Nicotinamide adenine dinucleotide (β-NAD+) is a primary metabolite involved in fundamental biological processes. Its molecular structure with characteristic functional groups, such as the quaternary nitrogen of the nicotinamide ring, and the two high-energy pyrophosphate and nicotinamide N-glycosidic bonds, allows it to undergo different reactions depending on the reactive moiety. Well known as a redox substrate owing to the redox properties of the nicotinamide ring, β-NAD+ is also fundamental as a substrate of NAD+-consuming enzymes that cleave either high-energy bonds to catalyse their reactions. In this study, a panel of novel adenine-modified NAD+ derivatives was synthesized and biologically evaluated against different NAD+-consuming enzymes. The synthesis of NAD+ derivatives, modified in position 2, 6 or 8 of the adenine ring with aryl/heteroaryl groups, was accomplished by Suzuki-Miyaura cross-couplings. Their biological activity as inhibitors and/or non-natural substrates was assessed against a selected range of NAD+-consuming enzymes. The fluorescence of 8-aryl/heteroaryl NAD+ derivatives allowed their use as biochemical probes for the development of continuous biochemical assays to monitor NAD+-consuming enzyme activities. The introduction of different substituents in position 8 on the adenine ring allowed the modulation of their fluorescence, resulting in the development of more sensitive and alternative probes compared to the known fluorophore ε-NAD+. The different substitutions introduced on the adenine ring also allowed us to probe the active site of an NAD+-dependent bacterial DNA ligase. The selective activity of 8-aryl/heteroaryl NAD+ derivatives against different NAD+-consuming enzymes offers excellent opportunities for their application as tool compounds in in-vitro/in-vivo studies, and as inhibitor templates for drug discovery.

Cancer is uncontrolled cellular proliferation devoid of normal biological regulatory mechanisms. Therapeutic treatment of cancers relies on overcoming such proliferation, to allow cytotoxic destruction of cancer cells. Many of the recent therapeutic breakthroughs have been in the targeting of various blood cancers: eg imatinib has effectively eradicated chronic myeloid leukaemia (CML) morbidity. However, acute myeloid leukaemia (AML) remains essentially incurable for the vast majority of patients. The current gold-standard treatment for AML has remained relatively unchanged for decades, consisting of the antimetabolite cytarabine (ara-C) and the anthracycline cytotoxic antibiotic daunorubicin (DNR). The cytoprotective gene haem oxygenase-1 (HO-1) has been found regulated in various forms of cancers (including AML) and implicated in chemotherapeutic drug-resistance. HO-1 protects AML cells from induced apoptosis via its transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2). We show a role for HO-1 in regulating apoptosis in AML cells in response to ara-C and DNR. HO-1 expression was increased in response to both cytotoxic agents. Upon micro RNA (miRNA) silencing of HO-1 expression, both ara-C and DNR stimulated greater apoptotic responses in these silenced AML cells. A concurrent induction in reactive oxygen species (ROS) generation was observed. Studies with ara-C-resistant AML cell lines (THP1araC(1)) showed there to be significantly suppressed levels of Nrf2 and HO-1. A miRNA screen in these resistant cells revealed reduced basal miR-196a expression. One of miR-196a’s targets is the Nrf2-inhibitory protein Bach1 (BTB and CNC homology 1), which was found to be elevated in these cells. Exogenous replacement of miR-196a with the introduction of a miR-mimic, suppressed the Bach1 overexpression, elevated HO-1 expression, and reintroduced sensitivity towards ara-C. These findings suggest HO-1 inhibition in conjunction with chemotherapy would improve the number of cases who reach complete remission (CR).

The in-situ analysis of pharmaceutical solid dosage forms films is of great interest to pharmaceutical research, especially with the drive towards continuous processing compared to batch processing requiring either in-line or on-line analysis of pharmaceuticals. The size and shape of many solid dosage forms impedes their in-situ analysis, particularly when using thermal analysis techniques such as differential scanning calorimetry (DSC). Thermal probe methods, which combine atomic force microscopy (AFM) with thermal analysis, permits imaging and characterisation of solid dosage forms in-situ. The goal of this thesis is to perform the in-situ analysis of film coated minitablets through the use of thermal probe methods. Minitablets, which are small diameter tablets, were coated with Opadry I, Opadry II or Surelease, and blends of Surelease and Opadry I and Surelease and Opadry II. In addition the same systems were prepared as cast free films to ascertain differences resulting from the two methods of producing the films. Distinct morphologies were observed between the film coated minitablets and cast free films, particularly for the polymer blends. Thermal analysis by modulated temperature DSC (MTDSC) and localised thermomechanical analysis (L-TMA) of film coated minitablets coated with polymer blends indicating miscibility. However the novel technique of heated tip tapping mode was employed, which was able to resolve separate domains of the polymer blends, which appeared as nanophases. Heated tip tapping mode was further employed to determine changes in the surface morphology of film coated minitablets which had undergone curing. It was successfully used to observe the further coalescence of Surelease and the alteration in the phase distribution of the polymer blends after film coated minitablets had been cured. Polymer films with incorporated pigments were also analysed by thermal probe methods with differences in pigment distribution being observed between film coated minitablets and cast free films. Tapping mode AFM was able to determine the location of pigment particles and the novel use of L-TMA performed in tapping mode allowed thermal analysis without changing AFM mode. Overall the use of AFM combined with thermal probes achieved the aim of performing in-situ analysis of polymer film coats; however further research to correlate in-vivo behaviour of film coats would be of value.

