The objective of this research is to investigate the role of ultrasound-guided foam sclerotherapy (UGFS) in the treatment of chronic venous disease (CVD). UGFS was found to be a safe and effective treatment for both primary and recurrent great saphenous vein (GSV) and small saphenous vein (SSV) incompetence, assessed by occlusion of treated veins on duplex ultrasound (DUS), and by disappearance of visible varicose veins (VV) on clinical examination. There was some evidence that healing of chronic venous ulcers (CVU) may be improved by UGFS when combined with compression bandaging. When compared with patients undergoing superficial venous surgery (SVS), UGFS was associated with significantly less pain, bruising and analgesia requirement, and a quicker return to work and driving. Significant improvements in both generic physical and disease-specific health-related quality of life (HRQL)were observed following UGFS, and were sustained for 12 months after treatment. UGFS significantly improved lower limb physical symptoms (pain, itching, restlessness, swelling, heaviness, cramp and tingling), cosmetic appearance, and provided life-style benefits in the majority of patients. Furthermore, the great majority of patients who expected such benefits had their expectations met or exceeded.

This thesis explores the ethics of conditional and directed deceased organ donation. It uses an empirical bioethics approach that uses empirical data to inform and enhance philosophical analysis. An initial philosophical analysis of the key ethical considerations was undertaken, and it is argued that the policy prohibiting most conditional and directed donations is wrong. The concept of altruism, in particular, is poorly conceived and applied in transplantation policy. Qualitative data obtained by interviewing relevant stakeholders are presented. The data suggest that although there are concerns about the consequences of accepting conditional and directed donations, many participants thought these donations should be accepted in some circumstances. The data also provide lines of argument against conditional and directed donations, and these are considered. Using this data, and making some reasonable assumptions, it is argued that it is better to accept conditional and directed donations than it is to reject them. The thesis culminates with 8 recommendations for policy regarding conditional and directed donations, and argues that a trial period of accepting these donations should be implemented so that the effects can be accurately observed.

The costimulatory receptor CD28 and its inhibitory counterpart CTLA-4 share the same ligands and comprise a crucial checkpoint in T cell activation. CTLA-4 removes its ligands from antigen presenting cells by trans-endocytosis, which reduces the availability of costimulatory ligands for CD28 engagement to regulate T cell activation. This project examined the functional implications of reducing the availability of costimulatory molecules for CD4 T cell responses, and investigated the use of trans-endocytosis by other T cell receptors. Surprisingly, it was revealed that PD-1 and OX40 can also internalise their ligands, although perhaps not via the same mechanism as CTLA-4 trans-endocytosis. It was also shown that altering the availability of CD28 ligands affects the extent of T cell proliferation, suggesting that CTLA-4 trans-endocytosis can finely tune the T cell response. Furthermore, it was observed that CD28 costimulation is not always required for T cell activation and proliferation, but CD28 engagement is required for the optimal upregulation of a number of effector proteins and for TH2 cytokine production. Interestingly, T cells activated in the absence of CD28 signalling were not classically anergic. Strikingly, it was also found that memory T cells are dependent on CD28 costimulation.

Mental retardation (MR) affects 23% of the population; those due to X linked mutations commonly result in moderate to severe MR. The OPHN1 gene (Ophn1 in mice) has been implicated in X linked mental retardation (XLMR) and encodes the RhoGAP protein, oligophrenin 1. Loss of function mutations alter Rho GTPase dependent signalling pathways and result in altered actin cytoskeleton dynamics which are important in dendritic spine structure, the site of neurotransmission. Here, using electrophysiological recordings combined with intracellular staining techniques and dendritic morphological analysis, I characterise synaptic (dys)function in neocortical and hippocampal neurons from the Ophn1 mouse model of MR. This study demonstrates an excitatory synaptic deficit in neocortical neurons combined with region specific changes in dendritic spine morphology. Inhibitory transmission was normal in both neocortical and hippocampal neurons. Kainate induced gamma oscillations were unaltered whereas spontaneous oscillations were reduced which lead to changes in synaptic function in CA3. Morphometric analysis showed ventriculomegaly in Ophn1 deficient mice that was associated with reduced cortical thickness. This study shows the loss of several previously reported phenotypes, including, altered inhibitory transmission, gamma oscillations and vesicle dynamics. Their loss, but preservation of morphological deficits, suggests that the model may be susceptible to genetic drift.

Wolfram Syndrome (WS) is a rare autosomal recessive disease characterised by insulin-dependent diabetes mellitus, optic nerve atrophy. The current study demonstrated that neuronal cells depleted of WFS1 protein, showed significantly elevated expression of the ER stress markers indicating an enhanced ER stress response. This was accompanied by increased apoptosis and impaired cell cycle progression. WFS1 depletion also resulted in decreased expression of Na+/K+ ATPase beta1 subunit and Vacuolar ATPase (V-ATPase) V1A subunit. The elevated expressions of ER stress markers, but not the decreased expression of pump subunits, were reversed by adenoviral over-expression of BiP/GRP78. Protein degradation assays showed more rapid degradation of both pump subunits in WFS1 depleted cells compared with controls. A novel interaction between WFS1 and the V1A subunit of the vacuolar-type H+-ATPase (proton pump) was demonstrated by co-immunoprecipitation in overexpression system and with endogenous proteins. The interaction was mapped to the WFS1-N terminal, but not the C-terminal domain. In conclusion, this study shows that WFS1 has a specific interaction with the V1A subunit of H+ V-ATPase. Chemical and molecular chaperones TUDCA, GRP78 and the histone deacetylase inhibitor VPA showed promising results in providing protection against ER stress induced apoptosis in WFS1 depleted neuronal cells.

