Japanese encephalitis virus (JEV) is a mosquito-transmitted Flavivirus that is closely related to other emerging viral pathogens including dengue (DENV), West Nile (WNV) and Zika viruses (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout South East Asia, the Pacific islands, and the Far East, causing around 68,000 cases worldwide each year. There is no specific treatment for JEV. This study aims to determine the molecular structure of new potential drug targets for JEV. In this study, the JEV non-structural protein 1 C-terminal β-ladder domain (C-NS1) is presented at 2.1 Å resolution. The crystal structure of C-JEVNS1 shares a conserved fold with flavivirus C-NS1 domains. The surface charge distribution of C-JEVNS1 is similar to WNV and ZIKV but is significantly different from DENV. Analysis of the C-JEVNS1 structure, in silico molecular dynamics simulations and experimental solution small angle X-ray scattering, indicate extensive loop flexibility on the exterior of the protein. It is proposed that this together with charge distribution on the exterior of the protein influence NS1-host protein interaction specificity which may impact on pathogenicity. These factors may also affect the interaction with the monoclonal antibody, 22NS1, which is protective against WNV infection. Liposome and heparin binding assays indicate that only the N-terminal region of NS1 participates in the interaction with lipidic membranes and that sulphate binding sites are not the glycosaminoglycans binding interfaces. For the first time, the crystal structure of the JEV capsid protein at 1.98 Å is also reported and compared to the existing flavivirus capsid protein. JEV capsid shows helical secondary structure (α helixes 1-4) and protein folding similar to DENV and WNV capsid proteins. It forms a homodimer by antiparallel pairing with another subunit (‘), α helix 1-1’, 2-2’, and 4-4’. The capsid dimer is believed to be the building block of the nucleocapsid. The flexibility of the N-terminal α helix 1 of the capsid could be important for its function. This dimer model agrees with a previous suggestion that the capsid protein interacts with RNA via the basic rich C-terminal, α4-α4’, and associates with lipid bilayers at the opposite hydrophobic, α2-α2’.

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BACKGROUND: According to the World Health Organization, long-term illness will account for almost three-quarters of global deaths by 2020. Evidence supports the use of both self-management and mindfulness-based interventions (MBIs) for those who live with long-term illness. While this research is promising, a major barrier with traditional MBIs is the amount of time they require (eight weeks) and the necessity of a trained specialist. OBJECTIVE: The aim of this thesis is to develop and evaluate a brief MBI for those with long-term illness, through a literature review, a qualitative study and a pilot study. METHODS: A systematic review of studies using brief MBIs for health-related outcomes was the initial stage. This provided a basis for a qualitative study, which was conducted to assess views of long-term illness patients (i.e., persistent pain, chronic obstructive pulmonary disease, cardiovascular disease) on the acceptability of a brief (10 minute) MBI, to define population suitability and to refine delivery and assessment. A pilot randomised controlled trial (RCT), informed by results from the first study, was then conducted with persistent pain patients. A brief mindfulness body scan audio was compared to an active control and immediate effects of the intervention were assessed with brief measures for perceived pain severity, distress and distraction. Feasibility of a definitive RCT was assessed in relation to recruitment and retention rates. At baseline, one week and one month, assessments included: mindfulness, anxiety, depression, health-related quality of life, pain catastrophizing, pain self-efficacy, activities of daily living limitations, and ratings o f‘usefulness’ and ‘likelihood to recommend’ the intervention. RESULTS: The review identified 71 eligible studies of brief MBIs, including 70 RCTs. Sixty-seven studies observed a positive effect on at least one health related outcome. There was high heterogeneity for both types of MBI and health-related outcomes and low use of clinical populations. Results from the qualitative study, with 14 patients, suggested that a brief MBI audio was acceptable and was most suited to a persistent pain population. Patients tended to prefer an MBI of 15 minutes rather than 10 minutes. In the pilot study of a 15 minute MBI audio, of 220 patients referred, 147 were randomised and 71 completed all assessments. There were no significant immediate effects of the MBI, although there was a tendency for a marked improvement in both groups. Significant effects were found for ‘usefulness’ at one week and self-efficacy at one month in the MBI group compared with the control. Levels of recruitment were acceptable and attrition rates were high. CONCLUSIONS: The findings of the systematic review demonstrate that brief MBIs can have a positive impact on a range of health-related outcomes and that further studies are required, especially with clinical populations. Qualitative findings confirm the acceptability of a brief MBI, particularly for a persistent pain population. In the pilot study, lack of immediate effects could be due to the potency of the control and a less engaging control needs to be considered. A definitive trial is required in which retention of patients is optimized, for example, by delivering the MBI alongside existing rehabilitative programmes.

