Hepatitis C virus is an infectious disease affecting millions of people worldwide and causing chronic liver disease. The current standard of care is not only long but causes numerous side effects. Due to incomplete virological response and poor tolerability, only 50% of the patients are cured, with variability by genotype. Despite the development of non-enzymatic viral protein inhibitors, new therapies target mainly enzymes responsible for viral replication or translation. Being commonly used for antiviral and anticancer therapy, nucleosides analogues have played an important role as anti-HCV agents. Despite their potency and selectivity, nucleoside analogues appear to be poor substrates for metabolic enzymes. In particular, the first essential phosphorylation step is often rate-limiting thus, resulting in poor bioactivation to the active triphosphate form. Hence, monophosphate prodrug strategies have been applied to efficiently deliver intracellularly the key monophosphate derivatives. Such strategies have been successfully used for anti-HCV therapy and the phosphoramidate ProTide INX-08189, discovered in our lab, is one such example. Aiming at developing back-up molecules of INX-08189, we report in the present work, the synthetic strategies to obtain several modified β-2’-C-methyl-6-O-methyl guanosine and other modified β-2’-C-methyl purine nucleoside analogues. The phosphoramidate ProTide approach and the phosphorodiamidate approach were applied to these modified nucleosides. In-vitro, and sometimes in-vivo evaluation against HCV replication is reported, and the mechanism of bioactivation to their corresponding monophosphate species is discussed. Enzymatic experiments using carboxypeptidase Y and Huh-7 cell lysates were carried out to investigate the release of the monophosphate forms. We also investigated the hydrolysis of the 6-O-methyl group at the nucleoside level with adenosine deaminase enzyme, and at the monophosphate level using molecular docking in adenosine deaminase like protein-1. Eventually, the intracellular putative mechanism of activation of the ProTides was studied using molecular modeling with cathepsin A enzyme and human Hint-1 phosphoramidase.

Healthcare associated infections (HCAIs) are a large and growing problem. Bacterial infections of patients and on the medical devices used to treat them represent a significant source of morbidity and mortality. There is also a significant economical impact to the healthcare system attributed to HCAIs. While bacterial infections per se are not a novel problem, the discovery of an adherent polymicrobial phenotype called a biofilm is. A biofilm is defined by its structure and the community of bacteria therein. This study investigated bacteria biofilms in a number of pertinent clinical scenarios. To achieve this, samples were taken from five different but related clinical areas where biofilms are known to infect or are suspected to, namely endotracheal tubes, tracheostomy tubes, burn wounds, chronic wounds and chronic wound dressings. Samples were analysed using microbiological and molecular analysis techniques, the latter included polymerase chain reactions, species-specific PCR and denaturing gradient gel electrophoresis to assess microbial diversity. Fluorescent in-situ hybridization was used subsequently to analyse species orientation and biofilm structure within the biofilm. This study showed a diverse bacterial population in all the samples, with the presence of oral biota in the ETT specimens, changing to commensal bacteria over time. Large threedimensional biofilm structures were present in the specimens confirming the presence of biofilms, and within one of the chronic wound dressings where a complex biofilm was visible within the matrix of the dressing itself. These findings have considerable significance clinically, not only in demonstrating the need for biofilm targeted diagnostic techniques, but also in highlighting the need for specific biofilm treatment modalities in critical care, burn services and chronic wound management.

The work described in this thesis is concerned with the design and evaluation of microsphere-based systems for drug delivery into the colon. In initial experiments, techniques were devised for the preparation of microspheres from two sustained-release acrylic polymers, Eudragits RL and RS, using emulsification-solvent evaporation techniques. For Eudragit RS microspheres containing the drug 5-aminosalicylic acid, the rate of drug release could be controlled by the type and concentration of surfactant used for preparation. Consequently, formulations could be produced which released encapsulated drug instantaneously or over many hours. The surfactants may have been altering the structure of the microsphere drug-polymer matrix. Two novel analytical techniques were employed to characterise Eudragit RS microspheres containing sulphasalazine. Fourier transform-Raman spectroscopy was successfully used as a non-destructive method for qualitative and quantitative microsphere characterisation. The technique provided good agreement with a UVspectrophotometric method in quantifying the amount of drug in microsphere samples. X-ray photoelectron spectroscopy was used to estimate the concentration of sulphasalazine at the microsphere surface for samples produced with or without the use of surfactant. Across a wide range of microsphere drug loadings, the surface drug content remained remarkably constant, but was consistently lower in the samples produced using surfactant. In a parallel programme of work, using gamma scintigraphy, the transit rate of different sizes of radiolabelled materials through the human colon was investigated to determine whether there was an optimal size to maximise colon residence. There was no clear evidence of any difference in the colon residence time of 0.2 mm particles, 5 mm tablets, or 8.4 mm tablets, under normal conditions and during accelerated transit. In healthy subjects, 50% of a dose of 0.2 mm particles resided in the ascending colon for an average of 11 hours. Finally, an in vivo biopharmaceutical evaluation of sulphapyridine-containing Eudragit RS microspheres in the human colon was undertaken. For this study, a neutron activation technique was developed for microsphere radiolabelling. Microspheres could be successfully radiolabelled by incorporation of samarium oxide followed by neutron irradiation. However, it was necessary to minimise the period of irradiation and the amount of incorporated samarium oxide, since high levels of both were found to adversely affect microsphere performance. The in vivo investigation revealed that the colonic bioavailability of sustained-release microencapsulated sulphapyridine was less than 50% of unencapsulated sulphapyridine powder. This shortfall was possibly due to an interaction of the drug with colonic bacteria or in vitro/in vivo differences in microsphere drug release characteristics.

