Haematopoietic stem cell transplantation (HSCT) is currently the only curative treatment for many patients with malignant and non-malignant haematological diseases. However, its success is greatly reduced by the development of complications, including graft-versus-host disease (GVHD), infection and relapse. Human leukocyte antigen (HLA)-matching of patients and donors is essential in HSCT, but does not completely prevent such complications. Thus, it is extremely likely that non-HLA genes also have an impact. Naturally occurring polymorphisms within non-HLA genes have been suggested to contribute to some of the genetic disparity that exists between transplant patients and donors, consequently, they may have the potential to influence HSCT outcome. As a result, this study investigated the significance of non-HLA polymorphisms in HSCT. Polymorphisms in the genes of interleukin (IL) 2 (IL2, -330 T/G), 4 (IL4, -590 C/T), 6 (IL6, -174 G/C) and 10 (IL10, -592 A/C, -1082 G/A), IL-1 receptor antagonist (IL-1Ra (IL1RN), intron 2 VNTR), tumour necrosis factor α (TNFα (TNFA), intron 3 (GA)n), TNF receptor II (TNFRII (TNFRSF1B), -196 M/R), interferon γ (IFNγ (IFNG), intron 1 (CA)n), vitamin D receptor (VDR, intron 8 A/C, exon 9 T/C) and oestrogen receptor (ESR1, intron 1 A/G, intron 1 C/T) have been studied extensively in HLA-matched sibling transplantation, however, relatively little is known about their role in unrelated donor HSCT. This study therefore examined these polymorphisms in the unrelated donor setting. VDR intron 8 A/C, IFNG intron 1 (CA)n and IL6 -174 G/C were found to associate with GVHD in our cohort. The VDR intron 8 A,A and IFNG intron 1 3,3 genotypes correlated with severe acute GVHD (grades III-IV), whilst the IL6 -174 G,G genotype correlated with chronic GVHD. No genetic associations were demonstrated with any other clinical outcome. This study also examined the +2044 G/A polymorphism in the gene encoding IL-13 (IL13), an immunoregulatory cytokine that has been implicated in GVHD. This polymorphism has been widely studied for disease associations; however, its role in HLA-matched sibling and unrelated donor HSCT is currently unknown. In our transplant cohort the IL13 +2044 A allele was found to correlate significantly with both acute and chronic GVHD. Higher IL-13 expression was also observed in patients with these conditions, although this altered expression could not be directly attributed to IL13 +2044 G/A. The prognostic significance of pre-transplant IL-13 serum levels was also investigated. No associations were observed between IL-13 expression and GVHD. Elevated pre-transplant IL-13 serum levels did, however, correlate with post-transplant relapse and Hodgkin disease in two independent cohorts. Again, this altered expression could not be directly attributed to the IL13 +2044 G/A polymorphism. This study clearly demonstrates that non-HLA polymorphisms influence the outcome of both HLA-matched sibling and unrelated donor HSCT. Thus, in the future, genotyping with respect to a panel of non-HLA polymorphisms may be used to complement HLA typing, increase the ability to predict the risk of transplant complications and allow post-transplant treatment strategies to be tailored to an individual.

Kidney transplantation is the gold standard renal replacement therapy for patients with end-stage renal disease. Despite advances in immunosuppressive therapy, chronic allograft dysfunction remains the commonest cause of renal allograft failure in living recipients. The typical pathology of this disease includes chronic inflammation with tubular atrophy and interstitial fibrosis. Although the origin of the excess fibroblasts and myofibroblasts remains controversial, the process of epithelial to mesenchymal transition might play a role. This study was designed to test the linked hypotheses that the immunosuppressive drug, Cyclosporine A and graft infiltrating T cells can induce allograft pathology by alteration of the bioavailability of fibrogenic TGF-β. An initial series of experiments examined the induction of mesenchymal transition by treatment of cultured human renal tubular epithelial cells with immunosuppressive concentrations of Cyclosporine A. Drug treated cells showed characteristic morphological changes associated with increased expression of the mesenchymal marker S100A4 and reduced expression of the epithelial marker E-cadherin; similar changes were induced by the addition of TGF-β1. The phenotypic change induced by Cyclosporine A was not the consequence of an increased response to autocrine TGF-β and could not be inhibited by specific blockade of the ALK5 component of the TGF-β receptor. Further studies showed in vitro that contact between activated T cells and renal tubular epithelial cells could induce mesenchymal transition by a mechanism that was dependent on activation of the TGF-β receptor complex. A final series of experiments defined a mechanism by which T cells activate latent TGF-β allowing subsequent receptor stimulation leading to either T cell or epithelial cells differentiation. The latency associated peptide binds to and inhibits native TGF-β but can be displaced by both thrombospondin-1 and neuropilin-1, producing active TGF-β. In this study it was shown that cytoplasmic thrombospondin-1 is exported and expressed on the surface of activated T cells following brief interaction with renal tubular epithelial cells; neuropilin-1 was also expressed by a mean 18% of activated human T cells. Inhibition of these two molecules with a blocking LSKL peptide sequence inhibited the normal response of activated human T cells to latent TGF-β1. This study demonstrated that both Cyclosporine A and T cells can induce renal epithelial to mesenchymal cell transition. However, the former process seems independent of TGF-β whilst the latter requires TGF-β receptor stimulation and might be regulated in vivo by T cell-mediated activation of latent TGF-β within the allograft.

