To achieve the necessary objective of an increasingly integrated and cost effective healthcare service organised, as far as possible, on prevention rather than cure will require out of the ordinary policy changes. The purpose of Medicine Use Reviews (MURs) is to assist patients to use their medicines more effectively, improve their safety and reduce medicine wastage occasioning reduced costs to the provider. A one-year trial of MURs in Guernsey commenced on 1st November 2007, two and half years after their introduction in the United Kingdom. (The States of Guernsey subsequently approved their permanent adoption). The processes leading to the implementation of the MUR service in Guernsey were evaluated employing the Context, Mechanism and Outcome (CMO) model of Pawson and Tilley (2004). Using a mixed methods approach both quantitative and qualitative methodologies were applied to collect and analyse the data. The provision of healthcare services is strictly controlled by laws and regulations, in many instances, specific to the island and its defined community. Thus, two significant strengths of this study were the availability of accurate data which enabled the creation of precise statistics and the ease of access to stakeholders. However, there were limiting factors, primarily those of confidentiality and the small number of GPs (4) and community pharmacists (3) who agreed to be interviewed. At an early stage in the project the researcher became conscious of the emergence of three significant and inter-related concepts, namely those of time, training and tools. All tasks require time to be accomplished, every task requires training and no task can be completed without the correct tools (facilities). Also conspicuous was the frequency the significance of mutual trust was specified by interviewees. The indications are that the concept of the MUR is sound and that, in collaboration with other healthcare professionals, pharmacists have a public health role to play, especially in promoting health education and offering advice. However, one of the major issues currently facing pharmacists is the lack of trust placed in their capabilities by other healthcare professionals and patients. Nonetheless, given the necessary time, tools (facilities) and an enhanced training coupled with access to patient medical records, community pharmacists can carry out a useful and vital role in public health promotion.

No description available.

3,4-Methylenedioxymethamphetamine (MDMA) use has increased dramatically and more intensive patterns of use such as binging have become common. This thesis pays particular attention to the translation of animal data to humans by examining low doses and binge type repeated regimen of MDMA in the rat. The functional effects especially acute and long-term effects on memory have been investigated together with the measurement of changes in 5-HT and dopamine to investigate the possible link of these neurotransmitters and the functional effects of MDMA. The acute effect of single low doses of MDMA on memory was initially examined and it was shown that MDMA (3 mg/kg) acutely disrupted novel object discrimination when given 30 min before the test. However there was no change in 5-HT and dopamine in the hippocampus, striatum and frontal cortex 150 min after MDMA administration. The combined techniques of radiotelemetry and in vivo microdialysis were used to examine effects of 'binge-type' repeated low dose MDMA administration (3 or 6 mglkg i.p. x 3 every 2 h). Locomotor activity, body temperature and 5-HT release in the hippocampus were simultaneously measured in the same animal during MDMA administration. MDMA (3 x 6 mglkg) increased locomotor activity after each injection. In addition MDMA (3 x 3 mg/kg) produced hypothermia following each injection while MDMA (3 x 6 mg/kg) changed thermoregulation as it decreased body temperature after the first injection and then increased body temperature after the second to a maximum of + 1.3 °C after the third injection. Both 'binge' doses of MDMA however increased extracellular 5-HT in the hippocampus after each injection and there was no correlation between 5-HT release in the hippocampus and changes either in locomotor activity or body temperature. The long-term effect of repeated administration of low doses of MDMA (3 or 6 mg/kg i.p. x 3 every 2 h) on memory was investigated using novel object discrimination 2 weeks after treatment. To imitate the single housing condition used in radiotelemetry experiments, rats were individually housed during drug treatment. MDMA (3 x 6 mglkg) caused impairment of novel object discrimination but there was no change in 5-HT, dopamine and their metabolites in the hippocampus, striatum and frontal cortex 2 weeks after MDMA treatment suggesting no contribution of either 5-HT or dopamine loss to the MDMA-induced memory impairment. The effects of housing conditions on MDMA-induced changes in body temperature and subsequent 5-HT neurotoxicity were determined. Group housed rats showed a similar pattern of changes in body temperature to singly housed rats measured by radiotelemetry following MDMA (3 x 6 mg/kg) suggesting no effect of the housing condition on MDMA-induced changes in body temperature. MDMA (3 x 6 mg/kg) given to group housed rats however produced loss of hippocampal 5-HT 2 weeks after treatment indicating that MDMA-induced hyperthermia is not an essential factor for MDMA-induced neurotoxicity. The influence of tyrosine on MDMA-induced 5-HT neurotoxicity was determined by depletion of brain tyrosine availability by giving a tyrosine-free amino acid mixture (1 glkg twice 1 h apart) to Dark Agouti rats before and after MDMA administration (12.5 mglkg i.p.). A small increase of tyrosine in the hippocampus and striatum occurred in rats treated with MDMA alone. Although the tyrosine-free amino acid mixture decreased tyrosine in the hippocampus and striatum by more than 50% 2 h after administration, this did not protect against MDMA-induced acute hippocampal and striatal 5-HT depletion and long-term 5-HT loss in the hippocampus indicating no effect of tyrosine on MDMA-induced 5-HT neurotoxicity. Overall the results of the present study provide extensive evidence for acute and long-term memory impairments following single and 'binge-type' repeated low dose MDMA administration and that these effects may translate effectively to human conditions. The memory impairments appeared to have no link with 5-HT and dopamine thus it is important to focus on other factors involved in the mechanism of MDMA-induced memory impairments.

