The work presented in this thesis was undertaken to increase understanding of the intracellular mechanisms regulating acid secretion by gastric parietal cells. Investigation of the effects of protein kinase C on secretory activity induced by a variety of agents was a major objective. A further aim was to establish the sites at which epidermal growth factor (EGF) acts to stimulate prostaglandin E2 (PGE2) production and to inhibit acid secretion. These investigations were carried out by using the HGT-1 human gastric cancer cell line and freshly isolated rat parietal cells. In HGT-1 cells, the cyclic AMP response to histamine and to truncated glucagon-like peptide 1 (TGLP-1) was reduced when protein kinase C was activated by 12-0-tetradecanoylphorbol 13-acetate (TPA). Receptor-binding studies and experiments in which cyclic AMP production in HGT-1 cells was stimulated by gastric inhibitory polypeptide, cholera toxin and forskolin suggested that the effect of TPA was mediated by uncoupling of the histamine H2 receptor from the guanine nucleotide regulatory protein Gs, possibly by phosphorylation of the receptor. An involvement of protein kinase C α in this effect was suggested because an antibody to this isoform specifically prevented the inhibitory effects of TPA on histamine-stimulated adenylate cyclase activity in a membrane fraction prepared from HGT-1 cells. Carbachol-stimulated secretory activity in parietal cells was specifically inhibited by Ro 31-8220, a bisindolylmaleimide inhibitor of protein kinase C. Thus protein kinase C may play a role in the activation of the secretory response to carbachol. In parietal cells prelabelled with [3H]-arachidonic acid or [3H]myristic acid, EGF did not affect [3H]-fatty acid or [3H] - diacylglycerol content. No evidence for effects of EGF on phosphatidylinositol glycan-specific phospholipase C, phospholipase A2 or on low Km cyclic AMP phosphodiesterase activities were found.

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The application of fluorogenic and chromogenic enzymatic substrates in culture media continues to offer huge potential for accessible bacterial detection and identification. However, their clinical use poses two major disadvantages: the presence of commensal and/or coliform bacteria camouflages the detection of bacteria of interest, and false results may arise due to secretion of a similar enzyme by a different species (or strain of) that acts upon the same enzymatic substrates. In the current work, selective antimicrobial agents or suicide substrates were developed and investigated in order to prevent the growth of these interfering bacteria, thus improving the integrity of the detection and diagnosis of common clinical infections. This project has been carried out under collaboration between University of Sunderland (UK), bioMérieux (France) and Freeman Hospital, Newcastle-upon-Tyne (UK). The L-alanine analogues D/L-fosfalin and -chloro-L-alanine were synthesised, both of which target alanine racemase, and different peptide derivatives based on these moieties were prepared using the well-established IBCF/NMM coupling approach with a series of protection/deprotection methods. Two dipeptide derivatives based on para-aminobenzoic acid (PABA) were also investigated. A series of synthetic derivatives were subjected to microbiology evaluation using 20 different clinical strains of clinical bacteria at the Freeman Hospital. The stability of -Cl-L-Ala-D/L-Fos was investigated at pH 6.1 – 9.1; hydrolysis products were formed above pH 6.1. The minimum inhibitory concentration (MIC) of L-Ala-L-Ala-D/L-Fos was double the concentration of L-Ala-L-Ala-L-Fos, showing that only the diastereoisomer with L-fosfalin exhibited significant inhibitory activity against the bacteria. The introduction of -chloro-L-alanine into the phosphonopeptide derivatives enhanced the antibacterial activity; for example the growth of commensal bacterium, Escherichia coli, was inhibited at a low MIC (0.125 – 0.5 mg/L) by most X--Cl-L-Ala-D/L-Fos tripeptide analogues, except when X: sarcosine (MIC: 4 mg/L). However, no significant antibacterial activity was found in the PABA derivatives. These di- and tri-peptide fosfalin-containing derivatives, especially with the inclusion of -Cl-L-alanine, offer improved selectivity for the detection and identification of pathogenic bacteria in clinical samples by restricting the over-growth of commensal bacteria.

