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The C-terminal tensin-like (Cten) gene is a member of the tensin family and is localised at the cytoplasmic tail of β-integrin. It is involved in various biological events although the role of in the development of colorectal cancer (CRC) is uncertain. In order to study this, the expression of Cten during the development of CRC was initially evaluated using immunohistochemistry (IHC) on colorectal adenomas and carcinomas. Positive immunoreactivity for Cten was observed in 317/342 (92.6%) of CRC and 19/20 (90%) of colorectal adenoma. High Cten expression was significantly associated with advance Dukes stage (p=0.001), lymph node metastasis (p<0.001), extra-mural vascular invasion (p=0.001) and distant metastases (p=0.008). Survival analysis demonstrated that patients with high Cten expression had significantly shorter disease free survival (DFS) on univariate analysis (p<0.001) a trend towards Cten expression as an independent predictor of DFS on multivariate analysis (p=0.071). To further test the association with metastasis, the role of Cten in metastasis was tested by (a) intrasplenic injection of CRC cells stably transfected with Green Fluorescent Protein (GFP) tagged Cten into nude mice and (b) testing a series of primary CRCs and their metastases by IHC. Compared with control mice (injected with cells transfected with GFP empty vector), mice injected with cells expressing Cten developed larger tumours in the spleen (p=0.03) and liver (p=0.05). Compared with primary tumours, the metastatic deposits had a significantly higher frequency of nuclear localisation of Cten (p=0.002). To further investigate the potential role of Cten in metastasis, in-vitro models were used to investigate Cten function. Ectopic expression of Cten in the HCT116 CRC cell line (which expresses low levels of Cten) caused changes in cell morphology and increased cell motility (both migration and invasion). Conversely, the reciprocal Cten knock-down experiments in SW620 CRC cell line (which expresses high levels of Cten) resulted in inhibition of both cell migration and invasion. Since Cten is in complex with integrins at focal adhesions, its interactiosn with integrin-linked kinase (ILK), focal adhesion kinase (FAK) and CD24 were tested. Cten was shown to regulate ILK, FAK and CD24. Moreover, inhibition of CD24 after forced expression of Cten abrogated the Cten-mediated effects on both cell motility and protein levels of ILK and FAK. The studies were expanded and Cten expression was tested by IHC in another cancer model i.e. breast cancer (BC).Consistent with the data from CRC, increased Cten expression in BC was found to be associated with poor prognostic variables and shorter disease free survival. In conclusion, Cten expression is associated with poor prognosis in CRC and BC. This may be consequent to an ability to enhance metastasis which is related to promotion of cell motility. The activities of Cten are probably consistent across different tumour models.

Host-microbial interactions are of major importance in the pathogenesis of inflammatory bowel disease (IBD). Toll-like receptors (TLR) are pattern recognition receptors which recognise conserved molecular patterns derived from micro-organisms. Crypt intestinal epithelial cells (IEC) were isolated form mucosal specimens of healthy controls and patients with IBD (ulcerative colitis, UC, and Crohn's disease). A population of IEC enriched for intestinal stem cells (ISC) were identified using Hoechst dye exclusion and by their adherence to cultured primary intestinal myofibroblast cell monolayers. Compared to healthy control colon, TLR2 and TLR4 mRNA and surface protein were significantly up-regulated in crypt IEC isolated from the inflamed mucosa of UC and Crohn's colitis. Compared to healthy control ileum, TLR4 mRNA was significantly up-regulated in crypt epithelial cells isolated from the inflamed mucosa of Crohn‟s ileitis. TLR2 and TLR4 mRNA expression from histologically normal and inflamed colonic mucosa in UC did not significantly differ, and expression of TLR4 transcripts was significantly greater in crypt IEC isolated from histologically normal proximal colonic mucosal samples compared to healthy controls. Myofibroblast-adherent crypt cells expressed TLR2 and TLR4 protein to a greater level than the underlying myofibroblasts. Hoechst-effluxing putative intestinal stem cells expressed both TLR2 and TLR4 transcripts and protein, and TLR3 and TLR5 transcripts. In conclusion, crypt intestinal epithelial cells up-regulated TLR2 and TLR4 expression in UC and Crohn's colitis and up-regulated TLR4 expression in Crohn's ileitis. TLR2 and TLR4 was expressed constitutively in crypt IEC from histologically normal mucosa, suggesting differential TLR expression may in part be a primary event in UC. This provides further insights into the pathogenesis of IBD. Putative intestinal stem cells expressed TLR2, TLR3, TLR4 and TLR5, suggesting that direct microbial sensing by ISC may be important in maintaining intestinal homeostasis and in regulating ISC function.

