Higher education in the twenty-first century is experiencing transformational change due to the advances in technology, with this period referred to as the Fourth Industrial Revolution – the Information Age. Just as the three previous revolutions created step changes in society so will this one, and as the changes now are occurring over a much shorter time period academics, educators and universities have less time to understand and respond to these events. The three key technological changes are firstly the availability, power and pervasiveness of computers, secondly the development of the Internet and finally how these factors have affected knowledge and learning, in the new millennium. These changes in the Information Age have influenced learning theories and learners, with the rapidity meaning there is less time to consider and investigate how technology can be used to enhance student learning in higher education. The opportunities technology provide to improve student learning in higher education range from the design of small educational resources to overarching curricula and educational organisations themselves. This work investigated the design of small educational resources called learning objects and in particular, the storyboard creation aspect of this process and then the educational gains achieved from using said resources. The established knowledge of resuscitation was a suitable vehicle to investigation the design of learning objects as it has a strong internationally accepted theoretical foundation and nurses are required to learn this knowledge as part of their pre-registration education. The Storyboard Workshop (phase 1) of this research investigated how learning objects are designed by nursing students (n=7) and by tutors (n=6), by applying Tuckman’s stage of group development model revealing how each homogenous group functioned and what twelve pedagogical factors student-designers and tutor-designers felt important when analysed using the Learning Object Attributes Metric (LOAM) Tool. In the Learning and Evaluation (phase 2) of this investigation, novice nursing student were randomly assigned to view either the student-designed (n=58) or tutor-designed (n=61) learning object to acquire established resuscitation knowledge with the learning gain and acceptability of the resource viewed, assessed. The results of phase 1 revealed student-designers and tutor-designers generally discussed similar LOAM pedagogical factors though students spent more time discussing navigation and tutors focussed on the objective. When Tuckman’s model was applied the student-designers spent significantly less time forming and storming and significantly more time performing than the tutor-designers, suggesting when designing learning objects on established knowledge, students focus on the task whereas tutors may refer to professional experience that may distract from the design process. Phase two demonstrated irrespective of the designers, viewing either the student-designed or tutor-designed learning object conferred significant learning gains when pre and post viewing (knowledge, student-designed 4.3 to 8.3, p=.000; tutor-designed 4.4 to 8.2, p=.000 and confidence in knowledge, student-designed 5.4 to 7.5, p=.000; tutor-designed 5.3 to 6.9, p=.000) was assessed. However, the difference in confidence in knowledge significantly favoured the student-designed resource (2.1 v 1.5, p=.042), though both resources were very positively evaluated. In the design of a learning object it may be the student-designers are more attuned to their peers needs, and this effect could be exploited by ensuring students are integral in the design of a learning object for novice student to use when acquiring established knowledge. In addition, this effect may be applicable with projects to design learning objects for novice learners to acquire established knowledge, whether this has a clinical focus or for novice students in non-healthcare disciplines.

A nonsynonymous single polymorphism (SNP) in G protein-coupled receptor kinase 5 (GRK5) was discovered in 2008 that changes the amino acid at the position 41 from Glutamine (Q) into Leucine (L) (Liggett et al., 2008). The putative functions of the GRK5-L41 polymorphism were reported to be involved in faster desensitisation of both b1 and b2 adrenergic receptors in vitro and the cardiac protective functions by improving the survival rate of patients with heart failure conditions or transplantation in vivo. Nevertheless, the mechanisms underlying these processes are still poorly understood, which is the purpose of this thesis. This thesis presents the establishment of the first human cardiac models of GRK5- L41 polymorphism using human pluripotent stem cells (hPSCs) and their derived cardiomyocytes (CMs). The thesis is distributed into four main themes, containing (1) the formulation of monolayer cardiac differentiation protocols; (2) the establishment of human induced pluripotent stem cells from lymphoblastoid cell lines bearing the GRK5- L41 sequence; (3) the development of footprint-free and shortcut CRISRP/Nickase approach that allowed generating the gene-edited human embryonic stem cells expressing GRK5-Q41, GRK5-Q/L41, and GRK5-L41; and (4) the evaluation of GRK5- L41 functions using cardiac functional analysis assays. Relating to disease modelling and cardiovascular biomedical research, the ability to differentiate the hPSCs into CMs plays a critical role by providing an unlimited resource of human CMs for in vitro testing and experiments. Here, three main monolayer cardiac differentiation protocols, including E8-AB, mTeSR-AB, and mTeSR-CHIR, were described in details and proven to be highly consistent, efficient, robust, and reproducible. Additionally, these protocols