Introduction: Pneumonia contributes significantly to the morbidity and mortality in stroke patients, especially those fed via nasogastric tubes. Methods: This project was conducted in two steps; 1. A randomised controlled trial: The efficacy of prokinetic agent metoclopramide was tested in a double-blind randomised controlled trial. Acute stroke patients with no pneumonia needing nasogastric feeds were randomized to 10 mg metoclopramide or placebo three times daily via the nasogastric tube for 21 days or until feeds discontinued. Participants were examined daily for clinical evidence of pneumonia. 2. A secondary analysis of data collected for the diagnosis of pneumonia. This was performed to identify early diagnostic markers of post-stroke pneumonia. Results: 1) For the MAPS study 60 patients (mean age 78 years, mean NIHSS 19) were randomized, 30 in each group. Pneumonia was diagnosed in 26/30 and 08/30 in placebo and treatment group respectively (p < 0.001). 2) Signs and symptoms of 47 radiologically confirmed pneumonia revealed that hypoxia, tachypnoea and inspiratory crackles were the most consistent signs in early pneumonia and pyrexia, cough, and purulent sputum were less commonly observed. CRP of 40 mg/l had the best predictive value for early diagnosis of post-stroke pneumonia. Conclusion: Treatment with metoclopramide treatment was associated with a significant reduction of aspiration and pneumonia. However, the incidence of pneumonia in the control gourp was very high and may have led to a false positive result. The findings of this study should therefore be confirmed in a larger study. A raised CRP of 40 mg/l or above was a senditive and specific diagnostic marker for post-stoke pneumonia.

One of the severe complications of a Plasmodium falciparum infection is cerebral malaria (CM). CM is characterised by the accumulation of mature infected red blood cells (RBC) in the brain microvasculature. One of the consistent detrimental effects of sequestration is the breakdown of the blood-brain barrier (BBB), often with a fatal outcome in children in endemic areas. This study investigates the mechanisms underlying BBB breakdown secondary to sequestration, using immortalised human brain microvascular endothelial cells (tHBEC) as an in-vitro model of BBB and ITG-strain Plasmodium falciparum. First, the tHBEC monolayer was co-cultured with Plasmodium falciparum infected red blood cell (PRBC) or uninfected red blood cells (uRBC) control for 20 hours and the supernatant was recovered for subsequent analysis. The co-culture supernatants showed upregulation of inflammatory mediators (MCP-1 and IL-8) and a member of metalloproteases (ADAMTS-1, ADAMTS-4, MMP-2 and MMP-9) in the PRBC-tHBEC co-culture supernatants. The PRBC-tHBEC co-culture supernatants induced loss of endothelial cell monolayer integrity, represented by real time reduction in the transendothelial electrical resistance, measured using Electrical Cell-Substrate Impedance Sensing (ECIS™). The same supernatants also increased the permeability of tHBEC monolayer to the fluorescently labelled 40 kDa dextran showing leakage across the tHBEC monolayer. Interestingly, the loss of barrier function of tHBEC monolayer is partially inhibited by the addition of protease inhibitors GM6001 and rhTIMP-3. Prolonged exposure to PRBC-tHBEC co-culture supernatants reduced the level of vinculin. This study demonstrates that the interactions between PRBC and tHBEC induces activation of tHBEC and the release of proteases that contribute to BBB breakdown in CM, and could be a potential drug target for adjunct therapy in CM.

The immune system, and in particular CD8+ T cells, have the potential to eliminate tumours and novel immunotherapies are giving encouraging results for cancer patients. This thesis focuses on the improvement of one such therapy, adoptive T cell therapy. The aim of this thesis was to genetically modify human CD8+ T cells to enhance their anti-tumour potential for adoptive T cell therapy. One approach was to mutate the SH2 domain containing protein tyrosine phosphatase (SHP-1) gene to reduce T cell checkpoint inhibition. A second approach was to express a shedding-resistant form of L-selectin in T cells to promote T cell homing. In this thesis, the rapid shedding of L-selectin within minutes after TCR stimulation was shown to be ADAM17 dependent. Furthermore, new data indicated a second proteolytic cleavage which is γ-secretase dependent. The shedding-resistant L-selectin (ΔM-N) was shown to resist proteolysis by both ADAM17 and γ-secretase. Previous work by our group and others has shown that the loss of SHP-1 leads to the enhanced entrance of T cells into proliferation and gives better protection against tumour growth in mice. To study the effect of SHP-1 loss in human CD8+ T cells, SHP-1 deficient tumour-specific T cells were generated by lentiviral delivery of a pair of SHP-1 specific zinc finger nucleases in conjunction with the Mel TCR. Populations of melanoma-specific CD8+ T cells containing a small fraction (< 8 %) of SHP-1 mutant cells showed no enhanced Mel526 target cell killing in vitro or in vivo in Mel526 xenograft bearing NSG mice. However, there was some suggestion of enhanced survival and/or proliferation in vitro of melanoma-specific CD8+ T cells containing a population of SHP-1 CD8+ mutant cells. Overall the proof of principle of gene-editing SHP-1 in T cell lines and primary CD8+ T cells was demonstrated which supports further exploitation of this approach for testing in adoptive T cell therapy strategies.

