This thesis describes the study of collagen from the human colonic wall in healthy subjects and in those subjects with colonic diverticulosis, in relation to aging of the tissue. This thesis also examines the effect of diet on the collagen of the colon of the laboratory rat, as a model for the study of the human subject. Chapters 1 and 2 describe the background to the study. Chapter 1 describes the structure and function of the human colon and some of the conditions which affect it. Chapter 2 describes the structure and previous studies on connective tissues and the realationship between structure and function. Chapter 3 describes the materials nad methodology used in the work in this thesis, for examining, both the effect of age and diet on the collagen of the colon. This involves biochemical and structural analysis. Chapter 4 presents data from the study of the effect of aging on the collagen from healthy colons and those with colonic diverticulosis. The chapter describes biochemical and structural evidence for altered colonic collagen in the aged tissue. Chapter 5 presents data from the study of the effect of diet on colonic collagen using the laboratory rat as a suitable model for the situation in humans. This chapter describes biochemical and structural evidence for altered colonic collagen as a consequence of a diet low in dietary fibre. This chapter also examines the effect of maternal diet on the health of the offspring. The chapter also describes the possible role of dietary fibre in protection against colonic diverticulosis. Chapter 6 discusses the techniques used and the results produced. The chapter discusses the two theories relevant to the aetiology of colonic diverticulosis, the way in which these theories conflict and run in parallel. The inevitability of colonic diverticula as a consequence of age is discussed. A possible mechanism for the development of colonic diverticulosis is described. The techniques and their potential in further studies is discussed.

The technological developments seen in recent years have facilitated remarkable progress in the field of flexible gastrointestinal (GI) endoscopy. Smaller high-resolution charge-coupled devices (CCDs) have facilitated the manufacture of ultrathin (UT) (<6mm) endoscopes, while the introduction of device assisted enteroscopy (balloon-assisted and spiral enteroscopy) has allowed endoscopists to access the deep small bowel (SB) without the need for recourse to major surgery. Furthermore, the application of double-balloon colonoscopy (DBC) has shown promise to improve outcomes in patients with 'technically difficult' colons. Although these 3 types of innovative endoscopic technologies all share the potential capacity to enhance minimally invasive patient care, research into their optimal role and effectiveness (particularly within UK clinical practice) remains limited. This thesis has examined the potential role of this selection of advanced flexible GI endoscopic technologies for the enhancement of minimally invasive patient care. The first study evaluated transnasal upper GI endoscopy in the UK and confirmed that within this clinical paradigm, transnasal endoscopy using UT endoscopes, is a feasible, effective and more acceptable alternative to patients than conventional oral upper GI endoscopy. The next series of studies were dedicated to device assisted enteroscopy (DBE in particular) and showed that DBE is capable of providing a safe and effective, minimally invasive alternative to major surgery in selected cases. A comparison of spiral enteroscopy as an alternative to DBE, showed that spiral enteroscopy (in its current, manual form), appears to be inferior to DBE in its ability to facilitate deep enteroscopy. The final study evaluated technically difficult colonoscopy and included the development and validation of a score for technical difficulty which may in the future be applied to routine clinical practice. This study also highlighted the usefulness of DBC as a potentially more effective tool than conventional colonoscopy for technically difficult cases.

This thesis reviewed and investigated several aspects of the management of enterocutaneous fistula (ECF). The best management of ECF, particularly complex fistulas, is in specialist units where there is a multidisciplinary structured approach. Surgical management of ECF at St. Mark's National Intestinal Failure Unit is safe and improving over time. The highest incidence of ECF development occurs following abdominal surgery particularly in the presence of Crohn's disease. The audit confirmed this in our unit. A retrospective audit of our unit revealed the majority of patients underwent definitive surgery. There were improved overall healing rates following both surgery and conservative management in the current cohort, with also improvement in the 30 day post fistula resection mortality compared with a previous series at our unit. Multivariate analysis revealed co-morbidity, source of referral and aetiology had significant associations with ECF healing. This thesis has included the first study and design of a scoring system for predicting ECF healing. The scoring systems devised were both validated. They can provide further information to aid clinical management and decision making for the multidisciplinary team and predict the possible long term outcome for patients at external institutions. Meta-analyses of randomised control trials has revealed somatostatin and octreotide increase the likelihood of fistula closure, reducing the time to fistula closure, and neither has an effect on mortality. 5 Immunological studies revealed an increased on-going production of cytokines, in particular TNF-α, in non-IBD ECF compared with control terminal ileum tissue. The data may provide evidence for the potential use of anti-TNF-α agents in the treatment of non-IBD ECF. A future pilot study has been designed with the aim to identify a potential novel therapy for patients with persistent enterocutaneous fistula not associated with inflammatory bowel disease. Any positive findings would equate to a major medical advance with a new use for anti-TNF-α agents.

