The opportunistic human pathogen Pseudomonas aeruginosa controls virulence, production of secondary metabolites, motility, biofilm formation, growth in anaerobic conditions, intracellular and intercellular signalling and the switch from an acute to a chronic mode of infection at the transcriptional and post-transcriptional levels by modulation of the Gac/Rsm system. Cell density-dependent signal accumulation and environmental stimulators such as pH changes and ion limitation activate the GacS/GacA two-component system which in turn triggers transcription of the small regulatory RNAs RsmY and RsmZ. These sRNAs sequester multiple copies of the RNA-binding protein RsmA, antagonising its function. The RsmA/CsrA proteins act as translational repressors by binding to the GGA-motifs in the untranslated region of target mRNAs and blocking ribosome binding. In this study, the biological function of RsmN, an RsmA homologue with a conserved RNA-binding pocket but a distinct protein folding, the predicted autoregulatory mechanism of RsmN, the nature of target transcripts of RsmN, and the cross-regulation between the two Rsm proteins were investigated. The positive control of proteolytic and elastinolytic activities and swarming motility by RsmN has been demonstrated using single and inducible double deletion mutants of rsmN. Furthermore, rsmN deletion increased microcolony formation during biofilm formation. Regulation by RsmN was most apparent in the absence of RsmA, when rsmN expression was induced via a multicopy plasmid and at temperatures lower than 37°C. The double deletion of rsmA and rsmN affected growth, diminished proteolytic and elastinolytic activities, triggered autolysis and led to the increased secretion of the type VI secretion system protein Hcp1. Moreover, the double deletion of rsmA and rsmN altered the colony morphology of P. aeruginosa. Mutagenesis of the functionally critical, conserved RNA-binding residue which is identified as Arg44 in RsmA and Arg62 in RsmN resulted in the loss of RsmN function. In a genome-wide analysis by RNASeq, target transcripts were co-purified with RsmN from 37°C and 34°C cultures of a wild-type strain expressing rsmN in multicopy numbers. RNASeq results indicated that small regulatory RNAs such as CrcZ, RsmY and RgsA are common targets of RsmN and RsmA, whereas PhrS is a target of RsmN only. Other common RsmA and RsmN targets included transcriptional regulators, heat shock proteins, proteases, starvation response proteins, components of the denitrification pathway, outer membrane proteins required for pore formation, type III and type VI secretion system proteins and RsmA. Transcripts of heat shock proteins, the tss operon genes and rsmA were enriched by RsmN at 37°C but not at 34°C whereas the lasB transcript was enriched by RsmN at 34°C but not at 37°C. Based on the list of common targets of RsmA and RsmN and the results obtained from phenotypic assays, induction of the lytic Pf4 prophage, accumulation of alkyl quinolone or c-di-GMP signalling molecules, imbalanced redox state, carbon starvation, increased membrane permeability, and aggregation of misfolded proteins are suggested as possible mechanisms triggering the excessive autolysis of the rsmNind ΔrsmA mutant under uninducing conditions. The data gathered so far suggests that rsmN is differentially expressed, with increased RsmN activity at temperatures below 37°C in comparison with RsmA, and, RsmA and RsmN collectively contribute to the regulation of secondary metabolite production, motility and microcolony formation in P. aeruginosa.

