Osteoarthritis (OA) is expected to become the fourth leading cause of disability worldwide by 2020. There is no cure, and joint replacement surgery becomes a final resort. Chronic pain associated with OA is poorly controlled by current treatments, and often involve chronic use of non-steroidal anti-inflammatory drugs (NSAIDs), which is associated with serious side-effects. OA is associated with alterations in endocannabinoid (EC), an attractive target for the control of pain. ECs are rapidly degraded by a number of enzymes, including cyclooxygenase-2 (COX- 2), the major target of NSAIDs. However, the role of COX-2 in EC-mediated effects on nociceptive transmission is not fully understood. The aims of this thesis were to investigate peripheral and spinal pain responses in a model of OA pain, understand the role of COX-2 inhibition on neuronal responses and the potential role of ECs in mediating these effects, and to establish the functional effects of the EC system in a model of OA pain. Effects of spinal and peripheral administration of the COX-2 inhibitor nimesulide (1-1 OOlJgin 501JL)on mechanically evoked responses of dorsal horn neurones in the naive, anaesthetised rat were measured, and the contribution of the CB1 receptor was determined with the antagonist AM251 (11Jgin 50IJL). Effects of nimesulide on spinal levels of ECs and related compounds were quantified using liquid chromatography-tandem mass spectrometry. Spinal, but not peripheral, injection of nimesulide significantly reduced mechanically evoked responses of dorsal horn neurones. Inhibitory effects of spinal nimesulide were blocked by the CB1 receptor antagonist AM251, but spinal EC levels were not elevated. Indeed, both anandamide and N-oleoylethanolamide were significantly decreased by nimesulide, highlighting a putative role for other oxidative enzymes of ECs in the generation of CB1-active metabolites. The monosodium-iodoacetate (MIA) model of OA pain has recently received much interest, but is not yet fully defined. Work in this thesis sought to further characterise this model. Cytokine levels in synovial fluid, spinal cord and hindpaw skin at early time-points post- intra-articular injection (1mg MIA in 50IJL, P.O. 3-24hr) were measured, and the later (P.O. day 28-31) effects on neuronal responses and pain behaviour were determined. Intra-articular injection of 1mg MIA produced stable and robust changes in two measures of pain relevant to clinical OA, and evidence for the presence of central sensitisation was demonstrated. It was also demonstrated that early-stage painful responses in this model are not associated with changes in cytokines in the joint. Effects of spinal and systemic administration of nimesulide (3-100IJg in 501JL)on mechanically evoked and post-stimulus responses of dorsal horn neurones in MIA-treated rats were also measured, as were the effects of spinal cannabinoid receptor antagonism with AM251 (0.1- 10IJg in 501JL)and the CB2 receptor antagonist SR144528 (0.001-0.1IJg in 50IJL). Spinal and systemic COX-2 inhibition in the MIA model attenuated spinal neuronal responses to both noxious and innocuous stimuli, demonstrating the importance of both spinal and peripheral COX-2 products in mediating neuronal responses in this model. Antagonism of the spinal cannabinoid receptors resulted in elevated spinal neuronal responses in MIA-treated rats, demonstrating a functional role for spinal EC-mediated modulation of nociceptive transmission in the MIA model, expanding on work in this lab which showed elevated spinal ECs in the MIA model of OA pain. This work therefore demonstrates that the central EC system may be an important target for the treatment of OA pain.

