The human BTG/TOB protein family comprises six members (BTG1, BTG2/PC3/Tis21, BTG3/Ana, BTG4/PC3B, TOB1/Tob, and TOB2) that display anti-proliferative activity in a number of cell types. They are characterised by a conserved N-terminal BTG domain that mediates interactions with the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylases. It was unclear whether the anti-proliferative activity of the BTG/TOB proteins was mediated through interactions with Caf1a (CNOT7) and Caf1b (CNOT8). To address this we further characterised the amino acid residues located along the BTG2 and TOB1 interaction surface with Caf1a (CNOT7)/Caf1b (CNOT8) to identify residues required for the interaction. We then analysed the role of BTG2 and TOB1 in the regulation of cell proliferation, translation and mRNA abundance using a mutant that is no longer able to interact with Caf1a (CNOT7)/Caf1b (CNOT8). We conclude that the anti-proliferative activity of BTG/TOB proteins is mediated through interactions with the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylase enzymes. We also demonstrate that recruitment of BTG2 and TOB1 to mRNA leads to reduced protein levels and mRNA degradation. Furthermore, we show that the regulation of mRNA abundance and protein levels is dependent on Caf1a (CNOT7)/Caf1b (CNOT8), but does not appear to require other Ccr4-Not components, including the Ccr4a (CNOT6)/Ccr4b (CNOT6L) deadenylases, or the non-catalytic subunits CNOT1 or CNOT3.

In recent years, two G protein coupled receptors have been de-orphanised which respond to long chain free fatty acids (FFAs), and so are able to mediate the signalling of these important nutrient molecules. FFA1 (GPR40) is predominantly expressed in pancreatic -cells, while the expression profile of GPR120 includes gut endocrine cells and adipose tissue. These distributions, together with the potential of both receptors to stimulate insulin and incretin hormone secretion, singled them out as potential drug targets for type 2 diabetes and obesity. The aim of this thesis was to evaluate the pharmacology of these receptors and their signalling properties, including the development of fluorescent FFA receptor ligands to evaluate agonist binding using imaging techniques. GPR120 has been identified to exist as two splice isoforms in humans, differing by a short insertion in the third intracellular loop, but no full isoform specific characterisation of receptor signalling and trafficking had been undertaken. This work therefore studied the GPR120S and GPR120L isoforms in terms of both G protein dependent and arrestin dependent signalling, and trafficking. It was found that the long GPR120L isoform exhibited reduced G protein signalling, but similar arrestin recruitment and lysosomal intracellular trafficking profiles as GPR120S. Potentially, expression of the long GPR120 isoform provides a mechanism to direct signalling to the arrestin pathway, for example to produce anti-inflammatory effects in macrophages. As the expression profile of GPR120 overlaps with that of FFA1, for example in colonic endocrine cells, a series of constrained GPR120 homo-dimers and GPR120:FFA1 heterodimers were created using irreversible bimolecular fluorescence complementation, and the potential for novel pharmacology was investigated by monitoring dimer internalisation. However, there was no evidence that such dimerisation altered the pharmacology of the ligand tested. Second, a model of the GPR120S ligand binding site was tested using point mutagenesis of the receptor. This mutagenesis validated key features of the model, including the role of Arg99 in co-ordinating the agonist carboxylate group, and interactions of the agonists with the conserved transmembrane VI Trp “toggle-switch” involved in receptor activation. Another mutation (Asn215) provided evidence for ligand-specific binding modes within the pocket. This study showed the complexity of testing mutants designed to interfere with ligand binding indirectly through signalling assays and highlighted the requirement for a FFA receptor binding assay to measure ligand affinity directly. In the absence of radioligands of suitable selectivity and affinity, a novel fluorescent ligand, based on the FFA1/GPR120 agonist GW9508, was used to successfully develop a whole cell FFA1 competition binding assay for the first time, obtaining FFA1 affinity estimates for a range of synthetic ligands. Fluorescent ligand binding was further investigated using fluorescence correlation spectroscopy and photon counting histogram analysis, defining the diffusion characteristics of FFA1 receptors in the membrane of single living cells, and providing preliminary evidence for their dimerisation.

