Severe acute malnutrition (SAM) affects approximately 13 million children under-five and is associated with over 1.5 million preventable child deaths each year. Case fatality rates in hospitals treating SAM remain at 20-30%, and coverage of those affected remains low. Training and support to improve centre-based management can reduce case fatality rates. However, an exclusive inpatient approach does not consider the many barriers to accessing treatment that exist for poor people in the developing world. Community-based therapeutic care (CTC) is a new approach for the management of SAM that uses Ready-to-Use Therapeutic Foods (RUTF) and triage to refer cases without complications to outpatient care and those with complications to inpatient treatment. This thesis aims to test the hypotheses that a CTC strategy can treat children with SAM effectively and can achieve better population treatment coverage than a centre-based approach. Five studies, using primary data, are presented. The first 3 studies evaluate the clinical effectiveness of CTC through examination of individual outcome data from research programmes in Ethiopia and Malawi. The fourth study examines the coverage of a CTC programme for SAM in Malawi and compares this with coverage of a centre- based programme. The final study is a multi-country evaluation of 17 CTC programmes implemented across Africa. Results from all studies that use the CTC treatment model show that outcomes can meet the international Sphere standard indicators of < 10% mortality and > 50% coverage. Coverage of a CTC programme in Malawi was three times that of a centre-based programme in the same region (73.64% (95% C.I. 66.0%, 81.3%) vs. 24.5% (95% C.I. 17.8%, 31.4%)). A number of factors were vital to achieving low mortality and high coverage in these programmes. These included decentralisation of outpatient treatment services and community mobilisation techniques to encourage early presentation, and the use of appropriate triage criteria, to identify children suffering from SAM with no complications that could be treated safely as outpatients. The use of triage did not appear to increase mortality (OR 0.51 95% CI 0.28, 0.94). This thesis suggests that CTC does not increase case fatality rates associated with SAM and could reduce them, and that it could increase the number of children receiving treatment.

The signal transducer and activator of transcription (STAT) proteins are a family of transcription factors which transduce extracellular signals from cytokines, growth factors and G-proteins to the nucleus. STATs become activated by phosphorylation and translocate to the the nucleus where they bind to specific target promoters. STAT1 has previously been shown to have a role in inducing apoptosis in the myocardium following ischaemia/reperfusion injury (I/R), however the role of STAT3 in myocardial apoptosis is less clear. Here it is shown that STAT3 is phosphorylated in cardiac cells both in vito and in vivo in response to I/R injury and plays a protective role by reducing the levels of apoptosis. Several modulators of STAT3 activity were found to be upregulated following I/R, including JAK2, SOCS3 and GRIM-19. STAT3 was also found to be important in regulating DNA damage and repair through altered activity of DNA damage response proteins. Administration of the antioxidant tempol in vivo, reduced infarct size in a rat model of I/R injury and this was accompanied by a reduction in STAT1 and STAT3 phosphorylation. Increasing STAT1 phosphorylation with IFN-• treatment abolished the protective effect of tempol, suggesting that inhibition of STAT1 phosphorylation may be a key protective effect of tempol infusion. Affymetrix microarray analysis of hearts from the in vivo I/R model identified several novel gene expression changes and uncovered transcriptional reduction in large numbers of genes involved in mitochondrial respiration and transport. In addition, this approach identified several possible new regulators of cardiac protection mediated by tempol and the urocortin hormones. The mitogen activated protein kinase (MAPK) family is involved in sensing cellular stress and play key roles in I/R injury and inflammation. MAPK activity is balanced by MAPK phosphatases (MKPs) such as MKP-1 and the role of MKP-1 in modulating the immune response was investigated. Mice deficient in MKP-1 were more susceptible to endotoxic shock and had elevated levels of serum cytokines. MKP-1 was found to be upregulated following toll-like recptor (TLR) stimulation and this was dependent on the signaling adaptors MyD88 and Trif. Macrophages deficient in MKP-1 had increased phosphorylation of p38 MAPK and JNK following TLR stimulation and secreted elevated amounts of the pro-inflammatory cytokines TNF-α, IL-12 and the anti-inflammatory cytokine IL-10. The temporal control and regulation of cytokine production in response to TLR stimulation was dissected using pharmacological inhibition of MAPKs. MKP-1 was not found to contribute to T cell differentiation but did have a role to play in the adaptive immune response as MKP-1- deficient mice failed to recover from an experimental model of multiple sclerosis.