The recently discovered 5-HT6 receptor has generated interest due to increasing evidence for its role in feeding, obesity, anxiety, depression and cognition. Initial studies utilising selective 5-HT6 receptor antagonists found pro-cognitive effects in various cognitive paradigms. In the last three years selective 5-HT6 receptor agonists have been developed and initial reports suggested they impaired cognition as predicted, but more recent reports have found paradoxical pro-cognitive effects in learning and memory tasks. The main aim of the current thesis was to determine the role of the 5-HT6 receptor in a conditioned emotion response (CER) task in rats. Both the effects of 5-HT6 receptor antagonists and agonists given alone, and their abilities to reverse a cholinergic- or glutamatergic-induced memory impairment were analysed. Secondly, to analyse the intracellular mechanisms involved in the behavioural effects exerted following treatment with 5-HT6 receptor ligands by examining changes in hippocampal protein expression. Pre-treatment with either the muscarinic receptor antagonist, scopolamine, or the NMDA receptor antagonist, MK-801, induced memory impairment in the 24 hour retention trial. Post-training administration of 5-HT6 receptor antagonist, SB-271046, and agonists, EMD 386088 and E-6801, had little effect on CER-induced behaviour when given alone, but both reversed the cholinergic- and glutamatergic-induced deficits. Western blot analysis revealed no significant difference between hippocampal BDNF and 5-HT6 receptor protein levels following any drug or shock treatment, but some interesting trends were observed. CER slightly increased BDNF expression, this was reduced by scopolamine and MK-801 which in turn was reversed with SB-271046 and EMD 386088. CER decreased 5-HT6 receptor expression, scopolamine caused further reduction, SB-271046 and EMD 386088 increased the expression following scopolamine. MK-801 increased 5-HT6 receptor expression, whilst SB-271046 further enhanced this expression, EMD 386088 reduced it. No significant results were observed in the proteomic studies. These findings provide further evidence for the exciting potential therapeutic use of 5-HT6 receptor compounds in the treatment of cognitive dysfunction.

The aryl hydrocarbon receptor is a ligand-dependent transcription factor that induces expression of a number of genes encoding drug metabolizing enzymes, such as CYP1A1, CYP1A2 and CYP1B1. Recently, it was suggested that the AhR signaling pathway may be involved in mediating the anticancer activity of novel 2-(4-aminophenyl) benzothiazole drugs in MCF-7 breast cancer cells. There is no direct proof of direct binding between these drugs and AhR, and it is also unclear how AhR signaling per se plays a role in the activity of these drugs. This study investigates the role of AhR in the mechanism of action of the benzothiazole drugs by determining the ability of these drugs to bind to the rat hepatic AhR, to induce CYP1A1 mRNA and to inhibit cell growth in rat hepatoma H4-II-E cells. The apparent binding kinetics of [3H]-TCDD to AhR in rat liver cytosol were, KD= 0.37nM and Bmax = 40 fmol/mg cytosolic protein. Using the standard assay conditions, 18 compounds competitively displaced [3H]-TCDD from specific sites, and are ligands for AhR. Induction of CYP1A1 mRNA by 5 compounds was determined in H4-II-E cells. The highest affinity ligand, IH445, was the most potent with an EC50 ~ 80-fold lower than that of TCDD (60 pM) with no detectable antagonistic activity in H4-II-E cells. The other high-affinity benzothiazoles tested were (30-100) x 103-fold less potent for inducing CYP1A1 mRNA than TCDD. The binding affinities of these compounds were 200-1000-fold higher than induction potency. For example, 5F 203 has a Ki value of 2.8 nM, induced CYP1A1 mRNA to similar maximal levels as seen with TCDD, and has an EC50 of 3 μM. The 1000-fold difference for 5F 203 between binding and CYP1A1 RNA induction was suggested to be a result of metabolism or that 5F 203 exhibits partial AhR antagonist activity. The time course effect on the CYP1A1/β-actin mRNA ratios by 5F 203 revealed that the response was increasing linearly in response to 5F 203 at 4 h treatment, indicating that the former possibilty is less likely to be a major factor. To address the second possibility, the antagonistic activity of 5F 203 on TCDD-induced CYP1A1 mRNA was investigated. H4-II-E cells were treated with increasing concentrations of TCDD ± 1μM 5F 203. The results demonstrated that 5F 203 shifted the EC50 of TCDD 100-fold to the right. Schild analysis on the antagonism of TCDD-induced CYP1A1 mRNA by different concentrations of 5F 203 provided a quantitative explanation for the 1000-fold difference between binding and induction for 5F 203. In contrast, the EC50 of 5F 203 in human MCF-7 cells was 2 nM, which is ~ 10-fold less potent than TCDD. Moreover, 5F 203 had no detectable antagonistic activity on TCDDinduced CYP1A1 mRNA. When 5F 203 was assessed for cell growth inhibition by MTT assay, it was found active in MCF-7 cells with a GI50 of 18 nM, but failed to elicit the same effect in H4-II-E cells. These results prove that 5F 203 is a potent agonist in MCF-7 cells, but a partial agonist in H4-II-E cells. The partial agonism observed with 5F 203 is a compound-specific property given that another analogue, IH 445, was found potent inducer of CYP1A1 mRNA with no antagonistic activity. The results of this study reveal species-specific partial agonism of the AhR. The potency of the cytostatic effect of 5F 203 parallel potency for inducing CYP1A1 mRNA in both cells. Moreover, both, the cytostatic effect of 5F 203 and partial agonism of AhR for inducing CYP1A1 mRNA is species-specific. Whether agonism/antagonism for the induction of CYP1A1 mRNA is related to the anticancer activity of 5F 203 remains to be elucidated.