Background: Microglial cells can be regarded as the macrophages of the central nervous system. Their activation has protective functions in the destruction of pathogens, removal of debris and release of neurotrophic factors, but excessive activation can exacerbate the effects of inflammation contributing to neurodegenerative conditions such as Alzheimers disease. The cannabinoids have a variety of anti-inflammatory properties and the main aim of this project is to determine the role of cannabinoids in modulation of microglial cell function using in vitro approaches, and to investigate the molecular mechanisms underlying such modulation. Methods: BV-2 murine microglial and primary murine microglial cells were activated using bacterial lipopolysaccharide (LPS). Nitric oxide (NO) was determined with a Griess assay. Western blotting was used to measure the expression of NFκB p65, Ikβα, inducible nitric oxide synthase (iNOS), COX-2, and total and phosphorylated forms of the MAP kinases, p38, JNK1/2 and ERK1/2; blots were analysed with an Odyssey imaging system (Li-Cor Bioscience), Expression of GPR55 mRNA was determined by RT-PCR. Phagocytosis was assessed in BV-2, HAPI and primary murine microglial cells and in RAW 264.7 monocyte/macrophages using fluorescent latex beads and the cells viewed by confocal microscopy. Fluorescent bead accumulation was quantified on consecutive image and rate phagocytic was calculated by normalizing the number of beads to the number of cells in each field. Western blotting was used to measure the expression of the receptor channels TRPV2 and TRPV1 and AKT. Immunocytochemistry was used to investigate the translocation of TRPV2, and the involvement of MLC11, PLCγ2, PKCα, ε in phagocytosis. Fura-2-based Ca2+ imaging of microglia was undertaken and migration was assessed using a novel “Compass” device. Result: BV-2 cells did not express CB1 or CB2 receptor mRNA; however, the cannabinoid receptor agonist CP55-940, the CB1 antagonist AM251and the non-psychoactive cannabinoid cannabidiol (CBD) all at 10μM produced significant inhibitions of lipopolysaccharide (LPS; 100ng/ml)-stimulated nitric oxide (NO) formation. The putative GPR55 receptor agonists VSN16R and O1916 (were without effect, as was the endogenous GPR55 agonist lysophosphatidylinositol (LPI). A number of other cannabinoid receptor agonists and antagonists and the phytocannabinoids (CBG, CBDV, THCV, CBDA and CBGA (Pertwee, 2008) (all 10 µM) were without. Cannabidiol inhibited LPS-enhancement of both iNOS and COX-2 expression. LPS significantly induced phosphorylation of the MAP kinases ERK 1/2, p38 and JNK and CBD inhibited both LPS-induced p38 and JNK phosphorylation but was without effect on phosphorylation of ERK1/2. The p38 inhibitor SB203580 (10 µM) also significantly reduced iNOS expression after 24 hours of LPS stimulation. LPS increased NF-KB p65 expression and this was significantly attenuated by CBD. LPS also stimulated NF-KB p65 translocation to the nucleus whereas CBD inhibited the effect. CBD-induced phagocytosis of BSA latex beads was similarly induced in HAPI, RAW264.7 and primary murine microglial cells. Inhibitors of Rho kinase (Y27632) and PI (3)kinase (wortmannin) inhibited basal but not CBD-enhanced phagocytosis. CBD increased intracellular calcium in BV-2 cells and the TRP channel blocker ruthenium red reversed CBD-induced phagocytosis. CBD increased expression of TRPV2 protein and mRNA and caused a translocation to the cell membrane. This was abolished in presence of cycloheximide and PI3K inhibitor. Other cannabinoids and phytocannabinoids (CBG, CBDV, THCV, CBDA and CBGA) were without effect. CBD also increased BV-2 cell migration. Conclusion: The data presented demonstrate that CBD, despite inhibiting NO formation, mediated by reduction of NF-kβ P38 MAPK, JNK and ROS activity, enhances microglial migration and phagocytosis. The mechanism of action appears to involve TRPV2 channel activation accompanied by increased protein synthesis and translocation to the cell membrane. Therefore, CBD might be developed as a useful treatment for neurodegenerative disease.

