Introduction: Acute necrotizing pancreatitis remains a potentially devastating disease with a significant morbidity and mortality, but remains without specific drug therapy, Increasing evidence indicates that calcium overload leading to mitochondrial injury is central to pancreatic acinar cell fate, The mitochondrial permeability transition pore (MPTP) forms in response to cellular injury and determines cell fate, Cyclophilin-D (CyP-D) is a key regulator of MPTP formation, The role of the MPTP in pancreatitis is a novel area of investigation, This study explores the role of CyP-D and the MPTP in both murine and human isolated pancreatic acinar cell responses to pancreatic toxins as well as in the severity of murine experimental pancreatitis,

Literature Review : The prevalence of bowel disease is high in the UK with colorectal resection often a preferred intervention, creating temporary or permanent stomas. Whilst improvements in laparoscopic techniques and Enhanced Recovery Programmes, with standardised procedural developments, have improved biomedical outcomes, there has been little explicit consideration given to the psychological impacts of colorectal surgery and stoma formation. A systematic review of published research was undertaken providing analyses of findings and quality appraisals of research using standardised measures to investigate impacts of stoma status on body image and psychological well-being following colorectal resection. Eight papers reviewed revealed significant negative impacts of stoma presence for psychological disturbances and suggested this relationship is mediated by body image disturbances in stoma patients. The clinical implications of the findings are discussed and possible areas for research identified. Research Report : Limited past research has identified psychological difficulties and body image changes occurring in colorectal resection patients who require the formation of a stoma, however predictors of these constructs are relatively unknown. The Self-Regulatory Executive Function (S-REF) model advances an explanation for these findings with specific reference to cognitive schemas and intense self-focus. A mixed methodology, cross-sectional survey design was utilised, with questionnaires were returned by 84 participants. Data were examined using correlation analysis and multiple regression. Patients requiring permanent stomas demonstrated significantly higher levels of psychological morbidity and body image disturbance compared with non-stoma patients and community norms. Aspects of chronic self-focus and appearance schematisation were significantly associated with psychological morbidity and body image disturbance. Findings advanced the S-REF model, providing partial explanation for the prevalence of psychological morbidity in colorectal resection patients and suggesting possible markers for identifying individuals likely to struggle with adjusting to stoma formation with possible clinical relevance. Critical Appraisal : Reflections on the research process are summarised and critically appraised.

The emergence of new treatments in Cystic Fibrosis (CF) has emphasised the need for sensitive, repeatable, responsive and feasible measures of lung disease. FEV I is limited as an assessment tool as it is insensitive to changes in the peripheral airways. Lung Clearance Index (LCI) is proposed as an outcome measure for the detection of early lung disease and use in CF clinical trials. The use of LCI in CF is supported by the large number of studies demonstrating its robustness. However, most studies have been performed in children, with only approximately 20% of studies including adults. The aim of this study was to examine the clinimetric properties of LCI and to assess the relationships between LCI and other physiological, clinical and microbiological markers of disease in a CF child and adult group. Results of this study show that LCI had good intra and inter-visit repeatability over a longer term period. LCI had superior sensitivity to lung function abnormality and Pseudomonas aeruginosa infection, compared to FEV 1 % predicted. On assessment of routine microbiological culture results, subjects with P. aeruginosa alone had the worst LCI. Extended culture methods to detect the entire airway micro biota composition, showed that subjects with a smaller load of anaerobic bacteria had a worse LCI. LCI also correlated with biomarkers of inflammation. These results suggest that LCI may be sensitive to differences in airway microbial community composition and inflammation. Compared to spirometry, LCI correlated with more patient reported symptom and health related quality of life questionnaire scores. However, the response of LCI with intravenous antibiotic treatment for pulmonary exacerbation was not statistically significant. FEV I % predicted may be more responsive across the disease severity range in this setting. Overall, this study has shown that LCI is a robust, sensitive and meaningful measure for use in clinical trials during clinical stability, across the age range in CF.