The prevalence of type 2 diabetes is increasing in the UK. Many people with type 2 diabetes require glucose-lowering therapy including insulin when lifestyle interventions fail to provide adequate glucose control. Some epidemiological studies report an association between insulin use in type 2 diabetes and an increased risk of serious adverse events when compared with other glucose-lowering therapies. However, these findings should be interpreted with caution due to the risk of confounding by indication. The aim of this thesis was to characterise the epidemiology of type 2 diabetes and to investigate the risk of serious adverse events associated with increasing insulin dose in people with type 2 diabetes prescribed insulin therapy. A series of retrospective, observational studies were conducted using data from the Clinical Practice Research Datalink. People with type 2 diabetes were identified and prevalence and incidence rates calculated. The risk of all-cause mortality, major cardiovascular events and cancer in people with type 2 diabetes who progressed to insulin with or without metformin were evaluated using multivariate models. Between 1991 and 2010, the estimated incidence and prevalence of clinically diagnosed and recorded type 2 diabetes increased three-fold. During the same period, the estimated number of people with diagnosed and recorded type 2 diabetes treated with insulin increased seven-fold. Estimated insulin dose was associated with an increased risk of all-cause mortality in people with type 2 diabetes receiving insulin monotherapy (aHR 1.54, 95% CI 1.32–1.78, for 1 unit/kg/day increase in insulin dose) and in those treated with insulin with or without metformin (1.48, 1.31–1.70). However, the use of metformin in combination with insulin was associated with a reduction in risk compared with insulin alone (0.60, 0.52–0.68). Due to the limitations associated with observational studies, further research is required in order to improve our understanding of the risks and benefits of exogenous insulin in type 2 diabetes.

Background: The HOPE trial showed that ramipril reduced cardiovascular morbidity and mortality in patients with peripheral arterial disease (PAD). However, evidence regarding the effect of angiotensin converting enzyme (ACE) inhibitors on walking distance, ankle brachial pressure index (ABPI), arterial stiffness and quality of life (QoL) in this group of patients is limited. Objective: The aim of this study is to investigate ACE inhibitors effect on clinical parameters of PAD, arterial stiffness and QoL in patients with intermittent claudication (IC). Methods: 33 patients (25 males, mean age: 65+/-7.8) with IC (Fontaine stage II or higher) were randomised to receive ramipril (5 mg once daily for 2 weeks increased to 10 mg once daily for 22 weeks, n=14) or placebo (n=19) for 24 weeks in a double-blind study. Walking distance was assessed using a standard laboratory treadmill test (2.5 km/hr at 10% incline). ABPI was assessed pre (r-ABPI) and post-exercise (t-ABPI). Arterial stiffness indices were measured using the SphygmoCor device. Results: After 24 weeks, ramipril improved maximum treadmill walking distance; adjusted mean change difference (95% confidence interval); by 130.5 (61.8 to 199.2) m longer than placebo (P=0.001), improved treadmill intermittent claudication distance by 121.9 (55.9 to 187.8) m longer than placebo (P=0.001) and improved patient reported walking distance by 159 (5.5 to 313) m compared to placebo (P=0.040). Ramipril reduced carotid femoral pulse wave velocity (a measure of arterial stiffness) by -1.47 (-2.4 to -0.57) m/s compared to placebo (P=0.002). However, r-ABPI and t-ABPI minimally changed in both groups (Ramipril 0.02 (-0.08 to 0.11)) vs. placebo 0.03 (-0.05 to 0.10, P=0.830) and (Ramipril 0.04 (-0.04 to 0.12)) vs. placebo 0.02 (-0.04 to 0.09, P=0.720), respectively. Ramipril had a slight insignificant effect on QoL physical domains compared to placebo. Conclusion: Ramipril improves walking distance in patients with IC; however, this improvement is not related to improvement in ABPI but might be due to ramipril ability to reduce arterial stiffness. ACE inhibitors effect on QoL needs to be validated in a larger randomised controlled trial.