Lipids are minor constituents of wheat flour, but are functional in bread making. As a result they have been extensively studied, but the published results are conflicting. It was therefore the objective of this study to understand better the impact of wheat lipids on wheat flour functionality and to propose approaches for positive modification of the flours. Initial work established quantitative and qualitative assessment methods for the non-starch lipids occurring in starch based systems. Seven solvent systems were compared for their ability to extract the non- starch lipids at ambient temperature. The choice of solvent system greatly influenced the total extractable amounts of lipid and there were differences in the relative composition. Bligh and Dyer methodology showed good extraction ability based on the total amount of non-starch lipids and there was good representation of the different lipid classes (neutral lipids, glycolipids, phospholipids). As this method was reasonable fast, with high extraction ability and showed low coefficient of variation (typically 16%) it was used in subsequent studies including the assessment of lipids in wheats of different variety and the levels and class of lipids segregating in the different mill fractions. As part of a larger study the impact of agriculture based variation in wheat flour chemistry and quality parameters was tested on high quality wheat varieties. Wheat variety was found as the major factor determining wheat flour properties, but environmental effects and post- harvest processing i.e. milling also had an impact. Correlations were made for the composition, flour quality parameters and the bread quality and results showed that whilst proteins are a major determinant for bread texture, lipids significantly influenced bread volume with non-polar lipids significantly and positively associated with bread volume, while the phospholipids reduced dough resistance and therefore may be associated with a decrease in loaf volume. Despite the acknowledged limitation of the data sets in some aspects, the wheats were tested for their “stability” within and over the growing sites. From these data, suggested wheat varieties worthy of further examination have been made. To see if positive modifications could be induced into wheat flours the technique of cold plasma (0.2-0.4 Wcm-2) was applied. It was shown that measureable free fatty acids and phospholipids significantly reduced after treatment and that there was a shift towards higher molecular weight proteins. Markers of oxidation increased and there was a significant increase in hydrogen peroxide and the formation of secondary oxidation products (n-hexanal). All the changes that occurred in the wheat flour during cold plasma treatment ultimately resulted in stronger dough. This suggests that cold plasma is an effective treatment for modifying functionality and enhancing the oxidative state of wheat flour.

Pre-eclampsia is a common hypertensive disorder of pregnancy that substantially affects maternal and neonatal morbidity and mortality worldwide. Despite decades of research, the aetiology of the disease remains poorly understood, and the clinical management of it is hampered by the lack of reliable diagnostic tests and effective therapy. Several screening tests have been suggested for the prediction of preeclampsia; however, none possess the sufficient specificity and sensitivity. Moreover, multiple pathways are known to be involved in the pathogenesis of pre-eclampsia, so it is very unlikely that a single or a small group of biomarkers will accurately predict the disease. In this thesis, two separate targeted LC-MS/MS methods were developed and validated to quantify plasma glycoproteins in pre- eclampsia to increase the pool of preeclampsia biomarker candidates and explore new pathways associated with the disease. The first method was hypothesis-driven and aimed to quantify the oxidation level of the plasma glycoprotein angiotensinogen (AGT), which has been proposed to be involved in the increased blood pressure characteristic of pre-eclampsia. The second method was hypothesis-generating and aimed to detect glycoprotein fold changes between different disease conditions using a simple and cost-effective conventional LC-MS/MS workflow. For both methods, a reproducible workflow for efficient glycoprotein/AGT extraction from human plasma was developed by coupling ConA lectin affinity chromatography with reversed-phase solid phase extraction fractionation (RP-SPE). Analysis of the enzymatically digested proteins was conducted using targeted LC-MS/MS working under the multiple reaction monitoring mode. For the quantification of the two distinct forms of AGT in the plasma (the sulphydryl-bridged oxidised form and the free thiol reduced form), a differential alkylation strategy was coupled with targeted LC-MS/MS to recognise and quantify the cysteine (Cys) peptides involved in the redox switch of AGT. The developed method enabled the reproducible detection of the two distinct