Rhythmic contraction of cardiac myocytes is maintained by precisely controlled Ca2+ efflux from intracellular stores mediated by the cardiac ryanodine receptor (RyR2). Mutations in RyR2 result in perturbed Ca2+ release that can trigger arrhythmias. RyR2- dependent ventricular tachyarrhythmia is an important cause of sudden cardiac death, the mechanistic basis of which remains unclear. RyR2 dysfunction has also been implicated in other cardiovascular disorders such as heart failure and cardiomyopathy, thereby becoming an important target for putative drugs. The massive size of RyR2 (~2.2 MDa) along with its intracellular location poses considerable challenges to studies aimed at understanding the mechanisms underlying channel dysfunction. Single channel studies of reconstituted RyR2 in artificial lipid bilayers have provided important insights into channel behaviour in response to various physiological ligands, toxins, drugs and biochemical modifications. However, the precise mechanisms by which RyR2 is activated by its primary physiological trigger, cytosolic Ca2+, and the structural determinants of channel gating are yet unknown. In this study, I aim to understand the actual physical reality of RyR2 gating behaviour using novel experimental approaches and analytical procedures. I have examined in detail, single channel kinetics of wild type purified recombinant human RyR2 (hRyR2) when activated by cytosolic Ca2+ in a precisely regulated minimal environment where the modulatory influence of factors external to the channel were minimised. This mathematical modelling of hRyR2 single channel behaviour will serve as a future experimental platform upon which the effects of disease causing mutations can be studied, as well as the influence of physiological modulators and potentially therapeutic compounds capable of stabilising mutant channel function. Single channel studies of hRyR2 when modified by its archetypal ligand ryanodine in the absence of Ca2+ have uncovered an unusual voltage sensitive gating behaviour in this ligand-gated channel, providing further insights into the mechanisms underlying channel modification.

The metastatic spread of cancer cells to distant sites in the body accounts for the majority of cancer-related death and significantly decreases patient survival. Whilst cell migration is a physiologically important process, when uncontrolled, it can be a contributing factor to the metastatic phenotype. Actin polymerisation enables the dynamic restructuring of the cytoskeleton which is fundamental to cell migration and is stimulated by the Arp (actin-related protein) 2/3 protein complex which in turn is activated by members of the WAVE (WASP Verprolin homologous protein) family. WAVE1 and 3 expression was targeted separately in the PC-3 cell line utilising ribozyme transgene transfection. In vitro experiments revealed a reduction in cell growth and invasion following WAVE1 or 3 knockdown in PC-3 cells. These experiments were also repeated with small molecule inhibitors targeting the Arp2/3 complex, ROCK and N-WASP independently. This inhibitor work implicates Arp2/3 as a facilitator of cell proliferation through which WAVE regulates. Inhibition of Arp2/3, ROCK or N-WASP in WAVE1 knockdown cells increased cell invasion which may be attributed to the regulatory role of WAVE3 on MMP activity. Co-localisation of WAVE1 and 3 with ARP2 and ROCK-I was observed in PC-3 cells whilst this affect was abolished with WAVE1 or 3 knockdown. Furthermore, WAVE3 and WAVE1 knockdown affected ARP2 and ROCK-II tyrosine phosphorylation, respectively. These results suggest WAVE1 and 3 proteins are involved in several metastatic traits that characterise PC-3 cells. Furthermore, the contribution of WAVE in the networks that influence these traits may also involve association with Arp2/3 complex, ROCK-I and –II and N-WASP. Additionally, it sheds light on the similarities between these two related proteins and also highlights their subtle distinctions in PC-3 cells. The data outlined here provides justification to futher explore WAVE1 and 3 as potential contributors of prostate cancer progression.

