This thesis centres on the development of arterial spin labelling (ASL) MRI, a non-invasive technique to image cerebral perfusion. In the first chapter I explain the principles of cerebral blood flow (CBF) quantification using ASL beginning with the original implementation through to the most recent advances. I proceed to describe the established theory behind the key additional MRI contrast mechanisms and techniques that underpin the novel experiments described in this thesis (T2 and T1 relaxation, diffusion imaging and half-Fourier acquisition and reconstruction). In Chapter 2 I describe work undertaken to sample the transverse relaxation of the ASL perfusion-weighted and control images acquired with and without vascular crusher gradients at a range of post-labelling delay times and tagging durations, to estimate the intra-vascular, intra-cellular and extra-cellular distribution of labelled water in the rat cortex. The results provide evidence for rapid exchange of labelled water into the intra-cellular space relative to the transit-time through the vascular bed, and provide a more solid foundation for CBF quantification using ASL techniques. In Chapter 3 the performance of image de-noising techniques for reducing errors in ASL CBF and arterial transit time estimates is investigated. I show that noise reduction methods can suppress random and systematic errors, improving both the precision and accuracy of CBF measurements and the precision of transit time maps. In Chapter 4 I present the first in-vivo demonstration of Hadamard-encoded continuous ASL (H-CASL); an efficient method of imaging small volumes of labelled blood water in the brain at multiple post labelling delay times. I present evidence that H-CASL is viable for in-vivo application and can improve the precision of δa estimation in 2/3 of the imaging time required for standard multi post labelling delay continuous ASL.

The tumour suppressor p53, is a key regulator in deciding between cell survival or cell death by its ability to induce cell cycle arrest and apoptosis. It is one of the most frequently mutated genes in human cancers and influences the efficacy of many cancer therapies. The newly identified ASPP family of proteins is a specific regulator of p53-, p63- and p73-mediated apoptosis. The family consists of three members, two pro-apoptotic proteins ASPP1 and ASPP2, which selectively stimulate p53's apoptotic function and one anti-apoptotic member, iASPP, which inhibits p53-mediated cell death. This thesis has focused on identifying changes in iASPP protein expression in response to chemotherapeutic drugs. Our data describes a substantial decrease in mobility of iASPP by Western blot analysis, following treatment of cells with arsenic trioxide and microtubule drugs such as nocodazole, taxol and colchicine. Importantly, the iASPP expression pattern correlated with the cellular physiology. More precisely, a small decrease in iASPP mobility was associated with cells arrested in mitosis, while a more substantial mobility shift was related to cell death. These observations were confirmed in a variety of cell lines derived from solid tumours and haematological malignancies. We found that this mobility shift was caused by post-translational modifications of iASPP at the N-terminus in a defined region spanning amino acids 91 until 125. Mass spectrometric analysis provided evidence for phosphorylation on specific serines. Moreover, analysis of antibody epitopes and iASPP point mutants, provided evidence for the involvement of other residues within this epitope, suggesting multiple modifications. Furthermore, the signalling pathways leading to this iASPP modification following treatment with arsenic trioxide and nocodazole involve oxidative stress, mitogen-activated protein kinases, nuclear factor-B, glycogen synthase kinase-3, prolyl hydroxylases and cyclin-dependent kinases. All these data leads to a better understanding of the regulation of iASPP expression and function in cancer cells under normal and stress conditions. Additionally, increased knowledge of the mechanisms of action of these drugs could lead to new applications or improved strategies for treating malignancies.

This thesis examined the mechanisms underlying small intestinal tumourigenesis in experimental models and humans with a mutation in the Adenomatous Polyposis Coli gene. Mutant APC alleles found in human intestinal carcinomas retain two 20 amino acid repeats (20AARs), required for beta catenin binding. Current APC mutant experimental model systems do not contain mutant APC proteins analogous to those found in human disease. I therefore generated and characterised a novel model of intestinal tumourigenesis in order to evaluate the effect of two 20AARs on optimal levels of nuclear beta catenin required for tumourigenesis. I developed a further model in order to examine the role of the C terminus of APC in intestinal tumourigenesis. I found that two 20AARs generated an intermediate level of nuclear beta catenin which was optimal for tumourigenesis and the N terminus alone was sufficient for tumourigenesis. The presence of two 20AARs conferred a more severe phenotype than models with zero 20AARs. An increase in the stem cell number and a high crypt fission index accounted for the observed difference in severity. I extended these findings to patients with duodenal Familial Adenomatous Polyposis (FAP) and found increased stem cell numbers and a high crypt fission index in these patients compared with patients without APC mutations. I examined normal human duodenum and found that niche succession, monoclonal conversion and crypt fission led to the spread of mutant clones in intestinal mucosa, using mitochondrial DNA analysis.

