This is a qualitative study investigating the impact of factors affecting the working lives of practitioners in Health and Education during the period 2006-2016. It was conducted in the context of the increasing evidence of low recruitment, low retention rates and a high incidence of stress amongst expert practitioners in these two public institutions. What emerges from the data is a cross-sector phenomenon identified here as a ‘cry for professional intimacy’, formed as these practitioners give voice to a strong desire to be allowed to refocus on the relational aspects of their work. A review of contextual literature and related research pointed to an argument that pressures from the application of neo-liberal principles to the public sectors of Health and Education have created a new era of commodification and business style assessment of practice. At the national level, a loss of trust in our educators and doctors has been increased by the media moral panic over events such as the Shipman and Mid Stafford cases in Health, and in Education by the Climbié case and the condemnatory PISA report by McKinsey (2007). Government statistics and surveys by professional bodies have documented the rising experience of stress amongst these practitioners, but there appeared to be a gap in research that focused on both the cross-service nature of this phenomenon and the voices of practitioners themselves. The aim of this research therefore was to address the absence of stakeholder voices and to capture data from senior practitioners. This is the first time that research has concurrently interrogated executive-level practitioners in Health and Education. Specific questions were: firstly, what changes are taking place in the working lives of expert practitioners in Health and Education? and, secondly, is there a shared pattern of experience between expert practitioners in both sectors? Structuration (Giddens 1979; 2005; 2013), multiple-level analysis (Jepperson & Meyer 2011) and the concept of self-efficacy (Bandura 1982) formed the theoretical framework for this investigation. This field study involved thirty-six semi-structured interviews of practising leaders with at least ten years of management experience in Health and Education. The sample included hospital consultants, nurse directors, general practitioner (GP) partners, head teachers, Advanced Skills Teachers (ASTs) and university course leaders. Interviews were conducted in thirty institutions, including National Health Service (NHS) hospitals, GP practices, state secondary schools and universities. Data analysis of the thematic coding showed a highly significant link (chi square p > 0.0001) between autonomy, relationality and a consequent perception of efficacy amongst these practitioners in both sectors. The results of this field study therefore serve as a powerful indicator of the cross-sectoral nature of the dissonance of expert practitioners in Health and Education in the UK. It indicates an urgent need for further research into the link between autonomy and relatedness to the efficacy and retention of practitioners in these public services.

Healthcare associated infection (HCAI) poses a serious threat to patients admitted into hospital as well as health care staff. Whilst recommendations for preventing HCAI exist, many research studies, primarily quantitative in nature, have reported serious concerns about the suboptimal infection prevention and control (IPC) practices adopted by healthcare workers (HCWs) within acute clinical settings. However, there remains a lack of understanding about why suboptimal practices persist. Although quantitative studies have identified poor staff compliance with the IPC recommended practices, attempts to tackle the problem have yielded limited success. It is suggested that a key reason for this is the failure to take into account the cultural context in which the non-compliant behaviours take place. This qualitative study, guided by ethnographic principles, uses a combination of focus groups and individual interviews with frontline staff and organisational leaders to explore cultural issues affecting staff compliance with recommended IPC practices in a ring-fenced acute hospital elective surgical ward (ESW). The study reveals that noncompliance with IPC policies and procedures in the ESW was legitimised and subsequently tolerated by both frontline and managerial staff, especially when the acute hospital was under stress. In particular, the ESW operational ring-fencing policy for protecting elective surgical patients from HCAI acquisition was repeatedly breached due to the conflicting pressures and competing demands of a busy hospital environment. The findings challenge the sustainability of the policy of ring-fencing the ESW as a discrete component of a busy acute hospital in order to protect elective surgical patients from HCAI in the context of the current healthcare system. It is highly likely that, as people live longer due to advances in medicine and technology, the demand for trauma and medical emergency beds will increase in the future, rendering the ring-fencing of any bed unsustainable in an acute hospital setting.

Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) is a protein belonging to the TNF family of ligands. TRAIL is able to induce apoptosis in tumour cells, whilst leaving normal cells unaltered, representing therefore an attractive anti-cancer agent. In breast cancer, however, different cell lines are resistant to TRAIL. Moreover, many studies have demonstrated that breast cancer stem cells (bCSCs), the cells that are the most responsible for relapses and metastasis development, are resistant to TRAIL. Cellular FLICE-Like Inhibitory Protein (c-FLIP) plays a crucial role in TRAIL-resistance due to its ability to interfere with the TRAIL pathway, preventing therefore the apoptosis. In the course of a previous study, the homology model of c-FLIP has been constructed and using a structure-based virtual screening of commercially available compounds, one hit compound (3) able to inhibit c-FLIP at micromolar concentrations has been identified. Starting from the structure of the hit compound, several new derivatives belonging to four different structural families were designed and synthesised: sulfonamide derivatives, amine derivatives, amide derivatives and methylene derivatives. All the newly synthesised compounds were tested in an in vitro assay for their ability to sensitise TRAIL-resistant MCF-7 breast cancer cell line to TRAIL. Different derivatives retained the ability to increase TRAIL sensitisation showing a similar or slightly improved activity compared to the original hit. Some of the most promising compounds were further evaluated in in vitro pharmacokinetic assays, dose-response and cytotoxicity studies. The results obtained from these studies suggested the identification of a novel hit compound (88) which showed the ability to increase TRAIL sensitisation with an IC50 value in the range of 15-19 μM and improved metabolic stability compared to 3. Additionally, molecular docking analyses suggested a potential ability of the newly synthesised derivatives to occupy the pocket of c-FLIP. Taking into consideration all these results, a series of novel potential small molecule c-FLIP inhibitors showing ability to increase TRAIL sensitisation in resistant breast cancer cells and breast cancer stem cells was developed.

Pulmonary hypertension is an abnormal physiological state associated with a variety of medical conditions. However, the ability to accurately phenotype disease subtypes within this heterogeneous syndrome is limited. In this thesis, I utilised advanced phenotyping techniques, guided by pathophysiological processes known to be dysregulated in pulmonary vascular diseases; immunity and metabolism. I used flow cytometry based immunophenotyping to study circulating leukocyte subpopulations and metabolomic analysis to study metabolite profiles in circulating blood. I hypothesised that there would be differences between disease and health, and differences between disease subgroups. In the immunophenotyping studies, I identified an immune cell signature in Idiopathic Pulmonary Arterial Hypertension (IPAH) and Heritable Pulmonary Arterial Hypertension (HPAH) characterised by increased frequencies of T follicular helper (Tfh) cells, plasmablasts and PD1-expressing CD8+ T cells. This signature was not found in Chronic Thromboembolic Pulmonary Hypertension (CTEPH). These findings support the hypothesis that dysfunctional immune activation may be implicated in IPAH pathobiology, and that IPAH and HPAH may have shared immunopathological mechanisms. In the metabolomic studies, I identified wide ranging metabolic changes in pulmonary vascular disease, including evidence of disrupted energy metabolism, increased cellular proliferation and reduction in antioxidant metabolites. Additionally, by comparing paired samples from different anatomical sites, it was possible to differentiate metabolic perturbations which are localised to specific anatomical sub-compartments. Key to the clinical applications of this research, I have demonstrated immunological and metabolic alterations which are a shared feature amongst different pulmonary vascular disease subgroups, but also some changes which are specific to disease subsets. Future advances in disease phenotyping may facilitate effective new targeted therapy for pulmonary vascular diseases.

Nodular thyroid disease is common (prevalence 2-6%) and is a significant risk factor for the development of thyroid cancer. My aim was to apply genome-wide- linkage-analysis to identify the defect in a large kindred with multi-nodular goitre (MNG) of adolescent onset progressing to papillary thyroid cancer (PTC). Genomic DNA from 18 individuals (8 affected) was hybridized to Affymetrix GeneChip® Human Mapping 10K 2.0 Arrays. Results were analysed with Affymetrix GTYPE software to produce a call rate of ~92%. Extensive quality control steps were performed (PLINK, GRR) prior to linkage analysis using Merlin software in multipoint non-parametric and parametric (dominant) model. A non-parametric LOD score of 3.01 was obtained on chromosome 20 across 20cM, the same region produced a dominant LOD score of 2.03. Haplotype analysis reduced the region of interest to 3.7 Mbp, (encodes 10 genes). Analysis of copy number variation in an affected individual (Illumina Human 660W-Quad) revealed an intronic deletion of ~1000 bp in one copy of Phospholipase-C β1 (PLCβ1), (the first in the 10 gene list), which is present in all affected family members and carriers. The deletion contained ‘ATAA’ at the junction site and this InDel was found in 1 of 105 healthy unrelated people, a similar variant was reported in the database of genomic variants in ~1% of Europeans . The deletion was not present in 70 unrelated PTC patients but was found in 4/81 with MNG (all European); the deletion frequency in the general population vs. MNG gives a X2 value of 5.076 (p=0.024). PLCβ1 expression was measured in thyroid tissues from affected family members and subjects free of the InDel and were significantly higher in the former (p< 0.02). In conclusion, the InDel identified in familial MNG occurs in a proportion of sporadic MNG. It predisposes to goitre formation, possibly by increasing PLCβ1 transcription and activating the diacyl-glycerol, PKC and MAPK pathways.

