The modified Rankin Scale (mRS) is the preferred outcome measure in stroke trials. Typically, mRS assessment is based on a clinician’s rating of a patient interview and interobserver variability is common. Meta-analysis suggests an overall reliability of k=0.46 but this may be less (k=0.25) in multi-centre studies. Mandatory training in mRS assessment is employed in most trials to mitigate this but the problem persists. Variability in assigning outcomes may lead to endpoint misclassification increasing the challenge of accurately demonstrating a treatment effect. We aimed to assess the impact of endpoint misclassification on trial power and explore methods to improve the use of the mRS in acute stroke trials. First we used the mRS outcome distributions of previous phase III randomised controlled trials (RCT) in stroke (NXY059 study and tPA NINDS study) to perform statistical simulations. We generated power estimates and sample sizes from simulated mRS studies under various combinations of sample size, mRS reliability and adjudication panel size. Simulations suggest that the potential benefit of improving mRS reliability from k 0.25 to k 0.5, k 0.7 or k 0.9 may allow a reduction in sample size of n= 386, n= 490 or n= 488 in a typical n=2000 RCT. We then developed a method for providing group adjudication of mRS endpoints and examined the feasibility, reliability and validity of its use in a multicentre clinical trial. We conducted a “virtual” acute stroke trial across 14 UK sites. Local mRS interviews were scored as normal but also recorded to digital video camera. Video clips were uploaded via secure web portal for scoring by adjudication committee reviewers. We demonstrated excellent technical success rates with acceptability to both participants and investigators. 370 participants were included in our “virtual” acute stroke trial and 563 mRS video assessments were uploaded for central review. 96% (538/563) of study visits resulted in an adjudicated mRS score. At 30 and 90 days respectively, 57.5% (161/280) and 50.8% (131/258) of clips were misclassified. Agreement was measured using kappa statistics (k/kw) and intraclass correlation coefficient. Agreement between the adjudication committee was very good (30 days kw 0.85 [95%CI 0.81-0.86], 90 days kw 0.86 [95% CI 0.82-0.88]) with no significant or systematic bias in mRS scoring in comparison to the local mRS. We demonstrated criterion and construct validity of centrally adjudicated mRS scores through comparison with the locally assigned mRS score and other measures known to affect stroke outcome including baseline NIHSS (bNIHSS), Systolic Blood Pressure (SBP), blood glucose and home time. We studied our cohort of mRS video clips to identify any features predictive of variability in mRS scoring. Patient specific variables included participant age, pre stroke mRS, baseline stroke severity as graded by baseline NIHSS (bNIHSS) and presence of language disorder. Interview specific variables included length of interview, poor sound quality, location of the interview, use of a proxy or discussion of prior disability. At both 30 and 90 days only “interview length” was a significant predictor of agreement in mRS scoring. Using a sample of mRS video clips in English and Mandarin, we conducted a pilot study to assess the effect of translation of mRS interviews on interobserver reliability. The interobserver reliability of the translated mRS assessments was similar to native language clips (Native (n=69) kw 0.91 [95%CI 0.86-0.99], Translated (n=89) kw 0.90 [95% CI 0.83-0.96]). We then incorporated a translation step into the central adjudication model using our existing web portal. Inter observer reliability seen in the modified clips (kw 0.85 [95% CI 0.74-0.95]) was similar to that seen in the original video files (kw 0.88 [95% CI 0.78-0.99]). Finally we aimed to investigate the ability of raters to detect more subtle degrees of disability within mRS ranks through blinded assessment of pairs of clips with matching mRS grades. These pairs contained either two clips with full agreement in mRS grade at initial group review or one clip with full agreement and one clip where scores were skewed in the direction of “more” or “less” disability. Pairs were randomly assigned to multiple raters. We could not identify any reliable pattern in identification of the “less disabled” mRS clip. More sensitive grading of the mRS with “good” or “bad” forms of each grade is not reliable on the basis of this exploratory study. Perhaps alternative methods of converting the ordinal ranks of the mRS scale into a more continuous distribution should be investigated; such as the use of a mean mRS score following multiple mRS ratings. Prior estimates of mRS reliability in multicentre studies are poor [k=0.25]. The risks of endpoint misclassification affecting trial power are substantial. Simulations suggest that the effect of improving interobserver reliability and multiple mRS assessments may reduce study sample size by 25%, resulting in substantial ethical and financial benefits. Agreement between our adjudication committee was good [k=0.59(95% CI:0.53-0.63), kw=0.86(95% CI:0.82-0.88)]. Central review may bring many additional potential benefits: “expert” review, quality control and improved blinding in complex trial design. Central adjudication of mRS assessments is feasible, reliable and valid, including the use of translated mRS assessments. This model of outcome assessment has been incorporated into four ongoing large clinical trials: CLEAR-3, MISTIE-3, EUROHYP-1 and SITS-OPEN.

