Introduction: Chronic heart failure (CHF) is associated with an increased incidence of atrial fibrillation (AF) and elevated levels of catecholamines and endothelin-1 (ET-1), each of which affects the atrial L-type calcium current (ICaL) and consequently action potentials. Hypotheses: ET-1 modulates the effects of isoproterenol (ISO) on ICaL and action potentials in human atrial myocytes. Methods: Atrial myocytes were isolated enzymatically from samples of right atrial appendage obtained from consenting patients in sinus rhythm undergoing cardiac surgery. The nystatin-perforated whole cell patch clamp technique was used at 37ºC to record ICaL and action potentials in voltage-clamp and current-clamp mode respectively. Results: The current-voltage relationship of ICaL was bell-shaped, peaking at +10 mV with a current density of -4.8±0.4 pA/pF (mean± s.e.m., n=89 cells, 34 patients). ISO, 0.1 nM to 1 µM, increased peak ICaL in a concentration-dependent manner (n=4-46 cells) with a maximum response of 250± 53% above control and an approximate EC50 of 0.06 µM. Isoproterenol at 0.05 µM significantly increased peak ICaL from -4.7± 0.4 to -12.2± 0.9 pA/pF (P<0.05, Students t-test; n=64 cells). This adrenergic effect was reversed by ET-1 at all concentrations tested from 0.01 to 10 nM and was partially reversible upon ET-1 washout and in the presence of the specific ET-A receptor antagonist, FR139317 (n=5-12 cells). Neither ET-1 alone nor the ET-B receptor agonist Sarafotoxin S6c, at 10 nM, had an effect on ICaL. Isoproterenol (0.05 µM) prolonged the action potential duration at 50% repolarisation (APD50) from 30± 7 to 46± 7 ms (P< 0.05, n=15 cells), but had no effect on APD90 nor the cellular ERP. These adrenergic effects on APD50 and SDs were also abolished by ET-1 at 10 nM (P< 0.05, n=15 cells). Superfusion with ET-1 (10 nM) alone had no significant effect on APD50, APD90, nor ERP (n=21 cells). There were no significant interactions between these electrophysiological effects and diseases states or chronic pre-operative drug therapy. Spontaneous activity, defined as a depolarisation occurring during phase 3 of action potential repolarisation or a depolarisation of greater than 3 mV amplitude during phase 4, frequently interrupted action potential recordings during, but not prior to, superfusion with ISO. Using a repetitive stimulation protocol, ISO at 0.05 µM produced spontaneous depolarisations in 5 of 7 cells studied (P< 0.05, chi-2 test). Endothelin-1 at 10 nM abolished these depolarisations in all 5 cells (P< 0.05). Superfusion with ET-1 (10 nM) alone was associated with spontaneous depolarisations in significantly fewer cells (P< 0.05, n=2 of 13 cells). In a retrospective univariate analysis, patient comorbidity and pre-operative drug therapy were not found to influence the electrophysiological effects observed. Conclusions: ET-1 reversed adrenergically induced increases in peak ICaL, APD50 and SDs in human atrial myocytes. This anti-adrenergic effect may be expected to influence the occurrence of AF in patients irrespective of comorbidity or pre-operative drug therapy.

