Metal ions such as silver (Ag+), mercury (Hg2+), zinc (Zn2+) and copper (Cu+/Cu2+) have a long history of antimicrobial usage and some, such as Cu+/Cu2+, Ag+ and Zn2+ compounds are still used as antimicrobials. Prior to the introduction of antibiotics, Ag+ was arguably the most important antimicrobial and with the rapid emergence of antibiotic resistance, interest in Ag+ and its compounds as alternative antimicrobials have recently been revived. However, resistance to Ag+-based compounds has been emerging, with initial reports of carriage of silver resistance on a Salmonella enetrica serovar Typhimurium multi-resistance plasmid pMG101 isolated from burns patients in 1975. The proposed model for the mechanism of Ag+ resistance encoded by the sil genes from pMG101 involves export of Ag+ ions via an ATPase (SilP), an RND family effluxer (SilCFBA) and a periplasmic chaperone of Ag+ (SilE). SilE is a periplasmic protein predicted to be intrinsically disordered until it binds Ag+ ions. This hypothesis was tested using structural and biophysical studies which showed that SilE is an intrinsically disordered and unstructured protein in its free apo-form, but folds to a compact, defined structure upon optimal binding of six Ag+ ions in its holo-form. Sequence analyses and site-directed mutagenesis established the importance of histidine and methionine containing motifs for Ag+-binding, and identified a nucleation core that initiates Ag+-mediated folding of SilE. The data show that SilE is a molecular metal sponge absorbing up to a maximum of eight Ag+ ions.

Natural killer (NK) cells reconstitute rapidly following allogeneic stem cell transplantation (allo-SCT) at a time when alloreactive T cell immunity is being established. Important differences are seen in the patterns of reconstitution between T cell deplete, T cell replete and umbilical cord stem cell transplants. 82 patients who received T cell-deplete allo-SCT were studied to determine the functional and transcriptional profile of the reconstituting NK cells and to assess the relationship with clinical outcome. NK cells at day 14 (D14-NK) were donor-derived, intensely proliferating and expressed chemokine receptors targeted to lymphoid and peripheral tissue. Spontaneous production of the immunoregulatory cytokine IL-10 was observed in over 70% of cells and transcription of cytokines and growth factors was augmented. D14-NK cell number was inversely correlated with the incidence of grade II-IV acute graft versus host disease (GVHD). These findings reveal that robust reconstitution of immunoregulatory NK cells by day 14 after allo-SCT is an important determinant of clinical outcome and suggest NK cells may suppress development of the T cell-mediated alloreactive immune response through production of IL-10.

Understanding drug-protein interactions and downstream effects of these interactions underpins much of clinical pharmacology. By studying the protein targets of a small molecule we can learn about the action of this compound in the body, and this information can lead to greater understanding of mechanisms and clinical effects. We probed the polypeptide interactions of five small molecules using a human vascular phage display library, with the intention to elucidate previously unknown protein targets for the small molecule in human vasculature. A method for studying the chemical nature of this interaction was also developed. The photochemical immobilisation system used - Magic Tag® - was developed as a means of immobilising small molecules without concealing any facet of the molecule from the interaction study. Five different photochemistries are displayed in a multi-well format, to maximise diversity in the display of the small molecule. A human vascular tissue T7Select® bacteriophage display library was prepared from internal mammary artery tissues donated from patients undergoing coronary artery bypass surgery. Biopanning of the immobilised small molecule against this library allowed hypothesis generating analysis of small molecule-polypeptide interactions. Because of the non-selective nature of the photochemical immobilisation of the small molecule, several regioisomers might be expected to form on the Magic Tag® surface. To be able to connect a protein interaction with a specific face of the small molecule, analysis of this regio-non-specific interaction must be undertaken. For this purpose a cleavable resin analogue of the Magic Tag® surface was prepared.

