No description available.

No description available.

No description available.

No description available.

Dicarbonyl compounds have been implicated in the process of skin sensitisation. It is widely accepted that the mechanism involved in this allergic reaction relies upon the reactions of these electrophilic compounds with the amino acid side chains of cellular proteins. The most likely points of reaction for dicarbonyl compounds are the side chains of the amino acids lysine and arginine. As such, model compounds were selected to mimic these nucleophilic groups. Butylamine was chosen to represent the lysine side chain. A series of mono- and di-alkylguanidine compounds were synthesised and their reactions with dicarbonyl compounds were evaluated. This led to butylguanidine being selected to mimic the arginine side chain. A series of 1,2- and 1,3-dicarboxyl compounds was selected which included some compounds for which biological data was available. Additional compounds were included in the series to investigate the effects of substituents on the carbonyl group. The majority of the 1,3-dicarbonyl compounds had to be synthesised. The reactions of these compounds towards the model nucleophiles were investigated and kinetic parameters were obtained. Reactions were monitored by HPLC. As part of the method development a study in to the effects of temperature and pH on the separation of 1,3-dicarbonyl compounds was undertaken. A short molecular modelling study was undertaken to investigate the dicarbonyl compounds. The parameters obtained were compared to the experimental orders of reactivity observed from the kinetic studies. Biological data was obtained for the additional compounds in the series. Both the kinetic and the computational parameters were then compared with this biological data. By using (Q)SAR approaches it was possible to identify correlations in the data and in the case of the 1,3-dicarbonyl compounds to develop a predictive model. This study also highlighted a possible structural alert associated with increased allergenic potency that could be used in rule-based prediction systems.