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder of the haemopoietic stem cell, defined by the Philadelphia chromosome (Ph) - the outcome of a balanced, reciprocal translocation between the long arms of chromosomes 9 and 22. The novel fusion oncogene generated on chromosome 22 as a result of this translocation is called BCR-ABL. In the majority of patients, this oncogene transcribes a 210-kDa constitutively active protein tyrosine kinase, often referred to as p210BCR-ABL, which is necessary for the transformation of the disease. The introduction of the orally available, tyrosine kinase inhibitor (TKI) - imatinib mesylate (IM) - marked a major advance in CML treatment in terms of efficacy and tolerability and has now become the first line of therapy. IM acts by competing with ATP to block ABL-kinase activity, resulting in the selective apoptosis induction of BCR-ABL+ cells. However, despite the success of IM as standard therapy for CML, only a small proportion of patients obtain a complete molecular response, where they become negative for BCR-ABL transcripts by RTPCR. It is hypothesised that this minimal residual disease is the result of a primitive quiescent subpopulation of leukaemic cells, which may be the cause for relapse at a later date. Another major clinical concern is the observation of molecular resistance in IM-treated patients. Proposed mechanisms of resistance include BCR-ABL amplification, decreased intracellular IM concentrations caused by drug efflux proteins such as multi drug resistance-1 and the development of point mutations within the ABL-kinase domain. In an attempt to overcome IMresistance, a second generation of BCR-ABL inhibitors has been developed. Dasatinib (BMS-354825, Sprycel) is a TKI developed for the treatment of IM resistant or -intolerant patients with Ph+ leukaemias, which has a 325-fold greater potency than IM against cells expressing wild-type BCR-ABL, and is effective against all IM-resistant BCR-ABL mutants tested in vitro, except T315I. Previously, we showed that dasatinib induced durable inhibition of BCR-ABL and impressive clearance of Ph+ cells, however, the primitive quiescent cell population did not appear to undergo apoptosis even after several days TKI exposure. Therefore, it was still not clear whether early CML progenitor cells depend on BCR-ABL for their growth and survival. In this study we have attempted to determine whether CML stem cells are dependent on BCR-ABL TK activity for their survival and/or proliferation using dasatinib treatment and aimed to characterise the cells which survived drug exposure. We found that 10% of the CML cells were able to survive the dasatinib treatment. We also showed that maximal BCR-ABL TK inhibition was achieved in the surviving CML cells, both in the bulk population of cells and the more problematic primitive stem cell population. Those cells which survived the dasatinib treatment were found to be primitive, residing mainly in the undivided cell fraction and the very early cell divisions. Since these BCR-ABL TK-inhibited, resistant cells were also able to grow when re-cultured in cytokines and form longterm culture-initiating cell (LTC-IC) colonies; these data suggested that ~10% of primitive CD34+ CML cells are not addicted to BCR-ABL TK activity for their survival. This also suggested that these primitive, resistant CML cells appeared to survive and proliferate by BCR-ABL-independent mechanisms. Therefore, the next experiments were then designed to investigate the cellular process of autophagy as a potential means of primitive CML cell survival. Analysis of the properties of CD34+ CML cells which remained viable following dasatinib treatment, revealed the existence of cytoplasmic autophagic structures determined by electron microscopy and significantly increased autophagosome-asociated LC3-II, particularly in the cells cultured without growth factors (GF)s. This suggested that autophagy is induced following GF deprivation of CML cells and is significantly increased within these cells, upon BCR-ABL inhibition following dasatinib treatment. Furthermore, we also found that the inhibition of autophagy greatly potentiated the effect of TKI treatment on the reduction of primitive CML progenitor cells, in terms of the effective eradication of functionally defined colony forming cells and LTC-ICs. Overall, this thesis has shown for the first time that the most TKI-resistant primitive CML cells are likely to be independent of BCR-ABL TK activity for their proliferation and/or survival. Furthermore, we have shown that these resistant CML stem cells rely on the BCR-ABL independent autophagy process for survival in response to stressful conditions, such as, GF deprivation and TKI treatment.

