The aim of this thesis was to optimise a method for the unequivocal identification of aluminium (Al) in human brain tissue. The second aim was to devise a protocol that was simple to implement and as such could be carried out without the need for sophisticated and expensive instrumentation. The presence of Al in brain tissue is linked to a number of neurodegenerative diseases and is the cause of dialysis encephalopathy. Therefore, it is of paramount importance to be able to both measure and locate Al in complex milieu such as human brain tissues. Multiple methods have been proposed for the identification of Al in biota including human tissues. However, there have been limitations within these methods such as specificity, selectivity and limits of detection. Herein, transversely heated graphite atomic absorption spectrometry (TH GFAAS) and fluorescence microscopy were used to measure and locate Al within brain tissues of donors who died with a diagnosis of familial Alzheimer’s disease (fAD) or autism. A fluorimetric method was optimised using surrogate human tissues so as not to use actual human brain tissue for method development. The results identified lumogallion as a selective and specific fluorophore for the identification of Al. The complex of lumogallion with Al produced characteristic orange fluorescence, which was specific to Al and for which there were no interferences from other metals which could be present in tissue, e.g. calcium, magnesium, iron. Congo red was used to identify the presence of amyloid, which was confirmed as being in a  sheet confirmation by the presence of apple-green birefringence. Lumogallion was used successfully and unequivocally to demonstrate the presence and location of Al within all four main lobes of brain tissue donated by individuals with a diagnosis of fAD. The combination of TH GFAAS and fluorescence microscopy was used to provide the first evidence of Al in the brains of individuals with fAD and these results should be considered in the light of the suggested role of Al in all forms of Alzheimer’s disease.

L-selectin (CD62L) is a type I transmembrane protein expressed by lymphocytes which directs their migration from the bloodstream into lymph nodes and infected tissues. Stimulation of the T cell receptor (TCR) activates the enzyme A Disintegrin and Metalloproteinase 17 (ADAM 17), which cleaves L-selectin at the ectodomain generating a metalloproteinase product (MP product) comprising of a transmembrane region and a 17-amino acid intracellular domain (ICD). ϒ-secretase is a multi-subunit protease that cleaves up to 90 identified type I transmembrane proteins in the intramembrane region following ectodomain proteolysis by metalloproteinases. Presenilin (PS), the catalytic component of γ-secretase is activated during an intramolecular cleavage called endoproteolysis separating the carboxy (C) and amino (N) termini. The catalytically active C-terminal fragment of PS then induces intramembrane proteolysis of substrates. The aim of my thesis was to firstly determine whether the MP product of L-selectin was a substrate for PS. Subsequently, I analysed whether stimulation of the TCR activates PS, inducing intramembrane proteolysis of the MP product releasing the ICD into the intracellular region. My data showed for the first time that in a resting T-cell, L-selectin forms a multi-component complex with both ADAM 17 and PS. TCR-activation induces ADAM 17 dependent proteolysis of L-selectin generating an MP product. Stimulation of the TCR also causes endoproteolysis of PS, where activated PS then cleaves the bound MP product. After PS cleavage, the released ICD was unstable and therefore difficult to detect, however I was able to block its formation using either PS inhibitor treatment or generating I351W mutated L-selectin, which was resistant to intramembrane proteolysis.

Inflammation is widely accepted as an initiator, and involved in the exacerbation of many diseases. Key modulators of inflammatory responses are oxygenated metabolites of polyunsaturated fatty acids (PUFAs). There are two main classes of PUFAs, Omega-3 (n-3) and Omega-6 (n-6). Arachidonic acid, a derivative of omega-6 is known to produce pro-inflammatory mediators known as prostaglandins (C20, n-6) via cyclooxygenase (COX). COX (a member of the myeloperoxidase (MPO) family) is a major therapeutic target of intervention in inflammatory disease. We investigated whether Drosophila could be used to model and dissect new novel targets of inflammation. Using an inflammatory model based on a JAK temperature sensitive, gain-of-function mutation (hop\(^T\)\(^u\)\(^m\)), we have characterised lipid mediator-signaling pathways in Drosophila. Dietary supplementation with PUFAs was able to modulate inflammation, with omega-6 fatty acids increasing, and omega-3 fatty acids reducing, the severity of the hop\(^T\)\(^u\)\(^m\) inflammatory phenotype. While COX and MPO inhibitors were able to reduce inflammation, LC-MS analysis of Drosophila extracts failed to detect the existence of prostaglandins or their precursor, arachidonic acid (C20). Interestingly, however, we have been able to detect C18 oxygenated metabolites known as hydroxy-octadecadienoic acids (HODEs). HODEs are generated by the oxidation of omega-6 (C18, n-6) fatty acid, linoleic acid. Chiral analysis via LC-MS indicated a higher proportion of the enzymatic chiral form is generated of both 9- and 13-HODE in Drosophila samples. Members of the MPO family are able to produce HODEs. We have identified 3 candidate Drosophila COX/MPO homologues: Pxn, Pxt and Irc that may generate HODE inflammatory lipid mediators. We have shown them to have both COX and MPO activity in vitro. Deletions of these genes reduced the inflammatory phenotype in the hop\(^T\)\(^u\)\(^m\) assay. Functional characterization using deletions of these genes showed affects on longevity as well as survival upon infection. We show that Drosophila could be used to investigate 9- and 13-HODE inflammatory effects in diseases such as atherosclerosis.