Stroke is a common disorder with dire consequences for the patient and for society and will increase in prevalence over the coming years. Following stroke, many patients unfortunately suffer a further stroke, and recurrent strokes account for approximately 25% of the total. Considerable scope therefore exists to improve both primary and secondary stroke prevention. This thesis has addressed several areas at key stages in the prevention of stroke by developing strategies to better identify those at highest risk, attempting to better target pre-existing anti-platelet therapy and by beginning the evaluation of xanthine oxidase reduction and uric acid lowering therapy in the prevention of stroke. A clinical scoring system to aid diagnostic recognition in those with suspected transient ischaemic attack (TIA) was successfully developed and has the ability to reduce the referral of those without cerebrovascular disease to busy TIA clinics. The score was developed on data from 3216 patients and included 9 clinically useful predictive variables. After adjustment to reflect the greater seriousness of missing true TIA patients, 97% of TIA and 22% of non-TIA patients were accurately identified. The results were confirmed during prospective validation. Use of the score could have a substantial effect on waiting times for assessment; there is potential double the numbers seen within the timeframe recommended by guidelines with no other change to services. This would be an important advance given the recent evidence that rapid assessment and treatment of those with TIA greatly reduces stroke risk. Aspirin resistance was found to be higher in those with cerebrovascular microembolic signals (MES) and carotid disease compared to those with equivalent carotid disease and no MES. This study included sixty-two patients who mostly had symptomatic carotid disease. Approximately a quarter had MES. The rate of aspirin resistance on at least one test was 25.8% (16 patients), with 13 (21%) resistant on PFA-100 testing, 8 (12.9%) using the Verify-Now system and 5 (8.1%) resistant on both. Aspirin resistance was more common in patients with MES (50% compared to 17.4% without, p=0.018 on Fishers exact test). This provides a link to a well established and robust surrogate marker of outcome and thus a useful model to further study the benefits of guided anti-platelet strategies. An interventional clinical anti-platelet trial based upon individual aspirin responsiveness in high risk individuals such as those with MES is now warranted. Aspirin resistance was also confirmed to be a common phenomenon in a case-control study of 180 patients. It was present in 34% of those with recent stroke and in 18% of those with risk factors but no established disease. However, the role of poor compliance with therapy as a cause in a substantial number of cases was established; it accounted for approximately half of those labelled resistant in the stroke group. Further, when only those with objective evidence of recent aspirin ingestion were considered, the prevalence of aspirin resistance was similar in both groups (at 26%). This suggests that objective measures to confirm compliance with aspirin therapy should be mandatory in future studies of aspirin resistance. Increasing serum uric acid was found to be a predictor of poor functional outcome following acute stroke but not in an independent fashion. In total, 852 patients were included in this study and greater uric acid levels were associated with increased odds of poor outcome on univariate but not multivariate analysis (OR 1.3, 95% CI 0.73-2.31). However, there was no evidence of an association with favourable outcome as other groups have found. Increasing serum uric acid was also shown to be predictive of increased risk of stroke, total, vascular and coronary mortality in treated hypertensive patients but interestingly, the relationship between stroke mortality and serum uric acid appears J-shaped and most apparent in females. A study of the use of allopurinol in those with diabetes showed that xanthine oxidase inhibition improves cerebral nitric oxide bioavailability suggesting a beneficial effect of allopurinol on cerebrovascular health. This study included 14 participants who had impaired baseline cerebrovascular nitric oxide bioavailability. Allopurinol led to a significant improvement in responses to NG-monomethyl-L-arginine (L-NMMA) when compared to placebo (p=0.032, median improvement in ICA flow reduction following L-NMMA of 3144 (95% CI 375 to 7143)) mls). L-NMMA is an inhibitor of endothelial nitric oxide synthase which reduces cerebral blood flow in healthy volunteers; the bigger the reduction, the greater the endothelial health. However, a study of the effect of allopurinol treatment on cerebrovascular reactivity (as measured by response to acetazolamide infusion) in a group of patients with recent subcortical stroke revealed no positive effect. Cerebrovascular reactivity was unchanged by treatment with allopurinol. This raises interesting questions regarding the longevity of any positive effect of allopurinol as this, and other studies of 3 month duration, have revealed no benefit. Further, subjects in this study did not, on balance, have elevated serum uric acid and it has recently been suggested that only those with significantly elevated levels benefit in the setting of congestive cardiac failure. Whether this is also true in those with stroke also requires to be clarified. A large study of the effect of allopurinol on carotid intima-media thickness, a robust and modifiable marker of vascular risk, in those with recent stroke is planned to address these questions. The studies in this thesis therefore include a number of pragmatic findings which could improve care at all stages in the prevention of stroke. The TIA scoring system could improve recognition of the high risk condition TIA, a useful model has been developed in which to study a population of patients truly resistant to aspirin and important lessons have been learned to aid further evaluation of xanthine oxidase inhibition; a promising therapy for the prevention of stroke.