Human cardiac ryanodine receptors (hRyR2) are major Ca2+ release channels in the heart, which form quaternary complexes with luminal proteins, calsequestrin (CSQ2), junctin (JUN) and triadin (TRD1). These proteins facilitate Ca2+ release during excitation-contraction coupling, modulating the response of hRyR2 to luminal Ca2+ changes. Catecholaminergic Polymorphic Ventricular Tachycardia is an arrhythmogenic disorder, caused by mutations in RyR2 and CSQ2 genes. Defective sensing of cytosolic/luminal Ca2+ by hRyR2 is a candidate mechanism underlying disease pathogenesis, likely caused by defective luminal protein regulation. Using a recombinant approach, this study aimed to evaluate if mutant (N4104K and A4556T) hRyR2 respond to or interact differently with, these accessory proteins. Expression constructs corresponding to CSQ2 and JUN were generated and expressed stably and transiently with wild-type (WT) or mutant hRyR2 in HEK293 cells. Immunofluorescent co-localisation confirmed successful co-expression and association of these luminal proteins with hRyR2 in situ. Ca2+ activation of wild-type/mutant hRyR2 in the absence/presence of CSQ2 and/or JUN was assessed by [3H]-ryanodine binding, while luminal Ca2+ effects were monitored using single-cell Ca2+ imaging - examining spontaneous Ca2+ release events. A4556T-hRyR2 displayed similar cytosolic and luminal Ca2+ dependence to wild-type channels, whilst N4104K-hRyR2 displayed a remarkably different Ca2+ activation profile, demonstrating functional heterogeneity between hRyR2 mutants. Ca2+ imaging revealed an inhibitory effect of CSQ2 on WT and N4104K-hRyR2 activity, both in the presence and absence of JUN. In line with this, CSQ2 was found to bind directly to hRyR2 by co-immunoprecipitation, an observation that has not been previously demonstrated in the literature. Immunofluorescence studies suggested reduced CSQ2 and JUN association with A4556T-hRyR2, but this could not be confirmed with co-immunoprecipitation. Ca2+ imaging investigations with this mutant however, suggested that CSQ2 wasn’t able to regulate these channels in the same way as WT, implying a change in functional effect.

Osteocytes are currently regarded as being pivotal in maintaining bone homeostasis. They differentiate from osteoblasts, are embedded in mineralised matrix and difficult to isolate. Current models for in vitro osteocyte studies are limited. Others have suggested that osteoblasts in 3-dimensional (3D) cultures differentiate to osteocytes. This study aimed to develop 3D cultures enabling differentiation of osteoblasts to osteocytes, which could be used for studies of osteocyte differentiation and responses to mechanical loading. Furthermore, the effects of external compounds on osteoblast differentiation in 3D were assessed. Mouse (MC-3T3, IDG-SW3) and human primary osteoblasts (hOBs) were maintained in type I collagen gels in either non-osteogenic or osteogenic media, and +/- compounds such as insulin-like growth factor-1 (IGF-1). Furthermore, mechanical loading (5 mins, 10 Hz, 2.5 N) was applied to 3D cultures and responses characterised. Cells were viable in collagen gels for 25 days, and expressed mRNA for mature osteocyte markers e.g. sclerostin in osteogenic medium. Furthermore, IGF-1 upregulated mRNA expression of osteocyte markers and other molecules (e.g. receptor activator of the nuclear factor kappa-β ligand - RANKL - 43-fold) in MC-3T3 cells, indicating modulation of cell differentiation and function. Osteocyte markers were expressed earlier in IDG-SW3 cells in 3D compared to published marker expression profiles in 2D monolayer cultures. Following mechanical loading, known mechanosensitive markers were modulated in IDG-SW3 cells in 3D, for example, RANKL and vascular endothelial growth factor (VEGF) up-regulated and sclerostin downregulated post-loading. This 3D model enables differentiation of osteoblasts to osteocytes in an environment akin to osteocytes in vivo. External compounds accelerated cell differentiation, and this was also accelerated in 3D compared to monolayer. Furthermore, the 3D model enabled osteocyte mechanical loading. This model can be used with human cells, will further our understanding of osteocyte differentiation, and inform on osteocyte function including their responses to mechanical loading.

Chronic Obstructive Pulmonary Disease (COPD) is one of the commonest long-term conditions worldwide. It is characterised by chronic airflow limitation, pathological changes in the lung and significant extra-pulmonary manifestations. The treatment of an acute exacerbation of COPD (AECOPD), involves glucocorticoids and bronchodilators supplemented by antibiotics if needed. In-hospital, oxygen, which has potential risks as well as benefits, and additional respiratory support can be given if the patient deteriorates. Clinicians need to decide which treatment to provide and who can be safely discharged. This has led to the advent of scoring systems to define severity in COPD This thesis examines the evidence base for the use of magnesium in airways disease and presents the results of the first randomised double-blind placebo-controlled trial using nebulised magnesium in the treatment of AECOPD. 116 patients were randomised, but after 3 nebulisations over 90 minutes, there was no significant difference in FEV1 compared to placebo (p=0.67). In a second study, the CRB65 score was retrospectively assigned to a cohort of patients presenting to the emergency department with AECOPD, using data collected from a previous audit. Patients with a CRB65 score of 0 or 1 had a low risk of in-hospital and 30- day mortality and could be considered for discharge, whereas those with scores between 2-4 required admission with mortality increasing with the score. The CRB65 score showed a similar utility in AECOPD as it does in pneumonia. Finally, 18 subjects with stable but severe COPD were randomised in a crossover study to two nebulisations with salbutamol and ipratropium over 15 minutes with a five minute interval between nebulisations, using air or oxygen as the driving gas. When oxygen was used there was a 3.1mmHg difference (p<0.001) at 35 minutes, compared to air, illustrating the potential risks of repeated nebulisations to those with severe COPD.

