Viral infections account for 25.4% of all human illnesses. Current anti-viral nucleosides are able to prevent viral replication; however, viruses evolve resistance to available treatments. Ribose nucleosides are ubiquitous; however due to the discriminatory nature of kinases, low intracellular concentrations of the active triphosphate is a significant factor in inactivity. In contrast, oxetane nucleosides have been shown to be phosphorylated by these kinases with high levels of the required active nucleotide triphosphate (NTP) resulting. Oxetane nucleosides, however, have been found to be inactivated by pyrimidine nucleoside phosphorylases (PNPs). Previous studies have illustrated that sulfur containing nucleosides are not hydrolysed by PNPs. Therefore in this programme a library of 9 novel 3,3-bis(hydroxymethyl)-thietan-2-yl nucleosides was synthesised using Vorbrüggen conditions starting from a fluorothietane precursor, over 8 steps. Cell viability studies using an XTT assay on the thymine (103% ± 4.9%), uracil (95% ± 2.3%), 5- fluorouracil (98% ± 7.5%) and 5,6-dimethyl uracil (82% ± 4.1%) derivatives showed no significant loss in cell viability at up to 100 μM concentration, suggesting they were suitable for further study in anti-viral screens. Steps towards the synthesis of a complementary library of 4,4-bis(hydroxymethyl)-thietan-2-yl nucleosides have been optimised with successful synthesis of a novel key intermediate, thietane-2-one, in a yield of 37% over 6 steps. 4’-Thiohamamelose nucleosides show promise as potential anti-viral compounds as previous studies have shown that 4’-thioribose nucleosides are stable to PNPs and have longer half-lives than ribose nucleosides. Increasing the stability of the 4’-thiohamamelose nucleosides could improve the potential for development of the anti-viral profiles of this class of compounds. Therefore the chemistry of 4’-thiohamamelose nucleosides has been explored, with an intermediate hamamelolactone being prepared in a yield of 44% over 5 steps. Small molecule nucleosides offer the greatest benefits as anti-viral compounds versus other small molecule anti-viral agents as nucleosides are able to directly halt viral replication. The sulfur containing nucleosides are potentially more stable than their ribose counterparts, leading to better pharmacological and pharmacodynamics outcomes.

An important molecular mechanism by which physical activity reduces cardiovascular risk is through regulation of plasma lipids. This study aimed to investigate whether low intensity exercise modulates monocytic lipid metabolism and the activity of the nuclear transcription factor Peroxisome Proliferators-Activated Receptor-γ (PPARγ). Furthermore, this study aimed to elucidate the molecular signalling mechanisms induced through PPARγ activation. Thirty-four sedentary adults, mean age 45.6 ± 11.1 years, participated in an eight week low intensity exercise programme consisting of walking 10,000 steps, three times a week. Compared to controls, there was a significant decrease in total cholesterol (pre-exercise: 5.73 1.39 mmol/L; post-exercise: 5.32 1.28 mmol/L) and a significant increase in high density lipoprotein (pre-exercise: 1.46 0.47 mmol/L; post-exercise: 1.56 0.50 mmol/L) after the exercise programme. There was also a significant increase in serum oxidised LDL (oxLDL) concentrations pre to post exercise (0 weeks: 554 107ng/ml; 4weeks: 698 134ng/ml; 8weeks: 588 145ng/ml). A significant increase in leukocyte mRNA expression for PPARγ (4 weeks: 1.8 0.9 fold; 8 weeks: 4.3 1.9 fold) was observed, which was reinforced by increased PPARγ DNA-binding activity post exercise (pre-exercise: 0.22 0.09 OD units; post-exercise: 1.13 0.29 OD units. A significant increase in gene expression was observed for the oxLDL scavenger receptor CD36 (4 weeks: 3.8 0.6 fold; 8 weeks: 2.7 0.5 fold). LXRα (8 weeks: 3.5 0.8 fold) and two LXRα regulated genes involved in RCT, namely ABCA1 and ABCG1 were significantly upregulated after eight weeks of exercise (8 weeks: ABCA1: 3.46 0.56 fold; ABCG1: 3.06 0.47 fold). The culmination of in-vitro evidence lead to the postulation of a two pathway molecular mechanism associated with oxLDL stimulation of CD36, via PPARγ in monocytic cells. A „short-term‟ pathway (<24 hours), upregulates PPARγ (2 fold) via a transient ERK1/2 and COX-2 dependent mechanism. Whereas a „long-term‟ pathway (>24 hours), involves the direct upregulation of PPARγ via ligands within oxLDL, which is ERK1/2 independent but still COX-2 dependent. Activation of PPARγ enables direct DNA-binding with CD36, facilitating the oxLDL to enter the cell, via CD36, exacerbating the effect and promoting the cellular clearance of oxLDL. However, between 24 and 72 hours the „short-term‟ pathway is required to upregulate PPARγ via COX-2 and hence induces upregulation of CD36. It is possible that over several weeks of low intensity exercise the more rapid molecular pathway can be supplemented by PPARγ ligands present within oxLDL, and hence directly stimulate PPARγ gene transcription. In conclusion this study proposes a novel molecular mechanism for low intensity exercise induced modulation of plasma lipids via cellular clearance of cholesterol that involves activation of the nuclear transcription factors PPARγ and LXRα.