forms of AGT in the plasma with CV% < 15%, and confirmation of the identity of the differentially alkylated Cys peptides was supported by LC-MS/MS. Analysis of clinical plasma samples using the developed method showed a significantly higher level of the oxidised AGT in pre-eclamptic women compared to gestational age-matched normotensive controls (P=0.008), whilst maintaining a similar total AGT level in the plasma. The research findings indicate that the elevated level of oxidised AGT rather than its total level might be a contributing factor to the hypertension characteristic of pre-eclampsia, and provide an extra line of evidence linking the oxidative state and the generation of reactive oxygen species with hypertension in pre-eclampsia. In the second part of the research, 54 clinically relevant glycoproteins were selected to be profiled by label-free targeted LC-MS/MS. Measurement of the analytical precision of the method revealed acceptable CV values for the majority of the assays (median CV 11.8%). Analysis of plasma samples collected from early- and late-onset preeclamptic women using the developed glycoprotein profiling methodology successfully identified significant changes in the level of several proteins in pre-eclampsia. Two of them, apolipoprotein D and kallikrein, are reported for the first time to be altered in the plasma of pre-eclamptic women suggesting that they could be further evaluated as novel biomarkers. Some pre-eclampsia-relevant pathways and biological processes, including iron transport and metabolism, coagulation, and lipid metabolism and oxidative stress were found to be altered in the disease. Moreover, different glycoproteins were changed in early-onset compared to late-onset pre-eclampsia which might reflect different pathophysiological mechanisms. Additionally, the method was applied to identify any altered glycoproteins in plasma samples from women with polycystic ovary syndrome (PCOS). These were subsequently compared with those found to be altered in pre-eclampsia, resulting in the proposal of possible underlying pathophysiological mechanisms that may explain the reported association between the two conditions, such as hypofibrinolysis and thrombophilia and iron overload. Moreover, the study detected, for the first time, significant changes in the plasma levels of vitronectin and insulin growth factor acid labile subunit, suggesting that these may also be further appraised as potential biomarkers for the diagnosis of PCOS. Taken together, the two targeted LC-MS/MS methods developed in this thesis provided relevant information regarding pre-eclampsia by identifying potential pre-eclampsia protein biomarkers. This sheds light on the different biochemical processes altered in the disease and points to possible pathophysiological mechanisms that might assist in explaining the link between PCOS and pre eclampsia, all of which should improve the understanding of the molecular mechanisms of the disease. The present work offers information that may play a key role in improving the health care of women with preeclampsia and serves as a foundational cornerstone for future work.

Chapter 1: Introduction This Chapter provides an introduction to the study of reactive intermediates and the specialised techniques which have been developed in order to study these extremely short lived species. In particular, it provides an overview of ultrafast Time Resolved Infrared Spectroscopy (TRIR). This key technique underpins a large amount of the work presented in this Thesis as well as a description of the apparatus and methods used across the other Chapters of this Thesis. Chapter 2: Ultrafast TRIR studies of rhenium complexes of thioether substituted hexaazatrinapthylene ligands The excited states and photophysical properties of mono, bi and trinuclear (Re(CO)3Cl) complexes of the 2,3,8,9,1314-hexa (octyl-thioether)-1,6,7,12,13,18-hexaazatrinapthylene ligand (HATN-S(C8H17)6) have been investigated using ultrafast TRIR spectroscopy. These measurements are supported by theoretical calculations and resonance Raman spectroscopic investigations of these compounds. The position and the intensity of the (CO) bands of the photoexcited species directly report on the electron transfer/distribution in the excited state. In all cases the observed product bands were blue shifted compared to the parent, this is consistent with electron transfer away from the metal centre. The observed shift for the thioether substituted HATN complexes studied here was smaller than that reported for the analogous HATN-Me6 complexes, indicating reduced electron transfer upon excitation. Red shifted product bands corresponding to “spectator” metal centres were also observed for the bi and trinuclear species, these bands were shifted by approximately the same amount as for the HATN-Me6 species, indicating a similar reduction of the HATN core upon excitation. These results are consistent with the formation of a dual charge transfer ILCT/MLCT excited state involving electron donation from