The aim of this thesis was to identify the EP-receptor subtype(s) mediating: - inhibition of the superoxide anion generation in human neutrophils stimulated by formyl methionyl leucine phenylalanine (FMLP), - inhibition of eosinophil cationic protein (ECP) release in a mixed population of PMN stimulated by FMLP, - inhibition of ECP release of superoxide anion generation in a pure population of human eosinophils stimulated by FMLP and C5a respectively, Another part of this study was to test the hypothesis that cAMP is the second messenger mediating inhibition of neutrophil and eosinophil activation, by PGE<SUB>2</SUB>. In the neutrophils, PGE<SUB>2</SUB> and the selective EP<SUB>2</SUB>-receptor agonists produced a concentration-related inhibition of superoxide anion generation. These results taken together with agents which interfere with the cAMP pathway suggested that inhibition of neutrophil superoxide anion generation by PGE<SUB>2</SUB> is mediated by stimulation of EP<SUB>2</SUB> receptors and subsequent activation of adenylate cyclase. From the work carried out using a mixed population of PMN it was not possible to identify the subtype of EP-receptor involved in eosinophil activation. However, since the presence of neutrophils in the preparation could influence the effects of agonists on eosinophil activation, experiments were repeated using a pure population of eosinophils. The results obtained would support the involvement of an EP<SUB>2</SUB> receptor mediating the inhibition of ECP release produced by PGE<SUB>2</SUB>. The study of the possible role for cAMP as a second messenger was mainly performed in a mixed population of PMN not enabling us to draw any conclusion. However, the measurement of cAMP levels in a pure population of eosinophils support this hypothesis, but further study is required in order to definitively conclude on this matter.

Caffeine (10 mM) stimulated the outputs of prostaglandin (PG) F<SUB>2α</SUB>, PGE<SUB>2</SUB> and PGI<SUB>2</SUB> (measured as 6-keto-PGF<SUB>1α</SUB>) from the day 7 and day 15 guinea-pig uterus superfused <I>in vitro</I>. Caffeine-induced PG production was unaffected by the removal of extracellular calcium, ryanodine (RY) and ruthenium red (inhibitors of calcium release from ryanodine receptor (RYR) channel, and calmodulin inhibitors (W-7 and trifluoperazine (TFP)). In fact, W-7 greatly potentiated the caffeine effect on PGF<SUB>2α</SUB> output. TMB-8, an intracellular calcium antagonist, inhibited the increase in PGF<SUB>2α</SUB> output produced by caffeine without preventing the increases in outputs of PGE<SUB>2</SUB> and 6-keto-PGF<SUB>1α</SUB>. Caffeine (1 mM but not 0.1 mM) and theophylline (Theo.; 10 mM) also stimulated PG outputs from the day 7 guinea-pig uterus superfused <I>in vitro</I>. Caffeine and RY both stimulated prostaglandin production by the perfused mesenteric vascular bed of the rat. Caffeine (10 mM) stimulated the output of PGF<SUB>2α</SUB> after 8 h, and the output of 6-keto-PGF<SUB>1α</SUB> after 2, 8 and 24 h from the day 7 guinea-pig endometrium in culture. Caffeine (10 mM) inhibited PGF<SUB>2α</SUB> output after 2 h, but stimulated PGF<SUB>2α</SUB> output after 24 h from the day 15 guinea-pig endometrium in culture. The amounts of PG released from cultured epithelial cells were between 100- to 200-fold and 300- to 1000-fold higher than those from cultured stromal cells from the day 7 and day 15 endometrium, respectively. Outputs of all three PGs from epithelial cells, but not from stromal cells, were 4.4- to 5.5-fold higher from cells obtained on day 15 than on day 7. PGF<SUB>2α</SUB> was the major PG released from the epithelial cells obtained from day 15 guinea-pig uterus. Caffeine had both a stimulatory and an inhibitory effect on PG output from endometrial cells cultured from the day 7 and day 15 guinea-pig uterus.

