Rheumatoid Arthritis (RA) is the commonest inflammatory polyarthritis in the UK, and is associated with significant symptoms, disability and premature mortality. Treatment options in 1980 were restricted to corticosteroids, non-steroidal anti-inflammatory drugs, and a small number of disease modifying anti-rheumatic drugs (DMARDs). There had been few large, well conducted randomised controlled studies such that our knowledge about the relative efficacy and safety of the drugs available was very limited. Trial design also left much to be desired, with inadequate methods of randomisation and/or concealment of allocation being commonplace. There was no consensus about which outcome measures ought to included in trials, and no composite measures of outcome had yet been developed or validated. The limited evidence base and restricted therapeutic armamentarium was reflected in the poor outcome of the disease for many patients: remission was rare, and patients often experienced increasing disability, orthopaedic intervention, work-related unemployment and premature mortality. Within 20 years, the prognosis for patients with newly diagnosed RA has dramatically improved. Modern management results in the majority of patients achieving low disease activity or even remission. The studies incorporated into this thesis have played an important role in the evolution of these management strategies. Early studies focussed on building the evidence base for the use of DMARD monotherapy, demonstrating that sulfasalazine and methotrexate were both safe and effective treatments. The Sulfasalazine-Auranofin trial contributed to the downfall of auranofin, a drug that is no longer manufactured. Contrary to early concerns, methotrexate was proven to be well tolerated, even in the socially deprived population of Greater Glasgow, and this drug had become the DMARD of first choice in the management of RA. DMARD monotherapy, however, is usually not sufficient to maintain good disease control in the long term. The Gold-Hydroxychloroquine study was one of the first studies to investigate the role of combination DMARD therapy in a well conducted, double blind randomised controlled trial (RCT). The results were negative, but a follow up trial demonstrated the superiority of stepping up to Methotrexate-Sulfasalazine combination therapy when compared to sequential monotherapy in the MASCOT study. The West of Scotland Early RA corticosteroid trial was a double blind RCT investigating the role of low dose oral corticosteroids in addition to sulfasalazine therapy. It failed to demonstrate any benefit of low dose steroids, a finding that is at odds with a growing literature that has established corticosteroids as a proven disease modifying therapy. The strategy trials (Tight Control of RA [TICORA] and Triple Therapy in Early RA [TEAR] studies) have been the most influential studies to have been performed in Glasgow. They did not primarily address the issue of whether a drug is effective or whether one drug is more effective than another. Rather, they sought to test a hypothesis drawn from observations in other biologic models (principally Type I Diabetes Mellitus): namely, that i) using currently available DMARDs ‘tight control’ can be achieved if patients are reviewed frequently, their disease assessed formally (using the disease activity score), and their treatment escalated if their disease remained active; ii) that the achievement of tight control would lead to improved outcomes. The studies provided strong evidence that dramatic improvements in symptom control, disability and radiographic progression can be achieved by pursuing this strategy of ‘Tight Control’. National and international clinical guidelines, and international consensus statements have embraced the results of TICORA (and subsequent confirmatory studies), such that regular, frequent assessment of the patient, use of composite measures of disease activity and the adoption of a ‘treat-to-target’ therapeutic strategy have become accepted as ‘best practice’ throughout the world.

The health benefits of the fruit of Punica granatum (pomegranate) have been recognised for many centuries. This thesis tested the hypothesis that a potent novel antimicrobial system could be developed based upon the activity of pomegranate rind extract (PRE), particularly when combined with a potentiating agent, and focussed on studying activity against Herpes simplex virus, types 1 and 2 (HSV-1 and HSV-2). High potentiated virucidal action of PRE against HSV-1 was observed when ferrous sulphate (FeSO4) was added; however, this activity diminished rapidly and also gave rise to an unsightly black byproduct. Data was obtained suggesting this was linked to the rapid oxidation of ferrous to ferric (Fe (II) to Fe (III)); however, no such oxidation was indicated when zinc sulphate (ZnSO4) was added instead, prompting an exploration of the effects of PRE + ZnSO4 combination on HSV. The potentiation of PRE by ZnSO4 in virucidal mode was comparable to that observed with FeSO4 (at the concentration examined for both metal salts). More in-depth investigation of the potentiation of PRE and ZnSO4 achieved over a 9000 fold increase in viral destruction in comparison to either agent alone. This activity was not transient and did not produce a (black) byproduct. Other salts of Zn (II) generally performed similarly to ZnSO4, with the main exception of ZnO which was too insoluble in water to test. When the antiviral properties of PRE were examined, the results were again potent and comparable to Aciclovir, the established treatment for Herpes simplex infections. Furthermore, PRE demonstrated even greater potency against Aciclovir-resistant HSV-2. When applied to ex vivo skin, PRE produced a 66% reduction in the level of cyclooxygenase-2 (COX–2) - an important inflammatory mediator - with the presence of ZnSO4 having no effect. PRE and ZnSO4 were formulated as a stable hydrogel, which was able to effectively deliver the biologically active compounds through the epidermal, buccal and vaginal membranes to the epidermal/dermal interface - the major sites of HSV-1 and HSV-2 vesicular clusters during a clinical infection.