Biliary tract cancer (BTC) is difficult to diagnose, in part related to the lack of reliable tumour markers. The aim of this project was to use whole genome RNA expression profiling in order to identify novel biomarkers for diagnosis and prognosis in biliary tract cancer. Chapter 1 summarises clinical aspects of BTC as well as current diagnostic and prognostic tests. Chapter 2 addresses the identification of circulating tumour cells for the diagnosis of BTC. It includes details of a study investigating measurement of circulating cytokeratin 19 fragments (CYFRA 21-1), demonstrating that CYFRA 21-1 is a more specific, but less sensitive diagnostic marker than CA19-9, and predicts a poor prognosis in BTC. Chapter 3 investigates the potential for using RNA isolated from archived formalin fixed, paraffin embedded (FFPE) surgical and explanted liver tissues from patients with primary sclerosing cholangitis (PSC) with and without cholangiocarcinoma, for use in whole genome RNA expression analysis. We demonstrate that, although technically possible, the rarity of samples and RNA degradation that occurs as a result of the tissue processing, are such that further evaluation using these materials is not feasible at this time. Chapter 4 addresses and validates methodology for isolating RNA from samples of biliary brushings taken at the time of endoscopic retrograde cholangiopancreatography (ERCP). We demonstrate that RNA isolated from biliary brushings is of low quantity and degraded, and that this degradation occurs in vivo. However, we demonstrate that such RNA is still useful for downstream applications such as quantitative real time PCR and is therefore suitable for whole genome RNA expression analysis using microarray technology. Chapter 5 describes the methods and results obtained from using whole genome RNA expression analysis using microarray of RNA isolated from ERCP biliary brushings. The results are presented as a shortlist of candidate genes requiring further validation. Chapter 6 provides results of qPCR studies performed in order to validate the gene expression profile identified by microarray. A selection of candidate genes are investigated using TaqMan Array and SYBR Green qPCR and demonstrate a high correlation with the pattern of expression shown by microarray. Chapter 7 investigates whether a selection of the genes identified in malignant biliary brushings are similarly upregulated in fresh frozen surgical resection material from patients with benign and malignant biliary diseases. In addition, we provide evidence for gene translation and upregulation at the protein level by immunohistochemistry for a selection of the protein products. Chapter 8 discusses the main conclusions drawn from the work as well as potential future studies.

Background and Aims: Hepatocellular carcinoma (HCC) often presents at a late stage which limits the use of curative therapy. Hence the pressing need for increasing research into newer therapies such as immunotherapy. Alpha-Fetoprotein (AFP) is an oncofetal antigen elaborated in most HCCs and is a tumour rejection antigen in animal models for HCC making it a target for the development of T cell based immunotherapy. The effect of AFP on antitumour immune responses in patients with HCC has not been explored in depth. We aimed to study the effects of AFP on the immune system cells including dendritic cells (DCs). In man, naturally occurring anti-AFP CD8 T cell responses have been detected in patients with HCC. One vital step for the design of epitope-based therapeutic vaccines is the identification and characterization of T-cell epitopes on AFP. Several AFPderived peptides have been identified and T cells recognizing these epitopes have been studied in patients with HCC. However, the role of anti-AFP CD4+ T cell responses (Th1 cells or regulatory T cells) in HCC patients has not been studied. Also, our aim was to study the role of Th1 cells in HCC patients and investigate any possible association between the expansion of these cells with clinical features of the disease such as stage of disease, serum AFP levels and tumour invasiveness. Results and Conclusions: In our first study, the treatment of monocyte-derived DCs with AFP led to DC dysfunction as detected by the down-regulation of surface molecules (CD40 and CD86) and inhibition of their T cell-stimulatory capacity. In addition, AFP treatment induced