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Numerous epidemiological studies indicate that flavonoid intake as part of a balanced diet confers beneficial health effects in man, including improved cardiovascular function, reduced incidence of cancer and amelioration of symptoms associated with inflammatory disorders (Boots et al., 2008). A recent area of interest that may be fruitful is the study of anti-inflammatory effects of flavonoids in combination with statins. Porcine coronary artery (PCA) segments were incubated overnight at 37°C in modified Krebs-Henseleit solution with or without 1µg/ml lipopolysaccharides (LPS), with either (0.1–10µM) quercetin, or 10µM quercetin 3′-suphate and 10µM quercetin-3-glucuronide, or with (0.01-10µM) epicatechins, 10µM catecchin and10µM epigallocatechin gallate. (0.03-3µM) simvastatins and 10µM pravastatin are also used in this study. In addition, since many quercetin-rich foods also contain significant amounts of myricetin, this flavonoid has also been examined. After 16 to 18 hours, segments were prepared for isometric tension recording in Krebs-Henseleit solution. The segments were then exposed to cumulatively increasing concentrations of KCl and then U46619. Responses are shown as milliNewton or calculated as the concentration causing 50% of the maximum effect (-log EC50). For nitrite measurement, segments of the PCA were incubated in DMEM at 37°C for 24 hours, with or without 1μg/mL LPS. The nitrite content (nmol) of the bathing medium was determined by spectrophotometry using the Griess reaction, while inducible nitric oxide synthase was identified immunohistochemically. Differences between mean values were assessed by ANOVA (post-hoc Dunnett test). Prolonged exposure to LPS caused hyporesponsiveness of the PCA associated with increase in nitrite production by a mechanism that appears to involve the induction of nitric oxide synthase. Nitrite content of the incubation medium increased 3 to 10-fold following exposure to LPS and inducible nitric oxide synthase was detected in the adventitia. The results indicated that all of the tested flavonoids and statins are able to suppress LPS-induced changes in vascular responses, nitrite production and expression of inducible nitric oxide synthase. While 10µM myricetin was inactive. In conclusion I have demonstrated that quercetin, and its principal human metabolites and catechins oppose pro-inflammatory events in both endothelial cells and vascular smooth muscle cells. Possibly through a mechanism involving inhibition of NFkB. Since pre-treatment of the PCA with statins reduced LPS-induced changes in vasoconstrictor responses, suppressed the induction of nitric oxide synthase caused by LPS and the associated increase in nitrite production. It is unlikely that the effect of the statin involves direct inhibition of NOS. These findings are consistent with clinical studies suggesting that prior use of statins may afford protection against bacterial sepsis.

*Please note: The abstract, acknowledgments, list of publications, table of contents and abbreviations in this PDF file appear in a different order to that of the print version of this thesis. Cancer patients, treated with systemic adjuvant chemotherapy, have described experiencing persistent deteriorations in cognition. The nature of these effects is unclear, and although a wide range of theories have been advanced, there is currently no treatment. This thesis uses an animal model to investigate the effects of a commonly prescribed chemotherapeutic agent, 5-fluorouracil (5-FU). The cognitive effects of 5-FU were examined using two behavioural tests, the object location recognition test (OLR) and the conditioned emotional response test (CER) both of which require input from the hippocampus, a brain region associated with memory. Memory consolidation by the hippocampus requires the continual production of new neurons (adult neurogenesis) from progenitor cells in the sub granular zone (SGZ) of the dentate gyrus. As an anti mitotic agent, 5-FU could be reducing the cell proliferation required for neurogenesis and this could be a cause of the cognitive deterioration. This hypothesis was tested by quantifying the numbers of proliferating cells (Ki67+) in the SGZ in sections together with the levels of doublecortin (DCX), a neurofilament expressed in developing neurons and brain- derived neurotrophic factor (BDNF), a factor required for new neuron survival and synaptic plasticity, by Western blotting. After developing the methodology (chapter 2); adult male Lister Hooded rats were given five i.v injections of 5-FU (25mg/kg) over a two week period and their behaviour and cellular aspects of the hippocampus compared with saline injected controls (chapter 3). 