Abnormalities of the cervical or intracranial circulation, termed arteriopathies are the leading mechanism of both cause and recurrence of childhood arterial ischaemic stroke (AIS). Approximately 20% of children with AIS will have stroke recurrence but there are currently no robust biomarkers to identify this high risk group, and hence identification of patients who may be amenable to secondary preventative strategies has not been possible. This thesis attempts to address this unmet need by studying novel biomarkers to distinguish patients at risk of stroke recurrence. Indices of endothelial injury, repair and hypercoagulability were compared between patients with recurrent clinical disease course and children with a single event. Circulating endothelial cells (CECs) were higher in children with recurrent AIS, compared to those with no recurrence and controls. Further evidence of endothelial injury and cellular activation was derived by examining circulating microparticles (MP) profiles. Plasma from patients with AIS recurrence contained increased numbers of endothelial, platelet and neutrophil derived MP compared to those with no recurrence. These MPs were highly prothrombotic due to phosphatidylserine exposure providing a platform for the assembly and activation of coagulation factors; and also expression of tissue factor on some MP. An efficient in vitro assay to assess MP-related hypercoagulability by quantifying MP-mediated thrombin generation was established. Children with recurrent AIS were shown to have an enhanced MP-mediated thrombin generation. Lastly, a disturbance in endothelial progenitor cells (EPCs) in children with AIS recurrence was observed suggesting that there could be impairment of endothelial repair in these patients. In conclusion, despite the wide spectrum of clinical and radiological presentation of childhood AIS, the studies undertaken in this thesis suggest that there is an unfavourable imbalance between endothelial injury and repair, and excess hypecoagulability in children with recurrent AIS. These novel observations provide unique insights into the pathophysiology of paediatric AIS.

may suffer negative outcomes following admission to hospital. Previous research in the USA, Finland, Sweden and Australia has shown that up to 54% of children exhibit problematic behaviours (PB) 2 weeks post-discharge and 16% at one month. Risk factors included higher child/parent pre-operative anxiety, child temperament, pre-surgical behavioural disturbances, younger age, premedication, and pain at home. The incidence of post-hospital PB in British children is not known and potential influencing variables have not been examined. The aims of this study were to describe and compare children’s post-hospital PB following day case or inpatient surgery and to examine the association of parent, child, pre-operative and in-hospital factors with parent and child anxiety, preparation for surgery and child posthospital PB and postoperative symptoms. A descriptive, repeated measures study design was used, involving self-report questionnaires, direct observation of behaviour and post-discharge questionnaire follow-up. Children, 2 to 12 years, scheduled for general, ENT and urology surgery under general anaesthesia were invited to participate. 73.3% children exhibited PB and 93.4% were in some pain (≥1, 0-10 NRS) on day 2 postdischarge from hospital. The incidence of PB and pain decreased significantly over the follow-up period with 31.8% children exhibiting PB and 25.2% experiencing pain at the end of week 4. PB and pain were associated with families taking additional time off work/school and increased follow-up healthcare. Following multivariate regression analyses, factors associated with PB were parents’ level of preparation for their child’s care at home, higher parent education, younger child age, the child’s previous pain experience, children who did not attend pre-admission clinics, child and parent anxiety, children who stayed overnight in hospital, and higher child pain intensity at home. The findings suggest that poorer parent self-efficacy in caring for their child in hospital and at home are associated with increased child negative outcomes at home.

Thymosin β4 (Tβ4) is a 43 amino acid peptide encoded by the Tmsb4x gene located on the X-chromosome. It has previously been shown to act as a secreted factor from the myocardium to the overlying epicardium of the developing murine heart, to mediate transformation of epicardial derived progenitor cells (EPDCs) into the coronary vasculature. This PhD project seeks to build on these studies and characterises the function of Tβ4 in the developing systemic vasculature, using the mouse as a model system. Expression analyses demonstrated specific localisation of Tβ4/Tβ4 in the endothelial cells of the embryonic vasculature. In order to ascertain the function of vascular Tβ4, global and endothelial cell specific in vivo Tβ4 loss of function models were examined. Both global and endothelial-specific Tβ4 mutant embryos displayed a reduced recruitment of vascular mural cells to developing blood vessels. Detailed phenotypic examination revealed that the mural cell deficit could be attributed to impaired differentiation of mature mural cells from undifferentiated mesoderm. This process was modelled in vitro, and it was discovered that treatment of the mural progenitor cell lines 10T1/2 and A404 with exogenous Tβ4 could promote their differentiation into mural cells. This process correlated with an increase in Smad phosphorylation and increased activity of the TGF-β pathway. Decreased levels of TGF-β target genes in vivo in Tβ4β null embryos indicated that TGF-β signalling was perturbed in the absence of Tβ4. These findings suggest a model whereby Tβ4 is secreted by the developing endothelium to stimulate the differentiation of uncommitted mesoderm into mature peri-vascular mural cells, via activation of the TGF-β pathway in the target cell population. As a consequence, Tb4 plays an essential role in vascular stability through mural cell support which has implications for vascular dysfunction in disease.

