The liver has a unique connection with the immune system; harbouring vast numbers of lymphocytes, able to instigate secondary lymphoid organ-independent naive T cell activation, and promoting potent immune tolerance. We set out to determine the effect of this unique microenvironment on the biology of CD4+ T cells at three key interaction points: following migration into the parenchyma, after short-term hepatocyte contact, and at long-term tissue-residency. Modelling transmigration through hepatocytes revealed intrinsic, disease-specific cytokine responses in blood-derived CD4+ T cells, not discernible through static co-culture. However, short-term co-culture did induce activation-independent CD69 upregulation, reliant upon cell-cell contact. This phenotype mimicked the similar hepatic CD4+ CD69INT cells that we discovered in liver tissue. Unlike CD69HI cells which represented the tissue-resident memory T cells (TRM) of the liver, CD69INT cells were the most activated population, likely able to migrate to many liver and gut niches, and singularly able to produce IL-4 and IL-10. By contrast, CD69HI TRM displayed a resting phenotype, marked for more restricted movement, and produced the best multifunctional TH1 responses following stimulation. These data demonstrate the importance of studying migration, and provide detailed characterisation of CD69HI TRM and novel CD69INT cells, along with their proposed roles and generation pathways.

Recent evidence indicates that immune cells within adipose tissues can drive the formation of ectopic lymphoid structures, known as Fat Associated Lymphoid Clusters (FALC). FALC support B-cell antibody production in response to infection and inflammation. This investigation explores the immune cell composition and role of different adipose tissues both in steady state and during immune responses in mice. Firstly, a detailed analysis of the immune cell composition of peritoneal adipose tissues was performed. To investigate the function and migratory properties of these tissue-resident cells, cytokine and chemokine receptor expression was then assessed. How immune cells in adipose tissues responded to infection was examined using an intestinal helminth infection with the parasite Heligmosomoides polygyrus. Adipose tissues predominantly contained regulatory T cells, invariant natural killer T cells and group 2 Innate Lymphoid Cells (ILC2s). Significant differences were observed in composition, cell surface markers and cytokine production of ILC2s between adipose depots and secondary lymphoid tissues, indicating that tissue specific signals can direct ILC2 responses. Finally, increases were observed in ILC2 and FALC numbers in the mesenteries of WT mice following parasite infection. These data indicate that immune cells within adipose tissues respond to infection and may contribute to immune responses.

Volume 1, the research component, includes a literature review examining the efficacy of interventions for parents of children with autism spectrum disorder (ASD) in relation to parent outcomes; an empirical paper, which presents findings of a quantitative study exploring parental illness perceptions, coping, wellbeing, parenting stress and parent perceptions of the child-parent relationship in parents of a child with pathological demand avoidance syndrome (PDA) and a public dissemination document. Volume 2, the clinical component, includes: a report detailing two psychological formulations; one from a cognitive behavioural perspective and one from a systemic perspective, for a 20 year old gentleman who was experiencing anxiety and depression, following removal of part of his bowel; a service evaluation report detailing an investigation of the extent to which a local respiratory service was addressing the psychological needs of COPD patients; a single case experimental design presenting an evaluation of a behavioural intervention for a 25 year old woman with a moderate learning disability, who presented with skin picking behaviours; a case study of a fourteen year old girl, who was under investigation for Crohn's disease and experienced anxiety and an abstract, reflecting on providing consultation within a looked after and adopted child's psychology service.

Intracellular Ca2+ and adenosine triphosphate (ATP) are the key elements needed for stimulant-evoked exocytotic enzyme secretion from pancreatic acinar cells. Physiological Ca2+ signals consist of repetitive spikes confined to the secretory granule region, which stimulate ATP production; whereas sustained global cytosolic Ca2+ elevations - toxic Ca2+ signals decrease ATP levels and cause necrosis leading to the inflammation of the pancreas - acute pancreatitis (AP), a life-threatening disease currently without specific therapy. The work presented in this thesis focuses on the mechanisms underlying the development of pancreatitis evoked by L-asparaginase and the potential ways to intervene asparaginase-associated pancreatitis (AAP). Asparaginase is an essential element of the chemotherapy regimen in the successful treatment of acute lymphoblastic leukaemia (ALL), the most common childhood cancer. But asparaginase treatment can lead to AAP, which is a side-effect of ALL treatment occurring in about 5–10% of cases. Following AP, further treatment with asparaginase is withheld to prevent recurrence of AP; however, withholding scheduled asparaginase is associated with a potential increase in ALL relapse. Understanding the pathogenesis of AAP could lead to effective therapies for this complication, potentially reducing toxicity and allowing re-exposure to continue treatment with asparaginase.

