Stem cells play fundamental roles in embryonic development, tissue homeostasis and have great potential in regenerative medicine. The main aims of this study were: i) to elucidate the properties and neural differentiation potential of somatic stem cells from different sources focusing on the analysis of stem cells with low immunogenicity and/or suitable for autologous cell therapy, amniotic fluid (AFSC) and adipose tissue-derived stem cells (ADSC), respectively; ii) to investigate a putative neural stem/progenitor cell niche in the choroid plexus (CP), organ that plays crucial roles in cerebrospinal fluid secretion and brain homeostasis. Unlike previously suggested, I found that AFSCs do not harbour significant neurogenic potential, as assessed by treatment with neurogenic small molecules, transplantation onto hippocampal organotypic cultures and within the chick nervous system. However, in a severe embryonic injury model grafted AFSCs reduced haemorrhage and significantly increased embryo survival via paracrine mechanisms. I then established and characterized ADSCs cultures derived from the fat of paediatric patients. They expressed markers of embryonic stem cells, mesenchymal and neural tissues, and displayed significant plasticity, as indicated by their ability to differentiate both into bone and cartilage upon appropriate stimulation, to home into the chick nervous system, and to be relatively rapidly reprogrammed to “induced pluripotent stem cells”. Altogether, though ADSCs seem more plastic than AFSCs, both provide valuable tools for developing novel therapeutic approaches and analyzing cell phenotype modulation. Finally, I showed the presence of neural precursors, neuroblasts and neuron-like cells within the CP in different species by analysis of neural markers and BrdU incorporation in vivo and in organotypic cultures, and demonstrated innervation of the CP at early developmental stages. Altogether these findings suggest the existence of a neural regulatory network within the CP that may play a crucial role in modulating its function in the developing and post-natal brain.

Children's immune systems develop over the first 20 years of life and are fundamentally different to adults' in terms of lymphocyte numbers, turnover rates and population structure. Children perinatally infected with the human immunodeficiency virus (HIV) can expect their infection and any antiretroviral therapy (ART) administered to affect the developing population dynamics of the immune system and long-term immunological health in adulthood. Describing, understanding and predicting these effects is important for guiding individualised treatment regimes, public health policy and future research. The work described here develops an empirical model for CD4 T-cell reconstitution in HIV-infected children starting ART based on a monophasic, asymptotic recovery function and applied to data in a nonlinear mixed-effects framework. The model quantifies effects of age on both pre-ART and long-term CD4 count, and an association between poor pre-ART and poor long-term immune status. It allows prediction, by extrapolation, of CD4 counts in adulthood given ART initiation at different ages and CD4 levels. These predictions suggest that current ART initiation guidelines preserve potential for good long-term CD4 numbers in young children, but that this potential is progressively damaged as age increases. The model also identifies groups of children with qualitatively different CD4 trajectories and illustrates fundamental differences between recovery in children and adults probably due to children’s more active thymuses. Next, a mechanistic model of T-cell homeostasis is described, aiming to develop a biological understanding of some of the effects identified using the empirical model. A model of homeostasis in HIV proves a valid, useful tool for understanding pathogenesis and the reasons for incomplete recovery on ART, and illustrates the power of mixed-effects modelling for gleaning information about differences between children’s T-cell dynamics. In the future, more complex and complete models building on this foundation will help understanding of the interactions between development, HIV and ART.

Necrotising enterocolitis (NEC) is an acute inflammatory condition of the preterm intestine associated with significant morbidity and mortality. The pathogenesis remains unclear. The Protein C (PC) pathway is the main anticoagulant system of the body with key anti-­‐ inflammatory, anti-­‐apoptotic and barrier stabilising effects on the microcirculation, cells of the innate immune system and epithelial cells of the lungs, intestine and skin. Neonates, and preterm neonates in particular, have a physiological deficiency in the PC pathway. This thesis was aimed at discovering the effects of NEC on the PC pathway. We demonstrated with immunohistochemistry that thrombomodulin (TM) and endothelial protein C receptor (EPCR) are strongly expressed in healthy preterm neonatal bowel; these receptors are the activation apparatus of PC. On examination of intestine affected by NEC, we discovered TM and EPCR expression was reciprocally reduced with increasing inflammation, but lost only in necrotic areas. Dual staining showed that intravascular deposits of fibrin occurred in vessels with weak or absent TM expression. The effect of NEC on the concentrations PC pathway proteins was assessed in an observational study. We report for the first time that activated protein C (APC) levels in healthy preterm infants are low; in keeping with the physiologically low levels of PC. In NEC the levels of PC, free and total PS are reduced. The levels of APC varied; some infants were able to boost levels 10-­‐fold while others remained unchanged. We studied the effect of APC and PC supplementation in an animal model of bowel ischaemia-­‐reperfusion injury designed to simulate prophylaxis and treatment schedules. APC given after reperfusion reduced intestinal injury; PC had less effect. The results presented in this thesis show that PC pathway activity is reduced in NEC and may contribute to the pathogenesis of the condition; introducing the possibility of new therapeutic options.