A pathophysiological study was made of spontaneously breathing dogs anaesthetised by a neurolept-analgesic technique and exposed to 100% oxygen at 2 ATA to demonstrate the time-course and mechanism of response. The animals remained apparently normal for some 18 hours, following which the majority developed a fulminating intra-alveolar oedema and died of hypoxaemia within a few hours. There was no evidence of systemic nor pulmonary capillary hypertension, and electron microscopy demonstrated complete absence of damage to the endothelial and Type 1 epithelial cells of the alveolar septum. Changes were detected in the Type 2 cells, and oedemagenesis was attributed to an oxygen-induced depression of surfactant activity.

The PhD project is about the Regulation of vascular tone by endothelium. It involves studying the effects of methylated arginine analogues such as monomethyl arginine (L-NMMA) and asymmetric dimethylarginine (ADMA) on nitric oxide (NO) activity in rat aorta using organ baths containing Krebs solution at 37 ˚C and gassed with 95% O2 and 5% CO2. These two inhibitors of endothelial nitric oxide synthase enzyme (eNOS) showed an anomalous action, as they inhibited basal NO activity (assessed by enhancement of phenylephrine-induced tone and by blocking relaxations produced by superoxide dismutase or the PDE5 inhibitor; T0156) but not that is stimulated by agonists like acetylcholine or the calcium ionophore A23187. After establishing these findings I will try to find the reason(s) for these paradoxical actions of these two endogenously synthesized inhibitors of eNOS.

Oil and emulsion vehicles have been shown to alter the oral absorption of many drugs. This may be due to enhanced lymph flow and/or altered gastro-intestinal motility in the presence of the oils. The oral absorption of a model compound (DOT) in the presence of three chemically different oils, arachis oil, Miglyol 812 and liquid paraffin was investigated in rats, the influence of lymphatic absorption and gastro-intestinal motility being determined. The findings were applied to the for.mulation of the'steroid prednisolone, in an attempt to produce elevated more uniform plasma drug levels by enhancing lymphatic absorption. The rank order for total DOT absorption from 1m! Volumes of different vehicles was arachis oil > Miglyol 812= water containing 6% Tween 80 > liquid paraffin. The concentration of DDT in lymph collected via thoracic duct cannblae in anaesthetised rats was greatest in the presence of arachis oil, there being no difference between Miglyol 812 and liquid paraffin. Using a gamma camera the gastric emptying rate and total intestinal transit of 9~c-sulphur colloid, an oil phase marker, was shown to be faster in the presence of 1ml liquid paraffin than the other two oils. The oral absorption of 3H-prednisolone was independent of the nature of the oily vehicle (30pl volumes) and was not selectively absorbed into the lymph. Esterification to 3H-prednisolone-21-palmitate increased the lipophilicity of the drug but did not stimulate selective lymphatic absorption and reduced oral absorption following administration in arachis oil. Lymphatic absorption of the ester was not promoted by administration in 1ml arachis oil. Only the lymphatic absorption of compounds exhibiting selective uptake into the lymph may be enhanced by the presence of a suitable lipid vehicle. Altered gastro-intestinal motility in the presence of lipids may have greater potential for enhancing the absorption of a wider variety of compounds.

This thesis describes synthetic studies directed towards a second generation total synthesis of ulapualide A. Ulapualide A is an extraordinary bioactive tris-oxazole based macrolide which was isolated from the egg masses of the marine sponge Hexabranchus sanguineus and exhibits potent antifungal activity with inhibition of leukaemia cell proliferation. The Introduction to this thesis includes an overview of natural product chemistry and draws attention to the 'ulapualide' family of secondary metabolites including their isolation, biological activity, biosynthesis and structural determination. Also included is a summary of a total synthesis of ulapualide A by our research group in Nottingham, together with a review of oxazole containing natural products. The Results and Discussion section of this thesis details our general strategy for an alternative design for the synthesis of the tris-oxazole based macrolide core of ulapualide A. A synthesis of a model system exemplifying this strategy is then described, together with a detailed discussion of polyoxazole ring formation. This is followed by application of the model study to ulapualide A itself, and includes a total synthesis of the polyol C26-C41 side-chain of ulapualide A. The section concludes by describing our synthetic efforts towards the remaining chiral fragment of this natural product, the bottom-chain. The thesis concludes with an Experimental section containing full details of the preparative work completed and listing spectroscopic and analytical data for all new compounds synthesised during the study. An Appendix contains a description of contemporaneous synthetic studies carried out by Panek et al during the course of my PhD studies. X-ray crystallographic and spectroscopic data, together with reprints of publications resulting from our work are also included.