It was decided to investigate the regulation of apoE by the LXR subfamily of nuclear receptors. LXRs have atheroprotective properties due to their ability to upregulate the expression of genes involved in the reverse cholesterol transport process. ApoE is a direct target of these ligand-mediated transcription factors and a clearer understanding of the LXR-mediated transcription of apoE would aid the development of potential therapeutic agents for atherosclerosis. Therefore, the involvement of signal transduction pathways in the LXR-mediated regulation of apoE in macrophages was investigated. Through the use of commercially available inhibitors, we firstly identified a novel role for the JNK/SAPK MAPK, P13K and CK2 pathways in the LXR-mediated induction of apoE mRNA protein and secretion in human THP-1 macrophages. This inhibition of apoE induction was also shown to occur in human primary monocyte-derived macrophages and further investigations demonstrated the potential role of these cell signalling pathways in the LXR-mediated regulation of ABCA1, ABCG1 and LXR?. It was also found, for the first time, that treatment of THP-1 macrophages with the oxysterol LXR ligand, 22(R)-hydroxycholesterol, induced JNK phosphorylation and kinase activity, and the subsequent phosphorylation of the c-jun transcription factor. Treatment of THP-1 macrophages with the LXR ligand also resulted in the phosphorylation and activation of Akt, a downstream component of PI3K signalling. In addition CK2 activity was found to be increased in THP-1 macrophages treated with 22(R)-hydroxycholesterol. In conclusion, the studies presented in this thesis demonstrated, for the first time, an important role for the JNK/SAPK MAPK, P13K and CK2 pathways in the activation of macrophage apoE gene expression by the LXR subfamily of nuclear receptors. A potential role for these cell signalling pathways was also implicated in the LXR-mediated regulation of ABCA1, ABCG1 and LXR?.