both a metal centre and the thioether moieties upon excitation. Chapter 3: Ultrafast TRIR studies of rhenium and platinum complexes of Pyridyl-1,2,3-Triazole and related ligands The excited states and photophysics of a series of substituted rhenium and platinum complexes that contain a 2-pyridyl-1,2-3-triazole ligand, an easily modified analogue of the common 2,2’-bipyridine (bpy)ligand, have been studied by TRIR spectroscopy. The compounds are found to be analogous of similar complexes with bpy, exhibiting MLCT or MLLCT exicted states (in the case of the (Re(CO)3X) and (Pt(CCHPh)2) complexes respectively). Changing the substitution of the triazole ring has little effect on the excited state band positions, this is attributed to the insulating effect of the triazole moiety. Complexes substituted with conjugated phenyl groups on the triazole ring exhibit greatly increased excited state lifetimes than those substituted with non-conjugated benzyl groups. This is attributed to the rigid rotor effect. The addition of an electron donating triphenylamine group to the pyridyl moiety results in the observation of ILCT states. Chapter 4: Towards long lived alkane and noble gas complexes from cationic piano stool complexes in the fluorous phase The synthesis and characterisation of a series of cationic transition metal tricarbonyl half sandwich complexes solubilised in perfluoroalkane solvents by means of the weakly coordinating, fluorous solubilising anion tetrakis(3,5-bis(perfluorohexyl)phenyl)borate (BArF64) is described. These complexes were photolysed in perfluoroalkane solutions doped with methane, heptane and xenon and followed by ultrafast TRIR spectroscopy with the aim of generating long-lived organometallic alkane and noble gas complexes suitable for study by NMR at room temperature. While no organometallic alkane or noble gas complexes were observed, several novel transition metal-BArF64 zwitterionic species are generated upon photolysis. These species are the result of -2 binding of one of the aryl moieties of the BArF64 anion to the vacant coordination sites generated following photoejection of a carbonyl ligand. While highly reactive they are long lived and appear to be permanent photoproducts. In the case of the rhenium complex an additional product band assigned as a dimeric species is also observed. Chapter 5: Towards the synthesis of fluorous phase soluble cationic piano stool complexes. The synthesis of fluorous tagged cationic transition metal tricarbonyl complexes is explored with the eventual aim of performing fluorous phase TRIR experiments utilising the more weakly coordinating (but less solubilising) anion tetrakis(perfluoro-tert-butoxy)aluminate in place of the previously studied BArF64 anion. Several routes to perfluoroalkyl arene ligands are explored and evaluated, as is the use of different lengths of alkyl spacer to insulate the ligand binding site from the strong electron withdrawing effects of the perfluoroalkyl moiety. This process lead to the synthesis of the new complexes [(m-(3-(perfluorooctyl)-propyl)n-benzene)manganesetricarbonyl] hexafluorophosphate for n =1-3. These compounds are soluble in the fluorous phase despite their charge. The use of the m-(3-(perfluorooctyl)-propyl)n-benzene ligand to solubilise complexes of rhenium and rhodium was also attempted. The addition of methyl groups to the ring was explored as a route to additional steric bulk and a more electron rich, strongly binding ligand. In addition to arenes, the fluorous tagging of other ligand systems was also investigated, including cyclopentadienyl and tris(1-pyrazolyl)methane.

Replication of DNA is a vital process for growth and cell division in all domains of life. The mechanisms of helicase loading as well as hand-off of nascent RNA primers from the primase to the replicative polymerases during DNA synthesis are of fundamental importance. The prototype structural/functional model provided by the widely studied Gram-negative Escherichia coli DNA replication system is not universally applicable. The current model for the Gram-positive Bacillus subtilis DNA replication suggests that PolC is the main processive replicative polymerase with DnaE playing an essential but minor role during DNA synthesis. Our results suggest that DnaE is a major polymerase important for DNA replication and DNA repair. DnaE polymerase activity is stimulated in the presence of SSB, the clamp DnaN and PolC polymerase, its error-prone synthesis is modulated by the aforementioned proteins and its errors are corrected in trans by the PolC exonuclease domain, in a template specific ternary DNA-DnaE-PolC complex. Hereby, we propose a new revised model for DNA replication in Bacillus subtilis, where DnaE is the major replicative polymerase on the lagging strand and PolC, which is the major polymerase on the leading strand, provides the crucial proof-reading activity in trans. These findings revise the current model in Bacillus subtilis and suggest a division of labour between DnaE and PolC polymerases.