<I>Aims of the thesis:</I> To study the mechanisms of chemokine production that may occur during hepatic disease, the role of chemokines in hepatic injury and repair following paracetamol poisoning and hepatic expression of chemokines in patients with liver disease. In addition, the roles of tumour necrosis factor alpha (TNF) and stem cell factor (SCF) were also studied following paracetamol poisoning. <I>Results:</I> Both CXC and CC chemokines were produced during monocyte adhesion with human hepatoma cell lines. IL-8 production was dependent on proinflammatory cytokine production. In contrast, CC chemokine production appeared to be dependent on free radical activation of NF-κB. Direct TNF stimulated IL-8 production was mediated by TNF RI receptors via a protein kinase C pathway and was inhibited by dexamethasone. Both CXC and CC chemokines were detectable in human liver biopsies in patients with hepatitis C, hepatic allograft rejection and alcoholic hepatitis. Hepatic CXC and CC chemokines were also induced following paracetamol poisoning, inhibition of MIP2 and MIP1 alpha increased 3 day mortality and augmenting MIP2 expression was associated with accelerated hepatic regeneration. Hepatic TNF expression was not induced following paracetamol poisoning and inhibiting TNF was not associated with protection from hepatic injury. SCF was present within the liver at high concentration and located in both hepatocytes and bile ducts. Paracetamol poisoning was associated with reduced hepatic SCF concentrations and inhibiting SCF was associated with delayed hepatic regeneration. <I>Conclusions: </I> Both adhesion mediated and direct proinflammatory cytokine stimulation induce hepatic chemokine production. Chemokines are expressed in liver tissue from patients with hepatitis and therefore may be implicated in the pathogenesis of hepatic inflammation.

This thesis has studied intracellular mechanisms involved in prostaglandin (PG) synthesis by and release from the guinea-pig uterus and placenta. Adenosine, ATP and its analogous stimulated prostaglandin output (particularly PGF<SUB>2α</SUB>) from the superfused guinea-pig uterus, and from the endometrium and myometrium cultured for 24 h. Adenosine and ATP-induced PGF<SUB>2α</SUB> output from the superfused guinea-pig uterus was inhibited by the adenosine (A) receptor antagonist, 8-sulphophenyltheophylline and the P2 purinoceptor antagonist, suramin, respectively. Adenosine and ATP-induced 6-keto-PGF<SUB>1α</SUB> output from the superfused guinea-pig uterus was unaffected by 8-sulphophenyltheophylline and suramin respectively. Therefore, adenosine and ATP-induced increases in PGF<SUB>2α</SUB> output from the superfused guinea-pig uterus appear to be receptor mediated responses, while the mechanisms involved in 6-keto-PGF<SUB>1α</SUB> production remain unclear. PG output, particularly PGF<SUB>2α,</SUB> from the guinea-pig placenta and sub-placenta cultured for 24 h increased significantly between days 22 and 29 of pregnancy. Metabolism of PGF<SUB>2α</SUB> by the placenta also increased between these two days suggesting that increased PG output was not due to decreased metabolism. Indomethacin, a non-selective inhibitor of PGHS, and NS-398 a PGHS-2 selective inhibitor, reduced PG output from day 22 and day 29 guinea-pig placenta and sub-placenta after 24 h of culture. PGHS-2 appears to be the predominant enzyme responsible for PGF<SUB>2α</SUB> synthesis by day 22 and day 29 guinea-pig placenta and sub-placenta, while both isoforms of the PGHS enzyme are involved in PGE<SUB>2</SUB> and 6-keto-PGF<SUB>1α </SUB>synthesis by day 22 and day 29 guinea-pig placenta and sub-placenta. The outputs of PGF<SUB>2α</SUB>, and PGE<SUB>2</SUB> from the placenta and sub-placenta and 6-keto-PGF<SUB>1α</SUB> output from day 29 guinea-pig placenta in culture were reduced by the inclusion of cycloheximide, puromycin and actinomycin D (protein synthesis inhibitors) in the culture medium.