Tamoxifen has long been used in the treatment of hormone responsive breast cancer. However, tumours frequently develop resistance within 2-5 years of treatment, characterised by the return of tumour growth. The epidermal growth factor receptor EGFR is an important contributing factor in allowing formerly tamoxifen sensitive tumours to grow in the presence of tamoxifen. High levels of EGFR in many tumours correlate with a poor prognosis and an increased resistance to cytotoxic drugs. It was the initial aim of this study to ascertain whether the increased EGFR signalling associated with tamoxifen resistance results in a phenotype more resistant to cytotoxic drugs, and to study the underlying mechanisms that may cause this. Rather than observing the expected increase in resistance to cytotoxic drugs upon the development of tamoxifen resistance, a greatly increased sensitivity to the radiomimetic drug bleomycin was observed. Inhibition of EGFR in either the tamoxifen sensitive or resistant cells had very little effect on bleomycin sensitivity, The rate of uptake of various drugs was measured, and found to be identical between tamoxifen sensitive cells and their tamoxifen resistant derivatives. Microarray analysis of mRNA levels of drug efflux proteins also showed no significant decrease in drug efflux pump gene expression, with two efflux pump genes MRP3 and MRP4 showing increased expression. Tamoxifen resistant cells displayed greatly increased sensitivity to the apoptosis inducer camptothecin, and showed a significant increase in the levels of activated caspases present. Immunocytochemistry revealed a significant downregulation of the anti-apoptotic protein bcl-2.. Sensitivity to bleomycin was also measured and was found to inversely correlate to bcl-2 status. Finally bcl-2 levels were modulated using oestrogens and antioestrogens, and with an siRNA directed against the oestrogen receptor. The effect on bleomycin sensitivity was examined. Reduction of bcl-2 expression by either method had no effect on bleomycin sensitivity

Purinergic P2X7 receptors are ligand-gated ion channels characterized by the formation of a membrane pore upon continuous activation. This receptor has been shown to be expressed and functional in osteoclasts. P2X7 receptor knockout mice studies suggested that this receptor has a role in bone formation. The aim of this project was to characterise the expression and function of P2X7 receptors in human osteoblasts and to investigate the modulation of P2X7 receptor expression and function by oestrogen and glucocorticoids. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to study P2X7 receptor expression at the mRNA and protein levels, respectively. P2X7 receptor pharmacology was studied using pore formation and YO-PRO 1 influx in the presence of different agonists and antagonists. The effects of P2X7 receptor activation on physiological aspects of osteoblast function, namely alkaline phosphatase (ALP) production, interleukin-6 (IL-6) and IL-lp release and mineralisation, were also studied. The general findings are: Human osteoblasts (cell lines and primary cells) express functional P2X7 receptors with higher expression seen in primary cells. P2X7 receptor protein is expressed in all the osteoblast-like cell line samples used (lysate, nuclei, and membranes). The functional form of the P2X7 receptor protein appears to correspond to the 67 kDa band and not the 85 kDa band as suggested by the Western blotting results. The pharmacology of P2X7 receptors in human osteoblasts is atypical as evinced by the low affinity of the agonist, benzoyl- benzoyl-adenosine triphosphate (DBzATP) and the weak action of the antagonists, KN62 and brilliant blue-G (BBG). 17p-oestradiol and dexamethasone modulate P2X7 receptor function probably by a non-classical, non-genomic mechanism. ALP production and IL-6 release are increased by P2X7 receptor activation. P2X7 receptor activation appears to decrease mineralisation in primary human osteoblasts. In addition to P2X7 receptors, human osteoblasts express P2X4 and P2X5 receptors. These findings suggest that P2X7 receptors play an important role in osteoblast bone formation and in osteoblast-osteoclast signalling. Targeting P2X7 receptors is a novel approach for the treatment of conditions such as osteoporosis and rheumatoid arthritis. However, further research is needed to develop selective agonists and antagonists.