Currently, the American College of Sports Medicine (ACSM) recommends that protocols for cardiopulmonary exercise testing (CPET) should last between eight and twelve minutes. However, the justification for these exercise durations rely on limited experimental data. These recommendations have a significant impact on the ability of frail patients to be assessed using CPET and should conform to evidence based practice. This thesis begins by assessing the validity of these recommendations in relation to maximal exercise responses before assessing the consequences of these recommendations on sub-maximal exercise measurements. These studies were conducted in a relatively large cohort (compared to the study that underpins the ACSM guidelines) of heterogeneous volunteers (they are both men and women, with a significant age range and varied functional capacity) to make the data more relevant to clinical exercise testing. The data presented in chapter three demonstrate that it is very difficult to obtain exercise duration conforming to the current ACSM guidelines by using a standardised ramp exercise protocol on both treadmill and cycle ergometer exercise. However, sub-group analyses for those subjects who achieved moderate (8-12 minutes) and short (less than 8 minutes) exercise durations. In addition, a separate analysis was carried out for a different sub-group of those who achieved moderate (8-12 minutes) and long (more than 12 minutes) of durations of exercise. Despite this, it was possible to demonstrate in sub-group analysis that there was no significant difference in peak oxygen uptake, peak carbon dioxide output, peak heart rate, peak ventilation and peak power output when exercise duration was less or more than that prescribed by the ACSM recommendations. In addition, the effects of long, moderate or short duration exercise per se were also analysed in this chapter and again exercise duration was shown to be without effect on the main maximal markers of exercise performance. In chapters four, five and six, the initial findings were extended to determine the effects of exercise duration on a range of clinically relevant sub-maximal markers of exercise performance. It was likely, since exercise duration did not affect maximal exercise that the physiological determinants of maximal performance were not significantly altered during short or long duration exercise and consequently it was likely that sub-maximal markers of functional capacity would not be affected. However, the quality of the data obtained during CPET can obviously influence the accurate measurement physiological responses during exercise and much of the analysis in these chapters focused on the validity of the data analysis. Chapter four investigated the limitations to measuring the break point in the relationship between oxygen uptake and carbon dioxide output during progressive exercise (the so called ventilatory threshold or ‘V-slope’). The accurate measurement of this break point was determined by standard gas exchange criteria and the effects of reducing the data available for analysis (by reducing the amount of breaths available for comparison at reduced exercise durations) were examined. The data showed that reducing the data available for analysis had an impact on the quality of the data (decreasing the goodness of fit) but no significant effect on the determination of the ventilatory threshold. Chapter five determined the effects of exercise duration on the oxygen uptake efficiency slope (OUES). As expected, the effects of exercise duration were not significant but additional investigation into the commonly employed data analysis procedures was performed. These data show that the log transformation of the relationship between ventilation and oxygen uptake allows reliable assessment of ventilatory efficiency in most cases, however, the impact of the lactate threshold on ventilation and the biological variability in where the threshold occurs as a proportion of functional capacity can impact on the sensitivity of this measurement to predict aerobic and/or anaerobic capacity. Chapter six determined the effects of exercise duration on the breathing reserve index and found no significant difference during short, moderate or long exercise duration exercise. Further analysis was performed to demonstrate limitations in the use of predicted maximum voluntary ventilation (rather than direct measurement). Taken together, these data demonstrate that the current ACSM recommendations for CPET are too restrictive and may limit the application of such testing in populations that cannot exercise for between eight and twelve minutes. The data further suggest that the testing and analysis procedures used during CPET are central to producing valid maximal and sub-maximal markers of functional capacity and the recommendations should focus include guidelines in relation to such aspects.

