Asthma is a chronic respiratory disease with a prevalence that has increased worldwide over the past 40 years. Longitudinal cohort studies have been designed to determine associations between early life events and asthma prevalence. One such cohort is the Merthyr Allergy Prevention Study (MAPS). MAPS recruited high risk subjects, before birth, from a deprived population in South Wales. The original study was a randomised controlled trial (RCT) of either normal diet for the first four months of life, or a cows’ milk protein exclusion diet with soya formula milk supplementation if subjects were not breast fed. Subjects were subsequently followed up as part of a cohort study. The findings presented are based on the final follow up at age 23 years. While there was a significant protective effect of breast feeding on wheeze at age 1, there was no evidence of an association with wheeze at age 23 years. The intervention arm of the RCT was associated with an increased risk of asthma and sensitisation at age 23 years. There was tracking of both total serum IgE and positive skin prick test results over the years but there was no clear relationship between these two measures of allergy. Although the prevalence of atopy was low in childhood, there was still a clear association with this and wheeze later in life. Wheeze at age 3 years or older was an important determinant of asthma at age 23 years. There was a significant association between those who wheeze from age of 3 to age 23 years and atopic status at age 7 years. In conclusion we have investigated a birth cohort from a relatively deprived area of South Wales and found characteristics in the first 7 years of life are critical in determining if asthma develops in early adulthood.

Background: Children with severe neurodisability (ND) commonly suffer with respiratory disease and this is the leading cause of premature death. The nature of this respiratory disease is however, poorly understood. The underlying aetiology is often multifactorial, but aspiration (direct or reflux) is likely to play a key role. Unfortunately, diagnostic tests for reflux or direct aspiration are limited. There is a clinical need for high quality research on children with severe ND to define underlying mechanisms of respiratory disease and guide management. Aims: In this study, the aims were to (i) characterise respiratory symptoms and their relationship with lower airway inflammation in children with severe ND, (ii) explore available bronchial lavage (BAL) biomarkers of reflux aspiration, assessing their validity and their relationship to clinical and airway inflammatory data, (iii) develop a novel assay for the accurate detection/quantification of pepsin in BAL, (iv) investigate the validity of an alpha-amylase assay in paediatric BAL and explore the relationship of alpha-amylase levels with lower airway inflammation and clinical symptoms, and (v) investigate the effects of pH alteration and pepsin exposure on airway epithelial cells (AEC). Methods: Clinical data and BAL samples were collected from children with severe ND at times of stability and respiratory deterioration and also from healthy controls. BAL differential cell counts and cytokine measurements (ELISA) were performed. Lower airway microbial colonisation/infection was assessed. BAL pepsin measurement was attempted using a number of methods, and the feasibility of inhibitor affinity enrichment and LC-MRM-MS techniques to identify and quantify BAL pepsin was explored using SDS PAGE gel and mass spectrometry analysis. BAL alpha-amylase activity was measured using an ethylidene-pNP-G7 based assay. BEAS-2B cells were cultured in monolayer and the cytotoxic/inflammatory effects of pH alteration and pepsin exposure were explored through trypan blue staining and cytokine assays. Results: Children with severe ND have burdensome chronic respiratory symptoms that impact on their quality of life and that of their families’. Symptoms may relate to lower airway inflammatory levels. A trend for greater airway neutrophilia and respiratory symptoms was seen in those with lower airway microbial positivity. Available ‘in house’ pepsin ELISA assays were not robust but Western Blot results indicated a higher frequency of BAL pepsin positivity in ND patients with acute respiratory deterioration. Notably, positivity was also found in some healthy controls, highlighting the need for a quantitative assay. The use of an immobilized inhibitor (pepstatin agarose) to recover pepsin from paediatric BAL samples was shown to be feasible. Subsequent digestion of pepsin and detection by LC-MSMS with selective ion monitoring was demonstrated. Significantly increased BAL alpha-amylase levels were seen in Elective-ND patients compared to healthy controls and furthermore, significant correlations were observed with BAL lower airway inflammatory markers. BEAS-2B cells were sensitive to mild acidification, with up-regulation of TGF Beta-1, IL-6 and IL-8 mRNA expression. A corresponding rise in protein secretion was not necessarily seen and in some cases, was significantly down-regulated. No significant cytotoxicity or decrease in cell viability was observed in any condition. Conclusions: In children with severe ND, chronic respiratory symptoms may be directly related to lower airway inflammation and bacterial colonization. Measurement and quantification of pepsin in BAL (as a marker of reflux aspiration) is difficult. However, an inhibitor affinity enhanced mass spectrometry based technique has potential as a ‘gold-standard’ method for identification and quantification of pepsin in BAL. BAL alpha-amylase activity shows promise as a biomarker of direct aspiration. Mild alterations in airway pH may directly contribute to the pathophysiology of inflammatory airway disease and specifically, reflux-aspiration related lung disease in this group. This requires further study as a potential novel therapeutic target.