The use of the Gompertz function, an accurate model of xenograft growth, could be used to predict subsequent tumour behaviour and thus identify responsive disease. The aim of this study was therefore to determine the relationship between tumour response to tamoxifen and changes in apoptosis and mitosis, to confirm their chronology and specificity in a model system, and to assess the possibility of identifying these changes in fine needle aspirates (FNAs) rather than biopsy material. In a cohort of 50 post-menopausal women with ER positive breast cancer treated with tamoxifen, the level of expression of both ER and the cell-survival gene bcl-2 predicted for tumour response. After three months' treatment there was a significant fall in proliferation in most tumours, but no overall change in apoptosis, as assessed by morphology. There was a significant trend for tumour response to correlate with the change in the apoptotic:mitotic ration (p < 0.05). To determine more accurately the timing of these changes, mice bearing xenografts of the ER positive ZR-75-1 and ER negative MDA-MB-231 breast cancer cell lines were treated with tamoxifen. After 2 days, an increase in apoptosis and the apoptotic:mitotic ratio was seen in the sensitive ZR-75-1 tumours (p < 0.05), and on day 7 there was also evidence for reduced proliferation (p < 0.05); however clear evidence of tumour regression only occurred after 14 days. On day 7 there was however a significant correlation between the apoptotic:mitotic ratio and the difference between the actual and the Gompertz-predicted tumour volumes (p < 0.02). These changes were not apparent in tamoxifen treated MDA-MB-231 tumours. No changes in bcl-2 expression were seen. Furthermore, although similar changes were seen in tumour FNAs, statistically they were not as significant, suggesting that they do not offer an alternative to repeat histological examination of the tumours. Early changes in the apoptotic:mitotic ratio during tamoxifen treatment of breast cancer may aid in the prediction of endocrine sensitivity.

The effects of <I>N</I>-methyl-D-aspartate (NMDA) receptor antagonists on learning behaviour have been studied extensively as they block long-term potentiation (LTP), the surrogate neural model for memory storage mechanisms. However, NMDA receptor antagonists also induce prominent changes in motor behaviour and the role of this drug in learning impairment by blocking LTP or by inducing behavioural abnormality remains ambiguous. Thus competitive NMDA receptor antagonists are less than satisfactory pharmacological tools for resolving the above question since the doses required for both effects are indistinguishable. Furthermore, the non-competitive NMDA receptor antagonists like MK-801 (dizocilpine) induce pronounced behavioural effects at lower doses than those required to block LTP and severe ataxia inhibits the ability of animals to perform learning tasks involving motor ability. These features greatly reduce its usefulness as a pharmacological tool. In an attempt to resolve this issue, the novel non-competitive NMDA receptor antagonist, FR115427 ((+)-1-methyl-1-phenyl-1,2,3,4-tetrahydroisoquinoline hydrochloride), was used in this thesis. Although its binding affinity to the NMDA receptors is about 10 times less than that of MK-801, it induces relatively mild ataxia which allows testing over a wider dose range. The faster kinetic profile of FR115427 has the additional advantage that it highlights the clear-cut time dependence of the drug's action. In conclusion, the action of FR115427 on LTP was temporally and dose dependently differentiated from that on spontaneous behaviour and learning. It is suggested that non-competitive NMDA receptor antagonists impair spatial learning by an unknown mechanism that need not be related to their action on LTP.