Poly(lactide-co-glycolide) (PLG) biodegradable microparticles were evaluated as an oral delivery system for immunisation against equine influenza virus. Model particulates (fluorescent polystyrene and gold labelled polystyrene) were used to characterise the route and mechanism of intestinal uptake. Peyer's patches were found to be the site of particulate intestinal absorption characterised by phagocytosis at the M cell surface. Microparticles were found within intercellular compartments indicating a paracellular route for the transit of microparticles from lumen to lymph. A quantitative method of counting the number of microparticles passing into the lymph in both the superior mesenteric and thoracic lymph ducts indicated levels of intestinal uptake high enough to deliver vaccines via this route. PLG microparticles encapsulating equine influenza virus were prepared by the process of solvent evaporation. The immune responses were evaluated in mice after either systemic or oral immunisation of Balb\c mice using formalin treated equine influenza (Prague 56 H7N7) either encapsulated in PLG biodegradable microparticles or free in solution. Using the single-radial- immunodiffusion test, the haemagglutinin integrity of the microencapsulated influenza was found to be preserved during the microencapsulation process. When administered systemically the microencapsulated virus induced raised levels of anti-influenza IgG antibody in serum that were comparable with those obtained with the virus in solution. Oral administration of the microencapsulated virus induced raised levels of anti-influenza IgA antibody in saliva as well as levels of anti-influenza IgG comparable to those obtained after parenteral injection. Raised levels (compared to preimmune levels) of anti-influenza antibodies in both the systemic circulation and mucosal secretions indicates that orally administered microencapsulalated equine influenza virus represents a practical method of immunisation against influenza.

Quorum sensing signalling molecules (QSSMs) are important to Pseudomonas aeruginosa virulence and biofilm development which aid establishment and persistence of these bacteria in the host. Recent progress in quorum sensing (QS) research has demonstrated that the two QSSMs, 3-oxo-C12-HSL and PQS interact with eukaryotic cells and modulate immune responses. Early research has indicated these two QSSMs are immunosuppressive, and because T cells play an important role in defending the host against the attack of P. aeruginosa (Stevenson et al., 1995), this warrants investigations into the interactions between QSSMs and T cells. Previous studies have shown 3-oxo-C12-HSL and PQS can exert differential immune-modulatory effects on mammalian immune responses, however, no studies have confirmed these activities using pure human T cells. The purpose of my PhD was to investigate for the first time, the effects of these two QSSMs on pure human T cells in a staged manner, beginning with mouse splenocytes, human peripheral blood mononuclear cells (hPBMCs) and finally pure T cells, then if successful, paving the way for gene array technology. This present work confirms inhibitory effects by QSSMs on mouse splenocytes stimulated to proliferate using the lectin concanavalin (ConA) or anti-CD3 antibody, and hPBMCs stimulated to proliferate using anti-CD3 and anti-CD28 antibody. In order to further understand interactions between QSSMs and the immune system, the effects of 3-oxo-C12-HSL and PQS on pure human T cell proliferation and cytokine production following stimulation of T cells with monoclonal antibodies directed against CD3 and CD28 were compared, using CsA as the positive control. All three compounds inhibited pure T cell proliferation. CsA and PQS were the more potent anti-proliferative compounds with IC50 values of 3.2±0.31 µM and 3.8±0.15 µM respectively compared to 19±1.62 µM for 3-oxo-C12-HSL, indicating the QSSMs ability to suppress T cell activity and therefore advantageous to P. aeruginosa. To further comprehend the mechanism of action of these two QSSMs, the effects of QSSMs on cytokine production were assessed. 