Controlling the interaction of cells with a material surface is of major interest in the field of biomedical material science. Plasma polymers are an attractive way to modify the surface chemistry of a material because this technique is versatile and can be applied to a wide range of different surfaces. The aim of the present work is to prepare a new chemical gradient tool using plasma polymerisation and assess its ability to provoke position dependent cell-surface interactions. A novel diffusion based approach is used to develop gradients from hydrophobic hexane (ppHex) to more hydrophilic allylamine (ppAAm) plasma polymers. The surface of the gradient and that of uniform control samples is characterised using WCA, XPS, ToF-SIMS and AFM. This data shows that the most distinct gradient was found in the wettability profile which can be controlled by changing the size of the opening through which diffusion of depositing species from the plasma occurs. The mechanism of the gradient formation is studied with channels of well defined cross sections. The deposition rate obtained on these samples shows a sharp drop off in the amount of ppHex deposited from the plasma starting 2 mm in advance of the opening. An estimation of the sheath dimensions indicates that this corresponds to the sheath thickness. It is suggested that plasma deposition through small openings such as pores depends on the relative dimensions of the sheath and the pore cross section. Inside the channels, oligomer formation is observed in the gas phase, presumably following a nucleophilic addition reaction mechanism. To study the stability of these plasma polymer surfaces in physiological conditions, surface analysis is also carried out on samples exposed to aqueous solutions. Some changes in the topography of the plasma polymer films are found. Most notably, uniform samples of ppHex deposited on top of ppAAm show the formation of blisters that are not observed on other samples. It is argued that these blisters are the result of water penetrating through the top ppHex layer and interacting with the more hydrophilic ppAAm or glass substrate. 3T3 fibroblasts cultured on the gradients show a gradual increase in cell density. This cell density gradient can be related linearly to the wettability gradient on the surface with non-linear relationships being observed with other surface parameters such as the ppHex thickness. The cell number on uniform ppAAm is much greater than on the ppAAm side of the gradient. Data from experiments with non-proliferating 3T3 fibroblasts indicates that the differences between the gradient and uniform ppAAm as well as the cell density increase along the gradient have their origin in a different number of cells adhered to the surface within the first 24 hours of cell culture. The adsorption of albumin and fibronectin on the plasma polymers demonstrate that displacement of the former by the latter takes place on the surface when adsorbed competitively. However, this displacement does not occur in different extents along the gradient surface, suggesting that protein displacement can not explain the increase in cell density towards the ppAAm end of the gradient.

Fenofibrate is a PPARα agonist, used to treat dyslipidemia. Unpublished work that has been previously carried out in our group has identified that fenofibrate also displays affinity for the cannabinoid receptors, CB1 and CB2. A dual receptor ligand, with the PPARα agonist activity of fenofibrate, combined with antagonist activity at the CB1 receptor, or agonist activity at the CB2 receptor, could reduce appetite, decrease plasma triglyceride levels, increase high density lipoprotein (HDL) levels, lower low density lipoprotein (LDL) levels and reduce atherosclerosis. This could enable the multi-symptomatic treatment of obesity with the advantage of avoiding side effects associated with taking multiple medications. However, whilst fenofibrate has affinity for the cannabinoid receptors, only its active metabolite; fenofibric acid (10b) activates PPARα. This project sought to develop novel ligands for the cannabinoid receptors that retain activity at PPARα. A series of amide derivatives of fenofibrate were synthesised, and pharmacological testing revealed that the piperidinyl (48g) and morpholino (48h) derivatives had agonist activity and a higher affinity for the cannabinoid receptors than fenofibrate. However these derivatives failed to bind and activate PPARα. Although a dual PPARα / cannabinoid receptor ligand has not been found with these amide derivatives, the compounds synthesised could provide a platform for the further development of cannabinoid receptor ligands of this novel class. This was demonstrated by further modifications to compounds (48g) and (48h) which indicated that activity at the cannabinoid receptors is tuneable.