In eukaryotic cells, the degradation of the mRNA poly(A) tail (deadenylation) is a crucial step in the regulation of gene expression. The Ccr4-Not complex is the major deadenylase enzyme involved in the mRNA deadenylation. The complex is composed by two subunits with ribonuclease activity (Caf1 and Ccr4) and at least six non-catalytic subunits. In vertebrate cells, the duplication of the catalytic subunits Caf1 (encoded by CNOT7 and CNOT8) and Ccr4 (encoded by CNOT6 and CNOT6L) lead to the heterogeneity of the complex. The non-catalytic subunits are organised in modules, each with a specific function, allowing the recruitment of the complex on specific mRNAs. Regulatory proteins interact with the deadenylase complex tethering it on specific mRNAs and activating mRNA degradation pathways and down regulating protein expression. This study discovered and explored the interactome of the catalytic subunits Caf1 and Ccr4 and detected new interacting partners that may recruit the deadenylase complex on specific mRNAs. Nowadays, genotyping patients using whole genome and next generation sequencing technologies, allows a wider but more accurate sight of the genomic contest of a specific disease. The alteration of the function or the structure of the Ccr4-Not complex in cancer was assessed investigating the structure and function of the variants of the components of the NOT-module (CNOT1, CNOT2 and CNOT3) and the nuclease sub complex (CNOT7, CNOT6L, CNOT1, and the regulatory protein BTG1) found in various types of cancers. Finally, phylogenetic analysis of 15 mammalian species identified different evolutionary rates acting on the paralogous deadenylase subunits.

In vertebrates, haematopoietic stem cells (HSCs) maintain the blood system throughout life. HSCs are believed to arise during embryogenesis from haemogenic endothelial cells (HECs) that undergo an endothelial-to-haematopoietic transition (EHT). Here, in order to trace the progeny of the embryonic ECs in zebrafish, an inducible CreERT2-LoxP system was used. Following short-term induction of the Cre recombinase during early embryonic stages, Cre reporter gene expression was observed in early larval haematopoietic cells (HCs). At adult stages, PCR revealed the presence of the recombined Cre reporter gene in HCs, demonstrating that adult HCs had originated from embryonic ECs. In zebrafish, HECs of the ventral wall of the dorsal aorta (vDA) are thought to form HCs by basal epithelial-to-mesenchymal transition (bEMT), a process that depends on the transcription factor Runx1. Here, making use of the recently identified gfi1aaqmc551Gt line, confocal microscopy showed that qmc551: GFP+ cells were HECs. While most GFP+ cells underwent bEMT, some displayed a novel type of apical departure. In runx1morphants, bEMT was abrogated and most GFP+ HECs remained in the vDA. Apical departure, however, was still observed in the absence of Runx1, suggesting a fundamental difference in the underlying mechanism. While gfi1aa expression was lost in vDA HECs of qmc551 homozygous embryos, EHT of GFP+ HECs was completely unaffected. Up regulation of its paralogue Gfi1ab suggested functional redundancy. To study this redundancy, the CRISPR/Cas9 system was used to mutate the gfi1ab gene. Here, the mutant gfi1ab alleles qmc552 and qmc553 were identified. Both alleles, as well as a third allele sh320 that was generated by a collaborator, encode truncated, most likely non-functional proteins. Initial data on gfi1aaand gfi1ab double mutant embryos showed a defect in definitive haematopoiesis. Whether HECs were affected and blocked in their ability to undergo EHT remains to be determined.