3-oxo-C12-HSL significantly inhibited IL-2 release while PQS enhanced the production of IL-2 even though suppression of T cell proliferation was observed, suggesting a cytostatic effect and demonstrating PQS may in fact act proximally to the IL-2 receptor (IL-2R) or downstream of the T cell signalling pathway, whereas 3-oxo-C12-HSL acts on early T cell signalling events. 3-oxo-C12-HSL also inhibited production of IFNγ in pure T cells and although results were inconclusive for PQS in pure T cell assays, both QSSMs were shown to have an inhibitory effect on IFNγ in mouse splenocytes, suggesting suppression of T cell proliferation is via Th1. Furthermore, 3 oxo C12 HSL suppressed IL-4, IL-5, IL-10 and TNFα while PQS suppressed IL 10 release at 3.12 µM and enhanced TNFα release, indicating these QSSMs may inhibit T cell proliferation by eliminating both Th1 and Th2 response. The immune suppressive properties of 3-oxo-C12-HSL and PQS show potential as future therapeutic entities. Immunosuppressive drugs such as CsA and rapamycin are routinely used to maintain transplants and treat auto-immune disorders. However, they can be non-selective and are limited by their side effects including nephrotoxicity and neurotoxicity. Despite recent developments of new immunosuppressants, there remains an unmet need for less toxic and more widely applicable immunosuppressive agents. 3-oxo-C12-HSL and PQS are worthy of attention as possible future immunosuppressive agents used in conjunction with or in place of present immunosuppressants. In summary, this study clearly demonstrates for the first time that the two structurally diverse QSSMs, 3-oxo-C12-HSL and PQS, can exert differential modulatory effects on pure T cells, opening a path for further study into their mode of actions within the T cell signalling pathways and their effects at an RNA level.

Cannabinoids show potential as new treatments for inflammatory bowel disease (IBD),exerting several favourable effects in the gut, including anti-inflammatory and antimotilityeffects. The main difficulty with cannabinoids is their psychotropic side effects,but access to the brain may be prevented by conjugating the cannabinoid to a bulkygroup such as a dendrimer. The aims of this thesis were to investigate the mechanism by which cannabinoids reduce gut motility and to investigate whether cannabinoids protect the intestine from inflammatory-damage. A further aim was to determine whether cannabinoids remain pharmacologically active when conjugated to a dendrimer. All cannabinoids used (apart from arachidonoylcyclopropylamide and (-)WIN 55,212-2) caused a concentration-dependent reduction in the size of electrically-stimulated contractions in the guinea-pig ileum. The responses were not blocked by CB1, CB2, CBe(putative endothelial cannabinoid receptor) or GPR55 antagonists, suggesting that none of these receptors were involved in mediating cannabinoid responses. PSN 375963 reduced carbachol-induced contraction, suggesting that the GPR119 may be present on ileum smooth muscle. (+)WIN 55,212-2 was shown to protect the guinea-pig ileum from hydrogen peroxide-induced damage but this protection was not blocked by CB1, CB2, CBe or GPR55 antagonists, suggesting that the protective effects were not mediated through these receptors. Conjugation of JWH007 to a spacer (GA003) abolished activity in the guinea-pig ileum and the conjugation of JWH007 to a spacer and dendrimer (GA006) was found to be toxic in the macrophage assay. These studies show that cannabinoid-mediated inhibition of guinea-pig ileum contractions is not mediated through the CB1, CB2, CBe or GPR55 receptor. These receptors were not involved in the (+)WIN 55,212-2 mediated protection against hydrogen peroxide-induced damage in the ileum. The approach of attaching a dendrimer to JWH007 to prevent central nervous system (CNS) penetration does not appear to be a feasible approach because the cannabinoid-dendrimer was unexpectedly cytotoxic.