Gastrointestinal (GI) nematodes are important helminth pathogens responsible for severe losses to livestock industries and human health throughout the world. Control of these infections relies primarily on chemotherapy; however there is rapid development of resistance to all available classes of anthelmintic drugs, and therefore new alternative treatments are urgently required. Plant cysteine proteinases (CPs) from papaya latex, pineapple fruit and stem extracts have been demonstrated to be effective against GI nematodes of rodents, chickens, pigs and sheep. The current study extended evaluation of different plant extracts and the factors affecting the efficacy of papaya latex supernatant (PLS) as an anthelmintic against GI nematodes in a mouse model system and formulation and delivery for use in ruminants. The study started with purification and concentration of CPs in PLS using different methods to determine which of them would provide high yield of CPs. It was found that concentration by dialysis provided a high yield of active enzyme in PLS. Storage of PLS at -200C and -800C retained more active enzymes for prolonged period of time than at ambient temperature and 4oC. Motility assay conditions showed to have no influence on enzyme activity. While the in vitro experiment results showed significant detrimental effect of pineapple fruit extract, stem bromelain and little effect of kiwi fruit extract against Heligmosomoides bakeri motility. In vivo experiments showed less efficacy of these enzymes than expected when compared with PLS. The first factor to be assessed in this study was the effect of fasting on the anthelmintic efficacy of PLS. The results showed that PLS was equally effective in reducing worm burdens whether mice were fasted before treatment or not, and by avoiding fasting the side effects of treatment were minimized. Comparison of efficacy in a range of mouse strains indicated that efficacy varied between mice of different genotype. At the dose used, the treatment was most effective in C3H mice ranging from 90.5% to 99.3% in reducing worm burdens and less effective in NIHS, CD1 and BALB/c strains (7.9%, 36.0% and 40.5% reduction respectively). However, host sex and body size were shown not to have any influence on the anthelmintic efficacy of PLS. Since CPs are particularly sensitive to pH, variation between mouse strains in gut pH was investigated but no significant differences in pH were found along the GI tract of the poor (BALB/c) and high responder mice (C3H) to PLS treatment and concurrent administration of the antacid cimetidine also did not improve efficacy. The study also explored the potential of formulation and delivery of PLS as an anthelmintic drug for ruminants. In vitro studies involving both immediate and slow release dosage formulations simulating the physiological conditions (pH, temperature and peristaltic movement) in the GI tract of the animal were conducted. In the slow release experiments, two hydrophilic matrices were tested, the xanthan gum and hydroxypropyl methylcellulose (HPMC) (both Methocel-LVCR and Methocel-CR). Methocel-CR provided better slow release results compared to the others. In the immediate release experiments 3 disintegrants (Primojel, L-HPC and Ac-Di-sol) were investigated and Ac-Di-Sol® was found to produce the faster immediate drug release rate. Preliminary in vitro studies also showed that PLS was highly effective against equine GI nematodes. Finally the empirical findings in this study provide useful information for improvement of formulation and delivery of these naturally occurring plant-derived enzymes for treatment of intestinal worm infections in humans and livestock, while achieving maximum efficacy and minimal side-effects.