have been ascertained to be effective in more than 27 hPSC lines routinely maintained in the lab regardless of the culture conditions (non-defined vs. defined culture conditions), cell types (human embryonic stem cells vs. human induced pluripotent stem cells), reprogramming methods, and somatic cell sources (in the case of induced pluripotent stem cells). Indeed, by using the E8-AB protocol, more than 1x107 CMs/line have been produced in this thesis, providing enough resource for functional assay analysis and mechanistic studies of GRK5-L41. Furthermore, two independent approaches were made to create the human cardiac model of GRK5-L41 polymorphism, involving the establishment of GRK5-L41 bearing hiPSCs from lymphoblastoid cell lines, and simultaneously introducing the GRK5-L41 sequence to the HUES7 genome to create the Q/L41, and L41 expressing HUES7 lines. In general, four hPSC lines were successfully generated in this thesis, including hiPSC-GRK5-L41, hiPSC-GRK5-Q41, HUES7-GRK5-Q/L41, and HUES7- GRK5-L41. The H-Fib-hiPSC, cell lines generated from HUES7-derived fibroblast, was the additional line obtained after testing the effectiveness of episomal plasmid. All cell lines were able to differentiate into CMs at high purity, approximately 85%, and were used for the development of functional assays. Four main functional experiments were developed focusing on the GRK5-related functions in the heart, consisting of contractility and hypertrophic response to catecholamine induction, especially during the chronic response. The effects of catecholamine, in this case, Isoprenaline (ISO), on the contractility of the CMs were measured by two assays, the CardioExcyte96 platform detecting the contraction rate and beating pattern in real-time, and the LANCE Ultra cAMP assay assessing the production of cAMP. These results indicated that extended culture of CMs in ISO (>30h) introduced the detrimental effects on the contractility and beating pattern of the CMs in vitro, generating the arrhythmias in GRK5-Q41 CMs. Interestingly, the GRK5-L41 CMs exhibited a high level of the beat rate in response to ISO and maintained it constantly during prolonged exposure to ISO similar to that of b-blocker treatments in GRK5-Q41 CMs. The Western Blot analysis of the cellular distribution of GRK5 spotted the localisation of GRK5 during ISO treatment for 72h. Further characterisation using immunofluorescence analysis of chronic exposure to ISO demonstrated the elevation of BNP level, a hypertrophic marker, indicating that ISO treatment duration (>30h) induced the hypertrophic response of hPSC-CMs in vitro. Taken together, the findings within this thesis has been the first step in a discovery process of the cardiac protective functions of GRK5-L41 polymorphism during heart failure. Despite the presence of limitation and difficulty, it manages to provide sufficient information to explore further the interrelationship between nuclear accumulation of GRK5, hypertrophic response, and contractility regulation mediated by either GRK5-Q41 and GRK5-L41 in hPSC-CMs in vitro.

Cardiovascular disease, of which ischaemic diseases such as stroke and peripheral arterial disease make up a large proportion, are the leading cause of death worldwide. The endogenous response to ischaemia is to upregulate growth factors to stimulate the growth of new vessels and in some cases, form a collateral network. The concept of therapeutic stimulation has therefore become a priority area of cardiovascular research. The collateral network formed must consist of intact, stable, non-leaky vessels that can respond appropriately to stimuli. While the potent angiogenic driver, vascular endothelial growth factor (VEGF) appeared to be a promising target, trials in peripheral ischaemia patients have been disappointing. Upregulated by VEGF during angiogenesis, the Notch ligand Dll4 is a key regulator of vessel maturation and function. Widely known to regulate tip and stalk cell selection during sprouting angiogenesis, its role in the recruitment and growth of mural cells, regulation of permeability, compliance and signalling is poorly understood. Inhibition of Dll4 results in non-functional, poorly perfused vessels with reduced pericyte coverage suggesting a greater role for Dll4 in the formation of mature, intact, operative vessels. I therefore tested the hypothesis that the physiological characteristics of the neovasculature are regulated by Dll4 during physiological neovascularisation. A recombinant human sDll4 protein was used to induce Notch signalling in an endothelial cell monolayer and resulted in increased expression of VE-Cadherin and cell-cell contacts. Using the Landis-Michel microvascular permeability technique, Dll4 signalling was shown to decrease the permeability of rat mesenteric vessels and then subsequent experiments showed that this could be prevented by the proteins kinase A (PKA) inhibitor H89 dihydrochloride. In the rat mesenteric angiogenesis model, inducing Notch with an adenovirus (Ad.) encoding sDll4 (Ad.sDll4) resulted in a less angiogenic vasculature and when added into arteriolargenesis stimulating adenovirus combinations it led to a more endogenous-like vasculature and a switch from sprouting to branching. In a mouse hindlimb ischaemia model, Ad.sDll4 hindered blood flow recovery to the hind paw but led to increase capillary and arteriolar density. These results show that Dll4 plays a key role in regulating vascular permeability and that this is through a cAMP/PKA dependent pathway and involves VE-Cadherin expression. They also demonstrate a role for Dll4 in arteriolargenesis in both physiological and pathological settings and the possibility of using Dll4 as part of an ischaemic therapeutic strategy cannot yet be ruled out.