Introduction: The main aim was to investigate Dukes B cancer prognosis using array comparative genomic hybridisation (aCGH). The cancers were typed for microsatellite instability (MSI), APC loss of heterozygosity (LOH) and ploidy. A secondary aim was the selection, on the basis of aCGH results, of potential colorectal cancer genes using In Situ Hybridisation (ISH). Sporadic adenomas were also investigated with aCGH to investigate the timing of chromosomal instability in tumourigenesis. Methods: Dukes B cancers collected in Oxford, Leeds and Harrow were examined using aCGH based on a set of 3452 BAC clones at ~1Mb spacing. MSI was assessed using BAT26 and D5S346. D5S346 gave LOH at the APC gene locus. Ploidy was assessed using flow cytometry (FACS). From the aCGH data genes in regions of copy number gain were chosen for ISH analysis. Results: 79 Dukes B cancers (43 good outcome, 36 bad outcome on the basis of 5 year survival) were investigated with aCGH. The most commonly gained chromosomes across all cancers were 13, 20 and 7, and the most commonly lost were 22, 18 and 14. Comparing survival groups; chromosome 6 was more often lost in cancers associated with good outcome, chromosome 16 was more often gained in microsatellite stable cancers associated with good outcome, and chromosome 22 more often gained in microsatellite stable cancers associated with poor outcome. Chromosome 13 showed greater than single copy number change significantly more often in bad outcome cancers. Several new areas of small genomic gain and loss were detected. Four candidate genes were identified (CDX2, RHOA, FLT1 and ARHGEF1). None showed a relationship with outcome. Large scale chromosomal changes were found in 10 of14 sporadic adenomas. Conclusions: Array CGH did identify some difference between good and bad outcome Dukes B cancers. However on the basis of this data, the technique could not be used as a useful clinical tool to predict prognosis.

Positron Emission Tomography (PET) with 18F-FDG has emerged as a powerful tool in oncology. Furthermore, recent advent of PET/CT and novel tracers are continually expanding its role. This thesis investigates its application in two solid cancer models. In the diagnosing of primary pancreatic cancer, 18F-FDG PET/CT was shown to be more accurate than conventional CT. It did not add information to locoregional staging of disease but impacted management of patients with potentially operable tumours, by accurately confirming the presence / absence of metastases. In the pre-operative staging of patients with colorectal liver metastases (CLM), 18F- FDG PET/CT was also superior to CT in assessing extrahepatic disease, where it again impacted management. The accuracy of detecting hepatic disease was similar for both. Compared to PET alone, PET/CT improved the accuracy of lesions localization and interpretation. Next, the feasibility of imaging with the novel thymidine analogue tracer 18F-FLT was investigated. Overall, 18F-FLT PET was less accurate than 18F-FDG in detecting lesions in both cancer types, thus suggesting it to be an unsuitable tracer for routine diagnosis and staging. In the cohort of pancreatic cancer patients, 18F-FLT uptake (SUVs) were found to strongly correlate with the immunohistochemical proliferation marker, Ki-67 antigen. This supported 18F-FLT‟s potential role as a surrogate marker of proliferation. The prognostic implications of these require further investigation. Finally, an in vitro model was use to examine early changes in 18F-FLT uptake in response to treatment with cytotoxics. At 2 hours following pulse treatment with 5-fluorouracil, (and before changes in cell numbers and cell cycle phase were seen), a dose dependent increase in 18F-FLT uptake was seen. No change was observed with 18F-FDG nor following Cisplatin treatment. This adaptive response may have a role as an early predictor of response to 5-FU (and potentially other antimetabolites), which requires further investigation.

Respiratory motion during cardiac Positron Emission Tomography (PET) Computed Tomography (CT) imaging results in blurring of the PET data and can induce mismatches between the PET and CT datasets, leading to attenuation-correction artefacts. The aim of this project was to develop a method of motion-correction to overcome both of these problems. The approach implemented was to transform a single CT to match the frames of a gated PET study, to facilitate respiratory-matched attenuation-correction, without the need for a gated CT. This is benecial for lowering the radiation dose to the patient and in reducing PETCT mismatches, which can arise even in gated studies. The heart and diaphragm were identied through phantom studies as the structures responsible for generating attenuation-correction artefacts in the heart and their motions therefore needed to be considered in transforming the CT. Estimating heart motion was straight-forward, due to its high contrast in PET, however the poor diaphragm contrast meant that additional information was required to track its position. Therefore a diaphragm shape model was constructed using segmented diaphragm surfaces, enabling complete diaphragm surfaces to be produced from incomplete and noisy initial estimates. These complete surfaces, in combination with the estimated heart motions were used to transform the CT. The PET frames were then attenuation-corrected with the transformed CT, reconstructed, aligned and summed, to produce motion-free images. It was found that motion-blurring was reduced through alignment, although benets were marginal in the presence of small respiratory motions. Quantitative accuracy was improved from use of the transformed CT for attenuation-correction (compared with no CT transformation), which was attributed to both the heart and the diaphragm transformations. In comparison to a gated CT, a substantial dose saving and a reduced dependence on gating techniques were achieved, indicating the potential value of the technique in routine clinical procedures.

