This thesis describes the use of mathematical modelling to analyse the treatment of patients with immune disorders; namely, Multiple Myeloma, a cancer of plasma cells that create excess monoclonal antibody; and kidney transplants, where the immune system produces polygonal antibodies against the implanted organ. Linear and nonlinear compartmental models play an important role in the analysis of biomedical systems; in this thesis several models are developed to describe the in vivo kinetics of the antibodies that are prevalent for the two disorders studied. These models are validated against patient data supplied by clinical collaborators. Through this validation process important information regarding the dynamic properties of the clinical treatment can be gathered. In order to treat patients with excess immune antibodies the clinical staff wish to reduce these high levels in the patient to near healthy concentrations. To achieve this they have two possible treatment modalities: either using artificial methods to clear the material, a process known as apheresis, or drug therapy to reduce the production of the antibody in question. Apheresis techniques differ in their ability to clear different immune complexes; the effectiveness of a range of apheresis techniques is categorised for several antibody types and antibody fragments. The models developed are then used to predict the patient response to alternative treatment methods, and schedules, to find optimal combinations. In addition, improved measurement techniques that may offer an improved diagnosis are suggested. Whilst the overall effect of drug therapy is known, through measuring the concentration of antibodies in the patient’s blood, the short-term relationship between drug application and reduction in antibody synthesis is still not well defined; therefore, methods to estimate the generation rate of the immune complex, without the need for invasive procedures, are also presented.

The prodiginine antibiotics are produced by eubacteria, in particular members of the actinomycete family. Interest in this group of compounds has been stimulated by their antitumour, immunosuppressant and antimalarial activities at non-toxic levels. Streptomyces coelicolor A3(2) produces two prodiginines: undecylprodiginine and its carbocyclic derivative streptorubin B, which are both derived from the two intermediates 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC) and 2-undecylpyrrole (2-UP). The red gene cluster of S. coelicolor contains 23 genes responsible for prodiginine biosynthesis. PCR-targeting was used to generate rapid in-frame deletions or replacements of several genes in the S. coelicolor red cluster. Using this method redI, redJ, redK, the A domain encoding region of redL, redT and redV were disrupted. Prodiginine production by these mutants was analysed by LC-MS allowing roles for the genes investigated to be hypothesised. A major focus was investigating the function of RedH (proposed to catalyse the condensation of 2-UP and MBC) and RedG (proposed to be responsible for the oxidative carbocyclisation of undecylprodiginine to form streptorubin B) by genetic complementation of existing mutants and heterologous expression of the genes in S. venezuelae coupled with feeding of synthetic MBC and 2-UP. The results of these experiments clearly defined the roles of RedH in the condensation of MBC and 2-UP and RedG in the oxidative carbocyclisation of undecylprodiginine. Streptomyces longispororuber is known to produce undecylprodiginine (like S. coelicolor) and a carbocyclic undecylprodiginine derivative called metacycloprodigiosin (streptorubin A), which contains a 12-membered carbocycle instead of the 10-membered carbocycle of streptorubin B. A S. longispororuber fosmid library was constructed, from which a clone containing a previously identified redG orthologue was isolated and partially sequenced. Expression of the S. longispororuber redG orthologue in the S. coelicolor redG mutant resulted in production of metacycloprodigiosin instead of streptorubin B showing that RedG and its S. longispororuber orthologue catalyse carbocyclisation reactions during prodiginine biosynthesis. Another aim of the work was to investigate redU, a gene from the red cluster that encodes a phosphopantetheinyl transferase (PPTase). PPTases are responsible for post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). A pre-existing redU mutant and two newly constructed mutants lacking PPTases encoded elsewhere in the S. coelicolor genome were analysed to investigate the role of PPTases in S. coelicolor metabolite biosynthesis. Production of prodiginines, actinorhodins, methylenomycins, calcium dependent antibiotics, coelichelin and grey spore pigment was investigated as ACPs and PCPs are involved in biosynthesis of these compounds. Different specific PPtases were found to be required to modify the ACP/PCP domains/proteins in the biosynthesis of these metabolites.

