ mutants causes synthetic lethality. In order to study phenotypes caused by loss of functional Rmi1, and to explore the genetic interactions between RMI1 and the MUS81, MUS81, SLX1 and SLX4 genes, a temperature sensitive mutant of RMI1 was isolated, named rmi1-1. Similar to rmi1Δ deletion mutants, rmi1-1 cells are highly sensitive to the DNA damaging agent, MMS and the replication inhibitor, HU. In addition, rmi1-1 mutants accumulate replication-associated branched DNA structures, and arrest in G₂/M after a transient exposure to MMS. These cells are proficient in DNA damage checkpoint activation. Deletion of SLX1, SLX4, MUS81 or MUS81 in the rmi1-1 strain causes synthetic lethality, which is associated with cell cycle defects. Following a transient exposure to MMS, rmi1-1 mutants accumulate homologous recombination intermediates. These intermediates are slowly resolved at the restrictive temperature, revealing a redundant resolution activity in the absence of functional Rmi1. This resolution depends upon Mus81-Mms4, but not on Slx1-Slx4 or Yen1. I propose that while the Sgs1-Top3-Rmi1 complex constitutes the main pathway for removal of homologous recombination intermediates following a perturbed S-phase, Mus81-Mms4 can act as a back up for resolution of these intermediates, which most likely represent double Holliday junctions. In this study, I also present screens for high copy suppressors of rmi1-1 phenotypes, and for novel Rmi1 interaction partners.

The aetiology of IBD is unknown, but available evidence suggests that an aberrant immune response towards the commensal microbial flora is responsible for intestinal inflammation in genetically susceptible individuals. Studies from animal models of intestinal inflammation have greatly advanced our understanding of the immunological basis of IBD. However, translation of results from animal research into human studies is essential in order to improve treatment options and patient quality of life. In this thesis we present the successful introduction of translational studies on human tissue in our laboratory. In particular, we evaluated the role of the IL23/IL17 pathway in the human immune response and its role in IBD. IL23-driven inflammation has been primarily linked to its activity on Th-17 cells; however, work from our laboratory has identified a novel population of IL23-responsive ILC, which are responsible for innate colitis in mice. Here we have analyzed the role of IL23-responsive innate cells in IBD. Our results show increased expression of Th-17 signature genes amongst intestinal CD3- cells in patients with IBD. Furthermore, we observed a marked and selective increase in IL17 producing CD56- ILC in the inflamed intestine of patients with CD. ILC may contribute to intestinal inflammation through secretion of cytokines, such as IL17A and IL17F, and recruitment of other inflammatory cells, representing a novel tissue-specific target for the treatment of IBD. In addition, we present here our preliminary data on the characterization of human intestinal and systemic DC populations. In particular, we aimed to evaluate if in the context of the intestinal microenvironment DC develop specific regulatory features, as observed in murine CD103+ DC. We show that human intestinal DC populations exhibit specific regulatory properties, such as expression of genes associated with TGF-β and RA activity. Furthermore, CD103+ DC are present in the human gut and are characterized by tolerogenic markers. Remarkably, patients with IBD have reduced frequencies of intestinal CD103+ DC, which display a more pro-inflammatory phenotype. Alteration in DC subset composition and functional activity may result in a distort balance between immune effector and regulatory responses, promoting the development of intestinal inflammation.