Background: Indiscriminate friendliness (IF) refers to a lack of reticence with unfamiliar adults and has been well documented in maltreated children. This risky behaviour is distinct from attachment insecurity and has been found to persist when care-giving quality improves. There is a lack of consistency in the literature regarding the importance of care-giving following adoption. Some studies suggest that care-giving quality is not related to IF, whilst others have suggested that the emotional availability of carers is predictive. This study aimed to establish if there is a relationship between EA and IF in a group of previously maltreated infants. Method: In a cross-sectional design, a subsample of infant-carer dyads (n = 55), that were recruited as part of an on-going RCT (Pritchett et al, 2013), were observed. Videos of meal and playtime activities were analysed using The EA Scales (Biringen, 1998). IF was measured, as part of the RCT, using a semi-structured interview. This tool also identifies children that in addition to IF, have no preferred attachment figure: IF (NA). Univariate correlation analyses and regression analyses were used to explore relationships between variables. Results: This study found that child emotional availability predicted indiscriminate friendliness, even when other associated factors (age and carer non-intrusiveness) were controlled for. A composite Carer EA score was not related to IF, but carer non-intrusiveness was significantly associated with IF. Conclusions: Child emotional availability is uniquely associated with indiscriminate friendliness in maltreated children. A specific care-giving factor (non-intrusiveness) was associated with indiscriminate friendliness. It is suggested that carer-child interactions are related to indiscriminate friendliness in maltreated children and may represent a useful target for intervention. Therefore, future research may wish to explore the amelioration of indiscriminate friendliness through an intervention focusing on the carer-child relationship.

Bladder cancer is the 5th most common and the 9th most lethal cancer in the UK. Based on histopathological and genomic analysis, a model of two independent pathogenesis pathways has been suggested, resulting in either non-invasive superficial or invasive urothelial tumours with potential to metastasise. Prominently, the fibroblast growth factor receptor 3 (FGFR3) is found mutated in up to 84% of non-invasive superficial tumours. Alterations in FGFR3 such as mutation or wild type receptor overexpression are also found in 54% of muscle-invasive tumours. FGFR3 is a tyrosine kinase receptor for fibroblast growth factors (FGFs), which stimulates both the RAS/MAPK and the PI3K/AKT pathways and regulates a range of cellular processes such as cell growth and division during development. In this study we examined the role of FGFR3 in bladder cancer by using mice as a model organism. Firstly, we addressed whether combination of Fgfr3 and Pten mutation, UroIICre Fgfr3+/K644E Ptenflox/flox, is able to drive non-invasive superficial bladder cancer. We observed that the thickness of the double mutant urothelium was significantly increased compared to singly mutated Fgfr3 or Pten, UroIICre Fgfr3+/K644E and UroIICre Ptenflox/flox. Moreover, several cellular abnormalities were detected that were accompanied by differential expression of layer-specific markers, which strongly suggested that they were caused cooperatively by Fgfr3 mutation and Pten deletion. The results supported the hypothesis that FGFR3 activation can play a causative role in urothelial pathogenesis of non-invasive superficial bladder cancer together with upregulated PI3K-AKT signalling. Secondly, we aimed to identify mutations that cooperate with Fgfr3 and with other common bladder cancer mutations such as Pten and Ras, in promoting urothelial tumourigenesis by Sleeping Beauty (SB) insertional mutagenesis in mice. The SB system may constitute an inefficient tool in the bladder to induce urothelial tumourigenesis, since it failed to produce bladder tumours in Fgfr3 as well as in Hras mutant mice. In mice with Pten deletion, one tumour was generated and general hypertrophy with cellular abnormalities was observed in all samples. No direct association between Fgfr3 and Pten mutations was found; however, SB mutagenesis supported that Fgfr3 and Pten cooperation may merge at the signalling downstream. Thirdly, we examined the role of the most common mutation in FGFR3, S249C, in the urothelium and in tumour progression and invasion by subjecting Fgfr3 mutant mice to a bladder-specific carcinogen, N-butyl-N-(hydroxybutyl)-nitrosamine (OH-BBN). We showed that FGFR3 S249C mutation by itself does not lead to