The emerging role of the multifunctional enzyme, Transglutaminase 2 (TG2) in Cystic Fibrosis (CF) has been linked to its increased expression and intracellular transamidating activity. However, a full understanding of the molecular mechanisms involved still remains unclear despite numerous studies that have attempted to delineate this process. These mechanisms include the NFκB and TGFβ1 pathway amongst others. This study reveals for the first time that the development of fibrosis in CF is due to a TG2-driven epithelial to mesenchymal transition (EMT) via a mechanism involving the activation of the pro-fibrotic cytokine TGFβ1. Using a human ΔF508/W1282X CFTR CF mutant bronchial cell (IB3-1), its CFTR corrected “add-back” cell (C38) as well as a primary human bronchial epithelial cell (HBEC), elevated TG2 levels in the CFTR mutant IB3 cell were shown to activate latent TGFβ1 leading to increased levels found in the culture medium. This activation process was blocked by the presence of cell-permeable and impermeable TG2 inhibitors while inhibition of TGFβ1 receptors blocked TG2 expression. This demonstrates the direct link between TG2 and TGFβ1 in CF. The presence of active cell surface TG2 correlated with an increase in the expression of EMT markers, associated with the CF mutant cells, which could be blocked by the presence of TG2 inhibitors. This was mimicked using the “addback” C38 cell and the primary human bronchial epithelial cell, HBEC, where an increase in TG2 expression and activity in the presence of TGFβ1 concurred with a change in cell morphology and an elevation in EMT marker expression. Conversely, a knockdown of TG2 in the CF mutant IB3 cells illustrated that an inhibition of TG2 blocks the increase in EMT marker expression as well as causing an increase in TEER measurement. This together with an increase in the migration profile of the CF mutant IB3 cell against the “add-back” C38 cell suggests that TG2 drives a mesenchymal phenotype in CF. The involvement of TG2 activated TGFβ1 in CF was further demonstrated with an elevation/inhibition of p- SMAD 2 and 3 activation in the presence of TGFβ1/TG2 cell-permeable/impermeable inhibitors respectively. The use of a comparative airway cell model where bronchial epithelial cells were cultured at the air liquid interface (ALI) confirmed the observations in submerged culture depicting the robustness of the model and reiterated the importance of TG2 in CF. Using a CFTR corrector combined with TG2 inhibitors, this study showed that the correction and stabilisation of the ΔF508 CFTR mutation in the mutant cell forged an increase in matured CFTR copies trafficking to the apical surface by circumventing proteosomal degradation. Thus the results presented here suggests that TG2 expression is elevated in the CFTR mutant bronchial cell via a TGFβ1 driven positive feedback cycle whereby activation of latent TGFβ1 by TG2 leads in turn to an elevation in its own expression by TGFβ1. This vicious cycle then drives EMT in CF ultimately leading to lung remodelling and fibrosis. Importantly, TG2 inhibition blocks TGFβ1 activation leading to an inhibition of EMT and further blocks the emerging fibrosis, thus stabilizing and supporting the maturation, trafficking and conductance of CFTR channels at the apical surface.