Polycystic kidney disease (PKD) is the most common inherited cause of kidney failure affecting 1 in 1000 adults and children worldwide. Many studies focus on the disrupted cellular functions within the cyst epithelia but promising therapeutic strategies have not yet translated to clinically approved treatments for human PKD. This thesis takes a different approach and hypothesised that cyst growth requires the support of the underlying renal microvasculature and that targeting vessels could be an alternative therapeutic strategy. The renal vasculature in cystic and wild-type kidneys was first characterised using a mouse model of autosomal recessive PKD (ARPKD). It was found that mRNA levels of markers of blood and lymphatic endothelium were altered in cystic kidneys compared with wild-type. By immunohistochemistry, there was disorganisation of the vasculature with expansion of lymphatic capillaries surrounding the smaller cysts and regression of the microvasculature surrounding the larger cysts. Overall, the peri-cystic microvasculature shifted from a blood to a lymphatic endothelial phenotype. To test the potential of targeting the renal vasculature as a treatment for PKD, vascular endothelial growth factor-C (VEGFC) was administered to mouse models of both ARPKD and autosomal dominant PKD (ADPKD). The treatment significantly reduced disease severity as measured by kidney/body weight ratio by at least 28% in both models. In the model of ADPKD, serum blood urea nitrogen was also significantly reduced by around 35% indicating an improvement in renal function. Measurement of cyst characteristics revealed improvements in percentage cyst area, total number of cysts per kidney and the average cyst size. The treatment also restored normal renal vascular patterning and decreased the number of CD206+ macrophage cells which contribute to cyst growth. This thesis suggests that targeting the renal vasculature could be a therapy for PKD, with the potential to enhance current approaches.

The majority of children with Cerebral Palsy (CP) have feeding difficulties, which result in chronic malnutrition and respiratory disease, reducing quality of life for caregiver and child, and causing early child mortality. In well-resourced countries, high and low-tech medical interventions, ranging from gastrostomy tube-feeding to parent training, are available. In Bangladesh the former is not viable and the latter is both scarce and its effectiveness not evaluated. The study aimed to evaluate the effectiveness of a training programme to improve the feeding practices of carers of children with CP, observing the impact on level of nutritional intake, risk of aspiration and distress caused to both during feeding. Thirty-seven caregivers and their children aged 1-11 with moderate-severe CP and feeding difficulties were invited to a six-session training programme. Pre and post measures (quantitative and qualitative) were taken during home visits in addition to giving brief advice. A control phase was evaluated for 12 of the participant pairs whilst awaiting training. A minimum of four training sessions was successful in significantly improving children’s nutritional intake and chest health, maximising independence in feeding, improving the experience of mealtimes for both child and caregiver, decreasing caregiver stress regarding their child’s feeding difficulties and improving child levels of cooperation. Catch-up growth was observed in 26% of the children. A significant difference in the outcomes between advice only and groups was observed. In conclusion, carers in Bangladesh, who have minimal formal education and live in abject poverty are able to change care-giving practices significantly after four training sessions, with positive consequences for both child and caregiver. Methods of providing affordable food supplementation need to be investigated and further steps must to be taken to lobby policy-makers in order to ensure that services have the motivation and capacity to address this area of need.