This thesis explores the impact of lifestyle factors on the development of metabolic syndrome (MS) in Saudi Arabian women. A survey of a snowball sample was used to recruit 258 female and explored factors influencing physical activity (PA) and food intake and their effects on BMI in women living in the KSA and the UK. Participants completed a self-reporting questionnaire relating to knowledge, attitudes, barriers and levels of PA, sedentary activity and eating habits. Excessive energy intake, physical inactivity and sedentary lifestyle were all prevalent in Saudi women, resulting in 80%, over the age of 35y, being overweight or obese. BMI was associated with both energy intake and PA, though the relationship with the former was stronger. The most common barriers to regular exercise were transportation and lack of time. Findings were generally similar between women living in Saudi Arabia and the UK. The efficacy of reducing energy intake, with or without increased PA, on risk factors associated with MS in overweight Saudi women was investigated in a pilot study. After a four-week program, incorporating dietary modification alone (D) or in combination with regular vigorous aerobic exercise (D+E), improvements were seen in body composition and a range of metabolic risk factors. Both groups lost weight, but, paradoxically, those in D lost significantly more than those in D+E (5.3 vs. 3.3%, p=0.016). Moreover, significant reductions were also found in blood pressure, plasma triacylglycerol, insulin, total and LDL cholesterol, with no significant differences between the two groups. Plasma glucose and HDL cholesterol remained unaltered. Overall, these changes led to a decline in the prevalence of MS from 20% to 5% and 21% to 7% for the D and D+E groups, respectively. Thus, reducing energy intake appears, at least in the short term, more important than increasing PA in reducing body weight and associated metabolic risk factors. These studies confirm that excessive dietary intake and physical inactivity both contribute to overweight and obesity in Saudi Arabian women. With appropriate support, it is possible to both reduce energy intake and increase PA, although, in the short -term, the former appears to be most important. It remains to be established whether longer-term improvements in PA would further improve metabolic health.