Neuronal death is induced by a series of pathogeneses in different neurodegenerative diseases, and one of them, which has been widely accepted previously, is the overload of intracellular calcium (Ca2+) in neurones. This study has investigated ryanodine receptor (RyR) on the endoplasmic reticulum (ER) and several potassium (K+) channels which might be neuroprotective, such as the large conductance Ca2+-activated K+ channel (BK) and adenosine-5’-triphosphate (ATP)-sensitive K+ channel, in both neuronal and astrocytoma cell lines. The reverse transcription (RT)-polymerase chain react (PCR) results in this Thesis show that the messages of RyR, BK channel and ATP-sensitive K+ channel (KATP) exist at the messenger ribonucleic acid (RNA) level in those cell lines. And, the expression of RyRIII message was found being increased in SH-SY5Y cells but decreased in NTERA-2 cells after differentiation. The BK channel was confirmed as functional in SH-SY5Y cells with patch clamp recording. Cell insults which increase intracellular Ca2+ ([Ca2+]i), such as cobalt (II) chloride (CoCl2), hydrogen peroxide (H2O2), beta-amyloid (Aβ1-42) and glutamate, were found to reduce cell number in those cell lines in cell proliferation MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assays, and the cells with higher expression of RyRIII message were more sensitive to CoCl2 and H2O2 insults. In cell proliferation assays testing RyR or K+ channel modulators in the presence of insults, it has been found that generic blockade of RyR or K+ channel might be neuroprotective, and the activation of RyR or BK channel and the blockade of KATP channel may aggravate the insults. Selective blockade of RyRI and RyRII cannot protect the cells, which probably indicates that RyRIII is the key target in neuroprotection. Activators of the KATP channel cannot protect the cells at a low dose of CoCl2 but might be protective at a high dose, although cromakalim was an exception. Hence, blockade of RyRIII and the BK channel, and activation of the KATP channel, could be possible neuroprotective strategies. Future study should measure [Ca2+]i and cell apoptosis through Ca2+ imaging and fluorescence-activated cell sorting (FACS) respectively, and design selective a RyRIII blocker.

Caffeine is often described as the most widely consumed psychoactive drug in the world. Despite a substantial amount of research examining the effects of caffeine on mood and cognition, there remain a number of unresolved issues in this field, two of which formed the focus of this thesis. The first pertains to whether caffeine has any behavioural effects beyond a reversal of withdrawal effects purported to exist in habitual consumers following caffeine deprivation. A second relates to the biobehavioural effects of caffeine when consumed in combination with other potentially psychoactive components, as is usually the case in dietary forms of caffeine. This thesis, therefore, firstly compared the cognitive and mood effects of acute administration of caffeine to habitual consumers and habitual non-consumers of caffeine. The effects of combining caffeine with other naturally concomitant compounds were then explored, firstly by examining the impact of combining caffeine with L¬theanine (an inhibitory amino acid found in tea) and then by exploring the effects of guaranâ (a caffeine-containing whole extract). Finally, following on from these latter studies, an attempt was made to establish the lowest active dose of caffeine. Each experiment followed a placebo-controlled, double-blind, balanced cross-over design. In each study, treatment-related changes in cognitive performance were assessed with computerised assessment tools (the Cognitive Drug Research battery, a sentence verification task and serial subtractions), and mood was assessed using both Bond¬Lader and specifically tailored caffeine research visual analogue scales. Where appropriate, salivary caffeine levels and autonomic activity were monitored. Performance was similarly improved for habitual consumers and habitual non- consumers of caffeine following caffeine administration. The administration of caffeine in combination with L-theanine led to some modulation of the effects of caffeine. This was also demonstrated when examining the effects of guaraná. A direct comparison of caffeine and guaranâ with matched caffeine levels revealed differences in the effects of the two treatments. Exploration of the lowest active dose of caffeine revealed (largely impairing) effects of caffeine at doses lower than those found in decaffeinated beverages. These findings may have important implications for caffeine research. Firstly, they suggest that behavioural effects of caffeine cannot be attributed wholly to withdrawal reversal. Secondly, they demonstrate that other components commonly co- consumed with caffeine are likely to modulate its biobehavioural effects. Finally, they suggest that levels of caffeine hitherto thought to be inactive may have (negative) psychoactive properties.

