Background Severe idiopathic constipation is a disorder that can significantly impair Quality of Life (QOL). Idiopathic constipation refers to the situation where no organic, biochemical, structural, endocrine or neurological explanations can be found to account for symptoms. Little is known about the factors that predict poor QOL in such patients. The pathophysiological processes involved are not fully understood, particularly with reference to subsets of patients defined by their symptoms (such as the urge to defecate). A proportion of patients remain unresponsive to treatment with life style changes, high fibre diet, laxatives and non-invasive therapies. They can be regarded as having severe refractory idiopathic constipation. They may ultimately be considered for surgical intervention. The main aims of this thesis were to examine three key themes regarding severe refractory idiopathic constipation; 1) To identify factors that can predict disease-specific QOL. 2) To explore the pathophysiology of the urge to defecate and determine whether there are differences between patient groups defined by this symptom. 3) To prospectively evaluate the efficacy of a novel therapy that is a potential alternative to surgery. Predictors of Disease specific QOL: This study involved gathering data with QOL questionnaires (PAC-QOL and SF-36). Before identifying predictors, it was necessary to evaluate the psychometric properties (validity and reliability) of the QOL measures. It was important to confirm that their use was appropriate in patients with severe refractory idiopathic constipation. Confirming the psychometric properties of the measures was a prerequisite secondary aim of the study. Determining predictors of poorer QOL could enable clinicians to identify patients in whom the impact and severity of constipation is greatest. Predictors could be used to target therapies to those with greatest need. The relationships between demographics, symptoms assessed by clinician, symptoms assessed by the patient, results of objective tests and the patient's perception of health were studied. Psychometric testing of the QOL and health perception measures validated their use in patients with severe refractory idiopathic constipation. Multiple linear regression suggested that symptom intensity assessed by the patient and patient perception of mental health were the main predictors of disease specific QOL. With these results in mind, it is possible that therapeutic strategies that reduce symptoms and address problems of mental health may improve QOL. Pathophysiology of the urge to defecate: Approximately 60% of patients with idiopathic constipation have a reduced rectal urge to defecate (RRUD). They may represent a distinct group with differences in the properties and motility of the anorectum including a reduced frequency of the sampling reflex. This reflex describes relaxation of the anal sphincter allowing rectal contents to come into contact with sensory receptors in the proximal anal canal prior to defecation. The frequency of sampling events in patients with reduced urge and normal rectal urge to defecate (NRUD) was measured in a prospective comparative study using solid state, semi-ambulatory continuous anorectal manometry. The mean frequency of sampling events was 8.91/hr (sd 5.9) in the normal rectal urge group and 8.77/hr (sd 7.2) in the reduced urge group (NS). No differences in anorectal sensation or colonic transit time were found. The explanation for the noticeable division of patients into those with NRUD and RRUD remains unclear. Frequency of sampling is no different between the groups and is unlikely that sampling dictates a RRUD. Further study is required to evaluate the role of other potential determinants such as rectal compliance, rectosigmoid motility and central processing of sensory perceptions. Evaluation of a novel therapy: Percutaneous Endoscopic Colostomy (PEC) in the left colon is a minimally invasive endoscopic technique that can be used to irrigate the left colon and relieve constipation. The technique offers a potential alternative to Quality of Life, physiological evaluation and novel treatment in refractory constipation: A study of patients from a specialist clinic in the North East of England. surgery in severe cases. The aim was to perform a prospective efficacy study in refractory idiopathic constipation. The initial step was a retrospective data analysis of patients who had PEC inserted at our unit over a four year period. This is presented in the thesis. The results were to be used to inform the design of a prospective study. Over the 4 year period 31 patients attended for PEC. Indications included idiopathic constipation, recurrent sigmoid volvulus, colonic pseudo-obstruction and neurological constipation. Although symptoms were improved in the majority of patients and recurrent sigmoid volvulus prevented, complications were common. Infection and abdominal pain necessitated the removal of PEC in the majority of patients. Patients with refractory idiopathic constipation were particularly susceptible to these complications. Two deaths occurred due to faecal peritonitis occurring after insertion. Insertion was associated with significant morbidity and mortality. The widespread use of PEC is not recommended and insertion should be restricted to specially selected cases. The results did not support the initiation of a prospective study of efficacy. Consequently, this aim of the thesis was not fulfilled. Conclusions By producing this thesis, predictors of disease specific QOL have been identified. These predictors could be studied in future hypothesis testing studies of QOL. Experience has been gained in the use of physiological techniques that could be utilised in further studies of pathophysiology in idiopathic constipation. Evaluation of PEC has added valuable information to the existing literature and has directly influenced clinical practice.