Gram-negative bacteria are the most common cause of the sepsis and lipopolysaccharide (LPS) a major component of Gram-negative bacteria is known to be of major importance in the development of sepsis. Human infection with Salmonella, a Gram-negative bacterium, is associated with a number of cases of sepsis and is particularly important in childhood sepsis. During salmonellosis, monocytes and macrophages produce a number of different pro-inflammatory mediators such as TNF-α, IL-1α, IL-12, IL-18, IFN-γ, reactive nitrogen species and oxygen species. Although the production of these inflammatory mediators is required for resolution of bacterial infections, they are contraindicated in diseases such as sepsis. In the initial (acute) phase of sepsis a Systemic Inflammatory Response Syndrome (SIRS) occurs in which inflammatory mediators are produced in high concentration, which can lead to organ failure and death. The SIRS phase is then replaced by a Compensatory Anti-inflammatory Response Syndrome (CARS) phase which leads to immunosuppression. The CARS phase can lead to secondary infection and subsequent mortality within 28 days of hospital admission. To date, several studies have evaluated the role of vasoactive intestinal peptide (VIP) as an anti-inflammatory agent that may have therapeutic potential in septic patients both in vitro and in vivo. VIP has been shown to inhibit production of inflammatory mediators produced by human monocytes in response to LPS. The aim of the work described in this thesis was to investigate the therapeutic potential of VIP in sepsis using an ex vivo human monocytes model infected with viable Salmonella Typhimurium 4/74 (rather than LPS). The study shows that VIP (10-7 M) stimulates an increase in the numbers of Salmonella recovered from infected human monocytes (MOI = 10). In addition, VIP also increases the survival rate of human monocytes infected with Salmonella. These two results may suggest a detrimental effect of VIP during bacteraemia and sepsis, since monocyte death may be beneficial during sepsis and bacterial overgrowth could lead to further increased LPS (and other antigen) stimulation of the immune system. However, VIP did significantly decrease Salmonella and LPS-induced TNF-α, IL-1β and IL-6 in monocyte supernatants. VIP also had a positive effect on IL-10 production in human monocytes infected with Salmonella or stimulated with LPS. Whether this suggests a possible detrimental effect of VIP is unknown but septic patients with high serum IL-10/ low TNF-α concentration ratio have previously been shown to have a poor prognosis. Higher IL-10 concentrations in infected monocytes (due to VIP) could also increase the CARS phase of disease with increased immunosuppression. Flow cytometry and qPCR analyses showed that of all of the VIP receptors, VPAC1 was expressed most highly during Salmonella infection, or LPS stimulation, of human monocytes. Administration of VIP inhibited VPAC1 has been shown by many studies to be the most important receptor by which VIP inhibits production of inflammatory immune mediators, or increases IL-10 production from murine macrophages. Results in this thesis, therefore, suggested that Salmonella infection may promote VPAC1 expression and so provide a mechanism of inhibiting the production of inflammatory mediators in infected cells. This could then increase intracellular survival of Salmonella and provide a means of greater dissemination of the infection. To ascertain how increased VPAC1 expression on the surface of monocytes may be achieved, analysis of the expression of known intracellular endosomal and exosomal constituents was performed. Confocal laser microscopy, using specific antibodies, showed that VPAC1 on the monocyte cell membrane was internalised within early endosomes (measured by co-localisation of VPAC1 and EEA1) rather than being degraded within lysosomes (measured by immunoreactivity to LAMP1). VPAC1 is then transported via a Rab11A recycling endosome and packaged in the Trans-Golgi network (TGN), shown by co-localisation of VPAC1/Rab11A and the TGN marker (TGN46). VPAC1 was then associated with Rab3a and calmodulin. The function of these latter two proteins in the docking of exosomes to the cell membrane is well known, thus suggesting that Salmonella induced VPAC1 was also recycled to the cell membrane within exosomes. VIP inhibited the expression of both Rab3a and calmodulin but not the co-localisation of VPAC1 with these two proteins. Further studies then showed that a calmodulin agonist (CALP1) increased VPAC1 expression on the surface of monocytes, while a calmodulin antagonist (W-7) decreased expression of VPAC1 on the surface of monocytes. In conclusion, this thesis does present hitherto unknown data regarding Salmonella infection of human monocytes and the effects of VIP on infected monocytes. VIP has potential as an anti-sepsis therapy since it reduces the production of inflammatory mediators by Salmonella-infected and LPS-stimulated monocytes. However, the fact that VIP increases survival of infected human monocyte and increased growth of Salmonella in human monocytes may preclude its use in sepsis.