This thesis examines the toxicity of metallic and organometallic nanoparticles which have applications in the medical field. Commercially available Silver particles} used to create bactericidal surfaces} are tested against two human in vitro cell models to investigate the sensitivity of the models and the toxicity of the particles. No toxicity to a human blood brain barrier in vitro cell model arises from concentrations of particles likely to be encountered. Significant toxicity however is demonstrated in a human placental in vitro cell model} raising concerns for maternal exposure to Silver particles and selecting the placental cell model for further study. A novel polymer nanoparticle drug delivery system capable of encapsulating a wide variety of lipophilic drugs is described. Extensive characterisation demonstrates successful encapsulation of fluorescent} water insoluble} Tris-(8-hydroxyquinolinato) Aluminium (III) (AlQ3) a molecule with antibiotic potential. The AlQ3 nanoparticles are tested against a human placental in vitro cell model} at physiologically relevant doses} finding no significant toxicity to the cell membrane} metabolism} nucleus or viability. Confocal experiments with concomitant organelle staining confirm cellular internalisation and examine the AlQ3 intracellular localisation. The antibacterial properties of AlQ3 nanoparticles are then demonstrated by the treatment of human keratinocytes infected with Methicillin Resistant Staphylococcus Aureus (MRSA). The polymer nanoparticle delivery system is then further examined by synthesising nanoparticles of the chemotherapeutic analogue} Tris-(8-hydroxyquinolinato) Gallium (lit) (Ga~). Ga~ having undergone phase I human clinical trials} provides an ideal example of an insoluble drug molecule whilst allowing for direct comparison to the AlQ3 nanoparticles. Characterisation confirms the formation of nanoparticles suitable for passive disease targeting with the potential for extended blood circulation. Toxicity testing against a human placental in vitro cell model shows significant toxicity to the cell membrane} metabolism} nucleus and viability. Confocal experiments confirm the GaQ3 nanoparticles follow the same intracellular pathway as the AlQ3 nanoparticles elucidating further the mechanism of toxicity.

It is an offence to drive a car whilst impaired due to use of a psychoactive substance. The incidence of illegal drug use has escalated in recent years, and this escalation is also represented in the driving population.. At present there is no objective tool which can be used at the roadside to assess the drug induced performance impairment of drivers suspected of illegal drug intoxication. A literature analysis highlighted that empirical investigation into the cognitive and psychomotor effects of illegal drug use on aspects of performance related to car driving was scarce. By drawing together empirical and epidemiological evidence it is possible to conclude that driver impairment, as a consequence of illegal drug use poses a significant threat to traffic safety. Psychopharmacology is based on the premise that drugs affect behaviour, and that this change in behaviour is measurable. Behaviour is assessed through the use of repeated psychometric assessment in the laboratory. The aim of this thesis was to demonstrate that by using a portable device it was possible to reliably assess drug induced performance impairment in field conditions, with just one set of assessments.

The failure of antibiotics to treat infections caused by multi-drug resistant bacteria is a significant problem in the field of antimicrobial chemotherapy. The characterisation and development of antibacterial agents displaying novel modes of action (MOAs) or the modification of existing antibiotic scaffolds may address this problem. This study therefore sought to identify antibiotic candidates, establishing their antibacterial activity, bacterial specificity, MOA and propensity for resistance development. From nearly half a million compounds which were screened in silica against RNA polymerase (RNAP), D-alanine: D-alanine ligase and peptidoglycan transglycosylases, no inhibitors with specific activity against their target were identified, which highlights the difficulties of developing novel antibacterial agents. However, targeted inhibition of the cell envelope and RNAP were observed for the type B lantibiotic derivative NVB353 and corallopyronin A, respectively. The former may show greater promise as a chemotherapeutic candidate, due to lower propensity for resistance development. In addition, a number of compounds which appear to damage the bacterial cell membrane specifically were identified, and which may be suitable for treatment of persistent bacterial infections. Transcriptional profiling of Staphylococcus aureus treated with a panel of known membrane damagers was also used to identify upregulafed genes which might be potential candidates for future development of biosensors solely responsive to membrane damage. These biosensors could be used to eliminate compounds which are likely to cause non-specific toxic side effects if administered to humans, but may also identify membrane damaging agents that could be developed for clinical use should they show bacterial specificity.