urothelial abnormalities. However, in OH-BBN-induced tumours the presence of S249C increased the number of animals that formed bladder tumours by 4.4-fold. Our results present for the first time an effect of FGFR3 S249C mutation in invasive bladder cancer. Lastly, we sought to establish methods to generate and assess invasive bladder tumours using in vivo and in vitro techniques. First we examined the effectiveness of a Cre-expressing adenovirus (AdenoCre) to generate mouse models of bladder cancer with different combinations of genetic mutations. p53 deletion or mutation together with Pten loss led to formation of aggressive bladder tumours; however the origin of these tumours was likely to be the bladder muscle. Hras activation in combination with Pten deletion did not produce tumours or any cellular abnormalities by 8 months. AdenoCre-mediated tumour induction was successful in the presence of β-catenin and Hras mutation. However, an issue of AdenoCre transduction was the frequent observation of tumours in various other tissues such as the pelvic soft tissue, liver, pancreas and lung. Using an optimised AdenoCre procedure, the technique would allow lineage tracing of cancer stem cells in a developing bladder tumour and potentially during metastatic spread. Secondly, we tested imaging techniques in the living animals and validated ultrasound as a functional method to detect bladder wall thickening, as well as to monitor tumour growth in vivo. Thirdly, with the aim to assess cell transformation, migration and response to drug treatment, we tested essential ex vivo techniques and assays such as 3D sphere culture, organotypic slice culture as well as a Collagen-I invasion assay. The 3D tumour sphere culture was successful with murine Wnt-activated tumours as well as with invasive human cell lines. The organotypic slice culture was assessed as a system to test the effect of therapeutic drugs on the tumour cells; however, an issue of tissue disintegration has yet to be overcome. The Collagen-I assay successfully recapitulated invasion of a human bladder cancer cell line; however, the system needs to be adapted to murine bladder tumours. Taken together, this study presents for the first time evidence that support the functional role of FGFR3 signalling in the early stages of non-invasive urothelial carcinoma as well as in tumour progression of established neoplasms in mice. Given the wide availability of inhibitors specific to FGF signalling, our FGFR3 mouse models in conjunction with optimised ex vivo assays and imaging systems may open the avenue for FGFR3-targeted translation in urothelial disease.

Background: In comparison to other medical appointments, consultations regarding haematopoietic stem cell transplant (HSCT) tend to be emotional and longer due to the high volume and content of information exchanged between the patient and consultant. HSCT offers the potential to cure the cancer but also carries a multitude of life-threatening side effects. Existing research has shown that patients immediately forget the majority of information they receive in medical consultations thus resulting in misunderstandings about treatment, adjustments and about coping post transplant. Despite this, no previous research has evaluated cancer patients’ level of recall and understanding considering the volume of information they receive from doctors during consultation for HSCT. Aim: To create, within a haemato-oncology setting, a coding framework capable of evaluating the interaction between the patient and doctor in the HSCT consultation. Methods: The medical consultations of five HSCT patients who were eligible for HSCT were recorded and these patients subsequently completed semi-structured interviews. Transcripts were analysed using directed content analysis. A recall and understanding information template (RUIT) with an associated coding framework was developed and piloted. Results: The procedures undertaken in developing the RUIT demonstrate strong inter-rater reliability and content validity. Further testing, through piloting the instrument, indicated that the RUIT coding system has strong inter-rater agreement. However, disparity in the classification of some categories was also revealed. Cancer patient’s viewed the consultation as informative but also felt that both the content and volume of information they received were difficult to process. Conclusions: This study is the first qualitative investigation of cancer patients’ recall and understanding of content from a HSCT consultation through the unique development of a coding framework. Future use of the RUIT in clinical practice and recommendations for further research are discussed in relation to the relevant literature.