Introduction: Rare diseases have gained recognition over the past decade as an important area for health service improvement. They affect 1 in 17 people over a lifetime, consume 14% of the NHS budget, and are an important cause of illness and death[1]. In 2013, the Department of Health published for the first time a “UK Strategy for Rare Diseases” which identified 5 key recommendations for improving the lives of people with rare diseases, and included the need for epidemiological studies. 75% of rare diseases are genetic in origin, and have onset in childhood. However, rheumatologists provide care for people with a number of rare diseases, amounting to an important component of the 25% of rare diseases which are believed to be non-genetic and present in adulthood. One of the main challenges in studying rare disease, is the difficulty in finding enough people with the disease to study. Recent linkage of databases of primary and secondary care health records in the UK provide an opportunity to study representative samples, large numbers of people, and the breadth of healthcare provision. In this thesis, I report 4 epidemiological studies in routinely collected healthcare data, conducted in an exemplar rheumatic disease, ANCA-associated vasculitis. Methods: I used routinely collected healthcare data from local hospitals, a large database of UK general practice records, and linked hospital episode statistics to identify cases of ANCA-associated vasculitis. The specific questions addressed in the studies were: 1. What are the current incidence, prevalence and mortality of ANCA-associated vasculitis in the UK, stratified by age, sex and ethnic group? I addressed this question in a local hospital cohort, and then in linked primary and secondary care records in England. I used projections of the population structure in 20 years’ time to predict the expected number of incident and prevalent cases. 2. What is the natural history of the most common type of ANCA-associated vasculitis (granulomatosis with polyangiitis) prior to diagnosis, and are there opportunities to diagnose it sooner in primary or secondary care? I conducted a case-control study in a large database of primary care records, and attempted to develop a model for GPs that predicted the risk of a person having granulomatosis with polyangiitis, such as is advocated by the UK Strategy for Rare Diseases. 3. What are the strongest aetiological factors in granulomatosis with polyangiitis in the UK? I compared the frequency of possible aetiological exposures between cases and population-based controls in a large database of prospectively collected primary care data. Results: 1. There were about 1300 new cases of ANCA-associated vasculitis in the UK in 2015, more than previously thought. The incidence has been stable since 2000, however the disease is more common in older people, therefore due to predicted aging of the UK population there will be 34% more cases in 2035. We are not able to detect any differences in incidence rates between people from different ethnic groups, but our studies lacked power for this question. 2. People with granulomatosis with polyangiitis consulted their GP more than healthy people prior to their diagnosis. However, they consulted with a wide variety of symptoms, and none were highly predictive of the diagnosis. In addition, the lower the prevalence of the disease, the lower the positive predictive value of a diagnostic model. Granulomatosis with polyangiitis is rare, so even with an exceptionally well-performing model (with a sensitivity of 100% and a specificity of 90%) only 2 of every 10,000 people flagged as ‘at risk’ would have granulomatosis with polyangiitis, and the rest would have false positive results. 3. People with granulomatosis with polyangiitis were 5 times more likely than population-based controls to have a previous diagnosis of bronchiectasis, and 2-3 times more likely to have a previous diagnosis of an autoimmune disease or chronic renal impairment. Conclusions: 1. Although incidence appears stable, commissioners need to expand services to diagnose and treat people with GPA, and other adult-onset rare diseases, over the next 20 years due to the predicted increase in the proportion of the population in the age-groups at highest risk. 2. In particular, over the next 20 years, the age structure of the Black and Minority ethnic population in the UK is predicted to change to have many more people in older age-groups, and the UK medical community need to be alert to an expected emergence of significant numbers of Black and Minority ethnic people with ANCA-associated vasculitis. 3. Computerised prompts to alert GPs to consider a diagnosis of rare disease are unlikely to work. Resources to improve early diagnosis and treatment of ANCA-associated vasculitis would be better targeted at secondary care where the majority of cases have contact in the year before diagnosis. 4. The novel association of bronchiectasis with developing granulomatosis with polyangiitis raises a new hypothesis for bronchiectasis as a possible aetiological factor in granulomatosis with polyangiitis.

