I investigated the impact of DARC expression on haematopoiesis using DARC-deficient (DD) mice and developed humanised transgenic DARC models expressing the \(FYB(ES)\) and \(FYB\) human genes. I developed murine irradiation chimeras with differential DARC expression and showed that the absence of erythroid DARC but continued DARC endothelial expression was associated with reduced peripheral blood neutrophil counts. FYB(ES)TG mice showed reduced peripheral neutrophil counts and expansion of BM myelopoiesis, and reduced lymphopoiesis and erythropoiesis, compared to FYBTG mice. FYB(ES)TG and DD mice had reduced GMP cells and LSK cells, and proliferation and apoptosis of these cells were reduced. Microarray analysis of differentially expressed genes showed increased myeloid gene expression in GMP and LSK cells. Mixed irradiation chimeras showed that DD BM retained these changes, suggesting an intrinsic cell effect. Analysis of BM and serum showed a significant increase in G-CSF and eotaxin. Morphological analysis of myeloid cells in femur sections revealed increased myeloid cell clustering in DD and FYB(ES)TG mice. These data suggest that neutrophils are preferentially retained in the DD BM, possibly as a result of loss of optimal chemokine gradients created by erythroblast DARC which may favour neutrophil egress and thereby leading to peripheral neutropenia.

Background: The non-invasive assessment of volume status in left ventricular systolic dysfunction (LVSD) is challenging. The main thesis objective was to establish the feasibility and potential clinical utility of repeated measures assessment of non-invasive biomarkers in defining changes in volume status within individual volunteers. Methods: Differential volume manipulation protocols were achieved in a three-staged plan of investigation, firstly, in patients with decompensated heart failure receiving intravenous furosemide, secondly, in normal volunteers receiving acute loads of oral water or intravenous saline, and thirdly, in stable LVSD patients on chronic furosemide dosing undergoing staged diuretic withdrawal and resumption. Repeated measures of biomarkers relevant to volume status including blood and urine biomarkers, echocardiographic and bioimepdance measures were performed to assess their sensitivity to the induced volume changes. Results Summary: I demonstrated the smallest variance for bioimpedance measures, and the largest variance for urine biomarkers. In the patients with decompensated heart failure, none of the biomarkers studied showed potential clinical utility at tracking acute volume response to intravenous furosemide. In the normal volunteers, a significant change in estimated blood volume was observed following intravenous saline, but not with acute oral water ingestion. Only whole-body and trunk bioimpedance measures, and to a lesser extent, mitral valve early peak velocity with and without the Valsalva manoeuvre, appeared sensitive enough to map these changes in volume status. In the stable LVSD patients, statistically significant increases in B-type natriuretic peptide, urinary creatinine, urinary kidney injury molecule 1, and in bioimpedance-estimated body water composition were observed with diuretic withdrawal, with levels of these markers reducing following diuretic resumption. However, the amplitude of the changes observed was either lower than the respective within-subject variance or too small to be potentially useful as a marker of volume change. This would thus limit the clinical utility of these biomarkers in the routine monitoring of volume status in LVSD. Conclusion: The repeated measures of biomarkers studied in response to different volume manipulations were interpreted in the context of their within-subject normal variance. None of the biomarkers studied appeared to have the ideal characteristics clinically for the monitoring of subclinical changes in volume status in stable LVSD or in response to acute diuresis in decompensated heart failure. The significant increases in urine biomarkers following diuretic withdrawal in stable LVSD suggested potentially beneficial renal effects of furosemide in stable LVSD.

his study investigated the underlying mechanisms of outer membrane homeostasis in Gram-negative bacteria. Using both the evolved laboratory strain E. coli K12 and the broad host range pathogen Salmonella enterica serovar Typhimurium, we have identified and characterised a series of non-essential genes responsible for the maintenance of the outer membrane barrier function. We have revealed their importance for bacterial pathogenesis suggesting their use as novel targets for drug development. This study has provided the first description of a pathway for phospholipid transport from the inner membrane to the outer membrane via the lipoprotein PlpA, a gene previously of unknown function. As several of the genes highlighted by our initial studies were associated with the biogenesis of virulence factors, we complemented our investigations by characterising the contributions of the S. Typhimurium Type V proteins to virulence in a murine model. These investigations have provided the first peer reviewed characterisation of a trimeric autotransporter from Salmonella, has identified a mechanism by which Salmonella can survive on tomatoes, and has highlighted the functional redundancy of these proteins in Salmonella infection. These findings have significantly advanced our understanding of the mechanisms mediating outer membrane homeostasis and the biogenesis and functions of virulence factors.