Aims: The current study had the following aims: 1. Establish an effective pulsatile human cadaver pulsatile flow model (PHCM). 2. Explore the acceptability of PHCM 3. Assess the face and construct validity of PHCM. 4. Compare the effectiveness and transferability of endovascular skills taught on PHCM versus a virtual reality simulator (VRS). 5. Examine the role of video-enhanced feedback during technical skills training. Methods: 1. Cadaveric experiments were conducted at a licensed research facility: Newcastle Surgical Training Centre (NSTC). 2. Structured questionnaires were used to explore public and professional opinion. 3. Face and construct validity were assessed in a standard manner using practitioners of varying levels of experience. 4. Novice candidates were recruited and completed the same training regime on PHCM or VRS before crossing over onto the alternate model to compare the effectiveness of PMCH and transferability of endovascular skills. All performances were recorded and scored by two blinded experts using a validated clinical scoring tool. 5. Novice candidates were assessed performing a basic suturing exercise before and after varying forms of feedback (including video enhanced feedback). Results: 1. A PHCM was successfully created. 2. Patients and professionals support cadaveric endovascular training but expressed some reservations over its feasibility. 3. Expert practitioners confirmed the models face validity. PHCM has construct validity in differentiating between novice candidates and both intermediate (p=0.000)* level and expert (p=0.000) practitioners (improved overall procedure score (OPS)). 4. PHCM training improved candidate’s quantitative parameters (Time p=0.000, Fluoroscopy p=0.026, Contrast p=0.008) and clinical performance scores (p=0.000)*. Both PHCM and VRS demonstrated transferability of basic endovascular skills. 5. Video feedback is superior to a structured lecture (OPS) and individualized feedback was not superior to unsupervised video-enhanced feedback (p=1.000*). Conclusion: PHCM is a feasible, valid and effective model for training basic endovascular skills. The role of unsupervised video feedback could further enhance technical skills training and warrants further investigation.

Background: Current management of patients presenting with acute coronary syndrome (ACS) includes aggressive and expeditious revascularisation, including surgical revascularisation. However, early surgery following ACS is associated with high mortality, as subsequent global ischaemia induced during surgery is imposed on infarcted myocardium. Emerging evidence suggests that matrix metalloproteinases (MMPs), especially MMP2, may have an important role in the acute myocardial dysfunction seen after global ischaemia-reperfusion injury, by targeting intracellular functional and structural proteins. Aims: To investigate whether MMP2 has a causative role in heart dysfunction when previously infarcted hearts were subjected to further global ischaemia-reperfusion, as occurs in cardiac surgery, with and without cardioplegic protection. Methods and Results: MI was surgically induced in male Wistar rats, (250-350 g body weight) by in vivo ligation of the left anterior descending artery. The animals were recovered for 7 days prior to excision of hearts and isolated Langendorff perfusion, followed by induction of further global ischaemia and reperfusion. The recovery of mechanical function (left ventricular developed pressure: LVDP) of the heart was assessed during reperfusion. MMP2 activity was also measured during the early reperfusion phase in the heart tissues. Infarcted hearts had less capacity to recover function after an additional period of global ischaemia, which was associated with higher MMP2 activity in the infarcted hearts compared to normal hearts. Inhibition of MMP2 improved recovery of function. When an MMP inhibitor was used as an adjunct to St Thomas’ Hospital cardioplegia, there was a trend towards improved recovery if the inhibitor was present before, during and after ischaemia. Conclusion: MMP2 has a role in causing cardiac dysfunction when infarcted hearts were subjected to further global ischaemia-reperfusion. Inhibition of MMP2 resulted in improved recovery of the function of the hearts during reperfusion. With cardioplegia, MMP2 inhibition before, during and after ischaemia was crucial to improve cardioprotection during early reperfusion.