The Drosophila melanogaster fat body, analogous to mammalian adipose tissue and the liver, is the primary organ of fat and glycogen storage as well as being responsible for the humoral immune response following infection. Normal functioning of the fat body is of critical importance to the survival of the organism, but many molecular regulators of its function remain ill-defined. Here, for the first time, we demonstrate that the Drosophila bromodomain-containing protein fs(1)h is essential in the fat body for normal lifespan as well as metabolic and immune homeostasis. Under physiological conditions, flies lacking fs(1)h in the fat body exhibit a severely reduced lifespan, abnormally high expression of immune target genes including antimicrobial peptides (AMPs) and cytokines, an inability to utilise triglycerides following periods of starvation, and low basal AKT activity, mostly resulting from systemic defects in insulin signalling. Loss of fs(1)h in the fat body also results in hypoglycemia and a dysregulation of several fat body-derived signals, indicating a role for fat body fs(1)h in the regulation of various systemic endocrine signals. Removing a single copy of the AKT-responsive transcription factor foxo ameliorates almost all the observed phenotypes, restoring lifespan, metabolic function, uninduced immune gene expression, and AKT activity suggesting many of the in vivo effects of fs(1)h in the fat body are foxo-dependent. However, survival is not rescued and AMP expression is still elevated following bacterial infection in fs(1)h knockdown foxo heterozygous flies, indicating some of the phenotypes observed are independent of the FOXO hyperactivation. We propose that the promotion of systemic insulin signalling activity is a key in vivo function of fat body fs(1)h.

Marginal Zone (MZ) cells are a subset of B cells. They reside the marginal zone outside the mantle zone of the spleen and circulate the blood in humans. They have also been identified in the crypt epithelium of tonsil, the sub-capsular sinus in lymph node and in the dome area of Peyer’s patches in gut-associated lymphoid tissue of human intestine. A monoclonal antibody (4D12) that recognises an antigen on MZ B cells and a subset of GC cells in tissue sections has been described previously but this has not been investigated in detail and features of the 4D12 antigen and the lymphocytes that express it are unknown. The re-establishment and culture of the 4D12 MoAb secreting clone after a long-term storage is described. A method to analyse the distribution of 4D12 antigen on subsets of B cells was devised. Subsets expressing 4D12 antigen included CD27+IgM+IgD+ cells, CD27+IgM+IgD- cells, transitional B cells and plasmablasts. Lower expression by mature naïve and class switched memory B cells was observed. 4D12 MoAb tended to recognise a greater proportion of CD27+IgM+IgD+ cells and CD27+IgM+IgD- cells in spleen compared to tonsil and blood. Intracellular staining showed that the 4D12 antigen is proportionally more presented in the cytoplasm than the cell surface and that cytoplasmic 4D12 antigen expression is not restricted to B cells. The identity of the antigen was sought by western blotting and mass spectrometry (by collaborators) and the output of the mass spectrometry was provided for this study. Mass spectrometry data was analysed here and a list of candidates for the identity of 4D12 antigen were selected and tested by flow cytometry.

Keloid scars form due to an over-exuberant, fibrotic wound response in the skin of predisposed individuals. There is currently no clear genetic link identified, and limited treatment options available. Epigenetic changes in fibrosis, and specifically in keloids, are beginning to be identified and this offers potential targets for treatment. This project explored the bromodomain and extraterminal (BET) family of proteins: Brd2, Brd3 and Brd4, which function as epigenetic regulators through their binding to acetylated lysine residues, predominantly on histone proteins. We hypothesised that they would be a target of therapeutic value given that their inhibition has shown anti-proliferative and anti-inflammatory properties in other cell types. We identified an overexpression of Brd2 in keloid tissue compared to normal controls, including a possible expression of a differential Brd2 isoform. We then used a small molecule bromodomain inhibitor, I-BET151 (GSK), to investigate the effect of BET protein inhibition in primary dermal fibroblast cultures from normal skin (NDFs) and keloid scars (KDFs). We confirmed previous literature findings, in other disease models, that BET inhibition decreased proliferation, as well as the expression of the inflammatory cytokine interleukin-6 (IL-6). In addition, we identified some more novel effects of BET inhibition, including a decrease in fibroblast contraction in a collagen gel model and decreases in proteolytic activity in fibroblast cultures, as well as a more complex ex vivo tissue model. Interestingly, we also observed profound differences between NDFs and KDFs in their signalling downstream of IL-6 in serum free environments. In particular, a strong induction of pAkt was observed in KDFs after 30 minutes of cytokine stimulation, which was not seen in NDFs, and BET inhibition abrogated this. In conclusion, BET inhibition appears to offer therapeutic value in keloid scars by targeting a number of disease-associated cell behaviours, including aberrant signalling responses, which may influence the fibrotic response. Some of these effects may be of relevance in other disease contexts including cancer, where these inhibitors are currently progressing in clinical trials.

