Boron neutron capture therapy (BNCT) is a method for selectively destroying malignant (normally glioma) cells whilst sparing normal tissue. Irradiation of 10B (large neutron capture cross-section) with thermal neutrons effects the nuclear fission reaction: 10B + 1n → → 7Li+ + α + γ; where the penetration of -particles and 7Li+ is only 8 and 5 μm, respectively, i.e., within a single cell thickness, assuming 10B can be preferentially located within glioma cells. Poor selectivity is the main reason why BNCT has not become a mainstream cancer therapy. Carboranes. a third generation of high boron-containing, low-toxicity, BNCT compounds, are currently being investigated. Towards the aim of increasing malignant cell targeting specificity, this thesis investigates monodispersed dipalmitoylphosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) SUV liposome formulations containing carboranes derivatised with delocalised lipophilic cations (DLCs), specifically the dequalinium bis nido carborane salt. AFM studies showed the loaded liposomes appeared stable (63 days, 4°C, if re-probing was employed). The integrity of the liposome membrane in serum, as reflected by %latency and %retention experiments using a fluorescent marker (calcein), was found to be high for both types of liposomes prepared using cholesterol. Successful entrapment of carboranes was demonstrated by the Nile Red method and by ICP-MS measurements. The liposomes were of sufficient size (80-100 nm) to pass through the blood brain barrier (BBB). The cationic moiety of the carborane salt allowed selective targeting of glioma mitochondria, thought to be due to differences in mitochondrial membrane potentials between malignant and non-neoplastic cells. Specific targeting of IN699 (glioma, WHO grade IV) and SC1800 (non-neoplastic astrocyte) cells with the carborane salt was evidenced by live cell (fluorescence) imaging. Spray drying was used as an alternative method of formulating agents for BNCT treatments for liver and lung cancers, where the larger (micrometre-diameter) particles do not need to pass across the BBB. Polyvinylpyrrolidone / o-carborane co-spray-dried microparticles were produced. 1H NMR studies revealed the high temperatures (180 °C) of the spray drying process did not degrade the PVP. Mean particle diameters (x90) were in the 2 – 10 μm range, with finer fractions being present (x10 ≅ 1 – 2 μm), and were therefore considered suitable for delivery to the lungs. SEM imaging showed the particles to be spherical, with dimples and cavities caused by the spray drier nozzle characteristics, as typical with the spray drying process. Some small irregularly-shaped crystalline particles, thought to be o-carborane, were observed by SEM, although the proportion accounted for less that than in the formulation (10 %w/w). An attempt was made to map the boron content in spray-dried powders on a surface using EDS, although the low atomic weight of boron made detection not possible. Cytotoxicity studies, using human glioblastoma U-87 MG (cancerous) and human fetal lung fibroblast MRC-5 (non-neoplastic) cells, revealed the PVP / o-carborane co-spray-dried particles to be non-toxic.

The spore forming bacterium, Bacillus anthracis is the aetiological agent of anthrax. The 2001 US anthrax letter attacks and the 2009‐2010 outbreak of injectional anthrax in the UK highlighted the importance of early detection and confirmation of this agent, both for patient outcome and forensic investigations. A reliable and consistent method was used in this study to safely simulate blood cultures with B. anthracis and used to determine the time to positive detection. This was performed with different strains and with varying concentrations of inoculum. An inverse linear relationship was observed with all strains and used to estimate the bacterial blood concentration of anthraxpatients based on data gathered from the literature and front‐line laboratories in the UK. The study explored a method to potentially reduce the turnaround times for the confirmation of B. anthracis at the national reference laboratory. Serum separator tubes were used to concentrate the bacteria from simulated blood cultures. A simple wash step was performed prior to performing confirmatory phenotypic tests and inactivation for rapid molecular detection. A comparison of test results with and without serum separator tube processing was made for B. anthracis and bacterial isolates referred during the outbreak of injectional anthrax. Simulated mixed blood cultures of B. anthracis and possible common contaminants were also tested. Compared to routine methods, confirmatory phenotypic test results were achieved 24 hours sooner using the method. The simple wash step and inactivation was sufficient to provide nucleic acid for molecular confirmatory assays and genotyping. A new ‘sample to answer’ platform, the Biofire Filmarray® was also trialled and correctly identified B. anthracis directly from simulated blood culture and provided results within one hour. Aspects relating to potential biosafety concerns for processing B. anthracis blood cultures were explored. The data generated suggests the aerosol risk is low for B. anthracis. Viability of material on microscopy slides was examined and the data supports the recommended use of alcohol fixation for slide preparation. There has been no previous evidence reported for sporulation occurring in blood culture bottles and the study findings suggest this is possible five days post positive detection. Interactive e‐learning modules have been produced to disseminate the study outcome. The e‐learning is intended for front‐line laboratories to raise awareness for the safe handling and laboratory identification of B. anthracis.