Human populations are at risk of exposure to constitutive androstane receptor (CAR) activators present in a range of substances, including pharmaceuticals, plasticizers and crop protection agents. What exposure to CAR activators means for human health is uncertain. Activation of CAR in rodents is associated with liver hyperplasia, increased proliferation and eventual hepatocarcinoma; however the effect in human hepatic cells is unclear. There are two methods by which a compound can achieve activation of CAR; directly or indirectly via cellular signalling pathways. Phenobarbital is a prototypical activator of CAR and does so in an indirect manner via suppression of epidermal growth factor receptor (EGFR) signalling. Direct activation of CAR in rodents also causes hepatocellular carcinoma but the human outcome is less clear. We have carried out microarray and miRNA analysis of CITCO (a potent and selective hCAR ligand) treated primary human hepatocytes. To mitigate the well documented effect of primary hepatocyte dedifferentiation primary hepatocytes were cultured in dynamic three dimensional culture in vitro. Gene expression changes indicate that direct activation of hCAR causes the promotion of a pro-proliferative and anti-apoptotic phenotype. The miRNA expression profile is crucially different to rodent data that is currently published. Despite the pro-proliferative phenotype shown there is no evidence that primary human hepatocytes proliferate in response to direct activation of CAR by CITCO. This leaves the possibility that a proliferative response may be observed in vivo or that the changes in gene expression are solely a human physiological adaptation to direct hCAR activation by CITCO and no proliferation would occur. The effect on human health and liver toxicity is unclear but this body of work has provided data that may be used to further understand the mechanistic effects of direct hCAR activation in human hepatocytes. A more complete understanding of this will help to inform the toxic potential of direct hCAR activation in vivo.

Significant opioid- and endocannabinoid-dependent changes occur within the periaqueductal grey (PAG), rostroventral medulla (RVM) and spinal cord (DH) during postnatal development of the rat (Sprague Dawley). These changes are involved in the differential descending control of spinal excitability between young and mature rats. Microinjection of the µ-opioid receptor (MOR) agonist DAMGO (30ng) into the PAG of rats increased spinal excitability and lowered mechanical threshold to noxious stimuli in postnatal day (P)21 rats, but had inhibitory effects in adults and lacked efficacy in P10 pups. A tonic opioidergic tone within the PAG was revealed in adult rats by intra-PAG microinjection of CTOP (120ng, MOR antagonist) which lowered mechanical thresholds and increased spinal reflex excitability. Spinal adminstration of DAMGO inhibited spinal excitability in all ages yet the magnitude of this was greater in younger animals than in adults. The expression of MOR and related peptides were also investigated using TaqMan RT-PCR and immunohistochemistry. Proopiomelanocortin (POMC) peaked at P21 in the ventral-PAG, and MOR increased significantly in the DH as the animals aged. CB1/CB2 receptor activation by WIN55212 (4µg, CB1/CB2 agonist) and HU210 (4µg, CB1/CB2 receptor agonist) in the PAG, RVM and DH was anti-nociceptive in both young (P10, P21) and adult rats, but GPR55 receptor activation by LPI (12µg, endogenous GPR55 agonist) and AM251 (2.77µg, CB1 antagonist, GPR55 agonist) was exclusively inhibitory in young rats. Micro-injection of LPI into the adult RVM facilitated spinal reflex excitability, suggesting that GPR55 receptor activation in mature animals is pro-nociceptive. The expression of cannabinoid receptors and endocannabinoid-synthesising enzymes was investigated with immunohistochemical and TaqMan RT-PCR techniques. Overall the expression of CB1 receptors and the anandamide synthesising enzyme NAPE-phospholipase D (NAPE-PLD) increased within the descending pain pathway with age, whereas the expression of the 2-AG synthesising enzyme Diacylglycerol lipase α (DAGLα) decreased. These results illustrate that profound differences in the endogenous-opioidergic and endocannabinoid signalling systems occur within the descending pain pathway throughout postnatal development.

