Serial Magnetic Resonance (MR) Imaging can reveal structural atrophy in the brains of subjects with neurodegenerative diseases such as Alzheimer’s Disease (AD). Methods of computational neuroanatomy allow the detection of statistically significant patterns of brain change over time and/or over multiple subjects. The focus of this thesis is the development and application of statistical and supporting methodology for the analysis of three-dimensional brain imaging data. There is a particular emphasis on longitudinal data, though much of the statistical methodology is more general. New methods of voxel-based morphometry (VBM) are developed for serial MR data, employing combinations of tissue segmentation and longitudinal non-rigid registration. The methods are evaluated using novel quantitative metrics based on simulated data. Contributions to general aspects of VBM are also made, and include a publication concerning guidelines for reporting VBM studies, and another examining an issue in the selection of which voxels to include in the statistical analysis mask for VBM of atrophic conditions. Research is carried out into the statistical theory of permutation testing for application to multivariate general linear models, and is then used to build software for the analysis of multivariate deformation- and tensor-based morphometry data, efficiently correcting for the multiple comparison problem inherent in voxel-wise analysis of images. Monte Carlo simulation studies extend results available in the literature regarding the different strategies available for permutation testing in the presence of confounds. Theoretical aspects of longitudinal deformation- and tensor-based morphometry are explored, such as the options for combining within- and between-subject deformation fields. Practical investigation of several different methods and variants is performed for a longitudinal AD study.

To explore the effect of endotoxin as a sensitising agent prior to neonatal hypoxiaischemia differing doses of endotoxin (E. coli lipopolysaccharide, LPS) were given to neonatal mice prior to hypoxia-ischemia, with sensitising effects noted at dosages of 0.3mg/kg of LPS or higher. Varying the time interval between endotoxin administration and hypoxia-ischemia demonstrated that LPS given between 4 and 12 hours before hypoxia-ischemia had a sensitising effect on subsequent hypoxia ischemia. In contrast, LPS given at the time of or 24 hours before hypoxia-ischemia did not. To help understand the mechanism by which this sensitising effect occurs, a dose-response study of LPS alone was undertaken. Here, a dose-dependent activation of microglia was demonstrated throughout the brain, particularly in the thalamus and cortex, by 12 hours following endotoxin administration. There was also evidence of vascular endothelial activation (ICAM1) as early as 4 hours after endotoxin administration. To study the role of the TNF cluster of cytokines (TNF alpha, lymphotoxin alpha and lymphotoxin beta), animals with a deletion of the entire TNF cluster were examined. Deletion of the TNF cluster was shown to abolish both endotoxin-mediated sensitisation of the developing brain to subsequent hypoxia, and to prevent upregulation of macrophage and vascular endothelium by endotoxin alone. This study also examined the effects of hypoxia-ischemia on intracellular pH. Increasing duration of hypoxia-ischemia resulted in a progressive intracellular acidosis within the brain, initially ipsilateral to the carotid ligation, but becoming bilateral with prolonged hypoxia. In the reoxygenation phase, there was a rebound intracellular alkalosis at 6 hours of reoxygenation across the whole forebrain. Previous studies have suggested that this alkalosis is mediated by a Na+/H+ exchanger. Blockade of this transporter with N-methyl isobutyl amiloride prior to hypoxia-ischemia was shown to confer neuroprotection in the developing brain.

Idiopathic Mendelian epilepsies have been typically identified as channelopathies. Evidence suggests that mutations in genes encoding GABAA receptors, GABAB receptors or voltage-dependent calcium channels (VDCCs) may underlie childhood absence epilepsy (CAE), an idiopathic generalised epilepsy with complex inheritance. The aims of this project were: i) Ascertainment of a patient resource ii) Investigation of candidate genes by linkage analysis iii) Mutation analysis by direct sequencing iv) Construction of single nucleotide polymorphism (SNP) based haplotypes in candidate genes v) Intra-familial association analysis using SNP based haplotypes DNA and clinical data were obtained from: 53 nuclear CAE pedigrees; 29 families including individuals with CAE and a broader „absence‟ epilepsy phenotype; 217 parent-child trios; a North American family in which absence epilepsy segregates with episodic ataxia type 2 (EA2) Sixteen calcium channel genes and seven GABAA and two GABAB receptor subunit genes were excluded by linkage analysis. Significant linkage was demonstrated for CACNG3 on chromosome 16p12-p13.1 for both CAE and the broader absence phenotype. Positive linkage was also obtained at the GABRA5, GABRB3, GABRG3 cluster on chromosome 15q11-q13. Non-parametric linkage analysis was significant at both the 16p and 15q loci. Two-locus analysis supported a digenic effect from these two loci. Sequencing of CACNG3 revealed 34 sequence variants, none clearly causal, although bioinformatic analysis provided supportive functional evidence. Association analysis showed significant transmission disequilibrium both for individual single nucleotide polymorphisms (SNPs) and SNP based haplotypes spanning CACNG3. This work has provided genetic evidence that CACNG3 and at least one of the three GABAA receptor genes are susceptibility loci for absence epilepsy. Linkage analysis performed in the family with absence epilepsy and EA2 was suggestive that the VDCC CACNA1A was the causative gene. This was subsequently confirmed by sequence analysis in collaboration with the Institute of Neurology, UCL. This is the first reported family in which a CACNA1A mutation that impairs calcium channel function cosegregates with typical absence seizures and 3Hz spike-wave discharges on EEG.