During sepsis and critical illness, a systemic inflammatory response can significantly produce reactive oxygen and nitrogen species, which can damage host tissues. Usually, several different antioxidant defences protect host cells from these reactive species. One of the important antioxidant defences is glutathione, a tripeptide synthesised from the precursor amino acids cysteine, glutamate and glycine. The aim of this study was to measure glutathione synthesis in vivo, using deuterated glycine as a tracer. Firstly, a new method for analysis of glutathione and glycine using both gas chromatography-mass spectrometry (GC-MS) and gas chromatography-isotope ratio mass spectrometry (GC-IRMS) was developed. The derivatives of both compounds were measured in a single chromatographic analysis, and the method was compatible with both GC-MS and GC-IRMS, and capable of measuring low carbon-13 or deuterium enrichment of glutathione in 50μl of erythrocytes. Erythrocyte glutathione synthesis was measured, using GC-IRMS, in critically ill infants and children who had been infused with deuterated glycine. Glutathione fractional synthesis rate (FSR) was not statistically different between septic and non-septic patients. In order to determine whether glutamine is able to increase GSH synthesis by acting as a glutamate precursor, erythrocyte GSH synthesis was measured using GC-IRMS in septic infants and children randomised to receive glutamine dipeptide or placebo. There was no significant difference in glutathione FSR between those given glutamine and those given placebo. Intestinal glutathione depletion is known to occur following intestinal ischaemia-reperfusion injury. I measured glutathione synthesis in a rat model of intestinal ischaemia-reperfusion injury, and found that hypothermic rats with ischaemia-reperfusion injury had a higher intestinal glutathione synthesis than normothermic rats, providing a potential mechanism by which hypothermia may maintain intestinal glutathione levels. In conclusion, the newly developed method of glutathione analysis using GC-IRMS was useful for compound specific isotope analysis of biological samples.

Post-transcriptional gene regulation is a very powerful and evolutionary conserved mechanism which allows cells to finely modulate gene expression by acting at the level of their mRNAs. In this thesis the post-transcriptional regulation of the Brn-3b transcription factor was analyzed as a case study. This protein belongs to the POUIV family and to the homeobox super-family of transcription factors which are involved in neuronal differentiation and body patterning. The data presented in this thesis show that regulatory sequences contained in the 3’UTR of Brn-3b mediate the degradation of its mRNA in some neuronal cell lines. Furthermore, the regions regulating this effect have been characterized and two different microRNAs, very short RNA molecules which are key players in the post-transcriptional regulation of mRNAs, have been found to be directly involved in regulating the levels of Brn-3b. As Brn-3b plays an essential role in the survival of retinal ganglion cells, it was also investigated whether its mRNA is similarly regulated in a retinal ganglion cell line (RGC-5) as it occurs in other neuronal cell lines. Intriguingly, possibly due to its pro-survival role, Brn-3b is protected from degradation by microRNAs in RGC-5 cells in contrast to its fate in other cell types. Furthermore, one of the two microRNAs involved in its regulation is not expressed in the RGC-5 cell line, paralleling its expression profile in primary RGCs. Following a brief analysis concerning the possible regulation of Brn-3b in primary retinal ganglion cells, the possible general role of post transcriptional gene regulation was assessed by evaluating the role of Dicer in the differentiation of primary retinal ganglion cells. This showed that Dicer, one of the key enzymes in the production of microRNAs, might be involved in the modulation of neuronal differentiation markers and might thus have a direct role in retinal ganglion cell differentiation. Overall this thesis provides evidences that post-transcriptional gene regulation is a mechanism which might modulate the levels of mRNA molecules depending on whether the encoded protein has a pro-survival role. Furthermore, preliminary evidences suggest that Dicer, a key enzyme in the production of microRNAs, might have a role in the modulation of retinal ganglion cells differentiation markers.

INTRODUCTION: Low post-operative death rates after cardiac surgery make mortality an inadequate outcome measure. As post-operative morbidity is more common, its measurement would be more sensitive. Accurate identification and quantification might also allow its aetiology to be addressed. The nine domain Post-Operative Morbidity Survey (POMS)(1) is the only prospective tool for standardised morbidity measurement in general surgical patients. I sought to develop and validate such a tool (cardiac- or C-POMS) for cardiac surgery. METHODS: Development: Morbidity was prospectively assessed in 450 cardiac surgery patients on postoperative days 1, 3, 5, 8 and 15 using POMS criteria and cardiac-specific variables (from an expert panel). Other morbidities were noted as free-text and included if prevalence >5%, missingness <5% and mean expert-rated severity-importance index score >8. Reliability/validity: assessed by expert panel review, using Cronbach’s alpha (internal consistency) and linear regression to test the ability of C-POMS to predict length of stay (LOS). Clinical utility: assessed by multi-professional teams at two hospitals. RESULTS: Development: Following item-reduction, C-POMS resulted in a 13 domain model. Reliability/validity: Internal consistency (>0.7) on D3-D15 permits use of C-POMS as a summative score of total morbidity burden. Mean C-POMS scores were 3.4 (D3), 2.6 (D5), 3.4 (D8) and 3.8 (D15). Patient LOS was 4.6 (p=0.012), 5.3 days (p=0.001) and 7.6 days (p=0.135) longer in patients with (compared to without) morbidity on D3, D5, D8 and D15, respectively. For every unit increase in C-POMS summary score subsequent LOS increased by 1.7 (D3), 2.2 (D5), 4.5 (D8) and 6.2 (D15) days (all p=0.000). Clinical utility: Demonstrated by C-POMS now being routinely collected at two hospitals. CONCLUSIONS: C-POMS is the first validated tool for identifying total morbidity burden post cardiac surgery. CPOMS identifies considerable morbidity in these patients and may assist in modelling causation and in identifying preventative and therapeutic targets.

