The Darlington Retrospective Outpatient Study (DROPSY) was carried out at the Darlington Memorial Hospital Darlington. From Jan 2002 to Dec 2007, 1041 patients were seen in the heart failure (HF) clinic. Of these 270 (26%) were diagnosed as having left ventricular systolic dysfunction (LVSD). Of the 771 patients who did not have systolic dysfunction, we identified 243 patients who fulfilled the study criteria for heart failure with preserved ejection fraction (HFpEF). The remaining 528 patients formed the non heart failure (Other) group. Patients with HFpEF were older and more likely to have hypertension and diabetes than the other two groups. The LVSD group had more men plus ischemic heart disease patients while the third group of non HF also had more females and a high number with COPD. Over the mean follow up of 7 years, the number of admissions to hospital per patient was similar in both the LVSD and HFpEF groups, but HFpEF patients had a significantly longer length of stay. In the HFpEF group, the use of beta blockers, ACE inhibitors, and a lower median resting HR of < 78 / min predicted better survival. All cause mortality was high in both LVSD and HFpEF groups, with strikingly different cause of death. Patients with LVSD had more cardiovascular deaths whereas HFpEF patients were more likely to die of non cardiovascular causes. Patients in the third group diagnosed as not having HF (Other group) also had high five year mortality. Conclusion Patients with LVSD and HFpEF have high mortality but different causes of death. The use of beta blockers, ACE inhibitors, and a lower resting heart rate in the HFpEF group was associated with better survival. Patients who were reassured as not having HF do badly as well.

Heart failure (HF) is a progressive, life-limiting illness affecting around 750,000 people in the UK, with a mortality rate of 50% within four years. A large body of qualitative research demonstrates variable quality of HF care at the end of life, difficulties in identifying the dying phase, poor communication, and a failure to achieve a ‘good’ death. However little is known about the assessment of the need for palliative care, the recognition of the last days of life, or the extent to which these considerations are communicated to patients and carers and to the wider multi-disciplinary team. Greater understanding of the transition points from HF diagnosis to death may inform future service planning, including the most appropriate model of palliative care to apply to this patient group. Thus, the aim of this study was to explore experiences of giving or receiving a prognosis and managing the transition point from diagnosis to palliative and end of life care for those with HF. The study involved two stages. The first was a systematic review of the uptake of the Liverpool Care Pathway (LCP) in order to assess current utilisation of end of life care pathways. The second utilised a modified constructivist grounded theory methodology to assess experiences of giving and receiving a prognosis, combining semi-structured interviews with clinicians, observations of clinic and home visit appointments, followed by a series of longitudinal semi-structured interviews with thirteen patients with HF and nine carers. The systematic review demonstrated that the LCP was utilised for less than half of all dying patients. Interviews with clinicians revealed frustration and uncertainty about the contested nature of HF diagnosis and prognosis. Most clinicians rejected the concept of HF as a terminal illness in their everyday practice, and expressed uncertainty about roles and responsibilities for end of life care, alongside a reluctance to actively plan for end of life for individual patients. In contrast, some clinicians demonstrated the ability to deliver problem-based, individualised care but sometimes felt constrained by the perceived lack of multi-disciplinary advanced care planning. Most patients and carers talked about death and dying in general terms but felt that HF specific end of life considerations did not apply to them. They placed much more importance on understanding the emergence of their symptoms and negotiating everyday restrictions. Most patients had not made any decisions about advance care directives, and reported no prognostic discussions with clinicians. Overall, the majority of participants rejected notions of HF as a terminal illness in favour of day to day management and maintenance, despite obvious deteriorations in disease stage and needs over time. This is the first known study exploring the experiences of prognostic communication at all stages of the HF disease trajectory. Findings raise questions regarding the pragmatic utility of the concept of HF as a terminal illness and have implications for future HF care pathway development. A key recommendation emerging from this study is that notions of prognosis should be ultimately rejected for HF care, and be replaced with a problem-based approach to care which combines elements of active and palliative care from diagnosis onwards, alongside regular assessments of communication preferences.