Medulloblastoma (MB) is the most common malignant brain tumour in children. MBs arise in the cerebellum, originating from neural stem cells progenitors. It has previously been shown that the lack of integrity of signalling pathways due to genetic alterations play a key role in MB chemosensitivity. An example is the p53 signalling, with p53 mutations and or deletions which are associated with drug resistance. Here, we explored the role of p53 induction by chemotherapeutic drugs such as etoposide in MB and the way to bypass the need for intact p53 to trigger apoptosis. We specifically demonstrated that a downstream miRNA target transcribed by p53, miR-34a, is able to reduce the number of viable cells in cultures of MB cells lacking functional p53, although the effect was limited. Beyond the genetic background of a tumour, it is more and more described that the tumour microenvironment plays a key role in drug resistance. MB is a solid tumour and hence will contain areas deprived in oxygen (hypoxic). It has been widely documented that hypoxia is associated with poor prognosis and resistance to treatment in other cancer models and we here aimed to investigate the specific role of hypoxia in MB response to etoposide. We demonstrated that MB (p53 WT) cell lines became more resistant in chronic but not acute hypoxia and this was associated with a decrease in the recruitment of double strand DNA damage sensing machinery and subsequent impairment to transactivate p53 and transcription of pro-apoptotic genes. A transcriptomic microarray profiling study further revealed that chronic hypoxia induced broad and significant changes in global gene expression affecting many biological pathways including stem cell maintenance, neuronal development and phosphoinositol-3 phosphate kinase.

Background: Disorders of lung development cause death and disability in the young and old: novel insights into developmental regulators can aid therapeutic strategies. The Ca2+ATPase SERCA, already implicated in asthma and cystic fibrosis, appears to play a key role in lung development. SERCA inhibition with cyclopiazonic acid (CPA) in vitro, reduces both airway branching and peristalsis reversibly and dose dependently, whilst also halting myogenesis. It is unclear however, whether changes in branching are mediated via SERCA dependent contractility, or whether SERCA is a direct regulator of airway branching. Aims: (i) to further explore the CPA-induced embryonic lung phenotype by assaying gene expression and cell proliferation; and (ii) to determine effects of genetic perturbation of SERCA function in vivo on airway branching morphogenesis, in the absence of contractility (using a Drosophila model). Methods: Embryonic mouse (E11.5) lung explants were cultured +/- CPA at an air/fluid interface. Standard techniques were used to rear Drosophila and SERCA expression manipulated using conditional, heat-sensitive mutants and RNAi targeted to the trachea. Positively labelled, loss-of-function ‘flip-out’ RNAi and mutant clones were produced using heat-shock induced FLP-recombinase. Gene expression was assayed using real-time RT-PCR and SERCA function assessed using calcium dyes and genetic indicators. Embryonic and larval fly airways were imaged using fluorescent proteins and immunostaining, with live or fixed-sample confocal microscopy. Immunofluorescent staining was used to assess protein expression and cell proliferation. Results: SERCA inhibition with CPA significantly up or down regulated mRNA levels of key genes involved in lung branching morphogenesis, myogenesis and angiogenesis in vitro. CPA treatment also reduced cell proliferation dose-dependently in the lung epithelium and mesenchyme. In the fly embryo, neither conditional SERCA mutants nor targeted RNAi significantly affected tracheal morphology. However, residual SERCA mRNA and protein function was evident at this stage of development. Tracheal maturation, in the form of gas filling was significantly impaired though, in embryos expressing a conditional SERCA mutation. In larvae, development of the dorsal air sac primordium (ASP) was severely disrupted by targeted SERCA RNAi and this phenotype could be reproduced when sufficient numbers of loss-of–function clones were present. SERCA inhibition reduced the number of mitotic cells in the ASP and correspondingly, SERCA deficient clones comprised fewer cells than control counterparts: SERCA regulation of airway cell proliferation was therefore evident across species. Fewer SERCA deficient cells reached the tip of the ASP during morphogenesis compared to controls, whereas a greater proportion remained in the stalk, findings that indicate a cell-autonomous defect in cell migration. Changes in morphology were independent of changes in expression of the key ASP signalling pathways MAP kinase and Notch. Expression of the ASP tip-cell marker escargot was expanded in SERCA deficient larvae, with a number of positive cells being abnormally present in the stalk. This finding could be explained by a failure of these cells to migrate to the tip, alternatively by changes in cell fate. Given key roles of tip cells in morphogenetic signalling, escargot may play a role in SERCA inhibition-induced dysmorphogenesis. Conclusions: SERCA has an essential, conserved role in airway branching morphogenesis across species: this role appears independent of contractility. SERCA regulates cell migration and proliferation processes in the airway, findings that may have wider relevance, e.g. in proliferative disease, metastasis and tissue regeneration. Given evidence in plants and fungi of Ca2+ cycling regulating budding, findings here may indicate a role for SERCA as a generic regulator of iterative branching across biology, with clear implications for further research.