The role of post translational histone modifications in stem cells has been of increasing interest in recent years, however, the heritability of histone modifications has not yet been determined, and as such their status as epigenetic remains in question. Here we have taken the novel approach of comparing the enrichment of histone modifications, across specific genes and how they are modulated through various phases of the cell cycle: in doing so we address this question of heritability from a new perspective. Highly dynamic fluctuations in the enrichment of histone modifications were observed across the cell cycle in embryonic stem cells. In cell cycle regulated genes the patterns of modification enrichment revealed an increase in active marks either pre-emptive or at the point of expression, indicative of highly dynamic regulation, not a stable heritable transmission, perhaps reflective of the plasticity of these cells. Following on from this embryonic stem cells were differentiated for seven days, allowing the enforcement of canonical cell cycle regulation and a more lineage specific transcription profile. At this point histone modifications displayed a variety of patterns including what appeared to be the stable and presumably heritable transmission of H3K4me3 and H3K27me3 across the cell cycle.

Non Cystic Fibrosis bronchiectasis is characterised by perpetual neutrophilic inflammation in the lungs. The ongoing vicious cycle of bronchiectasis leads to further damage to already damaged airways and is a culmination of repeated infection, inflammation and failure of the host response to maintain sterility of the airway, despite a sophisticated innate and adaptive immune system. Pseudomonas aeruginosa commonly colonises the lungs of patients with bronchiectasis. I hypothesised that the concept of inhibitory antibodies in the serum may be a feature of Pseudomonas aeruginosa colonisation through a specific interaction between the host adaptive immune system and strain specific features. Here I have identified a mechanism where some patients colonised with Pseudomonas aeruginosa produce IgG2 antibodies specifically against the O antigen of bacterial LPS, which rather than promote complement-mediated killing actually inhibits it.

Traumatic haemorrhagic shock (THS) is the most frequent cause of preventable death after severe injury. Shock is characterised by inadequate provision of oxygen and substrates to tissues in relation to their requirements, and it is within the microcirculation that this process is regulated. Investigation of the microcirculation is therefore key to understanding the pathological processes following THS. In Part I, some mechanisms of microcirculatory dysfunction following trauma are presented. Endotheliopathy of trauma is associated with poor microcirculatory flow, and occurs within minutes of injury. It is also associated with higher levels of circulating cell-free DNA (cfDNA), supporting the hypothesis that cfDNA is an aetiological factor in this pathological response. Both endotheliopathy and elevated cfDNA and are related to poor clinical outcomes. In Part II, clinical implications of microcirculatory monitoring are discussed for patients in the early phase following THS. It is safe and feasible to monitor the microcirculation following THS, and a novel point-of-care grading system has performed well, suggesting that targeted fluid resuscitation towards microcirculatory flow after THS may be possible. The optimal fluid strategy in this context is unknown, but physical properties (e.g. oncotic potential and viscosity) as well as endothelial restorative properties appear to be as important as oxygen-carrying capacity. Potential therapeutic interventions aimed at microcirculatory and endothelial resuscitation open intriguing possibilities for improving outcomes after THS.

B cells have been proven to have a significant role in liver fibrosis. We postulate that enrichment of B cell subsets in hepatic diseases may implicate this population in liver pathogenesis. When comparing total B cells from human immune and non-immune-mediated liver disease explants, we found an enrichment of CD20+ B cells in PBC. Furthermore, phenotypic characterization of 11 B cell subsets in matched liver and blood highlighted an enriched naïve peripheral population, and activated B cell subsets in livers. Newly identified CD19+CD24-CD38- and CD19+CD24-CD38int B cells were also augmented in livers compared to matched blood. Furthermore, CD24-CD38- B cells were elevated in PBC and formed aggregates in tissues, whereas CD24-CD38int B cells localized around bile ducts and along fibrotic tracts in PBC. CD24-CD38int B cells secreted pro-inflammatory (IL-6, IFN-γ) and immunosuppressive (IL-10) cytokines following stimulation with CpG compared to other B cell subsets, implying that CD24- B cells may play a role in liver disease pathogenesis. Our findings suggest that B cells may be influential in hepatic disease progression and pathogenesis. Elucidating their role further could provide possible therapeutic targets for prevention or treatment of chronic liver disease.

Late kidney transplant losses along with chronic kidney disease in non-renal solid organ transplantation pose major problem in this field. To address this I compared ischaemia, interstitial scarring and inflammation on the renal biopsies from failing renal transplants and chronic kidney disease outside renal transplantation using native CKD as a control group. For matched renal function, a similar degree of interstitial scarring was observed across the three study groups. Ischaemia was predominant in failing renal transplants; conversely inflammation was predominant in native CKD. The relationship between macrophages and endothelial cells indicated that, despite a lower macrophage load, there may be an allogeneic impact of macrophages on endothelial cells. In the biopsies from CKD, mRNA levels for iNOS, arginase, CX3CL1, BCL-2, MCP-1/CCL2 and FSP-1 were increased in association with an increasing macrophage load. Macrophage load correlated positively with arginase/iNOS mRNA ratio, suggesting M2 phenotypic transformation of the macrophages. Co-localization studies, demonstrated an increased number of macrophages within 5 microns from endothelial cells in failing renal transplants when compared to chronic kidney disease. Further studies directed towards manipulating macrophage-endothelial cell interaction may be potentially beneficial in improving the longevity of renal transplants.