Examining 20 human wound bed biopsies (pilonidal sinus and venous leg ulcers), significant differences were observed between the subpopulations of wound macrophages in acute healing and chronic non-healing wounds. The results exhibit that within the acute wound there is an accumulation of early stage macrophages (mean 20.8, SD 8.6) differentiating from monocytes which become activated and contribute to the wound healing process. There being few early stage macrophages within the chronic wound (mean 10.4, SD 6.7) (p≤0.01). Chronic wounds in comparison demonstrated a significant accumulation of tissue macrophages (mean 34.0, SD 10.5) when compared to acute wounds (mean 10.9, SD 4.4) with limited wound healing (p≤0.01). The dermagraft (DG) study comprising of 53 patients, showed that applying a biologically active dressing (1-12 dressings over 12 weeks) and compression to the wound bed, exhibited complete closure (76%) or reduction in the size of the wounds at 12 weeks, compared to compression alone. Changes in the extracellular components and an array of inflammatory cells and cytokines in fifty three paired wound bed biopsies (106) with and without DG were examined at week 0 and week 6 of treatment. On histological investigation, DG exhibited an increase in the amount of collagen present and angiogenesis in the wound at week 6 of treatment. Although there were no significant changes in the lymphocyte counts in response to the application of DG, it was possible to demonstrate a significant increase in the number of stage macrophages at week 6 of treatment (p≤0.05) and a significant reduction in the tissue macrophage counts, at week 6 of treatment (p≤0.05) in patients treated with 4 pieces of DG. The levels of different cytokine expression within the wound bed at week 6 exhibited some changes but this was not significant, in response to DG treatment. This could be to the possible presence of proteinases within the chronic wound bed hydrolysing the cytokines produced by DG. From the results attained, it was able to conclude, for clinical use 4 pieces of DG at regular dosing intervals were sufficient to achieve wound contraction or closure. This dose regimen has not been taken forward for further pivotal studies. This thesis thus represents some of the first evidence in human tissue that macrophages may play role in wound healing, and in chronic wounds, a subpopulation of macrophages can be modified to stimulate these wounds towards healing.