Schistosomiasis is a chronic parasitic disease, estimated to affect 237 million people worldwide. It is caused by infection with Schistosoma helminths, which spend part of their lifecycles in aquatic snails. The mortality, development and fecundity rates of the parasites and their intermediate host snails are very sensitive to water temperature. The distribution and prevalence of schistosome parasites are therefore likely to be affected by climate change, however the potential effects of this have been largely neglected. Only two mathematical models of temperature and schistosome transmission in Africa have previously been developed, and neither explicitly simulated all temperature-dependent stages of the parasite and snail lifecycles. The aim of this thesis is to advance understanding of the potential effects of climate change on S. mansoni transmission, using an agent-based modelling approach. A mathematical model of water temperature, snail population dynamics and S. mansoni transmission was developed. The model was parameterised using data from Biomphalaria pfeifferi, the most widespread intermediate host species in Africa, and the dynamics of the model were explored. Infection risk was shown to be highest (above 90% of the maximum) at a constant temperature of 15-19°C. Simulating diurnal variation in temperature and/or higher cercaria and miracidium removal rates increased the optimum temperature for transmission to 16-26°C. The effect of simulating different species of intermediate host snail was also investigated. Simulating Bi. alexandrina and Bi. glabrata increased the temperature at which infection risk was highest to 19-21°C and 20-26°C respectively. The model was run using climate projections for eastern Africa. Comparisons of model output at baseline with empirical data showed that suitable temperatures are necessary but not sufficient for both schistosome transmission, and for high prevalences of schistosomiasis. All else being equal, infection risk may increase by up to 20% over most of the area over the next 20 and 50 years. Increases may be higher in Rwanda, Burundi, south-west Kenya and eastern Zambia, and schistosomiasis may become newly endemic in parts of these areas. The results for 20 years’ time are robust to changes in simulated snail habitats. There is greater uncertainty about the effects of different habitats on changes in risk in 50 years’ time.

Mucoadhesive formulations have been used to increase the residence time and improve bioavailability of nasal dosage forms. The exact nature of the interplay between formulations and the mucus layer has not been defined, although theories have been proposed suggesting that certain characteristics are required for optimum mucoadhesivity. This thesis presents an investigation into the effects of the properties of excipients in nasal formulations on their mucoadhesive performance. The main factors that were investigated included molecular weight, concentration, crosslinking density, charge, and viscosity. It was established using rotational and oscillation rheology that the polymeric formulations with the highest molecular weight expressed the highest viscosity. Thixotropy, a vital property in mucoadhesion, was also assessed. The greatest thixotropy was found with polymers of increasing molecular weight whereas low molecular weight polymers exhibited little or no thixotropy. As expected, high molecular weight polymers produced strongly gelled networks; a requirement for mucoadhesion. Mucoadhesive interactions between polymers and mucin were analysed using standard rheology and microrheology. Greater synergy was found with high molecular weight, linear, ionic polymers; factors which allow for improved chain interactions. Texture analysis of the formulations confirmed that the adhesive forces increased for higher molecular weight, ionic polymers. In conclusion, it was found that a combination of a high molecular weight, increased viscosity, charge, and a moderate level of crosslinking are all favourable properties in a polymeric nasal spray. The formulation of a mucoadhesive dosage form with these characteristics may improve the retention time of the formulation within the nose, resulting in an increased opportunity for drug absorption and thus greater bioavailability.