Hypoxia, defined as low oxygen tension, is a common characteristic of growing tumours. Tumour cells adapt to their hypoxic microenvironment by inducing angiogenesis and escaping cell death. In doing so, tumour cells become resistant to radiotherapy and many forms of chemotherapy. Hypoxia signalling and angiogenesis is mediated by the hypoxia-inducible factor (HIF) transcriptional complex. HIF can crosstalk to the p53 tumour suppressor protein, a critical regulator of cell cycle and cell death responses to stress. This study aims to understand how cell death responses are regulated in tumour cells by HIF and p53, in normoxia and in hypoxia. Recently, activation of p53 by the small molecule RITA has been investigated. Flow cytometry, comet assays and western blot analysis have been used to reveal a novel p53-dependent DNA damage response that activates cell cycle checkpoints, and induces significant cell death of hypoxic tumour cells. Activation of p53 also achieves anti-angiogenic effects, both in vitro and in vivo by inhibition of HIF-1α protein synthesis and HIF target genes, including VEGF. The MEK-ERK MAPK pathway has also been investigated as a critical modulator of p53-dependent cell death in normoxia and hypoxia. Inhibition of MEK1/2 by the MAPK signalling inhibitor PD98059 significantly inhibits p53 induction and cell death responses by RITA. The anti-tumour effect of p53 activation in response to RITA is therefore dependent on MEK-ERK signalling. By understanding p53 interactions with HIF signalling, and the role of p53 in responding to DNA damage and apoptotic signals, this study has potential to improve the therapeutic targeting of resistant tumours with deregulated angiogenic and cell death pathways.

The aetiology of inflammatory bowel disease (IBD) combines genetic predisposition and environmental factors. In both ulcerative colitis and Crohn disease, patients perceive that diet affects the course of their disease. This thesis addresses the frequently observed compromise of the epithelial integrity of the gut in IBD and subsequent effect of the luminal content, which makes up the main part of the environmental stimulus, thus introducing the role of diet in IBD. Initially I conducted a survey, demonstrating the current practice of dietary manipulation and exclusion in IBD and irritable bowel syndrome, determining that advice given is generally empiric and that sensitivity testing is infrequently used in practice. A subsequent observational study compared the occurrence of serum IgG antibodies to foods in IBD patients compared to controls. It showed that IBD is associated with increased serum IgG antibodies to a wide range of foods but that this does not correlate with patient reported food intolerance. A further study investigated the colonic mucosal response to food antigen exposure, patient reported food intolerances, food specific serum IgG antibodies and intestinal permeability. The mucosal response did not correlate with patients' perception of food intolerance nor alterations in intestinal permeability. This work reinforces the importance of food intolerance in IBD and attempts to correlate those intolerances to available tests. While gastroenterologists do give dietary advice to their patients with IBD, the available evidence does not allow unequivocal advice. No objective relationship between patient-perceived food intolerance and hypersensitivity testing was demonstrated. Future studies should seek to clearly define the association between intolerance tests and patient symptoms, investigate the mechanisms by which such tests might predict intolerance, and investigate the most promising strategies in carefully designed and controlled studies of dietary intervention.