This thesis investigates structural and dynamic properties of drug recognition mechanisms to duplex and quadruplex DNA using primarily high field NMR techniques and molecular dynamics simulations. The mechanism of co-operative binding of Hoechst 33258 to the DNA minor groove of duplexes that contain two binding sites such as d(CTTTTGCAAAAG)2, d(GAAAAGCTTTC)2 and d(CTTTTGGCCAAAAG)2 has been studied. NMR and other titration techniques have evidenced co-operative binding and no detection of an intermediate 1:1 complex. High-resolution NMR structure determination showed no evidence of direct contact between Hoechst 33258 molecules or DNA structure deformation that would facilitate co-operativity, Molecular dynamics simulations based on NMR data, allowed us to calculate thermodynamic quantities of the two binding events, and lead us to conclude that ligand binding can induce changes in DNA conformational flexibility in sites of the structure distant from the binding site and result in more favourable second ligand binding. The results highlight the general importance of flexibility in determining the properties of ligand-DNA interactions. The relative importance of ligand isohelicity and phasing in DNA minor groove has been investigated by studying the structure and dynamics of the 1:1 complex of Hoechst IO-d(GCAAATTTGC)2. The results suggest that DNA sequence-dependent structure and flexibility have significant role for the strong binding of Hoechst 10 to the duplex. The formation, stability, structure and dynamics of the d(TTAGGGT)4 quadruplex structure, which contains the human telomeric repeat TTAGGG, have been studied. Characteristic features of the quadruplex structure were determined and this information was used for understanding drug-quadruplex interactions. The complex of the fluorinated polycyclic methylacridinium cation RHPS4, lead compound for telomerase inhibition, with the d(TTAGGGT)4 quadruplex structure has been investigated. RHPS4 forms a stable G-quadruplex complex by endstacking externally to the a-tetrads of the Apa and Gp'T steps. This study presents detailed properties of the complex and provides further information for lead optimisation studies.

Single doses of phenobarbitone decreased the turnover rate of dopamine (DA) and noradrenaline (NA) and increased the whole brain levels of 5- hydroxytryptamine (5-RT) and gamma-aminobutyric acid (GABA) . Habituation to phenobarbitone increased the levels of DA in striata and midbrain and decreased that of cerebral hemispheres, leaving the total amount unchanged. Habituation resisted the depletion otherwise caused by alpha-methyl-p-tyrosine (alpha-m.p.t.) in the striata for DA and in the cerebral hemispheres for both DA and NA. Withdrawal of phenobarbitone decreased the levels of DA in the striata and both catecholamines in the cerebral hemispheres. Withdrawal increased the depletion of DA in the striata and cerebral hemispheres and that of NA in the cerebral hemispheres and midbrain caused by alpha-m.p.t. Withdrawal convulsions increased the levels of DA in the striata and decreased it in the cerebral hemispheres, leaving the total amount unchanged. NA was less in this group than it was in controls. Alpha-m.p.t. protected animals from convulsions. This group showed less DA in the striata and in the cerebral hemispheres and less NA in midbrain. Habituation to phenobarbitone increased the levels of 5-HT and 5-HIAA. Withdraiwal returned both levels to control values. While withdrawal convulsions decreased the levels of 5-HT, 5-HIAA levels were increased. Single doses of phenytoin increased the levels of DA in striata and NA in midbrain. It also increased the l levels of 5-HIAA in whole brain and decreased the depletion of 5-HIAA caused by p-chlorophenylalanine (p-c.p.a.) or pargyline. Long term administration of phenytoin increa sed the levels of dopamine in the striata and the midbrain and decreased that of the cerebral hemispheres. It also produced an increase in the level s of NA in the cerebral hemispheres. Similar effects were observed after alpha-m.p.t. Whole brain levels of 5-HT and 5- HIAA were increased after long term treatment with phentoin. Single doses of carbamazepine increased the levels of NA in the midbrain and decreased the depletion of 5-HIAA after p-c.p.a. and pargyline. The long term treatment with carbamazepine increased the total brain levels of 5- HT and 5-HIAA and those of DA in the striata and cerebral hemispheres and for NA in the cerebral hemispheres and midbrain. The same effect was seen after alpha-m.p.t. While NA and GABA levels were decreased in the primary focal area oneweek after cobalt implantation, 5-HIAA levels were increased. The same effect was seen for NA and 5-HIAA levels two weeks after cobalt implantation.