Prostate cancer is the most common male cancer in the UK. Although incidence is increasing, prostate cancer mortality is decreasing, mainly owing to the over diagnosis of disease that would not have become clinically apparent during the patient’s lifetime. The gold-standard for prostate cancer diagnosis is transrectal ultrasound guided biopsy of the prostate. Whilst prostate biopsy can inform on diagnosis, it’s prognostic ultiltiy is poor. Currently clinicians lack pathological biomarkers to differentiate between patients with prostate cancer who have indolent disease that can be safely managed with surveillance strategies, and those who will go onto develop aggressive disease which requires early radical curative treatment. Phosphorylation of the androgen receptor has been extensively investigated in relation to prostate cancer development and progression. Androgen receptor phosphorylation has been shown to regulate cellular localisation, transcriptional activity, cell growth and sensitivity to androgens in prostate cancer. However, only a small number of studies have investigated the prognostic significance of androgen receptor phosphorylation, and only consider a limited number of serine residues in clinical specimens. The research presented in this thesis sought to investigate the prognostic and predictive significance of AR phosphorylation at serine 578 in hormone-naïve prostate cancer. It was hypothesised that pARS578 would be associated with poor outcomes in prostate cancer and may be utilised as a prognostic marker at diagnosis in prostate cancer and predict response to drug treatment with a PKC inhibitor. It was also hypothesised that PKC, the putative kinase for phosphorylation at serine 578, would be associated with poor outcomes and may offer a potential therapeutic target in prostate cancer. In the current study, the phosphorylation site of primary interest was serine 578. Scansite 2.0, an online kinase search tool, predicted that PKC is the putative kinase mediating phosphorylation at serine 578 on the androgen receptor. Phosphorylation of the androgen receptor at serine 578 has been linked with increased AR transcriptional activity, cell growth, nuclear cytoplasmic shuttling, modulation of other AR phosphorylation sites and DNA-repair mechanisms. The prognostic significance of androgen receptor phosphorylation at serine 81 was also investigated in this study. Serine 81 is phosphorylated in response to DHT via an alternative pathway to that of serine 578. Serine 81 phosphorylation is associated with increased androgen receptor transcriptional activity and increased cell growth in prostate cancer. It was therefore hypothesised that androgen receptor phosphorylation at serine 578 and serine 81 would be associated with poor outcome measures in prostate cancer. Immunohistochemical analysis was performed in a cohort of 105 hormone-naïve prostate cancer patients undergoing active surveillance, representing a cohort of patients with low-risk disease, as defined by current clinical markers such as PSA and Gleason score at diagnosis. Nuclear PKC expression was significantly associated with pARS578 expression in the clinical specimens, supporting the prediction of Scnasite 2.0 that PKC is the kinase responsible for phosphorylation of the AR at this site. High cytoplasmic expression of pARS81 was associated with decreased time to intervention (HR 2.76 (95% CI 1.1-7.3), p=0.032). There was no association between pARS578 and time to intervention in this cohort. Analysis of combined expression of both phosphorylation sites revealed an association between high dual expression of cytoplasmic pARS81 and cytoplasmic pARS578 and decreased time to treatment intervention (HR 2.35 (95% CI 1.2-4.6), p=0.031). These results suggest a synergistic prognostic effect when these two phosphorylation sites are combined and identifies a sub-population of low-risk prostate cancer patients who are at increased risk of disease progression. A second study was conducted to investigate if these results could be replicated in a cohort of prostate cancer patients with all stages of disease at diagnosis. Immunohistochemical analysis in 90 hormone-naïve prostate cancer patients found that high expression of nuclear pARS81 (HR 2.1 (95% CI 1.1 – 4.2), p=0.030), nuclear pARS578 (HR 2.24 (95% CI 1.0-4.9), p=0.036) and cytoplasmic pARS578 (HR 4.54 (95% CI 2.0-10.4), p= < 0.001) was associated with decreased disease survival. Furthermore, high expression of cytoplasmic pARS578 was associated with decreased time to biochemical relapse (HR 2.1 (95% CI 1.0-4.2), p=0.034) and decreased disease-specific survival following biochemical relapse (HR 3.2 (95% CI 1.0-9.9), p=0.034). Dual expression of nuclear, cytoplasmic and total pARS81 and pARS578 were all associated with decreased-disease specific survival, suggesting that there is a sub-population of prostate cancer patients who may benefit from dual targeted therapy with androgen deprivation therapy and PKC inhibitors. A validation cohort of 243 hormone-naïve prostate cancer patients with all stages of disease was utilised to verify the results of the second cohort. Unfortunately, due to technical issues and time constraints, IHC could not be completed for the phosphorylation sites of interest in all patients. Despite this, high expression of cytoplasmic pARS578 was significantly associated with decreased time to biochemical relapse (HR 2.9 (95% CI 1.0-8.2), p=0.037) and trended towards an association with decreased overall survival (p=0.076). Interestingly, dual expression of high cytoplasmic pARS81 and cytoplasmic pARS578 was associated with decreased overall survival (HR 2.1 (95% CI 1.3-3.3) p=0.001) despite neither phosphorylation site independently predicting decreased overall survival. Lastly, a study to develop a technique for isolation, propagation and characterisation of primary prostate cancer cells from TRUS biopsy specimens was undertaken. Two primary prostate cell cultures were developed which were confirmed to have a malignant luminal epithelial cell phenotype with a functional AR using flow cytometry, RT-PCR and immunofluorescence. This technique is of high translational relevance, as it provides a model with potential to identify biomarkers to predict individual patient’s response to prostate cancer therapies. Overall these results suggest that androgen receptor phosphorylated at serine 81 and serine 578 are associated with poor outcomes in prostate cancer and are potential targets for new drug therapies. Additional studies are required to validate these results in a larger multi-centre cohort of prostate cancer patients before either of these phosphorylation sites can be utilised as a biomarker in clinical practice.