Rationale: Coronary artery disease (CAD) is a complex phenotype with multiple genetic and environmental risk factors. The circulating monocyte plays a key role in CAD and contributes to atherogenesis, plaque progression and atherothrombosis, especially through its interactions with platelets. This study tested the hypothesis that gene expression profiling of monocytes in a resting state, and following platelet-mediated stimulation would identify novel molecules that may determine the inherited risk of CAD. Methods: Four groups of subjects were recruited: patients with a premature MI (PMI) <65 years (n=19) and age/gender matched healthy controls (n=19), healthy young men with a strong family history of PMI (n=22) and matched controls with no significant family history of CAD (n=17). Monocyte RNA was extracted before and after platelet-mediated stimulation (for 4 hours) from all subjects for whole genome microarray analysis. Differentially expressed genes were validated by QPCR and those genes with similar trends in expression in the PMI patients and the healthy young men with a family history of PMI were selected for further analysis. These were tested in silico in CARDIoGRAM, a large scale genome wide association study (GWAS) to identify genetic variants that showed either strong associations with CAD or with gene expression in monocytes (expression quantitative trait locus - eQTL). Results: This work revealed similar trends in differential expression of specific monocyte genes between PMI patients and healthy men with a strong genetic risk of PMI compared with their respective healthy controls. These include genes implicated in lipid metabolism (ACAD10), sterol transport (CYP27A1, ARV1) and inflammation (CCL3, EGR1). Of these, ACAD10 and CYP27A1 were the genes which were most statistically significant. In the follow-up analysis of these genes, a genetic variant (rs2238151) in ACAD10 showed a significant association with risk of CAD (risk allele frequency (RAF): 0.63, OR: 1.08, corrected p: 5.85x10[superscript -6]) and MI (RAF: 0.57, OR: 1.09, corrected p: 6.24x10[superscript -6]) in the CARDIoGRAM GWAS meta-analysis and a variant in CYP27A1 (rs933994) was noted to be an eQTL for CYP27A1 expression in monocytes (p=2.9x10[superscript-70]). Conclusions: Gene expression profiling in resting and stimulated monocytes from subjects with premature CAD and those with an increased genetic risk of CAD have revealed novel gene variants which associate with susceptibility to CAD.

Glucocorticoids form an essential part of the response to major surgery and critical illness. Both inappropriately low and excessively high levels of glucocorticoid in this context lead to raised morbidity and mortality. Controversy still exists over who these patients are, how they might be diagnosed and when, how and what to treat them with. Simple control of endogenous glucocorticoids is well known and is regulated by a negative feedback system comprising the hypothalamus, the pituitary and the adrenal gland (the HPA axis). It is widely known that cortisol is secreted in a diurnal rhythm; levels are low during periods of sleep and rise to a peak just before waking. Underlying this diurnal rhythm is an ultradian rhythm of discrete pulses. It was previously thought that these pulses were due to a 'pulse generator'. However, recent work has shown that it is inherent within the system and as a result of the feedforward-feedback properties of adreno-corticotrophic hormone (ACTH) and cortisol. Pulsatility is important; transcription of cortisol responsive genes 'pulse' in-time with pulses of cortisol and non-pulsatile cortisol replacement in those with absolute deficiency is associated with an excess mortality. No previous study has examined ultradian rhythms of cortisol at and around the time of major surgery. Coronary artery surgery can be performed with (on-pump) or without (offpump) the use of cardiopulmonary bypass (CPB). Off-pump surgery is associated with lower levels of systemic inflammation as measured by markers, although this does not translate into improved long-term outcomes. Previous work using point measures of cortisol and ACTH after cardiac surgery has shown that both cortisol and ACTH rise by the end of surgery, with cortisol remaining elevated, but ACTH being 'suppressed' by 24 hours post surgery - a so called 'disconnect' between the pituitary and adrenal glands.

A brain injury has the potential to unravel everything you once knew. This frightening uncertainty is what philosopher Catherine Malabou (2012) calls the 'destructive accident' where a trauma has the capacity to change our lives forever. When this occurs, care is paramount. But how do we care for our brains? Who knows best? This thesis is a reflection of ethnographical observations of a neuroimaging clinical trial and two neurorehabilitation centres aimed at providing targeted rehabilitation after a traumatic or acquired brain injury. Though much work has been done in the social sciences on the rise of the neurosciences and its potential implications for 'hope and hype' (see Rose 2007, Beaulieu, 2004, Slaby, 2012, Rose and Abi-Rached, 2013) I question what a theory of plasticity (Malabou, 2008) can bring to the debates around material agency, embodiment and hope after a trauma; can patients still learn, adapt or make meaning of the world when they are severely cognitively impaired? I will show that for many of the patients in this research, plasticity was a key contributor in how they viewed themselves and their attitude to recovery which shaped their practices in diverse ways. By taking into account moments of surprise, affect and even love as well as larger governance practices, I question how we might formulate more ethical care practices for brain-injured patients. More broadly, I show' how hope and human agency are deeply and immediately embedded in real medical and healthcare practices so I argue a cautionary stance against the easy assumptions of loss which frequently mark critical theory.