Introduction: Current guidance on the treatment of high blood pressure provides the advice that co-pathology should be taken into account when treatment decisions are made, but does not specify the approach in people with dementia. A relationship between high blood pressure and dementia, all be it complex and variable over time, does exist, making dementia a relevant co-pathology in decisions around the treatment of hypertension. No trial evidence exists however to guide clinical decision making in this specific context and clinicians with theoretical concerns over adverse events or varying priorities may act differently while remaining within the scope of current guidance. To inform the design of potential future research examining the repercussions of different treatment approaches, the way high blood pressure is currently treated in people with dementia and the adverse events they experience need to be understood. Aims: This thesis reports research which set out to describe the treatment of high blood pressure in people with dementia and the adverse events that this population experienced over a six month period. Methods: (i) A systematic literature review of observational studies describing the treatment of hypertension in people with dementia was performed. (ii) A multicentre cohort study, the Hypertension IN Dementia (HIND) study, of 181 participants, recorded information on dependency in activities of daily living (ADLs), cognition, medication, diagnoses, and healthcare use. It provided a detailed description of the treatment of high blood pressure in the study population and the adverse events experienced over a 6 month period. Results: Literature review: The prevalence of hypertension in people with dementia was 45% (range 36%-84%), of whom 73% (range 48%-85%) were taking at least one antihypertensive. 55% of people with dementia achieved target blood pressure in the one study that reported this. The review found no studies that specifically set out to describe the treatment of high blood pressure in people with dementia in the UK. Cohort study: 181 participants were recruited from general practices and via memory clinics. The rate of recruitment was low (8%) in the GP arm, resulting in potential selection bias. The study population were mildly cognitively impaired (median MMSE 23 (IQR 18-26)), 56% were dependent for at least one ADL, had a median of 5 (IQR 3-7) diagnoses and were treated with a median of 7 (5-9) medications. High blood pressure was treated in 87% (95% CI 82% - 92%) and target blood pressure was achieved in 57% (95% CI 49% - 64%) of those on treatment, no different from the general population (87% (95% CI 85% - 89%) treated and 52% (95% CI 49% - 55%) achieving target). ACEi/ARBs were the most frequently prescribed antihypertensive class (55%), followed by calcium channel blockers (33%), beta-blockers (30%) and diuretics (21%). Diuretics were less likely to be prescribed than in the general population (21% (95% CI 15%-26%) vs 34% (95% CI 31% - 37%)). During 6 months follow up the study population reported 475 GP appointments, 65 hospital admissions, 214 falls, 1 myocardial infarction, 6 strokes and 8 deaths. Heart failure, stroke, recurrent falls, falls with fractures, death and GP appointments were more common in the study population than in benchmark populations. Conclusion: In conclusion in an area where clinicians were acting without a firm evidence base and where there were theoretical concerns around the potential side effects of antihypertensive use, clinicians treated hypertension in people with dementia much as they did in people without dementia. The same classes of antihypertensives were used to maintain blood pressure at a similar level to that achievable in the general population. Despite a potential selection bias that may have over recruited fitter and milder people with dementia than the overall population, the study population reported a higher level of cardiovascular events, recurrent falls, fractures and adverse symptomatology than those without dementia in benchmark populations. Although this finding could relate to reporting bias or a higher intrinsic cardiovascular risk it raises the possibility that the benefits of antihypertensive treatment are attenuated, while the risks are increased, in people with dementia with implications for the risk-benefit ratio in this population. Future specific research, using an approach that avoids selection bias, to explore the risk-benefit ratio of antihypertensive treatment in people with dementia is outlined and advice is provided to clinicians managing high blood pressure in people with dementia.