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease characterised by extensive synovitis resulting in cartilage and bone erosions. Both the innate and adaptive immune pathways contribute to the initiation and the maintenance of the disease. Understanding the role of these pathways is central to develop new therapeutics. We have developed a murine model of RA where ovalbumin (OVA) specific Th1 cells induced a breach of self-tolerance and a transient monoarthritis. This thesis aimed firstly to create a model of chronic autoimmune polyarthritis and then to investigate the contribution of B cells and innate inflammation to the induction of arthritis. Relapse of arthritis was associated with the nature of the antigen (OVA) employed and the route of administration. The analysis of collagen specific B cell response revealed that anti-type II collagen antibodies titres rise during the induction of the relapse of arthritis and that they were directed against the epitope U1. Although typical RA autoantibodies were detected in OVA-mediated arthritis, a mild arthritis could be elicited in absence of antigen presenting B cells and in complete absence of mature B cells. B cells were not necessary in the induction of pathology even though their presence was associated with a higher joint histology score. Finally, this thesis describes that an innate inflammatory stimulus, such as LPS, elicited joint pathology but was insufficient to breach B and T self-tolerance. On the contrary, antigen-specific T cell activation led to arthritis and the production of several autoantibodies typical of RA. The relapse and spread of arthritis developed in this thesis provides a useful tool to investigate the contribution of the innate and adaptive immune pathways in the development of autoreactive responses. A better understanding of these mechanisms will hopefully help to design new therapeutic intervention aiming to re-establish immunological tolerance.

This project is the first to investigate blood perfusion in the human hamstrings during isometric exercise with a near infrared spectroscopy (NIRS). A Kin Com dynamometer has been used to fix the knee positions and to measure torques during contractions. Both the NIRS optodes and the electromyography (EMG) electrodes were attached to the skin over the hamstrings. Previous studies used a NIRS to measure muscle blood flow in the forearm, quadriceps and calf muscles. The changes in haemoglobin concentrations were calculated using Spike 2 software. A total of 46 male volunteers participated in the four series of experiments described in this thesis. The following overall conclusions can be drawn: perfusion decreases in the hamstrings during contractions and then returns to normal levels after a period of time, changing the limb position at which the contractions are made does not affect the perfusion, warm-up exercises increase in blood perfusion for 8 minutes at 30 and 40% of MVC. The perfusion did not significantly change during an episode of DOMS or in the injured and non-injured limbs. These conclusions show the importance of warm-up before sports activities but not necessarily avoid injury. It can be concluded that there is no association between such conditions with hamstring injuries. The maintained perfusion at different conditions is a positive finding as the perfusion is not restricted indicating good delivery of oxygen despite muscle injury.