Elevated free fatty acids (FFA) are a feature of ageing and a risk factor for metabolic disorders such as cardiovascular disease (CVD) and type-2 diabetes (T2D). Elevated FFA contribute to insulin resistance, production of inflammatory cytokines and expression of adhesion molecules on immune cells and endothelial cells, risk factors for CVD and T2D. Molecular mechanisms of FFA effects on monocyte function and how FFA phenotype is affected by healthy ageing remain poorly understood. This thesis evaluated the effects of the two major FFA in plasma, oleate and palmitate on monocyte viability, cell surface antigen expression, and inflammatory activation in THP-1 monocytes. Palmitate but not oleate increased cell surface expression of CD11b and CD36 after 24h, independent of mitochondrial superoxide, but dependent on de novo synthesis of ceramides. LPS-mediated cytokine production in THP-1 monocytes was enhanced and decreased following incubation with palmitate and oleate respectively. In a model of monocyte-macrophage differentiation, palmitate induced a pro-inflammatory macrophage phenotype which required de novo ceramide synthesis, whilst oleate reduced cytokine secretion, producing a macrophage with enhanced clearance apoptotic cells. Plasma fatty acid analysis in young and mid-life populations revealed age-related increases in both the SFA and MUFA classes, especially the medium and very long chain C14 and C24 fatty acids, which were accompanied by increases in the estimated activities of desaturase enzymes. Changes were independently correlated with increased PBMC CD11b, plasma TNF-a and insulin resistance. In conclusion, the pro-atherogenic phenotype, enhanced LPS responses in monocytes, and pro-inflammatory macrophage in the presence of palmitate but not oleate is reliant upon de novo ceramide synthesis. Age-related increases in inflammation, cell surface integrin expression are related to increases in both the MUFA and SFA fatty acids, which in part may be explained by altered de novo fatty acid synthesis.

Crohn’s Disease is a chronic inflammatory disorder of the bowel affecting approximately 1 in 800 people in the UK. The terminal ileum is most commonly affected and the mesentery becomes thickened, a phenomenon known as ‘fat wrapping’. The cause is not understood. Elemental feeding can induce remission in Crohn’s disease and polyunsaturated fatty acid (PUFA) content may be the cause of a reduction in inflammation. Particular attention has focused on n-­‐3 and n-­‐6 PUFA content of elemental feeds. The aim of this study was to further characterize mesenteric fat in Crohn’s disease and to examine the effects of different PUFA on mesenteric inflammation in vitro. Samples of adipose tissue were collected from patients undergoing intestinal resection for Crohn’s disease and from controls. These were cultured in media and elemental (E028 and Emsogen) feeds containing different concentrations of n-­‐3 and n-­‐6 PUFA. Significant findings were that mesenteric (MF) and omental (OM) adipose tissue released more inflammatory cytokines IL-­‐6, leptin and MCP-­‐1 when cultured in media rich in n-­‐6 PUFA compared to media rich in n-­‐3 PUFAs. OM mean IL-­‐6 concentrations were 18.6(3.1-­‐21.8)ng/mL in n-­‐6 PUFA vs 3.07(0.62-­‐19.10)ng/mL in n-­‐3 PUFA (p=0.018), MF IL-­‐6 concentrations were 3.77(0.76-­‐9.52)ng/mL in n-­‐6 PUFA vs 1.5(0.42-­‐2.61)ng/mL in n-­‐3 PUFA (p=0.03). OM Leptin concentrations were 0.42(0.08-­‐0.90)ng/mL in n-­‐6 PUFA vs 0.08(0.07-­‐0.14)ng/mL in n-­‐3 PUFA (p=0.006), MF Leptin concentrations were 0.27(0.13-­‐2.62)ng/mL in n-­‐6 PUFA vs 0.12(0.07-­‐0.31)ng/mL in n-­‐3 PUFA (p=0.033). OM MCP-­‐1 concentrations were 18.80(4.39-­‐31.5)ng/mL in n-­‐6 PUFA vs 1.83(0.69-­‐4.82)ng/mL in n-­‐3 PUFA (p=0.002) and MF MCP-­‐1 concentration were 4.59(2.20-­‐13.72)ng/mL in n-­‐6 PUFA vs 1.20(0.82-­‐3.39)ng/mL in n-­‐3 PUFA (p=0.006). These findings show that n-­‐6 PUFAs stimulate a greater inflammatory response from omental and mesenteric fat in vitro and may assist in formulating a more effective elemental feed for inducing remission in patients with active flares of Crohn’s disease.