The Wnt signalling pathway is involved in stem cell maintenance, differentiation and tissue development, and in so doing plays a key role in controlling the homeostasis of colorectal crypts. In response to an external Wnt stimulus, the intracellular levels of the protein beta-catenin are regulated by the proteins which make up the Wnt signalling pathway. Abnormalities in the Wnt signalling pathway have been implicated in the initiation of colorectal and other cancers. In this thesis we analyse and simplify existing models of the Wnt signalling pathway, formulate models for Wnt's control of the cell cycle in a single cell, and incorporate these into a multiscale model to describe how Wnt may control the patterns of proliferation in a colorectal crypt. A systematic asymptotic analysis of an existing ODE-based model of the Wnt signalling pathway is undertaken, highlighting the operation of different pathway components over three different timescales. Guided by this analysis we derive a simplified model which is shown to retain the essential behaviour of the Wnt pathway, recreating the accumulation and degradation of beta-catenin. We utilise our simple model by coupling it to a model of the cell cycle. Our findings agree well with the observed patterns of proliferation in healthy colon crypts. Furthermore, the model clarifies a mechanism by which common colorectal cancer mutations may cause elevated beta-catenin and Cyclin~D levels, leading to uncontrolled cell proliferation and thereby initiating colorectal cancer. A second model for the influence of the Wnt pathway on the cell cycle is constructed to incorporate the results of a recent set of knockout experiments. This model reproduces the healthy proliferation observed in crypts and additionally recreates the results of knockout experiments by additionally including the influence of Myc and CDK4 on the cell cycle. Analysis of this model leads us to suggest novel drug targets that may reverse the effects of an early mutation in the Wnt pathway. We have helped to build a flexible software environment for cell-based simulations of healthy and cancerous tissues. We discuss the software engineering approach that we have used to develop this environment, and its suitability for scientific computing. We then use this software to perform multiscale simulations with subcellular Wnt signalling models inside individual cells, the cells forming an epithelial crypt tissue. We have used the multiscale model to compare the effect of different subcellular models on crypt dynamics and predicting the distribution of beta-catenin throughout the crypt. We assess the extent to which a common experiment reveals the actual dynamics of a crypt and finally explain some recent mitochondrial-DNA experiments in terms of cell dynamics.

The development of systems for the long term in vitro culture of functional liver tissue is a major research goal. The central limitation of experimental systems to date has been the early de-differentiation of primary hepatocytes in cultures. Several factors including cell-cell interaction, cell-matrix interaction, soluble factors and 3D structures have been identified as the keys to overcome this limitation. The first aim of this project is to compare the established 3D model, co-culture of hepatocytes and hepatic stellate cells (HSCs) on PDLLA coated surfaces, to other best available systems using collagen and Matrigel. The hypothesis is that hepatocytes functionalities, established by cell-cell interaction, 3D structures and soluble factors, can be further enhanced by introduction of cell-matrix interaction. In order to test the hypothesis, rat hepatocytes were cultured in five different systems, including monoculture of hepatocytes on collagen gel, in collagen-Matrigel sandwich, co-culture of hepatocytes and HSCs on collagen gel, in collagen-Matrigel sandwich and on PDLLA coated surface. Hepatocyte specific function assays, namely albumin secretion, urea secretion, testosterone metabolism by HPLC and CYP activities by LC-MS-MS, were used to analyze cell functionalities. Homo-spheroids were only formed in monoculture on collagen gel, but hetero-spheroids were developed in all the co-culture systems. The results of function assays showed hepatocytes in collagen-Matrigel sandwich configuration had the best secretion of albumin and urea and best CYP activities during the culture period. These data demonstrated the hypothesis that hepatocyte functions of the established model can be further improved by introduction of cell-matrix interaction. In addition to establishment of rat hepatocyte culture systems, hetero-spheroids of primary human hepatocytes and primary human HSCs on PDLLA coated plates were developed successfully, due to the great improvements of isolation and culture of primary human HSCs. However, hepatocyte function assays have not been applied yet. Hepatic cell lines have several advantages that are not applicable to primary cultured human hepatocytes, namely unlimited lifespan and stable phenotype. The immortalized Fa2N-4 cell lines have recently been assessed as replacements of primary human hepatocytes in CYP induction studies. The second aim of this study was to simultaneously characterize CYP1A2, CYP2C9, CYP3A4 and CYP2B6 induction in Fa2N-4 cells through assessment of mRNA, protein and activity endpoints for a range of prototypical compounds (previously assessed in human hepatocytes) with known positive and negative induction potential. LC-MS-MS and RT-PCR were used for assessment of activity and mRNA endpoints respectively. As a result, it is considered that Fa2N-4 cells offer a substitute for primary human hepatocytes for CYP1A2 and CYP3A4 induction but not for CYP2B6 due to lack of cytosolic CAR expression.