Gene therapy vectors based on the HIV-1 lentivirus are an attractive option for clinical applications because they enter a broad range of target cells efficiently and deliver stable gene expression through integration into host chromosomes. However, lentiviral vectors are known to integrate preferentially within actively transcribing genes. Leukaemia-like expansions observed in gene therapy trials using gammaretroviral vectors and are thought to have been caused by disruption of host proto-oncogenes at or close to the vector integration site, and the safety of these vectors may be related to the pattern of vector integration with respect to genes. The safety of integrating gammaretroviral and lentiviral vectors is therefore a significant concern with respect to their use in gene therapy. In this study, this problem was addressed by developing a novel hybrid vector which combines the efficient cell and nuclear entry properties of lentiviral vectors with chromosomal integration by the Sleeping Beauty transposase. Unlike the HIV-1 integrase enyme, Sleeping Beauty transposase does not exhibit a preference for integration within active genes. Nonintegrating lentiviral vectors were developed to carry Sleeping Beauty transposon and transposase expression cassettes. These were able to deliver transient transposase expression to target cells, and episomal lentiviral DNA was found to be a suitable substrate for integration by Sleeping Beauty transposase. Importantly, integration with this novel vector was found to occur significantly less frequently within active genes than a standard lentiviral vector. Finally, it was shown that the transposase protein can be incorporated into lentiviral vector particles in a manner analogous to HIV-1 integrase. The development of vectors with safer integration patterns may lead to better clinical outcomes for patients treated with gene therapy.

The oral cavity is one of the most complex microbial habitats across the human body. A balance of endogenous and exogenous factors controls this environment; any disturbance of that balance will lead to diseases, such as caries and periodontal diseases. Therefore a comprehensive assessment study of the oral microbiome acquisition and development from birth to adolescent age will provide valuable baseline data and better understanding of the microbial ecology. Analysing the depth of oral microbiome ecology and the impact of chronological factors such as teeth eruption and puberty by means of Next Generation Sequencing was the main aim of this study. Supragingival plaque samples were collected from 80 subjects (aged 1 month to 17 years), divided chronologically in to four groups: edentulous infants, deciduous dentition, prepuberty and post puberty. Microbial composition of plaque was assessed by barcoded pyrosequencing of the V5-V6 hypervariable regions of the 16S rRNA. Pyrosequencing reads represented 8 phyla and 102 higher taxa (genus or more inclusive taxa when sequences could not be confidently classified to the genus level) of these 31 unclassified yet. Four phyla (Firmicutes, Actinobacteria, Prtoteobacteria and Bacteroidetes) predominated all age groups. In the infant cohort, formula feeding was found to be an important factor in altering the oral microbiome, since exclusively formula-fed infants showed higher prevalence of Prevotella melaninogenica, Neisseria polysaccharea, and Granulicatella adiacens. However, there was no significant difference between the two groups with respect to phyla level. Moreover, no conclusion could be drawn regarding the correlation with dmft/DMFT in the pre-puberty and post-puberty groups due to the discrepancy in the sample size. However, at genera level Abiotrophia (mainly A. Defectiva) and Capnocytophaga (mainly C. gingivalis) were detected more frequently in the healthy group. Other major sequences involve (Streptococcus, Neisseria, Lautropia, Fusobacterium, Leptotrichia, Porophyromonas, Prevotella, Actinobaculum, Actinomyces, Corynebacterium, Rothia) were all recorded to be higher in dmft/DMFT group.

Cataract is a potentially reversible cause of childhood blindness which is responsible for at least 15% of the world’s blind children. Primary intraocular lens (IOL) implantation is the most important recent innovation in the management of childhood cataract, and has been widely adopted despite unanswered questions regarding best practice, visual benefits and adverse outcomes. In order to answer these questions, an epidemiological study was undertaken through systematic, standardised data collection through a national clinical network, the British Isles Congenital Cataract Interest Group. At the time of submitting this thesis, data are available for 236 children. IOL implantation was undertaken in the majority of children over 6 months old, but aphakia was the preferred option for younger children, due in part to the higher than anticipated frequency of other ocular anomalies. Overall primary IOL implantation conferred no visual benefit for children with unilateral cataract, but may be associated with better visual outcome following bilateral cataract surgery, whilst increasing the risk of the need for further surgical procedures under general anaesthetic, which may adversely impact on future cognitive development. 16% of all children developed glaucoma during the first postoperative year with age at surgery being the most significant factor. The potential eventual burden of aphakic and pseudophakic glaucoma is considerable, and these findings should encourage debate about the balance between the risk of amblyopia and the risk of glaucoma, as well as future research on this blinding complication Refractive planning and outcome in early life pseudophakia is highly variable. There is a pressing need for standardisation of refractive planning and continuous national monitoring of refractive outcomes, similar to that which exists in adult cataract surgical practice. Follow up studies of this unique inception cohort will provide further information on longer term outcomes and their impact on educational and personal development.