Breast Cancer continues to be the most common form of cancer in women. The ability of tumour cells to spread from primary and metastatic tumours is the primary cause of death in patients with cancer. Thus, it rightly follows that significant research is dedicated to the pathways and mechanisms controlling metastases in order to guide therapeutic approaches. Wiskott Aldrich Syndrome [WAS] is an X-linked recessive condition with immunodeficiency as the clinical manifestation. It is caused by mutations of the Wiskott Aldrich Syndrome [WAS] gene, which codes for a cytoplasmic protein with multiple functions. Two major complexes that are linked to the NWASP family, namely the ERM family and Rho GTPases are aberrantly expressed in human breast cancer. Additionally, X chromosome inactivation which silences gene expression from one of the two X chromosomes in females, is usually random. Skewed X inactivation has been shown to occur more frequently in breast and ovarian cancer patients. Individuals with WAS are known to have skewed X inactivation. In addition, they are more susceptible to certain forms of malignancy, primarily haematological. This formed the basis of the present study, which sought to elucidate the role of WAS protein in human breast cancer, and to determine if it plays a role as a tumour suppressor. We also attempted to determine its biological role and association with clinical outcome in patients with breast cancer. We examined the correlation of NWASP with human breast cancer in vitro, in vivo and in human breast cancer tissue. Immunohistochemistry studies of frozen sectioned human breast cancer tissues revealed that breast cells stained positively for NWASP and that cancer cells in tumour tissues stained very weakly. Quantitative RT-PCR revealed that breast cancer tissues had significantly lower levels of NWASP compared to normal background breast tissue. Although no significant correlation was found with tumour grade and TNM staging, lower levels of transcript were seen to correlate with clinical outcome following a 10 year follow up. The invasive human breast cancer cell line, MDA-MB-231 was used to over-express NWASP, with over-expression resulting in cells with reduced motility and invasion, increased adhesion to the basement membrane and more significantly, reduced tumour growth in vivo. This has important implications in understanding the mechanism whereby cancer cells become more motile and presents an interesting tool in analysing the progression of human breast cancer.

Background and Aims: The WIMAT colonoscopy suitcase is an ex-vivo, porcine, polypectomy simulator. This has been developed in response to the increasing demand for polypectomy training following the introduction of the National Bowel Cancer Screening Programme. The aims of this thesis are to establish if the simulator is a valid form of polypectomy skills training and to identify if this model can be used to develop objective parameters for polypectomy assessment. Materials and Methods: A series of clinical trials were systematically conducted to test the validity of the WIMAT colonoscopy suitcase. This included evaluating its content, construct and concurrent validity and conducting a skills transfer study comparing the WIMAT colonoscopy suitcase with a virtual reality simulator. Objective assessment parameters were examined by measuring the accuracy of self-assessment and using video coding software to analyse the hand movements performed during simulated polypectomy tasks. Results: Content validity was demonstrated by experts who scored the model’s anatomical, mechanical and visual realism favourably across multiple parameters (p=<0.01). Construct and concurrent validity were confirmed by participants performing simulated polypectomy in accordance with their “real-life” level of expertise (p=<0.01). Skills transfer to the clinical setting was demonstrated in a pilot randomised controlled study. Self-assessment following simulated polypectomy is inaccurate as experts tend to overestimate ability whereas novices underestimate ability (p=>0.05). The ratio of rotational hand movements to endoscopic tip angulation (RoTA) was significantly different when comparing novices to experts (p=<0.05). Discussion: The WIMAT colonoscopy suitcase is a valid form of polypectomy skills training. The simulator can be used to address the increasing demand for training in this procedure. Further work is needed to assess the reliability of the RoTA score at different stages of the polypectomy procedure before it is used as an assessment tool.