Adverse drug reactions (ADRs) in children are common but their predictors are not fully characterised. It is known that both increasing age and number of concomitant medicines increase ADR risk in children, and there is also some evidence that off-label and unlicensed medicine use may contribute. The purpose of the thesis was to characterise ADRs in children, focusing on known risk factors, which have not been adequately evaluated in the literature. The contribution of off-label and unlicensed prescribing to ADR risk in children was assessed in two large prospective studies. In the first study, which evaluated ADR-related hospital admissions, off-label or unlicensed medicines were more likely to be implicated in an ADR than authorised medicines (relative risk 1.67, 95% CI 1.38, 2.02, p < 0.001). In a multivariate analysis, patients admitted under the care of oncology were more likely to have experienced an ADR (odds ratio (OR) 25.70, 95% CI 14.56, 45.38, p < 0.001). The following risk factors were also associated with increased ADR risk: increasing age (OR 1.04, 95% CI 1.00, 1.08, p = 0.045), number of authorised medicines (OR 1.25, 95% CI 1.16, 1.35, p < 0.001) and number of off-label or unlicensed medicines (OR 1.23, 95% CI 1.10, 1.36, p < 0.001). In a sub-group analysis which excluded oncology patients, age and number of authorised medicines predicted ADR risk (OR 1.05, 95% CI 1.01, 1.09, p = 0.023 and OR 1.33, 95% CI 1.23, 1.44, p < 0.001 respectively) but the number of off-label and unlicensed medicines did not (OR 1.04, 95% CI 0.89, 1.12, p = 0.627). The second prospective study examined ADRs occurring in paediatric inpatients. Again, off-label or unlicensed medicines were more likely to be implicated in an ADR than authorised medicines (OR 2.25, 95% CI 1.95, 2.59, p < 0.001). Medicines licensed in children but given to a child below the minimum age or weight recommended had the greatest risk of being implicated in an ADR. Multivariate analysis showed that increasing age (HR 1.04, 95% CI 1.02, 1.05, p < 0.001) and receipt of a general anaesthetic (HR 5.30, 95% CI 4.42, 6.35, p < 0.001) were positive predictors of ADR risk. Both the number of authorised (HR 1.22, 95% CI 1.17, 1.26, p < 0.001) and the number of off-label or unlicensed (HR 1.27, 95% CI 1.20, 1.34, p < 0.001) medicines were predictors of ADR risk. ADR detection in the above studies was based on intensive surveillance. One possible method of detecting ADRs may be through the ICD-10 clinical coding system but this has not been investigated for paediatrics. Only 31.5% of the 241 ADRs evaluated from the prospective admissions study were coded correctly using at least one ICD-10 code. The clinical coding system could contribute to pharmacovigilance if deficiencies in how ADRs are recorded in the case notes and the clinical coding system can be addressed. An important ADR detected in the admissions study was the occurrence of haemorrhage post-tonsillectomy which has been attributed to the use of dexamethasone. In order to analyse this further, a systematic review and meta-analysis of dexamethasone and non-steroidal anti-inflammatory drug (NSAID) use in paediatric tonsillectomy was undertaken. Although there were a large number of randomised controlled trials and observational studies in this area, analysis of all of these led to the conclusion that there was insufficient evidence to rule out an increased risk of haemorrhage with dexamethasone use whether in combination with NSAID or not (Peto odds ratio for dexamethasone versus another intervention 1.41, 95% CI 0.89, 2.25, p = 0.15). Further, well powered, well designed studies are needed in this area. An important ADR detected in the in-patient study was post-operative nausea and vomiting. More detailed analysis was therefore undertaken to identify the risk factors for post-operative vomiting (POV), with a view to developing a risk score. The following were all identified as predictors of POV risk: age (OR 1.06, 95% CI 1.03, 1.10, p<0.001), duration of anaesthesia (OR 1.00, 95% CI 1.00, 1.01, p <0.001) and the use of intra-operative analgesics (OR 2.22, 95% CI 1.58, 3.12, p < 0.001). However, it was not possible to develop a robust model to predict the risk of POV because of the heterogeneity of the patient groups, the types of surgery, and the different clinical practices between different anaesthetists in terms of anti-emetic (choice, timing and doses). The use of off-label and unlicensed medicines in children is common but necessary and these medicines are frequently associated with ADRs. The rational prescribing of medicines is an important measure in the reduction of ADR risk and a solid evidence-base is a pre-requisite. The aim should be that the minimum number of medicines is used safely and effectively, at the lowest dose possible, for the minimum duration necessary.