Patients on dialysis are subject to a hugely elevated risk of cardiovascular mortality. Incidence and prevalence of, and mortality and morbidity from heart failure is significantly higher in the haemodialysis population than the general population as a whole. This thesis describes research work focusing on the large scale haemodynamic changes that occur during haemodialysis and how they may negatively impact on the cardiovascular system. Our results show that the haemodynamic disturbances which occur during haemodialysis are capable of causing a reduction in myocardial blood flow sufficient in magnitude to induce myocardial ischaemia. This is associated with a matched reduction in regional left ventricular (LV) function and is entirely in keeping with other published work describing haemodialysis induced myocardial stunning reflecting subclinical myocardial ischaemia (myocardial stunning). In addition, we now know that this phenomenon of haemodialysis induced myocardial ischaemia and stunning is common and associated with both short and long term complications including ventricular arrhythmias, left ventricular dysfunction, an increased hazard of death and time to first cardiovascular event. This is pertinent as in non-dialysis patients repeated episodes of myocardial stunning lead to chronic heart failure, and in dialysis patients the presence of LV dysfunction dramatically increases the risk of death. We also identified a number of factors associated with the presence of myocardial stunning including age, raised biochemical markers of cardiac damage (troponin-T), higher ultrafiltration volumes and lower intradialytic blood pressure. This is of crucial importance as ultrafiltration volumes and intradialytic haemodynamics are potentially modifiable risk factors that could provide targets for dialysis based interventions aimed at improving cardiovascular outcomes in the haemodialysis population.

Generally urban drainage systems are built to protect urban property and control runoff. Moreover, these systems collect the runoff for storage purposes to serve society through sufficient water supply to meet the needs of demand, irrigation, and drainage. Urban environments are exposed to risks of extreme hydrological events. Therefore, urban water systems and their management are critical. Precipitation data are crucial, but may be prone to errors due to the lack of information e.g., short length of records. In this thesis, a Monte Carlo simulation and regional frequency analysis based on L-moments approach were utilized during the research in order to estimate the uncertainty in the Intensity Duration Frequency (IDF) curves by using historical precipitation data from Environment Canada (EC) weather stations and simulating a new series of data through a weather generator (WG) model. The simulations were then disaggregated from daily into hourly data for extraction of the annual maximum precipitation for different durations in hours (1, 2, 6, 10, 12, and 24). Regional frequency analysis was used to form the sites into groups based on homogeneity test results, and the quantile values were computed for various sites and durations with the return periods (T) in years (2, 10, 20, and 100). As a result, the regional frequency analysis was used to estimate the regional quantile values based on L-moment approach. Moreover, the box and whisker plots were utilized to display the results. When the return periods and durations increased, the uncertainty slightly increased. The historical IDF curves of London site falls within the regional simulated IDF curves. Furthermore, 1000 runs have been generated by using the weather generator.

Generally urban drainage systems are built to protect urban property and control runoff. Moreover, these systems collect the runoff for storage purposes to serve society through sufficient water supply to meet the needs of demand, irrigation, and drainage. Urban environments are exposed to risks of extreme hydrological events. Therefore, urban water systems and their management are critical. Precipitation data are crucial, but may be prone to errors due to the lack of information e.g., short length of records. In this thesis, a Monte Carlo simulation and regional frequency analysis based on L-moments approach were utilized during the research in order to estimate the uncertainty in the Intensity Duration Frequency (IDF) curves by using historical precipitation data from Environment Canada (EC) weather stations and simulating a new series of data through a weather generator (WG) model. The simulations were then disaggregated from daily into hourly data for extraction of the annual maximum precipitation for different durations in hours (1, 2, 6, 10, 12, and 24). Regional frequency analysis was used to form the sites into groups based on homogeneity test results, and the quantile values were computed for various sites and durations with the return periods (T) in years (2, 10, 20, and 100). As a result, the regional frequency analysis was used to estimate the regional quantile values based on L-moment approach. Moreover, the box and whisker plots were utilized to display the results. When the return periods and durations increased, the uncertainty slightly increased. The historical IDF curves of London site falls within the regional simulated IDF curves. Furthermore, 1000 runs have been generated by using the weather generator.