Background There is no method in clinical use for measuring intraspinal pressure (ISP) and spinal cord perfusion pressure (SCPP) after traumatic spinal cord injury (TSCI) in humans. I hypothesised monitoring ISP and optimising SCPP may improve spinal cord function after injury. The thesis was performed in three stages. Methods (1) I ascertained the views of consultant neurosurgeons and neuroanaesthetists on the acute management of TSCI , via a survey. (2) A pressure probe was placed subdurally at the injury site in 18 patients with severe TSCI. Recording commenced within 72 hours of injury and continued for up to one week. Spinal cord blood flow was assessed using indocyanine green fluorescence, and spinal cord function using a limb motor score, motor evoked potentials (MEPs) and an index of autoregulation (sPRx). I explored the effect of different treatments on SCPP. (3) 134 magnetic resonance (MR) scans from 93 TSCI patients were analysed. In 14 patients with motor complete TSCI, I evaluated the effect of laminectomy on ISP, SCPP and compensatory reserve (sRAP). Results (1) The acute management of TSCI by U.K. neurosurgeons and neuroanaesthetists is highly variable, both surgically and in intensive care. (2) There were no procedure related complications with ISP monitoring. ISP was higher in 18 TSCI patients compared to 12 subjects without TSCI. Changes in PC02, sevoflurane concentration and mannitol administration had no significant effect on ISP or SCPP. Inotropes increased ISP with a net increase in SCPP. Increasing SCPP increased MEP amplitude and ICG fluorescence in some patients. (3) On MR, 26% TSCI patients had dural compression. Compared with intact lamina patients, the laminectomy group had lower ISP, comparable SCPP and comparable sRAP. In the laminectomy group, ISP remained high (>20mmHg) 41% of the time, and SCPP low «60mmHg) 24% of the time. Conclusions I provide proof-of-principle that subdural intraspinal pressure at the injury site can be measured with low risk after TSCI. Optimising SCPP improves motor function in some patients. The dura is responsible for spinal cord compression in a quarter of patients. Though bony realignment with laminectomy reduces ISP, it does not effectively decompress the spinal cord and does not increase spinal cord perfusion.

Background The most likely mechanisms of neurological injury following transcatheter aortic valve implantation (TA VI) and aortic valve replacement (A VR) are cerebral embolisation and hypoperfusion. Primary aim was to compare the potential mechanisms of neurological injury following TA VI and A YR. Methods 127 consecutive high risk patients with severe aortic stenosis who underwent TA VI (n=85) or A VR (n=42) were studied. Transcranial Doppler (TCD), cerebral oximetry, diffusion-weighted MRI (DW -MRI) (before, 6 days and 3 months following procedure) and neurocognitive assessment before and at 3 months were perfonned. Results Neurological injury was not significantly different between TA VI and AVR at one (1.1 % vs. 2.2%, p=0.25) and three months (4.7% vs. 2.2%, p= 1). At 3 months, overall cognitive score was higher in A VR compared with TA VI when adjusted for baseline score; the estimated difference between groups was 0.63 (95% Cl 0.87 - 1.17; p=0.02). Cerebral embolic load was 212 (123-344) during A VR and 134 (76-244) during TAVI, (p=0.07). Cerebral oxygen de saturation during AVR (7.56 ± 2.16) was higher compared to TAVI (5.93 ± 2.47) (p<0.01). Ischemic lesions measured by DW-MRI occurred in 76% ofTAVI and 71% of AVR patients at 6 days (p=0.69) and 63% and 39% at 3 months (p= 0.11). No significant association was found between cerebral emboli, cerebral oxygen desaturation, brain ischemic lesions and general cognitive score. Copclusions At 3 months follow-up, overall cognitive score was higher in A VR compared to TA VI, adjusted for baseline score. However, there was no difference in cerebral embolic load, ischemic lesions and oxygen desaturation.

This study assessed the contribution of inherited factors to the development of ESRD in a case-control study population comprised of exclusively White first time renal transplant recipients and their respective donors. A range of molecular methods were utilised to examine the genomic and epigenomic profiles of over 7,000 individuals, examining 2J06 SNPs, across a range of studies varying from a single SNP to single genes, gene families right through to interrogation of the major histocompatibility complex as a targeted genomic region. Stringent quality control measures were applied throughout, with replication and meta-analysis used where possible. While no single SNP locus was found to be a strong marker for ESRD, several regions within the major histocompatibility region are worthy of further investigation for association with ESRD, especially with regards to DNA methylation. The large scale consortium based collaborations which are becoming more commonplace will ultimately help to shed more light on both genetic and epigenetic factors which influence ESRD susceptibility.