Presynaptic adenosine A<SUB>1</SUB> receptors, at synapses between Schaffer collateral commissural (SCC) fibres and hippocampal CA1 neurones, were studied using the whole cell blind patch clamp technique and determining the effects of an adenosine agonist and antagonist. The selective adenosine A<SUB>1</SUB> receptor agonist 2-chloro-N<SUP>6</SUP>-cyclopentyladenosine (CCPA) and the antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no effect on the apparent input resistance (R<SUB>m</SUB>) and membrane potential (E<SUB>m</SUB>) of the postsynaptic cell and thus unlikely to alter the magnitude of synaptic response postsynaptically. Two approaches were employed to examine the role of the adenosine A<SUB>1</SUB> receptor in the regulation of glutamate release. Firstly, spontaneous miniature EPSPs (mEPSPs) were recorded and the actions of either CCPA or DPCPX were studied. Neither drug altered the mean amplitude of the mEPSPs compared to control. CCPA decreased the frequency of the mEPSPs from control, an inhibition which was completely reversed by co-application of DPCPX DPCPX alone increased the mean frequency of the mEPSPs from control. This suggested that the CA-1 neurones are subject to a tonic inhibition by endogenous adenosine. Secondly, single and pairs of evoked responses were measured during the activation of the SCC fibres using low intensity stimulation before and after exposure to either CCPA or DPCPX. After exposure to DPCPX the amplitude distributions of the singly evoked EPSPs and EPSCs were significantly shifted to the right when compared to control. This was accompanied by an increase in the mean amplitude. In paired pulse experiments, carried out under current and voltage clamp, exposure to CCPA produced a leftwards shift in the amplitude distributions of both the EPSPs and EPSCs and significantly decreased the mean amplitude of both the first and second responses. After exposure to DPCPX the mean amplitude of the EPSPs and EPSCs increased, but not significantly, and their distribution was shifted to the right.

This thesis focuses on the clinical efficacy, pharmacokinetics and pharmacodynamics of artemisinin based drugs and their combinations for their treatment of P. falciparu11l malaria. The aims of the thesis are to get a better understanding of the basic pharmacology of artemisinin type drugs and their combinations. Chapter I is introduction and general knowledge about malaria disease, antimalarial drugs, treatment and drugs resistance. Chapter 2 determines parasite drug susceptibility and drug interaction of dihydroartemisinin and the quinoline type drug, piperaquine, used together as a fixed dose drug combination against P. falciparu11l in vitro. The results show that P. falciparu11l is highly susceptible to these two drugs even though when used in combination they show slight antagonism. Using a set of genetically manipulated parasites the data also showed a clear role for mutations in pferl to confer cross resistance to piperaquine and chloroquine. Chapter 3 focuses on the development of methods to accurately measure artesunate and dihydroartemisinin levels in human plasma. The method was highly sensitivity, robust and importantly reproducible. This method was subsequently used for th.e measurement of artemisinin type drug concentrations throughout my research.

No description available.

This study investigated polyamines conjugated to either the nitrogen mustard chlorambucil, or to a fluorescent analogue (MANT). De novo syntheses were used where full control over regioselectivity was required, but derivatisation of naturally occuring polyamines, exploiting the regioselectivity of the BOC protecting group, was used where possible. The structures of two key compounds spermidine-chlorambucil (1) and spermidine-MANT (2), are shown. The reaction kinetics of chlorambucil and spermidine-chlorambucil were investigated in buffered aqueous solution and found to be first order under all conditions. Both compounds were subject to a common ion effect in the presence of chloride. Their rates of reaction were pH independent over the pH range 8 to 3 .5, but dropped rapidly below pH 3.5, corresponding to the pKa's of the aryl amine groups. The rates of reaction of both compounds were shown to be sensitive to the polarity of the medium, with similar decreases in rate in 50% aqueous acetone, and in solutions of the non-ionic surfactant Triton X-100. In the presence of charged surfactants, the compounds showed different rates of reaction, implying a specificity of interaction with surfactant head groups. Interactions with DNA were studied by fluorescence quenching and DNA crosslinking assays. The results suggest that conjugates interact with DNA similarly to free polyamines as binding is primarily electrostatic and dependent on polyamine charge. Transport across cell membranes and intracellular location of spermidine-MANT was demonstrated by fluorescence microscopy. Uptake was i) polyamine-mediated, ii) inhibited by the addition of spermidine, iii) tolerant of structural modification, and iv) saturatable, indicating an active mechanism. The fluorescent conjugate was located within cytoplasmic vesicles, and co-localised with lucifer yellow, which enters endosomes. These results show polyamine conjugates enter cells via an active polyamine transporter, possibly by receptor-mediated endocytosis.