Background and Aims: The interleukin 18 (IL-18) system consists of a proinflammatory cytokine (IL-18), a naturally occurring inhibitor (IL-18BP), and a dimeric receptor. IL-18 has been implicated in many autoimmune conditions, with elevated plasma levels predicting future risk of heart disease in healthy individuals. IL-18 also appears to have metabolic consequences, with IL18 -/- mice having higher bodyweight, attributable to increased feeding. The central hypothesis of this work was that genetic variation within both IL-18 and IL-18BP, that influenced their circulating levels, would be associated with individual risk for both heart disease and obesity. This was tested by measuring IL-18 levels in several study groups and accessing their correlation with numerous single nucleotide polymorphisms (SNPs) (chosen using a tagging SNP (tSNP) methodology) within both genes. Any effects (either single SNP or haplotypic) were then assessed directly using quantitative PCR (QPCR). Results: For both IL-18 and IL-18BP, tSNP sets were chosen that captured greater than 90% of the common genetic variation seen in a representative European Caucasian sample. Their association with levels of IL-18 and interleukin 18 binding protein (IL-18BP) were assessed in healthy and diseased individuals; genetic variation in IL-18 only was associated with differences in plasma IL-18 (and free IL-18 (fIL-18)) levels in all cohorts. Carriage of the rare allele of IL-18-5848T>C was associated with, on average across all study groups, 44% higher IL-18 levels than TT homozygotes. There was also a haplotypic effect, with a common haplotype - hGTATA (frequency of 20%) - being associated with 30% lower IL-18 levels. The effect was consistently of greater magnitude in diseased than healthy individuals. Despite IL-18 levels being elevated in those individuals who went on to suffer a myocardial infarction (MI) over 15 yr of follow-up (277.6 pg/ml vs. 239.6 pg/ml, p=0.05), there was no significant difference in genotype, or haplotype, frequencies in those who had heart disease compared to those who did not. No consistent association between BMI/obesity and IL-18 levels was observed, however, in two of the study groups genetic variation in IL-18 was associated with significant differences in body mass index (BMI). Overall, hGTATA was associated with a 9% higher BMI, equating to a 7 kg greater body weight for an average male. In in vitro studies of IL-18 expression in healthy, male hGTATA carriers, no significant difference in IL-18 messenger RNA (mRNA) concentrations were observed when compared to hGCATA controls. Conclusion: The data presented here disagrees with previously published results, showing no role for variation within IL-18 in establishing heart disease risk. However there are novel findings that suggest it may play a role in weight control. Given that these effects appear not to be mediated through plasma IL-18 levels, the identification of individuals through genetic testing may be especially relevant.

Rationale: 1. To characterise and quantify the cytokine response to critical illness and relate this response to common metabolic features of the illness; 2. To determine whether these immunologic and metabolic responses can be modified using enteral immunonutrition. Methods: Circulating cytokine concentrations (TNFα, IL-6, IL-10, TGF-β1, IL-lra, sTNF-RI and sTNF-RII), c-reactive protein and cortisol levels were determined using the ELISA technique. Cytokine genotypes (IL-10, TNFα and TNFβ) were determined following the amplification of DNA using polymerase chain reaction (PCR) then by using sequence specific oligonucleotide probes (SSOP) or Ncol restriction digest. Energy expenditure was measured continuously in mechanically ventilated patients by indirect calorimetry. The modified enteral formula, supplemented with glutamine, arginine, fish oil, nucleotides, fibre and antioxidants, was compared with an isocaloric, isonitrogenous control feed as part of a prospective, randomised, double-blind, controlled trial. Results: The frequency of IL-10, TNFα and TNFβ genotypes, between the ICU population and controls differed significantly. There were fewer high IL-10 producers in the ICU population (6% versus 30%, p<0.001) and fewer intermediate TNFα producers (6% versus 23%, p<0.001). The distribution of TNFβ genotypes was also significantly different between patients and controls (p=0.008). A high admission IL-6 level (>1000pg/ml) or an increase in IL-6 concentration during ICU admission was associated with an increased risk of mortality (p=0.005 and p=0.036, respectively). Elevated admission levels of IL-1ra, sTNF-RI and sTNF-RII were also associated with increased mortality risk (p=0.039, p=0.05 and p=0.05, respectively). Soluble TNF-RI was significantly correlated with measures of protein catabolism; urinary nitrogen (adj r²=0.166, p=0.002) and percentage energy derived from protein oxidation (adj r²=0.138, p=0.009). Both cortisol and c-reactive protein levels were weakly, but statistically highly significantly, correlated with anti-inflammatory cytokines IL-10 and TGFβ. Immunonutrition did not influence either pro- or anti-inflammatory cytokine responses in this ICU population. There was however a tendency to reduced mortality rate (32% versus 22%, p=0.522); reduced ICU length of stay (10 days versus 12 days, p=0.915) and reduced number of MODS days (5.5 days versus 7 days, p=0.843) with the trial formula. Conclusions: Genetic variation in IL-10 and/or TNFα production may be protective of admission to the ICU. Admission levels of the pro-inflammatory cytokine, IL-6, and to a lesser extent soluble receptors and the receptor antagonist (sTNF-RI, sTNF-RII and IL-1ra) can provide an indication of patient outcome. Overall cytokines correlate weakly (but statistically highly significantly) with metabolic and clinical features of critical illness. A randomised controlled trial of enteral immunonutrition has failed to demonstrate any statistically significant benefits in this general ICU population with MODS.

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