apoptosis of DCs and reduced their ability to produce TNF-alpha and IL-12. Our ex vivo results showed that the ability of monocytes, isolated from patients with elevated levels of serum AFP (>1,000 ng/ml), to produce TNF-alpha was impaired. In the second study we identified an AFP-derived T cell epitope that was recognized by circulating CD4+ T cells from patients with HCC and normal or mildly elevated AFP level in the early stage of the disease. This response was absent in healthy donors and in patients with chronic liver disease, which suggested that this response had been previously expanded in vivo in response to the tumour. The induction or activation of regulatory T cells (T-regs) by tumours or pathogens may suppress protective immunity. In the third study, we demonstrated that AFP contained an epitope which activated the expansion of inducible TGF-beta producing T-regs. In our fourth study we revealed that CD4 T-cell response expanded in the early stages of disease (Child–Pugh A score), which is usually associated with low concentrations of serum AFP. Furthermore, CD8 T cell response was largely detected in HCC patients with a Child–Pugh B or C score. Necrosis of tumour cells can activate both innate and adaptive antitumor immunity. In a further study by our group, development of higher frequencies of AFP-specific CD4+ T cells after embolisation therapy was noted. Necrosis produced by Transarterial Chemoembolization (TACE) or Chemoembolization (TAE) unmasks tumour rejection Antigen-specific T cell responses. This represented a method of in situ immune response induction that could be combined with immunotherapy to increase the frequency of AFP-specific T cells with the aim of controlling tumour growth and improving survival. Also, two further HLA-DR-restricted AFP-derived CD4+ T cell epitopes were detected. From our studies thus far, we concluded that predictive factors for detecting an AFP-specific Th1 response in patients with HCC included a serum AFP of <1000 ng/ml, Okuda stage 1 and treatment with TACE/TAE.

Introduction: The endothelial mucosal cell adhesion molecule (MAdCAM-1) is considered to be critically important in recruiting lymphocytes expressing the α4β7 cell surface integrin. In addition to its well-characterised role in the normal gastrointestinal tract, there is emerging evidence of its role in liver and gastrointestinal inflammation. The ability to detect MAdCAM-1 has thus far been challenging, hindering progress into studies to explore its modulation. Aims: (i) To characterise MAdCAM-1 in the liver and gut, (ii) establish an in vitro model system of MAdCAM-1 and (iii) investigate the factors leading to its expression and subsequent modulation. Methods: I have described novel methods of detecting MAdCAM-1 by: 1. Characterising its presence in the human liver, gut and associated tissues e.g. pancreas. 2. Developing a reverse transcriptase–polymerase chain reaction (RT-PCR) technique so as to detect MAdCAM-1 in the gastrointestinal system and thus quantify its expression using Real-Time RT-PCR analysis. 3. Developing an in vitro cell culture system using the endothelial SVEC4-10 cells to express and subsequently modulate the expression of MAdCAM-1. Results: Using immunohistochemical methodology I found that in end stage chronic liver disease, MAdCAM-1 is expressed primarily on the peribiliary plexus and lymphoid aggregates, where it may facilitate lymphocyte egress. MAdCAM-1’s constitutive expression was confirmed in histologically normal gut tissue and its upregulation was demonstrated in ulcerative colitis and Crohn’s disease, particularly localised to the venular endothelium of the lamina propria and follicular dendritic cells. MAdCAM-1 mRNA from human gut was measured by a RT-PCR technique in which a 94 base pair product consistent with human mucosal vascular MAdCAM-1 was detected in normal large bowel. Real Time analysis confirmed that MAdCAM-1 was upregulated in end stage liver disease. In a cell culture system MAdCAM-1 was shown to be upregulated by TNFα on SVEC4-10 using immunofluoresence studies and its expression was further modulated by steroids and anti-sense oligonucleotides. Conclusion: The importance of MAdCAM-1 in the gastrointestinal system is emphasised throughout. Our in vitro culture system utilising the SVEC endothelial cell line provides the basis for studying the modulation of MAdCAM-1 expression.

