Enteroaggregative Escherichia coli (EAEC) utilises an atypical type I secretion system, AatPABCD, for the secretion of dispersin (Aap). AggR is known to regulate the transcription of the aat system and aap in EAEC 042. Using RNA-Seq, the transcriptomes of EAEC 042 and EAEC 042ΔaggR were compared for differential gene expression. As well as new targets for AggR activation, aap was found to have the greatest difference in transcription. Whilst the function of Aap is understood, the role of the homolog, CexE, in enterotoxigenic E. coli H10407 is not. It was found that CexE could not complement the aap mutant phenotype instead having the opposite effect, resulting in an increase in aggregation. Aggregation can indicate a role in biofilm. It was found that a cexE mutant could not form a biofilm. The investigation of CexE structure revealed that CexE could be secreted by the Aat system. Therefore, Aat-mediated secretion of CexE was investigated. A modification by AatD of Aap/CexE proteins was identified and a new model for Aat secretion is proposed. In summary, the function of Aap and CexE are different but they are both secreted by the Aat system.

Background and Aims: Glucocorticoids (GC) have been implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Tissue GC levels are regulated by the enzyme 5α-reductase (5αR), which mediates GC breakdown, as well as converting testosterone to dihydrotestosterone (DHT). The aim of this study was to investigate the role of 5αR in the modulation of NAFLD. Methods: i) Human liver tissues from patients with NAFLD were used for immunohistochemical/qPCR and microarray analysis; ii) a cellular model of steatosis was developed to assess the effect of 5αR manipulation in-vitro; and iii) the effect of genetic 5αR knockdown on development of NAFLD, hepatic lipid metabolism and other metabolic parameters was assessed in a murine model of NAFLD. Results: In human liver, 5αR1 and 5αR3 expression correlate with histological severity of NASH. In a murine model of NAFLD, 5αR1-/- mice demonstrate increased steatosis but no significant difference in inflammation or fibrosis, suggesting that the deleterious effect of increased hepatic GCs on fat accumulation may be subsequently counteracted by their anti-inflammatory effect in NASH. 5αR1-/- also appears to protect against the development of hepatocellular dysplasia/carcinoma, which may result from the effect of reduced DHT levels on hepatic progenitor cell proliferation.

This thesis considers the design of paediatric allergy services in the West Midlands (WM) region. It is presented in 3 parts: the ftrst discusses a systematic review of allergy service delivery pathways across the UK and the rest of the world; The second quantifies the burden of paediatric allergy across the WM and compares it with the rest of the country using data from the health information network (THIN) database for primary care and the hospital episodes statistics (HES) database for secondary care. The final part ascertains the experiences of parents in the WM region who have accessed these services through a qualitative study and elicits parental preferences for these services using a discrete choice experiment. The main argument put forth in this thesis is that services in a financially constrained environment should be planned efficiently and be responsive to the needs and preferences of the local population. A systematic way method to achieve this is presented along with recommendations for improving the efficacy and efficiency of paediatric allergy pathways in the WM region.