The study of anaerobic bacterial growth associations plays an important role in the analysis of the toxicity and effect of pollutants. These associations can be found in the environment, for example in groundwater or wastewater treatment plants or even in the gut of animals. The aims of this study were to compare the toxicity exhibited on anaerobic growth associations by two similar alkylphenolic compounds, o-cresol and 2,4-dimethylphenol (DMP), individually and combined, and examine the specific effect these compounds had on individual bacterial groups within the consortium. Through the use of anaerobic batch cultures, the inhibitory concentrations and effects of o-cresol and 2,4-dimethylphenol on anaerobic processes were analysed. Concentrations of o-cresol = 1.85mM were shown to have an inhibitory effect on sulphate reduction, acetogenesis and methanogenesis. At concentrations = 7.4mM complete inhibition of sulphate reduction was noted and the acetogenic and metha nogenic processes were greatly reduced, concentrations = 9.3mM appear to cause complete inhibition of methane production. 2,4-dimethylphenol inhibited bacterial groups at concentrations =0.41mM; complete inhibition of sulphate production was observed at concentrations of DMP =3.28mM. Although methane production was significantly reduced at the highest concentration (4.1mM) complete inhibition was not observed. It was found that DMP was more toxic to all the bacteria and archaea within the consortium than o-cresol. Use of continuous flow systems allowed for the separation of the microbial groups within the consortium allowing for the analysis of the direct effect of o-cresol and 2,4- Dimethylphenol. Analysis of the results indicated that the compounds inhibited all the microbial groups, although with varying x degrees of success. It was observed that acetoclastic methanogenic bacteria are more resilient to the toxic effect of the compounds than h methanogens and sulphate reducing bacteria. The combined toxicity of these compounds was analysed utilising batch cultures. The combined toxic effect on the growth consortium was observed after addition of both of these compounds to batch cultures. The results showed that, when combined, these compounds exhibited a toxic effect on the consortium, which was synergistic in nature. This study has highlighted the toxic effects of o-cresol and DMP on anaerobic consortia and the importance of the synotrophic relationships between the different bacterial and archaeal groups. It is important that work in this field is carried on to develop a better understanding of the implications of toxic chemical spills and their impact on the groundwater environment.

An arteriovenous fistula (AVF) is a vein graft which is created to permit vascular access allowing haemodialysis to be performed. AVFs are associated with failure rates as high as 50% at 6 months. Failure is principally due to vascular smooth muscle cell proliferation, leading to the development of neointima causing stenosis and impaired blood flow. The aims of this study were to; 1) explore the inflammatory and proliferative characteristics of AVF stenosis and the role of TLR-4, 2) assess the ability of anti-inflammatory diclofenac to inhibit VSM cell proliferation, 3) develop a novel model of AVF in the rabbit to assess the impact of cannulation injury and the effect of topical diclofenac and 4) investigate the mechanisms responsible for diclofenac mediated activity. Human stenotic AVF segments and cell explants taken from haemodialysis patients vs. healthy long saphenous vein controls were shown to have significantly higher TLR-4 expression and activation of the downstream kinase IRAK-4. Also associated with AVF stenosis was an increased expression of pro-inflammatory cytokines including MCP-1. VSM cell explants derived from stenosed AVF had a significantly increased capacity to proliferative vs. healthy controls, which was inhibited by diclofenac treatment. Using a novel rabbit AVF model, cannulation injury was shown for the first time to drive stenosis. Topical diclofenac significantly inhibited this injury response, reducing mean vein wall width from 46.8±5.7μM to 15.8±1.8μM, comparable to 16.7±1.6μM in the non-injured AVF. In addition to previously well defined COX inhibition, evidence was generated in this study to implicate AMPK in the anti-proliferative activity of diclofenac. Therefore, activation of TLR-4 in AVF stenosis appears to play a significant role in the generation of an inflammatory and proliferative VSM cell response. Cannulation injury, which undoubtedly causes a pro-inflammatory response, significantly contributes to AVF stenosis which is inhibited by prophylactic topical diclofenac via the activation of AMPK.