Progress made in the palliation of those with terminal cancers has allowed most symptoms to be controlled if not completely alleviated. For many in this situation now, the most overwhelming and unpleasant ongoing problems are related to the accompanying cachexia. Cachexia - (Greek,kakos-bad, hexis-condition) a wasting syndrome that causes weakness and a loss of weight, fat, and muscle(1) Cachexia has direct and tangible consequences such as reducing independence to a level where the patient requires institutional end of life care rather than being able to die in their own home or a place of their choosing. It also reduces survival independent of the primary disease histology, stage or the patient’s performance score(2) Upper gastrointestinal adenocarcinomas (i.e. oesophagus, gastric, pancreas, ampullary) often result in profound cachexia. Indeed unexplained weight loss is often the presenting symptom. There are often few other symptoms and consequently diagnosis is often made relatively late in the disease process, when the tumour has spread beyond the possibility of surgical cure. For a proportion of patients, chemotherapy and radiotherapy can offer substantial improvements but side effects often outweigh benefits. Many then either decide never to take these options or elect to stop taking them during the treatment course. A substantial proportion of people in this situation have no acceptable treatment options available to them. Previous attempts to medically manipulate this condition have been largely unsuccessful. Early small trials using thalidomide have pointed to a possible role in reducing loss of lean body mass but none have demonstrated a functional benefit or investigated underlying mechanisms. In this trial we aimed to draw definite conclusions as to whether patients with terminal upper gastrointestinal adenocarcinomas would benefit from taking thalidomide. We also investigated the likely biological mechanisms underlying any effects. One of our major challenges was identifying a practical method for measuring lean body mass in a clinical setting. Measurement of lean body mass by Dual Energy X-ray Absorptiometry (DEXA) is accurate but expensive, bulky, immobile and entails a small radiation dose. Our comparisons between methods of lean body mass measurement showed that anthropometry was a reasonable alternative to DEXA scanning (Gold Standard) but that bio-impedance produced an even more accurate result. We concluded that bioimpedance could be used as a valid alternate to DEXA in this population with the proviso that it as lean body mass falls it will tend to be underestimated by bioimpedance. We found thalidomide to be well tolerated but to offer no clinical benefit overall. In fact, at the three month visit those in the thalidomide group had a significant greater reduction in measured grip strength and the functional aspect of their quality of life as measured by questionnaire. Neither change was sustained at the six month visit. There was no measurable change in lean body mass between groups. The average survival was slightly higher in the placebo group but this difference was not significant (mean survival thalidomide group 83 days, placebo group 88 days). There was also a suggestion of some benefit of thalidomide therapy in those presenting with a more inflammatory disease as measured by plasma IL-6 and CRP. Sub-group analysis revealed that the thalidomide treatment group had a significantly longer survival over the placebo treated group if they presented with a higher than average IL-6 and a reduction in weight loss. Survival was significantly shorter with thalidomide treatment for those presenting with a lower than average IL-6. Thalidomide led to a significant suppression of plasma IL-6 levels over time. Unfortunately the survival advantage seen with thalidomide treatment in those with a more inflammatory state was not associated with any improvement in quality of life. The reduction in grip strength and functional quality of life was less marked in the thalidomide treated group but still present. It may be that thalidomide treatment leads to an overall reduction of muscular strength through its known side effect of somnolence leading to inactivity but that in treating the inflammatory component of cachexia it is able to improve survival. We suggest that future clinical trials of cachexia include measurement of peripheral cytokine measurements which seem to be strongly associated with outcomes. There may be a different anti-inflammatory medication or combination of treatments that could successfully treat the cachexia without the same side effect profile of thalidomide. Clinical benefits may be dictated by the degree of inflammation present at presentation and patients may need to be stratified for future therapies.