Restricted expression of contractile protein gene isoforms contributes to the functional distinction between cardiac and skeletal striated muscle types, with these genes often misregulated in myopathic disease. The homeobox transcription factor Prox1 is expressed in both cardiac and skeletal muscle, and ablation of Prox1 specifically within cardiomyocytes resulted in the abnormal upregulation of the fast-twitch skeletal isoforms of the contractile protein genes troponin T (Tnnt3), troponin I (Tnni2), and MyLC 1 (Myl1) from early in heart development. Similar loss of function studies in skeletal muscle using a Myf5-driven Cre revealed overexpression of these same fast-twitch genes specifically in slow-twitch skeletal muscle, identifying Prox1 as the first transcription factor known to directly repress a program of fast-twitch skeletal genes across both cardiac and skeletal striated muscle types. Furthermore, loss of Prox1 expression in skeletal muscle was sufficient to cause a switch from a slow to a fast-twitch fibre-type, which has not previously been reported following knockout of a single transcription factor. Intriguingly, aberrant expression of Tnnt3, Tnni2, and Myl1 has been observed in other muscle-specific knockout models, including HDAC1/2 and miR-208a (cardiac), and Sox6 and miR-208b/miR-499 (skeletal), where the transcription factor directly responsible for their misexpression remains to be demonstrated. Recent work has revealed a lack of conservation in the regulation of these genes from the zebrafish, suggesting that in the mouse this role may be fulfilled by Prox1. This study has additionally uncovered novel putative regulatory relationships between Prox1 and miRNAs implicated in cardiac and/or skeletal muscle development, function, or disease, using a high-throughput in vitro screen. Herein is also the first description of the heart phenotype of survived cardiac-specific Prox1 knockouts, which develop severe dilated cardiomyopathy that is fatal within the first 14 weeks of life. Consequently, these mice model the functional impact of loss of Prox1 function on the adult heart, including the effect of ectopic fast-twitch contractile protein gene expression, and the molecular pathways that underlie the development of dilated cardiomyopathy. An improved understanding of the function of Prox1 in the developing and adult heart may provide new opportunities for therapeutic intervention.

Intestinal ischaemia and reperfusion (I/R) contributes to the pathogenesis of numerous clinical conditions in all age groups. Many of these diseases, including neonatal necrotizing enterocolitis (NEC), result in significant morbidity and mortality through multiple organ dysfunction, and available treatment is currently limited to supporting vital functions. My aims were: to investigate novel therapeutic strategies such as moderate hypothermia and peroxynitrite decomposition catalyst FeTMPyP [5,10,15,20- tetrakis(N-methyl-4'-pyridyl)porphyrinato iron (III)] in experimental models of adult and infant intestinal I/R; and to characterise the inflammatory process in human NEC, evaluating its relationship with clinical outcome. In an adult rat model, total-body moderate hypothermia applied throughout ischaemia and reperfusion counteracts oxidative stress in both the intestine and distant organs. This suggests that hypothermia could be beneficial as a preventative measure when intestinal ischaemia can be foreseen. However, in clinical practice therapy can usually be commenced only after ischaemia has occurred. In two sets of experiments, I showed that rescue hypothermia applied after mesenteric ischaemia improves outcome in both adult and neonatal rats, and this benefit is maintained after rewarming. Hypothermic protection could result from prevention of multiple organ dysfunction through several different pathways, including modulation of hepatic phosphoenergetics, pulmonary inflammatory infiltrate, cardiac energy metabolism, and systemic oxidative stress. Administration of peroxynitrite decomposition catalyst FeTMPyP as a rescue therapy at reperfusion also exerts a protective effect in neonatal rats, possibly via inhibition of adhesion molecule expression, leukocyte recruitment, and lipid peroxidation in the intestine, leading to prevention of systemic oxidative stress. In a study conducted on human specimens from neonates with NEC, tissue injury seems to be mediated via increased expression of endothelial adhesion molecules ICAM-1 and P-Selectin, leading to macrophage and neutrophil infiltration. Endothelial E-Selectin is expressed exclusively in NEC patients, and appears to be a marker of rapidly evolving disease and distant organ failure.