Despite intensive research, cancer remains one of the major causes of worldwide morbidity. It is widely believed, however, that if currently available anti-cancer drugs could be delivered specifically to tumours then the disease would have been mastered. The delivery of prodrug converting enzymes by clostridial spores specifically to the anoxic centres of tumours is one potential delivery mechanism. This is due to the extreme selectivity of spores to germinate solely in the hypoxic regions of tumours. Once germinated, the expression of a prodrug converting enzyme converts a systemical1y administered prodrug to a highly toxic drug only in the tumour. Previous studies using Clostridium acetobutylicum and Clostridium beijerinckii as the delivery vehicle highlighted that prodrug converting enzyme expression is only found in tumours. However, no significant anti-tumour affect was observed. Two possible reasons were evolved. Firstly, expression of the prodrug converting enzyme may be low, and/or, secondly, the tumours may not be colonised sufficiently to promote an antitumour effect. Preliminary studies identified that Clostridium sporogenes NCIMB 10696 may represent a more suitable host. Higher spore titres could be prepared and, once administered, higher cell counts are found in the colonised tumours. Prodrug converting enzymes with improved kinetics over pre-existing enzymes have also been identified. Once effective gene transfer systems and expression systems had been developed, suitably high levels of several different prodrug converting enzymes, in particular nitroreductases, were obtained. Initial in vivo studies on one of the early recombinant strains identified a definite anti-tumour effect. Since those initial studies, further improvements to expression have been made. It is hoped that a more significant anti-tumour affect would result from using these improved strains. It is the ultimate aim of CDEPT to have the prodrug converting enzymes integrated into the host genome so as to negate the use of antibiotics. Towards this, studies on the use of both classical and novel integrative technologies have been investigated.

The aim of this work was to enhance the immune responses to ovalbumin (OVA) following its oral administration, by the association of the protein with colloidal carriers, which may protect the protein from degradation in the gastrointestinal tract and/or facilitate its uptake across the intestine. An enzyme linked immunosorbent assay (ELISA) was established for the determination of rat anti-OVA antibodies and an immunisation protocol was established to induce a statistically significant salivary antibody response to OVA in the rat. A radioimmunoassay for the determination of rat anti-OVA antibodies was also established, to confirm the ELISA results. Methods were established to determine the extent of incorporation or adsorption of OVA into or onto the colloidal carrier formulations. OVA was incorporated into liposomes and polyacrylamide microparticles, and adsorbed to poly 2-butylcyanoacrylate particles, and gastrically intubated into separate groups of experimental rats. The primary and memory immune responses, both sera and saliva, were compared for each formulation with suitable control and blank groups. All colloidal carriers induced enhanced immune responses to OVA following oral administration in the rat, when compared with the respective control group responses. However, the enhancement for the liposomal group was not statistically significant when assessed in an Unpaired Student 't' test. The effect of particle size on the immune responses was assessed by the oral administration of 100 nm and 3pm poly 2-butylcyanoacrylate particles with adsorbed OVA. An electron microscopy study was undertaken with gold labelled poly 2-butylcyanoacrylate particles in an attempt to demonstrate the uptake of particles by M-cells overlying the Peyers' patches in the rat intestine.