Alcoholic liver disease (ALD) is a significant and growing global health problem, responsible for over 10000 deaths per year in the UK alone. Clinical liver failure can result from gradual, chronic depletion of the hepatocyte pool and replacement with fibrous tissue in cirrhosis or from rapid, acute hepatocellular dysfunction secondary to inflammation in acute alcoholic hepatitis (AAH) which carries a mortality of up to 35% on first presentation. Corticosteroid therapy has shown some benefit in AAH but its utility is limited by uncertainty in patient selection and poor clinical response in a proportion of cases. Our current understanding of AAH pathogenesis attributes hepatocellular dysfunction to the action of supra-physiological concentrations of proinflammatory cytokines. Evidence from animal and human studies suggests that the major source of cytokine release is the hepatic macrophage or Kupffer cell responding to an increased concentration of bacterial endotoxin in portal blood following an ethanol-mediated increase in gut permeability. However, this enhanced and sustained inflammatory response is at odds with the normal response in the liver in which endotoxin tolerance allows bacterial components to be cleared from the blood without an inflammatory response. This study set out to investigate factors that determine the enhanced inflammatory response in AAH and its response to therapy. Genetic analysis revealed a single nucleotide polymorphism in a component of the endotoxin response pathway (the Toll-like receptor adapter molecule MAL) associated weakly with advanced disease in both ALD and the related condition non-alcoholic steatohepatitis. Different alleles associated with advanced disease in the two conditions, suggesting divergent importance of signalling pathways in their pathogenesis. Assays in AAH patients demonstrated that their lymphocyte steroid sensitivity was impaired relative to normal controls, correlated with clinical markers of steroid responsiveness, improved in recovery and could be improved by ex vivo supplementation with theophylline, a known recruiter of histone deacetylases. The role of histone modifications in the enhancement of inflammatory responses in ethanol was investigated in a human macrophage cell-line model which revealed increased histone acetylation at pro-inflammatory cytokine gene promoter regions associated with potentiated cytokine responses to endotoxin after culture in ethanol or its metabolite acetate. This effect was abrogated by knockdown of acetyl-coA synthetases, suggesting that increased synthesis of acetyl-coA from acetate is crucial for histone acetylation and consequent increased cytokine production after ethanol exposure. These findings suggest that while genetic predisposition may have some effect on innate immune responses in the pathogenesis of alcoholic liver disease, the more significant contribution is likely to come from gene-environment interactions such as modulation of histone acetylation by products of ethanol metabolism. This epigenetic relationship between metabolism and gene expression in inflammation, mediated by histone deacetylases such as the sirtuin proteins, may be a novel therapeutic target in ALD and potentially also in other inflammatory conditions.