Percutaneous coronary intervention (PCI) improves the blood supply to the heart by unblocking narrowed coronary arteries. Implantation of a coronary stent is usually required to scaffold the artery and improve long-term vessel patency. Drug-eluting stents (DES) have been developed to decrease the incidence of stent renarrowing, known as in-stent restenosis (ISR), the main limitation of bare metal stents (BMS). DES release potent drugs into the artery wall to inhibit cell division and attenuate ISR. However, this strategy can also impair vascular healing and increase the risk of stent thrombosis, which is a serious concern. Novel approaches to this problem are urgently required. Oxidative stress reflects a state in which reactive oxygen species (ROS) prevail over antioxidant defences. PCI causes a major release of ROS from the injured artery wall and these molecules appear to play an important role in critical signalling pathways involved in vascular repair. Numerous animal studies have found that oral antioxidants may reduce ISR and improve healing, yet these strategies have not been effective in humans. Stent-based delivery of antioxidants may offer more efficacious, targeted protection against oxidative stress than oral administration. The role of oxidative stress in endothelial repair mediated by bone marrow-derived endothelial progenitor cells (EPCs) in patients with coronary heart disease is also poorly defined. The main aims of this thesis were: to determine the in vitro effects of oxidative stress on key aspects of thrombosis and vascular healing; to evaluate a novel antioxidant-eluting stent in an in vivo porcine model; and to examine the relationship between oxidised low-density lipoprotein (oxLDL), EPCs and coronary endothelial function in patients with stable angina. Oxidative stress, generated by the xanthine/xanthine oxidase reaction, inhibited whole blood aggregation in a concentration-dependent fashion. This was probably due to an excess of ROS which impaired, rather than stimulated, thrombosis. Healthy endothelial cells (ECs) also inhibited whole blood aggregation, but this was not mitigated by oxidative stress. EC migration was assessed using an in vitro endothelial wound scratch assay. Oxidative stress was highly toxic to ECs and inhibited migratory activity. Nitrone D, a novel spin trapping antioxidant, was evaluated for its suitability as a novel DES coating. Nitrone D displayed weak antithrombotic effects, but markedly inhibited EC migration. Nitrone D was therefore unsuitable for a DES that was intended to improve re-endothelialisation. Oral probucol has established efficacy in animal models of restenosis, but not in humans. Probucol has been successfully incorporated as a dual DES coating with rapamycin in clinical trials. Succinobucol is a novel derivative of probucol with more potent antioxidant, anti-inflammatory and antiproliferative effects. A novel polymer-free succinobucol-eluting stent (SES) and succinobucol/rapamycin-eluting stent (SRES) were developed and compared to a commercially available polymer-free rapamycin-eluting stent (RES) and BMS. Pharmacokinetic studies demonstrated optimal drug elution from the SES. However, in a porcine coronary model, the SES significantly increased neointimal thickness and aggravated ISR. The RES reduced neointimal thickness non-significantly, whereas the SRES caused no difference in neointimal thickness, compared with the BMS. The SES was associated with greater inflammation and persistent fibrin deposition around the stent struts, which are signs of defective healing. There were no significant differences in endothelial regeneration between the groups. Subsequent cell culture studies found that succinobucol was toxic to ECs and smooth muscle cells. In the clinical study, circulating levels of EPCs were strongly correlated with coronary endothelial function, which is a novel finding. Plasma oxLDL levels were not correlated with EPCs or coronary endothelial function. In conclusion, ROS reflect a large array of molecules released after PCI that are multi-faceted regulators of platelets and vascular cells. As such, they represent a complex target for novel DES technologies. Excessive ROS may inhibit thrombus formation and delay re-endothelialisation. However, potent antioxidants delivered to injured arterial tissue after PCI may not necessarily encourage the physiological processes required to accelerate vascular repair. At high dose, local delivery of antioxidants may actually promote inflammation and aggravate ISR. Although oxLDL is known to induce endothelial dysfunction, it is not correlated with the number of circulating EPCs. These findings underline the complicated role of oxidative stress in vascular repair after PCI. Further studies are required to clarify whether antioxidants will ever provide advantages over existing options in the rapidly evolving field of interventional cardiology.

GPR40, GPR41 and GPR43 are a small family of GPCRs. Fatty acids were identified as the ligands for these receptors in 2003. High levels of circulating fatty acids have been linked to a variety of diseases, including type 2 diabetes mellitus, cancer, and high levels of fatty acids are produced by anaerobic bacteria at the site of infection. High levels of GPR40 have been detected in the beta cells of the pancreas and all three receptors have been shown to be expressed in immune cells. Due to the recent identification of the ligand for these receptors, their pharmacology, function and oligomerisation was investigated during this study. A [35S]GTPgammaS binding assay was developed to monitor the activation of GPR41 using a GPR41-Gαi3 Cys351Ile fusion protein. Using the fusion protein improved the signal to background ratio and allowed the potency of a variety of short chain fatty acids to be determined. A GPR40-Gαq fusion protein was also