Introduction: Asthma and chronic obstructive pulmonary disease (COPD) affect 8% and 5% of the population, respectively, in the United Kingdom (UK) and the United States (USA). A variety of medicines are available but how they are utilised in real practice is not fully understood. The aim of this work was to describe and compare the treatment of asthma and COPD in the UK and USA. Methods: Three retrospective databases (two administrative and one electronic health record datasets) were formed from sources in National Health Service (NHS) Scotland, NHS Forth Valley, Scotland, and Kentucky, USA. Several analyses were conducted, including mapping and evaluation of national medicine utilisation, evaluation of adherence/persistence with chronic therapy, classification of therapy against guideline recommendations, and appraisal of inhaled corticosteroid (ICS) prescribing. Results: National medicine utilisation figures indicated an increasing preference over time for combination therapy wit h ICS and long-acting beta agonist (LABA) inhalers. Therapy for asthma demonstrated some unanticipated trends, with widespread use of high-dose combination therapy in up to one-third of patients and a lack of standardised therapy approach by clinicians at step 2/3. For COPD, spirometry data was unable to verify diagnosis in up to a quarter of patients, and approximately one-third of patients received unlicensed doses of combination therapy. Adherence and persistence with chronic medicine in both databases was better amongst women, with advancing age and with oral therapy. Direct comparisons between the UK and USA were difficult due to the different healthcare structures and methods for data collection, but doses of ICS in children appeared more aggressive in the USA. Conclusion: The treatment of respiratory disease can be optimised in several clinical areas, most notably with ICS prescribing. Further research and quality improvement measures are needed to improve the care of respiratory disease.

A series of selected essential hydrophobic amino acids, L-Valine, L-Leucine and L-Isoleucine were studied to prepare novel co-crystalline forms for comparison with their pure forms. The crystals of these amino acid complexes were characterised by various techniques including spectroscopy, thermal and X-ray diffraction. The analysis has confirmed the formation of three new co-crystals of L-Leucine-L-Isoleucine (compound 1), L-Valine-L-Leucine (compound 2) and L-Valine-L-Isoleucine (compound 3), the first co-crystals of naturally occurring hydrophobic amino acids with the same chirality. The second experimental chapter describes the use of a Continuous Stirred Tank Crystalliser (CSTC) for bulk preparation of a multi-component system via an anti-solvent crystallisation process. The results indicate that the process conditions used to fabricate co-crystals of amino acids with anti-solvent crystallisation are reproducible with regard to the final product properties, purity and yield. In the last theme, a host-guest complex is shown wherein the c(RGDfV), a cyclic pentapeptide [c(ArgGlyAspD-PheVal], forms an inclusion complex with β-cyclodextrin. In addition, the batch to batch variation between two batches of the pentapeptide procured from the same source is highlighted. The c(RGDfV) and the inclusion complex were characterised by differential scanning calorimetry, fourier transform infrared spectroscopy, circular dichroism, powder X-ray diffraction and nuclear magnetic resonance spectroscopy. Based on ¹H nuclear magnetic resonance (NMR) experiments, the possible mode of interaction of betacyclodextrin with the c(RGDfV) is proposed. Different methods to crystallise c(RGDfV) batch (B-1) and its self-assembly to form hydrogel is also presented.