As the incidence of type 2 diabetes rises exponentially in the early part of the 21st century, the global healthcare and financial burdens associated with this chronic condition rise in tandem. As such, it becomes increasingly important to characterise this disease further, and unravel the molecular mechanisms which lead to hyperglycaemia and, ultimately, premature vascular disease. Insulin resistance, while pathognomonic of type 2 diabetes, is not confined to this condition. It has become clear that insulin resistance lies at the centre of a constellation of cardiovascular risk factors, which include hypertension, obesity and dyslipidaemia. To date there are no large prospective studies that have examined the role of insulin sensitivity in the development of vascular disease, however epidemiological evidence suggests that there is a relationship. Thus, studies exploring the factors that determine insulin sensitivity are of considerable clinical importance. In this thesis, a series of studies is described which were designed to measure insulin action in the whole body and in isolated tissues. Insulin sensitivity was measured in the whole body using the hyperinsulinaemic euglycaemic clamp, though also in adipose tissue by measuring insulin-mediated suppression of lipolysis and in human resistance vessels by measuring insulin-mediated vasodilation. In association with this, the effect of activation of endogenous hormonal axes that may influence insulin sensitivity was investigated, not only in the whole body but also in individual tissues. As an overview of the experimental chapters: i) Insulin-mediated suppression of lipolysis; development and application of an assay of insulin sensitivity; ii) Dietary sodium restriction and insulin sensitivity; iii) Angiotensin II and insulin sensitivity in human adipocytes; iv) Glucocorticoids and insulin sensitivity: dissociation of insulin's metabolic and vascular actions. Conclusions. From the above experimental chapters, several conclusions were made: i) Insulin sensitivity in isolated human adipocytes is reduced in obese, though otherwise healthy, females with normal fasting plasma glucose. ii) Dietary sodium restriction is associated with a reduction in systemic insulin sensitivity. iii) Intracellular crosstalk between angiotensin II and insulin does not result in insulin resistance in human adipocytes. iv) Dexamethasone treatment is associated with a significant reduction in systemic insulin sensitivity, though no change in insulin action in the vasculature.