This mixed method research study originally contributes to understand the role covered by Supported Employment Agencies (SEAs) in promoting positive health behaviours of employees with learning disabilities. Employment is rarely experienced by people with learning disabilities. Supported Employment Agencies provide a service to facilitate the employment experience for people with learning disabilities, through supporting in finding, keeping and maintaining a job. While many studies on employment have highlighted health benefits for the general population in employment, data is lacking for people with learning disabilities. This study wants to understand if and how Supported Employment Agencies support the health of their clients with learning disabilities. The quantitative phase of this study involved managers of Supported Employment Agencies completing a web-survey to understand the strategies used by Supported Employment Agencies preventing health risk behaviours and promoting health. In the qualitative phase of this study Grounded Theory Method was used to discover the role played by Supported Employment Agencies in supporting the health of their clients with learning disabilities. For this purpose interviews with mangers and job coaches of Supported Employment Agencies and interviews with employees with learning disabilities were held. Results from this study reveal Supported Employment Agencies to influence the health of employees with learning disabilities in several ways, both informally and formally. Indeed, health was a key element in all phases of supported employment, even if Supported Employment Agencies were not formally committed and funded to promote health. The thesis highlights the potential for SEAs to capitalise on their role as employment mediators to promote health outcomes and healthy lifestyles for employees with learning disabilities.

This thesis was an observational study to retrospectively examine the prevalence and incidence of DR and its associated risk factors from two screening services (Wales and Johannesburg, South Africa) in order to determine safe screening intervals in persons without evidence of DR at initial screening based on digital photography. Between 2005 and 2009 a total of 135,152 persons with diabetes over 12 years of age in Wales were screened. However, a total of 43,759 persons were excluded from analysis as they did not have their type of diabetes recorded (29,807) or where it was recorded it was outside of the pre-specified age at diagnosis of diabetes range of ≥30 years for type 2 DM and <30 years for type 1 DM (13,952). In the Centre for Diabetes and Endocrinology, Johannesburg, South Africa a smaller population of 5,565 were screened between 2001-2010. A total of 50 persons were excluded from this analysis as they had a type of diabetes recorded other than type 1 DM or type 2 DM. Therefore, data from 91,393 (86,390 T2DM, 5,003 T1DM) persons from the Wales screening service and 5,515 (3,978 T2DM, 1,537 T1DM from South Africa, were analysed. In Wales, the prevalence of any DR was 31.0%, background DR (BDR) 26.6% and referable DR (RDR) 4.4% in T2DM at baseline. The prevalence was higher in T1DM at 56.2%, 39.8% and 16.4% respectively. Increased duration of diabetes was independently associated with increased prevalence and incidence of any DR, BDR and RDR for T2DM and T1DM as well as treatment modality in T2DM. The four year cumulative incidence of RDR was 1.6% for T2DM and 5.6% for T1DM. At the Centre for Diabetes and Endocrinology Johannesburg the prevalence of any DR was 21.6%, BDR 14.8% and RDR 6.7% in T2DM at first screening and higher at 36.9%, 27.2% and 9.7% respectively for T1DM. Glycaemic control (HbA1c) and duration of diabetes were significantly associated with the prevalence and incidence of any DR, BDR and RDR in both T2DM and T1DM. Ethnicity and hypertension were also risk factors. The seven year cumulative incidence of RDR was 4.7% for T2DM and 5.0% for T1DM. Risk factor analysis indicated that the screening intervals could be extended beyond annual based on type of diabetes, duration of diabetes, HbA1c, ethnicity, hypertension and treatment modality in T2DM. This analysis adds to an increasing evidence base for considering extending the screening intervals beyond annually for those at low risk with no DR at initial screening. Limitations of this study included the need to exclude a high number of persons in order to ensure the quality of the data; the numbers lost to follow up (meaning all results from 3 years should be interpreted with caution) and the fact that the visual impairment and blindness within the programmes could not necessarily all be attributed to DR as recordings of other lesions were not included in the datasets.

The individual, familial, societal and economic impact of psychological distress following stroke is well established. Nevertheless, validated treatments to reduce psychological distress and enhance well-being in community-living stroke survivors is limited. Recently, Acceptance and Commitment Therapy (ACT) has demonstrated promising results for improving psychological wellbeing in a variety of presentations. However, there is a lack of research exploring ACT with adults who have survived a stroke. The current thesis examines the efficacy of a four-session ACT intervention that was delivered to both stroke survivors and their carers via a third-sector charity organisation. A quasi-experimental design was used with 69 participants assigned to either the ACT intervention or waiting list control group. Outcome variables captured levels of psychological distress, post-traumatic growth and quality of life at three time points (pre-intervention, post-intervention and two month follow-up) and were analysed using linear regression whilst controlling for baseline levels. A mediational analysis examining specific ACT processes – psychological flexibility and goal directed thinking - were also examined. Additionally, a one-hour focus group including seven individuals who completed the group was analysed by inductive thematic analysis to gain greater insight in to the personal experiences of the group. Group assignment predicted lower psychological distress in favour of the ACT group at post-intervention and at follow-up. There were no significant differences found for post-traumatic growth or quality of life. The mediational analyses suggest that the ACT intervention did not significantly alter levels of psychological flexibility, or goal directed thinking compared to the waiting list control group, and these measures did not appear to mediate the changes in psychological distress. The qualitative analysis supported the positive gains of the ACT group. The results are discussed in terms of developing ACT intervention for stroke survivors and carers.