Patient nonadherence to medication harms patient outcomes and raises costs via wasted and unnecessary treatment (Osterberg and Blaschke, 2005). However, current adherence measures are far from optimal (Vitolins et al., 2000), and adherence enhancing interventions rarely successful (Haynes et al., 2008). This may be a reflection of inadequate patient targeting and adherence measurement. This thesis describes the development of questionnaires intended to be clinically useful by predicting patient risk of nonadherence. A scoping review with meta-analysis was undertaken to identify predictors objectively shown to be associated with nonadherence. Any pre-existing questionnaires to measure the selected predictors were identified via literature review. Pre-existing questionnaires incorporated were the Beliefs about Medicines Questionnaire (Horne et al., 1999), Perceived Stress Scale (Cohen et al., 1983), Patient Health Questionnaire (Kunik et al., 2007), and the Patient-Doctor Relationship Questionnaire (Van der Feltz-Cornelis et al., 2004). Novel items were developed to measure patient demographics, health literacy, mental health, risky health behaviours, beliefs about medicines, self-efficacy , social support, and access to medicines. These scales were incorporated into two novel questionnaires. The Patient and Lifestyle Scale (PALS), and the Wellbeing and Medications Scale (WAMS). A feasibility study was conducted with 16 patients at a GP surgery to identify limitations in research design and perform preliminary psychometric assessment. Issues with participant identification were highlighted, however, indications were that PALS and WAMS could be used to predict self-reported and prospective refill adherence. A practitioner focus group appraised the clinical utility of the questionnaires whilst acceptability and validity were assessed via six participant interviews. The PALS and WAMS were perceived to be potentially clinically useful and most items were considered acceptable. Findings also indicated that mental distress is associated with nonadherence and that long term adherence may depend more upon integrating medicines into every day habits than rational cost-benefits appraisals.

Lipid-based delivery systems offer many advantages on enhancing the bioavailability of protein/peptides. Gelucire 50/13 is a complex mixture of glycerides and PEG. It is mainly used in solid oral formulations for delivering small molecular weight drugs. The purpose of this project was to develop novel uses of Gelucire as a liquid crystalline-based gel-forming material for protein/peptide delivery. A thorough physical and mechanical characterisation of the gels (with and without lysozyme as a model protein) was conducted using a combination of analytical techniques including ATR-FTIR, DSC, relaxometry NMR, rheological and texture analyser, and imaging analyses (SEM, AFM, and cryo-TEM). The results demonstrated the sophisticated microstructures of the gels due to the formation of various liquid crystalline phases that change with the gel water content. The gels with low water contents are characterised by highly restricted diffusion of water molecules in the gels, while water-rich and lipid-rich phases are present in the gels with medium to high water contents. The ordered liquid crystalline structures with lipid-rich and water-rich domains provide excellent carrier properties for hosting proteins/peptides. The effect of water content on the microstructure, physical properties and in vitro performance of the gels prevails on other effects such as gel preparation method and protein incorporation. The wide range of microstructures of the gels enables the mucoadhesive properties and release profiles of lysozyme from the gels to be controlled. Highly stable disc-shaped nanoparticles were produced from the Gelucire gels using a single-step and solvent-free method without the addition of stabilisers. In vitro cell culture studies revealed high tolerance to and rapid uptake of the gel nanoparticles by Caco-2 cells. The good protein encapsulation efficiency and the retained biological activity of lysozyme indicates considerable potential for these nanoparticles to be a new class of safe, low-cost and effective carriers for protein/peptide delivery.