10-50% of individuals with cleft lip and palate (CLP) may undergo maxillary osteotomy in adolescence. The surgery can impact on speech including articulation, resonance and velopharyngeal function. A systematic review of the literature using levels of evidence and power calculations was undertaken. This showed that the evidence base at present for CLP is weak or non-existent due to poor speech and study methodology. The aims of the PhD were to examine the impact of the surgery on speech and to identify valid predictors of acquired velopharyngeal deterioration post-operatively. The prospective study recruited a consecutive series of CLP patients undergoing maxillary osteotomy within the North Thames Regional Cleft Service (UK) and two control groups: a non-CLP group undergoing the surgery and normal controls. CLP patients were seen for perceptual speech assessments, speech acoustics, nasometry, lateral videofluoroscopy, nasendoscopy and pressure-flow. The data were analyzed using perceptual clinical ratings and/or specialized computer software. Reliability studies for all outcomes were undertaken. Statistical analyses using SPSS included within- and between-subject analyses of variance, planned comparisons to look at differences across time points, and correlational analyses. Several multiple regression models were tested to identify valid predictors of acquired velopharyngeal deterioration. The main findings of the study indicate that maxillary osteotomy affects CLP and non-CLP patients differently. In CLP, the surgery has a positive impact on articulation but affects resonance and velopharyngeal function negatively. Although /s/ improves, it is not normalized even a year post-operatively. Results also indicate that resonance changes at 3-months post-operatively appear to be permanent. Initial multiple regression analyses identified pre-operative closure ratio and proportion of palate contacting the posterior pharyngeal wall as valid predictors of hypernasality at 12-months post-operatively. A speech care pathway for patients with cleft lip and palate undergoing advancement surgery, encompassing key elements of assessment, review and management, is proposed.

Although contemporary treatment cures most children with ALL, with survival rates in the region of 90%, treatment outcomes in certain cytogenetic subgroups remain poor. Patients with such abnormalities have a greatly increased risk of relapse and re-treatment responses are poor. Understanding the mechanisms of resistance in these patients is therefore a priority in the search for improvements in chemotherapy. The translocation t(17;19)(q22;p13) is a rare cytogenetic abnormality, which occurs in less than 1% of childhood ALL and is associated with a very poor prognosis and chemotherapy resistance. The translocation results in the fusion of E2A on chromosome 17 with HLF on chromosome 19. It has been commonly assumed that, following relapse, ALL cells will divide more rapidly than at presentation. To test this hypothesis, competitive transplantation studies were performed using paired presentation and relapse samples from a case of t(17;19)-positive ALL in a NSG xenograft mouse model. Surprisingly mice engrafted with the presentation cells survived significantly shorter (p<0.05, logrank test) than those with relapse cells, indicating the aggressiveness and rapid proliferation of presentation cells, while the survival curves of mice engrafted with various proportions of presentation and relapse cells had intermediate levels of survival. Administration of dexamethasone prolonged the survival of mice engrafted with the presentation cells and those with various proportions of cells, with no effect on survival of mice with relapse cells. Flow cytometry analysis showed that a high percentage (70%-95%) of human leukaemic cells engrafted in bone marrow and spleen across all groups tested. RT-PCR amplification identified this t(17;19) case to be a Type 1 E2A/HLF fusion. In order to determine the genes responsible for chemo-insensitivity, whole genome SNP array analysis was performed on the matched presentation and relapse samples which demonstrated deletion of glucocorticoid receptor (NR3C1) gene in relapse cells which is not seen in presentation cells, confirmed by fluorescence in situ hybridisation. This deletion was used to identify the relapse cells (R) in mice engrafted with mixtures of presentation and relapse cells. FISH analysis showed the percentages of presentation cells (P) were higher than the ratios of the cell initially transplanted. In concordance, real-time PCR analysis showed high levels of NR3C1 in all mix cells ratios. These results indicated that presentation cells proliferate more rapidly and outgrow relapse cells in competitive clonal repopulation experiments. Dexamethasone treatment reduced the percentages of presentation cells in all mix populations with high significance (p<0.01, t-test) in the 30%-P+70%-R ratio. Levels of NR3C1 in all mix cell populations were significantly depleted (p<0.05, t-test) by dexamethasone. To be able to physically track the proliferation of leukemic cells populations in vivo and to follow progression of the disease, two lentiviruses pSLIEW (expressing firefly luciferase and green fluorescent protein) and pSRLICW (expressing Renilla luciferase and mCherry fluorescent protein) were generated. pSRLICW was just successfully generated but has not yet been tested. Lentivirus pSLIEW functionality was validated by transducing various leukaemic cell lines. By assessing the percentage of green fluorescence (gfp) cells, transduction efficiencies in SEM and RS4;11 (ALL cell lines) were 45% and 5% respectively, whereas transduction efficiencies in Kasumi-1 and SKNO-1 (AML cell lines) were 72% and 98% respectively, higher than the ALL cell lines. Transduced SEM and Kasumi-1 lines were sorted for higher gfp populations for xenograft purposes. Real time bioluminescence imaging on mice xenografted with sorted transduced SEM cells showed rapid progression of the disease in the systemic while Kasumi-1 cells xenografted in mice produced localised tumours and progressed much slower. These lentiviral-transduced cell lines xenografts have proven the in vivo monitoring capability by real time luminescence imaging. The information gained from this project study provides novel insights into the mechanisms of relapse in childhood ALL. The growth characteristics of the leukaemic blasts should be considered in assigning patients to different therapeutic options.