Bleeding following cardiac surgery requiring cardiopulmonary bypass (CPB) is associated with increased morbidity. Identification of patients at increased risk of bleeding might allow intervention to prevent bleeding developing. In this thesis, clotting factors, anticoagulants and calibrated automated thrombin generation were investigated as potential methods for identifying such patients. Post-CPB FXIII, fibrinogen and platelet count were significantly lower in those who bleed more than 2 mL/kg/hr for two consecutive hours and in those who bleed in excess of 1 litre at 24 hours. ROC analysis demonstrated these had modest predictive value. Calibrated automated thrombography was unable to identify patients at risk of bleeding. Calibrated automated thrombography was also used to investigate the effects of haemostatic treatment (FFP, rFVIIa, PCC and TFPI inhibition) on thrombin generation in vitro. Blocking the effect of TFPI produced the greatest improvement in thrombin generation. The effect of CPB on platelet phospholipids was investigated using mass spectrometry. Post-CPB the ability to externalise phosphatidylethanolamine and phosphatidylserine was impaired. The ability to externalise and synthesise 12-HETE-PC and 12-HETE-PE in response to both thrombin and collagen post-CPB was also reduced. The effect of these phospholipids on thrombin generation and the ability to identify patients at risk of bleeding was then investigated. Thrombin generation using liposomes containing 12-HETE-PC or 12-HETE was lower in patients who required haemostatic treatment for post-CPB bleeding compared to those who did not. This suggests there are variations between individuals in the way their coagulation factors interact with oxidised phospholipids and that this may influence bleeding. Finally a cell based model of thrombin generation was developed using monocytes as a source of tissue factor and incorporating the observed changes in phospholipids, clotting factors and anticoagulants. This model provides a basis to further investigate the influence of different TF expressing cells on thrombin generation which may affect bleeding.

Hirschsprung’s disease causes a life-threatening bowel obstruction in children shortly after birth, because the stem cells that will form the enteric nervous system (ENS) have failed to colonise the entire length of the gut, creating an absence of neuronal cell bodies – the aganglionic region. Although there are numerous surgical techniques to deal with this problem, many children go on to have severe problems with constipation and faecal incontinence, and some require a permanent colostomy. It was hypothesised that clinical outcomes in children with Hirschsprung’s disease could be improved by transplanting ENS stem cells into the defective bowel, as previous studies has already shown it was possible to produce such stem cells from embryonic bowel . The aim of this thesis was to develop a method of isolating such cells from the bowel of children with and without Hirschsprung’s disease, characterise these cells and investigate whether they could be successfully transplanted in an in vitro model. In the first part of this thesis, a method was developed to take samples of postnatal bowel (both small and large) and dissociate it to produce a cell suspension that will allow ENS stem (or progenitor) cells to grow in the form of neurospheres. These neurospheres are characterised in terms of growth and composition, and it is shown that they contain reproducing cells that are capable of forming neurons on subsequent differentiation. When the neurospheres are transplanted into aganglionic embryonic mouse bowel grown in tissue culture, neurons and glia from the neurosphere colonise the bowel wall. In the second part of this thesis, the neurospheres were further characterised in terms of their response to the ENS growth factors glial cell derived neurotrophic factor (GDNF) and endothelin 3 (EDN3) and this was compared to the response of neurospheres derived from embryonic and neonatal mice. It was shown that postnatal human ENS neurospheres require the addition of GDNF to produce significant neurite outgrowth. Subsequent studies then demonstrated that the human neurospheres could be dissociated and regrown in culture, exponentially increasing their numbers whilst retaining the ability to produce neurons and glia on transplantation into aganglionic embryonic hindgut. In the final part of this thesis the functional effect of neurosphere transplantation into aganglionic embryonic hindgut was investigated. Firstly it was shown that the transplanted neurons integrate closely with the recipient smooth muscle and gut pacemaker cells (the interstitial cells of Cajal). Secondly it was shown that aganglionic embryonic mouse hindgut has a higher rate of contraction than ganglionic bowel cultured in the same conditions, and that human and mouse ENS neurosphere transplantation restores the contraction frequency of aganglionic bowel to that of ganglionic bowel. Taken together the results presented in this thesis demonstrate that it is possible to generate neurospheres from postnatal human bowel samples that contain ENS stem cells which are capable of producing a restorative functional effect when transplanted into an in vitro model of Hirschsprung’s disease. This is a step toward the development of a successful stem cell treatment for this condition.