Metal-on-metal hip resurfacing arthroplasty (MoMHRA) has undergone a recent resurgence as an alternative treatment option for young and active patients with significant hip osteoarthritis. Despite the satisfactory short-term implant survivorship, abnormal periprosthetic soft-tissue masses relating to the hip joint (pseudotumours) are being increasingly reported. These were found to be locally destructive, requiring revision surgery in a high proportion (75%) of patients, the outcome of which is poor. However, there is currently no definitive evidence of potential mechanisms involved in the occurrence of such destructive soft tissue masses. The broad aims of this thesis were: 1) to determine the extent of this emerging clinical complication (pseudotumour) with a high revision burden; and 2) to determine whether pseudotumour results from a local biological reaction to an increased wear debris burden generated by excessive MoMHRA implant wear. A clinical study investigated the prevalence of, and association between, pseudotumours and serum metal ion levels in 158 MoMHRA patients (201 hips). The overall prevalence of pseudotumour was found to be 4%, considerably higher than previously reported. The study also established that pseudotumour only occurred with elevated metal ion levels, suggesting the underlying cause is increased wear. It was postulated that pseudotumours are likely to be a biological reaction to the large amount of metal debris generated in vivo due to excessive wear. Two potential biological reactions mediating the occurrence of pseudotumours were then investigated. An in vitro clinical study demonstrated that systemic hypersensitivity type IV reaction, mediated by lymphocyte reactivity to these metals, is not the dominant biological response involved in pseudotumours. A further in vitro experiment demonstrated that metal particle-induced cytotoxicity is likely to be an important factor leading to pseudotumours. Subsequent research focused, firstly, on assessing the magnitude of bearing surface wear that occurs in pseudotumour patients, and secondly on elucidating the potential wear mechanism responsible for the increased implant wear in these patients. A retrieval study demonstrated that MoMHRA implants revised due to pseudotumour were associated with significantly greater linear wear rates. This combined with the metal ion data confirmed that pseudotumour is associated with increased wear at the MoM articulation. An edge-loading wear pattern was always found in the pseudotumour group. An in vivo study was then developed to investigate whether the edge-loading occurs during functional activities. Edge-loading in the pseudotumour group was found to occur with up to 4-fold increase in duration and up to 7-fold increase in force. This in vivo finding supported the in vitro retrieval study finding of an edge-loading wear pattern in the pseudotumour group, suggesting that edge-loading during functional activities is an important in vivo mechanism responsible for localised high wear and subsequent elevation of metal ion levels in MoMHRA patients with pseudotumours. It is concluded that pseudotumour is likely to result from a local biological reaction to increased metal debris load, generated by excessive MoMHRA implant wear due to edge-loading. In susceptible patients, the dose-dependent cytotoxicity of wear debris leads to subsequent necrosis of periprosthetic soft tissues. Clinicians need to be aware of pseudotumours and surgeons should undertake MoMHRA with great care in the knowledge that even with satisfactory component positioning, the problem can occur. In evaluating MoMHRA patients with unexplained symptoms with normal plain radiographs, further investigation with soft-tissue imaging modalities such as ultrasound or MRI is recommended.

Stroke is the third leading cause of death in the developed world and is the leading neurological cause of disability with a massive impact on personal life and society. Large artery atherosclerosis is one of the main causes of ischaemic stroke. However, in several aspects of this condition there is still a significant amount of uncertainty about its prevalence, appropriate investigation and possible treatment. Reliable data on epidemiology are therefore necessary to provide clinicians and researchers with crucial information to guide diagnostic and therapeutic management as well as further research. With this thesis I aimed to provide useful information about the prevalence of large artery disease in certain groups of patients, and to contribute to investigation- and managementstrategies using data from a large population based study, the Oxford Vascular Study (OXVASC). OXVASC is a prospective, population-based incidence study of vascular disease in Oxfordshire, UK, which started in 2002 and is ongoing. The study population comprises all 91,106 individuals registered with nine general practices and uses multiple methods of case ascertainment to identify all patients with vascular events. Firstly, I have shown that the prevalence of ≥50% vertebral or basilar artery stenosis in posterior circulation TIA or minor stroke is more than twice as high as the prevalence of ≥50% carotid stenosis in patients with carotid territory events, and is associated with a very high early risk of stroke of 22% and TIA of 46%. Furthermore, severe vertebral and/or basilar artery stenosis is associated with multiple TIAs at first presentation. Secondly, I have shown that early risk of stroke was higher after posterior circulation TIA, with a 1-year risk of 16%, than after carotid territory TIA, with a 1-year risk of 9%. In addition, I was able to show for the first time, that the ABCD2 score was predictive of early stroke not only in patients with carotid circulation TIA but also in patients with vertebrobasilar TIA. Thirdly, in a pilot feasibility study about arterial spin labelling magnetic resonance imaging in patients with large artery disease in the vertebrobasilar circulation I have shown that patients with severe large artery disease have significantly impaired occipital brain perfusion. My results suggest that this new technique might be a useful tool to identify suitable patients for interventional treatment of vertebrobasilar large artery disease. Fourthly, I was able to show that the risk of ipsilateral stroke and TIA in patients with an asymptomatic carotid stenosis is very low with contemporary best medical treatment alone, suggesting that routine carotid endarterectomy for asymptomatic carotid stenosis might not longer be feasible. Finally, I have clarified that lower rates of intervention for moderate to severe symptomatic carotid stenosis in women than in men can be explained by sex-differences in the populationbased incidence of carotid large artery disease and not due to under-investigation or reluctance amongst women to undergo investigation or treatment.

M.tuberculosis, the causative agent of human tuberculosis, is able to achieve long-term persistence within host organism macrophages. This persistence is achieved via the ability of the mycobacterium to prevent phagosomal-lysosomal fusion. The mechanisms by which fusion is inhibited remain incompletely understood. Here we provide evidence supporting a mechanistic link between infection with pathogenic mycobacteria and the cellular pathway defective in the rare lysosomal storage disorder Niemann-Pick disease type C (NPC). We observed that NPC phenotypes, including lipid storage and reduced lysosomal calcium release, can be induced in wild-type murine and human macrophages by infection with pathogenic mycobacteria. This phenotype induction did not occur following infection with the non-pathogenic M.smegmatis. Phenotype induction could be achieved in the absence of the mycobacteria using lipids from the mycobacterial cell walls. The importance of mycobacterial cell wall lipids to mycobacterial virulence has been well-documented. This lipid-mediated inhibition likely occurs through the NPC1 protein. Susceptibility to phenotype induction was inversely proportional to levels of functional NPC1, whilst a pre-existing dysfunction in the NPC pathway (either stemming from mutation or pharmacological inhibition) rendered cells less able to clear non-pathogenic mycobacteria. Finally, we demonstrate that therapies for NPC, particularly curcumin, are able to promote clearance of mycobacteria from infected macrophages. NPC therapies may hold promise for a new approach to the treatment of tuberculosis.