Myocardial infarction is a medical emergency and needs an immediate treatment with reperfusion therapy. But, reperfusion itself causes damage to the myocardium by overproduction of reactive oxygen species (ROS). These excessive ROS generated during reperfusion lead to oxidative stress in the myocardium and damage to the cardiomyocytes. Reperfusion injury has been suggested to be responsible for up to 50% of the final infarct size of the myocardium and this resultant infarct size is highly related with the development of chronic heart failure. Although both the superoxide anion and hydrogen peroxide are elevated following myocardial infarction, the levels of their endogenous scavenger enzymes, superoxide dismutase and catalase, decrease even more. Catalase is a major antioxidant enzyme in the body’s endogenous antioxidant defence systems and this enzyme is considered to be responsible for most of the peroxidase activities in the cardiomyocytes. Delivery of active proteins such as catalase to the myocardium is challenging as they can easily be inactivated by proteases, aggregation and natural inhibitors. Delivery of therapeutic proteins using polymeric nanoparticles can offer an attractive approach. No studies on evaluation of the cardio-protective effect of catalase-loaded nanoparticles in conserving cardiomyocytes from the oxidative stress induced by reperfusion injury have been reported yet. Poly(lactic-co-glycolic acid) (PLGA) is a US Food & Drug Administration (US FDA)-approved biocompatible and biodegradable polymer, and this polymer was used in this study to prepare catalase-loaded nanoparticles. In the double emulsion method of fabricating protein-loaded nanoparticles, emulsification process induces denaturation of proteins. No reports have showed the effects of trehalose and bovine serum albumin on the encapsulation of catalase into PLGA nanoparticles and comparison of their effects yet. This study showed that trehalose and bovine serum albumin added to the initial aqueous phase as a stabilizer of the catalase activity were observed to be effective in reducing loss of enzymatic activity of catalase during emulsification steps of the enzyme encapsulation process in the double emulsion method. Surprisingly, unlike BSA-stabilized catalase-loaded PLGA nanoparticles, trehalose-stabilized nanoparticles showed very low enzymatic activity which was comparable to the catalase activity of the unstabilized nanoparticles. Freeze-drying is a useful technique to stabilize the nanoparticles and proteins. But, the process of freeze-drying itself exerts stresses resulting in aggregation of the nanoparticles and denaturation of the proteins. Cryoprotectants are added to protect the nanoparticulate systems from the damage induced by the freeze-drying process. Trehalose is a well-known cryoprotectant. No studies on the cryoprotective effect of trehalose in reducing the loss of enzymatic activity of the encapsulated catalase upon freeze-drying of the catalase-loaded PLGA nanoparticles have been reported yet. In this study, trehalose added as a cryoprotectant minimized the loss of enzymatic activity of the encapsulated catalase of catalase-loaded nanoparticles in a dose-dependent manner. Cryoprotected catalase-loaded nanoparticles were found to be effective in reducing oxidative stress-induced damage in a widely recognized hydrogen peroxide-induced cellular model of oxidative stress in HL-1 cardiomyocytes. This protective response was dose-dependent. The nanoparticles also demonstrated efficacy in reducing apoptosis that plays an important role in myocardial cell death during reperfusion of the myocardium and is linked with the development of heart failure following myocardial infarction. Structural study of the encapsulated catalase of catalase-loaded nanoparticles was performed by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy and fluorescence spectroscopy which showed that trehalose could reduce the encapsulated enzyme’s structural alterations induced by freeze-drying of the nanoparticles. The promising cardioprotective effect demonstrated by the cryoprotected catalase-loaded PLGA nanoparticles in rescuing cardiomyocytes from oxidative stress, makes this catalase-loaded nanoparticle a valuable candidate for the treatment of reperfusion injury and warrants further investigation including animal trials.