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Although the pathogenesis of migraine remains to be fully elucidated, recent studies have implicated the involvement of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) and also magnesium. This thesis has further explored the role of 5-HT and magnesium in migraine pathogenesis and also the effects of red wine, believed to trigger migraine in some individuals. Central serotonergic functioning was investigated in migraine and tension-type headache patients by neuroendocrine challenge test, using oral fenfluramine, which stimulates the release of 5-HT from hypothalamic neurones, causing a dose dependent release of prolactin from the pituitary. Plasma prolactin was significantly raised in tension-type headache patients compared with controls, suggesting a central serotonergic supersensitivity. Fenfluramine triggered a headache in most migraine and tension-type headache patients and headache scores positively correlated with prolactin response. Temperature, also under partial control by central 5-HT mechanisms, was significantly raised in migraine patients compared with controls. These findings suggest supersensitivity in the central 5-HT system in tension-type headache, but not in migraine, however, further investigation is required in migraineurs. Plasma prolactin and cortisol measurements were used to determine if red wine acts on 5-HT centrally. No changes were found following red wine administration in controls and migraineurs, suggesting it does not. Migraineurs exhibited significantly lower mean phenolsulphotransferase (PST) P and M levels compared with controls, levels in diet sensitive patients being further reduced compared with non-diet sensitive patients and controls. Red wine is known to inhibit PST in vitro. This research demonstrated that red wine also significantly inhibited PST P in vivo in controls. Urinary magnesium (Mg[sup]2+) excretion was found to be significantly increased during attack compared with migraineurs not in attack and controls. The effect of red wine on Mg[sup]2+ homeostasis was investigated in controls by examining the effects of red wine ingestion on serum Mg[sup]2+, which did not alter, and urinary Mg[sup]2+ levels which significantly increased, implying a general release of Mg[sup]2+ from body stores. However, this also occurred after vodka and white wine ingestion, suggesting this effect is not specific to red wine. Stress is a commonly quoted migraine precipitant. Urinary Mg[sup]2+ was investigated in controls when stressed and stress free, no significant difference was found. In conclusion, this thesis has provided many observations which require further research. A significant supersensitivity in the central serotonergic system has been demonstrated in tension-type headache, but not in migraine. Furthermore, centraI release of 5-HT has been shown not to be involved in the mechanism of a red wine induced migraine attack. This thesis has also provided the first evidence of a reduced PST M and P activity in specifically red wine sensitive migraineurs. Further indication as to the general depletion of magnesium from the body during a migraine attack has also been provided.

The main objective of the study was to investigate the bioactive components, their effect on HL60 leukaemic and Caco-2 colon carcinoma cancer cell lines and their bioavailability from a decoction of a Chinese Herbal Remedy OldenIandia diffusa, using a Caco-2 monolayer as a mimic for intestinal absorption. The HPLC separation method was set up by investigating parameters such as column type, mobile phase, isoeratic/gradient elution, flow rate, pH of buffer and detection wavelength. Using this method eleven fractions (F1-F11) were collected. Results from a cytotoxicity investigation using the CyQUANT NF, trypan blue and neutral red uptake assays showed that the decoction has a cytotoxic effect on HL60 (V=13.5±4.3%, n=3) and Caco-2 (V=50.0±1.4%, n=3) cancer cell lines. The most cytotoxic active fraction was F9 (V=62.2±7.3% (HL60) and V=32.l±7.9%, n=3 (Caco-Zj). DAPI staining and Western blotting (detection of cleaved-PARP) studies on the decoction and F9 fraction indicated that mode of cell death was apoptosis, which was mediated by a caspase cascade. Fraction F9 was separated into eight further fractions (compound- fraction-l to 8) by optimisation of the HPLC method. By using liquid-liquid extraction with ethyl acetate, eleven sub fractions (compound-fraction-l to 8 and ethyl acetate fractions (FEA-l to 3) were collected purely. All collected fractions were analysed by high resolution-MS and their MWs were determined as 268.07,238.07,242.24,280.38,256.24,282.25, 284.27, 456.36,550.17,328.22 and 330.24. In the most cytotoxic fraction F9, oleanolic acid and ursolic acid were isolated and identified in concentrations of 0.068 mg/g and 0.166 mg/g, respectively. FEA-l to 3 also showed cytotoxic effects on these cancer cells (V=53.3±2.2%, 55.4±4.4% and 50.6±11.3% (HL60) and V=63.4±13.5%, 45.8±5.9%, and 59.5±9.7%, respectively (Caco-2), Fraction FEA-l was identified as E-6-0-p-coumaroyl seandoside methyl ester (MW 550.17). Cytotoxicity assessment results showed that it has a growth inhibition effect on both cancer cell lines. Bioavailability after ingestion of the decoction was studied using 21-day grown Caco-2 monolayers. The post absorption sample (PAS) of the decoction and fraction F9 were shown to have a good permeability (P[sub]app=3.575xlO[sup]-6 cm/s). The PAS also has a cytotoxic effect on cancer cells (V=67.0±7.5%, n=3). When analysed by LC-MS, the most of the compounds that had previously been seen in the original fractions were again observed.