Major vascular surgery is associated with a substantial risk of cardiovascular events and death. This risk is of increased importance in prophylactic elective open Abdominal Aortic Aneurysm (AAA) repair, where a balance of risk of rupture and postoperative outcome is assessed prior to management decisions. Further, the UK Small Aneurysm Trial has shown that prophylactic repair of an AAA has no survival benefit for the first three years due to the major adverse cardiac event (MACE) rate of 5-15%. There is however no ideal method of predicting this risk. Cardiac Troponin I (cTnI) is a contractile protein that is a highly sensitive and specific marker of myocardial necrosis. A few case reports have commented on the finding of preoperative asymptomatic elevated cTnI levels and poor outcome in a small number of patients undergoing major vascular surgery. There are however no studies looking at its incidence in the vascular surgical population or its utility as a preoperative marker. Several studies have noted that B-type natriuretic peptide (BNP), a diagnostic and prognostic marker of heart failure, may have a role in predicting MACE in settings including major vascular surgery. There are no studies that have investigated this role in AAA repair alone. The aim of this thesis is to investigate the incidence of, and to determine a possible role for, preoperative elevated cTnI in major vascular surgery. The further aim is to determine if a single preoperative BNP level correlated with MACE and all-cause mortality in elective open AAA repair in both the short and long-term. Comparisons to current accepted risk indices in AAA, and a possible role for BNP in EndoVascular Aneurysm Repair (EVAR) will also be investigated. Patients were recruited in two cohorts: Firstly, a prospective, 2 year observational single centre cohort study of all patients undergoing a vascular procedure, with an expected cardiac event rate >5%, recruited patients who had no clinical or ECG evidence of myocardial ischaemia. Preoperative cTnI was performed in all and postoperative screening (clinical assessment, ECG and cTnI) for cardiac events was performed at days 2, 5 and 30. 213 patient were recruited, of whom 11 (5.2%) had an asymptomatic elevated preoperative cTnI (>0.02 ng/ml). Eight of these patients proceeded directly to theatre, and 2 were delayed but later underwent surgery with a persistently elevated cTnI. Of these 10 patients, 5 (50%) died and 4 (40%) suffered MACE. The remaining patient was delayed due to the poor outcome of the preceding patients, and later underwent an uncomplicated aortic bifurcation graft with a normal cTnI level which had been preceded by coronary intervention. Secondly, a prospective, 2 year observational multi-centre cohort study in the 3 largest vascular units in Glasgow (Gartnavel General Hospital, Glasgow Royal Infirmary and Southern General Hospital) was performed between August 2005 and August 2007, recruiting all patients who were admitted for both elective open AAA repair and EVAR. Preoperative BNP levels were performed and batch analysed at the end of the study. Postoperative screening for cardiac events was performed as described above. Data was collected to allow calculation of risk indices associated with outcome in AAA repair (Glasgow Aneurysm Score [GAS], Vascular physiology only Physiological and Operative Severity Score for enUmeration of Mortality [V{p}-POSSUM], Vascular Biochemical and Haematological Outcome Model [VBHOM], Revised Cardiac Risk Index [RCRI] and Preoperative Risk Score of the Estimation of Physiological Ability and Surgical Stress Score [PRS of E-PASS]). Follow-up was continued to a minimum of 3 years, where possible, with cause of death recorded. 106 of 111 patients were recruited. The median [interquartile range] BNP concentrations in the 16 patients (15%) who suffered immediate postoperative MACE was 206 [118-454] vs 35 [17-61] pg/ml in the remainder (p=0.001). ROC analysis indicated a BNP concentration of 99.5 pg/ml best predicted MACE (area under the curve 0.927), with sensitivity of 88% and specificity of 89%. The BNP in patients who suffered cardiac death was significantly higher than in those that did not (median BNP 496 [280-881] vs 38 [18-84] pg/ml, p=0.043). ROC analysis revealed a cut-off of 448 pg/ml (AUC 0.963), with sensitivity 80%, specificity 100%, positive predictive value 100% and negative predictive value 99%. Not only did higher values of BNP predict MACE, but it was also found to predict all-cause mortality in the immediate (median BNP 100 [84-521] vs 35 [17-81], p=0.028), intermediate (median BNP 201 [97-496] vs 35 [17-73], p<0.001) and long-term (median BNP 98.5 [58-285] vs 32 [17-71.5], p<0.001) postoperative periods. ROC analysis revealed decreasing BNP levels to predict outcome over time, with a BNP of >60.5 pg/ml (AUC 0.761) found to best predict death at 3 years. Whilst BNP was found to predict outcome, most risk indices performed poorly. The GAS, VBHOM and RCRI performed poorly and did not predict any outcome measure. V(p)-POSSUM was, however, found to predict all outcome measures (p=0.028, p=0.030, p=0.038 for MACE, cardiac death and all-cause mortality respectively). The PRS component of E-PASS was found to predict MACE (p=0.019) and cardiac death (p=0.017). BNP performed better than any risk index. During the study period only 40 of 42 patients admitted for elective EVAR were recruited. Of these 40, only 3 suffered a non-fatal MI and 1 died of respiratory failure. BNP was not found to predict MACE or death in this cohort, and due to the small number of patients, and events, no strong conclusions could be drawn. Whilst preoperative elevated cTnI was found to identify patients that were at an increased risk of both postoperative MACE and death following their major vascular surgical procedure, its use in elective open AAA repair is limited due to infrequent occurrence. Preoperative serum BNP concentration, however, predicted postoperative MACE, cardiac death and all-cause mortality in patients undergoing elective open AAA repair on immediate, intermediate and long term follow-up. Further, BNP performed better than any current risk index for elective open AAA surgery. This simple blood test, therefore, offers valuable information regarding risk stratification of prospective surgical patients and should be considered a part of routine preoperative assessment in this prophylactic procedure.

The aims of the thesis are to elucidate the incidence of post-operative supraventricular arrhythmias (SVA) in patients that undergo thoracotomy for lung or oesophageal resection in the local population and to determine risk factors associated with the development of SVA. Further objectives are to compare methods of SVA detection and to prospectively evaluate the role of magnesium sulphate as a preventative measure. The incidence of post-thoracotomy SVA in the local population was 25.5% which was found to be higher than reports in the literature. Yield of arrhythmia detection did not differ between continuous electrocardiogram monitoring and 12 lead electrocardiogram recordings. Risk factors identified were increasing age, the presence of ischaemic heart disease and hypertension, and undergoing a pneumonectomy. There was a trend towards a higher mortality in the group that developed an arrhythmia, in the pilot study. The amount of lung resected was also found to be significantly associated with an increasing incidence of SVA post-operatively. The prophylactic, peri-operative administration of intravenous magnesium sulphate did not reduce the incidence of SVA post-thoracotomy overall. The peripheral administration of magnesium sulphate was not well tolerated secondary to the minor side effect of stinging, although no major side effects such as respiratory depression were observed. However, it did significantly reduce the incidence of SVA in the higher risk cohort pneumonectomy subgroup. Further work is anticipated in this group along with further research into risk factors and aetiology.