The work presented in this thesis explores the use of electrohydrodynamic atomisation as a one-step method for the fabrication of monodispersed poly(lactic acid) and poly(lactic-co-glycolic) nanoparticles designed for selective delivery of anti-cancer drugs. Following optimisation studies of the relevant electrospray parameters, a range of PLA and PLGA nanocarriers loaded with the anti-cancer drug doxorubicin and with folic acid (aimed at achieving tumour targeting) was prepared and characterised using dynamic light scattering, electrophoretic mobility measurements, and confocal and atomic force microscopy. Unloaded and/or selectively loaded nanoparticles were also fabricated using the same technique and employed as controls. It was found - for all nanoparticles tested - that sensible yield, minimal size and polydispersity were obtained when using dimethylsulphoxide and dichloromethane with an optimal collection distance of 15 cm, applied voltage 9.2 – 9.6 kV and flow rate 10 μL/min. Nanoparticles were further tested in vitro for their interactions with human cells in terms of toxicity, tumour selectivity and cellular uptake, by using a range of techniques that include cytotoxicity assays, confocal microscopy, live-cell imaging and flow cytometry. When compared to the results obtained with normal human cells (16HBE), those attained using human cancer cells that overexpress folate receptors (CALU-3) indicated an increased cytotoxic effect of the loaded nanoparticles. Furthermore, cellular uptake studies demonstrated significant selectivity of the nanoparticles loaded with both doxorubicin and folic acid for the CALU-3 cell line compared to normal 16HBE cells.

Gastrointestinal endoscopy is a vital tool for the detection and treatment of early neoplasia in the upper and lower gastrointestinal tracts. Survival from gastrointestinal cancer is largely dependent on the stage at diagnosis – thus detection of early lesions, or better still treatment of pre-cancerous lesions is vital to improve outcomes. The world of endoscopy is changing rapidly with the development of dye-based and digital enhancement techniques with the aim of improving the detection and characterisation of neoplastic lesions. This thesis reviews the development of all the main advanced imaging techniques and comprehensively reviews the evidence for the use of these in the colon. Chapter 4 describes the development of a new classification system for characterising small colonic polyps using the Pentax i-Scan digital enhancement system. Chapter 5 describes a prospective cohort study using i-Scan for the in-vivo characterisation of small colonic polyps in 87 patients. No differences in the accuracy of polyp characterisation between high-definition white light endoscopy, i-Scan and chromoendoscopy were found when performed by an expert endoscopist. All 3 modalities met the ASGE criteria for management based on optical diagnosis. Chapter 6 describes a retrospective study looking at factors influencing polyp and adenoma detection in a large UK Bowel cancer screening cohort. Endoscope definition (standard vs high definition) was examined in particular. In this group of patients, endoscope definition was found to have no impact on any outcome measure, but endoscopist and bowel preparation were consistent predictors of key quality outcomes. Chapter 7 describes a randomised controlled clinical trial recruiting 126 patients of a pre-endoscopy drink containing water, n-acetyl cysteine and simeticone. This was shown to significantly improve mucosal visibility compared to water alone, or no preparation, and also significantly reduced the need for procedural fluid flushes. Chapters 8 & 9 describes two studies examining the baseline performance and impact of training modules on the accuracy of colonic lesion and polyp characterisation amongst non-endoscopists and endoscopists with varying degrees of experience. Baseline performance of experienced endoscopists was found to be no different to that of inexperienced endoscopists and novices in both studies, but training improved accuracy in all groups. These studies highlight the need for training in lesion characterisation to become part of the formal training programme for all endoscopists.

Tumour invasion is the key element in the high rate of mortality and morbidity in glioma patients. Increased expression, in neoplastic cells, of receptor tyrosine kinases (RTKs) of the TAM family (comprising Tyro3, Axl and MerTK), has been reported in several cancers including gliomas, in which they play a key role in tumour invasion. Axl RTK, with molecular weight of 120-140 kDa, mediates glioma cell adhesion and invasion through a variety of ways. Within the scope of this thesis, Western blotting, quantitative polymerase chain reaction (qRT-PCR) and glioblastoma multiforme (GBM) tissue microarray revealed an upregulation of Axl and Tyro3 in brain tumours and two adult GBM cell lines, SNB-19 and UP007. Both cell lines were treated with the TAM ligand Gas6 and/or the specific Axl small molecule inhibitor BGB324, and analysed in assays for survival, 3D colony growth, motility, migration and invasion. Western blotting was used to detect protein expression and signal protein phosphorylation. In both cell lines, BGB324 inhibited specifically phosphorylation of Axl as well as Akt kinase further downstream. BGB324 also inhibited survival and proliferation of both cell lines in a concentration-dependent manner, as well as completely suppressing migration and invasion. Axl inhibition by BGB324 also sensitised GBM cells to golden standard chemotherapeutic agent temozolomide. Furthermore, novel, unconventional activation mechanisms for the TAMs in human GBM cells were investigated. With the use of Western blotting, co-immunoprecipitation and in vitro kinase assays, Axl was shown to both interact with and be activated by the RTK EGFR. With the aid of qRT-PCR screens, EGFR was shown to promote GBM cell invasion through the Axl/TIMP1/MMP9 signalling axis. Additionally heterodimerisation of Axl with its sister RTK, Tyro3, in GBM cells was confirmed using co-immunoprecipitation assays; the functional significance of this complex was determined to be promotion of GBM cell survival. In conclusion, this thesis demonstrates the importance of TAM signalling in GBM, identifies novel molecular pathways employed by GBM cells for their survival, growth and spread, and thereby further strengthens the case for targeting TAM receptors as a novel therapeutic approach to combat both primary tumours as well as secondary tumours arising from drug resistance.