The cannabinoid CB1 receptor is a G protein-coupled receptor (GPCR) which is important in the regulation of neuronal function, predominately via coupling to heterotrimeric Gi/o proteins. The receptor has also been shown to interact with a variety of other intracellular signalling mediators, including other G proteins, several members of the mitogen activated kinase (MAP) superfamily and β-arrestins. The CB1 receptor is recognised by an array of structurally distinct endogenous and exogenous ligands and a growing body of evidence indicates that ligands acting at GPCRs are able to differently activate specific signalling pathways, a phenomenon known as functional selectivity or biased agonism. This is important in future drug development as it may be possible to produce drugs which selectively activate signalling pathways linked to therapeutic benefits, while minimising activation of those associated with unwanted side effects. The main aim of this thesis, therefore, was to investigate ligand-selective functional selectivity at the cannabinoid CB1 receptor both endogenously and exogenously expressed in a variety of cell lines. Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293) cells stably transfected with the human recombinant CB1 receptor and untransfected murine Neuro 2a (N2a) cells, were exposed to a number of cannabinoid receptor agonists, including the endogenous agonist anandamide, the phytocannabinoid Δ9-THC, and several synthetic ligands. Ligand affinity was determined using competition radioligand binding assays. Regulation of several CB1 receptor-coupled intracellular signalling mediators was investigated; G protein activation was measured using the [35S]-GTPγS binding assay; phosphorylation of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK) and p38 MAP kinases was measured using the immunocytochemical In-cell Western technique. Modulation of cAMP accumulation and β-arrestin recruitment were measured using DiscoveRx detection assay kits. Using concentration-response data, agonist bias was analysed using equimolar comparison plots, and by comparison of intrinsic relative activities for all agonists, calculated relative to the high potency cannabinoid receptor agonist HU-210 for the transfected cells or WIN 55,212-2 for the Neuro 2a cells. The PPARα agonist fenofibrate was identified as a previously unrecognized cannabinoid receptor agonist, exhibiting modest selectivity for the CB2 receptor subtype (~25-fold). In addition to its functioning as an orthosteric agonist, fenofibrate, at high concentrations, appeared to act as a negative allosteric modulator at the CB1 receptor expressed in CHO cells, identified by radioligand binding assays, and non-competitive inhibition of the orthosteric agonist CP 55,940 in the [35S]-GTPγS binding assay. All three cell lines showed CB1- and Gi/o protein-dependent activation of ERK and inhibition of forskolin-stimulated cAMP accumulation: however, the magnitude of these responses differed between the three cell lines, with the responses in the Neuro 2a cells being markedly smaller than in the recombinant cell lines. The synthetic agonists WIN 55,212-2 and ACEA both exhibited bias towards ERK activation, in comparison to inhibition of cAMP accumulation, in both the CHO and HEK cell lines. HU-210, Δ9-THC, methanandamide and fenofibrate all exhibited bias towards ERK activation in the Neuro 2a cells, but only the HU-210 bias was quantified, as the other three agonists did not couple to cAMP formation or inhibition in this cell line. In addition, time-course experiments revealed further novel patterns of agonist bias in ERK signalling, with CP 55,940 alone producing a second phase sustained ERK response in CHO cells, while higher concentrations of WIN 55-212-2 produced a CB1-receptor-dependent reversal of ERK phosphorylation in HEK cells at 20 min, but not 5 min, agonist stimulation. Additional cell-dependent responses were observed with HEK cells alone, exhibiting Gi/o protein-independent ERK activation and increase in cAMP levels. Despite reports in the literature to the contrary, none of the cell lines tested showed receptor-mediated activation of JNK or p38 MAP kinase. In conclusion, this thesis has demonstrated functional selectivity at the cannabinoid type 1 receptor in a number of cell lines, expressing both native and transfected recombinant receptors. These findings contribute to our increasing understand of the complexity of GPCR signalling, and potentially allow for the development of more targeted drugs able to selectively engage with beneficial signal transduction pathways.