Mammary gland involution proceeds through massive, highly controlled epithelial cell apoptosis and tissue remodelling. Thus, mammary gland represents an ideal physiological environment in which to study apoptosis. Recently, the Smad gene family has been identified as mediators of TGF-P superfamily signaling and has been implicated in mediating epithelial cell apoptosis. The Smad family of signal transduction proteins focuses around a central mediator, Smad4, and this report presents a number of analyses which have been undertaken to investigate the potential apoptotic role of Smad4 in the mammary gland. To investigate the role of Smad4 I have utilised an over-expressing Smad4 transgenic mouse. This transgenic mouse has been designed to increase Smad4 protein in the secretory epithelial cells of the mammary gland during pregnancy and lactation by using the biological properties of the BLG promoter. Histological investigation of Smad4 slides indicated accelerated involution and significant increase of apoptosis at day 2 and 3 of involution. Molecular analysis through Western blots for STAT3 and STAT5a levels showed a deregulation of both proteins at the same time points. The mechanism with which transgenic mice regulated apoptosis was prove by Western blots to be via the p27Kipl molecular pathway and independent of p21wa or Box. Microarray analysis for day 3 involuting mice showed an over expression of Vitamin D Receptor. A result which was confirmed by both semi quantitative RT-PCR and Western blot analysis. Raw microarray data showed a down regulation of Methyl Binding Domain 2 (MBD2) in Smad4 transgenic mice indicating that MBD2 could play a role in mammary gland involution. However, analysis of conditional mammary knockout MBD2 mice at day 3 involution did not show any involvement of MBD2 in the regulation mammary gland. Another way of investigating Smad4 was by passing the Smad4 construct to Stat3 mammary knockout animals with the hypothesis that Smad4 could restore STAT3's delayed involution. Based on the deregulation of STAT3 protein levels in the smad4 transgenics the above hypothesis was investigated at day 3 of involution. Phenotypic investigation showed that over expression of smad4 is not able to restore the delayed phenotype of STAT3 knockouts. My final investigation was to identify molecules whose expression is altered by a conditional deletion of STAT3 in the mammary gland. This investigation was approached through microarrays which demonstrated an over expression of the Bone Morphogenic Protein receptor, a result which demonstrates once more the synergistic cooperation of Smads and STATS.

This thesis describes work carried out to characterise cardiac responses to cannabinoids. A particular focus of the work was to study the mechanism by which cannabinoids protect hearts from ischaemia-reperfusion injury Studied in rat isolated right atria, anandamide (an endocannabinoid) induced a limited positive chronotropy. As this response was blocked by indomethacin and not mimicked by methanandamide (a non-hydrolysable analogue), the mechanism of action appears to involve conversion and release of products of cyclooxygenase-2. Conversely, negative chronotropy was observed when baseline rate was elevated by a fi-adrenoceptor agonist. The pharmacological profile of this anti-adrenergic response indicated the involvement of CB2 receptors. Potential protection afforded by cannabinoids against rat isolated hearts subjected to ischaemia-reperfusion was studied in two different models. In the first, hearts were subjected to global, no-flow ischaemia-reperfusion with agonists introduced 5 min before ischaemia. The second method subjected hearts to regional ischaemia-reperfusion with agonists introduced 35 min into ischaemia. Antagonists, where used, were present throughout the protocol. In the global, no-flow ischaemia-reperfusion model both anandamide and methanandamide significantly reduced infarction but in the regional ischaemia-reperfusion model only methanandamide significantly reduced infarction. In both models the infarct limiting action was lost in the presence of both the CBi receptor antagonist SR141716A and the CB2 receptor antagonist SR144528. However, CBi- or CB2-receptor agonists, used alone or in combination, were ineffective at reducing infarct size. These results suggest that neither CBi nor CB2 receptors, alone or in synergy mediate cardioprotection. These results suggest that one or more novel sites of action, possibly a new cannabinoid receptor subtype, might mediate cardio-protection in rat hearts. These findings demonstrate the potential for cannabinoids to be used as adjuncts to thrombolytics. However, the in vivo effects of cannabinoids in situations of compromised cardiac function needs to be assessed as they could potentially cause excessive hypotension due to peripheral vasodilatation.