Background Liver disease is a serious problem both in the UK and globally. While the incidence and mortality from several chronic diseases are decreasing, mortality from liver disease is increasing. As well as the medical sequelae for an individual with liver disease, in the UK the increase in chronic liver disease poses particular problems with respect to increasing hospital admissions, mortality and significant costs to the public both in terms of treatment and in loss of productivity. The increase in society of several risk factors for chronic liver disease, notably alcohol intake, obesity and type 2 diabetes, mean that these problems are likely to increase in the future. Despite these apparent problems there are surprisingly few reliable sources of data on the occurrence of chronic liver disease (cirrhosis) in the general population of the UK and the rate and consequence of disease progression particularly among ambulatory patients. Nor are their robust estimates of the prevalence of abnormal liver function tests (which may represent undiagnosed liver disease) and their associations with mortality. This thesis utilises two distinct datasets to examine separate areas of interest in the epidemiology of liver disease in the UK. The first three studies contained within this thesis are concerned with the epidemiology of cirrhosis in the general population of the UK. The second group of three studies focuses on the prevalence of elevated liver function tests in a population of older people in the UK, the demographic, clinical and lifestyle factors associated with such and the mortality following an elevated liver function test. Objectives 1. To estimate the incidence and prevalence of cirrhosis in the population of the UK 2. To describe the mortality associated with cirrhosis compared with the general population and the disease progression of cirrhosis 3. To estimate the prevalence of elevated liver function tests among people aged 75 and over in the UK 4. To describe the association between elevated liver function test and demographic, lifestyle, clinical characteristics and mortality among people aged 75 and over. Methods To examine objectives 1 and 2 I utilised the General Practice Research Database (GPRD) constructing a population based cohort of 4537 subjects with cirrhosis and 44,403 age, sex and practice matched controls. I used Poisson regression to estimate incidence rate ratios and describe trends in alcoholic and non-alcohol-related cirrhosis. Using Cox regression within an historical matched cohort design I estimated the absolute excess mortality rates and hazard ratios for mortality in people with cirrhosis compared to the general population. I described the probability of progressing from one disease state to another. To examine objectives 3 and 4 I accessed data from one arm of the Medical Research Council (MRC) Trial of Assessment and Management of Older People in the Community, a representative sample of community dwelling people aged 75 and over, totalling 15,308 participants. The prevalence of abnormal liver function was described as the proportion of study participants with elevated aspartate transaminase, alkaline phosphatase or serum bilirubin. Associations between elevated liver function and demographic, lifestyle and clinical factors were examined using multivariable logistic regression. I determined the absolute mortality rates and hazard ratios for all-cause and cause-specific mortality using a Cox proportional hazards model. Findings Epidemiology of cirrhosis (GPRD) These studies have shown an increasing trend in both the incidence and prevalence of cirrhosis in the UK with an estimated 45% increase in incidence of cirrhosis in the 10-year period studied. I estimate that 76 per 100,000 people were living with cirrhosis in 2001. Just over half of all cirrhosis was associated with alcoholism. Disease progression with cirrhosis among this mainly ambulatory population was rapid with a rate of decompensation in people with compensated disease of 5% per year and 1 in 10 dying in the first year following diagnosis. This figure increased to 25% of people dying within one year for those with decompensated disease. Mortality in subjects with compensated and decompensated cirrhosis was 93.4 and 178.0 per 1000 person years compared with only 19.2 per 1000 person years in the general control population. Following adjustment for age and sex people with compensated and decompensated disease were respectively 5 and 10 times more likely to die than the general population. Epidemiology of abnormal liver function tests (MRC cohort) Abnormalities in liver function were common with roughly 1 in 6 people aged 75 and over having at least one elevated liver enzyme, although most of these elevations were mild. A single elevated measurement of aspartate transaminase was associated with an increased consumption of alcohol and a lower age in contrast with that of a single measurement of alkaline phosphatase which showed an association with higher age and lower alcohol consumption. An elevated bilirubin measurement was strongly associated with being male. Having a single elevated liver function test was associated with a modest increase in the hazard of death compared with people with normal liver function tests (adjusted hazard ratio for death 1.27 (95% CI[1.19, 1.36]). As well as an unsurprising increase in the hazard ratio for death from liver disease, elevated aspartate transaminase or alkaline phosphatase were both