5-FU treated animals showed significant impairments in their performance of both the OLR and CER behavioural tests. Animals were sacrificed after the behavioural tests were performed and analysis showed they had significantly reduced numbers of dividing cells in the SGZ and non significant reductions in the levels of BDNF and DCX within the hippocampus. These results demonstrate that 5-FU treatment can produce cognitive impairments in this animal model which are similar in nature to those described by patients after chemotherapy. These behavioural changes are correlated with a reduction in the cell proliferation required for hippocampal neurogenesis providing support for the hypothesis that chemotherapy drugs are affecting this aspect of hippocampal function. In order to develop a treatment for the cognitive effects of chemotherapy the antidepressant fluoxetine was co-administered with 5-FU (chapter 4). This approach was based on recent evidence that fluoxetine can increase neurogenesis and protect neurons after damage. As with the experiment described above, performance in the CER test was impaired by five injections of 5-FU (25 mg/kg) as compared with saline treated controls. Similarly, animals treated with six injections of 5-FU (20mg/kg) were unable to discriminate between objects in novel and familiar locations in the OLR task. However co-administration of fluoxetine in drinking water (10mg/kg/day) for three weeks, starting a week before 5-FU treatment, prevented the impaired performance of this task found in the 5-FU only group. 5-FU chemotherapy caused a significant reduction in the number of proliferating cells in the SGZ compared to controls but this reduction was eliminated in the group co administered with fluoxetine. Fluoxetine on its own had no effect on proliferating cell number or behaviour. Moreover hippocampal BDNF or DCX protein levels in the co-treated group (5-FU+fluoxetine) were significantly increased compared to the 5-FU only treated group. These findings suggest that while 5-FU can negatively affect cell proliferation and hippocampal dependent memory, these deficits can be reversed by co- administration of fluoxetine. To understand the long term effects of chemotherapy, the cellular effects of 5-FU treatment were quantified one day, 2 and 6 weeks after the end of two weeks of 5-FU (20mg/kg) treatment (chapter 5). The results showed that 2 weeks of 5-FU treatment did not significantly reduce cell proliferation in the SGZ when quantified one day after the end of treatment. However proliferating cell numbers were significantly reduced compared to controls two and six weeks after the end of treatment. This suggests that 5-FU has a delayed effect on cell proliferation with its maximum effect two weeks after the end of treatment. Cell survival was quantified by BrdU labelling cells immediately prior to 5-FU treatment, and quantifying the numbers of BrdU positive cells at the different time points. BrdU+ cell numbers were significantly reduced at the end of treatment and continued to decline at 2 weeks but stabilised by 6 weeks. These results demonstrate that 5-FU has prolonged effects on neurogenesis after the end of chemotherapy treatment. The effects of 5-FU on cognition and neurogenesis are discussed and correlated with chemotherapy treated patient reports of continued cognitive impairment for months or years after completion of chemotherapy treatment.

Beta-Adrenoceptors (B-ARs) mediate vasodilatation by activating various mechanisms that collectively contribute to vascular smooth muscle (VSM) relaxation. It has been shown that B2-AR stimulation in cultured cells results in activation of extracellular signal-regulated kinase (ERK). As the functional relevance of this was not known, the aim of the current investigation was determine the role of ERK in beta-AR-mediated vasodilatation. Isoprenaline-induced relaxation of porcine coronary artery (PCA) segments pre-contracted with the thromboxane mimetic U46619 was significantly enhanced by inhibition of ERK activation. Relaxations to the beta2-AR agonist salbutamol, but not those to the beta1-AR agonist xamoterol or the adenylyl cyclase activator forskolin, were also enhanced. The intermediate-conductance Ca2+-activated K+ (IKCa) channel blocker TRAM-34 prevented the enhancement of beta2-AR-mediated responses. Taken together, the data indicate that ERK inhibits beta2-AR-mediated vasodilatation by interacting with a cyclic 3’, 5’-adenosine monophosphate-independent relaxation pathway involving K+ channels. This may occur through a direct regulatory action on the IKCa channel via phosphorylation. Furthermore, the finding that increased ERK activation in a rat model of Type II diabetes was associated with significantly impaired beta-AR-mediated vasodilatation raises the possibility that ERK may represent a promising therapeutic target in the treatment of disease states characterised by abnormal vascular function.