Background: Identifying Crohn’s disease recurrence in symptomatic patients after ileocaecal resection is difficult as symptoms may reflect the effect of surgery rather than active disease. The aim of this study was to evaluate faecal concentrations of granulocyte degradation products (faecal calprotectin and faecal lactoferrin) in this post-operative setting. Methods: A post-operative cohort of 104 patients (median follow up of 24 months) provided a single stool sample. A second cohort of 13 patients was followed prospectively for 1 year with regular faecal calprotectin (FC) and faecal lactoferrin (FL) measurements. Faecal measurements were compared with symptom diaries, the Harvey Bradshaw Index (HBI), endoscopic examination, C-reactive protein (CRP) and platelet measurement. Results: Both faecal calprotectin and faecal lactoferrin correlated significantly with Harvey Bradshaw Index (r = 0.532, P< 0.001, r = 0.687, P< 0.001 respectively). Twenty eight patients with severely clinically active disease had high mean (s.e) levels of faecal calprotectin (661.1(119.1) μg/g) and faecal lactoferrin (116.6(32.2) μg/g); and forty three patients with clinically inactive disease had low levels of faecal calprotectin (70.2(27.1) μg/g) and faecal lactoferrin (5.9(2.4) μg/g). In patients with mild to moderately clinically active disease, faecal calprotectin and faecal lactoferrin identified individuals with and without recurrent inflammatory disease. In the uncomplicated course, both markers (faecal calprotectin and lactoferrin) normalized within 2 months. Faecal markers were more accurate at predicting clinical disease activity than C-reactive protein, platelet count or endoscopic appearance. Conclusion: Faecal calprotectin and faecal lactoferrin are non-invasive tests that can help to identify disease recurrence in symptomatic post-operative patients [1].

Fibrosis is characterised by the excessive accumulation of extracellular matrix (ECM) proteins, resulting in a loss of tissue architecture and function. Central in liver fibrosis development is the transdifferentiation of hepatic stellate cells (HSCs) to a myofibroblast phenotype, responsible for increased deposition of ECM. Presently there are no treatments available for fibrosis. Critical to the discovery of novel anti-fibrotics is the development of a non-invasive imaging modality to accurately diagnose fibrosis severity. The hepatic myofibroblast specific single chain antibody (scAb) C1-3 was conjugated with a fluorophore and administered to mice with liver fibrosis prior to IVIS imaging to diagnose fibrosis severity. The expression of C1-3’s target antigen (synaptophysin) was confirmed by quantitative real-time PCR (qRT-PCR) and immunocytochemistry (ICC) in both quiescent and activated HSCs. The anti-inflammatory effects of PXR agonists were investigated utilising an in vivo model of liver fibrosis. Finally the pro-inflammatory properties of hepatic myofibroblasts were studied in vitro and in vivo. There was a statistically significant increase in fluorescence detected ex vivo in fibrotic livers versus the controls. qRT-PCR confirmed that quiescent HSCs (qHSCs) and hepatic myofibroblasts express similar levels of synaptophysin. The PXR agonist PCN significantly reduced the level of liver inflammation (NF-κB activity) following liver injury in vivo, 24 hours after its administration. The data presented indicates hepatic myofibroblasts release a pro-inflammatory soluble factor and induce NF-κB activity when injected in vivo. These findings suggest that hepatic myofibroblast number is an indicator of fibrosis severity. Hepatic myofibroblasts possess pro-inflammatory characteristics, which may contribute to fibrosis development. Finally PXR agonists exhibited anti-inflammatory properties that may be beneficial in the treatment of liver fibrosis.