The hunt for biomarkers for anti-VEGF agent bevacizumab is ongoing since last decade with no success. Identifying robust biomarkers for stratifying patients and for monitoring response is important for the future use of bevacizumab in breast cancer. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) analysis and genome wide gene expression analysis are two promising approaches to understand the molecular mechanisms and search for biomarker of anti-angiogenic therapy. Firstly, with the retrospective pilot study, a close link between DCE-MRI findings and the molecular mechanisms assisting cancer survival and metastasis was established. Secondly, the prospective window of opportunity study conducted using single cycle of bevacizumab given before neoadjuvant chemotherapy and by performing detailed pharmacodynamic analyses with DCE-MRI and gene expression before and two weeks after bevacizumab had shown a wide variation in responses to bevacizmab both at genomic and imaging level. A close link between changes in DCE-MRI and the changes in gene expression profile was further established suggesting DCE-MRI has potential to serve as non-invasive biomarkers of antiangiogenic therapy. Tumours with high baseline values of forward transfer constant K^trans showed the maximum response as assessed by DCE-MRI after bevacizumab. By performing biopsy after single cycle of bevacizumab, the changes in genes related to immune response, metabolism and cell signalling were observed that gives a useful insight into mechanisms governing response and resistance to bevacizumab. Also the certain gene expression changes observed with post bevacizumab biopsies, such as down regulation of endothelial cell specific molecule-1 (ESM1), cyclin E1 (CCNE1) and up regulation of pyruvate dehydrogenase kinase 1 (PDK1), cyclic GMP-inhibited phosphodiesterase B (PDE3B) could be helpful in decision-making about future therapy with bevacizumab at an early stage. This study has suggested that using bevacizumab in combination with other targeted agents could overcome resistance.

Regulation of the growth and differentiation of trophoblast cells is critical for successful embryo implantation and placentation. Cytokines are key players in these processes, as well as modulating the maternal immune response to prevent rejection of the conceptus. This thesis focused on the investigation of the cytokine interleukin (IL) - 33 and its receptor, ST2. ST2 has two isoforms, a functional cell surface receptor (ST2L) and a soluble decoy receptor (sST2). Previous work in this laboratory had shown that the human placenta expresses both IL-33 and sST2 at term. The aim of this thesis was to investigate IL-33 and ST2 in early pregnancy, the time when trophoblast is at its most active, with a view to better understanding their role. IL-33 and ST2 mRNA and protein were examined in 14 first trimester placentas from 6-12 weeks of gestation. IL-33 was localized to cells in the villous stroma, whereas ST2 was present in the syncytiotrophoblast, villous cytotrophoblast and the invasive extravillous cytotrophoblast of the cell columns. Secretion of sST2, but not IL-33, by the placenta was found. Investigation of pre-implantation embryos showed the presence of ST2, but not IL-33 protein. Decidualized endometrium was investigated as a potential source of IL-33 and sST2 at the maternal-fetal interface and, although mRNA for both was present, no protein could be found. The key finding was that sST2, rather than ST2L, was the predominant isoform in the placenta. This led us to reconsider the hypothesis that IL-33/ST2 interactions in the placenta are important for successful pregnancy and raised the possibility that they may have independent roles. Using trophoblast cell lines as a model, it was shown that sST2 binds to trophoblast cells, significantly inhibits their proliferation and stimulates their invasion in vitro. This is the first report of this novel role for sST2 in pregnancy. Thus these studies have shown that sST2 may play an important role in implantation and placentation through controlling trophoblast invasion.