Resistance to anthelmintics used to treat parasitic nematodes of veterinary importance represents a serious welfare and economic problem for the livestock production industry. Research into the mechanisms by which parasites develop resistance is necessary to prolong the life of the available drugs and to minimise development of resistance to new classes. Metabolism of anthelmintic compounds by parasites is a possible mechanism of resistance that has received little research, despite there being precedence in the case of insecticide resistance. Due to the more advanced molecular tools available and comparative ease of manipulation; we have used the model nematode Caenorhabditis elegans to investigate the metabolism of two important anthelmintic drugs, ivermectin and albendazole. Whole genome microarrays and RT-QPCR were used to identify clusters of genes, which are significantly up-regulated upon exposure of C. elegans to anthelmintic. The transcriptomic response to albendazole is characterised by genes potentially involved in xenobiotic metabolism. These include members of the cytochrome P450 family and the UDP-glucuronosyl/ glucosyl transferase family. In contrast, the response to ivermectin appears to represent a fasting response caused by the phenotype of drug exposed nematodes. Recombinant worms carrying GFP reporter constructs of several genes of interest demonstrated their expression in the intestine, which is thought to be the main site of xenobiotic detoxification in nematodes. HPLC-MS techniques have definitively shown that C. elegans is able to metabolise albendazole to two glucose conjugates. These metabolites are compatible with the transcriptomic response to the drug and are similar to albendazole metabolites produced by the parasitic nematode Haemonchus contortus. No ivermectin metabolites were identified in the current study. The data presented confirms the ability of the nematode C. elegans to respond to and metabolise anthelmintic compounds. In addition, the study validates the use of C. elegans as a model organism for parasitic nematodes and provides a platform upon which to investigate nematode metabolism further.

The strategy of suicide gene therapy in cancer is based on the idea of enabling tumour cells, by gene transfer, to convert a non-toxic pro-drug into a toxic product. Previous work has shown that the combination of herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) transfer with the pro-drug ganciclovir (GCV) to be a promising suicide gene therapy in cancer. Unlike several other gene therapy systems, early-phase clinical trials of this strategy have shown encouraging results. Therefore, methods to improve its therapeutic efficacy are urgently sought. The thymidine analogue 5-iodo-2’-fluoro-2’-deoxy-1-ß-D-arabino-furonosyluracil (FIAU) is an alternative substrate of the HSV1-TK enzyme. The iodine atom of FIAU can be substituted with radioactive iodine, for example; [123I]-iodine, and thereby utilised for the delivery of ionising radiation into tumour cells expressing the viral tk gene. The aim of this study was primarily to investigate the therapeutic potential of combining HSV1-tk gene transfer and [123I]FIAU for the targeted radiation cytotherapy of prostate cancer cells alone or in combination with GCV. The HSV1-tk gene was cloned into the plasmid vector pcDNA3.1. This plasmid, driven by the ubiquitous promoter of CMV, was then used to transfect the prostate cancer cell line DU145 and the glioma cell line UVW. A viral TK positive, commercially available cell line derived from osteosarcoma (143B-TK) along with its TK-negative clone were also used for comparison. The viral tk gene transfection efficiency was assessed by three independent methods. Firstly, the uptake of [123I]FIAU normalised to the uptake of tritiated thymidine ([methyl-3H]TdR); secondly, GCV sensitivity, assessed by the MTT assay; and thirdly, by the detection of HSV1-tk gene by RT-PCR. The highest specific activity of [123I]FIAU was obtained by the use of a no-carrier added method of synthesis. The cytotoxicity of [123I]FIAU was assessed by clonogenic assay after incubating monolayers of parental and TK-positive clones of the cell lines with a range of doses of [123I]FIAU for the periods of 4 h, 8 h and a period equal to their doubling times. The effect of this treatment on cell cycle progression was assessed by FACS analysis after staining the cellular DNA with propodium iodide. Combination therapy using GCV and [123I]FIAU for the treatment of TK-positive clones of the prostate cancer cell line DU-145 and the osteosarcoma cell line 143B was assessed by the method of median effect analysis and combination index. Monolayers were treated with a constant ratio of various doses of [123I]FIAU for 4h or GCV for 72h. The combination therapy followed three different timing schedules of GCV-before-[123I]FIAU, [123I]FIAU-before-GCV, or simultaneous therapy. The expression of HSV1-tk gene by the three cell lines was confirmed by the three methods described above. For instance, the TK positive clone of the cell line DU145 exhibited 