The human myocardium is a metabolic omnivore and utilises fatty acids, glucose, ketones, amino acids and lactate to produce energy. Altered metabolism results in cardiac muscle dysfunction and can play a potentially significant role in development of heart failure. Metabolic modulators like Perhexiline are potentially significant new treatments in the management of heart failure and coronary artery disease. Diabetes is a metabolic disorder that results in altered high energy phosphate kinetics in the myocardium. We demonstrate that microvascular disease plays little role in the development of impaired cardiac energetics in young patients with uncomplicated type 1 diabetes. We have shown an increase in left ventricular torsion in these patients with normal ejection fraction. Coronary microvascular disease and rotational deformation delay play a significant role in the development of increased torsion in these individuals which counteracts the early diastolic dysfunction. Furthermore the left atrial contribution to left ventricular filling is increased in these individuals. We demonstrate that Perhexiline has a differential action on insulin sensitivity in subjects with and without diabetes. It also increased plasma ketones and triglycerides in these patients. Finally we demonstrate that Perhexiline can be safely used and provides good relief of symptoms when used clinically in subjects with refractory angina and heart failure.

The headroom method proposes to offer medical device developers a simple way to integrate health economics into the decision of whether or not to develop a device, so that only commercially viable innovations are pursued. The aim of this PhD research is to evaluate the headroom method on this basis. A mixed-methods approach is used to assess both its prognostic ability and its usability by developers. Two prospective case studies demonstrate the method’s early application, whilst efficacy is assessed by twenty further case studies where the headroom method is applied retrospectively. The headroom method predicted NHS uptake with a sensitivity of 92% and a negative predictive value of 67%; however, results reflect the close-to-market context of the study sample. When numerical headroom assessments were considered alongside qualitative factors identified (relating to the clinical and market context), the method generally offered a good indication of future market potential. Interviews with twelve potential users of the method identified practical issues around time, expertise and objectivity, which varied according to the participant’s involvement in the innovation process. The headroom method is demonstrated to be a flexible approach, which could ground development choices in the value that a device might offer the health service.

Angiogenesis- growth of capillaries from a pre-existing network- can be induced in cold-acclimated fishes, where torpor onset and increased oxygen availability, suggests that the primary stimulus for angiogenesis is not metabolic. It was hypothesised that cold-induced angiogenesis was due to increased blood viscosity, therefore endothelial mechanotransduction of high shear stress, and that warm-induced capillary rarefaction was due to reduced shear stress. The reversal of elevated shear stress by vasoconstriction using the nitric oxide synthase inhibitor, L-NNA, and cyclooxygenase inhibitor, indomethacin had different effects. L-NNA administration hinted towards capillary regression at low temperatures but there was a trend towards increased capillarity at intermediate and high temperatures, whereas indomethacin had no effect. Neither warm acclimation nor vasodilatation using the α-adrenoceptor antagonist, prazosin, had an effect on capillarity. Investigation of the effects of NO on heart rate at high temperature showed NO may reduce heart rate at high temperature. However, this does not explain the trend towards an increase in capillarity with L-NNA at high temperature. Evidence is presented for the absence of eNOS in fishes suggesting either nNOS-derived NO or prostanoids are responsible for vascular tone. Microarray analyses were used to identify signalling pathways that would explain the discrepancies, but proved inadequate to reveal significant endothelial responses to cold acclimation.