Lung cancer is the most common cause of cancer death in the UK. In suitable cases, the best chance of cure is surgical resection. Due to high levels of co-morbidity seen in this population, lung resection is associated with high cardio-respiratory complication rates. One such complication is the development of Acute Lung Injury / Acute Respiratory Distress Syndrome (ALI/ARDS). ALI/ARDS is reported to occur in four to 11% of patients undergoing lung resection and is the major cause of hospital mortality following lung resection. ALI/ARDS occurring following lung resection is widely interpreted to be a variant of ALI/ARDS and follows an identical clinical and pathophysiological course to that seen in the wider critical care environment. The pathophysiology of lung injury following lung resection is complex and can be broadly conceptualised as occurring secondary to insults specific to both the ipsilateral (surgical) lung, the contralateral (anaesthetic) lung in addition to those insults common to both lungs. Increased recognition of the role of ventilator induced lung injury, and peri-operative fluid prescribing in the pathogenesis of lung injury in this population has brought the prevention of lung injury to the attention of the thoracic anaesthetist. Though high quality evidence is lacking, expert opinion widely favours the adoption of lung protective ventilatory strategies and restriction of peri-operative fluids in patients undergoing lung resection. This thesis presents the rationale, methodology and results of four discrete studies concerning the development of lung injury in the thoracic surgical population undergoing resection of primary lung cancer. Investigation I is a survey of contemporary UK thoracic anaesthetic practice when anaesthetising for thoracic surgery and lung resection, with specific reference to strategies designed to prevent lung injury. Though implementation of the techniques described is far from universal, the survey results suggest that aspects of lung protective ventilation are widespread within UK thoracic anaesthetic practice. Investigation II seeks to examine the impact of increased adoption of such strategies over time. A random effects meta-analysis and meta-regression analysis was performed to examine the trends in the incidence of and mortality from ALI and/or ARDS over time. The main findings of this study are that whilst there is no evidence to suggest the incidence of ALI and/or ARDS post-lung resection is falling, mortality due to ARDS (but not ALI) does appear to be falling over time. Investigations III and IV examine the utility of two clinical monitoring methodologies which have potential to provide bedside clinical monitoring of lung injury development in the thoracic surgical population in order to guide clinical decision making, monitor patient progress and serve as a surrogate end point in future clinical studies. Investigation III examines the utility of a single lung injury biomarker (long chain Pentraxin 3 – PTX3) and a panel of multiple lung injury biomarkers in the early post-operative period following lung resection. The properties of the ‘ideal’ lung injury biomarker are defined, against which PTX3 and the multiple biomarker panel are compared. PTX3 compared favourably to properties of the ‘ideal’ lung injury biomarker and appeared to identify a population of patients with elevated post-operative Lung Injury Score with high sensitivity. Conversely there is no evidence from the results presented that a ‘risk of lung injury score’ derived from a panel of 7 candidate lung injury biomarkers (as previously defined in a cohort of critically ill patients with ALI/ARDS) has any utility in the lung resection population. Investigation IV tests the reproducibility and construct validity of transpulmonary thermodilution derived measurements of extravascular lung water and pulmonary vascular permeability index in patients undergoing lung resection. The study’s findings are largely supportive of the reproducibility and construct validity of extravascular lung water measurement and pulmonary vascular permeability measurements after lung resection. In combination, it is hoped that the studies presented provide greater insight into the syndrome of post lung resection lung injury. More accurate definition of standard anaesthetic practice and the incidence of and mortality from ALI/ARDS following lung resection should serve to inform future clinical studies seeking to prevent, treat, or better understand this important clinical syndrome. The biomarker PTX3 and transpulmonary thermodilution derived measurement of extravascular lung water and pulmonary vascular permeability index are presented as surrogate endpoints suitable for use in such studies.