Chronic hepatitis C virus infection is a massive worldwide healthcare burden, which is estimated to cost in the USA alone over $5 billon per annum. Until recently, the only current effective treatment was combination therapy with peg-interferon alpha and ribavirin (peg-I FNα+RBV), which is expensive and has significant side-effects, and is only effective in up to 50% of those infected with HCV genotype 1. In this thesis, a method was evaluated for detecting genotype 1b infections using a 5’non coding region sequence based approach (TRUEGENE). This allowed the identification of genotype 1b patients who could then be analysed for mutations in key regions of the genome associated with interferon resistance. A total of 14 patients with HCV genotype 1 were prescribed peg-IFNα+RBV and subjected to detailed sequence analysis of their interferon sensitivity determining region (ISDR) Following PCR amplification and sequence analysis there was no evidence for significant mutations occurring in the ISDR in patients who did not respond to therapy either at the start of therapy of during therapy. The recognition that host genetics may play a role in HCV infection and the success of treatment with interferon based therapies is now firmly established through the identification of single nucleotide polymorphisms (SNPs) in the IL28 gene which encodes a lambda interferon. I used an allele specific SNP detection system for two loci in the IL28B gene region (rs12979860C/T and rs8099917G/T) in a large population of Gulf region, and particularly Saudi Arabian, HCV infected patients (n=315) some of whom were co-infected with either HBV (n=1 01) or HIV (n=1 00). The results showed that the genotype distribution at both SNP loci were different to other studies based on North American and European populations. In addition, when the patients were separated into responders, non-responders and transient responders, marked differences in genotype frequencies was observed together with associations between specific alleles and HCV load. The inclusion of co-infected individuals allowed me to show that IL28B polymorphisms at these two loci are not evenly distributed between patients with HCV mono-infection and co-infections. In the final phase of the thesis, I used a proteomic approach to indentify novel proteins that may be useful as biomarkers for treatment response. After optimization of the electrophoretic procedure the patients who had been used for the earlier ISDR studies were analysed. Although preliminary, the data identified haptoglobulin as a protein whose expression pattern inversely correlated with the control of replication after therapy. In conclusion this thesis has attempted to identify viral and host genetic markers that contribute to treatment success and failure in HCV and has shown that differences in the prevalence of IL28B polymorphisms exist between Saudi Arabia and elsewhere and between patients harbouring dual infections compared to single HCV infection.

Plasma Apolipoprotein AI (ApoAI; 28-kDa) is the major constituent of HDL and is atheroprotective. A natural variant, ApoAI-Milano (ApoAI-M), has an Arg-173Cys substitution and is “super-atheroprotective”. The Cys residue allows formation of disulphide-linked homodimers, as well as heterodimers with ApoA-II, and confers HDL with unique structural and functional properties. ApoAI-Paris (Arg-151Cys; ApoAI-P) is also a natural variant with similar anti-atherogenic properties to ApoAI-M. The aim of this thesis was to inhibit development of atherosclerotic lesions by expressing high, sustained levels of human ApoAI and variants from livers of hyperlipidaemic ApoE-/- mice. First, I cloned wild-type (wt) ApoAI, ApoAI-M and the double mutant, ApoAI-MP, into self-complementary, adeno-associated virus serotype-2 (scAAV2)-based plasmids. Liver-specific expression was driven by the strong, but short, promoter LP1 containing a hepatic control region and the alpha-1-antitrypsin gene promoter/ enhancer. I verified construct viability by transfecting cultured cells invitro, which confirmed secretion of monomer ApoA-I proteins and, with ApoAI-M and ApoAI-MP variants, formation of dimers (>50-kDa). In-vivo hydrodynamic tail-vein injections of plasmids further confirmed transgene expression. Next, I generated wtApoAI, ApoAI-M and ApoAI-MP viral vectors by cross-packaging with serotype-8 capsid, which has high hepatic tropism. Tail-vein (liver-directed) injection of viruses demonstrated increasing secretion of recombinant proteins into plasma over a 12 week period for ApoAI and both variants. At termination (12 weeks), atherosclerotic lesion size was determined by aortic en face analysis. Expression of wtApoAI reduced progression of atherosclerosis by 47% (P=0.001) compared to control. In contrast, ApoAI-M reduced progression by only 17% (P=0.07), although the reduction with ApoAI-MP was 41% (P=0.002). These findings were consistent with the increases in plasma HDL, which were wtApoAI > ApoAI-MP > ApoAI-M. In conclusion, liver-directed scAAV2/8LP1-mediated gene transfer of wtApoAI significantly reduced atherosclerotic lesion progression in ApoE-/- mice, whereas ApoAI-M was less potent. Interestingly, the ApoAI-MP variant, which has increased dimer formation compared to ApoAI-M, also significantly reduced atherogenesis, suggesting it has potential as an anti-atherogenic candidate gene.