Successful kidney transplantation transforms outcome for patients with end stage kidney disease. Delayed graft function (DGF) following Ischaemia Reperfusion Injury (IRI) is a major problem, is hard to predict or monitor, and preventative or therapeutic strategies are lacking. Ischaemic Preconditioning (IPC) may limit IRI, but results are variable and potential mechanisms are not well defined. The aims of this thesis were to study the role of microRNAs, which are post-transcriptional regulators of gene expression vital in many physiological and pathophysiological processes, in the context of IRI, IPC and DGF. An in vivo model of IRI and IPC was developed, and histological, biochemical and mRNA kidney injury marker analyses were undertaken. MicroRNAs were then profiled using both Next Generation Sequencing (NGS) and hybridisation arrays, and changes in selected microRNAs confirmed by RTqPCR. Histology scores, serum creatinine and expression of kidney injury markers were significantly reduced in IPC compared with IRI. Microarray and NGS analysis identified a highly reproducible IRI signature, which was attenuated by IPC. Subsequently, microRNAs were profiled using Taqman Low Density Array (TLDA) and validated by RT-qPCR, from urine samples of kidney transplant patients with and without DGF. A DGF microRNA profile was uncovered, with overlap to the results from the IRI model. These data have identified a microRNA signature of IRI that was attenuated by IPC, which also improved outcome. Urinary microRNAs also showed a promising capability to predict DGF in human kidney transplantation. MicroRNAs thus show significant promise as biomarkers and potential therapeutic targets in this context.

Obesity is associated with an increased risk of developing type 2 diabetes and women with diabetes have a higher relative-risk of cardiovascular mortality than men. Polycystic ovary syndrome (PCOS) is a common condition that is associated with obesity and confers a particularly high risk of diabetes. There is a need to identify cardiovascular dysfunction in the pre-diabetic stage because, once diabetes is evident, it is difficult to improve the prognosis. Left ventricular hypertrophy (LVH) and diastolic dysfunction are well-established consequences of obesity, but the mechanisms which underpin these findings are not well-defined. Measurements of ventricular-arterial coupling characterise the load which the ventricle must overcome to eject blood. This thesis investigated whether quantitative measures of ventricular-arterial coupling could explain the development of LVH and diastolic dysfunction in young women at risk of diabetes. Two methods of quantifying ventricular-arterial coupling were used (i) a comparison of arterial and ventricular end-systolic elastance, and (ii) the amplitude and timing of wave reflections in the carotid artery using wave intensity. Increases in elastance were associated with general and central obesity. In contrast, increased wave reflections were predominantly associated with fat around the organs and with worse metabolic health. Arterial elastance and wave reflections were independent contributors of left ventricular mass but did not independently contribute to diastolic function. Women with PCOS had similar cardiovascular risk, elastance and diastolic function to matched controls. However, they had a lower odds-ratio of LVH which appeared to be explained by lower amplitude wave reflections. There may be an aspect of PCOS which mitigates the effects of obesity and pre-diabetic states on the pulsatile loading of the left ventricle. The quantification of ventricular-arterial coupling provides new insights to the effects of obesity and pre-diabetic states on sub-clinical cardiovascular disease.

Cardiovascular disease is a major cause of death and ill health across the world. Psychosocial factors are increasingly being recognised as potential cardiovascular risk factors, contributing to the development and progression of atherosclerotic disease. However, the pathways between psychosocial factors and cardiovascular disease are not yet fully understood. The aims of this thesis were to explore the associations between psychosocial factors (both chronic and acute stress) and measures of subclinical vascular disease, and to further develop and validate, a new method of assessing vasomotor function. Women with both depression and anxiety were found to have increased carotid intima-media thickness and this relationship was found to be influenced by the presence of dyslipidaemia. When looking at the inflammatory responses to an acute mental stress challenge it was shown that those participants who had an elevated fibrinogen response 45 minutes after the stress challenge had poorer endothelial function 3 years later, as assessed by flow-mediated dilatation. These findings suggest that both chronic and acute stress may play a role in the development of cardiovascular disease. Good reproducibility was demonstrated with the new method for assessing vasomotor function following further development of the protocol for the method. The technique was able to detect differences in vasomotor function between a group of patients with Familial Hypercholesterolaemia compared with age and gender matched controls. In addition it was also able to detect improvement in vasomotor function following a single lipoprotein apheresis treatment in patients with Familial Hypercholesterolaemia undergoing long-term treatment. These studies demonstrated the potential of this method for use in non-specialist vascular research laboratories and out in the field for the assessment of vasomotor function.