Information about the research activity Foundation degrees (FDs) for pharmacy technicians combine academic and work-based learning to provide skills and knowledge for enhanced pharmacy technicians’ practice. Nine of these programmes have been developed nationwide since the introduction of the qualification in 2000. Only three of these FDs continue to run at the time of writing this report and low student numbers within these courses make their future uncertain. With a population of potential students in excess of 15,000 and increased demands for enhanced pharmacy technicians’ practice, lack of success of these FDs can be due to how the qualification is perceived. This research represented the first systematic exploration of the perceptions of stakeholders with involvement in a FD for pharmacy technicians that evaluated one such programme: the FD in Medicines Management run by the University of Portsmouth. The study focused on a single course to provide an in-depth account of events and processes. It involved gathering the views of students, graduates, employers, and people involved in course delivery and development. The research had a dual purpose: to describe experiences and to feedback findings into pedagogic practice within the Portsmouth course. Research questions In order to carry out this evaluation, the following broad research questions were investigated: • What do people think of the FD qualification? • What is the experience of being involved with a FD like? • What effect does a FD have on pharmacy technicians? • What opportunities does a FD bring for role extension and development? • Can a theoretical model be developed to represent relationships between the experiences of stakeholders involved in a FD? Plan of Investigation The project was carried out over a period of three years from September 2007 to June 2010. It followed a qualitative evaluation research approach. Data was collected from one-to-one and group interviews, which were recorded, transcribed verbatim and subject to thematic analysis. The analysis aimed to identify significant concepts, themes and categories, building a theoretical model that would represent relationships between the experiences of the stakeholders. Within each of these, different views were compared and contrasted. Key findings A theoretical model was developed that represents the relationships between the experiences of stakeholders involved in a FD for pharmacy technicians; a number of key themes were also found. Firstly, it was established that there was a general lack of awareness of the availability and scope of FDs. While study participants perceived them as valuable qualifications, they reported that often this was not the case within their workplace. FDs were considered demanding qualifications from a pedagogic point of view, as course content had to be continuously updated to reflect changes in practice. They contributed to the personal and professional development of pharmacy technicians and provided skills and knowledge to enable individuals to have more independent roles. Pharmacy technicians’ motivation to acquire skills and knowledge to improve patient care was the main driver for participation in a FD. The new capabilities of pharmacy technicians led to increased aspirations for the development of their role, which were not always fulfilled. Different factors were accountable for this, which merit further research in order to fully describe this phenomenon. Action points As a result of this research, the following action points were undertaken to improve pedagogic practice within the Portsmouth FD in Medicines Management: • Development and implementation of a marketing strategy. • Review and update of the programme syllabus. • Implementation of new approaches to teaching, learning and assessment.

Background: The reported incidence of prostate cancer (PCa) has increased in recent years due to the aging of the population and increased testing; however mortality rates have remained largely unchanged. Studies have shown deficiencies in predicting patient outcome for both of the major PCa diagnostic tools, namely prostate specific antigen (PSA) and trans rectal ultrasound ‐guided biopsy (TRUS). Therefore, serum biomarkers are needed that accurately predict prognosis of PCa (indolent vs. aggressive) and can thus inform clinical management. Aim: This study uses surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI‐TOF‐MS) analysis to identify differential serum protein expression between PCa patients with indolent vs. aggressive disease categorised by Gleason grade and biochemical recurrence. Materials and Methods: A total of 99 serum samples were selected for analysis. According to Gleason score, indolent (45 samples) and aggressive (54) forms of PCa were compared using univariate analysis. The same samples were then separated into groups of different recurrence status (10 metastatic, 15 biochemical recurrence and 70 nonrecurrences) and subjected to univariate analysis in the same way. The data from Gleason score and recurrence groups were then analysed using multivariate statistical analysis to improve PCa biomarker classification. Using gel‐electrophoresis technique, candidate biomarkers were separated and identified by LC‐MS/MS and validated using optimised Western blot (WB) immunoassay against 100 PCa serum samples from the Wales Cancer Bank (50 as indolent group & 50 as aggressive group). Results: The comparison between serum protein spectra from indolent and aggressive samples resulted in the identification of twenty‐six differentially expressed protein peaks (p<0.05), of which twenty proteins were found with 99% confidence. A total of 18 differentially expressed proteins (p<0.05) were found to distinguish between recurrence groups; three of these were robust with P<0.01. Sensitivity and specificity within the Gleason score group was 73.3% and 60% respectively and for the recurrence group 70% and 62.5%. Four candidate biomarkers (categorised by Gleason score) were identified using a novel 1 D LC‐MS/MS technique. The candidate biomarker with m/z of 9.3 kDa was found to be upregulated in aggressive PCa patients, and was identified as Apolipoprotein C‐I (ApoC‐I). Another three candidate biomarkers (22.2, 44.5 and 79.1 kDa) were found downregulated in the aggressive group and up‐ regulated in the indolent group and identified as apolipoprotein D (ApoD), putative uncharacterised protein (PUP) and Transferrin (TF), respectively. The utility of the putative biomarkers was examined by Western blot (WB) analysis of 100 blinded PCa serum samples. None of the three SELDI identified biomarkers were able to statistically identify PCa patients’ progression. Conclusion: The use of SELDI to identify potential PCa progression biomarkers has been confirmed in PCa patients. However, immunovalidation of prospective biomarkers in blinded PCa serum samples was unsuccessful. This study demonstrates the importance of validation in ascertaining the true clinical applicability of a cancer biomarker.