Early detection methods are key to reducing morbidity rates from digestive tract cancer which is currently one of the fastest growing cancers in the World. Capsule endoscopes (CEs) are a new technology that can be used to improve early detection of the gastrointestinal (GI) tract disorder. The device integrates the technologies such as image processing, optoelectronic engineering, information communication, and biomedical engineering. The capsule is the size and shape of a pill and contains an optoelectronic camera, antenna, transmitter, battery and optoelectronic illuminating light emitting diodes (LEDs). The small size of these devices enables them to offer many advantages over conventional endoscopes such as accessibility to the entire intestine and minimising the risk of perforation, particularly for patients with difficult anatomy (e.g. post-operative scar tissue). Currently used devices are passive and can only follow the natural transit of the intestines, and hence there is considerable interest in methods of controlled actuation for these devices. In this thesis, a novel actuation system based on magnetic levitation is designed, developed and implemented, utilizing a small permanent magnet embedded within the capsule and an arrangement of digitally controlled electromagnets outside the body. The proposed approach is that the magnet can be moved and oriented by DC magnetic force and torque produced by coils placed outside of the human body, with a suitable position feedback sensor enabling closed-loop control. Theoretical analyses of the proposed actuation system are presented which model the magnetic field, force and torque exerted by electromagnetic coil on the embedded magnet. Based on the distribution of the magnetic field, an optimal geometry for the coils is proposed in order to achieve a levitation distance which is realistic for the inspection of the GI tract. Two types of systems are investigated in the thesis, namely single-input single-output (SISO) and multi-input multi-output (MIMO), and the dynamics of these systems are modelled in state space form and hence linear controllers are designed for capsule actuation. The controllers are simulated using Matlab/ Simulink tools to realize the mathematical analysis of the system, and then implemented digitally in real-time using Texas Instruments (TI) TMS320F2812 Digital Signal Processor (DSP) to validate the proposed actuation system. In the SISO system, a linear one degree of freedom (1DOF) proportionalintegral- derivative (PID) controller is designed to move the inserted magnet in the vertical dimension within an area around the operating point and to maintain it at a desired position. A realistic simulation model is designed and implemented to evaluate the proposed controller. Simulation results have shown that the controller is able to successfully hold the embedded magnet in the desired position. For practical validation, the PID controller is implemented in real-time on the DSP system, where pulse width modulation (PWM) is generated to control the coil current, and Hall effect sensors are used for position feedback. Experimental results are obtained under step and square wave input demand. In the proposed system, high frequency noise on the position sensor is initially rejected by hardware implementation of resistor capacitor-low pass filter (RC-LPF) circuit. The accuracy of the position feedback is increased by calibrating the DSP’s on-chip analogue-digital converter (ADC) in order to reduce conversion error due to inherent gain and offset errors. To further reduce the influence of the position feedback noise, an average of ten repeated samples based on mean filter is implemented by the DSP in order to reduce the influctuation of the sensor reading. The tracking performance of the actuation system based on two Hall effect sensors on the opposite coil’s poles is investigated under step trajectory input. In an improved actuation system, position feedback is provided by using an AC magnetic field to obtain the capsule position information, decoupling this from the DC actuation field. The noise of the position feedback in the improved system is reduced by replacing the PWM current drive with a linear power amplifier driven from a digital to analogue converter (DAC), hence reducing AC interference. Positioning sensor noise was found to be further reduced by implementing digital filtering based on a coherent detector using the DSP, without increasing response time. The performance of the actuation system using these position sensors is compared based on settling time, overshoot, steady-state error, and control input parameters in order to validate the proposed improvement in the position feedback. The experimental results have shown that the controller based on both sensing strategies satisfactory control of the magnet’s position. However, the response of the system based on AC position sensing has the shortest settling time, smallest overshoot value and steady-state error. In the MIMO system, several linear controllers such as pole placement (PP), Entire Eigenstructure Assignment (EEA), and linear Quadratic regulator (LQR) techniques are designed and their tracking performances are compared. Simulation results have shown that, based on acceptable control inputs, the LQR controller has the fastest response with minimal overshoot value and steady state error. However, the LQR controller based on 2DOF is unable to maintain stable control of the magnet due to the insufficient position feedback from the two coil sensors. Specifically, it is not possible to achieve a stable 2D system since the orientation angle of the magnet is not resolvable. Therefore, the position feedback is improved by obtaining the device position and orientation information from a pair of 3-axis orthogonal coils. A realistic simulation model for the 3DOF LQR controller is designed and implemented to evaluate the developed system. Simulation results have shown that this controller is can achieve the necessary stability. In conclusion, based on the results from the 1D control system, the thesis shows that the DC magnetic field, which is used for capsule movement, can be also used to provide the controller acceptable position feedback. However, the use of AC magnetic field for positioning purpose provides more accurate position information. In order to implement 2DOF control system successfully, two 3-axis orthogonal coil sensors are considered which are used to provide the actuation algorithm with more accurate feedback of position and orientation information.