Our knowledge of the released human ovulatory components, the cumulus-oocyte complex (COC) and follicular fluid, and their physiological effects on spermatozoa and roles in fertilisation remain poorly characterised. The aim of this study was to use a multi-pronged approach to begin to unravel these interactions and their relation to fertilisation success. Experiments designed to better replicate the physiological environment of the female tract showed environmental modulation of sperm motility. Mean sperm velocity values VSL, VAP and VCL increased by 12.4%, 15%, 16.5% respectively, when exposed to cumulus cells from pregnancy-positive donors, compared to pregnancy-negative donors. Follicular fluid elicited a [Ca2+]i response in spermatozoa that was independent of treatment outcome. The response of spermatozoa exposed to follicular fluid at a 50% (v/v) dilution was a large spike on the front of the ‘classical’ progesterone transient response, which has not been previously reported. Human sperm-zona binding (SZB) studies are hampered by the shortage of oocytes, and thus zona pellucida (ZP) available for research. As a possible source of ZP this study investigates the development of an in vitro model for oogenesis, utilising follicular fluid and cumulus cell co-culture with human embryonic stem cells. The feasibility of SPR technology, using both native and recombinant sources of ZP, to measure SZB and identify possible binding candidates is also assessed. The data in this study addresses just some of the potential effects of the COC and follicular fluid on spermatozoa. Further developments within this area may lead to better diagnostics and treatments for patients undergoing ART, in addition to providing targets for novel contraceptives.

Extra-renal synthesis of vitamin D3 has been reported in many tissues and cells including barrier sites where it induced immunomodulatory effects. I investigated local synthesis of vitamin D3 and its role in the induction of host defense peptides (HDPs) in human ocular barrier epithelial cells. I also examined the association between vitamin D receptor (VDR) single nucleotide polymorphism (SNP) with intermediate uveitis (IU) in Caucasians. Human corneal endothelial (HCEC-12), non-pigmented ciliary body epithelial (ODM-2), and adult retinal pigment epithelial (ARPE-19) cell lines, expressed mRNA and protein for VDR and vitamin D3 pathway elements and can locally synthesise 1,25(OH)2D3 in vitro. These cells upregulated mRNA, but not protein expression of HDPs in response to vitamin D3. IL-1β and TNF-\(\alpha\) did not synergise with vitamin D3 to upregulate HDPs in ocular barrier epithelial cells. VDR single nucleotide polymorphisms BsmI (rs1544410) is significantly associated with IU. Allele rs1544410-T and genotype rs1544410-CT were higher in IU patients than healthy controls. The results show that that extra-renal production of active vitamin D3 occurs in ocular barrier cells and may contribute to immune regulation in the eye. The association with SNP in the vitamin D3 receptor gene and intermediate uveitis suggests that genetic control of vitamin D3 may be linked to ocular inflammatory disease.

This thesis demonstrates that the Proline Rich Homeodomain transcription factor (PRH/HHEX) plays an important role in regulating the proliferation and migratory behaviour of breast cells. In tumourigenic MCF-7 breast cells, shRNA knockdown of PRH results in a pro-invasive and pro-proliferative phenotype. Key genes regulated by PRH in MCF-7 cells include TP53, endoglin (ENG) and e-cadherin (CDH1), which regulate migration/invasion in breast cells. Significantly, exogenous PRH functions as an inhibitor of cell proliferation/survival and migration/invasion in all breast cell types examined. Furthermore, the effects of exogenous PRH on cell proliferation/survival are dependent on the DNA binding activity of PRH. This work provides an explanation for the finding that PRH expression is associated with increased overall survival in breast cancer patients. In contrast with this work, MCF-7 xenograft experiments reveal that expression of exogenous PRH in MCF-7 cells is oncogenic. Furthermore, shRNA knockdown experiments in MDA-MB-231 cells show that endogenous PRH increases proliferation of these cells. This thesis therefore demonstrates that the role of PRH can differ dramatically between breast cell types and between ex vivo and in vivo conditions.

The doctoral research forming this thesis used mixed-methods to explore methodological and ethical issues associated with patient-reported outcome (PRO) measurement in clinical trials. A qualitative study of trial staff suggested there are perceived inconsistencies in the administration and management of PROs in some UK trials which could undermine PRO trial results and introduce bias. In addition, the study found that staff reported intermittently encountering ‘concerning’ PRO data in trials, but were unsure how it should be managed. A large-scale survey of UK-based trial staff and management demonstrated the genralisiability of these findings. A systematic review of PRO literature for front-line data collection staff found guidance was lacking. A large-scale review of PRO-specific literature for trial protocol developers suggested guidelines were inconsistent and difficult to access. Finally, using a novel PRO protocol checklist, a systematic review of trial protocols found that PRO information was commonly absent, even where a PRO was the primary outcome. In conclusion, the thesis highlights a need for the development of comprehensive consensus-based PRO guidelines addressing protocol development, training and the management of ‘concerning’ PRO data in trials; aiming to facilitate improvements in PRO protocol content and PRO assessment, whilst protecting the interests of trial participants.