Eisenmenger syndrome is characterised by severe, lifelong hypoxaemia and pulmonary hypertension. Despite this, patients do surprisingly well and report a reasonable quality of life. This thesis describes a series of experiments investigating the adaptations that occur in these patients in response to the chronic hypoxaemia. Patients with Eisenmenger syndrome have severely limited exercise tolerance when assessed using cardiopulmonary exercise testing. However, they appear to maintain aerobic metabolism until late on in exercise. Studies using skeletal muscle 31P MRS during and throughout recovery from exercise showed that these patients have similar mitochondrial oxidative capacity compared to healthy controls. Echocardiography showed that patients with Eisenmenger syndrome have preserved right and left ventricular systolic function. However they have evidence of right ventricular diastolic dysfunction as evidenced by impaired early diastolic relaxation. The cardiac 31P MRS study demonstrated that despite the normal systolic function shown on echocardiography, there is impairment of septal energetics as revealed by a reduction in PCr/ATP ratio. The results presented in this thesis indicate that adult patients with Eisenmenger syndrome have undergone beneficial adaptations to the severe hypoxaemia that they are exposed to from infancy.

Oral keratinocyte behaviour were analysed in two and three dimensional cultures of an immortalised human H400 cellline and primary rat keratinocytes (PRKs) using a novel method of quantitative microscopy, RT-PCR data and immunohistochemistry profiles. Monolayer cultures were established in high and low calcium media at different cell densities and analysed prior to generating 3D organotypic cultures (OCs) onde-epidermalised dermis (DED), polyethylene terephthalate porous membrane (PET) and collagen gels for up to 14 days.H400 and PRKs proliferation in monolayer cultures was greater in low calcium medium compared with high calcium medium.Gene expression analysis indicated that adhesion and structural molecules including E-cadherin, plakophilin, desmocollin-3, desmogleins-3 and cytokeratins-1, -5, -6, -10, -13 were up-regulated by days 6 and 8 compared with day 4in high calcium medium. Immunohistochemical profiles and gene expression data of OCs on DED recapitulated those of normal oral epithelium. The final thickness of OCs as well as the degree of maturation/stratification was significantly greater on DED compared with other scaffolds used. Quantitative microscopy approaches enabled unbiased architectural characterisation of OCs and the ability to relate stratified organotypic epithelial structures to the normal oral mucosa. H400 and PRK OCs on DED at the air liquid interface demonstrated similar characteristics in terms of gene expression and protein distribution to the normal tissue architecture.

The use of haemostatic materials that could be used to mitigate against the effects of the chemical warfare agent soman (GD) on contaminated personnel that may also present with wounds were investigated. To support the in vitro diffusion cell component of this work, the penetration rate of \(^1\)\(^4\)C-GD into different receptor fluids was evaluated to enable determination of the most appropriate receptor fluid to use as a sink for GD. Of the receptor media evaluated only 50% aqueous ethanol was able to maintain sink conditions. A number of haemostatic materials were shown to retain haemostatic efficacy in the presence of blood contaminated with GD, and were also shown to irreversibly sequester GD. The lead candidate, WoundStat™, was shown to be as effective a decontaminant as the current in service countermeasure fullers’ earth. Complementary in vivo studies using damaged ear skin in a terminally anaesthetised large white pig model showed that whilst use of WoundStat™ was not 100% effective in the prevention of mortality after GD poisoning, it did increase the therapeutic window where further nerve agent-specific medical countermeasures could be employed. Perhaps most importantly, application of WoundStat™ onto GD contaminated damaged skin did not increase the toxicity of GD.

Two major reasons for the failure of central nervous system axon regeneration are (i) lack of neurotrophic factors available to CNS neurones and (ii) the presence of molecules that inhibit the growth of axons. In this study a gene therapy approach using adeno-associated virus 8 (AAV8) was used to manipulate these two factors. The following major aims were addressed: (i) confirm the bioactivity of transgenes that would be packaged into the AAV8 vector; (ii) assess the cellular tropism of AAV8 in the dorsal root ganglion (DRG); (iii) evaluate the inflammatory responses of the nervous system to AAV8 after intra-DRG and intrathecal injection; (iv) determine the axon regenerative effect of AAV8-mediated delivery of nt-3 (a neurotrophic factor) and shRNA\(_{RhoA}\) (a disinhibitory therapy) to dorsal root ganglion neurones after spinal cord injury in the rat. Delivery of the nt-3 transgene in vitro resulted in production of high levels of NT-3 protein. Transfection of shRNA\(_{RhoA}\)-containing plasmids into cell lines resulted in a marked decrease in the amount of RhoA detectable in cell lysates. AAV8 was found to preferentially transduce large diameter, proprioceptive DRG neurones (DRGN) but in the context of a significant inflammatory response after intra-DRG injection 28d following intra-DRG injection. Axon regenerative effects of AAV8-mediated transgene delivery before lesioning were ambiguous and further work need to be undertaken to clarify this matter.