Sphingosine kinase/sphingosine 1-phosphate (SK/S1P) signalling interacts with major cellular pathways controlling cell proliferation, migration, survival, and resistance to chemotherapeutics. Moreover, extensive research has shown that the SK/S1P signalling is up-regulated in numerous human cancers (e.g. stomach cancer, colon, rectal, glioblastoma, ovarian, renal, lung and breast) making S1P signalling an important candidate as a biomarker and a key player in promoting cancer progression. Several inhibitors of SK/S1P signalling pathway have been identified and have shown to inhibit cancer cell survival and resistance to chemo- and radio-therapies. In this study, human breast cancer tissue microarrays at various tissue histological grades of ERα negative breast tumours were analysed for the expression of S1P signalling proteins (e.g. SKs and S1P₁₋₅ receptors) to identify the impact of expression level of these proteins on clinical outcomes. High SK1 and S1P₄ receptor tumour expression is associated with poor cancer prognosis in ERα negative breast cancer patients. Moreover, high SK1 and S1P₄ receptor expression in these tumours was also associated with cancer recurrence and this was dependent on the HER2 receptor expression. Indeed, the SK1/2 dual inhibitor SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) abrogated the S1Pstimulated ERK-1/2 activation in ERα-/HER2+ MDA-MB-453 cells suggesting that SK1 activity is required for the S1P₄/HER2-mediated ERK-1/2 activation that is known to promote cancer progression. In addition, a diverse array of kinases and transcription factors e.g. c-RAF-1, ERK-1/2, AKT, LYN, SRC family kinases (SFKs) and NFκB (p50 RelA) were analysed in combination with SK1 and S1P receptors to discover novel prognostic interactions that drive cancer progression in ERα positive breast cancer patients. High tumour SK1 expression in combination with high expression of either S1P₁ receptor or S1P₃ receptor or phosphorylated ERK-1/2 or phosphorylated AKT or phosphorylated NFκB or phosphorylated RAF-1 or Y416 phosphorylated SFK or LYN is associated with shorter disease-specific patient survival and disease-free cancer recurrence. Similarly, high S1P₁ receptor tumour expression in combination with high expression of either Y216 phosphorylated SRC or c-RAF-1 or ERK-1/2 or AKT kinase is associated with shorter disease-specific patient survival and disease-free cancer recurrence. High S1P₂ receptor tumour expression is associated with prolonged patient survival and this is enhanced in combination with high expression of c-SRC and Y416 phosphorylated SFK in ERα positive breast cancer tumours. Finally, high tumour S1P3 receptor expression in combination with high expression of LYN or c-RAF-1 kinases is associated with shorter disease-specific patient survival and disease-free cancer recurrence. Lastly, a new signalling pathway involving SK2, Y416 phosphorylated c-SRC, S1P₄ receptor and S1P₂ receptor was identified using pharmacological agents/gene silencing in ERα negative MDA-MB-231 breast cancer cells. In this pathway, SK2 possibly through 'inside out' S1P signalling activates the S1P₄ receptor that promotes cellular growth by preventing the nuclear accumulation of S1P₂ receptor. Moreover, SK2 activity also prevents the accumulation of Y416 phosphorylated c-SRC into the nucleus that might be crucial for tumour growth. Thus, this study shows that the high tumour expression of S1P signalling proteins is associated with poor disease prognosis in both ERα positive and ERα negative breast cancer patients. However, cancer progression is mediated by distinct set of S1P signalling proteins in different types of breast cancer. Hence, different treatment regiments including SK inhibitors and S1P receptor antagonist must be employed in treatment of ERα positive and ERα negative breast cancer patients.

This thesis presents an investigation into a 9th-10th type III fibronectin (FN) domain pair (FIII9-10) focused on the characterisation and functional activity of the protein. Initial work attempted to establish whether there was a binding interaction between FIII9-10 and IGFBP-5. Surface plasmon resonance failed to demonstrate an interaction and so thereafter the programme examined the use of FIII9-10 as a protein scaffold for cell adhesion. Surface-induced unfolding of proteins results in a loss of function. To improve the conformational stability of FIII9-10, Pro-Pro pairs were introduced. A previously developed multimeric FIII9'10 α5β1 ligand was also studied. FIII9-10 cDNA constructs were expressed in E. coli. Purity, fold and molecular weight were confirmed using SDS-PAGE, circular dichroism (CD) and mass spectrometry. Changes in conformational stability generated by the mutations were assessed by equilibrium chemical denaturation. An automated CD system was used to follow the secondary structure changes generated by thermal denaturation. Adsorption induced structural changes were investigated using a novel 'solid state' CD technique for a range of surface energies. Neutron reflectometry was employed to estimate the surface coverage and packing arrangement of the FIII9-10 mutants. Calculation of the Gibbs free energy demonstrated a two-fold increase in stability compared to wild type with a 9-11°C increase in the Tm. The mutated proteins showed enhanced stability when adsorbed to silica, but lost structure as surface hydrophobicity increased. This loss was less than that for the wild-type. ELISA studies verified that the mutation of the FIII9-10 did not compromise the receptor binding affinity. Immunofluorescence microscopy of baby hamster kidney on protein coated substrates displayed improved cell adhesion and spreading. The enhanced structural stability of the FIII9-10 mutants showed promise for use in influencing and controlling the interactions between medical implants and host tissue, mimicking the role of the extracellular matrix and improving biocompatibility.