Pannexins are newly discovered proteins that were first discovered by Panchin in 2000. The pannexin family has three isomers, i.e. pannexin-1, pannexin-2, and pannexin-3. In 2011, Billaud et al suggested that pannexin1 channels contribute to the spread of vasoconstriction after activation of α1D-adrenoceptors present on the surface of vascular smooth muscle cell (VSMCs) of thoracodorsal resistance arteries (TDA) isolated from mice. Phenylephrine acting upon the α1D-adrenoceptors activated pannexin1 channels present in the cell to release ATP, which in turn activated P2Y receptors on neighbouring cells to produce a co-ordinated contractile response. This aim of this work was to further investigate the role of pannexin in the regulation of contractile responses in the vasculature. To this end, the present study examined the presence and function of pannexins in the porcine splenic artery (PSA) and the rat aorta (RA), in which α1A- and α1D-adrenoceptors are present respectively. The role of pannexin channels and ATP (via activation of P2 purinoceptors) in the response to exogenous NA-induced contractile responses in the PSA and the RA were investigated, as were responses to sympathetic nerve activation in the PSA. The involvement of pannexin channels was studied using several pannexin inhibitors, i.e. mefloquine (a non-selective pannexin inhibitor), probenecid (a selective pannexin1 inhibitor at low concentrations), carbenoxolone (a selective pannexin1 inhibitor) and Brilliant Blue FCF (a selective pannexin1 inhibitor). Additionally, the involvement of ATP in NA-induced responses was examined using P2 purinoceptor antagonists (PPADS, suramin and NF449). Further experiments examined the role of pannexins in contributing to endothelium-dependent responses in a large vessel i.e. the porcine coronary artery (PCA). The results showed that both pannexin1 and pannexin2 are present in the PSA and the RA. In the PSA, mefloquine and probenecid reduced the responses to both NA-induced contractions and the frequency-dependent response curves generated to sympathetic nerve stimulation, whereas carbenoxolone, suramin and PPADS had no effect on responses to either exogenous NA or those caused by activating the sympathetic nerves. In the RA, mefloquine and probenecid reduced the response to NA-induced contractions, whereas BB-FCF had no effect. Purinoceptor antagonists (suramin, PPADs and NF449) had no effect on responses mediated by either α1A–adrenoceptors in the PSA or α1D–adrenoceptors in the RA, arguing against the role of ATP (via activation of P2 receptors) in mediating NA-induced responses in either the PSA or the RA. Conflicting results were obtained, in some cases, upon the use of three different pannexin inhibitors. The most likely reason for this is that mefloquine demonstrated non-selective inhibitory actions on contractile responses since it was also shown to inhibit responses to KCl, 5-HT, U46619 (the thromboxane mimetic), and responses to re-addition of calcium to depolarised preparations, suggesting that it acts to block L-type Ca2+ currents. Both mefloquine and probenecid demonstrated non-selective inhibitory effects when used at relatively high concentrations. Therefore, mefloquine and probenecid should only be used in low concentrations as pannexin1 inhibitors. It has been suggested that pannexin proteins may be involved in mediating endothelium-derived hyperpolarizing factor (EDHF) responses (Gaynullina, Shestopalov et al 2015). Bradykinin (BK) was shown to induce relaxation in PCA and to a lesser extent in the PSA after inhibition of NO-synthase and cyclooxygenase. The evidence for the involvement of pannexin in mediating an EDHF response was limited in both the PSA and the PCA, since neither carbenoxolone nor probenecid had any effect. While mefloquine reduced EDH-mediated responses to bradykinin in the PCA, the questions about its selectivity make this observation difficult to interpret. The present work therefore provided some evidence for the involvement of pannexin channels in conducting responses to NA-induced α1-adrenoceptor-mediated vasoconstriction in blood vessels in PSA and the RA, although great care must be taken in interpreting this data on the basis of a lack of selectivity of the pharmacological agents currently available as pannexin inhibitors. In addition, there was no evidence that activation of α1-adrenoreceptors causes the release of ATP from inside cells to act as an intercellular messenger, leading to P2 receptor-mediated contractions.