Liver failure remains a disease with a high mortality and with the exception of transplantation therapeutic options are limited. The liver however has regenerative potential, and strategies based not only at supporting the failing liver, but promoting its recovery would be a significant evolution. Plasma from patients with liver failure contains toxic molecules that have many effects on the liver including loss of cell viability. These factors represent a significant barrier to stem cell transplantation, bioreactor function and autologous liver recovery, suggesting removal or antagonism of these factors may be appropriate therapeutic strategies. Since apoptosis has been implicated in the pathogenesis of a number of liver diseases including liver failure we proposed that it may be one of the mechanisms by which plasma is toxic to hepatocytes. We developed and validated a model using primary human hepatocytes to investigate if plasma from patients with acute and acute-on-chronic liver disease was pro-apoptotic. Compared with normal plasma, acute liver failure plasma induced apoptosis whereas plasma from patients with acutely-decompensated chronic liver disease did not. Having identified that acute-liver failure plasma was pro-apoptotic we investigated the pathway via which the apoptosis was mediated by using specific inhibitors of caspases, key components of the death receptor and mitochondrial pathways. We found that apoptosis was induced via a pathway involving caspase 8 and caspase 3, suggesting involvement of the death-receptor pathway. We investigated the effects of Caspase inhibition as a therapeutic option in acute liver failure by using an established animal model but did not find an improved outcome in treated animals. We also investigated the effects of treatment with molecular adsorbent dialysis (MARS) on the pro-apoptotic effects of plasma and found MARS dialysis improved biochemical parameters, indicating effective removal of albumin-bound molecules, but the apoptotic effects of the patients' plasma were unchanged.

Purpose: Neuroendocrine Tumours (NETs) are rare and therefore poorly understood. Their slow progression and general poor response to standard chemotherapy regimes implies that their tumour biology is significantly different from most common cancers. There have been major advances in the past few decades in the diagnosis and management of these tumours which are perceived to be associated with improvements in quality and length of life. However, since most NETs have metastasised extensively by the time of diagnosis it is a major challenge indeed to try and effect tumour regression – the ultimate goal of any anti-cancer therapy. Chapters: 2 & 3: The epidermal growth factor receptor (EGFR) is commonly expressed in human tumours and provides an important target for therapy. Several classes of agents including small molecule inhibitors and antibodies are currently under clinical evaluation. These agents have shown interaction with chemotherapeutic agents in vitro and in vivo. However, the mechanisms of these interactions are not clearly understood, particularly in regards to NETs. The purpose of this study was to investigate the expression of EGFR in NET tissue and to determine its mechanisms of action, as well as the effects of modulation of EGFR activation. Experimental Design (Chapter 2): Paraffin-embedded tumour tissue was available from 98 patients with NETs (39 foregut, 42 midgut, four hindgut, five paragangliomas, and four of unknown origin). Immunohistochemical evaluation was performed for the expression of EGFR, p-EGFR, p-Akt, and p-ERK1/2. Results: Ninety-six percent of tumour samples were positive for EGFR expression; 63% were positive for activated EGFR; 76% were positive for activated Akt; and 96% were positive for activated ERK1/2. Importantly, the histological score for the activation of Akt and ERK1/2 correlated with the