Poly (butyl 2-cyanoacrylate) nanoparticles have been prepared with a range of particle sizes by varying the nature and concentration of stabiliser added to the polymerisation medium. Particle size analysis was performed by photon correlation spectroscopy. The range of diameters produced using dextran stabilisers was found to be approximately 100 to 800nm. This could be extended to 3ym using j3 -cyclodextrin and to 20nm using polysorbate 20. The results infer that the nanoparticles are sterically stabilised. The molecular weight of the cyanoacrylate polymer formed during nanoparticle production was found to be dependent on the type of stabiliser used together with the polymerisation pH and monomer concentration. The bulk of the polymer had a relatively low molecular weight (<2000) which indicates that nanoparticles are formed by an aggregative mechanism. Dextran was found to copolymerise with the monomer to give an interfacial layer of the polysaccharide attached by covalent linkages. By using dextrans bearing charged functional groups it was possible to alter the electrophoretic behaviour of the resulting nanoparticles. Partial oxidation of the surface dextran introduced aldehyde groups which were capable of covalently binding a simple amine, aniline, thereby enhancing the uptake and decreasing the release rate of this compound. This technique may be applicable to the covalent coupling of antibodies or cytotoxic agents to the nanoparticle surface. Nanoparticles were radiolabelled with a technetium-99m-dextran complex and the biodistribution of this colloid determined in rabbits by gamma scintigraphy following intravenous injection. Most of the nanoparticle suspension (approximately 50%) was cleared by the liver and spleen. Coating the nanoparticles with non-ionic surfactants (poloxamer 338 or Tetronic 908) failed to alter significantly this distribution pattern.

Assigning a fail grade, particularly when it results in the termination of a career goal, is a taboo and taken-for-granted aspect of an assessor's role. Hermeneutics provided the main framework for interpreting the subjective and objective experiences of both academic and fieldwork supervisors during this process. An incremental research design, using a principal and two supplementary methods was used to investigate the minutiae of assessing whether a student has achieved the required standard of competence. Focused interviews were conducted with 25 academic and 5 fieldwork supervisors to compare the perspective of staff with different roles, relationships and responsibilities. These were preceded by two questionnaire surveys with trained, experienced fieldwork supervisors. On the first survey 64% (n=72) ranked 'failing a student' as their most problematical responsibility. The second survey comprised immediate and follow-up evaluations of five 'failure workshops' attended by 101 supervisors. They highlighted the importance of an assessor's affective response, reinforced effective supervisory strategies and the professional obligation to act as a gatekeeper of future standards. The results revealed a diverse array of individual factors, institutional rituals and external pressures which seemed to facilitate or sabotage the quality of the process and outcome. These included an assessor's inexperience, the conflict in values and roles between therapist and educator; characteristics of the student particularly the pastoral relationship and stage of training; the valued impartiality of an external examiner or fieldwork organiser; and the threat to reputations and course viability if results provide the primary performance indicator. An understanding of the complex constellation of factors which may influence an assessor's ability and confidence to fairly judge both initial and ongoing competence is important for all 'caring' professions to ensure only safe practitioners are registered to work with vulnerable clients.

Existing anti-rejection drug regimes are inadequate since patients receive drugs despite serious side effects and poor response. New drugs are being developed which ultimately may allow for prescribing of rational, patient-specific immunosuppressive drug protocols. During this thesis the investigation of lymphocyte responses from renal transplant recipients to the immunosuppressant drugs Cyclosporin A (Cy A), FK506 and SDZ RAD were explored to understand the variation in sensitivity of lymphocytes to Cy A and FK506, the development of drug resistance, including resistance mechanisms, and the interactions between FK506 and SDZ RAD. Cy A and FK506 are substrates for P-glycoprotein (P-gp), the product of the multidrug-resistance (MDR1) gene in man. A hypothesis established during this thesis was that P-gp dependent mechanisms explain variations in lymphocyte sensitivities to Cy A and FK50. Lymphocytes from renal transplant recipients were assessed for their sensitivity to Cy A and FK506 and subsequently for P-gp expression and functional activity by flow cytometry. In further lymphocyte cultures the effect of the specific P-gp inhibitor, PSC 833 on sensitivity was investigated. Finally, the effects of the combination of FK506 and SDZ RAD in lymphocyte cultures were analysed. Results demonstrate a wide range in lymphocyte sensitivity to both Cy A and FK506, with the development of selective resistance to the drug used for treatment. All patients demonstrated P-gp functional activity but P-gp expression was not demonstrable. P-gp function did not account for the variation in lymphocyte sensitivity. There was no evidence of antagonism of effect of SDZ RAD in combination with FK506. In conclusion, these results suggest that non-P-gp mechanisms account for variations in lymphocyte sensitivity to Cy A and FK506. Combination therapy with SDZ RAD and FK506 is unlikely to be antagonistic in future treatment protocols.