Progesterone has been shown to be neuroprotective in a number of central nervous system injury models, including cerebral ischaemia. There is still a lack of understanding behind progesterone’s neuropotective properties, and the purpose of this project is to clarify some of these issues. Osmotic mini-pump infusion was hypothesised to more effective in delivering progesterone to the target organ of the brain, when compared to a bolus intraperitoneal injection. Progesterone pharmacokinetic profiles were compared between different dosing regimes. Intraperitoneal progesterone injection had a short half-life in both plasma and brain, while osmotic mini-pumps delivered higher concentrations of progesterone in plasma and particularly in brain, over a longer period, which supports the hypothesis. It was hypothesised that progesterone will reduce NO production and cell death in in vitro. Progesterone reduced nitric oxide production after challenging microglia with LPS, which supports our hypothesis and the nuclear progesterone receptor was found not to have a major role in nitric oxide attenuation. Neither of the microglial cell lines, BV-2 and HAPI cells produced elevations in NO formation in ischaemic conditions. The in vitro oxygen and glucose deprived model of ischaemia, reduced viability in both microglial and neuronal cells. Also, high pharmacological concentrations of progesterone exacerbated ischaemic injury, which does not support the hypothesis of progesterone in reducing cell death. Progesterone administration, via osmotic mini-pump infusion, was hypothesised to have a better outcome compared to vehicle treatment. After the onset of experimental stroke, progesterone delivery via osmotic mini-pump with loading dose was found to be beneficial in terms of neurological deficit score in adult male mice, which supports the hypothesis. Also, we hypothesise that co-morbidity can affect the efficacy of progesterone treatment in outcomes. Aged animals have an increased sensitivity to experimentally induce stroke and did not display, in the outcomes measured, any benefit from progesterone treatment. NOD/ShiLtJ mice had severe symptoms, resulting in high mortality after surgery and are not recommended as a model of diabetes for experimental stroke. Hypertensive BPH/2 mice are a potential hypertensive model and had better functional outcomes after treatment with intraperitoneally administered progesterone, compared to non-treated hypertensive animals in our small preliminary study. This supports our hypothesis that co-morbidity can affect the efficacy of progesterone treatment in outcomes. The gold-standard for assessing intervention effects across studies within and between subgroups is to use meta-analysis based on individual animal data. We hypothesise meta-analysis would reveal progesterone to reduce lesion volume, but also discover other effects in different subgroups of animals. Progesterone significantly reduced lesion volume, it also appeared to increase the incidence of death following experimental stroke. Furthermore, this negative effect appears to be particularly apparent in young ovariectomised female animals. These findings support the hypothesis that progesterone reduces lesion volume and progesterone having other effects in different subgroups. This investigation has clarified some issues and expanded our understanding on the neuroprotective properties of progesterone. However, these findings indicate further investigation is still required before progesterone can be considered for use in clinical trials as a neuroprotectant in stroke.

Medication errors are a significant global concern and can cause serious medical consequences in children. Double checking of medicines by two nurses is one strategy used by many children's hospitals to prevent errors from reaching paediatric patients. This thesis involves different studies that evaluated the effectiveness of the double checking process in reducing and preventing medication administration errors in a children's hospital. In addition, a systematic review was conducted of medication errors studies in the Middle East. A systematic review was also conducted of published studies of double checking. Six electronic databases were searched for articles that assessed the double checking process during the administration of medicines. Sixteen articles were identified. Only one of them was a randomised controlled clinical trial in a clinical setting. Only one study was conducted in a children's hospital. The review found that there is insufficient evidence to either support or refute the practice of double checking and more clinical trials are needed to evaluate the double checking process in children's hospitals. Based on the findings that were highlighted from the systematic review, a prospective observational study of paediatric nurses using the double checking process for medication administration was undertaken. The study aimed to evaluate how closely double checking policies are followed by nurses in different paediatric areas, and also to identify any. medication administration errors during the study period. 2,000 drug dose administration events were observed. There was variation between paediatric nurses adherence to double checking steps and different medication administration errors were identified. Based on the observational study, a semi-structured questionnaire study was developed. It was designed to explore the paediatric nurses' knowledge and opinions about the double checking process. The study showed that many nurses have insufficient knowledge on the double checking process and the hospital policy for medication administration. A simulation study was conducted to examine whether single or double checking is more effective in detecting and reducing medication errors in children. Each participant in this study was required to prepare and administer medicines in scenarios for two "dummy patients" either with another nurse (double checking) or alone (single checking). Different confounders were built into each scenario (prescribing and administration) for nurses to identify and address during the administration process. Errors in drug preparation, administration and failure to address confounders were observed and documented. The main findings from this study were that the double checking process is more likely to identify medication administration errors and contraindicated drugs than single checking. The time taken for drug administration was similar for both processes. Another systematic review was conducted to identify the published medication errors studies that have been undertaken in the Middle East. The review identified 45 studies from 10 Middle Eastern countries. Nine of the studies focused on medication errors in paediatric patients. Educational programmes on drug therapy for doctors and nurses are urgently needed in the Middle East. These studies have contributed to the field of medication safety by providing more information about double and single checking medication administration processes in paediatric hospitals. More educational and training programmes for nurses about the importance of double checking and improving their adherence rate to the double checking steps during medication administration are required to improve its effectiveness.

