The transdifferentiation of pancreatic acinar cells towards hepatocytes is an event that occurs in vitro and in vivo in rodents. The B-13 cell line is a model for studying this phenomenon in vitro; it readily transdifferentiates into hepatocyte-like cells in response to glucocorticoids such as dexamethasone (DEX). The transdifferentiation event is dependent on a transient suppression of Wnt signalling followed by induction of Serine/threonine-protein kinase 1(SGK1) via interactions with the glucocorticoid receptor. This thesis has aimed to further explore pancreatic to hepatic transdifferentiation, using the B-13 cell as a model and also investigated the phenomenon in human cells. As hepatic stellate cells are involved in liver regeneration and may support the progenitor niche in liver, coculture experiments were conducted to assess their effects on B-13 transdifferentiation. Transdifferentiation was enhanced in cocultures and found to be dependent on cell-cell interaction that resulted in further suppression of the Wnt signalling pathway by myofibroblasts. B-13 transdifferentiation was shown to be able to take place in vivo for the first time; cells were found to engraft only into the liver and pancreas of NOD/SCID mice. Interestingly, only cells within the liver environment showed expression of hepatocyte-specific genes. B-13 cells were also cultured in 3D bioreactor devices where they transdifferentiated into functional hepatocyte-like cells with gene expression at levels comparable to primary rat hepatocytes. Elucidating the mechanisms involved during B-13 transdifferentiation will support the isolation of an equivalent human pancreatic cell. Studies with a human cell line and primary exocrine cells demonstrated that glucocorticoids also induce hepatocyte-gene expression, and thus the generation/isolation of a human equivalent to the B-13 is a realistic goal.

Macronutrient digestion is a major factor in health and metabolic diseases such as obesity and diabetes and presents a huge global challenge. Modulating macronutrient digestion with food additives and pharmaceuticals has been shown to be a fruitful approach to the treatment of obesity (Orlistat) and diabetes (Acarbose). Previous work has shown that bioactive agents have novel modulatory effects on the major enzymes of digestion, and work in this lab has shown that specific alginates can inhibit pancreatic lipase up to 70%. Alginates are now being investigated as a potential anti-obesity agent. The purpose of this thesis was to develop in vitro methodologies and an analytical approach for investigating the effects of exogenous compounds on the major digestive enzymes; -amylase, pepsin, trypsin, and lipase. A 3-step process was developed consisting of; higher-throughput single enzyme analysis, selected enzyme kinetics and model gut analysis. Alginates were shown to inhibit the action of pepsin, but have no effect on trypsin activity in vitro. The structure of alginate is key to the inhibition of pepsin, and rheological and viscometric data suggested that this effect was due to a pH dependent interaction between alginate and protein substrate as well as direct enzyme-inhibitor interactions. A similar effect was observed with Fucoidan and sulphated carrageenans. In the model gut analysis, these effects manifested as inhibition of proteolysis in the simulated gastric phase, but not in the small-intestinal phase. Alginates were shown to increase the activity of α-amylase during in vitro single enzyme analysis, but have no significant affect on carbohydrate digestion in a model gut simulation. Fat digestion in the model gut simulation was inhibited by specific alginates, adding further weight to the potential use of alginates as a therapeutic treatment of obesity.

Campylobacter jejuni is an important food-borne pathogen, and a major cause of bacterial gastroenteritis. Despite this, relatively little is known regarding the way in which C. jejuni colonises hosts, causes disease or survives in the environment during transmission between hosts. C. jejuni responds to a number of biological molecules found in the human intestinal environment such as bile salts leading to the induction of Campylobacter invasion antigens (Cia), and norepinephrine in response to which the bacterium shows increased virulence. In this study we investigated the response of C. jejuni to mammalian pancreatic α-amylase. The results of this study show that C. jejuni responds to pancreatic α-amylase with production of a mucoid colony phenotype that results from increased secretion of extracellular polysaccharide (EPS) identified as an α-dextran, further to this it was found that the amount of extracellular protein produced by C. jejuni was also increased, the proteins where identified using liquid chromatography mass spectrometry (LC-MS), a number of proteins associated with virulence were identified in the samples grown in the presence of α-amylase. In response to pancreatic α-amylase C. jejuni forms significantly increased biofilm and exhibits increased virulence in the Galleria mellonella and Caco-2 epithelial cell models. Furthermore C. jejuni pre exposed to amylase show significantly increased colonisation of chickens and increased resistance to stresses. It is also shown that C. jejuni is able to utilise α-amylase as a nutrient source, this was determined by growing the bacterium on a defined minimal media with the α-amylase as the only source of carbon present. The ability of C. jejuni to respond pancreatic amylase was shown to require proteolytic activity of Cj0511.

No description available.