associated with modest increases in the hazard of death from cancer (adjusted hazard ratios of 1.56 (95%CI[1.21, 2.01]) and 1.61 (95%CI[1.39, 1.86]) respectively). Elevated alkaline phosphatase was additionally associated with increases in the hazard of death from respiratory disease (adjusted hazard ratio 1.58 (95%CI[1.32, 1.90])) and cardiovascular disease (adjusted hazard ratio 1.34 (95%CI[1.17, 1.55])). Conclusions From my work on the incidence and prevalence of cirrhosis I estimate that a minimum of 31,000 people in the UK are living with cirrhosis, a figure which is likely to rise given increasing trends in the incidence of cirrhosis described in this thesis. The significant mortality and disease progression associated with cirrhosis means that more needs to be done to combat both the incidence and progression of this disease both on an individual and population level. Elevations in enzymes regarded as reflecting liver function are common in people aged 75 and over and in most people these abnormalities are less than 2x the upper limit of normal for the assays used. These elevations I observed are associated with both a modest increase in all-cause mortality and also with an increase in death due to specific causes. Rather than simply a marker of liver function the investigation of people with elevated liver function tests, particularly those with severely elevated tests, may lead to the identification of potentially treatable conditions that underlie death.

Background There have been many conflicting changes in the prevalence of the risk factors for upper gastrointestinal bleeding and therefore it is not clear what the current trends in mortality or incidence are, nor which factors are important in driving these trends. As populations in many countries are ageing with an increasing burden of co-morbidity, this thesis investigates whether the relationship between non gastrointestinal co-morbidity and upper gastrointestinal bleeding might be an explanation for current trends. I hypothesised that non gastrointestinal co-morbidity was responsible for a large proportion of bleeds in the population and the deaths that occur following a bleed. Methodology Large scale routine population based data records were used to assess the current incidence and mortality trends of upper gastrointestinal bleeding in England, as well as more in depth studies of predictors of its occurrence and subsequent mortality. The databases were examined and compared to external sources to assess their representativeness, and methods for defining cases in linked primary and secondary care were developed. The specific questions addressed in the studies were: 1. What are the current trends and variations in occurrence of upper gastrointestinal bleeding? Incidence rates and adjusted incidence rate ratios were calculated by quintiles of socioeconomic status, age group, sex, region, and calendar year. 2. Has there been an improvement in 30 day mortality following upper gastrointestinal bleeding? A nested case control study using Hospital Episodes Statistics from England 1999-2007 examined mortality trends by age, sex, co-morbidity and type of bleed. 3. Does non gastrointestinal co-morbidity predict upper gastrointestinal bleeding? A matched nested case control study used the linked Hospital Episodes Statistics and General Practice Research Database to examine non gastrointestinal co-morbidity as a risk factor adjusted for other known risk factors for bleeding. Sequential population attributable fractions were calculated to estimate what each risk factor contributed to the disease burden. 4. What are the excess causes of death following upper gastrointestinal bleeding? Causes of death by ICD 10 category were extracted following a bleed from the linked Office for National Statistics death register. Crude mortality rates and excess cumulative incidence functions were calculated; the latter adjusted for the competing risks between different causes of death. Results 1. A higher incidence of upper gastrointestinal bleeding was observed in the north of England, but this variation was dwarfed by the variation associated with deprivation. Areas of greater deprivation had 2-3 fold higher rates of hospitalisation for upper gastrointestinal bleeding than areas of less deprivation suggesting that strong modifiable risk factors exist. 2. Over the last decade there was a 20% improvement in 28 day mortality following upper gastrointestinal bleeding, and those admitted with bleeding were increasingly older and had more co-morbidity. 3. A combined measure of non gastrointestinal co-morbidity was a significant independent predictor of upper gastrointestinal bleeding and explained a greater proportion of the burden of bleeding (19%) than any other risk factor in the population, including medications such as aspirin and NSAIDs. 4. More than half the absolute excess risk of death was due to co-morbidity not related to the upper gastrointestinal tract. Conclusions Non gastrointestinal co-morbidity both strongly predicts an event of upper gastrointestinal bleeding, and is responsible for a large proportion of the subsequent long term mortality. The magnitude of the association in the population explains both why its incidence had not decreased, and why the improvements in mortality were observed irrespective of endoscopic management or bleed type. Furthermore a bleed can be an indicator for a re-assessment of the severity of co-existing non gastrointestinal morbidity.