Agonists of hormone receptors possess affinity (the ability to bind) & efficacy (the ability to stimulate effect). In this thesis, alternative expressions of efficacy by recombinant prostanoid Chemoattractant Receptor Homologous molecule of TH2 cell (hCRTH2) receptors have been studied using a variety of assays and pharmacological techniques. When expressed in CHO cells, either with or without co-expression of chimeric G alpha 16z49 G-proteins, CRTH2 receptor-mediated calcium mobilisation pharmacology was found to be as published. Coupling of receptor activation to calcium elevation involved G beta gamma i/o mediated PLC beta -dependent mobilisation of both intra- & extra- calcium. In chimera-expressing cells, an additional coupling mechanism was observed which was presumably G alpha 16z49-mediated. The relative expression of receptor and G-protein molecules in both cell types was investigated but because of deficiencies in the methods employed the relative expression is essentially unknown. Because G alpha 16z49 & G beta gamma i/o represent different classes of PLC beta -activating G-proteins, simultaneous activation of them may have produced a synergistic response in chimera-expressing cells which may have affected the observed receptor pharmacology. When the G alpha 16z49 component was isolated in PTX-treated chimera-expressing CHO G alpha 16z49 cells, reversals of potency order were observed with respect to responses in untreated cells. These were most striking for 17 phenyl PGD2, 15 R 15 methyl PGF2 alpha, 15 deoxy delta 12,14 PGJ2 and 15 R 15methyl PGF2 alpha. Alterations of potency order were also observed in non-chimeric cells (G beta gamma i/o coupling) compared with PTX treated chimera-expressing cells. These were most striking for indomethacin, 16,16 dimethyl PGD2, delta 12 PGJ2 and 9,10 dihydro 15 deoxy delta 12,14 PGJ2. In [35S]-GTP gamma S accumulation assays using membranes prepared from non-chimeric cells and presumably reporting G alpha i/o coupling, agonist pharmacology was similar to G alpha 16z49 mediated calcium mobilisation data. However, the data were markedly different from G beta gamma i/o-mediated calcium mobilisation data generated in non-chimeric cells. These differences were most apparent for 13,14 dihydro 15 keto PGD2, 15 deoxy delta 12,14 PGJ2 and indomethacin. Desensitisation of agonist-stimulated calcium mobilisation was also studied. PGD2 produced rapid & long-lasting desensitisation of hCRTH2 receptors in a biphasic manner suggesting that two desensitisation mechanisms may operate. At low concentrations of PGD2 desensitisation was PTX-insensitive suggesting that a non-Gi/o-protein mediated mechanism may be responsible. Other CRTH2 receptor agonists inhibited responses to subsequent PGD2 EC80 exposure in calcium mobilisation assays. Interestingly, a group of molecules devoid of agonism in the calcium assay also inhibited PGD2 responses. This group of molecules included 19 hydroxy prostaglandins A2, E2 & F2 alpha , and PGE2 and appeared to mediate their effects through a mechanism that did not involve a competitive interaction with PGD2. The data generated here show that CRTH2 receptor agonist pharmacology is critically dependent on G-protein coupling partner and assay methodology, and are strongly indicative of agonist-directed stimulus trafficking. The data are consistent with the notion that G beta gamma subunit activation is not a passive "on-off" event but is rather an active event triggered by agonist- and GTP-dependent conformation changes in both receptor and G alpha subunit molecules.

The therapeutic benefits of cannabis have been known for centuries of years. Yet it has only been in the last 40 years that an understanding of the system by which its works in our bodies has begun to be defined. This has in turn led to the discovery and understanding of the endogenous cannabinoid (eCB) system, alongside its main synthesizing and hydrolysing enzymes as well as the endogenous ligands. The use of synthetic cannabinoid receptor (CBR) ligands for therapeutic use has provided problems regarding the natural endogenous regulatory tone of the eCBs, which in turn has resulted in unwanted side effects. Part of the reason of this is due to synthetic agonists producing the well documented psychotropic effects at CB t receptors. Alternative targets for the manipulation of the eCB system for therapeutic benefits have been explored. One remains to be the use of FAAH inhibitors, which in turn potentially increase levels of eCBs in the system, hence potentiating their effects at the CBRs, or at other receptor sites. Therefore we have developed two HTS assays for the identification of potential inhibitors of FAAH and MAGL. They prove to be robust, cheap and facile and provide a clear indication of inhibitable levels of FAAH and MAGL activity. The FAAH assay can be further used to establish concentration-response curves of initial `hit' compounds. Yet, the HTS MAGL assay requires further characterization for use in construction of concentration-response curves, as they are not assays specific for MAGL acitivity and include hydrolysis of the substrate 4-NPA by non-specific esterases. Z-factor scores were calculated for both assays, indicating excellent assays, which can potentially be applied to industrial lab robotics for screening of compound libraries.