Hypertension is a major risk factor for all cardiovascular disease, which is the largest known cause of global mortality. Essential hypertension-that is hypertension of unknown cause-is thought to have genetic and environmental risk factors. The best studied genetic system is that concerning corticosteroid biosynthesis. In humans, the principal glucocorticoid is cortisol, the main function of which is the control of intermediary metabolism; the major mineralocorticoid is aldosterone, which affects electrolyte and acid-base homeostasis. These steroid hormones are produced in the adrenal cortex through a series of biosynthetic reactions and under the influence of multiple regulatory factors. The final step in cortisol and aldosterone production involves, respectively, the cytochrome p450 enzymes, 11β-hydroxylase and aldosterone synthase. These are encoded by the CYP11B1 and CYP11B2 genes which have a similar sequence and are highly polymorphic and lie, in tandem, on human chromosome 8. Regulation of CYP11B1 and CYP11B2 mRNA abundance and of aldosterone and cortisol production have been extensively investigated. These studies have identified that there are several polymorphisms located across the locus which are associated with an increased aldosterone to renin ratio (ARR; used as an indicator of aldosterone regulation), inefficient 11β-hydroxylation and essential hypertension. However, to date, no underlying mechanism for these associations has been established. Regulation of expression by transcription factors has been widely studied but, in this thesis, it is the role of a novel regulator, microRNA (miRNA) that is central. miRNAs are short, non-coding RNAs which negatively regulate mRNA abundance They are transcribed from endogenous loci, then undergo a series of enzymatic maturation reactions that result in the production of a single-stranded molecule of approximately 20 nucleotides. They function by associating with a group of proteins known as the RNA-induced silencing complex (RISC) and targeting the 3’ untranslated region (3’UTR) of specific target mRNAs which they bind with imperfect complementarity. There are approximately 1100 human miRNAs, which have been implicated in the regulation of a range of target mRNAs and in several pathologies including cancer and cardiovascular disease. The aim of this project was to investigate what role, if any, miRNAs have in the regulation of CYP11B1 and CYP11B2 expression and in corticosteroid production. The studies in Chapter 3 investigated miRNA regulation of corticosteroidogenesis in the adrenal cell line H295R. miRNA levels were universally reduced by targeting Dicer mRNA, a key component of the miRNA synthetic pathway, with short interfering RNA (siRNA). This study identified all of the CYP450 enzymes of the corticosteroidogenic pathway (CYP11A1, CYP17A1, CYP21A1, CYP11B1 and CYP11B2) as likely candidates for miR-mediated regulation based on mRNA and steroid analysis. The study also suggested that StAR, 3βHSDII and 11βHSDII are not modulated by miRNAs. To determine whether apparent miRNA regulation of CYP11B1 and CYP11B2 expression occurs by direct action at their 3’UTRs, reporter constructs were generated and tested. Under both basal and stimulated (AngII) conditions, these studies support a regulatory mechanism involving the 3’UTR of CYP11B1 and CYP11B2. This chapter therefore provides evidence for miRNA-mediated regulation of corticosteroidogenesis. In Chapter 4, putative miRNA target sites in the CYP11B1 and CYP11B2 3’UTR were identified using bioinformatic prediction algorithms and the miRNA expression profile of the normal human adrenal, as determined by microarray analysis. Based on miRNA target site prediction and analyses of the 3’UTR sequences (including such parameters as relative length, predicted sequence conservation and RNA secondary structure), in silico methods indicated the possibility that miRNAs can target CYP11B1 and CYP11B2 mRNA. Furthermore, the expression of 107 miRNAs in the normal adrenal gland was confirmed. Cross-referencing of microarray expression and bioinformatic data identified 16 adrenal miRNAs predicted to bind putative sites in CYP11B1 and 16 predicted to bind CYP11B2; 12 of these miRNAs were common to both genes. These formed the basis of the miRNA target validation studies in Chapter 5. Sixteen adrenal miRNAs identified by bioinformatic analysis were tested individually in vitro. This was achieved by measuring mRNA expression, steroid production and 3’UTR reporter construct activity following artificially induced increases or reductions in the levels of specific miRNAs. These studies identified some miRNAs as being false positive predictions, while certain others were validated. The miRNA that gave the most striking and consistent results, for targeting both CYP11B1 and CYP11B2, was miR-24, which significantly decreased mRNA levels and steroid production. Analysis of adrenal miRNAs predicted only to target the CYP11B2 3’UTR confirmed miR-125a-5p and miR-125b as novel regulators, although effects on steroid secretion remain to be assessed. The studies in this chapter are the first to report of miRNA-mediated regulation of CYP11B1 and CYP11B2 expression. Finally, in Chapter 6, the miRNA expression profiles of four aldosterone producing adenoma (APA) samples were generated and compared to those of normal adrenal gland. Analysis identified 67 miRNAs expressed within the APAs; 54 were also present in the normal tissue. The levels of several miRNAs, including miR-24 and miR-125a-5p, were shown to be differentially expressed between the tissue types. This chapter also describes polymorphisms within the 3’UTR of the CYP11B1 gene, generated from 26 normotensive patients. No novel SNPs were identified, but three are located in putative miRNA-binding sites. Previously, sequence analysis of the CYP11B2 3’UTR had been used to map miRNA binding sites, this identified two miRNA-binding sites which mapped to a known SNP. Taken together, the studies in this chapter provide a foundation for exploring altered miRNA function and/or expression within the adrenal gland. In summary, the results presented in this thesis support a role for miRNA mediated regulation of corticosteroidogenesis through actions on CYP11B1 and CYP11B2 expression. It demonstrates that miRNA are present in the adrenal gland, that miRNA-binding sites are present on the 3’UTR of relevant mRNAs, and that miRNAs are capable of post-transcriptional regulation that significantly alters mRNA abundance and steroid production. My findings describe a novel regulatory mechanism of corticosteroidogenesis. Whether this mechanism is altered in diseases such as essential hypertension remains to be elucidated. If so, miRNAs could, in the longer term, be used as targets for novel therapies or as biomarkers to classify more precisely specific pathologies.