Drug induced liver injury (DILI) due to the isoxazolyl β-lactam antibiotic, flucloxacillin, is a rare idiosyncratic adverse drug reaction. The underlying mechanism remains unclear but a recent association with the human leukocyte antigen class I allele, HLA-B*57:01, indicated a possible T-cell mediated reaction. This study aimed to identify further genetic determinants conferring susceptibility to this form of DILI and to study metabolic and immune mechanisms of this toxicity. Flucloxacillin DILI cases (n=150) and matched population controls (n=282) were genotyped for HLA-B*57:01, confirming the previous association with disease (OR = 40.1, 95% CI 22.7 – 70.7). Cases negative for HLA-B*57:01 (n=26) were genotyped for HLA-B alleles and this analysis showed a borderline significant association with HLA- B*13:02 (p = 0.0376). Genotyping of all cases for additional immune-related candidate genes such as KIR3DL1/KIR3DS1 and for variants detected in limited exome sequencing studies, performed by others, resulted in confirmation of a significant difference in frequency compared with community controls (n=235) for a Caspase-5 polymorphism (rs45483102) (OR = 2.39 95% CI 1.22 – 4.68). Reporter gene studies were performed to further investigate the ability of flucloxacillin to act as a ligand for the xenobiotic-sensing nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR). No flucloxacillin activation of CAR was observed but flucloxacillin was confirmed to be a PXR ligand and studies comparing PXR activation by the isoxazolyl penicillin’s cloxacillin and dicloxacillin showed dicloxacillin to be a stronger PXR agonist than flucloxacillin. DILI cases due to cloxacillin (n=3) and dicloxacillin (n=2) were found not to possess the HLA-B*57:01 allele suggesting that the mechanism for DILI due to these drugs is different. Flucloxacillin metabolism pathways were studied using human liver microsomes, recombinant cytochrome P450 isoforms and rat B13/H cells which differentiate to a hepatocyte-like phenotype, with flucloxacillin penicilloic acid the major metabolite detected. Despite previous reports, formation of the metabolite 5'-hydroxymethyl flucloxacillin which was believed to be CYP3A4-mediated, could not be confirmed in any of the systems studied. Preliminary studies on T-cell mediated responses to flucloxacillin, by exposure of peripheral blood mononuclear cells (PBMCs) from HLA-B*57:01 expressing flucloxacillin-DILI patients and from HLA-B*57:01 positive and negative flucloxacillin-naïve donors to flucloxacillin, indicated increased expression of interferon-γ at the RNA level in 2 out of 3 of the patient samples but not in controls. This finding was generally consistent with reported findings by others. In summary, a novel HLA-B association involving some flucloxacillin DILI cases has been detected, HLA-B*57:01 does not appear to be an important risk factor for DILI due to other isoxazolyl penicillin’s and the ability to act as a PXR agonist appears to be a general feature of these penicillin’s so may not be directly relevant to the mechanism for flucloxacillin DILI. The confirmed association with caspase 5 may represent a minor additional risk factor for flucloxacillin DILI.