The prevalence of obesity worldwide has more than doubled since 1980. The World Health Organisation (WHO) estimates that more than one in ten adults in the global population is obese. Cardiovascular and metabolic health can be improved with moderate weight loss; losses of 5%–10% have been found to improve conditions such as diabetes, hypertension and cholesterol and low-density lipoprotein (LDL) levels. Within the UK, a number of weight management programmes that depend on lifestyle intervention (tier 2) and others that supplement this with drug therapy (tier 3) and surgery (tier 4) are available. The guidelines produced by the Scottish Intercollegiate Guideline Network (SIGN) advocate that weight management programmes address changes to diet, physical activity and behaviour. For patients with a body mass index (BMI) ≥30 kg/m2 or ≥28 kg/m2 in patients with comorbidities, orlistat can be considered as a drug intervention on a case-by-case basis following a full risk and benefit assessment. The objective of the Glasgow and Clyde Weight Management Service (GCWMS), a specialist weight-loss programme, is for patients to lose at least 5 kg. There are a number of metabolic disorders that are associated with obesity. One such disorder is type 2 diabetes mellitus, where weight loss is a standard recommendation to improve blood glucose control. Randomised controlled trials (RCTs) of orlistat indicate that the drug is effective in promoting weight loss and improving metabolic control for those patients with the comorbidity of type 2 diabetes and obesity. There are several different groups of anti-diabetic drugs that can be used to manage diabetes. The effects of the different medications on body weight are considerable. Some, such as biguanides (metformin), dipeptidyl peptidase-4 inhibitors (DPP-IV), Glucagon-like peptide-1 agonist (GLP-1) and sodium-glucose co-transporter-2 inhibitors (SGLT2), either have no effect on weight or can cause weight loss. Others, such as sulfonylureas (SUs) and thiazolidinediones (TZDs) can lead to weight gain. This thesis explores the impact of lifestyle interventions in weight management services, and the impact of drug interventions, on weight loss and glycaemic control. It is supported by the results of five complementary studies that reviewed the effect of orlistat on type 2 diabetes and assessed the impact of the prescription patterns of anti-diabetic drugs in addition to the effects of these pharmacological interventions on weight change in comorbid patients. The first aim of this thesis is to review the evidence of the effects of orlistat on diabetic outcomes. The second aim is to evaluate the lifestyle interventions, and phase 2 of the GCWMS. Finally, the third aim is to determine the prescribing patterns of anti-diabetic drugs, and to observe the association between anti-diabetic medications and weight change. This thesis addresses the following objectives: 1. To undertake a systematic review and meta-analysis of published studies in order to review the evidence of the effects of orlistat on weight loss, specifically concerning glycosylated haemoglobin (HbA1c) and fasting plasma glucose (FPG), using the Cochrane review methodology; 2. To investigate the proportion of patients losing 5 kg of weight, commencing from their entry into the GCWMS programme, until the end of the lifestyle phase of treatment, for individuals of different ages, genders, and socioeconomic groups; 3. To study the proportion of patients losing 5 kg of weight, commencing from their entry into the GCWMS programme, until the end of phase 2, with the three different interventions of orlistat, low-calorie diet (LDL), and further weight loss (FWL); 4. To investigate the proportion of patients referred to the GCWMS on weight-neutral, mixed, and weight-gaining anti-diabetic medications; 5. To investigate the effect of baseline anti-diabetic medications on weight change for patients within a weight management programme. Chapter 2 presents the first study, which was a systemic review that considered the evidence collected in RCTs on the efficacy of orlistat for type 2 diabetes and weight loss. The effects were considered at the biochemical level and included the levels of glycosylated haemoglobin (HbA1c) and fasting plasma glucose (FPG) in people with overweight and obesity. The results, collected from 2,802 participants in 12 trials, were combined into a meta-analysis. The overall finding was that a combination of orlistat and lifestyle intervention yielded superior results. When the results were compared, it was evident that patients who are overweight or obese who were subjected to combined lifestyle and drug intervention lost more weight and had better glycaemic control than patients who were subjected to lifestyle interventions only. Chapter 3 presents the second study which appraised the effectiveness of a real-life NHS lifestyle weight management intervention in reducing body weight by ≥5 kg. The study followed 23,650 patients referred to the GCWMS, of whom 7,329 attended at least two lifestyle intervention sessions. Those individuals had either a BMI of ≥30 kg/m2, with obesity-related comorbidities, or a BMI of ≥35 kg/m2 and were aged ≥18 years. The lifestyle interventions included a combination of a 600 kcal deficit diet, exercise, and behavioural changes. 30% of the overall group succeeded in losing ≥5 kg. Out of those who completed the programme, however, a considerably higher number (46%) lost ≥5 kg. The greatest losers were men, those aged ≥40 years, those with a BMI ≥50 kg/m2, and those from areas that are more affluent. Chapter 4 presents the third study which focused on patients who lost ≥5 kg in phase 2 of the treatment provided by GCWMS which comprised a low-calorie diet (LCD), orlistat 120 mg, three times a day, or further weight loss (FWL). Participants on LCD were prescribed a 1,200 or 1,500 calorie plan; however, those on FWL repeated the lifestyle phase. There were 3,262 participants who attended at least two sessions in phase 2; these were divided into three categories: 536 who took orlistat, 1,043 who followed a LCD and 1,683 who were selected FWL. By the end of phase 2, the levels of success in terms of weight loss across the groups varied from 31% of participants in the orlistat group to 22% of participants in the LCD group and 83% of participants in the FWL group who lost ≥5 kg. Chapter 5 presents the fourth study, which evaluated the pattern of anti-diabetic drug prescriptions for comorbid patients referred to the GCWMS. The study also looked at the proportion of patients who were referred prior to and after the publication of updated SIGN guidelines for the prescription of anti-diabetic medication. In total, the study enrolled 3,063 participants who received anti-diabetic medications, of whom 47.8% received weight-neutral medications, 39.4% had mixed-effect medications and 12.7% took weight-gaining drugs. Prior to the publication of the SIGN guidelines, 11.6% of participants were on weight-gaining drugs, a proportion that did not change significantly one year after the release of the guidelines. Weight-neutral drugs were more commonly prescribed to women, those with a higher BMI and young people. No relationship was observed between the Scottish Index of Multiple Deprivation (SIMD) and anti-diabetic drug prescriptions. Weight-gaining drugs such as SUs and TZDs were more commonly prescribed to older patients and those with lower BMIs. Chapter 6 presents the fifth and final study, which investigated the effect on body weight of anti-diabetic medications in 998 participants following the lifestyle phase of the GCWMS. By the end of the programme, patients who were on weight-neutral anti-diabetic drugs achieved a mean weight change of -3.3 kg (95% confidence interval [CI]: -3.8 to -2.9 kg) and those on weight-gaining drugs achieved a mean weight change of -2.5 kg (95% CI: -3.2 to -1.8 kg), p =0.05. / Among those who completed the programme, the difference was statistically significant (p =0.005). The association between weight change and anti-diabetic drug type was not explained by differences in sex, initial BMI or age. To conclude, there was a clinically and statistically significant change in weight, HbA1c and FPG in patients with obesity and type 2 diabetes who used orlistat. Of the patients following the GCWMS lifestyle phase, less than 50% succeeded in losing at least 5 kg, with patients who completed the programme being more successful. Participants who lost weight in the lifestyle phase were selected for FWL and experienced the greatest weight loss by the end of phase 2. Those who were unsuccessful in losing 5 kg through the lifestyle programme, were offered orlistat and LCD. The large sample size increased the precision of the results, while the stratification for potential confounding factors increased the study’s validity. A higher proportion of patients were prescribed weight-neutral medications, compared with mixed and weight-gaining anti-diabetic medications. The proportion of patients on weight-gaining diabetes drugs referred to the GCWMS did not alter appreciably following the release of the SIGN guidelines. By the end of the lifestyle treatment phase, patients receiving weight-neutral drugs (metformin, DPP-IV, GLP-1, and SGLT2) were more successful in losing weight than those receiving weight-gaining drugs (SUs, TZDs, and any combination including insulin). The main recommendation from this research are, that further studies are carried out to better establish the best timing of use of orlistat within a weight management programme, that the intensity of phase 2 of the GCWMS is increased, and that prescribers take account of a patient’s current BMI prior when prescribing anti-diabetic medication, especially when recommending weight loss and referring to a weight management programme.