Polyphenols are known to be major inhibitors of dietary non-haem iron bioavailability, mainly through their action as iron chelators. In this present study Caco-2 cells were used to investigate the influence of quercetin, the most abundant flavonol in the diet, on non-haem iron bioavailability using 55Fe, and the gene expression of intestinal iron transporters as measured via qPCR. Chronic exposure to quercetin (24 hours) had no significant effect on iron uptake but iron efflux was significantly decreased. Consistent with this, qPCR analysis revealed a significant decrease in basolateral transport genes ferroportin (FPN) and hephaestin expression suggesting polyphenols may have direct gene regulatory effects. Exploring the cellular mechanisms underlying quercetin-induced FPN down-regulation, transfection of 5’FPN promoter constructs showed quercetin did not affect activity but did decrease FPN1A mRNA whilst increasing FPN1B expression; this suggests that although FPN1B is specific to intestinal epithelial cells, FPN1A remains the major isoform. FPN 3’UTR miRNA array analysis identified candidate hsa-miR-17-3p to be significantly activated by quercetin (1.5 fold) and qPCR validation confirmed up-regulation of 101 ± 25.1 -fold (p<0.01). This represents a novel mechanism of quercetin-induced miRNA-mediated regulation of FPN. In HepG2 cells quercetin stimulated hepcidin expression and inhibited ferroportin gene expression; this may provide an additional means of regulating systemic iron levels. Quercetin was shown to be both pro- and anti- proliferative/apoptotic dependent on the concentration used which may have beneficial consequences for liver pathology of iron-overload diseases. In contrast to findings in Caco-2 cells, in Thp1 macrophages quercetin caused a significant dose-dependent increase in FPN expression. Furthermore, quercetin induced both FPN1A and 1B promoter activities. This strongly implies that quercetin acts at the level of the FPN promoter to increase FPN expression - an effect specific to macrophages only. This demonstrates that quercetin has cell-specific effects and its actions on FPN are differentially regulated dependent on cell/tissue type. The results show that quercetin can have multiple effects on iron homeostasis. Given its relatively long half-life in the circulation, repeated dietary intake of quercetin could lead to plasma accumulation in vivo. This may have important consequences for conditions that are low in iron such as anaemia; alternatively it has therapeutic potential for iron overload diseases such as haemochromatosis. By deducing the mechanisms of how dietary polyphenols interact with our intake of essential nutrients such as iron, intake can be optimised to harness the potential benefits polyphenols have to offer.

No description available.

Dietary fibre is a complex material made up of plant cell polymers which are not digested by gastrointestinal enzymes of man but partially fermented by the commensal bacteria in the colon. Since in man most of the colon is not readily accessible, indirect methods must be sought to study dietary fibre metabolism and its effects on colon function. In this project gas chromatographic measurements of two of the end products of fermentation, H2 and CH^, which are expired in the breath were used to monitor fibre degradation in the colon. Effects of fibre on colon function were assessed by measuring changes in stool weight, faecal constituents, transit time and serum lipids. The effect of dietary fibre ingestion on bile acid metabolism and the role of the colon in this process was further studied in the rat. Preliminary studies on the normal excretion pattern of the two gases showed that breath H2 was excreted at a level of less than 1.0umol/l and follows a regular daily pattern of excretion. The concentration was decreased by fasting. CK^ excretion was limited to certain individuals, about a third of the subjects studied, though the proportion of excretors increased with age. In those who excreted CH^ there was no regular daily variation in the excretion and levels were unaffected by diet. Test meals of the dietary fibre components, in the manner administered here, reached the caecum within three hours. Short term separate administration of chemical isolates of fibre resulted in raffinose, stachyose and hemicellulose increasing total breath H2 excretion from a base line level of 1.73 + O.73umol/l to levels of up to l6.36umol/l. Cellulose, pectin and lignin did not alter the excretion level. CH^ excretion was unaffected by any of the polymers. Difference in physical properties of the same chemical polymer appear to have no influence on the or CH^ production. The ingestion of 200g raw carrot daily for three weeks increased ^ evolution significantly but had no effect on the CH^. There was a lag period of a few days between the start of carrot intake and the increase in breath H2 excretion. Carrot decreased the serum cholesterol concentration from a control level of 6.6+0.5 to 5.9 + 0.3mmol/l (P < 0.05) and more than doubled bile acid excretion, particularly primary bile acids. The effect on cholesterol and bile acids remained the same three weeks after cessation of carrot intake. The effect on stool weight was modest and made no impact on the transit time. The addition of cereal fibre to the diet of rats affected the metabolism of bile acids in the gastrointestinal tract both quantitatively and qualitatively and their synthesis in the liver. It is concluded that dietary fibre because of its physical properties of absorption, adsorption and cation exchange and the partial degradation of some of its component polymers by bacteria in the colon, alters the distribution of organic and inorganic substances in the gut which change the luminal content of the colon. This change in luminal content affects the activity of the bacteria and influence some physiological functions of the colon like motility, water and electrolyte absorption and excretion, bile acid metabolism and reabsorption and cholesterol degradation|§nd excretion. Different polymers of dietary fibre have different effects on each of these variables. It is possible to .examine indirectly fibre metabolism and its effects on colon function by measuring expired gases and stool constituents.