Peritoneal dialysis (PD) is a life-saving form of renal replacement therapy for those with End Stage Kidney Disease. Peritoneal fibrosis is a considerable problem for PD patients, and mesothelial cells, which line the peritoneal cavity, play a central role in response to injury and fibrogenesis within the peritoneum. Mesothelial cells may undergo mesothelial to mesenchymal transition (MMT) contributing to peritoneal fibrosis and treatment failure. miRNAs are important regulators of fibrosis but their roles in peritoneal fibrosis are largely unknown. Here, a detailed characterization of the MMT process was performed in primary human mesothelial cells (HPMCs) in response to Transforming Growth Factor beta-1 (TGF-β1). Hybridization array showed mesothelial miR-21 and miR-31 expression was up-regulated by TGF-β1 which was validated by RTqPCR in different PD associated MMT models. Mesothelial cells cultured ex vivo from PD patients exhibited phenotypic changes consistent with a progressive MMT process that correlated with an increase in miR-21 and miR-31 expression. Association of miRNA expression and MMT markers in 33 peritoneal biopsies from patients undergoing PD treatment and in PD effluent from 230 PD patients confirmed these results. In silico analysis combined 4 target prediction algorithms (Targetscan, miRanda, miRDB and Diana-microT) for miR-21 and integrated the resulting outcome with mRNA arrays comparing omentum vs PD effluent-derived HPMCs with epithelial (E) and non-epithelial (NE) phenotype. 13 possible miR-21 targets during the MMT process associated to PD therapy were identified and model scrutinized. Four of these were confirmed to be miR- 21 targets. Functional gene analysis indicated that selected targets may be downstream modulators of Snail and cooperate driving MMT during peritoneal fibrosis. Taken together, these data provide a detailed characterisation of mesothelial miRNA expression and responses to TGF-β1, and identify miR-21 and miR-31 as promising biomarkers for peritoneal fibrosis associated to PD therapy.

Background: Percutaneous procedures are now the predominant tracheostomy technique within the critical care setting. Complication rates for various techniques appear to be equivalent to those achieved with surgical tracheostomy. There is a paucity of data when comparing percutaneous procedures, particularly when considering late complications (tracheo-innominate artery fistulae (TIF)), tracheooesophageal fistulae (TOF) and tracheal stenosis (TS). Given the severity of illness and associated mortality in many of these patients the incidence of these complications remains difficult to define. Confounding factors present in survivors of critical illness may present difficulties in diagnosis such that underlying tracheal pathology may go undiagnosed. Aims: To determine: The incidence of common early and late complications of percutaneous dilatational tracheostomy (PDT) in relation to surgical tracheostomy (ST). The role of peri-operative events that may contribute to the aetiology of late complications of TS, TIF and TOF. The incidence of early and late complications in relation to percutaneous tracheostomy to define the safest percutaneous technique. The utility of adjunctive techniques (bronchoscopy & ultrasound scanning) in reducing complications of PDT. The prevalence of sub-clinical TS following PDT using the single tapered dilator technique (STD). Aetiological factors for sub-clinical TS. Whether sub-clinical TS may present atypically in critical illness survivors. Methods: We have conducted a systematic review of all prospective studies reporting late complications after tracheostomy performed in the critically ill. We have also extracted data to assess the role of peri-operative events and monitoring in causing or preventing late complications. We have undertaken an eleven-year review of all PDTs performed within our unit to define the incidence of complications arising within our own population. Finally, a prospective study to identify the prevalence of sub-clinical TS and identify atypical presenting features in survivors of critical illness has been performed. Results: All surgical and percutaneous techniques are broadly similar in terms of early and late complications. There is a higher incidence of wound infection when comparing ST to the multiple dilator PDT. There are few studies assessing late complications between percutaneous techniques. The TS rate varies from 2.8 to 0.6% for ST and the STD technique respectively. Due to limited data we were unable to identify peri-operative events that may lead to late complications. There is a very low rate of complications attributed to the STD technique with only 9 significant late adverse events. The rate of sub-clinical TS is low with doubtful clinical significance. Conclusions: We have not found a significant difference in the incidence of TS between PDT and ST. Our pooled proportions meta-analysis may indicate a tendency toward a higher rate of stenosis for ST. The reported complication rates presented within our cohort study may indicate that the STD PDT is one of the safer techniques available. The rate of sub-clinical stenoses following STD PDT is low and of doubtful clinical significance. Further work is required to define the role for percutaneous tracheostomy outside the critical care setting and to gather qualitative data to assess the patient’s perception of tracheostomy in the critical care setting.