Schizophrenia is highly heritable, indicating that a large proportion of one’s susceptibility to developing the disorder is attributable to genetics. Recent large-scale genomic studies have revealed that genetic variants in patients with schizophrenia affect genes involved in synaptic plasticity processes, which are required for learning and memory, including genes encoding protein complexes associated with the NMDA receptor and the postsynaptic density. Further evidence suggests that associative learning may be particularly affected, although it is unclear which components of this cognitive process are implicated in schizophrenia. The present studies investigated the relationship between particular phases of associative learning, represented by the consolidation, retrieval and extinction of contextual fear memory in rats, with genetic variants, psychoactive drugs and postsynaptic density proteins associated with schizophrenia. I tested associative learning-related gene expression datasets for enrichment in genetic copy number variants from a large cohort of patients with schizophrenia and demonstrated that only genes associated with extinction learning are enriched in patient variants (Chapter 3). I report that fear extinction in rats was impaired by administration of the NMDA antagonist and psychotomimetic, ketamine (Chapter 4). The expression of activity- induced, postsynaptic density products of the Homer1 gene, which has been linked to psychiatric disease, was differentially regulated in specific hippocampal subregions following extinction learning (Chapter 5), and the effect of a partial knockdown of these genes during different phases of associative learning was investigated (Chapter 6). These results build on clinical studies linking abnormalities in associative and, specifically, extinction learning with schizophrenia and support the notion that genetic variants associated with the disorder impact particular cognitive domains. My findings are consistent with the theory that altered inhibitory-type learning processes contribute to the manifestation of schizophrenia.