generated, but basal levels of [35S]GTPgammaS binding in Gαq immunoprecipitates was high and did not increase significantly upon the addition of long chain fatty acids. Treatment of membranes expressing GPR40-Gαq with fatty acid free BSA reduced the basal [35S]GTPgammaS binding in a concentration dependent manner and allowed the responsiveness of GPR40 to fatty acids to be uncovered. This also allowed the potency of a variety of thiazolidinediones and small molecule agonists to be determined. It was found that troglitazone was the most potent thiazolidinedione tested. Rosiglitazone also acted as an agonist of GPR40, albeit with lower potency than troglitazone, whereas ciglitazone and pioglitazone displayed little potency. Using clones of Flp-In TREx HEK293 cells where GPR40-eYFP was cloned into the Flp-In locus, the expression of GPR40-eYFP could be controlled by the addition of doxycycline. Using this cell line confirmed that GPR40 mediated the rise in [Ca2+]i induced by the addition of troglitazone. The expression of GPR40 was detected in the rat beta cell line, INS-1E. Addition of lauric acid or troglitazone to these cells induced a large, transient rise of [Ca2+]i. Membranes prepared from INS-1E cells also displayed high basal [35S]GTPgammaS binding, which could be reduced by fatty acid free BSA. The combination of fatty acid free BSA and lauric acid or troglitazone increased [35S]GTPgammaS binding. The high levels of [35S]GTPgammaS binding observed in membranes expressing GPR40 may reflect the binding of an endogenous ligand to GPR40. Homology models of all three receptors based on the crystal structure of rhodopsin indicated that a conserved Arg residue in TM5 may co-ordinate the carboxylate group of the fatty acid. To confirm this observation a series of mutant receptors were generated, with the Arg residue in TM5 mutated to alanine. Neutralisation of the charge in TM5 in GPR41 and GPR43 resulted in receptors that were unable to respond to short chain fatty acids. Further mutants of GPR43 were generated in which the Arg in TM5 was replaced by Lys, Leu or Ser. None of these mutants were able mediate a rise in [Ca2+]i in response to short chain fatty acids. The equivalent mutation in GPR40 did not abolish the receptors ability to respond to long chain fatty acids. There are two further conserved basic residues in TM regions of the receptors; an Arg/Lys in TM2 and a His in TM4. These residues were also mutated to alanine. In GPR41 and GPR43, both mutants had similar function to the wild type receptors. Both GPR40 mutants were also able to respond to a variety of fatty acids measured by their ability to mediate a rise in [Ca2+]i in a FLIPR based assay system. A series of further GPR40 mutants were generated where two of the three basic TM residues were mutated. The most striking observation was found with the mutant in which the His in TM4 and Arg in TM5 mutated to alanine, this mutant was unable to mediate a rise in [Ca2+]i in response to a variety of saturated fatty acids. This may indicate that upon loss of charge in TM5 the His in TM4 compensates and vice versa. None of the small molecule agonists or thiazolidinediones were able to activate the TM4 or TM5 mutants. This indicated that the synthetic agonists of GPR40 require a more conserved binding pocket which may be due to their more rigid structure. The ability of GPR40 and GPR43 to form homo-oligmers was also investigated. It was found that GPR40 and GPR43 formed homo-oligomers as monitored by co-immunoprecipitation, FRET in single, living cells and by time resolved FRET.

Heart failure (HF) and chronic obstructive pulmonary disease (COPD) are common partners with common problems. Both are chronic systemic disorders incurring significant morbidity and mortality. Although around one third of patients with HF have concurrent COPD,1 remarkably few reports have addressed this often ignored combination. The systematic review presented within this thesis defines the diagnostic challenges, prevalence and prognostic implications of HF with coexistent COPD. I then critically appraise the twin controversies of β-blockade in COPD and β-agonists in HF. The two are inextricably linked, each therapy exerting the reverse pharmacologic activity of the other. The evidence for symptomatic or prognostic benefit from either therapy is limited, and in the case of β-agonists adverse consequences appear more likely. A Cochrane meta-analysis concluded that long term cardioselective β-blockade is safe and well tolerated in patients with moderate to severe or reversible COPD.2 Although often cited,3 these conclusions are simply not true. Of the 20 randomised controlled trials included in the meta-analysis, 11 involved single doses and only one lasted longer than a month. The 9 ‘long term’ studies (defined as more than a single treatment dose) involved 147 young, predominantly male patients with moderate airways obstruction (mean forced expiratory volume in 1 second (FEV1) 1.8 litres). The effect on health status has never been assessed in any cohort with COPD. The long term impact of β-blockade on pulmonary function, symptoms and quality of life is therefore largely unknown. Most importantly, no study has included patients with HF. I randomised 27 patients with HF and coexistent moderate or severe COPD to