Postoperative insulin resistance (POIR) is a hallmark feature in patients having major abdominal surgery. Surgical stress may induce changes in metabolic pathways that perturb glucose homeostasis, resulting in stress hyperglycaemia. The studies described in the present thesis set out to examine the evidence behind (1) the ‘Enhanced Recovery After Surgery’ pathway (2) preoperative carbohydrate drinks (CHO), in attenuating the surgical stress response and (3) to investigate the mechanistic basis of metabolic response to surgery and the development of postoperative insulin resistance in patients having major abdominal surgery. Meta-analysis of randomised studies in patients having major abdominal surgery showed that ERAS pathway was associated with a significant reduction in length of hospital stay and postoperative complications. Meta-analysis of randomised studies using CHO was also associated with reduced length of stay and decreased POIR in support of reduced fasting times and CHO, before surgery. The study in patients having major abdominal surgery showed that surgical trauma was associated with increased skeletal muscle interleukin-6 and pyruvate dehydrogenase kinase isoform-4 (PDK4) mRNA and protein expression. Increased PDK4 was associated with a concomitant reduction in pyruvate dehydrogenase complex (PDC) activity that controls the rate of muscle carbohydrate oxidation in mitochondria, and contributed to impaired glucose tolerance and decreased mitochondrial ATP production postoperatively. One hypothesis is that by maximising the contribution of carbohydrate derived oxidative ATP regeneration by activating the PDC with the synthetic PDK4 inhibitor dichloroacetate (DCA) and/or by increasing muscle glucose uptake using CHO may reverse the changes in carbohydrate oxidation seen postoperatively. The results from the study showed that administration of either CHO or CHO with DCA attenuates the impairment of CHO oxidation and the development of POIR, induced by surgical stress. Furthermore, DCA increased mitochondrial CHO oxidation above that achieved by CHO alone, which waned by 48 hours after surgery.