Background Chronic obstructive pulmonary disease (COPD) is a multimorbidity disease associated with increased risk of cardiovascular events, arterial stiffness and changes in body composition, potentially features of premature ageing and frailty. The aim of this thesis was to assess the change in aortic pulse wave velocity (PWV) over two years and its contributing factors in COPD. In addition, this thesis also aimed to examine the concept of frailty in patients with COPD and its change over a two-year follow-up. Methods Aortic stiffness and frailty were assessed cross-sectionally in 500 patients with COPD and 150 comparators using aortic PWV and frailty index (FI). Other assessments included spirometry, body composition, handgrip strength, Timed Up and Go test (TUG) and systemic inflammatory biomarkers. After two years, 143 consecutive patients were reassessed to examine the changes in aortic PWV and FI. Results In the cross-sectional data, patients with COPD had greater aortic PWV than comparators similar in age, gender and BMI, independent of traditional risk factors. After two years, the patients demonstrated a significant increase in aortic stiffness, independent of age, lung function, blood pressure and inflammation. In addition, a subset of patients was identified to have an accelerated aortic stiffness by 1.6 m/s. At the initial visit, the patients were more frail than comparators similar in age and gender. After two years, the patients had an increase in the FI, independent of lung function and inflammation. The progression of frailty was related to loss of muscle mass and strength, and prolonged TUG time. Conclusion The longitudinal findings of this thesis suggest that COPD is associated with a rapid increase in aortic stiffness, independent of conventional risk factors. Frailty is a clinical feature of COPD and its progression is dependent on loss of musculoskeletal mass and strength and prolonged TUG time.

Chronic lung disease (CLD) is a common respiratory sequalae of infants born extremely premature (< 32 weeks gestational age). A persistent and poorly-resolving neutrophilic lung inflammation has been strongly implicated in the development of CLD. Pathways of resolution of lung inflammation have been poorly characterised in preterm infants. Interleukin-8 (IL-8) is an key neutrophil chemokine implicated in the pathogenesis of CLD. Longer, and less functionally potent, isoforms of IL-8 predominate the preterm circulation but their expression and function in the preterm lungs is not known. The complex of interleukin-6 (IL-6), along with the soluble IL-6 receptor (sIL-6R), initiates IL-6 trans-signalling which experimentally has demonstrated downregulation of IL-8 during acute inflammation. Expression of IL-6 trans-signalling cytokines and their functional activity in the preterm lungs have not been studied. My work shows that the concentration of sgp130, a specific inhibitor of IL-6 trans-signalling, was significantly increased in the bronchoalveolar lavage fluid (BALF) of preterm ventilated baboons, and human infants developing CLD later, compared to infants who did not. Although total IL-6 activity was detected in a specific functional assay, IL-6 trans-signalling activity could not be detected from the BALF samples or by using a complex from recombinant cytokines. I have shown that the long isoform of IL-8, IL-877 was a minor proportion of total IL-8 in the preterm ventilated BALF, although significant expression of IL-877 was detected in vitro from lung cells. Preterm BALF efficiently converted exogenously added recombinant IL-877 to shorter isoforms, mainly by the activity of serine proteases from neutrophils and the clotting cascade. BALF from CLD infants converted significantly more IL-877, compared to BALF from No-CLD infants. Among the neutrophil serine proteases, proteinase-3 (Pr-3) converted IL-877 to functionally more potent isoforms; Pr-3 antigen and thrombin activity was also significantly increased in BALF from CLD infants, making them attractive targets for intervention.

In the absence of a routine ovarian screening programme, ovarian cancer symptom awareness is a potential route to earlier symptomatic presentation and disease diagnosis. However, materials to support this strategy may need to be tailored according to risk. The work presented in this thesis identified the contributors to anticipated symptomatic presentation for women at increased genetic risk of ovarian cancer. A mixed-method approach was used to identify determinants of anticipated symptomatic presentation, and included a systematic search of existing ovarian cancer symptom awareness tools, cross-sectional surveys with two risk populations and qualitative interviews with women at increased genetic risk. Additionally, a systematic search and a virtual reference group were used to identify symptom content. Cognitive interviews were undertaken to pilot the draft tool for acceptability and usability with a sample of potential users and providers. Endorsing more benefits than barriers to presentation was associated with earlier anticipated presentation in both risk populations; however, differential effects of underlying health beliefs on anticipated presentation were also identified. In those at increased genetic risk, emotional (worry) rather than cognitive aspects of risk perception predominate in influencing earlier anticipated presentation. Interviews with women at increased genetic risk revealed that personal experience with ovarian cancer shaped beliefs about the disease. The identified health beliefs were incorporated into OvSTAT (ovarian cancer symptom awareness tool), with core content applicable for women from the general population and tailored content to address the specific needs of women at increased genetic risk. OvSTAT was well received in user testing. Overall, the findings suggest that the emotional representation of risk distinguishes earlier anticipated presentation in women at increased genetic risk from that in the general population. OvSTAT could be a mechanism through which appropriate symptomatic presentation is improved, by helping women to manage worry associated with their increased genetic risk status.