Enzymatic hydroxylation of varied cellular substrates is catalyzed by the 2-oxoglutarate and Fe(II) dependent 2-oxoglutrate (OG) oxygenase group of proteins. These enzymes control gene expression, from epigenetics to splicing and translation. The 2OG oxygenase JMJD4 has been shown to catalyse the hydroxylation of the eukaryotic omnipotent termination factor 1 (eRF1), and is essential for optimal translational termination. In this thesis, we expand on previous work by examining two further potential binding partners of JMJD4, GTF2I and TCP1-γ. Subsequently, we find that depletion of JMJD4 and eRF1 is associated with growth reduction in cancer cell lines in 2D and 3D. The transcriptomic changes in response to eRF1 depletion are then assessed by RNA-Seq. Among the potential pathways identified, downstream targets of the transcription factor ATF4 were most prominent. Upregulation of ATF4 and its downstream targets was validated in an eRF1 rescue system and the contribution of specific subdomains of eRF1 to the transcriptional response assessed, indicating multiple arms of the unfolded protein response being upregulated downstream of defective translational termination. The implications of our findings and their relevance in wider biological and disease contexts, including cancer, is finally discussed.

The aim of this project was the study of neutrophil recruitment and reverse transmigration using murine and human in vitro models of inflammation. Murine in vitro models of inflammation were developed using an immortalised microvascular cell line (MCEC-1) and primary murine vascular endothelial cells (mEC) isolated from heart and lung. We found that MCEC-1 could recruit murine neutrophils without the requirement of cytokine stimulation, although efficient transmigration did require such a stimulus. Primary cells required cytokine stimulation to recruit mEC. Interestingly, and in contrast to human EC, mEC were relatively insensitive to TNF-α stimulation, although IL-1β was a good stimulus for adhesion and migration. Using the IL-1β driven system we generated reverse migrated murine neutrophils and their phenotype and prolonged survival were assessed. The effect of shear stress and nitric oxide on the regulation of the process of reverse migration was examined. Using adoptive transfer strategies we investigated the fate of mRPMNs in vivo. A significant part of this work involved the study of human reverse migrated neutrophils at a proteomic level using two-Dimensional Fluorescence Gel Electrophoresis methodology to identify changes in neutrophils associated with reverse migration process. We found that murine reverse migrated neutrophils had a very similar surface phenotype to human reverse migrated cells. They also showed prolonged survival. However, our preliminary data on trafficking in vivo did not give a clear indication about their fate upon adoptive transfer into recipient mice. In vitro studies showed that flow generated shear stress and nitric oxide delayed, but did not inhibit, the process of reverse migration. Finally, the proteomics study revealed a number of metabolic, cytoskeletal and regulatory proteins that were differentially expressed in human reverse migrated neutrophils although the functional significance of these changes is yet to be explored.

No description available.

One of the main results of Rb-E2F pathway disruption is deregulation of the E2F family of transcription factors, which can lead to inappropriate proliferation, oncogenic transformation, or the induction of apoptosis. Given the potential negative biological effects associated with deregulated E2F activity, it is of great importance to study E2F targets that mediate these effects. In Part I of this manuscript, we identify the RhoBTB2 putative tumor suppressor gene as a direct physiological target of the E2F1 transcription factor. We find that RhoBTB2 is highly upregulated during mitosis due in part to E2F1, and that overexpression of RhoBTB2 increases the S-phase fraction and slows the rate of proliferation. We also find RhoBTB2 similarly upregulated during drug-induced apoptosis due primarily to E2F1 and that knockdown of RhoBTB2 expression via siRNA slows drug-induced apoptosis. Taken together, we describe RhoBTB2 as a novel direct target of E2F1 with roles in cell cycle and apoptosis. In Part II, we independently identify from cancer cell lines two novel variants from the promoter of E2F1 target MCL-1---MCL-1 +6 and +18---as initially published by Moshynska et al (1). In contrast to Moshynska et al., we find the variant promoters identically present in both cancerous and adjacent noncancerous clinical lung samples, suggesting that the variants are germ-line encoded. We also find the variant promoters prevalent in genomic DNA derived from healthy control samples and present at frequencies similar to that observed in cancerous cell lines. In further contrast, we find the activity of the MCL-1 +6 and +18 promoters approximately 50% less than the common MCL-1 +0 promoter---both during normal cellular homeostasis and under conditions that actively induce Mcl-1 transcription. Given our results and those of others, we conclude that the MCL-1 +6 and +18 promoters are likely benign polymorphisms and do no [sic] represent a reliable prognostic marker for CLL as reported by Moshynska et al.