Initially, prostaglandins (PGs) were considered to only exist as free acid mediators. Although, formation of PG glycerol esters and PG ethanolamides by cellular cyclooxygenase (COX)-2 has been reported, generation of complex oxidised lipids via COX-1 has not been considered. In this study, formation of sixteen unique PG-containing phospholipids generated by agonist-activated human platelets is demonstrated using lipidomic approaches. Precursor scanning-tandem mass spectrometry identified a group of specific lipids comprising PGE2, PGD2 and two previously undescribed PG-like molecules (named PGb and PGc), attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/ and 18:0a/). PGb and PGc were also detected as free eicosanoids and their structures remain to be characterised. These novel lipids formed within 2-5 minutes of platelet activation by thrombin, collagen or ionophore and required activation of several intracellular signalling intermediates, including cytosolic phospholipase A2 (cPLA2), p38 mitogen-activated protein kinase (MAPK), src tyrosine kinases, phospholipase C (PLC) and cytosolic calcium. Unlike free PGs that are secreted, PG-PEs remain cell associated, suggesting an autocrine mode of action. Aspirin supplementation in vivo (75 mg/day) or in vitro (1 mM) blocked their generation, indicating that COX-1 is required. Pharmacological studies using inhibitors of fatty acyl re-esterification significantly reduced formation of PG-PEs. Furthermore, purified COX-1 was unable to directly oxidise PE in vitro. Collectively, these indicate that PG-PEs are initially formed as free PGs via COX-1, and then rapidly esterified into PEs. In summary, this is the first demonstration of acute generation of PG-PEs in agonist-activated human platelets from endogenous substrate via COX-1. These unique lipids may represent additional bioactive molecules from this key platelet enzyme.

Hepatitis C virus (HCV) infects 3% of the global population and HCV-related liver inflammation is a major cause of liver failure and hepatocellular carcinoma. Current treatments are based upon long courses of interferon-α (IFNα) injections, which have significant side effects and are only effective in 40-80% of individuals depending on viral genotype. Natural killer (NK) cells are innate lymphocytes, which can kill virally infected cells and are stimulated by IFNα. To establish a chronic infection HCV must evade immune responses. I hypothesised that NK cells are important for the successful eradication of HCV and that chronic HCV infection impinges upon NK cell function to prevent viral clearance. I found that NK cell function was reduced in chronic HCV and correlated with the proportion of NKp46+ NK cells in vitro. In keeping with these findings NKp46-rich intrahepatic NK cell populations were more activated and the proportion of these cells correlated with liver inflammation. During interferon-α treatment individuals who had the greatest increase in NK cell function in response to increasing stimulation had the fastest rate of viral clearance and were most likely to successfully clear the virus. Using a novel adenovirus vector expressing HCV proteins I have discovered that NS5B protein reduces NK cell cytotoxicity and cytokine production. Therefore, in this thesis I have described novel insights into the mechanisms of HCV immunoevasion, HCV-related disease pathogenesis with implications for viral eradication therapy.