Aim: We aimed to identify the antitumoral mechanism of lycopene on human cancer cells of common solid cancers. Methods: The effect of lycopene was assessed by MTT (Methylthiazoltetrazolium) assay at various times and concentrations on human cancer cells of lung (AGS), breast (MCF-7 and MDA-MB-231), colon (WiDR), prostate (PC-3 and LNCaP) and stomach (AGS).  Mechanisms of action were assessed by apoptosis (DNA ladder formulation, DAPI strain, TUNEL assay and western analysis of p53, bcl-2 and bax) and cell cycle study (flow cytometry, BrdU assay and western analysis of cyclinD1, p21 and p27). Results: The results confirmed statistically significant (p&lt;0.05), dose-dependent <i>in vitro </i>inhibitory effect of lycopene on all the cancer cells studied (maximum 54% inhibition on WiDR colon cancer cells). Although a small late apoptosis was noted (10-17% at 96 h), the results were not significant.  Cell cycle arrest in Gl phase was observed (up to 142%, most well seen at 48 h), reciprocated by a decrease in the cell population in S phase.  A decrease of cyclin D1 and an increase of p27 were also noted.  No changes were noted in the expressions of bcl-2 and bax.  Although p523 was slightly decreased, there was no change in the level of its downstream protein p21. Conclusions: Lycopene inhibited growth of common human solid cancer cells <i>in vitro.  </i>Maximum inhibition was noted on WiDR colon cancer cells, mediated through G1 cell cycle arrest and altered expressions of proteins cyclin D1 and p27.  The mechanism was independent of p53 pathway and not mediated by apoptosis.

The effects of endocannabinoids on human, mouse and rabbit bone cells were investigated.  At high concentrations anandamide and 2-arachidonyl glycerol (2-AG) inhibited human osteoclast formation with no effects at lower concentrations. The inhibition was not attenuated by antagonists for the CB₁, CB₂ or TRPV1 receptors, indicating a non-receptor mediated effect.  Conversely, anandamide and 2-AG increased mouse osteoclast formation.  The effect of anandamide was enhanced in cells from fatty acid amide hydrolase (FAAH)-null mice and abolished in cells from CB_1/2 knockout mice.  The effect of 2-AG was not eliminated in CB_1/2 knockout cells, indicating a non-CB₁/CB₂ action.  The CB₁ antagonist, AM251, and the CB₂ antagonist, AM630, both inhibited mouse osteoclast formation.  These effects were not rescued in the CB_1/2-knockout mouse cells.  Both anandamide and 2-AG stimulated actin ring formation and osteoclast activity in human and rabbit osteoclast.  This was prevented in the presence of AM630 but not AM251, indicating a CB₂-mediated response.  The endocannabinoids and the cannabinoid receptor antagonists do not have a regulatory action on osteoblast activity. The effects of the novel cyclooxygenase-2 (COX-2) metabolite of 2-AG, prostaglandin E₂-glycerol ester (PGE₂-G), on human osteoclasts were examined.  Treatment with PGE₂-G inhibited formation and ERK phosphorylation of human osteoclasts.  These effects were attenuated by a selective EP₄ antagonist and mimicked by PGE₂ alone, indicating that PGF₂-G is rapidly metabolised into PGE₂ in human osteoclast cultures.  However, PGE₂-G treatment elevated intracellular calcium levels in human osteoclasts, through a phospholipase C (PLC)- and IP₃- dependent mechanism, indicative of a G-protein coupled receptor effect.  This was not mimicked by PGE₂, or prevented by the EP₄ antagonist, but blocked by a putative PGE₂-G receptor antagonist, PDA-94 indicating that PDA-94 may be a PGE₂-G receptor antagonist.