Peptide immunotherapy (PIT) is a specific immunomodulatory treatment aiming to tip the balance from pro8inflammatory attack to restoration of immune tolerance. It has been successfully translated into clinical practice in the field of allergy, stimulating ongoing research in autoimmune diseases including type 1 diabetes. The safety and mechanistic effects of proinsulin C198A3 (PI C198A3), a HLA8DR4 restricted, naturally processed and presented peptide, was assessed through a multicentre placebo controlled double8blind clinical trial of 2 or 4 weekly intradermal doses of 10µg peptide for 6 months, with a 6 month follow8up period. Overall, treatment was well tolerated with no evidence of local or systemic hypersensitivity or disease acceleration. In some PIT8treated subjects there was retention of C8peptide. To address the question of whether and how PIT impacts upon immune function, over the 6 month treatment period, T cell receptor (TCR) clonotyping and gene expression analysis were performed on peptide8specific activated CD4 T8cells. TCR β8chain clonotyping revealed shared β8chain clonotypes between patients; however, these were not apparently linked to peptide immunotherapy. Gene expression changes were explored but no antigen8 specific changes related to treatment identified. This study concludes that peptide immunotherapy using PI C198A3 is a safe and well8 tolerated treatment in newly8diagnosed adults with type 1 diabetes; deployment of novel mechanistic studies to examine alterations in antigen8specific effector CD4 T cells do not reveal any linked changes in TCR or gene expression.

In this study, we investigate the impact of ovarian carcinoma associated ascites on dendritic cell (DC) activation by Toll-like receptor (TLR) agonists in vitro. DC have the potential to instigate a tumour-specific adaptive immune response, but their ability to induce differentiation of naïve lymphocytes into effector cells in lymphoid tissues is dependent on their activation status. Here, we examine whether DC activation by TLR agonists is impeded by ovarian carcinoma environment and if so, how these effects can be alleviated. Our results show that ascites reduces the TLR-mediated up-regulation of the co-stimulatory molecule CD86 and partially inhibits the production of the pro-inflammatory cytokines interleukin-6 (IL-6), IL-12 and tumour necrosis factor α (TNFα) in monocyte-derived DC from healthy donors. We further observe an impaired T cell stimulatory capacity of monocyte-derived DC upon activation with TLR agonists in the presence of ascites, indicating that their function as antigen-presenting cells is affected by the immunosuppressive factors. Selective neutralization of IL-10 and prostaglandin E2 (PGE2) in vitro alleviates the suppressive effects of ovarian carcinoma associated ascites. However, our results show that autocrine IL-10 contributes to the observed suppression. The role of autocrine PGE2 is yet unclear, as we have no indication that this protein is produced by TLR-activated monocyte-derived DC. We have established and present here an elegant method to dissect the relative contributions of ascites-derived versus autocrine IL-10 and PGE2, and experiments to this effect are ongoing. The findings of this study can enhance the understanding of the ovarian carcinoma environment and its influence and relevance in the context of DC-based vaccines and other immunotherapeutic intervention strategies in ovarian carcinoma.

The programmed cell death-1 (PD-1) receptor is a key regulator of T cell activation and cytokine production. Multiple studies performed in PD-1-deficient mice demonstrate its importance in preventing autoimmunity. Evidence suggests that PD-1- mediated regulation is reduced during chronic inflammation in human diseases, such as rheumatoid arthritis (RA). In this thesis, the role of inflammation in influencing PD-1-mediated regulation of human CD4+ T cells is further characterised. First, PD-1 and PD-L1 expression, as well as the functional consequences of PD-1 ligation in rheumatoid (RA) and psoriatic arthritis (PsA) were analysed. Using flow cytometry and analysis of existing gene expression arrays, it was determined that the percentage of PD-1+ cells within the CD4+ and CD8+ T cells compartment was increased in RA and PsA synovial fluid (SF) compared to paired peripheral blood (PB). Upon in vitro T cell receptor (TCR) stimulation of HC CD4+ T cells in the presence of plate-bound PD-L1fc chimera, significantly decreased proliferation and interferon (IFN)-γ secretion was observed. In contrast, RA and PsA PB- and SF-derived CD4+ T cells appeared resistant to such PD-1-mediated inhibition. Second, it was investigated whether proinflammatory cytokines modulate PD-1-mediated regulation of healthy CD4+ T cells (HC). Addition of proinflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β, which were increased in RA and PsA SF compared to HC and osteoarthritis (OA) controls, consistently abrogated PD-1- mediated suppression in HC CD4+ T cell cultures. Inhibitors of these cytokines reversed this effect. Finally, it was evaluated whether soluble PD-1 (sPD-1) negatively regulates the PD1/PD-L1 interaction. Soluble PD-1 (sPD-1) levels were increased in cell culture supernatants from TNFα and IL-6-stimulated cultures compared to untreated controls, and also in RA and PsA, but not in OA, serum and SF nor in HC serum. qPCR analysis of HC CD4+ T cells from TNFα- and IL-6-stimulated cultures also revealed increases of the PD-1Δex3 splice variant. Functionally, addition of sPD- 1fc counteracted PD-1-mediated suppression of HC CD4+ T cells, increased T cell proliferation in HC CD4+ T cell/monocyte co-cultures but had no effect on HC CD4+ Treg cell-mediated suppression. Together, the data presented in this thesis provide new evidence that the inflammatory environment of the RA and PsA joint compromises PD-1/PD-L1 mediated T cell regulation.