Introduction: The P2Y14 receptor, with its unique pharmacologic profile, is the only member of its P2Y family that is activated by UDP-sugar nucleotides as well as MRS2690, the synthetic analogue of UDP-glucose. Its role in the immune and inflammatory systems is well documented; however its role in the cardiovascular system and, particularly, the vasculature is beginning to emerge. Aims and Objectives: The aim was to provide evidence for the expression and the role of the P2Y¬14 receptor in porcine coronary arteries. The objectives of the study were to characterize the P2Y14 receptor response profile in the presence of a P2Y14 receptor antagonist, PPTN, in the porcine coronary artery using organ bath pharmacology. The effects of non-selective P2 receptor antagonists, suramin and PPADS and the P2Y¬6 receptor antagonist MRS2578, on responses to UDP-glucose and MRS2690 were investigated. P2Y14 receptor protein expression and receptor localization was explored using Western blot and immunohistochemistry. P2Y14 receptor coupling to the inhibition of adenylyl cyclase/cAMP activity, using a vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) bead assay, in porcine coronary arteries was determined. Human isolated red blood cells (RBCs) and platelets as sources of UDP-glucose were investigated. Hypoxia-induced release of UDP-glucose from porcine coronary arteries was also investigated. The function of the P2Y14 receptor as an anti/pro-inflammatory stimulus in porcine coronary arteries was also explored. Results: UDP-glucose, UDP-N-acetylglucosamine and UDP-glucuronic acid elicited concentration-dependent contractions in the porcine coronary artery; MRS2690-induced contractions were 10-fold more potent than those of the UDP-sugars. The contractile responses to MRS2690, but not UDP-glucose, were significantly inhibited by PPTN (3 µM) (in presence of U46619 and forskolin to precontract and raise cAMP, respectively). The contractile responses to UDP-glucose, but not MRS2690, were altered (either inhibited or potentiated) in the presence of suramin and PPADS. MRS2578, a P2Y6 receptor antagonist, did not change the contractile responses to UDP-glucose or MRS2690 in U46619 preconstricted coronary arteries. MRS2578 did not change the contractile responses to MRS2690, but did to UDP-glucose (in the presence of forskolin plus U46619). Immunoblotting showed that the P2Y14 receptor protein exists in two isoforms, sized 41 kDa and 61 kDa in porcine coronary arteries. Immunohistochemistry revealed localization of P2Y14 receptors in the vascular smooth muscle cells and endothelium of porcine coronary arteries. UDP-glucose and MRS2690 significantly decreased VASP-P in coronary arteries, an effect which was partially reversed in the presence of PPTN. Red blood cells induced contractions in preconstricted porcine coronary arteries; the response remained unchanged in the presence of PPTN. Platelets induced relaxations and contractions in the absence and presence of L-NAME (inhibitor of nitric oxide synthase), respectively, in porcine coronary arteries; the response remained unchanged in the presence of PPTN. PPTN does not change hypoxia induced relaxation in preconstricted coronary arteries. UDP-glucose and MRS2690 had no significant effect on the release of cytokines/chemokines from segments of coronary arteries. Conclusion: The action of UDP-sugars and MRS2690, P2Y14 receptor agonists, as inducers of contraction is consistent with an involvement of functional P2Y¬14 receptors in porcine coronary arteries. Inhibition by PPTN, a selective P2Y14 receptor antagonist, of contractile responses to a selective P2Y14 receptor agonist, MRS2690, further provides evidence of P2Y14 receptors in porcine coronary arteries. Release of purine and pyrimidine nucleotides is known to be exaggerated in pathophysiological conditions, such as shear stress, endothelial cell damage and hypoxia. This may elicit chronic activation of P2 receptors including P2Y14 receptors, consequently causing increased vasoconstriction of porcine coronary arteries. The patho/physiological sources and stimuli for local UDP/UDP-glucose release remain to be determined since PPTN did not block responses to RBCs, platelets and hypoxia under the conditions of the present study. The P2Y14 receptor may be a novel target to control abnormal vascular contraction under pathophysiological conditions.