The work described in this thesis relates to gastro-oesophageal reflux (GOR) and extra-oesophageal reflux (EOR) in the cystic fibrosis (CF) population. This thesis describes the characteristics of GOR and EOR and their association with pulmonary function, gastric aspiration and inflammation in the airway. In particular the study explores the increasing prevalence of overweight/obesity in CF and its association with GOR/EOR. In a sub group of patients (n=12) a longitudinal investigation took place to observe the effects of a new CFTR potentiator (Ivacaftor 150 mg/12h) on GOR/EOR symptoms. 72 CF adults recruited from a CF outpatient clinic consented to the study (39M/33F; median age 21 (16-60) years) and completed questionnaires to characterise symptoms of GOR (DeMeester score 0-9; < 1 normal) and EOR (Reflux Symptom Index (RSI) score 0- 2 45; < 13 normal). Patients were measured for BMI kg/m and grouped according to the 2 2 following BMI categories: underweight <18.5 kg/m , normal weight 18.5-25.0 kg/m , 2 2 overweight 25.1-30.0 kg/m and obese >30.0 kg/m . An expectorated sputum sample was provided and analysed for biomarkers of reflux (pepsin n=69 ELISA) and inflammation (IL-6 n=62 and IL-8 n=64 ELISA). Pulmonary function (FEV and FVC % predicted) and 1 genotype were recorded at the time of data collection. Statistical relationships were assessed using the Kruskal-Wallis statistical test followed by the Mann Whitney U test. GOR symptoms (DeMeester) were identified in 42% of patients and EOR symptoms (RSI) in 63% of patients. Pepsin was detected in 48 (70%) patient samples (median: 330ng/ml; range 80-1150ng/ml) and not correlated with GOR/EOR symptoms. GOR/EOR symptoms and gastric aspiration did not associate with pulmonary function, nor was GOR associated with inflammation of the airways. CF patients can be overweight/obese (16.7%), and this was associated with better lung function; they have less reflux. Obesity didn't show a relationship to F508del/F508del status or to gender. Ivacaftor treatment was associated with reduced symptoms of GOR and EOR (6 weeks to 12 month post medication) accompanied by positive effects on pulmonary function. GOR is common in CF patients and EOR symptoms are very prevalent in CF and more so than GOR. Microaspiration of gastric content into the airway was not correlated with pulmonary function and occurred across the spectrum of disease severity. Overweight/obese patients experience less EOR symptoms. The CFTR potentiator Ivacaftor reduced GOR and EOR symptoms after 6 weeks of treatment.

Increasing physical activity and reducing sedentary behaviour are important targets in the prevention of chronic diseases. The current evidence, which is based on self-reported measures, shows low levels of physical activity in Maltese children. In the absence of objective data on a nationally representative sample of Maltese children, there is no evidence on which to base policies and no baseline data to monitor changes following interventions. Furthermore, Maltese children are considered to be amongst the most obese in the world. However, statistics are also based on self-report. The aim of this thesis is to examine physical activity, sedentary time and weight status, using robust methods, in a representative sample of 10-11-year-olds, and to study any associated correlates. Initial data from focus groups indicated unique leisure patterns in Maltese children while a pilot study using objective measures indicated high levels of inactivity and sedentary time, combined with high levels of obesity and an association between activity levels and obesity. A nationally representative survey showed extremely high rates of overweight (24.2% of boys, 16.4 % of girls) and obesity (14.8% of boys, 13.6% of girls), with no strong social or geographical patterning. The study also showed a low proportion of children (39% of boys, 10% of girls) meeting the public health guideline of 60 minutes of MVPA per day. The children also spent a lot of time per day being sedentary (560 minutes for boys, 579 minutes for girls). A strong association between physical activity and obesity was evident, particularly after school hours, while no association was observed with sedentary time. Obese boys spent considerably more time using computers on weekdays than children in other weight categories. Multivariable regression analyses of objectively measured and self-reported physical activity and screen time with socio-demographic factors and BMI identified specific subgroups in need of interventions.

Background: Histopathology provides the gold standard assessment of colorectal biopsies. Numerous techniques for improving lesion detection at colonoscopy and classification are emerging. This thesis applied two complementary methods of vibrational spectroscopy which can provide a unique molecular 'fingerprint'; Raman and Fourier Transform Infrared (FTIR). Hypotheses: 1. An 'in-house' constructed flbreoptic Raman probe which fits through the accessory channel of a colonoscope can classify different colorectal pathologies. 2. FTIR spectroscopy can classify colorectal pathologies within tissue sections and determine biochemical composition. Methods: Biopsy samples were collected at colonoscopy, snap frozen and later thawed for experimentation. Point Raman spectra were measured from biopsies using the probe system. Subsequent formalin fixation enabled histopathological comparison. Principal component analysis (PCA) and linear discriminant analysis (LOA) with leave one out cross validation (LOOCV) correlated Raman spectra with histopathology. FTIR spectral maps were measured across tissue sections using an infrared imaging system in transmission mode. Contiguous haemotoxylin and eosin stained sections were compared by a pathologist. Spectra from epithelia were classified using PCA, LOA and LOOCV. Reference spectra from purchased biochemicals were measured. Dot product and ordinary least squares analyses estimated the relative biochemical signal contribution.