Introduction: Perthes’ disease is an idiopathic osteonecrosis of a juvenile hip that frequently precipitates premature osteoarthritis. The year 2010 marked a century since Perthes’ disease was first described, but the aetiology and mechanism remain unknown. The incidence of Perthes’ disease varies widely, and it has been suggested that differential exposure to adverse socioeconomic circumstances may be a key precipitant. This work seeks to further the understanding of the distribution and determinants of Perthes’ disease, by exploring temporal and geographic patterns using a case register from Merseyside, discharge data from Scotland and the world’s largest community disease register. Analytical studies are then used to test hypotheses and investigate a disease mechanism. Methods: The descriptive studies were based on data from the Merseyside Perthes’ Disease Register (1976 – 2008), the General Practice Research Database (1990 – 2008) and hospital discharge data for Scotland (2000 – 2009). A systematic review of the published literature was used to explore international variations in incidence. Two case-control studies were used to test hypotheses. The first used a community population derived from the General Practice Research Database to investigate comorbid disease associations. The second used a hospital population to examine tobacco smoke exposure, anthropometric markers of prenatal androgenisation, hyperactivity and impaired endothelial function as a possible disease mechanism. Results: There was a graduated North-South divide in the UK incidence of Perthes’ disease, with rates in Scotland more than twice those in London. All three descriptive studies demonstrated a sustained fall in disease frequency across the study periods. There was a marked association with area deprivation, which was independent of the urban environment. Internationally a North-South divide persisted with equatorial regions being relatively unaffected by disease, and Northern Europe having the highest disease incidence. The international disease distribution was primarily a function of race, although latitude remained an independent predictor of incidence. Analytic studies revealed an association with congenital genitourinary tract anomalies and an association with asthma. There was no clinically apparent hyperactive tendency, though a more subtle abnormality in behavioural profiles was apparent. Exposure to tobacco smoke was a notable risk factor for Perthes’ disease, which was independent of individual or area deprivation measures. Arterial caliber was reduced amongst cases with a corresponding generalised reduction in arterial flow, though endothelial function appeared normal. Conclusions: Within the UK the incidence of Perthes’ disease is in decline. The geographic distribution suggests that variation in disease incidence is related principally to deprivation, though the underlying determinant(s) remain unclear. Latitude may hold an additional ‘risk’ in the disease aetiology, though the specific component(s) of latitude similarly remain unclear. Additional independent risk factors include sex, ethnicity, genitourinary disease and tobacco smoke exposure. Factors acting early in the development of the child appear to offer important insights into the disease aetiology, with particular interest on the influence of prenatal sex hormones. Reduced arterial caliber may have a role in the disease mechanism, and wider implications to vascular health. The aetiological factor in Perthes’ disease remains elusive, but it is likely that unraveling this enigma will unlock additional secrets relating to the fetal origins of diseases.