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.

Marfan Syndrome is a genetic, cardiovascular disease caused by a defect in the fibrillin 1 gene on chromosome 15. This defect causes abnormal deposition of elastin throughout the body. Elastin is found in many organs including the aorta. Marfan Syndrome is diagnosed by the Ghent criteria. The mean age at death is 44 years for men and 47 years for women, and about 70% die from acute cardiovascular complications, mainly aortic dissection. The assessment and treatment of the aortic complications of Marfan Syndrome has not changed for many years. Serial echocardiography is performed to measure the aortic root diameter. If thought to be increasing in size, beta blockers are prescribed to delay aortic dilatation and surgery, and to prevent aortic dissection or rupture despite the paucity of good research data. I have investigated three novel diagnostic tools: Tissue Doppler Imaging, Applanation Tonometry and Wave Intensity Analysis which have potential advantages in the assessment of the left ventricle and aorta and their interaction in Marfan Syndrome. I also investigated three drugs a beta blocker, an angiotensin converting enzyme inhibitor and a calcium channel blocker to look at their impact on some of the parameters measured by these three novel tools in a double-blinded, randomised cross-over trial. I conclude that these three novel tools would be useful adjuncts in monitoring Marfan Syndrome and their response to treatment. I also found that beta blockers may still have a role to play in delaying and preventing aortic complications when given together with an angiotensin converting enzyme inhibitor, calcium channel blocker or angiotensin receptor blocker. There are, however, other issues that need addressing to improve the management of the cardiovascular complications of Marfan Syndrome. This includes a multi-team approach to this multi-system disease and improvements in the standard of research.

P2Y12 antagonists are pharmacological agents used clinically in advanced stages of coronary artery disease in order to inhibit ADP-induced activation and aggregation of platelets and prevent deadly thrombotic events. Of the orally-prescribed P2Y12 antagonists available clopidogrel is the most established, it exhibits an excellent safety track record and is a popular drug, and was accredited for years the second-best selling drug in the world. However, since clopidogrel was introduced to the market in 1997 many pleiotropic effects have been noticed, which suggest other off-target yet beneficial effects in addition to its anti-platelet effects. The overall hypothesis being tested in this body of work was that P2Y12 antagonists, clopidogrel in particular, have the positive influence on vascular NO signalling. A vascular model was set up using isolated rabbit aortae in which clopidogrel enhanced NO donor-induced vasorelaxation. Although the precise mechanism was not found, the effect was independent of P2Y12 receptors and possibly linked to decreased superoxide production and improved anti-oxidant/inflammatory status in vessels. This finding might be relevant for patients receiving concomitant therapy with organic nitrates and clopidogrel. In vitro studies revealed novel S-nitrosation properties of P2Y12 antagonists, surprisingly without the need for metabolism to their active form. Newly synthesized SNO derivatives of clopidogrel and prasugrel were more potent in inhibition of platelet aggregation and induction of vasodilation than their parental forms. Although the formation of drug-SNO species has to be confirmed in vivo, they have a potential to increase NO bioavailability in patients. Clopidogrel administration to coronary artery disease patients resulted in upregulated plasma levels of nitrite and cGMP after 2 h-intake of a loading dose, which were further increased after 3 days of a maintenance therapy. This effect was never shown before in man and most likely reflects improved endogenous NO production, but also providing additional protection from the effects of nitrite at the same time. Taken together, the results of this thesis clearly demonstrate the influence of clopidogrel on vascular response to NO as well as NO production, metabolism and bioavailability. It is important to identify these alternative pathways especially in the current era with alternative P2Y12 antagonists that overcome some of the limitations of clopidogrel but may not share all the beneficial properties.

In summary, these observations demonstrate that lung function appears to be impaired by mucus secretion in an experimental animal model of chronic allergic asthma. The guinea pig model of chronic asthma may be utilised to investigate the development of a mucus hypersecretory phenotype in asthma or identify potential targets for the development of novel therapies aimed at reducing goblet cell mucus production or secretion.