Pannexins (Panx) are membrane bound channels which allow the exchange of molecules, particularly ATP, between the cytoplasm and the extracellular space. Panx channels have been shown to be expressed in the vasculature, and there is evidence suggesting that they are involved in the regulation of vasoconstriction in small arteries. In mice thoracodorsal resistance arteries Panx1 channels are co localized with α1D-adrenoceptors on smooth muscle cells, and have been reported to open after α1D-adrenoceptor stimulation to release ATP which is then responsible for causing vasoconstriction by activating P2 purinoceptors (Billaud et al., 2011). This thesis examined the expression and the role of Panx channels in rat mesenteric and cerebral arteries. Western blotting was used to probe for the presence of pannexin proteins in rat mesenteric and cerebral arteries. The functional role of pannexin channels in the mesenteric and cerebral arterial vasoconstriction was studied using pressure myography. The effects of the pannexin channel inhibitors, mefloquine, probenecid, and carbenoxolone were tested against sympathetic nerve mediated vasoconstriction in 2nd order mesenteric arteries. The effects of purinoceptor antagonists, suramin, and NF449 as well as apyrase an ectonucleotidase that degrades ATP and αβ-MeATP which desensitises P2X1 purinoceptors and mefloquine, probenecid and carbenoxolone were examined against responses to noradrenaline (NA) and potassium chloride (KCl) in pressurized 2nd order mesenteric arteries. The effects of increasing concentrations of pannexin channel inhibitors, mefloquine, probenecid, and carbenoxolone, and purinoceptor antagonists, suramin, and NF449 were also examined against the myogenic tone of 3rd order mesenteric arteries and cerebral arteries. Panx1 siRNAs were used to try to knock down the Panx1 protein in isolated mesenteric arterial smooth muscle cells and mesenteric arteries. Panx1 and Panx2 proteins were detected in rat mesenteric and cerebral arteries, but no Panx3 protein was detected. The activation of the sympathetic nerves in the 2nd order mesenteric arteries resulted in a frequency-dependent vasoconstriction, which was reduced in the presence of all the pannexin channel inhibitors (mefloquine, probenecid and carbenoxolone). Mefloquine caused a significant difference in the vessel diameter, the vessel diameter was 91.00 ± 36.19 µm in the absence of mefloquine and 1.00 ± 0.70 µm in its presence (Student’s t test, p > 0.05). Probenecid caused a significant change in the vessel diameter, at 10Hz the vessel diameter was 60.75 ± 4.59 µm in the absence of probenecid and 26.25 ± 2.83 µm in the presence of probenecid (Student’s t test, p>0.05). Similarly NA and KCl caused a concentration dependent contraction of the 2nd order mesenteric arteries. All the agents tested against the NA mediated responses, pannexin channel inhibitors, purinoceptor antagonists (suramin, NF449), and αβ-MeATP except apyrase resulted in the reduction in the contraction. The concentration that caused a 20% reduction in the vessel diameter in the absence of carbenoxolone was -6.82 ± 0.09 M and it was significantly different in the presence of carbenoxolone, it was -6.22 ± 0.16 M (student’s t test, p>0.05). The concentration that cause a 20% reduction in the vessel diameter, in the absence of suramin was -6.72 ± 0.18 M and it was significantly different in its presence -6.00 ± 0.15 M (Student’s t test, p>0.05). However, mefloquine and probenecid also inhibited the contraction produced by raising extracellular KCl. The myogenic tone of 3rd order mesenteric arteries was reduced by both pannexin channel inhibitors and purinoceptor antagonists. With the exception of mefloquine, none of the agents had any effect on the myogenic tone of cerebral arteries. The attempt to knock down the Panx1 protein in isolated mesenteric arterial smooth muscle cells and mesenteric arteries was unsuccessful, despite demonstrating that the delivery systems worked. In conclusion, Panx1 and Panx2 proteins are expressed in both mesenteric and cerebral arteries. Some of the observations in this study provide evidence that support the role for pannexin channels in sympathetic nerve responses and those to exogenous NA, these include the inhibitory effects seen with the P2 purinoceptor antagonists, suramin, and NF449 as well as αβ-MeATP on exogenous NA. This effect was also mimicked by the pannexin channel inhibitors, mefloquine, probenecid and carbenoxolone which also inhibited the nerve mediated contractions. Thus the data is consistent with the hypothesis that transduction of responses to NA involves the opening of pannexin channels and the release of ATP as an intercellular messenger, to enable synchronized vasoconstriction of the vascular smooth muscle cells. However, some caution needs to be applied since mefloquine and probenecid also reduced the response to raised extracellular KCl indicating that they may not be selective. In the case of the myogenic tone major differences in the effects of the pannexin channel inhibitors were seen between the cerebral and mesenteric arteries showing that probenecid and carbenoxolone were acting selectively, however mefloquine was non-selective. Thus the effects obtained with probenecid and carbenoxolone coupled with those obtained with P2 purinoceptor antagonists, suramin and NF449 on the myogenic tone of mesenteric arteries suggests a role for pannexin channels as the stress/pressure sensor in mesenteric arteries.