4.25 ± 0.15 times higher [123I]FIAU/ [methyl-3H]TdR uptake ratio and 43 times higher sensitivity to GCV compared with the parental cell line. The three cell lines demonstrated sensitivity to radiolabelled FIAU, which was significantly enhanced by HSV1-tk gene expression. This sensitivity was time-, dose-, and proliferation-dependent. Maximum cell kill was achieved when the monolayers were exposed to [123I]FIAU for a period equavelant to the cellular doubling time. For example, the sensitivity enhancement factor by tk gene expression of the cell line DU145 increased from 5.2 to 7.6 when the treatment period was prolonged from 4 h to 26 h (doubling time of DU145). Following the treatment with [123I]FIAU for a period equal to the doubling time, cells were arrested at G2/M phase of the cell cycle. For instance, 49% of DU145-TK cells treated with 1 MBq/ml for 26 h were at G2/M phase compared with 21.9% of the untreated cells. In contrast, incubation of DU145-TK or 143B-TK cell lines with lethal doses of [123I]FIAU for 4 h and GCV for 72 h had no significant effect on cell cycle progression. Comparison of the effectiveness of [123I]FIAU in the monolayer and spheroid cultures indicated that clonogenic cell kill resulting from Auger electron bombardment was restricted to targeted rather than bystander cells. The combination therapy of [123I]FIAU and GCV of the cell line DU145-TK resulted in antagonistic effect throughout the examined dose range of the schedules of FIAU-before-GCV and simultaneous therapy and the low toxicity concentration range (lower surviving fractions) of the GCV-before-FIAU schedule. The high toxicity concentration range of the latter schedule has shown evidence of additive effect. For the osteosarcoma cell line 143B-TK, synergistic effect was observed at the high toxicity concentration range of the three combination schedules and antagonism at the low toxicity concentration range of the combinations. We concluded from this in vitro study that the combination of HSV1-tk gene transfer and the delivery of radiolabelled FIAU is a promising strategy for targeted radiation cytotherapy of prostate cancer. This proliferation-dependent therapy has caused significant cell cycle arrest that warrants further investigation. Furthermore, the combination of GCV and radiolabelled FIAU for the treatment of tumour cells expressing the gene of viral TK resulted in a dose- and schedule-dependent synergism. We believe that these encouraging results should be substantiated by in vivo experiments in the near future.

Multidrug-resistant Gram-negative infection is an important cause of mortality and morbidity. Management of these infections is often dependent upon “treatment of last resort” "small molecule" antibiotics which suffer from significant toxicity and an indiscriminate volume of distribution. The aim of this study was to develop a prototype polymer-antibiotic conjugate that may be customised by polymer modification and binding chemistry to afford selective, controlled release at an infected site. These studies employed the biodegradable, naturally-occurring polymer, dextrin, and a polymyxin antibiotic, colistin, as the first model combination. Physicochemical characterisation of a library of succinoylated dextrins and dextrin-colistin conjugates demonstrated that conjugation of dextrin to colistin was feasible and reproducible, resulting in masking of colistin's amino groups through incorporation in peptide bonds. Exposure to physiological �-amylase activity resulted in controlled degradation of the dextrin component, leading to sustained colistin release. Following exposure of the conjugates to physiological concentrations of �-amylase, minimally-modified, low molecular weight dextrin, conjugated to colistin, demonstrated significantly earlier, maximal release of colistin and subsequent reinstatement of antimicrobial activity. At maximum unmasking, the lead conjugate reported equivalent antimicrobial activity to the current clinical formulation of colistin (Colimycin®)against a range of MDR organisms including: A. baumannii, K. pneumoniae and E. coli. A static two-compartment dialysis bag model was developed under infinite sink conditions, which demonstrated that the conjugates were able to suppress bacterial growth over a significantly greater duration than colistin sulfate. Ex vivo studies of infected human wound fluid samples confirmed that colistin could be readily liberated from conjugate in infected sites. Significantly higher amylase activity in these wound fluid samples supported the notion of locally-triggered, enzymatically-mediated unmasking. An in vivo intravenous, pharmacokinetic model in rats demonstrated the increased half-life associated with conjugation and succinoylation. Moreover,the dextrin-colistin conjugates were better tolerated than colistin sulfate at higher concentrations. These studies have demonstrated the feasibility of developing this new class of “nanoantibiotics” and highlighted their potential usefulness as bioresponsive nanomedicines for the treatment of MDR Gram-negative infection.