Podiatrists have developed an informal specialism within their profession which enables them to treat MSK injuries with functional orthoses. The podiatric biomechanics theories, on which this treatment is based, have been called into question and may have little scientific basis. Research evidence into functional orthoses suggests they may not be as successful at treating MSK injuries as it first appeared. This has lead to debate within MSK podiatry about which theories and orthoses to use in relation to the treatment of MSK injuries. It suggests that podiatrists may be unable to apply easily the principles of evidence-based practice in MSK podiatry. The aim of this thesis is to explore how MSK podiatrists interpret and use evidence in clinical practice; what influences their beliefs, their perceptions of the interpretation of research and other forms of evidence and it examines their lived experience of using evidence in clinical practice. A hermeneutic approach was used to conduct semi structured interviews with seventeen podiatrists who use orthoses to treat MSK injuries in a variety of practice settings. Interpretative phenomenological analysis was utilised to analyse the data. Ethical approval was given by the University School ethics panel and relevant permissions were granted by employers. The findings reveal there is confusion about evidence in MSK podiatry. The participants understand the concept of evidence-based practice to be the use of quantitative research evidence in practice. In reality there is little definitive knowledge from research and the main source of evidence used in practice is patient feedback supported by a process of active experimentation. Clinical knowledge in MSK podiatry is a form of practical wisdom, gained through the lived experience of practice. The uncritical acceptance of evidence based practice principles together with an emphasis on randomised controlled trials suggest other forms of evidence are devalued. Thus, MSK podiatrists have fears their practice is based on weak evidence. The discourse of MSK podiatry has changed, participants use a Tissue Stress approach with the biomechanics theories forming a framework to understand gait and as the basis of their active experimentation. The discourse has become more vague and individual. The language used by the participants reinforces the ambiguity, for example there is a wide range of terms to describe orthotic designs and modifications. This could be a way to maintain the status of MSK podiatry. The concluding discussion examines the findings in the context of current literature that MSK podiatry is a practical and experiential specialism. It could be described as a patient centred approach based in a practical wisdom. The participants are not undertaking EBP as they describe it because the lack of propositional knowledge undermines the research undertaken in this area. Therefore it is recommended that a national outcome study is needed to confirm the effectiveness of orthoses and to gain understanding about the orthoses being prescribed and the MSK conditions being successfully treated. There needs to be further exploration to establish understanding of the language of MSK podiatry . The professional body for podiatrists should consider a mentorship programme for practitioners to allow the individual knowledge of MSK podiatry to be shared and to maintain the community of practice in these changing times for healthcare provision in the UK.

Background: A video-based 'digital legacy' is a selection of videos which document a person's life, memories, achievements, or special family events. The videos are copied to a digital source to be specifically given to a child or young person to use in the future. A video-based digital legacy may either be purposefully recorded by the person living with MND (plwMND), or, compiled later by bereaved family members. To date, there is little published research about how children and young people are affected when a family member has MND and subsequently dies. As such, there is a dearth of literature on how to best support these young people. Objective: This research is investigating the views, perceptions and experiences of digital legacies with people affected by MND. Methods: The study is underpinned by Interpretative Phenomenological Analysis (IPA) meaning a small homogeneous sample was required using purposive methods of recruitment. Interviews were conducted and audio recorded with four plwMND regarding their experiences of creating a purposeful digital legacy for a child or young person in their family. Interviews were also conducted with three bereaved young people regarding their experiences of using a video legacy of a parent who had died from MND. Also, a sample of twenty healthcare professionals, specialists and experts were interviewed from across the United Kingdom regarding their perceptions on the use of digital legacies with plwMND, and, young people who are bereaved. Ethics: Ethical approvals were obtained from a Faculty of Research Ethics Committee at Edge Hill University (FREC), the Health Research Authority (HRA), and the National Research Service for Scotland. Discussion: 'The Model of Reciprocal Bonds Formation' and coining of the term 'autobiographical chapter' has been developed from this study. Creating a digital legacy provides a number of mutual challenges and benefits for both plwMND, and bereaved young people. Recommendations are provided regarding i) optimal 'windows of opportunity' in which the digital legacy is recorded/used; ii) actionable solutions for current policy/practice; iii) future directions for research.