Despite 30 years of research, a prophylactic vaccine against HIV-1 is still lacking and is urgently needed in order to control the global AIDS pandemic. The discovery of broadly neutralising antibodies (BNAbs) was an important step for HIV-1 research but no vaccine candidate tested so far has been able to reproduce responses containing such antibodies, and it remains unclear how this could be achieved via immunisation. In this thesis, I attempted to explore this gap of knowledge in two ways. First, certain features (‘signatures’) of the Env protein that were associated with a broadly neutralising response were identified through machine learning. Further characterisation of these signatures revealed several ways by which these naturally-occurring mutations might alter the immunogenicity of the Env protein that could result in the elicitation of a broadly neutralising response. The incorporation of such signatures in future vaccine design could be useful as the Env protein might adopt a conformation that encourages the elicitation of a broadly neutralising response. Second, 3 novel vaccination approaches were proposed aiming to induce a BNAb antibody response. The development of 2 approaches proved to be difficult and was not continued. For the third approach, non-neutralising immunogen-derived antibodies were used to mask immunodominant epitopes on the Env protein (i.e. ‘antibody-shielding’), thus allowing the antibody response to be focused to the highly conserved CD4 binding site (CD4bs). Subsequent immunisation of the antibody-shielded gp120 proteins in mice and rabbits demonstrated that antibody-shielding was able to significantly dampen the V3-specific antibody response while retaining the CD4bs-specific response. However, the antibody response to the V1/V2 loop was enhanced upon V3-dampening which indicates that further optimisation of the antibody-shield is needed in order to eliminate any antibody response towards the immunodominant regions. In conclusion, these results are the first description of a number of novel vaccination ideas and provide valuable insights into how these approaches could be optimised to become effective HIV-1 vaccines that can lead to the elicitation of a broadly neutralising antibody response.

Background: Endometriosis is a common condition in women of reproductive age, causing pelvic pain and subfertility, but little is known about its impact on health-related quality of life (HRQoL) and work productivity worldwide. Methods: In 10 countries across five continents, this study recruited 1,418 women, aged 18-45, without a previous surgical diagnosis of endometriosis, scheduled to undergo a laparoscopy to investigate symptoms suggestive of endometriosis or to be sterilised. Pre-operatively, women completed a standardised questionnaire assessing symptoms, diagnostic delay, HRQoL and work productivity using validated instruments. Surgeons completed a standardised questionnaire incorporating findings at laparoscopy including endometriosis stage according to revised American Fertility Society criteria. Results: There was a mean delay of 9.2 years (SD 8.8), principally in primary care, between the onset of symptoms and diagnostic laparoscopy. This diagnostic delay was longer in centres where healthcare was predominantly state-funded (12.8 vs. 7.6 years; p<0.001). In multivariate analyses, the delay was positively associated with the number of pelvic symptoms (chronic pelvic pain, dysmenorrhoea, dyspareunia and heavy periods; p<0.001) and a higher body mass index (p<0.001). Physical HRQoL was significantly reduced in affected women compared to those with similar symptoms and no endometriosis (p=0.012). Not being in paid employment, severe pelvic pain and moderate-severe disease were associated with reduced physical HRQoL (all p<0.001). Each affected woman lost on average 10.0 hours (SD 10.6) of work weekly, due mainly to reduced effectiveness while working. The annual indirect cost of endometriosis associated with work productivity loss ranged from US$399 per woman in Ibadan (Nigeria) to US$18,586 per woman in Boston (USA). Conclusions: Endometriosis significantly impairs HRQoL and work productivity across countries and ethnicities, yet women continue to experience diagnostic delays in primary care. A higher index of suspicion is needed to expedite specialist assessment of symptomatic women. Future research should seek to clarify pain mechanisms in relation to endometriosis severity.

Human macrophages are one of the main targets for HIV-1 infection, despite their moderately low surface expression levels of the main HIV-1 receptor, CD4. The site of HIV-1 fusion can occur at the surface or following uptake through an endosomal pathway and it might be anticipated that the site would affect the progress of HIV-1 through the cell to the nucleus. Previous pharmacological studies provide one line of evidence for an endosomal entry route which is dependent on Detergent Resistant Membranes (DRMs). However, these findings need confirmation using a genetic approach, as small molecules may have multiple non-specific effects. For this study, a novel genetic approach was developed to manipulate sub-cellular CD4 distribution and investigate whether it determines the HIV-1 entry pathway in macrophages. This was achieved by transducing human embryonic stem cells (hESC) with lentiviral vectors and differentiating these cells into homogeneous genetically modified macrophages. This cellular system by-passes the challenges posed by the refractoriness to direct genetic manipulation of heterogeneous primary macrophages. Firstly, as proof of principle, a short hairpin RNA targeting CD4 was expressed in hESC-macrophages, resulting in knockdown of CD4 and, as anticipated, strong inhibition of HIV-1 infection. Secondly, expression of LCK in hESC-macrophages effectively tethered CD4 at the cell surface, and sequestered HIV-1 into an unproductive pathway, presumably through surface fusion, rather than progressing successfully to the nucleus. Thirdly, endogenous CD4 was substituted with CD4 mutants designed to be excluded from DRMs, which resulted in reduced successful HIV-1 entry versus substituted control CD4. The results support the model in which the productive entry pathway of HIV-1 in macrophages occurs via fusion after a raft-dependent endocytic uptake pathway, and requires CD4 localization to lipid rafts.