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This research set out to develop a framework to evaluate the consultation skills of healthcare practitioners undertaking medication-related consultations. A medication-related consultation framework would facilitate the teaching and evaluation of consultation skills and provide a structured format for feedback. Furthermore, it would allow to identify practitioners' learning needs in order to target areas for improvement. The aims of this research were (i) to develop a standardised framework outlining key competencies that should be undertaken in a medicationrelated consultation, (ii) to assess the framework's psychometric properties (validity, reliability), and (iii) to produce guidelines to accompany the framework to facilitate its use and educational impact. To achieve these aims the research was divided into three parts. The first was concerned with the generation of the framework competencies. A critical review of relevant healthcare consultation literature identified key components of patient-centred consultations. The second part involved the testing of the framework's psychometric properties. Face and content validity were explored using a systematic approach to gain views of experts in the field of practitionerpatient consultations, student pharmacists, 'expert patients' and a framework development panel. Discriminant validity, inter and intra-assessor reliability and internal consistency were investigated using data obtained from 150 assessments following the application of the framework by ten assessors to fifteen video-taped simulated consultations of varying quality (good, satisfactory, poor). Any issues which arose as a result of the assessors' use of the framework were collated and addressed in the guidelines developed in part three of the study. The final consultation framework consisted of forty-six key competencies divided into five main sections. These were (A) Introduction (6 items), (B) Data Collection & Problem Identification (15 items), (C) Actions & Solutions (8 items), (D) Closing (3 items), and (E) Consultation Behaviours (14 items). Appropriate adjustments were made following the initial systematic review to improve its face and content validity. Use of the framework resulted in the assessment of the quality of a consultation on three levels; a rating for each individual competency (1 =not at all to 4=very good), a global rating for each section (5-point scale with the middle and extreme points anchored by explicit descriptors) and an overall rating for the entire conSUltation (5-point scale, 1 =poor to 5=very good). Additional space for qualitative comments was provided. The framework was found to discriminate between the rating of consultations at the overall level, i.e. between good, satisfactory and poor (Kruskal Wallis Chi-square=12.5; df=2; p<0.01) and to have moderate to high inter-assessor reliability at this level (rho=0.49 to 0.76). Inter-assessor reliability was low to moderate on the global assessment level (rho=0.26 to 0.68) and consistently low on the individual competency level (rhosO.39). Intra-assessor reliability was found to be generally higher than inter-assessor reliability with high agreements on the overall level (rho=0.59-0.95) and moderate to high on the global level (rho=0.42 to 0.94). The agreements on the individual competency level were inconsistent and ranged from low to high (rhosO.39 to ;::0.70). The framework's internal consistency was found to be acceptable for each section as indicated by moderate to high positive correlations between individual competencies and the corresponding global rating (rho=0.40 to 0.94) and by satisfactory Cronbach's alpha coefficients (ranging from a=0.58 to 0.97). This framework meets key criteria necessary for a formative assessment instrument in that it possesses good face, content and discriminant validity. Whilst the framework demonstrated acceptable inter-assessor and intra-assessor (test-retest) reliability on the overall assessment level and moderate agreement on the global assessment level, this was not the case on the individual competency level. This is acceptable for instruments used for formative assessments where the emphasis is placed on the identification of a practitioner's relative strengths and weaknesses and where specific strategies for improvement are to be fed back to the practitioner. However, in summative assessments where 'pass' or 'fail' decisions about a candidate's performance are made, the possession of high validity and reliability at all assessment levels is important. Further work is needed to test whether the use of the specific guidelines developed to support the framework and additional assessor training improves the framework's reliability when used by multiple assessors. Additionally, further validation studies need to be undertaken.

The role of insulin-like growth binding-protein-3 (IGFBP-3) was investigated in primary cultures of hyperplastic prostate cells in the hope of identifying novel drug targets. Benign prostatic hyperplasia (BPH) is a disease of the ageing male, the aetiology of which is unknown. Growth factors, such as insulin-like growth factor (IGF) are implicated in stimulating the prostate growth which causes hyperplasia. IGFBP-3 is an inhibitory binding protein, which functions to sequester IGF away from its receptor, but also has an IGF-independent action once fragmented. Cellular proteases, including prostate specific antigen (PSA), fragment IGFBP-3, and the resulting fragments have actions independent of the intact protein. The inhibitory action of fragmented IGFBP-3 has been documented in prostate epithelial cell lines, but there are no reports of its action in stromal cells, which is investigated here. Initially, the endogenous production of IGFBP-3 by primary cultures of stroma and epithelium was investigated. RT-PCR showed expression of IGFBP-3 mRNA in both stroma and epithelium, and immunocytochemistry, and Western blotting on cell lysates, indicated IGFBP-3 protein production in both cell types. The localisation of IGFBP-3 in primary cultures was the same as that observed in sections of hyperplastic and malignant prostate tissue. Western blotting on stromal and epithelial conditioned medium showed fragmentation of endogenous IGFBP-3 by cellular proteases. Proteolysis of IGFBP-3 by PSA produced fragments of 22-25kDa and 15kDa. ELISAs showed a large differential in concentration of IGFBP-3 produced by primary stomal and epithelial cells which affected subsequent growth experiments using exogenous protein. Proliferation experiments showed a non-dose dependent inhibitory response to IGFBP-3 by epithelial cells, probably due to sequestration of IGF. No response was observed in stomal cells to intact or fragmented protein, however, due to the high concentration of endogenous IGFBP-3 produced.