There has been an escalating interest in the medicinal use of Cannabis sativa in recent years. Cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC), the main constituents of Cannabis sativa, have well documented immunomodulatory effects in vitro and following administration of high doses to animals. However, these effects have not been clearly evident in humans following oral administration of cannabinoids, probably due to low systemic bioavailability. To note, cannabis and cannabis-containing medicines are currently used for symptomatic relief in autoimmune diseases, such as multiple sclerosis (MS), and in cases of immunodeficiency, such as in cancer patients on chemotherapy regimens. In this thesis, we aimed to elucidate the impact of enhancing the transport of orally administered cannabinoids to the intestinal lymphatic system, the major host of immune cells, on the immunomodulatory effects of cannabinoids. Oral administration of lipophilic cannabinoids with long-chain triglycerides (LCT) was investigated as a simple approach to enhance the intestinal lymphatic transport. The effect of LCT on the intraluminal processing of orally administered cannabinoids was assessed by means of in vitro lipolysis model. The results of in vitro lipolysis demonstrated that at least one-third of CBD dose would be solubilised and readily available for absorption to the enterocytes when orally administered in LCT-formulation. The association of CBD with chylomicrons (CM) in the enterocytes and subsequent intestinal lymphatic transport was estimated using an in silico model, in vitro association by artificial CM-like lipid particles, and ex vivo uptake by plasma-derived CM from rats and humans. The results of CM association studies revealed high intestinal lymphatic transport potential for CBD in rats and humans. Similar high lymphatic transport potential was also reported for THC in our laboratory. Oral co-administration of CBD and THC with LCT to rats increased the systemic exposure by 3-fold and 2.5-fold, respectively, compared to lipid-free formulations. The underlying mechanism of increased bioavailability is likely to enhanced intestinal lymphatic transport and decreased pre-systemic metabolism in the liver. The results of biodistribution experiments indicated that the intestinal lymphatic transport of CBD and THC was, indeed, enhanced following oral co-administration of lipids as denoted by the dramatic increase in the concentrations recovered in MLN and intestinal lymph. The concentrations of CBD and THC in intestinal lymph fluid were in the range of 120 and 60 µg/mL compared to 0.5 and 0.6 µg/mL in plasma, respectively. Moreover, CBD and THC showed dose-dependent immunosuppressive effect on lymphocytes isolated from rats and peripheral blood mononuclear cells (PBMC) isolated from humans as assessed by lymphocyte proliferation assay and flow cytometry analysis of inflammatory cytokines. These effects were only significant at concentrations achieved in the intestinal lymphatic system, but not in plasma, following oral co-administration of cannabinoids with LCT. CBD showed more immunosuppressive effects on lymphocyte proliferation and the expression of inflammatory cytokines comparing to THC. Also, PBMC from MS patients were more susceptible to the immunomodulatory effects of cannabinoids than PBMC from healthy volunteers and cancer patients on chemotherapy. In conclusion, oral administration of cannabinoids with lipids can enhance the intestinal micellar solubilisation and augment the systemic exposure to cannabinoids by enhancing intestinal lymphatic transport. The concentrations of lipophilic cannabinoids recovered in the intestinal lymphatic system were extremely high and exceeded the immunosuppressive threshold of CBD and THC. The increase in systemic exposure to cannabinoids in humans is of potentially high clinical importance as it could turn a barely effective dose of orally administered cannabis into highly effective one, or indeed a therapeutic dose into a toxic one. In addition, administering cannabinoids, in particular CBD, with a high-fat meal, as cannabis-containing food, or in lipid-based formulations could represent a valid therapeutic approach to improve the treatment of MS, or other T cell-mediated autoimmune disorders. However, in immunocompromised patients, administration of cannabinoids in this way may deepen the immunosuppressive effects.