Obesity and overweight have reached epidemic proportions in the United States (WHO, 1988). Despite the various treatment programs, the problem is getting worse, more people are becoming obese, and it is estimated that 90-95% of those who lose weight will regain it (Legro, 2000). Current treatment programs fail to take into consideration emotional variability within this population. The purpose of the present study was to examine the relationships between attachment, BMI, and psychopathology. Obese participants (N = 101) seeking treatment for weight loss completed 3 questionnaires: 1) Experiences in Close Relationships-Revised, 2) Millon Clinical Multiaxial Inventory-Ill, and 3)Questionnaire on Eating and Weight Patterns-Revised. Results indicated that attachment moderated the relationship between BMI and dysthymic symptoms, major depressive symptoms, and alcohol dependency symptoms. 'Secure'ly and 'Fearful'ly attached participants at high BMI were generally more distressed than 'Securely and 'Fearful'ly attached participants at low BMI. However, 78 avoidant and 'Preoccupied' participants at high BMI were generally less distressed than avoidant and 'Preoccupied' participants at low BMI. Such findings suggest that increases in BMI are associated with distress or lack of distress dependent on attachment classification. Assessment and treatment implications are elaborated.

Pancreatic β-cell dysfunction plays a central role in the pathogenesis of type 2 diabetes. This dysfunction is characterised by secretory defects in the β-cells and the loss of β-cell mass, at least in part secondary to increased β-cell apoptosis. Although the mechanisms through which β-cell dysfunction develops are unclear, accumulating evidence suggests that elevated levels of circulating free fatty acids (FFAs) as can occur under conditions of obesity, play a role in the pathogenesis of type 2 diabetes. It has been suggested that endoplasmic reticulum (ER) stress and the resulting unfolded protein response (UPR) play a role in FFA induced β-cell dysfunction. This thesis was aimed at investigating the role of obesity induced ER stress in the development of β-cell dysfunction in type 2 diabetes. The UPR was induced in MIN6 β-cells in response to both the saturated fatty acid (FA) palmitate, and unsaturated fatty acid oleate. Palmitate however induced a more marked ER stress response in comparison to oleate. Although both FAs induced ER stress, only palmitate evoked apoptosis in the β-cells, indicative of the differential signalling by unsaturated and saturated fatty acids. ER stress and evidence of functional adaptation was also observed in islets obtained from Zucker and Zucker diabetic fatty (ZDF) rodent models of obesity. The development of β-cell dysfunction in the progression from obesity to obesity associated type 2 diabetes in the ZDF rats was however not accompanied by a further increase in ER stress markers. This suggests that ER stress signalling does not play a significant role in the development of β-cell dysfunction. In conclusion, the studies outlined in this thesis demonstrate that ER stress is induced in in vitro and in vivo models of β-cell lipotoxicity. It is however apparent, that ER stress does not contribute significantly to β-cell dysfunction and perhaps, only plays a small insignificant role in β-cell apoptosis in the pathogenesis of type 2 diabetes. It is hypothesised, that β-cell dysfunction develops in type 2 diabetes as a result of the inability of the β-cell to mount an additive UPR in response to ER stress.