Mild platelet-based bleeding disorders are among the most complex bleeding disorders to understand, due to the absence of a ‘gold standard’ test for diagnosis and the significant overlap with the bleeding phenotype observed in healthy individuals. The work in this thesis is focussed on patients with a clinically diagnosed mild platelet disorder for which no acquired cause was identified by the referring expert clinician. ADP and thromboxane A2 (TxA2) are key secondary mediators of platelet aggregation and function in synergy to facilitate robust platelet activation in the event of vascular damage. Less than ten function-disrupting inherited gene defects in the ADP P2Y12 receptor and only one in the platelet TxA2 receptor have been reported, with none in the UK. Over a period of nearly 4 years, patients with a diagnosis of platelet dysfunction made at UK Comprehensive Haemophilia Care Centres were investigated using platelet aggregation and secretion assays alongside controls and reference curves to nine platelet agonists to exhibit an abnormal bleeding phenotype in response to different platelet agonists, focusing in searching on P2Y12 receptor defects. In addition, the P2Y12 ADP receptor from 140 subjects diagnosed with mild type 1 von Willebrand Disease (VWD) from the EU MCMDM-1VWD study was sequenced in view of the similarity in bleeding phenotype of patients with type 1 VWD and mild platelet disorders and the fact that both conditions show incomplete penetrance consistent with a multifactorial basis for each disorder. The sequencing was performed by Dr Martina Daly in Sheffield. The work in this thesis has led to the identification / characterisation of a patient who is homozygous for an early missense mutation in the P2Y12 ADP receptor and two patients with heterozygous point mutations in the P2Y12 ADP receptor (who also has type 1VWD) and in the TxA2 receptor. In addition, I studied platelet aggregation and secretion, along with a number of more specialised assays, in nearly 80 other patients during the course of the thesis and have subdivided these on the basis of the observed defect. Interestingly, in nearly one third of the patients, a platelet defect was not found.

There is potential for haemorrhaging injuries that become contaminated with toxic chemicals e.g. sulphur mustard (SM). There are no specific medical countermeasures for such injuries at present. It is proposed that haemostats could simultaneously stop bleeding and decontaminate wounds. Products must be able to clot SM-contaminated blood and reduce SM percutaneous absorption to be considered suitable. Sulphur mustard did not significantly affect coagulation in vitro or in vivo. Overall SM did not affect the pro-coagulatory function of haemostats. However, clot strength in WoundStat™ treated blood was adversely affected by SM in vitro. Superficial damage to the skin significantly increased the percutaneous absorption of SM with good agreement between in vitro and in vivo studies. Moreover, the altered toxicodynamics imply that the latent period associated with SM pathology is due, in part, to rate of absorption and not solely caused by biochemical pathways. Application of WoundStat™ significantly reduced the amount and rate of SM penetration through the skin in vitro. Furthermore, WoundStat™ reduced SM percutaneous absorption and pathology in vivo. Microarray analysis has identified that SM exposure via damaged skin causes changes in gene expression which may warrant further investigation as potential therapeutic targets.

Donor organ shortage is a major barrier to the progress of liver transplantation; options to widen the donor pool include use of marginal donor grafts and those from donors after circulatory death (DCD), despite risks of early graft failure. This thesis studies the key metabolic feature differences between DCD and from donors after brain death (DBD), using combination of microdialysis for tissue fluid sampling, and colourimetry, Coularray and Fourier Transform ion Cyclotron Resistance - mass spectrometry(FTICR-MS) as analytical platforms. The initial study proved feasibility of above methods to identify metabolic changes through cold storage to reperfusion, and the involvement of energy and amino acid metabolism pathways. Comparison of DCD and DBD grafts by microdialysis combined with colourimetry proved energy depletion, and increased lactate/pyruvate ratio in DCD grafts. Metabolomic studies consolidated the findings of primary impact on energy metabolism pathways during cold storage. Both CEAD and FTICR-MS identified key biomarker differences and the effect on tryptophan and kynurenine pathway, and this finding was reproduced in all three metabolomic studies conducted. Over expression of these metabolites in DCD grafts and failed allografts may be related to energy metabolism, and tryptophan and kynurenine could potentially be developed as biomarkers predicting liver graft function.