Introduction: Previous research has shown that patients with Parkinson’s disease (PD), multiple sclerosis (MS) and muscular dystrophy (MD) are known to be at risk of dysphagia and could benefit from dysphagia screening. The aim of this study was to describe the use of dysphagia screening and assessment procedures amongst patients with neurological conditions when they have an unplanned admission to hospital. Methods: Two methods of data collection were used in this study. The first method was a prospective observational study to determine the use of dysphagia screening and assessment procedures amongst patients with Parkinson’s disease, multiple sclerosis and muscular dystrophy. The second, qualitative study examined clinicians’ perceptions of the factors that influenced the decision to screen for dysphagia in people with neurological conditions and the difficulties experienced. Data were collected from clinicians using semi-structured in-depth interviews. Potential interventions to improve the management of dysphagia in these conditions were identified. Results: Two hundred patients were recruited to the observational study. Thirty four percent (n=68) of this group underwent a swallow screening assessment (SSA) during the first week of admission and 93% (n=63) of these were judged to have dysphagia. Amongst those who were not assessed initially (n=132), a further 77% (n=101) were found to have dysphagia. Twenty people took part in the interview study including doctors, health care assistants, nurses and therapists. Clinicians reported that the factors which underpinned their decision to screen for dysphagia included pre-existing swallowing difficulties, recognition of symptoms, staff/relative anxiety, communication difficulties and the presenting complaint and diagnosis. However, clinicians reported that their limited knowledge, clinical competencies in swallow screening, a lack of confidence and resources, affected their practice and use of dysphagia screening and assessments. Clinicians noted a number of interventions that could improve the management of dysphagia when patients are admitted to hospital and these included: training in dysphagia screening; development of dysphagia pathways or guidelines; provision of an alert system, introduction of on-call speech and language therapy services; and research funding. Discussions and Conclusions: The findings of this study suggest that screening for dysphagia does not occur routinely when patients with neurological conditions are admitted to hospital for an acute condition. This means that opportunities to detect treatable causes of potentially life-threatening complications are being missed. Many inter-related factors were reported to account for this practice and these related primarily to limited knowledge and confidence and the limited accessibility of speech and language therapists outside usual working hours. Interventions to improve routine dysphagia screening should help to reduce the incidence of avoidable complications and perhaps shorten length of stay. Dysphagia pathways or guidelines are needed to support effective management in acute hospital settings.

Background Since its introduction in the early 1990s, minimally invasive gastro-oesophageal surgery for cancer has been growing in popularity. Despite this, published evidence on this type of technique is weak and its role in the management of gastric and oesophageal cancer remains controversial. Aims The aim of this thesis was to test the hypothesis that: minimally invasive gastro- oesophageal cancer surgery has superior outcomes compared to control studies of conventional open surgery; but current studies are methodologically inadequate to confirm this. Methods The first study (chapter 3) is a systematic review of the literature on minimally invasive gastro-oesophageal cancer surgery, outlining the differences between literature published in Eastern and Western countries. The following 3 chapters outline and use a phase II surgical study to obtain data on minimally invasive gastro-oesophageal cancer (MIGOCS.) The MIGOCS group was set up in 2005 amongst UK surgeons. An online database was developed to enable data collection and comprises 5 sections: demographics; pre-operative staging and assessment; surgical intervention; post-operative course; pathology and clinical outcome. The first study is retrospective collecting data up to December 2006; the second study is prospective with data obtained between December 2006- July 2008 from centres around the UK utilising the MIGOCS database. Chapter 7 involves analysis of the learning curve in laparoscopic gastro-oesophageal cancer surgery using CUSUM (continuous surveillance monitoring) assessment. By studying operative time at each centre, improvement or deterioration in quality were detected. Results The systematic review of minimally invasive gastro-oesophageal surgery consists in the majority of case reports, with no randomised controlled trials of oesophagectomies and 4 (low quality) randomised controlled trials of gastrectomies. It demonstrates a mortality and morbidity of 2.3% and 46.2% respectively for oesophagectomies; 0.1% and 12.7% respectively for gastrectomies. Data from this review suggests that the minimally invasive approach is beneficial compared to open surgery in terms of reduced mortality, respiratory complications, blood loss and quicker return to a good quality of life (but not reduced hospital stay as expected.) There are currently 60 MIGOCS member consultant surgeons from over 40 UK centres. The retrospective study obtained data from 7 UK centres with an overall mortality and morbidity of 6.0% and 57% respectively for oesophagectomies and 7.7% and 13% respectively for gastrectomies. The prospective study collected data from 7 UK centres, comprising a total of 258 minimally invasive oesophagectomies and 33 minimally invasive gastrectomies. Overall mortality and morbidity were 2.5% and 56.6% respectively for oesophagectomies and 10.8% and 27.3% respectively for gastrectomies. CUSUM analysis varied considerably between centres. The two larger volume centres however demonstrated an improvement in their operative time with experience, with a possible pateau at around 30 procedures. Conclusions Published data suggests that the minimally invasive approach to gastro-oesophageal cancer has advantages over conventional open surgery. Data collected in this thesis does not overwhelmingly support published evidence, but does demonstrate that this technique is both safe and feasible even during the early part of a surgeon's learning curve. It is the first study to provide an insight into outcomes of this type of surgery in a multicentre setting in the UK; and has made progress towards a randomised controlled trial.