histological score for activated EGFR. These data provide a rationale for considering EGFR inhibitors in the treatment of NETs. Additionally, direct inhibition of Akt and ERK1/2 may provide further therapeutic options in the treatment of NETs in the future. Experimental Design (Chapter 3): The effects of the EGFR inhibitors gefitinib and erbitux were determined in several NET cell lines. The modulation of DNA-PK activity by these agents was quantitated using a variety of techniques including immunoprecipitations, immunoblotting, cellular fraction extractions and immunohistochemistry. Results: Prolonged EGFR inhibition leads to reduction in DNAPKcs concentration in all tested NET cell lines except RIN, which displays the lowest levels of EGFR expression. This reduction in DNAPKcs is likely to lead to sensitisation of NET cells to ionising radiation, which causes double-strand DNA breaks, DNAPKcs being crucial to their repair. There are direct EGFR/DNAPKcs interactions in NET cell lines, which can be enhanced by EGFR inhibition. EGFR inhibition leads to transfer of DNAPKcs from the nucleus to the cytoplasm in SHP and BON cell lines but not in CRI, RIN, or NCI cell lines. This difference in the observed outcomes may be due to the lack of or mutations in intermediary proteins, though proof is needed for this hypothesis. The re-distribution of DNAPKcs is likely to lead to sensitisation of NET cells to ionising radiation, which causes double-strand DNA breaks, DNAPKcs being crucial to their repair. Chapter 4: Surgery is at present the only therapy with the possibility of achieving a cure. Therefore optimising diagnostic modalities in order to discover the tumours early or to discover all the tumour lesions prior to surgery would be of use. The purpose of this project was to assess the role of 99mTc-depreotide in patients with negative or weakly positive OctreoScan® (Krenning score \geq 1; measured on a scale range 0-4). To determine the usefulness of 99mTc-depreotide scintigraphy for highlighting lesions that may be missed by OctreoScan® and/or CT/MRI imaging. Experimental Design: Prospective analysis of 25 NET patients, with negative or weakly positive ¹¹¹In-pentetreotide scans, who were consecutively enrolled to undergo ¹¹¹In-pentetreotide and 99mTc-depreotide imaging. The results were compared with either CT or MRI scans. Results: Histology was available for 20 of 25 patients: of these 40% had high grade tumours (cellular proliferation marker Ki67 score > 20%), a further 35% had intermediate grade tumours (Ki67 2-20%), and the remainder 25% had low grade tumours (Ki67<2%). 52% of patients had completely negative and 48% had weakly positive OctreoScan®. 32% of these same patients had significantly positive 99mTc depreotide scans (Krenning score \geq 2), with the histology demonstrating intermediate or high grade tumours. Chapter 5: Some NETs follow an indolent course compared to others which display a rapidly progressive course with only short-lived response to therapy. Being able to confidently predict the long-term prognosis is obviously desirable. The aims of this project were to determine the diagnostic and prognostic value of serum alphafetoprotein (AFP) and human chorionic gonadotrophin beta (hCG\beta) in NETs. Patients and methods: a database containing biochemical, histological, and survival data on 360 NET patients was constructed. This data was statistically assessed, using SPSS statistics package, to determine the utility of commonly measured tumour markers with particular emphasis on AFP and hCG\beta. Results: AFP and hCG\beta were raised in 9.5% and 12.3% of patients respectively, and jointly raised in 9.1% of patients in whom it was measured. AFP levels associated strongly and positively with tumour grade, serum CgA, hCG\beta levels and worse survival. hCG\beta levels also associated strongly and positively with serum CgA, AFP levels and worsening survival.