Traditionally, in older children and adults, spirometry has been an integral part of the clinical assessment of respiratory diseases; however these tests are relatively novel for preschool children. Like most medical observations, reliable interpretation of spirometry testing in very young children are accompanied by a relative dearth of suitable reference data. To address the lack of appropriate spirometry reference data for young children, this thesis aims to collate available spirometry data from healthy children to develop updated reference centiles for this age group. The Asthma UK collaborative group contributed >10 000 spirometry measurements in healthy children between 2-10 years. These data were used to develop more spirometry centiles using the LMS method with the GAMLSS technique. To address the discontinous paediatric and adult equations, the highly flexible modelling techniques were also applied to develop the first continuous and smoothly changing spirometry centiles between 4-80 years. In this case, the American NHANES Ill dataset was extended with paediatric data and re-modelled. The resulting centiles improved the interpretation of spirometry during childhood and early adolescence. Application of these centiles in clinical practice should facilitate more accurate detection and quantification of lung disease in both children and adults. Furthermore, this thesis provides a foundation from which future international studies can be established to maintain an up-to-date database of reference data for lung function.

Non-typhoidal Salmonella is an important bacterial pathogen causing worldwide morbidity and mortality. Early interactions between Salmonella Typhimurium and intestinal epithelium have been demonstrated in animal models, but little is known in humans. The aims of this thesis were to a) establish in vitro and ex vivo models to study such interactions, b) seek evidence of Salmonella invasion in human tissues in vivo, and c) identify virulence genes responsible for Salmonella adherence, invasiveness, and intracellular growth using a high throughput screening model based on transposon mediated differential hybridisation (TMDH). HEp-2 cell cultures were validated as a reproducible in vitro model at different early stages and for investigating host cell cytokine responses and signalling pathways. The model discriminated between different wild-type and characterised mutant strains; it also demonstrated the ability of probiotic lactobacilli to inhibit IL-8 expression in IL- 1β-pretreated and Salmonella-co-treated cells. This was not via IκBα degradation, phosphorylation of NF-κB p65 or its nuclear translocation. Lactobacilli did not postinfectiously affect intracellular Salmonella proliferation or IL-8 production. Polarised Caco-2 cells were employed to investigate host responses to various bacterial pathogen associated molecular patterns (PAMPs). Exposure to Salmonella Typhimurium DNA triggered IL-8/hBD-2 mRNA expression and infection with its FliC-deficient mutant ΔfliM stimulated IL-8 mRNA expression. Thus, both flagellin and bacterial DNA could elicit pro-inflammatory responses. Histology and immunohistochemistry of in vivo and ex vivo human tissues showed bacterial/Salmonella invasion in epithelial cells, macrophages, and neutrophils. After HEp-2 cell infection and extraction of genomic DNA using 1,440 transposon mutants of Salmonella Typhimurium, Transposon Directed Insertion-site Sequencing (TraDIS) identified 47 genes responsible for bacterial colonisation, among which 5 genes (intergenic sucD-cydA, glyA, yqiC, wzxE, and rfaI) account for bacteria-cell association. Furthermore, we discovered that speG is responsible for intracellular replication. Selected mutants will be re-characterised for pathophysiology investigations and prospective adaptation to prevention/treatment in non-typhoidal salmonellosis.

Reversible phosphorylation of tyrosine residues on proteins is a cornerstone of cell to cell signalling, and is achieved through the concerted action of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), which catalyse the addition and removal of phosphate moieties respectively. While the contribution of PTKs to oncogenesis and tumour maintenance has been well documented in a range of human cancers, knowledge of the specific role of PTPs has generally lagged behind. This thesis examines that role in neuroblastoma, a common and deadly sympathoadrenal tumour of infancy. We provide evidence that vanadium-­‐based PTP inhibitors cause synergistic hyperactivation of Akt and Erk, which drives an irreversible differentiation and senescence program that is independent of p53, p16INK4A and PTEN. It is then shown that in another group of tumour cell lines, such compounds used alone can induce selective caspase-­dependent, p53-independent apoptosis that is associated with activation of Akt and altered redox homeostasis. Lastly we examine the expression profile of the PTPome in a wide panel of neuroblastoma cell lines, providing preliminary evidence for candidate PTP oncogenes and tumour suppressors, as well as mediators of the effects of vanadium compounds.

