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.

Neutrophils are efficient phagocytic cells that form the body’s first line defence against entry of foreign infectious microorganisms, but also contribute to tissue damage and non-infectious, chronic inflammation. Neutrophils contain a variety of separate classes of proteinase containing granules: primary (azurophilic) granules [containing serine proteinases elastase (NE), proteinase 3 (Pr3), and cathepsin G (Cat G)], secondary granules [containing metalloproteinase 8 (MMP-8)] and tertiary granules [containing metalloproteinase (MMP-9)]. These proteases are important molecules in immune and inflammatory processes. Sustained inflammation is associated with accumulation of these proteinases and is assumed to contribute to normal parenchymal damage and pathology. Regulation of proteolysis induced by these proteases is crucial to avoid self-induced damage. In the first part of my PhD thesis, I sought to examine the surface expression of Pr3 and CD177 (a surface receptor of Pr3) on neutrophils, in presence of physiological inhibitors of proteases (alpha-1-antitrypsin; AAT). I have demonstrated that membrane-bound Pr3 (mPr3) is still detectable on the surface of neutrophils in the presence of purified inhibitors and autologous serum as a source of physiological inhibitors. The interaction between CD177 and Pr3 was also examined by expressing CD177 cDNA on the surface of non-neutrophil cells (CHO cells), as was the ability of purified AAT and serum to interfere with these interactions. AAT was able to remove Pr3 from the surface of CD177-CHO cells, and similar results were observed for AAT removal of Pr3 binding to CD177-expressing neutrophils. In the second part of this thesis, I examined the change in neutrophil proteinases (Pr3, MMP-8 and -9) expression following neutrophil transmigration. In vitro transmigrated neutrophils showed no significant change in mPr3 expression compared to un-migrated neutrophils and both CD177-positive and CD177-negative subsets were able to migrate across HUVEC cells. In addition, intracellular Pr3 and MMP-8 also showed no change after in vitro transmigration. For comparison I also examined the CD177 and proteinase expression in salivary neutrophils (in vivo low inflammation transmigration) relative to matched volunteer blood neutrophils. In contrast to the in vitro data, I found only CD177-positive (with Pr3 bound to the surface) neutrophils present in the saliva of healthy individuals. I also found that levels of MMP-8 and MMP-9 were completely depleted in salivary neutrophils, relative to the matched levels in blood neutrophils from the same donor. In the third part of this thesis I used confocal microscopy to examine the intracellular distribution of neutrophil proteinases within different granule subsets. It was found that selected neutrophil proteinases co-localised with two different granule markers; showing their location in either azurophilic granules (CD63) or secondary granules (CD66b). However, the relationship with Pr3 revealed some discrepancies compared to previous reports. Some neutrophil proteins were co-localized both before and after neutrophil stimulation; whereas others were co-localisation before but not after stimulation. This suggested that degranulation of subsets of granules had occurred.

Polycystic ovary syndrome (PCOS) is a common condition characterised by hyperandrogenism, oligo/anovulation and defects in insulin secretion and sensitivity. PCOS patients also have an increased prevalence of hypertension, dyslipidaemia and endothelial dysfunction, a state associated with decreased nitric oxide bioavailability and increased oxidative stress. Using women with PCOS as a model of predisposition to cardiovascular disease (CVD), the aim of this thesis was to provide a clearer understanding of mechanisms that may predispose individuals to endothelial dysfunction, and ultimately CVD. PCOS patients were compared to healthy controls in an observational study, which involved a comprehensive assessment of biochemical nitro-oxidative stress indices and a detailed characterisation of circulating microparticles (MPs). There was little evidence to suggest that women with PCOS have an increased oxidative stress compared to age/BMI-matched controls. However, PCOS patients did display elevated levels of annexin V positive MPs that were predominantly derived from platelets. In vitro studies investigated the effect of several metabolic stressors akin to those found in PCOS on endothelial-derived MP characteristics and function. Human endothelial (HECV) cells were exposed to oxidative, hypoxic, hyperandrogenic and metabolic stressors. Each metabolic stressor affected MP generation uniquely, suggesting MP characteristics and function reflect parental cell conditions. In order to determine whether circulating MP levels could be modulated in a clinical cohort, the effect of apheresis on circulating MP levels was investigated in patients with established CVD (familial hypercholesterolaemia). Apheresis decreased circulating levels of MPs and was associated with a decreased thrombin generation capacity in these patients. The data in this thesis thus provide evidence that young women with PCOS have an elevated concentration of annexin V positive MPs, even though there is little biochemical evidence for nitro-oxidative stress. Further studies are needed to assess the effect of this increase in circulating MPs on cardiovascular clinical end-points. In vitro experiments showed that the cellular stress condition is reflected in the MP characteristics, whereby each pathological stressor resulted in a unique MP phenotype. Furthermore, in patients with established CVD, apheresis reduced circulating levels of MPs. In conclusion, an elevated annexin V positive MP population may represent a novel mechanism by which cardiovascular risk is increased in patients with PCOS. These findings could have future implications for use as biomarkers, in diagnosis and therapeutics.