Background: Poor adherence to inhaled corticosteroids (ICS) is known as the main cause for therapeutic failure in asthma treatment and associated morbidity. Adherence is complex and can have many causes, which will vary between conditions, treatments and patients. To improve adherence, it is vital to understand what effects a patients adherence, so appropriate interventions can be developed and targeted, both for the patients who would benefit most and at the most important points in treatment. Very few studies have characterised the variables associated with poor adherence and how these differences may change over time, and the most appropriate methodology for investigating this relations have not previously been defined. Aims and objectives: The aim of the PhD study was to investigate what characteristics associated with a patient’s adherence to ICS, and to investigate whether these relationships change over time using a large primary care dataset. The objectives included the development of a longitudinal measure of asthma patients’ adherence to ICS, then to investigate the time dependent relationship between adherence and other available patient variables by trialling a number of different methods. In addition, the effect of adherence on clinical outcome in asthma was tested, since counter intuitively many studies have not previously found a clear relationship between the variables. Methods: A retrospective longitudinal study using a large cohort was conducted using primary care data from the Clinical Practice Research Datalink (with Hospital Episodes Statistics data) between 1997 and 2010. Asthma patients aged between 12 and 65 years, without a diagnosis of chronic obstructive pulmonary disease were included in the study cohort. ICS prescriptions were extracted and used to calculate the annual prescription possession ratio (PPR). Several definitions of the PPR measure were tested to develop a proxy measure to represent adherence. Variables related to clinical outcomes and other characteristics were also identified for each patient in the cohort. A two-way analysis was conducted to compare the relationship between adherence and each patient variable with time, and then four methods were used to further investigate the relationship between adherence and patient exacerbations including; (1) comparing adherence in the year before and after an exacerbation; (2) descriptively exploring the clinical outcomes associated with different adherence levels; (3) identifying the relative risk of an exacerbation associated with adherence defined by different cut off levels of PPR; and (4) descriptively exploring the effect of adherence on outcome and outcome on adherence over time. Finally, the available variables associated with adherence (including previous adherence and clinical outcomes) were analysed in a dynamic panel model to understand explore the effect of variables on patients’ adherence to ICS which allows for the feedback effects of previous adherence and clinical outcome and the effect of time on adherence. Results: Many patient variables were found to effect adherence. When modelling the effect of patient variables on adherence, adherence was found to be lower in younger patients (+0.11%/year), patients with fewer years in the study (+0.25%/year), with more severe asthma (step 5 patients had a 3.32% lower PPR than step 2 patients), with good control (5.21% lower), with lower previous adherence (-0.51% per % PPR), and who had not previously experienced an exacerbation (0.87% lower compared with patients who had experienced no primary car exacerbation and 1.45% lower for those who experienced no secondary care exacerbation). Adherence increased with patient year, consistently across most subgroups, with the following exceptions; the 20-25 year old age group had lower initial adherence (53.9%) than the younger patients (58.3%), patients registered in the East Midlands had the lowest adherence (57.7%), but increased over time to become the highest (90.7%) and in the first year of the study the adherence for patients treated at step 2 of the guidelines was the lowest (57.5%) but it increased over time to become the second highest step (85.7%). Conclusion: This longitudinal follow-up study using electronic patient records over time was useful to identify the effect of multiple patient variables on adherence. The main characteristics associated with poor adherence were the characteristics that we would associate with better health, or less severe asthma. Therefore, the interventions to improve adherence or to review the appropriateness of treatment should be developed to target younger patients, early on in treatment before they have experienced an exacerbation of their asthma symptoms. The PPR measure developed was useful to measure changes in adherence over time, as a measure of the maximum amount of medicine that the patient had available to them, expressed as a percentage of their recorded prescribed dose. However there are some important limitations to the PPR measure including most importantly that adherence must be measured against a routinely prescribed daily dose of ICS and medicine prescribing and not medicine taking is measured, meaning that adherence is likely to be overestimated. The methods used to analyse the adherence measure had not previously been used to assess adherence in asthma. By using the results from each analysis method, information about different parts of the relationship between adherence and other patient variables including their exacerbation risk and time could be combined, which uniquely allowed the longitudinal measurement and analysis of adherence in asthma over extended study duration.