An observational study with an emergent design of two parts, consisting of quantitative and qualitative analysis, looked at communicative interactions between carers and children. The children were drawn from three groups: Cerebral Palsy, Down syndrome and typically developing. The first part of the study utilised a secondary data set of observations of play sessions of carers with children who have Cerebral Palsy and carers of children with typical development. A continuous observational data-logging method of four verbal and six non-verbal behaviours was piloted and used with this data, and also used with observations of a further research group of carers with children who have Down syndrome, Measurement and comparison of frequencies of verbal and non-verbal behaviours was carried out. The behaviours were: verbal - questions and replies, descriptive commenting and directives; non-verbal - attention-directing, demonstrating, physically orienting, attending and exploring. Between the groups, similarities and differences were found in the frequencies of particular behaviours. Although the greatest frequency of questions and replies were by carers and children in the typically developing group, frequencies of descriptive commenting were similar across the groups. The second part of the study consisted of a qualitative analysis of two carer-child pairs from each of the three groups. Differences in individual style were found between and within the groups. Characteristics and strategies of carer's communicative interactions, and their functional relation to the attentional and responding capacities of the children, were identified and described. A clinically interesting finding was made, that the questions asked by carers of the children with Cerebral Palsy and Down syndrome, were, in the absence of replies by the children, transformed into descriptive comments. The findings of the study are discussed. The methodology, and issues of interpretation, clinical significance and future research are critically evaluated.

This thesis had two main aims. Firstly, to develop separate questionnaires for children with asthma and children with diabetes and their parents, which assess children's adjustment to the illness and treatment adherence. Secondly, to test the hypothesis of an association between children's adjustment and treatment adherence. The essence of asthma and diabetes treatment is self-care and consequently children with asthma or diabetes have to learn to cope with the long-term demands and responsibilities of complying with a strict and complex treatment regimen. It is currently recognized that a major problem in paediatrics is poor treatment adherence, which can result in serious health consequences. This led to a shift in paediatric medicine, from focusing only on the physical treatment of the illness to exploring the psychological impact of the illness and how it affects children's socio-emotional adjustment. However, there is a shortage of adjustment and treatment adherence measures; existing ones have major limitations. Thus, the new questionnaires aimed at assessing both children's adjustment and treatment adherence. Four interlinked studies utilising qualitative and quantitative methods were carried out. Study 1 and study 3 were parallel but separate studies and involved interviewing a group of 15 children with asthma and 15 children with diabetes, their parents and paediatric nurses about the children's experiences and feelings in a range of contexts. The interviews showed that there were commonalities in stressors across children but differences in adjustment and treatment adherence levels. On the basis of these interviews separate questionnaires for children with asthma (study 2) and children with diabetes (study 4) and their parents were developed and administered to a sample of 60 children and their parents. The new questionnaires proved to be reliable and valid and confirmed the hypothesis of a significant relation between children's adjustment and treatment adherence. The development of a new assessment tool involves several steps: This work represents the first steps in developing a new assessment tool. As with any new assessment instrument, further development will be required to examine its validity and reliability.

The Lidcombe Program (LP) is a parent based behavioural treatment for early stuttering. A series of quantitative studies have shown it is an effective treatment for eliminating stuttering in children of six years and younger. This study explores parents' experiences in order to understand more about the process of doing the LP. Twenty-one in-depth qualitative interviews took place between March 2004 and March 2006 with parents of 14 children who showed a range of responses to treatment. Six parents were interviewed twice so that changes over time could provide further insights into the daily implementation of this approach. The data was analysed using an inductive approach supported by NVivo® qualitative software. This interview data suggests that parents' experiences of the LP are influenced by their ways of making sense of the principles and procedures of the program and how these fit with their understanding of stuttering, parenting and their children who stutter. Parents experience the LP as satisfying and relatively simple when therapy proceeds straightforwardly. When progress is slower or erratic parents perceive the treatment as more complex and confusing. Failure to adequately address conflicts or difficulties in implementing the LP can reduce its therapeutic impact. Continued attempts to the use the LP when there is little or erratic progress can lead to parental distress and concern that the procedures may harm their children. The parents' perspective provides knowledge about the daily implementation of the LP that is not recorded in the quantitative studies published so far. The range of experiences described by these parents increases our understanding of the process of doing the LP and indicates how parental beliefs and children's vulnerability to stuttering might influence outcome.