The growing numbers of putative receptors and allosteric sites in which cannabinoids have been observed to act upon has called in to question the established action of cannabinoids through solely the CB1 receptor in the guinea-pig ileum myenteric plexus. Previous work within our laboratory demonstrated cannabinoid inhibition of nicotine induced currents in cultured myenteric neurones through a mechanism expressing different pharmacological properties to those acknowledged as being ('. possessed by the CBI· receptor. To examine-this the effects of cannabinoid receptor ligands were examined on nicotine evoked contractions of the guinea-pig ileum myenteric plexus-longitudinal muscle preparation and on nicotine evoked currents in primary cultures ofmyenteric neurones. Contraction evoked by 100 JlM nicotine was inhibited by the cannabinoid agonist CP 55,940 (ICso 215 nM) but inhibition was not blocked by the CB1 receptor selective antagonist SR141716. Nicotinic log concentration-response curves showed a combination of some rightward displacement and decrease in maximal contraction ,. upon exposure to CP 55,940 suggesting non-competitive inhibition. In the presence of the voltage-gated sodium channel antagonist tetrodotoxin (TTX), both CP 55,940 and SR141716 inhibited contraction and acted additively when applied in conjunction to produce greater inhibition of nicotine evoked contraction. Some degree of stereoselectivity was observed for the cannabinoid agonist WIN 55,212-2 over the stereoisomer WIN 55,212-3, but in the presence ofTTX both stereoisomers displayed equal potency in inhibiting nicotine evoked contraction. Contractions evoked in the presence of the nicotinic receptor antagonist tubocurarine and those evoked by 10 JlM nicotine showed greater CP 55,940 inhibition (ICso 0.74 and 0.55 nM respectively) than that observed with 100 JlM nicotine. CP 55,940 inhibition of contraction evokedby 10 IlM nicotine displayed SR141716 sensitivity with log concentration-response. curves showing a parallel rightward shift (lCso 19.5 nM). Whole cell patch clamp of cultured myenteric neurones confirmed cannabinoid inhibition of nicotine evoked currents and displayed similar levels of inhibition by both active and inactive WIN 55,212 stereoisomers. Blockade of the adenylate cyclase/cAMP intracellular messaging system by 8-Br-cAMP did not affect CP 55,940 inhibition. Results suggest that cannabinoids may act through both CBI receptor dependent and independent mechanisms to inhibit nicotine evoked contraction in the myenteric (' plexus. The mechanisms through which cannabinoids act demonstrated some degree of selectivity dependent upon the level of stimulus evoked by nicotine, with contraction evoked by 100 IlM nicotine demonstrating CBI receptor independence whilst contraction evoked by 10 IlM nicotine showed SR141716 sensitivity. Blockade of action potential propagation by TTX showed a CB1 receptor independent inhibition of contraction occurring within the nerve endings of motor neurones. Whole cell recording confirmed previous work with the lack of WIN 55.212 stereoselectivity corroborating CBI receptor independent inhibition. The inhibition by cannabinoids of electrically induced contraction of the guinea-pig is well accepted. However, the intracellular mechanisms through which cannabinoid receptors act have yet to be established. The effects of cannabinoid receptor ligands on Ca2+ currents were examined in primary cultures of guinea-pig ileum myenteric AH neurones using the whole-cell patch clamp technique. Membrane depolarisation evoked a high-voltage activated calcium current (lca) that was reduced by 65.6% by c.o-conotoxin GVIA (100 nM). Exposure to the cannabinoid agonists CP 55,940 (10 JlM) and WIN 55,212-2 (10 JlM) reduced peak 1ca by 79.7% and 74.7% respectively. The inhibitory effects of CP 55,940 were concentration . dependent over the range 0.3 - 10 JlM with an ICso of 0.54 JlM, and blocked by pertussis toxin (lOO ng.mrl , 18 hr equilibration). WIN 55,212-3 (lO JlM), the (-)- enantiomer of WIN 55,212-2, evoked significantly less inhibition tha~ that produced by WIN 55,212-2, reducing peak lea by 43.1 %. The CBl receptor selective antagonist, SR141716 (l and 10 JlM), had no effect on CP 55,940 (l and 10 JlM) inhibition, whilst another CBl receptor selective antagonist, AM251 (lO JlM), and the CB2 receptor selective antagonist SR144528 (1 Jl~) produced only slight blockade of CP 55,940 (1 JlM) inhibition. In combination, SR141716 (1 JlM) and SR144528 (0.1 JlM) produced complete blockade of CP 55,940 (1 JlM) inhibition. When tested on their own, SR141716 (lO JlM) and the putative 'silent antagonist' 0-2050 (lO JlM) reduced lea by 53.0% and 82.3% respectively. Results suggest that in primary cultures of guinea-pig ileum myenteric AH neurones, cannabinoids inhibit N-type calcium channels through Gilo coupled receptors, comprising of either a combination of CBt and CB2 receptors, or involve a novel ,. cannabinoid receptor. Cannabinoids and opioids have been shown to possess similar pharmacological properties, including analgesia, addiction and euphoria. The powerful analgesic effects of opioids make them vital in the treatment of patients, but their undesirable effects limit their use. The interaction of cannabinoids and opioids opens the possibility of achieving similar or greater beneficial effects whilst reducing undesirable effects through the use oflower amounts ofdrug. The interaction between cannabinoid and opioid receptor ligands.was examined on electrically evoked contractions of the guinea-pig ileum myenteric plexus-longitudinal muscle preparation, and their action in both primary myenteric and NG108-15 neuronal cell cultures investigated. Further examination of the action of cannabinoids on opioidwithdrawal evoked contractions ofthe whole ileum was performed. Both morphine and CP 55,940, when applied independently, produced a concentration-dependent inhibition of electrically evoked contraction of the MPLM preparation. With prior application of CP 55,940, at concentrations of 1 and 10 nM which produced inhibition of 12.0 ± 2.9% (n = 7) and 26.8 ± 5.6% (n = 11) respectively, morphine displayed similar co?~entration-dependent characteristics as displayed in the absence of CP 55,940, suggesting a purely additive interaction. 100 nM CP 55,940 inhibited all morphine evoked inhibition. An inhibition of 40.8± 7.5% (n = 9) was produced by 100 nM CP 55,940 in the absence of morphine, whilst in combination with 1 JlM morphine, which produced maximum inhibition, an inhibition of 46.8 ± 6.8% (n = 9) was observed. Maximal inhibition produced by morphine in the presence of 100 nM CP 55,940 (46.8 ± 6.8%, n = 9) was smaller that that produced in the presence of 10 nM CP 55,940 (62.7 ± 3.4%, n = 11). Prior application of 100 nM and 1 JlM morphine prevented 1 JlM WIN 55,212-2 inducing inhibition of contraction. SR141716 (100 nM) significantly increased the size of electrically evoked contraction and increased inhibition produced by morphine. Opioidwithdrawal contractions of whole ileum were not inhibited by CP 55,940. Both potassium and calcium currents were not affected by morphine or DADLE. In NG108-15 neurones calcium currents were inhibited by DADLE and WIN 55,212-2 in a concentration-dependent manner. In the presence of a concentration of WIN 55,212-2 that produced maximal inhibition, DADLE, also at a concentration producing maximal inhibition, produced a significantly reduced inhibition.