Current transplant immunosuppression regimens have numerous limitations. Recent evidence suggests histone deacetylase inhibitors (HDACis) may represent a class of drug with immunosuppressive properties. This study initially assessed a panel of experimental and established HDACis, identifying a novel HDAC6-specific inhibitor (KA1010) to compare with cyclosporin and the pan-HDACi suberoylanilide hydroxamic acid in models of alloreactivity. Proliferation and MLR-based assays were used to determine the immunosuppressive effect of compounds, and a murine model of allogeneic skin transplantation was adopted to assess the in vivo effects of HDAC6 inhibition. KA1010 displayed superior inhibitory effects on the activation of PBMCs using in vitro models of transplantation. In a one-way MLR, KA1010 (5μM) reduced parent cell proliferation from 92% to 64% (p=0.001). A two-way MLR, adopting IFN-γ production as a marker of alloresponse, resulted in up to 91% reduction. Dose-response curves revealed dose-dependent profiles with a greater potency of HDACis over CyA. Mice treated with KA1010 displayed no significant features of skin allograft rejection upon histological analysis at 70 days and graft survival of upto 80%. Immunological assessment, revealed a significant increase in regulatory T cells (from 18% to 25%, p=0.0002) and a corresponding reduction in CD4\(^+\) T cells (from 58% to 42%, p=0.0009). HDAC6 may represent an optimal target for future immunosuppressant therapeutics with a particular role in transplantation. In this thesis, we have demonstrated a superior immunosuppressive effect of KA1010 over both CyA and SAHA, in the models of allotransplantation adopted.

Title: A study of some of the psychological issues affecting women undergoing treatment for urinary incontinence. Background: There is evidence suggesting that effective clinical treatment of urinary incontinence (UI) is not reflected in patients’ quality of life reports or in psychological well-being. There may be other psychological issues that are not routinely captured but may be influential in recovery. Aims and objectives: To explore women’s experiences of UI and identify what aspects are not altered by stress incontinence (SUI) surgery, to identify themes with which to develop a treatment-planning tool and to identify measures to improve patient satisfaction. Methods: Qualitative interviews and repertory grids were performed on women with UI. Themes generated from these women, together with themes from a literature review and a focus group, form the basis for future questionnaire development. Data were scrutinised for additional potential to improve satisfaction. Results: Wide-ranging themes were identified. Psychosocial aspects are not fully addressed by traditional UI treatment. Many suggestions for improvements in management were identified. Conclusions: There is much that can be done to try to improve satisfaction for women with UI. Many themes have been identified as a basis for the further development of a treatment-planning tool.

OBJECTIVE: Patients undergoing open repair of asymptomatic abdominal aortic aneurysms (AAA) demonstrate a prothrombotic state that initially deteriorates in the peri-operative period before improving beyond the pre-operative state. We hypothesised that a similar haemostatic improvement occurs following endovascular AAA repair (EVAR) and that the initial prothrombotic derangement may increase the risk of myocardial injury. METHODS: 60 patients[57 men; median(IQR) age, 77 (72-82) years] underwent EVAR. Patients were assessed at baseline, 24-hours and 1-month post-procedure. Thrombin-antithrombin III-complex (TAT), tissue-plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1), and soluble (s) P-selectin levels were assessed as biomarkers of coagulation, fibrinolysis and platelet activity, respectively. Cardiac Troponin T (cTnT) levels were assessed as a biomarker of myocardial injury (MI). RESULTS: An increase in sP-selectin levels occurred between baseline[median(IQR), 80.5(68-128) ng/ml], 24-hours[median(IQR), 89.5(73-112)ng/ml; p=0.003] and 1-month[median(IQR), 110(89-143)ng/ml; p=<0.0001] post-EVAR. There was a trend towards increased TAT levels at 24-hours[median(IQR), 21.65(13-33.1)μg/l; p=0.069] compared to pre-operation[median(IQR), 7.15(4.7-31.3)μg/l] followed by a significant decrease at 1-month[median(IQR), 8.1 (5.4-14.85)μg/l; p=<0.0001]. cTnT levels were raised (>0.03ng/ml) in 16% of patients. There was a positive correlation between cTnT and TAT levels at 24-hours post-EVAR(r=0.38, p=0.039, Kendall-tau-B=0.26) CONCLUSION: These novel data suggest that the peri-operative pro-thrombotic state following EVAR may be associated with an increased risk of MI.