Background: Glucose is a major fuel for brain function, and persistent and recurrent hypoglycaemia of any aetiology can cause brain injury. Children with hyperinsulinaemic hypoglycaemia (HH) are at a high risk of brain injury, as the inappropriate secretion of insulin during hypoglycaemia inhibits lipolysis thereby reducing the availability of ketone bodies (KB) that are an important source of alternate fuel for the brain during hypoglycaemia. In contrast children with ketotic hypoglycaemia (KH) are believed to be neurologically protected, due to the presence of abundant ketone bodies during hypoglycaemia. However, in both these groups of children, a comprehensive assessment of the neurocognitive profile with correlation to neuroimaging is not available. Aims: The aims of this thesis are 1. To understand the magnitude and rate of ketone body response during a diagnostic fast between children with KH and a ‘control’ group (children with suspected or previous history of hypoglycaemia that were subsequently normoglycaemic on the diagnostic fast). 2. To investigate and compare the neurocognitive profiles of children with HH and KH (used as a control group), and to correlate the profile of deficits in the HH group to the underlying structural abnormalities. The studies on children with KH (biochemical, neurocognitive and neuroimaging) were utilised to understand the neuroprotective role of KB during hypoglycaemia. Methods: 30 children with KH and 74 children from the ‘control’ group underwent a diagnostic fast with measurements of plasma glucose, lactate, catecholamines and serum insulin, cortisol, growth hormone, non-esterified fatty acids (NEFA) and 3-βhydroxybutyrate (ketone body) concentrations at the beginning, middle and end of fast. The neurocognitive profile of 21 children with HH was compared to a group of 14 children with KH, using a combination of standardised tests to investigate IQ, memory, attention, academic attainment, movement, emotion and behaviour. The structural integrity of the brain was evaluated using conventional neuroradiological assessments, hippocampal volumetry and diffusion tensor imaging (DTI). Results: Fasting studies have shown that children with KH demonstrate a significant increase (87% per hour) in the rate and magnitude of 3-βhydroxybutyrate concentrations relative to ‘controls’. Cortisol concentrations are a significant predictor of KB concentrations at the end of the fast. The performance of KH children as a group was within the normal range for the neurocognitive measures. However four children in the KH group scored in the borderline (77-79) and low average range (81-84) for full scale IQ, and memory scores in one KH child was lower than predicted by 19 points. Neuroimaging revealed small hippocampi in one child, focal white matter lesions in two children and diffuse white matter lesions in two children with KH. Children with HH underperformed significantly (relative to KH and standard population means) in the tests for intelligence (especially perceptual reasoning), memory, and sustained attention and manual dexterity. Memory impairments in children with HH did not correlate with hippocampal pathology. However, analysis of DTI studies has revealed the presence of white matter microstructural deficits that correlate with IQ and perceptual reasoning indices in these (HH) children. The genu and splenium of the corpus callosum were highlighted as specific white matter regions vulnerable to injury in the HH group. Conclusions: Children with hypoglycaemia are at risk of white matter injury. Children with HH manifest widespread cognitive deficits that are partly explained by the white matter microstructural deficits noted in the DTI studies. The increased rate and magnitude of KB response in KH group during fasting supports increased metabolic utilisation and a glucose sparing effect. However some chidren with KH in this study exhibit white matter injury and a wide variation in the neurocognitive scores is also noted, indicating the presence of neurocognitive impairment in certain children with KH.

Abstract Background: Lung function tests (LFT) provide valuable insights into respiratory physiology, and have been proven to be useful outcome measures in both clinical research and the clinical management of children with lung disease. Standardised methods and appropriate interpretation are, however, essential if these measurements are to be applied reliably. The aims of this thesis were to improve the application and interpretation of LFT’s in children and to determine the extent to which ethnic differences in lung function (LF) occurred between healthy Black and White children after adjusting for height, sex and age. Methods: A series of investigations using four commercially available LFT (Impulse oscillometry (IOS), specific airways resistance (sRaw), plethysmographic lung volumes, and spirometry) involving 400 healthy children (214 Black and 186 White) aged 4-12y were undertaken. Upon determining the most appropriate methods for interpreting LF in health, the LFT’s and a respiratory health questionnaire were applied to children with Sickle Cell Disease (SCD) to determine the extent to which each outcome measure identified LF abnormalities in these children. Results: Reference data for measurements of sRaw in children were developed as well as recommendations for interpreting spirometry and plethysmography in Black children. Despite the relatively high proportion of respiratory symptoms reported in SCD, the proportion of children with LF results falling outside the limits of normal was relatively small, and a pattern of restrictive lung disease was observed. Of the outcomes assessed in this thesis, spirometry appeared to be the most robust outcome measure for routine assessment of LF in SCD. Conclusions: Results from this thesis contribute to the literature that SCD is primarily associated with restrictive lung disease. Furthermore, the new interpretation strategies developed during the work for this thesis prevented significant misinterpretation of LF in Black children, and improved the standards for using these LFT’s in children.