receive bisoprolol or placebo, titrated to maximum tolerated dose over 4 months. Patients were elderly and predominantly male. Cardiovascular comorbidity, smoking history and pulmonary function were similar in each group (mean FEV1 1.37L vs 1.26L). There were several key findings. A reduction in FEV1 occurred after 4 months following treatment with bisoprolol compared with placebo (–70 ml vs +120 ml, p=0.01). Reversibility following inhaled β2-agonist and static lung volumes were not impaired by bisoprolol. All measures of health status exhibited a consistent non-significant improvement, including the Short Form 36 physical and mental component scores, Minnesota Living with Heart Failure Questionnaire, and Chronic Respiratory Questionnaire. The mean number of COPD exacerbations was similar in the bisoprolol and placebo groups. Although recruitment was limited, the results pose crucial questions and provide direction for larger randomised controlled trials. I analysed cross-sectional data from 61 primary care practices (377,439 patients) participating in the Scottish Continuous Morbidity Recording scheme. The prevalence of COPD in patients with HF increased year on year from 19.8% in 1999 to 23.8% in 2004. These changes may previously have been attributed to an ageing population or increasing age of presentation. However, the trend remained significant after age standardisation. A clear socioeconomic gradient was observed, with prevalence greatest in the most deprived. Consultation rates for HF or COPD in those with both conditions were greater than disease specific contact rates in patients with either condition alone. Cardiovascular comorbidity was similar in HF patients with and without COPD, despite differences in smoking history (respectively 76% vs 47%, p<0.001). This is concerning and suggests that common cardiovascular conditions are being under diagnosed (and likely under treated) in patients with HF and COPD. Although overall β-blocker prescribing increased over time, the adjusted odds of β-blocker prescription in patients with COPD was low (odds ratio 0.30 [95% CI 0.28–0.32], p<0.001). Whether the gap between patients with and without COPD is improving was previously unknown. Despite the overall improvement in beta-blocker prescribing, the relative difference in prescribing between those with and without COPD remained unchanged. By 2004, only 18% of individuals with HF and COPD were prescribed β-blockers. COPD is consistently an independent predictor of death and HF hospitalisation in patients with HF. However, the causes of increased mortality were unclear. I examined the relationship between COPD and cardiovascular outcomes in patients with myocardial infarction (MI) complicated by heart failure, left ventricular systolic dysfunction (LVSD), or both enrolled in the Valsartan in Acute Myocardial Infarction (VALIANT) trial. COPD was an independent predictor of mortality, largely due to increased non-cardiovascular (HR 1.86 [1.43–2.42]) and sudden death (HR 1.26 [1.03–1.53]). However, after multivariate adjustment COPD was not an independent predictor of atherosclerotic events (MI or stroke: HR 0.98 [0.77–1.23]). This is an important finding, as atherosclerotic consequences of chronic systemic inflammation in COPD have been postulated. These appear of limited clinical significance, at least during intermediate follow-up. Part of the adverse risk associated with COPD may be attributable to bronchodilators. The prognosis of patients with HF prescribed bronchodilators is however ill defined. I examined the prognostic implications of bronchodilator use in patients with HF enrolled in the Candesartan in Heart failure Assessment of Reduction in Mortality and morbidity (CHARM) programme. The diversity and magnitude of adverse outcomes associated with bronchodilator therapy was surprising. Bronchodilator use was associated with increased all cause mortality (HR 1.26 [1.09–1.45]), cardiovascular death (HR 1.21 [1.03-1.42]), death due to HF progression (HR 1.40 [1.07-1.82]) and HF hospitalisation (HR 1.49 [1.29-1.72]). Although association is not causation, it is possible that bronchodilators compound maladaptive remodeling and further depress myocardial function. Finally, β-blockers were independently associated with better survival in both VALIANT and CHARM. No significant interaction was observed between either COPD or bronchodilators and β-blockade with respect to mortality. Furthermore, β-blocker use was not associated adversely with any pre-specified outcome in patients with COPD or those prescribed bronchodilators, including non-cardiovascular mortality. Although recruitment bias and the absence of spirometry limit inference to patients with severe or reversible airflow obstruction, the results should encourage β-blockade in patients with COPD. In summary, the studies presented in this thesis extend our understanding of HF with concurrent COPD. Only large randomised controlled trials will solve the quandary of β-blockers and β-agonists. Justification for these trials evolves from observational data and smaller prospective studies such as my own. In the meantime, I hope the evidence presented will stimulate physicians to re-evaluate the management of patients with HF and COPD.

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.