Liver fibrosis is a wound healing response to chronic hepatocellular damage. The activated pregnane X receptor has been shown to exert an anti-fibrogenic effect in rodent chronic liver injury in vivo models. The aim of this study was to determine the effects of the human pregnane X receptor on hepatic stellate cell trans-differentiation to a pro-fibrogenic phenotype in vitro. Primary hepatic stellate cells were isolated from resected human liver and cultured under conditions in which they trans-differentiated into a pro-fibrogenic phenotype with increased expression of extracellular matrix components, cytokines and chemokines. Here we have demonstrated that the pregnane X receptor was expressed in primary cultures of hepatic stellate cells as well as a hepatic stellate cell line at the level of mRNA and protein, and was transcriptionally functional as determined by increased ER6-dependent reporter gene expression. Short term treatment of hepatic stellate cells with pregnane X receptor ligands such as rifampicin resulted in an increase in interleukin-6 secretion as well as an inhibition in DNA synthesis. Interleukin-6 promoter studies in primary hepatic stellates and the LX-2 hepatic stellate cell line suggested that the activated protein-1 site and nuclear factor interleukin-6 sites were required for pregnane X receptor-mediated regulation. Chronic ligand treatment over several weeks resulted in reduced proliferation and trans-differentiation of hepatic stellate cells with no obvious effect on the rate of apoptosis. We therefore conclude that the pregnane X receptor is transcriptionally active in human hepatic stellate cells and that activators inhibit the trans-differentiation and proliferation in culture. The pregnane X receptor may therefore be an effective target for anti-fibrotic therapy.

Proliferative diabetic retinopathy (PDR) is a major cause of blindness. In the present study the possible role of systemic growth factors on the migration and proliferation of retinal endothelial cells (EC) was examined. These studies show that patients with active PDR have a serum activity which stimulates the migration but not the proliferation of retinal EC, and that this activity is not diminished in plasma-derived serum (PDS). A strong correlation was observed between migratory activity in the serum and serum platelet-derived growth factor (PDGF) levels, while antibodies to PDGF partially inhibited the stimulatory effect of PDR serum. However, retinal EC failed to respond to purified PDGF in isolation. These was also an attempt to characterise the activity in PDR sera. The results indicate that the activity (a) is retained in a fraction of serum with a molecular weight between 10 and 30kd, (b) can be eluted from a Q-Sepharose column with 20-30% NaCl (c) has a pI of 5.8 and (d) is retained by passage down a Heparin-Sepharose column from which it could be eluted with 1.2M NaCl. The hypothesis that serum migration activity is due to synergism between PDGF and the HBGF, FGF was tested, and the <I>in vitro</I> results show that constant concentration of PDGF (25pM) combined with aFGF (1.0-1000fg/ml), but not, bFGF synergised to stimulate EC migration. It is suggested that systemic cells such as lymphocytes exposed to non-physiological glucose levels can be activated to secrete a wide range of growth factors which could be detected in the serum. These factors may include both the HBGF and PDGF which may synergise and contribute to the pathogenesis of PDR.

Senescence and its associated secretory phenotype have been investigated in several vanishing bile duct syndromes. The current study evaluated the presence of senescent biliary epithelial cells (BEC) in acute cellular rejection of human liver allografts to ascertain whether senescent cells contribute to human disease progression in liver transplantation. There was a significant correlation between senescent BEC and grade of rejection. Furthermore there was a significant correlation between grade of rejection and BEC undergoing epithelial to mesenchymal transition (EMT). There was never any overlap between senescence and EMT markers in BEC. Further investigation of the association between senescence and EMT in vitro using both primary and immortalised human BEC exposed to oxidative stress showed, for the first time, that TGF-­b2 is part of the Senescence Associated Secretory Phenotype (SASP) in liver disease. Blockade of TGF-­b signalling by inhibition of the TGFbR, prevented any of the oxidative stress-­induced changes in BEC. Blockade of integrin aVb6 integrin also showed a variable ability to prevent TGF-­b mediated changes in BEC. HGF and its mimetic, 1K1, were able to prevent oxidative stress induced EMT in BEC. Furthermore 1K1 showed a smaller induction of autophagy than HGF and was able to prevent up regulation of senescence markers. The paradigm of oxidative stress-­induced senescence leading to EMT was assessed in the current study, with identification of powerful therapeutic agents able to prevent these changes. This suggests that premature cellular ageing (senescence) in acute liver disease could be pharmacologically prevented from occurring. Inhibition of senescence prevents disease promotion by the effects of the SASP, blocking the progression to chronic disease; this may well apply to all organ systems.