Impaired uterine blood flow (UBF) leads to fetal growth restriction (FGR), one of the most challenging obstetric complications. FGR is associated with stillbirth, long-term neurological impairment and adult onset cardiovascular disease; there is no treatment currently available. Previously, it was shown that sustained local over-expression of Vascular Endothelial Growth Factor (VEGF) in the uterine arteries (UAs) of pregnant sheep using an adenoviral vector results in increased UBF as measured by Doppler sonography, reduced vascular contractility and increased vascular relaxation 4-7 days after administration. The aim of this thesis is to examine the long-term effects on UBF, UA vascular reactivity, and the possible mechanism of action. Telemetric transit-time flow probes were implanted around the UAs of mid-gestation pregnant sheep (n=10), and a telemetric blood-pressure sensitive catheter was inserted in the maternal (n=5) or fetal (n=4) carotid arteries. After obtaining baseline values for 7 days, we injected adenovirus vectors (5x1011 particles) encoding the VEGF-A165 gene (Ad.VEGF) into one UA, and a reporter β-galactosidase gene (Ad.LacZ), contra-laterally. UBF and maternal haemodynamics were measured daily until term, when the UAs were harvested and their vascular reactivity studied. There was a significant increase in UBF in the Ad.VEGF transduced side (36.53% v/s 20.08%, p=0.02), a reduction in UA contractility but no difference in relaxation. A significantly greater number of adventitial blood vessels were observed in the Ad.VEGF treated UA. There were no significant changes in maternal or fetal blood pressure post-injection. Similar effects were observed with injection of an adenovirus encoding another member of the VEGF gene family, VEGF-DΔNΔC (n=5). Endothelial cells (ECs) were isolated from the UAs of control midgestation pregnant sheep, cultured and infected with Ad.VEGF or Ad.LacZ vector. Protein extracted from UAECs infected ex vivo was assayed for eNOS and phosphorylated eNOS (Ser1177) levels by Western blotting. eNOS and phosphoeNOS levels increased with rising Ad.VEGF concentrations in UAECs; Ad.LacZ vector transduction had no effect. Local over-expression of VEGF effects a long-term increase in UBF by upregulation of eNOS, with neovascularization of the adventitia and reduced UA contractility. These changes may benefit pregnancies complicated by severe FGR. With clinical translation in mind, the last section of this thesis describes the optimization of a technique of gene targeting to the utero-placental circulation of growth-restricted guinea pigs, using a thermo-sensitive pluronic gel. Having optimized this technique, VEGF over-expression in the uteroplacental circulation is now being tested in this animal model of FGR.

Positron emission tomography (PET) suffers from a degradation in quantitative accuracy due to a phenomenon known as the partial volume effect (PVE). The effects are due to the limited spatial resolution of the scanner. Methods that correct for PVEs are known as partial volume correction (PVC) techniques and are either data-driven or make use of anatomical information from other modalities such as magnetic resonance (MR) imaging. This thesis reports investigations into PVC techniques for improving the quantification of brain amyloid PET tracers. These tracers image amyloid plaque aggregation in-vivo, which is a pathological hallmark of Alzheimer’s disease. An extension to existing anatomy-based PVC methods is reported. Region-based voxelwise (RBV) correction has been shown to reduce PVE-induced regional bias and variance when compared to commonly applied PVC techniques. This has been proven in phantom studies and observed in clinical data. In addition, RBV has been used to demonstrate that white matter variability exists in two different amyloid tracers. This finding has implications for the application of PVC in amyloid imaging and also how scans should be normalised. Alternative reference regions were investigated in two amyloid PET tracers. The brain stem, in combination with PVC, was found to result in the strongest agreement between tracers. Anatomy-based PVC techniques rely on parcellations of structural images. These parcellations are not necessarily representative of the PET data. A further extension to RBV is proposed which iteratively modifies the parcellations to find an optimal PVC in terms of the observed PET data. This novel technique reduces quantification errors due to PET-MR mismatch and has the potential to provide an additional parameter of ‘functional volume change’ in longitudinal studies.

Radiologists often need to localise corresponding findings in different images of the breast, such as Magnetic Resonance Images and X-ray mammograms. However, this is a difficult task, as one is a volume and the other a projection image. In addition, the appearance of breast tissue structure can vary significantly between them. Some breast regions are often obscured in an X-ray, due to its projective nature and the superimposition of normal glandular tissue. Automatically determining correspondences between the two modalities could assist radiologists in the detection, diagnosis and surgical planning of breast cancer. This thesis addresses the problems associated with the automatic alignment of 3D and 2D breast images and presents a generic framework for registration that uses the structures within the breast for alignment, rather than surrogates based on the breast outline or nipple position. The proposed algorithm can adapt to incorporate different types of transformation models, in order to capture the breast deformation between modalities. The framework was validated on clinical MRI and X-ray mammography cases using both simple geometrical models, such as the affine, and also more complex ones that are based on biomechanical simulations. The results showed that the proposed framework with the affine transformation model can provide clinically useful accuracy (13.1mm when tested on 113 registration tasks). The biomechanical transformation models provided further improvement when applied on a smaller dataset. Our technique was also tested on determining corresponding findings in multiple X-ray images (i.e. temporal or CC to MLO) for a given subject using the 3D information provided by the MRI. Quantitative results showed that this approach outperforms 2D transformation models that are typically used for this task. The results indicate that this pipeline has the potential to provide a clinically useful tool for radiologists.