Multiple Sclerosis (MS) is one of the commonest disabling neurological diseases affecting the young in the developed world. It affects the central nervous system (CNS) and is characterised by inflammation with neuronal demyelination /degeneration and failure of remyeli nation/regeneration. Inflammation is present throughout the course of the disease though it differs qualitatively and/or quantitatively in different phases. T cells are the central players in the immune processes and more so in an autoimmune disease like MS. Autopsy studies continue to add to our understanding of CNS inflammation. I studied the role of pathogenic and regulatory T cells in MS. I have demonstrated that there is a peripheral immune dysregulation as seen in some previous studies involving the Th17 cell axis. I observed the presence of raised frequencies of non-classic Th1 cells and dual positive Th17-Th1 cells in MS. Dual expression IL-17+1L-22+ cells were also raised in MS. Results included raised frequencies of Th22 cells in the blood in MS. This suggests the role of Th22 in pathogenesis of MS. The chemokine receptor CCR6 is considered Th 17 lineage associated. I noted the expression of CCR6 not only on Th 17 cells, but also on Th1, Th22 and Th21 which alludes to the role of these cells in causing CNS inflammation via the entry into the CNS though CCR6-CCL20 trafficking axis. I observed reduced frequencies of CTLA-4+ T cells which points to their limited regulatory capacity in MS. These findings provide important clues towards the pathogenesis of MS. These findings however, do not correlate with the disease activity and disease severity of MS at this stage. The plan is to explore the clinical correlation and contribution of these findings in the pathogenesis of the disease in further studies.

During fertilisation the egg undergoes a series of remarkable activation events including a prolonged series of intracellular Ca2+ oscillations. The Ca2+ oscillations are necessary and sufficient to trigger egg activation and support pre-implantation embryo development. The mechanism of the signal trigger is well established and involves a sperm factor released into the egg upon gamete fusion. Phospholipase C zeta (PLCζ) appears to be the mammalian sperm factor responsible for triggering Ca2+ oscillations during fertilisation. Consistent with this idea it has been found that, some cases of male factor infertility have been attributed to either qualitative or quantitative deficiencies in PLCζ. Intra-cytoplasmic sperm injection (ICSI) has overcome many cases of male factor infertility. However, some ICSI cases still see partial or total fertilisation failure. To overcome such failures, artificial egg activation using various protocols has been introduced. Ionophores are the main agents used to trigger artificial egg activation in human eggs. The work in this thesis provides evidence that the human PLCζ protein is effective and more efficient in triggering egg activation and in supporting pre-implantation embryo development in mouse eggs than other activators. That was achieved via a direct comparison of various activation protocols in a mouse model of ICSI fertilization failure. Furthermore through the use of ICSI and Ca2+ measurements in mouse eggs my studies have provided further evidence that the number of Ca2+ transients during activation can influence development to the blastocyst stage. Moreover, my work also investigates what methods could be used for injecting PLCζ protein within a clinic setting. . Other work has attempted to investigate how Ca2+ oscillations during fertilisation may influence later embryo development by testing the use of probe of hydrogen peroxide in mouse eggs. Overall the studies provide an important step in the development of new therapies for treating patients having problems conceiving with ICSI treatment.

There is an increasing need to develop anti-cancer vaccines that elicit strong and durable immune responses and are able to break immune tolerance to cancer antigens. Cytomegaloviruses (CMV) induce one of the strongest immune responses of any viral vector – as much as 20% of all CD8+ T cells can be specific for a single epitope, thus making them an exciting candidate for vaccine development. The majority of work, however, has been based on replication-competent viruses. In humans, the use of replication-competent human CMV (HCMV) vectors would not be permitted due to safety concerns, however replication-deficient vectors may not induce immune responses of the same magnitude as wildtype HCMV. In an attempt to make CMV vectors safer, I utilised a tetracycline repressor (tetR)-based system and generated a total of 55 mouse and human CMV vectors in which viral replication was dependent on inducible expression of multiple different viral genes. Having analysed the ability of the vectors to replicate in vitro and in vivo, I generated three vectors which replicated well in the presence of doxycycline, but in its absence replicated poorly in vitro, and did not appear to replicate in vivo. Importantly, in a direct comparison, the level of control was more stringent than viruses previously characterized in the literature. Adenoviruses are one of most widely used vectors in cancer immunotherapy, induce strong T-cell responses, and could be used in a prime-boost regiment with CMV vectors. However, we found that in a prophylactic regimen, a recombinant human adenovirus (Ad) encoding human 5T4 tumour-associated antigen alone did not protect against 5T4-expressing tumour challenge. We tested the immunogenicity of 5T4 in two mouse strains and established three 5T4-expressing cancer models in which vectors can be tested. In future work, we hope to use our inducible CMV/Ad vaccine regimen in these models.