Recently human liver microsomes have been found to underpredict in vivo human intrinsic clearance (CLint); this has led to an increase in the use of fresh and cryopreserved human hepatocytes as an alternative in vitro system for the prediction of in vivo human CLint. The aim of the work in this thesis was to assess the utility of human hepatocytes for the prediction of in vivo drug metabolic clearance. It was found that fresh and cryopreserved human hepatocytes also underpredict in vivo human CLint (6 and 6 fold respectively). The effect of including the binding to the in vitro system in the clearance prediction was assessed and even though the prediction improved (from 6 fold to 4 fold) the underprediction remained. Investigations were undertaken to explain this underprediction. These involved investigating the metabolism of the probe substrates, tolbutamide and dextromethorphan in different incubation vessels. It was found that there was no significant difference in the CLint of Tolbutamide between 96 well and 24 well plates. The effect of using a preservation solution for the transport of fresh liver tissue was also investigated. Two probe substrates were investigated, namely Midazolam and Clonazepam. It was found that the CLmax of Midazolam in 2 out of the 3 rat livers preserved for one hour in a preservation solution was reduced 2 fold when compared to the CLmax freshly isolated rat hepatocytes. The activity of the CYP3A4 enzyme over the course of a typical metabolism incubation period was also assessed. Studies in cryopreserved human hepatocytes indicated that there was an exponential loss of the CYP3A4 enzyme over the incubation period. The activity of the CYP3A4 enzyme in human liver microsomes remained constant for the first 60 minutes of the incubation. In the case of terfenadine the clearance when measured by the substrate depletion approach was observed to have a biphasic profile in cryopreserved human hepatocytes. This biphasic profile was explained by the loss of the activity of the CYP3A4 enzyme over the incubation period of the assay in cryopreserved hepatocytes. When the CLint was adjusted for the loss of enzyme activity the underprediction was reduced 2 fold. The rate of enzyme activity loss was not apparent in cryopreserved hepatocytes incubated in WME. This may be due to the presence of essential amino acids in WME that are essential for glutathione maintenance in the cell and subsequent protection of the enzyme from reactive oxygen species attack. The underprediction observed in human hepatocytes may be explained by one or a number of the above factors but it most likely that the underprediction is due to a combination of each of the above factors thus efforts to improve the underprediction requires further investigation.

Pt-based drugs are important cytotoxic agents that are used in the chemotherapeutic regimes of ~50% of all cancer patients. However, the efficacy of these drugs is often limited by drug toxicity and tumour resistance. Determination of the cellular pharmacokinetics and pharmacodynamics of Pt-drugs is important for understanding their molecular mechanisms of action and toxicity, and may be used, therefore, to predict the outcome of the treatment. ICP-MS is the most sensitive technique for the determination of Pt in biological samples and can offer robust, fast and accurate quantitations for studying pharmacokinetics and pharmacodynamics of Pt-drugs in patients. This thesis describes the development of a set of ICP-MS based assays for the determination of Pt-DNA adducts and Pt sub-cellular distribution in leukocytes of cancer patients and human cancer cell lines following treatment with Pt-based chemotherapy. It is ultimately aimed to use these assays in the clinic to predict the effectiveness and toxicity of Pt-based chemotherapy in individual patients, and offer those who would respond to the treatment personalised drug doses. Alternatively, patients who would not benefit from these drugs would be offered other forms of treatment. Pt DNA adduct formation was determined in leukocytes from patients undergoing Pt-based chemotherapy demonstrating significant inter-patient variability and excellent reproducibility of the assay. The sensitivity of the technique enabled quantitation of as little as 0.2 Pt adducts per 106 nucleotides using 10 µg of patient DNA. It was shown that Pt/P ratio was robust against DNA matrix effects, and was considered more reliable approach, with Eu as internal standard, for estimating Pt adducts per nucleotide compared to using Pt data in combination with DNA concentration measured by UV. Comparison of in vivo Pt-DNA adduct formation with the patients clinical notes suggested possible correlation between the adduct formation in leukocytes and toxicity. Speciation methods employing HPLC with complementary ICP-MS and ESI-Ion Trap-MS detection were developed and used for characterisation of oxaliplatin bi-functional adducts with mono-nucleotides and di-nucleotides. Further, a fast and sensitive LC-ICP-MS assay was developed and used for the quantification of oxaliplatin GG intra-strand adducts in human cancer cell lines. The assay, which has a detection limit of 0.22 Pt adduct per 106 nucleotides based on a 10 μg DNA sample, is suitable for in vivo assessment of the adducts in patients undergoing oxaliplatin chemotherapy. Combining the ICP-MS quantitation with a cell fractionation procedure allowed, for the first time, the detailed quantitation of entire sub-cellular Pt-drug partitioning in patient leukocytes in vivo, and in human cancer cell lines in vitro, following exposure to variety of Pt-drugs. The studies showed that Pt broadly follows the total protein content of the individual sub-cellular compartments with the majority being scavenged in the cytosol compartment.