Damage induced by ionising radiation (IR) is mainly repaired by classical non homologous end joining (D-NHEJ), but a small subset of DSBs is repaired with slow kinetics in an ATM (Ataxia telangiectasia mutated) and Artemis dependent manner. In addition, a PARP-1 dependent NHEJ backup pathway (B-NHEJ) was described, which is thought to function in the absence of D-NHEJ. Using ATM, Artemis or DNA ligase IV deficient mouse embryonic fibroblasts (MEFs) as a model system, the effect of the potent and specific PARP-1/-2 inhibitor KU-0059436 upon clonogenic survival after various types of damage that induce different spectra of SSBs and DSBs was measured. In Artemis or ATM deficient MEFs no sensitising effect of KU-0059436 was detected after neocarzinostatin (NCS) treatment; however PARP inhibition increased sensitivity to IR and methylmethane sulphonate (MMS) markedly. In these cell lines no specific single strand break repair (SSBR) defect was observed, and radio-sensitisation by KU-0059436 was replication dependent. Furthermore PARP inhibition led to increased formation of DSBs, an effect which was augmented in Artemis deficient cells. PARP inhibition in DNA ligase IV deficient cells led to increased sensitivity after damage induction to all agents. However, radiosensitisation by KU-0059436 was replication independent. Results show that PARP inhibition increases the dependence on Artemis and ATM after SSB induction, which is consistent with a model whereby DSBs that arise from SSBs during DNA replication in the presence of a PARP inhibitor require ATM and Artemis for their repair. In cells deficient for DNA ligase IV, PARP inhibition causes additional replication independent sensitisation both by abrogating B-NHEJ and promoting accumulation of replication independent DSBs.

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Many athletes consume caffeine for its known ergogenic effects. Since being legitimised by its 2004 removal from the World Anti-Doping Agency prohibited list of substances, caffeine s ability to enhance performance has led to its widespread use amongst the athletic population. However, despite caffeine s prevalence, little research has focused on the effect of caffeine ingestion on immune function both at rest and in response to exercise in humans. Therefore, the aim of this thesis was to investigate the influence of typically-used doses of caffeine (typical daily intake in training and competition doses) on aspects of innate and acquired immunity in response to prolonged exercise. At rest both a high (6 mg kg-1) and low dose (2 mg kg-1) of caffeine had little effect on antigen-stimulated T (CD4+ and CD8+) or natural killer (NK) lymphocyte activation, while a high dose of caffeine only increased the number of antigen-stimulated NK cells expressing CD69 1 h following caffeine ingestion (Chapter 4). In response to prolonged high intensity continuous cycling both high and low doses of caffeine increased the natural state of activation as well as the antigen-stimulated activation of NK cells 1 h after exercise cessation (Chapters 5 and 6). However, at the same time-point a high dose of caffeine decreased CD4+ and CD8+ cell activation (Chapter 5). One hour after high intensity intermittent shuttle running, a high dose of caffeine attenuated the exercise-induced increase in NK cell activation both in terms of the number of cells expressing CD69 and their geometric mean fluorescence intensity expression of CD69 (Chapter 7). These effects did not occur in response to intermittent exercise when 2 mg kg-1 caffeine was instead ingested in 3 repeated doses throughout the day (Chapter 7). In conclusion, the findings of this thesis demonstrate the complex actions of caffeine on antigen-stimulated T and NK lymphocyte activation 1 h after prolonged intensive exercise. However, the biological significance of these findings in terms of caffeine s potential to alter an individuals susceptibility to infection following prolonged high intensity exercise are yet to be determined.