RNA interference (RNAi) provides a specific and efficient way to silence gene expression; therefore, it is an attractive tool to be used in basic research on gene function as well as gene therapy. Despite the enormous potential of RNAi, delivering the small interfering RNA (siRNA) to the cells is one of the main hurdles. Previously there have been reports showing effective plasmid DNA delivery to cells and such systems could be used to deliver siRNA to cells due to the similarity of the delivery criteria between plasmid DNA and siRNA. Therefore, I hypothesise that a successful siRNA delivery system will have similar biophysical characteristics as a successful DNA delivery system. The aim of this study is to identify the important criteria to establish a promising siRNA delivery system by comparing the criteria of successful DNA delivery systems such as linear and branched polylysines and linear and branched PEIs. In order to deliver nucleic acid to a cell, a vector system should be able to bind and form a positively surface-charged nano-sized complex with the nucleic acid for cellular binding and uptake. Inside the cell, the vector should be able to dissociate from the nucleic acid for gene expression or silencing. Therefore, the important parameters to investigate are the binding and dissociation properties of the vector components to the nucleic acid and the size and surface charge of the complex. From the results, generally all the polylysines and PEIs can bind, dissociate and form a positively charged nano-particle with plasmid DNA, which can mediate gene expression. Despite the ability of all the polylysines and PEIs to bind to and dissociate from siRNA, only branched polylysines, linear and branched PEI, but not linear polylysines can form positively charged nano-particles with siRNA. Indeed, branched PEI behaves similarly towards siRNA and DNA biophysically. Interestingly, only branched PEI and siRNA complexes can mediate cellular uptake and 60% target gene knockdown. Branched polylysines or linear PEI siRNA complexes cannot mediate gene silencing in spite of the formation of positively charged nano-particles. This could be due to poor cellular uptake of these complexes or degradation of siRNA upon uptake. Therefore, to improve the design of the siRNA vector system, there is a need to research the cellular binding and uptake of siRNA complexes in the future.

Collagen induced arthritis (CIA) is a mouse model of autoimmunity that closely resembles human rheumatoid arthritis (RA), a debilitating disease with no cure. This study has been undertaken to generate antigen-specific tolerance to an autoantigen implicated in RA and immunodominant in animal models. Gene therapy protocols for RA that have gone to clinical trial have been designed to drive expression of therapeutic molecules at the site of inflammation, but not to modulate the immune response to key autoantigens. This study has shown that lentiviral vectors (lvv) expressing fusion proteins (FP-lvv) confer antigen-specific tolerance in CIA. Fusion proteins comprised of an endosomal-targeting domain coupled to the immunodominant CII259-273 peptide and an eGFP tag, were expressed in APCs. Confocal microscopy revealed substantial colocalisation with endosomes and lysosomes. Expression of the fusion proteins in APCs resulted in MHCII-presentation of the immunodominant CII259-273 peptide to CII259-273-reactive CD4+ T cell hybridomas. Furthermore, co-transduction with a lvv expressing the enzyme lysyl-hydroxylase 3 enhanced glycosylation of the expressed CII construct. Administering mice iv with FP-lvv one month prior to disease induction reduces by half the arthritic score during the first two weeks of clinical symptoms in CIA, providing partial but significant protection. The use of suitable controls showed that this effect is antigen-specific and measurements of α-CII IgG show a significantly lower titre in treated animals. This study provides evidence that lvv-mediated MHCII-presentation can be tolerogenic and hence, this approach could form an important part of future treatments for autoimmune diseases.