Background Prader-Willi Syndrome (PWS) is a complex genetic syndrome associated with hyperphagia and behavioural problems. Recent research suggested a link between hyperphagia and behavioural and emotional problems in PWS such as anger and anxiety. The current study aimed to explore this relationship further. Method Through parental report postal questionnaires, data was collected on the age, gender, weight, hyperphagia and behavioural and emotional problems of 105 children with PWS aged 4-18 years (M: 9.63 years). Results Following preliminary analysis, a series of multiple regressions were performed. Hyperphagic drive significantly predicted antisocial/disruptive behaviour, anxiety, social relating problems, communication disturbances and self-absorbed behaviours. Whilst hyperphagic behaviour did not significantly predict any behavioural/emotional problems. Conclusions This study reinforces research which has suggested an association between hyperphagia and non-food related behaviour in PWS. This has implications for the understanding of PWS and the development of psychological interventions for behavioural and emotional problems.

Development of the human brain occurs in a number of complex pre- and postnatal stages which are governed by both genetic and environmental factors. Aberrant brain development due to inherited defects may result in a wide spectrum of neurological disorders which are commonly encountered in the clinical field of paediatric neurology. In the work for this thesis, I have investigated the molecular basis and defined the clinical features of three autosomal recessive neurological syndromes. I studied a cohort of children with early onset epileptic encephalopathy and, in one family, identified a novel homozygous pathogenic mutation of PLCB1. I have also utilised autozygosity mapping techniques to study consanguineous families with a complex motor disorder, infantile parkinsonism-dystonia, and identified loss-of function mutations in the gene encoding the dopamine transporter (SLC6A3). Subsequent acquisition of a cohort of similarly affected children allowed detailed clinical and molecular characterisation of this novel disorder, dopamine transporter deficiency syndrome. Finally I have delineated the clinical and genetic features of PLA2G6-associated neurodegeneration. The identification of disease-causing genes contributes greatly to understanding the disease mechanisms underlying such early-onset disorders, and also provides novel insights into normal human neurodevelopment.

Paper partition chromatography has been used extensively to study the aminoacid excretion in the urine of healthy children, of newborn infants and of patients suffering from eight different metabolic disorders. The purpose of this investigation was to discover the aminoacid pattern in the urine and the extent to which it is characteristic of each condition, and to establish the differences between the various aminoacid patterns. In order to gain some insight into the mechanism of the various forms of aminoaciduria a comparison was made between the aminoacid pattern of the plasma and that of the urine. In some cases microbiological assay has been used to establish the plasma level of certain aminoacids more exactly than was possible by paper chromatography. [From p92, Summary and conclusions] The main diseases investigated were: Lignac-Fanconi disease Cystine-lysinuria Phenylpyruvic oligophrenia Liver cirrhosis and hepatitis Steatorrhoea Galactosaemia Wilson's disease [From p4, Introduction]

Introduction: Early-life differences in cardiovascular risk factors could contribute to ethnic differences in cardiometabolic disease in adulthood. The aim of this thesis was to investigate ethnic differences in lifestyle factors, adiposity and blood pressure among 5-6 year old children in the UK. Methods: Cross-sectional data on blood pressure, anthropometric measures, sociodemographics, dietary intake, ethnicity, and objectively-measured physical activity, were analysed (n=1470 consented children; 45% White British, 30% South Asian, 8% Black African/Caribbean). Results: Compared with White British children, South Asian children had higher, and Black African/Caribbean children had similar or lower, levels of total and central adiposity. Pakistani and Black African/Caribbean boys did more moderate-vigorous physical activity, whereas South Asian girls did less compared with their White British peers. South Asian and Black African/Caribbean children had lower or similar blood pressure compared with White British children. Sodium intake was highest among Black African children. Sugar intake was lower among all minority ethnic groups compared with White British children. Conclusions: The findings highlight several early-life ethnic differences which could plausibly contribute to cardiovascular health inequalities in adulthood. Early childhood might offer a key opportunity to prevent or reduce ethnic differences in cardiovascular and metabolic disease later in life.