In this thesis, I explored the relationships between motor ability, psychopathology and cognition in children with 22q11.2 deletion syndrome (22q11.2DS). Firstly, I established the prevalence of coordination difficulties in a sample of children with 22q11.2DS and investigated if coordination difficulties were related to psychopathology or cognitive ability. I found that rates of coordination difficulties were very high (~80%) in children with 22q11.2DS and that poorer coordination was related to psychopathology, IQ and attention performance. Second, I investigated sensorimotor performance in children with 22q11.2DS and its relationships with psychopathology and cognition. I found that children with 22q11.2DS had deficits in sensorimotor performance and that sensorimotor performance was related to attention, spatial planning and spatial working memory ability, but not psychopathology. Third, I investigated coordination using occupational therapy assessments in 10 children who previously screened positive for coordination difficulties, to assess how well a questionnaire measure captured coordination difficulties in this population. Eight of ten of the children assessed were assigned a diagnosis of developmental coordination disorder. In addition, I describe a pilot intervention study in two individuals with 22q11.2DS, which attempted to help improve their coordination skills. Finally, I investigated the brain structure of children with 22q11.2DS and how coordination is related to brain structure. The results showed that children with 22q11.2DS have changes in cortical surface area and volume of the parietal lobe and a larger caudate than unaffected sibling controls, but no relationship was found with coordination. Using diffusion imaging, I investigated the integrity of the cerebellar input and output tracts and found differences in the structure of the inferior cerebellar peduncle. These changes were not related to coordination scores. These results have potentially important implications for our understanding of the relationships between coordination difficulties and other commonly seen psychiatric disorders in 22q11.2DS.

The precursor of miR-140 is located on chromosome 16q22.1 and produces two mature single strands named miR-140-3p and miR-140-5p, which have been associated with several cancers including non-small cell lung cancer (NSCLC). I aim to investigate the differential expression of these two miR-140 strands in NSCLC and their roles in the invasion of lung cancer cells. My hypothesis is that these two miR-140 strands suppress the progression of the NSCLC by targeting specific gene transcripts. MiR-140-3p and miR-140-5p were downregulated in tissues from NSCLC patients and lung cancer cell lines (A549 and SK-MES-1). The sequencing of the miR-140 precursor indicated two DNA variants in SK-MES-1 cells but not in A549 cells. This might affect the biogenesis of its mature strands because the expression levels of both miR-140-3p and miR-140-5p were lower in SK-MES-1 than in A549 cells. Mimics of miR-140-3p and miR-140-5p reduced the invasive properties of A549 cells and enhanced their adhesion to laminin and collagen, two of the main components of the extracellular matrix. In SK-MES-1 cells, both miR-140-3p and miR-140-5p mimics reduced invasion but only miR-140-5p decreased the migration of the cells. The conditioned media from lung cancer cells treated with the miR-140-3p mimics impaired the tubule formation of primary endothelial cells, suggesting its role as an angiogenesis inhibitor. By using bioinformatic tools, the integrin β 3 (ITGB3) was predicted as a novel target of miR-140-3p, which was validated by Dual-Luciferase miRNA Target Expression Vectors and western blotting. By combining the global data from Kinex™ Antibody Microarray (with 878 antibodies) and RNA-Sequencing, I speculate that miR-140-3p inhibits lung cancer invasion and limits the angiogenic potential of the endothelial cells through the epidermal growth factor receptor (EGFR) signalling pathway. In contrast, miR-130-5p may target cyclin-dependent kinase 3 (CDK3) and sulfatase 2 (SULF2) in NSCLC.