Excessive neutrophilic inflammation of the airways in response to infection is characteristic for patients with CF. There is also an important but not fully understood role for platelets. Previous studies established increased copper levels in the circulation and in the sputum in CF. Inhaled tobramycin was suggested to have an anti-inflammatory effect beyond eradicating Pseudomonas aeruginosa. This study tested the hypothesis that tobramycin has anti-inflammatory and anti-oxidant efficacy due to its ability to bind copper into a copper-tobramycin complex. A copper-tobramycin complex was synthesised and the UV-VIS spectrum analysed. Neutrophil migration through a TNF-α-stimulated human lung microvascular endothelial cell layer towards thrombin-activated platelets was measured. The role of CFTRinh-172 on neutrophil transendothelial migration was assessed. Endothelial tobramycin uptake and CFTR expression were assessed using immunocytochemistry. Endothelial oxidative stress was measured using a fluorescent indicator. Neutrophils were stimulated to measure reactive oxygen species (ROS) production and neutrophil elastase (NE) activity, spectrophotometrically. Platelet and endothelium-derived NAP-2 and IL-8, respectively, contributed to neutrophil transendothelial migration. Copper-tobramycin was shown to be more effective than tobramycin in limiting migration of neutrophils. Both, tobramycin and copper-tobramycin accumulated in endothelial cells via a heparan sulphate-dependent mechanism, decreased intracellular ROS and increased endothelial surface CFTR expression. CFTRinh-172 failed to create an inflammatory profile in endothelium. Copper-tobramycin decreased extracellular superoxide released by activated neutrophils, and displaced NE from sites of encryption, making it more susceptible to inhibition by α1-antitrypsin. The antibiotic tobramycin was demonstrated to be a multi-potent drug with additional anti-inflammatory and anti-oxidant properties. These effects, desirable in CF treatment, are due to copper binding.

Cathodic delamination failure is believed to be caused by the production of OH- at the polymer/metal interface via oxygen reduction reactions. In cable connector assemblies, the use of manufacturing processes and materials that are resistant to cathodic delamination failures is highly desirable. Also, there is a need for an accelerated testing regime for assessing the resistance of polymer-to-metal composites to cathodic delamination failures. Cable connector assemblies are employed in marine environments for structural terminations and circuit interconnection of electrical power and signal cabling systems. They are found on offshore sea beds, ROV’s, vessels and submarines and are prone to cathodic delamination related failures particularly when the cable assemblies are coupled to dissimilar metal appurtenances in seawater, e.g., zinc anodes. The resistance to cathodic delamination failures of selected metal substrates and polymeric materials used in the manufacture and sealing of cable connector assemblies have been investigated. Materials and methods of surface preparation by grit blasting to enhance polymer-to-metal adhesion, thus increasing the service life-time expectation of cable connector assemblies have been elucidated. Methods of accelerated testing of polymer/metal bond durability using salt spray test and the validation of the results in seawater immersion tests and potentiostatic experiments have been described. Also, a novel accelerated test chamber for cathodic delamination tests was designed and manufactured. Applied potential was observed to be the factor that had the most negative effect on test samples while silicon carbide grit was found to produce the most effective surface cleanliness and roughness combination required for durable polymer-to-metal bonds.