This thesis initially reviews the ancient and modern literature on the oesophagus, and its investigation. The current literature on reflux oesophagitis is evaluated, and modern concepts of aetiology are discussed. The five year endoscopic experience of reflux disease for the Portsmouth gastroenterologists is quantified, and it is shown that this is a widespread condition affecting all adult age groups, with a predilection for the elderly. The current gold standard test for acid reflux - 24 hours for pH testing - is evaluated. A new method of interpreting pH test results (the area under the curve) is compared with the more usual cumulative acid index. It is shown that the new method is no more accurate than the old, when trying to separate normal from abnormal pH test results, suggesting that factors other than acid may also be involved in the aetiology of reflux disease. Measurements are performed on samples of human oesophageal refluxate, looking at three other factors which may be implicated in reflux oesophagitis, namely bile salts, trypsin and pepsin. These factors are compared with the pH of the refluxate. It is shown that significant quantities of all three substances are present in the samples, across the pH range, rather than in the narrow pH bands expected. Bile salts are selected for further study. The development of a system for measuring bile reflux using an external gamma detector and <SUP>75</SUP>SeHCAT labelled bile is outlined, and tested. It is shown that this system in insensitive as a measure of bile reflux, but is capable of monitoring the enterohepatic circulation. The developmental program for a new, internal gamma probe and portable monitoring system for use within the gastrointestinal tract is outlined. <i>In vitro</i> studies show this to be highly sensitive to <SUP>75</SUP>SeHCAT, and <SUP>99</SUP>Tc<SUP>m</SUP>HIDA, despite its small size. The internal gamma probe is validated as a measure of bile reflux in human volunteers using a gamma camera for comparsion of results. In all cases studied, correlation between counts from the internal probe and gamma camera are shown to be positive, and are strongly suggestive that bile reflux is being measured. This system can therefore be regarded as the first effective portable bile reflux detector, with significant potential for the further study of reflux related disease.

This thesis describes an observational study of the development of feeding behaviour in the breast and bottle fed human infant from birth to six months. The main aim was to explore the capacity of the infant to express satiety behaviourally. Naturally occurring behaviours were identified and categorised according to various objectively defined criteria during an initial period of observation on approximately 60 mother-infant pairs. A further sample of 20 mother-infant pairs was then used to examine more systematically a selected group of these behaviours. Each pair was visited at monthly intervals from birth to six months, and it is the data drawn from the results of these visits which forms the basis of this thesis. In the exploration of the infant's capacity to express satiety, an attempt was first made to identify potential satiety signals. Behaviours were observed in the neonate which had this potential, but the nature of their expression was found to depend upon age and feeding technique. The value placed on these potential signals was then assessed through the mother's behaviour, where it was found that her behaviour varied according to the feeding technique. Differences were described in terms of the concept of mother or infant control. Discussion of the findings brought out the possible influences that milk composition and the nature of mother control may have on the opportunities available for development in the infant's feeding behavioural repertoire.

The escalating levels of obesity highlight the need to better understand the mechanisms underlying energy intake and energy regulation. The blood glucose response (GR) has been shown to significantly influence short term food intake and therefore energy balance. Regulating the GR is also important in diabetes and impaired glucose tolerance; conditions which are also closely linked with obesity. Poor glycaemic control has moreover been shown to increase risks of other chronic diseases such as cardiovascular disease (CVD). Factors affecting the GR will therefore impact both on energy regulation and chronic diseases. A large number of factors influence the GR. A complete understanding of all these variables is essential if successful regulation of the GR is to be achieved. The studies presented in this thesis focused on two factors affecting the GR that have hitherto received little research attention. These are the physical state (liquid-sold nature) of food and habitual mastication (ingested particle size). The first study investigated the effects of the physical state and showed that it affected the shape and amplitude of the GR and insulin response (IR) curves but not the total metabolic response. The response pattern implied that liquids were satiating for a shorter length of time compared to solids. The subsequent study then investigated the effect of carbohydrate-based energy containing beverages on satiety and short-term food intake and found that they were detected by the physiological energy regulatory systems and suitably compensated for. However, there was a notable gender-wise variation in compensation efficiency. Whilst consuming a carbohydrate beverage does not appear to affect short-term energy balance of males it could induce a positive energy balance in females. Using both in vitro and in vivo models, other studies forming this thesis showed that the degree of particle size breakdown during habitual mastication influenced the magnitude and pattern of the GR. Therefore, habitual mastication appears to be a significant contributor to between-individual variations in the GR. It was noted, however, that these effects were only observed with rice but not spaghetti. The thesis also showed that salivary - amylase could potentially be a significant contributor to the GR, at least in those who spend a longer time masticating. The final study in the thesis showed further that the particle size of ingested food correlated inversely with the GR, IR and rate of gastric emptying. Differences in between-individual variations in the GR, IR, gastric emptying and post-gastric digestive aspects when ingesting food with varying particle sizes are also discussed.