Background Acetylcholine (ACh) and the calcium ionophore A23187 are both known to trigger EDHF-type responses in the rabbit iliac artery via endothelial cell hyperpolarization mediated by the opening of calcium-dependent potassium channels (Kca). In addition, ACh and A23187 also stimulate the release of hydrogen peroxide (H2O2) from the endothelium. Aims of study 1) To determine the relative contribution of different Kca channel subtypes to ACh- and A23187-evoked responses. 2) To determine whether there is a connection between the activation of endothelial Kca channels and the release of H2O2. 3) To identify the source of endothelium-derived H2O2 in the rabbit iliac artery. Major findings 1) Immimohistochemical investigations demonstrated expression of SKca, IKca and BKca channels in the endothelium of rabbit iliac arteries. 2) Mechanical studies with the SKca inhibitor apamin, the IKca inhibitor l- (2- chlorophenyl)diphenylmethyl -lH-pyrazole (TRAM-34) and the BKca inhibitor iberiotoxin demonstrated that all three Kca channel subtypes participate in ACh- and A23187-evoked EDHF-type relaxations. 3) Mechanical investigations with catalase and the catalase/SOD-mimetic manganese porphyrin (MnTMPyP) demonstrated that responses to ACh and A23187 both included a significant H2C>2-dependent component, that could be inhibited by combined Kca channel blockade. 4) Investigations with the NADPH oxidase inhibitor apocynin, the xanthine oxidase inhibitor oxypurinol and the inhibitors of the mitochondrial electron transport chain rotenone and myxothiazol indicated that mitochondria are likely to be the main source of H2O2 in the endothelium of the rabbit iliac artery. Conclusions The study has highlighted the concerted role of different Kca channel subtypes in ACh- and A23187-evoked EDHF-type relaxations in rabbit iliac arteries. It has also demonstrated that both responses consist of an H202-dependent component which is attenuated when Kca channels are inhibited. However, the evidence provided is not sufficient to prove that H2O2 release in the endothelium is coupled to Kca activation Additional studies aimed to identify the intracellular compartment that produces H2O2 upon stimulation with ACh and A23187. It has been demonstrated that under the current experimental conditions the most likely source of H2O2 is the mitochondrial electron transport chain.

The antitumour activities of 18 benzoic acid and 2-hydroxybenzoic acid analogues were investigated in HT-1080 fibrosarcoma cell line. Several approaches were used to identify the most effective apoptotic agents capable of inhibiting cell population expansion of HT-1080 cells mostly at a concentration of 0.4mM. Techniques used in this study included: cell viability assays (MTT, direct count and time-lapse tracking images), morphology (DAPI, haematoxylin-eosin, methyl green-pyronin y, and SEM), immunocytochemistry (Annexin V, caspase-3) and pharmacology (2-hydroxybenzoate uptake). The results indicated that most of these compounds showed antiproliferative activities at specific concentrations (range 0.025-8mM), with an incubation time of 2-180 hours. It is evident that zinc 2-hydroxybenzoate was the most effective antiproliferative agent at 0.3 and 0.4mM. Other analogues, mainly calcium, also showed antiproliferative activities but at higher concentrations (up to 8mM). The growth inhibitory effect on HT-1080 cells population after treatment with either calcium or zinc 2-hydroxybenzoates was identified as the occurrence of apoptosis. This was confirmed by the morphological techniques as well as by immunoassay including annexin V and caspase- 3, measured by flow cytometry. Although strong evidence has been presented here for apoptosis, the genetic mechanism remains uncertain. Neither the expression of the six proteins p53, p21, Bax, Bcl-2, histones and TNF-a, nor the cell cycle analysis was able to fully elucidate the mechanism of action of calcium and zinc 2-hydroxybenzoate on HT-1080 cells. Nonetheless, calcium and zinc 2-hydroxybenzoate-induced apoptosis clearly involved caspase-3 through Bax and p53/p21, respectively, and displayed the properties of potentially therapeutic compounds.