This thesis investigates the prevalence and pathophysiology of gastrointestinal involvement in systemic sclerosis (SSc). The primary pathologies within the gastrointestinal tract affect the mucosa, vasculature, smooth muscle and enteric nervous system. The aim of this thesis was to conduct experiments to assess these pathologies within a well-characterised SSc patient cohort. Introduction: A review of the current understanding of the pathophysiology of gastrointestinal disease in systemic sclerosis. Prevalence of GI symptoms: A prospective questionnaire study of 400 patients in order to assess gut disease burden and review of patient disease characteristics. Anorectal involvement: Extensive anorectal physiological assessment of symptomatic and asymptomatic systemic sclerosis patients compared with incontinent controls in order to assess aspects of neuropathy and myopathy. Nutritional effect as an assessment of mucosal involvement: Nutritional assessment of patients with and without gastrointestinal symptoms through anthropometric assessment, indirect calorimetry and bioelectrical impedance. The pathophysiology of gastrointestinal involvement in systemic sclerosis was further investigated in an established mouse model of scleroderma. This transgenic mouse model expresses a kinase deficient type II TGFβ receptor (TβRIIΔk) in fibroblasts and the mice develop skin fibrosis as well as pulmonary fibrosis and a structural vasculopathy. Gastrointestinal tissue from these mice was examined histologically and the contractile activity of gut tissue was examined in vitro.

Background: There is a paucity of research related to the problem of pressure ulcer in paediatrics. Variable incidence and prevalence rates have been reported, although, critically ill paediatric patients have proved to be at higher risk than those in general wards. Few investigations of contributing factors have been based on rigorous methods, and most existing risk assessment scales are either adult-based or depend simply on experience or observation. Objectives: Two separate studies were conducted as part of this research. A prevalence study aimed to measure the prevalence, location and categories of pressure ulcer, as well as pressure ulcer patients' characteristics in general inpatient paediatric wards. An incidence study was set up to measure the incidence, most affected locations, and categories of pressure ulcer, as well as significant risk factors for pressure ulcer development in critically ill children and neonates. It also aimed to compare the predictive validity of the Braden Q and the Glamorgan RASs in critical care areas. Design: One point prevalence study with a descriptive cross-sectional design and one observational cohort incidence study with longitudinal prospective design were conducted. Setting: All paediatric in-patient wards for the prevalence survey, and four paediatric critical care units (PICU, NICU, GIMU, and GICU) were surveyed in one university-affiliated hospital in Jordan. Paediatric patients in burn, isolation, and psychiatric wards were excluded. Sample: A total of 107 paediatric patients aged from birth up to 18 years old for the prevalence survey, and a total of 212 critically ill paediatric patients without pre- existing pressure ulcer for the incidence study, were recruited. Methods: All patients who met the inclusion criteria were included and assessed for pressure ulcer existence in one day for the prevalence study. Patients eligible for the incidence study were observed up to three times a week for two weeks, then once a week until critical care unit discharge, death, or when the eight week follow-up period ended. In both studies, data was collected by the primary investigator. Main Results: All identified pressure ulcers in both studies were categorised according to the European Pressure Ulcer Advisory Panel classification system. Eight patients (7.5%) had 13 PUs in the prevalence study and, of these, the majority were inpatients in critical units (87.5%, n= 7), had device-related ulcers (75%, n= 6), were female (62.5%, n= 5), younger than one year old (62.5%, n= 5), and had experienced longer stays hospital than pressure ulcer -free patients (Median (IQR)= 11 (27) vs. 4 (7)). Most of the ulcers seen were of partial thickness (category I and II) (n=6, 75%), while only two patients developed category III ulcers (25%), and none had category IV ulcers. If category I PUs were excluded, this would result in a prevalence rate of 2.8% (n= 3). The sites most frequently affected by pressure ulcer were the face (38.5%, n= 5), followed by the occiput and 'neck and shoulders', each with 15.3% prevalence (n=2). In the incidence study, 19 patients (9%) developed 29 ulcers, and as low as 5.2% when category I ulcers were excluded. Forty one per cent of pressure ulcers were category I, 48.3% category II, while only 10.3% were category III and none were category IV. The 'chest and shoulders' were the most affected areas with ulcers (20.7%, n= 6), followed by areas labelled 'other' (which included the arms, back and buttocks, as well as ears) (17.2%, n= 5), and four ulcers were located in each of the mouth, nose, 'feet and ankles' areas concurrently (13.8% for each). Based on a multivariate analysis, significant predictors of pressure ulcer were shown to be the mobility sub-item of the Glamorgan scale, and being on mechanical ventilation for 4 days or longer. The Glamorgan scale was more sensitive yet less specific than the Braden Q scale; however, neither of the scales was superior to the other in terms of its predictive validity. Conclusion: Pressure ulcers do exist in Jordanian paediatric patients, and with higher rates among those who are critically ill, thus would have its impact on changing the practice of Jordanian nurses to prevent or reduce its occurrence. Critical care unit paediatric patients most at risk include those who are supported on mechanical ventilation for longer periods, and those who are immobile. Both the Glamorgan and the Braden Q risk scales are valid tools to predict pressure ulcer among critically ill children, but neither is clearly superior to the other.