This thesis examines the uses of Electronic Health Records (EHRs) and challenges faced in their implementation in the context of England’s National Programme for IT (NPfIT), the biggest civil IT programme in the world (Brennan, 2007). Despite the huge investments and high visibility that characterised the NPfIT, its aim of national level EHRs was not achieved and the programme was dismantled after being in operation for nearly a decade. The concepts of ‘organising visions’ (Swanson and Ramiller, 1997), ‘boundary objects’ (Star and Griesemer, 1989), ‘technology frames’ (Orlikowski and Gash, 1994) and ‘professional hybridisation’ (Noordegraaf, 2007) are employed to explain findings from this research. The study uses qualitative research methods, drawing on documentary sources and 51 semi-structured interviews. Responding to the limitations of using solely organising visions (Swanson and Ramiller, 1997), this thesis studies the NPfIT using a dual lens combining organising visions and boundary objects to understand the dynamics between stakeholders of the NPfIT organising vision. This thesis presents the EHR itself as a boundary object, and illustrates the knowledge sharing capacity of EHRs across clinical boundaries. A key emergent finding is the presence of clinician-IT hybrid professionals - a group that has neither been subject to empirical research nor been given sufficient attention in critical projects such as the NPfIT despite their unique position that bridges the clinical and IT domains. This study presents key findings discussing the factors that support and discourage the emergence of clinician-IT professional hybrids.

This thesis presents two novel approaches for performing subgroup analyses or identifying subgroups in an individual patient data (IPD) meta-analyses setting. The work contained in this thesis originated from an important research priority in the area of low back pain (LBP); identifying subgroups that most (or least) benefit from treatment. Typically, a subgroup is evaluated by applying a statistical test for interaction between a baseline characteristic and treatment. A systematic review found that subgroup analyses in the area of LBP are severely underpowered and are of a rather poor quality (Chapter 4). IPD meta-analyses provide an ideal framework with improved statistical power to investigate and identify subgroups. However, conventional approaches to subgroup analyses applied in both a single trial setting and an IPD setting have a number of issues, one of them being that subgroups are typically investigated one at a time. As individuals have multiple characteristics that may be related to response to treatment, alternative statistical methods are required to overcome the associated issues. Tree based methods are a promising alternative that systematically search the entire covariate space to identify subgroups defined by multiple characteristics. In this work, a number of relevant tree methods, namely the Interaction Tree (IT), Simultaneous Threshold Interaction Modelling Algorithm (STIMA) and Subpopulation Identification based on a Differential Effect Search (SIDES), were identified and evaluated in a single trial setting in a simulation study. The most promising methods (IT and SIDES) were extended for application in an IPD meta-analyses setting by incorporating fixed-effect and mixed-effect models to account for the within trial clustering in the hierarchical data structure, and again assessed in a simulation study. Thus, this work proposes two statistical approaches to subgroup analyses or subgroup identification in an IPD meta-analysis framework. Though the application is based in a LBP setting, the extensions are applicable in any research discipline where subgroup analyses in an IPD meta-analysis setting is of interest.

In this thesis, we consider interim sample size adjustment in clinical trials with multiple co-primary continuous endpoints. We aim to answer two questions: First, how to adjust a sample size in clinical trial with multiple continuous co-primary endpoints using adaptive and group sequential design. Second, how to construct a test in order to control the family-wise type I error rate and maintain the power, even if the correlation ρ between endpoints is not known. To answer the first question, we conduct K different interim tests, each for one endpoint and each at level α/K (i.e. Bonferroni adjustment). To answer the second question, either we perform a sample size re-estimation in which the results of the interim analysis are used to estimate one or more nuisance parameters, and this information is used to determine the sample size for the rest of the trial or the inverse normal combination test type approach; or we conduct a group sequential test where we monitor the information, and the information is adjusted to allow the correlation ρ to be estimated at each stage or the inverse normal combination test type approach. We show that both methods control the family-wise type I error α and maintain the power and that the group sequential methodology seems to be more powerful, as this depends on the spending function.