Triple-negative breast cancer (TNBC) is an aggressive cancer subtype that displays poor prognosis due to a lack of targeted therapies and an early pattern of spread. Recent evidence also points to a correlation between cancer “stem-like” cells (CSCs) and the inherently aggressive traits of TNBC. As such, targeting signalling pathways which support metastasis and CSC populations may represent an important therapeutic strategy to treat these tumours and improve current patient outcomes. The non-receptor tyrosine kinase FAK (focal adhesion kinase) is known to influence cancer development and progression, with its upregulation common in several cancer types. Indeed, FAK can regulate various cellular processes associated with disease progression including, cell survival, migration and stem-like behaviours. Therefore, we explored the influence of FAK in TNBC cells and the potential benefit of its targeting in this subtype. Whilst assessment of FAK expression and activity across a panel of breast cancer cell lines representing the major clinical subtypes revealed that FAK was not significantly augmented in MDA-MB-231 cells (model of TNBC) versus other models, MDA-MB-231 cells displayed a FAK-dependent migratory and invasive behaviour involving FAK-mediated activation of Akt and STAT3. These observations also extended to cell proliferation, with pharmacological or genetic FAK inhibition leading to perturbed cell cycle progression. Whilst FAK did not contribute to the maintenance of a CSC subpopulation, FAK was necessary for their anoikis resistance and mammosphere self-renewal, the latter regulated by FAK-dependent modulation of β-catenin through GSK3β and interaction between the FAK/Wnt signalling pathways. Using computational modelling, several novel FAK inhibitors that targeted FAK kinase-independent scaffolding function were developed and screened to assess in vitro efficacy in TNBC cells. Of all 45 compounds, ‘compound 9’ showed significantly improved ability to reduce cell proliferation and migration versus the lead compound, chloropyramine. As expected, this agent had little effect of FAK phosphorylation but appeared to reduce focal-adhesion targeting and subcellular distribution of FAK and significantly inhibited cell migration and growth. Our in vitro data support a case for FAK as a promising therapeutic target in TNBC with an ability to suppress both tumorigenic events and those associated with metastasis. Targeting FAK scaffolding function may represent a novel approach to developing FAK inhibitors that can circumvent resistance traditionally associated with kinase inhibitors.

Human inflammatory/innate immune responses lie at the core of resistance to infectious disease and determine the nature of pathophysiological reactions of hematopoietic cells like leukaemia and allergy. The crucial step in these events is the ability of immune cells to function properly, which depends on their adaptation to stress caused by pro-inflammatory stimulation. The mechanisms underlying this crucial biochemical dogma, and its role in normal and pathological cross-links between immune cells, are not well understood. This PhD programme was devoted to investigation of these important problems. We found that the inflammatory mediator interleukin 1 beta (IL-1β), derived from human innate immune cells, triggers production of the major hematopoietic growth factor, the stem cell factor (SCF) in MCF-7 human epithelial cells. This process is controlled by the hypoxia-inducible factor 1 (HIF-1) transcription complex, which regulates cellular adaptation to inflammatory/hypoxic stress by promoting angiogenesis and glycolytic degradation of the glucose. Translational mechanism, which is majorly dependent on the mammalian target of rapamycin (mTOR) kinase pathway underlies IL-1β-induced HIF-1 accumulation and also contributes to SCF biosynthesis. The effect is applicable in both in vitro and in vivo systems. Further experiments demonstrated the involvement of this biosynthetic mechanism in the differential control of normal and pathological functions of inflammatory cells including monocytes, basophils and mast cells. Our results also demonstrated possible biochemical mechanisms regarding the cross-talk between inflammation and SCF-dependent blood cancer (leukaemia), which remains a serious medical burden worldwide. Finally, the pathways investigated could be further considered as potential therapeutic targets for pharmacological correction of human inflammatory reactions and treating cancer/leukaemia by classic and principally novel approaches, such as utilisation of gold nanoparticles.

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