In the present study, the function and the mechanism of action of RGS4, a member of a family of proteins called Regulators of G protein Signalling (RGS) was investigated. A C-terminal fluorescent tag on RGS4 confirmed that transiently transfected RGS4 was predominantly cytosolic and underwent translocation to the plasma membrane of HEK293T cells following co-expression of Gi1, the 2A-adrenoceptor, or agonist activated 2A-adrenoceptor. This translocation of RGS4 to the plasma membrane was most pronounced with the co-expression of the constitutively active GTPase deficient Gi1 Q204L. High-affinity GTPase experiments indicated that RGS4S30C had enhanced GAP activity towards Go1 compared to wild type RGS4. This approach also demonstrated a simultaneous significant decrease in potency of both adrenaline and UK14304 to increase 2A-arenoceptor-activated high-affinity GTPase activity of Go1 in the presence of RGS4 and a further significant decrease in potency of both ligands in the presence of RGS4S30C. This enhanced GAP activity and observed decrease in agonist potency was also transferable to RGS16, an RGS protein closely related to RGS4. The selectivity of the G subunit was also investigated. The enhanced GAP activity and simultaneous significant decrease in potency of adrenaline and UK14304 to increase 2A-arenoceptor-activated high-affinity GTPase activity of RGS4S30C and RGS16S30C was selective for Go1 over Gi1. RGS4S30K and RGS4S30F also demonstrated higher GAP activity than wild type RGS4 but no consensus side chain could be identified that conferred a specific enhancement or loss of GAP activity. The ability to inhibit intracellular calcium release by an activated 1b-adrenoceptor-G11 fusion protein was used in order to investigate the GAP activity of RGS4N88S, RGS4N128A and RGS4N88S,N128A. All three mutants had ablated GAP activity towards G11 and therefore failed to inhibit intracellular calcium release. A novel role for the RGS insensitive mutation G188S was also observed when despite similar expression, G11 G188S significantly reduced agonist-stimulated [35S]GTPS binding compared to wild type G11. RGS4 represents a novel target for pharmaceutical drug development and the study of its regulation of signal transduction is an important area of investigation. These results highlight specific areas of RGS4 research with great pharmaceutical potential.

The kinesins are a class of microtubule based motor proteins which have extensive involvement in the orchestration of the mechanics of mitosis. The most studied of these is the kinesin spindle protein Eg5, which is crucially involved in the establishment of the bipolar spindle in prometaphase. Inhibition of this protein results in monopolar mitotic spindles and subsequently mitotic arrest, which can lead to apoptosis in cancer cell lines. S-Trityl L-cysteine (STLC) was identified as a selective small molecule inhibitor of Eg5 which binds to an allosteric pocket formed by the loop L5 of Eg5. In this thesis, I present the structure based design and development and optimisation of the STLC scaffold to produce orally available potential drug candidates. This was accomplished by optimising the lipophilic binding interactions of the trityl group, and investigating a number of hydrophilic optimisation vectors from the same moiety. The L-cysteine tail was optimised to improve the potency and metabolic stability, and fluorination as a means of altering the lead candidates’ drug like properties investigated. In order to improve efficacy in multi-drug resistant (MDR) cell lines overexpressing the P-glycoprotein transporter, I also investigated a number of strategies related modifying to the terminal α-carboxylic acid. The optimised candidates display growth inhibition ≤ 50 nM across multiple tumour cell lines, and possess favourable metabolic, toxicological and physicochemical attributes. Evaluation in vivo confirms their anti tumour activity, and finally strategies for the further progression and development of the lead series in targeting haematological malignancies are discussed.