The aim of this thesis was to determine whether the blood flow velocities in the peripheral vascular system measured using phase contrast magnetic resonance imaging, PC-MRI, techniques could be used in the same way that blood flow velocities measured using spectral Doppler ultrasound are used to aid in the diagnosis of peripheral vascular disease. Specifically, we aimed to investigate the measurement of maximum velocities and the use of maximum velocity ratios; an area of investigation which has been neglected in studies of PC-MRI blood flow quantification to date. A series of optimisation and comparison studies were carried out using in-house developed test phantoms. Key to the in-vitro work was the establishment of a dual modality flow test system which would allow comparison of identical flow conditions measured using ultrasound and MRI. The work was complemented by in-vivo studies in healthy volunteers. A 4D PC-MRI commercial work-in-progress protocol and software package became available during the study and was evaluated in-vitro and in-vivo using similar methods as for the 2D PC-MRI studies. The main findings of the thesis were that 2D PC-MRI measurement of maximum velocities significantly underestimated those measured using spectral Doppler ultrasound. However, if corrections were applied to account for the overestimation of ultrasound maximum velocity due to spectral broadening, then the two methods were in agreement. In contrast, the use of maximum velocity ratios showed no difference between spectral Doppler ultrasound and 2D PC-MRI measurements. It was noted that one of the potential problems with the use of 2D PC-MRI in the measurement of the maximum velocity at a stenosis is the accurate positioning of the 2D velocity encoded slice in the stenotic jet. 4D PC-MRI, with a time resolved velocity encoded volume dataset, offers a potential solution to this. However, our evaluation of 4D PC-MRI showed that it can significantly underestimate both maximum velocities and maximum velocity ratios in comparison with 2D PC-MRI and spectral Doppler ultrasound and requires further development before it can be used for peripheral vascular applications.

A prospective randomised controlled clinical trial of treatment decisions informed by invasive functional testing of coronary artery disease severity compared with standard angiography-guided management was implemented in 350 patients with a recent non-ST elevation myocardial infarction (NSTEMI) admitted to 6 hospitals in the National Health Service. The main aims of this study were to examine the utility of both invasive fractional flow reserve (FFR) and non-invasive cardiac magnetic resonance imaging (MRI) amongst patients with a recent diagnosis of NSTEMI. In summary, the findings of this thesis are: (1) the use of FFR combined with intravenous adenosine was feasible and safe amongst patients with NSTEMI and has clinical utility; (2) there was discordance between the visual, angiographic estimation of lesion significance and FFR; (3). The use of FFR led to changes in treatment strategy and an increase in prescription of medical therapy in the short term compared with an angiographically guided strategy; (4) in the incidence of major adverse cardiac events (MACE) at 12 months follow up was similar in the two groups. Cardiac MRI was used in a subset of patients enrolled in two hospitals in the West of Scotland. T1 and T2 mapping methods were used to delineate territories of acute myocardial injury. T1 and T2 mapping were superior when compared with conventional T2-weighted dark blood imaging for estimation of the ischaemic area-at-risk (AAR) with less artifact in NSTEMI. There was poor correlation between the angiographic AAR and MRI methods of AAR estimation in patients with NSTEMI. FFR had a high accuracy at predicting inducible perfusion defects demonstrated on stress perfusion MRI. This thesis describes the largest randomized trial published to date specifically looking at the clinical utility of FFR in the NSTEMI population. We have provided evidence of the diagnostic and clinical utility of FFR in this group of patients and provide evidence to inform larger studies. This thesis also describes the largest ever MRI cohort, including with myocardial stress perfusion assessments, specifically looking at the NSTEMI population. We have demonstrated the diagnostic accuracy of FFR to predict reversible ischaemia as referenced to a non-invasive gold standard with MRI. This thesis has also shown the futility of using dark blood oedema imaging amongst all comer NSTEMI patients when compared to novel T1 and T2 mapping methods.

The aim of this thesis is ‘’Improving the Assessment of Exercise Capacity and Cardiorespiratory Fitness in Patients Attending Exercise-Based Cardiac Rehabilitation’’. Cardiorespiratory capacity is an important predictor of morbidity and mortality in cardiac patients, due to the prognostic power, is an essential outcome to measure in cardiac patients in clinical practice. In cardiac rehabilitation programmes the assessment of cardiorespiratory capacity (by field tests or treadmill test) is an essential practice supported by U.K., European and U.S.A. guidelines, which gives support to patients risk evaluation and stratification, setting individual patients goals, exercise prescription, and evaluation of the same. Overall, the findings of this thesis, which were generate by meta-analysis, crosssectional studies and laboratory research, provide an nsight into the factors associated with patients’ initial performance, and oxygen cost in functional capacity tests. Together, this data may improve the application, interpretation and patient understanding of these test results. One aim of CR is to improve patients’ functional capacity; we provide a standard value for ΔFitness, and information on factors which clinicians may need to consider when setting patient goals and interpreting changes in functional capacity, or ΔFitness due to CR.