The ability to predict the reactivity and selectivity of cytochrome P450 en- zymes using computational methods has the potential to reduce the number of late- stage failures in the drug discovery process. The antiinfiammatory drug diclofenac and anticancer therapy tamoxifen provide interesting test-cases for in silica predic- tion of selectivity in P450-mediated drug metabolism because the CYP2C9 enzyme selectively aromatically hydroxylates diclofenac at the 4'-position and tamoxifen at the 4-position, whereas other P450 isozymes produce 5-hydroxydiclofenac and 4' - hydroxytamoxifen. Molecular Dynamics (MD) simulations have been performed to identify the key residues involved in the orientation of diclofenac and tamoxifen in the CYP2C9 active site. The MD simulations were also used to assess the flexibility of different regions of the enzyme. Combined quantum mechanical/molecular mechanical (QM/MM) methods have been applied to calculating the energy barriers to formation of 4'- and 5- hydroxy- diclofenac in CYP2C9. The calculated energy barrier to 4'-hydroxylation was con- sistent with experimental kcat values but the energy barrier to 5-hydroxylation was found to be lower than that to 4'-hydroxylation due to formation of stabilising internal hydrogen bond in the 5-hydroxy transition state. It was found that the orienting effect of a hydrogen bonding interaction between the Argl08 residue of CYP2C9 and diclofenac causes the selectivity for 4'-hydroxylation, and that this in- teraction is also responsible for preventing formation of an internal hydrogen bond in the 4'-hydroxylation transition state. QM/MM methods were used to model the 4- and 4'-hydroxylation of tamoxifen by CYP2C9. The energy barriers to each reaction were found to be similar and so it is likely that the relative binding-free energies of 4- and 4'- reactive orientations is responsible for the observed selectivity for 4-hydroxylation of tamoxifen by CYP2C9.

Mu-opioid receptor (MOPr) agonists such as morphine are amongst the most important therapies for the acute relief of moderate to severe pain. However, their clinical efficacy is often compromised due to considerable inter-individual variability, the associated side effects and the development of tolerance and dependence. Numerous molecular mechanisms have been proposed to mediate tolerance to opioids. Although a definitive mechanism remains to be elucidated, desensitization, internalization and resensitization of the MOPr have all been repeatedly implicated as contributing factors. Like most G protein-coupled receptors (GPCRs), MOPr activity is tightly regulated by rapid desensitization and resensitization in response to activation by high efficacy opioid agonists. In contrast, morphine is typically reported to induce little or no acute desensitization or internalization. Howev~r, repeated or chronic administration of morphine results in considerable tolerance that is often greater than that induced by highly efficacious opioids. Despite intensive research into the regulation of the MOPr at the molecular, cellular and systems levels, much still remains to be learned about the mechanisms that underlie tolerance to opioids. The aim of this thesis was to investigate agonist-induced MOPr trafficking at the molecular and cellular level and its relationship to cellular morphine tolerance.

The epithelium of the small intestine provides a physical and biochemical banner that restricts the transcellular absorption of orally dosed drugs into the bloodstream through mechanisms including P-glycoprotein (Pgp) and Cytochrome P450 3A4 (CYP3A4). P-glycoprotein is constitutively expressed on the apical membrane of the intestinal lumen where it can act to limit xenobiotic absorption by excreting drugs that have reached the cell interior back into the intestinal lumen. CYP3A4, the most abundant phase one drug metabolising enzyme in humans, can metabolise over fifty percent of commercially available drugs and is located apically within the enterocytes where it can restrict bioavailability by metabolising xenobiotics.