α-dystroglycan (α-DG) is a peripheral membrane protein that is an integral component of the dystrophin-glycoprotein complex (DGC). In an inherited subset of muscular dystrophies known as the dystroglycanopathies, α-DG has reduced glycosylation that results in lower affinity binding to a subset of extracellular matrix (ECM) proteins including laminins. The aim of this work was ultimately to increase the current understanding of α-DG glycosylation to better comprehend disease pathology and to aid in the development of targeted therapeutics. To achieve this, I first utilised flow cytometry to identify potential imino sugar compounds capable of increasing α-DG glycosylation, and six were found to have this effect. These six compounds significantly increased the level of α-DG glycosylation with sufficient selectivity and potency to make them attractive targets for drug development. Secondly, I optimised a flow cytometry method to determine the level of glycosylated α-DG in cell lines from confirmed and putative dystroglycanopathy patients to use as a complementary diagnostic aid alongside immunohistochemistry and immunoblotting. A total of twenty one dystroglycanopathy patient fibroblasts were assessed, as well as fibroblasts from three healthy controls and seven pathological controls. All cell lines assessed had detectable levels of glycosylated α-DG, and the level detected in dystroglycanopathy patient cell lines was significantly reduced (as measured as measured by the mean fluorescence intensity of an antibody recognising the epitope and the percentage of cells positive for the epitope) compared to that detected in healthy control fibroblasts (p<0.0001 for both values). Additionally, the results indicated that the amount of α-DG glycosylation in patient fibroblasts is comparable to that in patient skeletal muscle. Lastly, I was involved in the functional characterisation of variants in the glycan processing enzymes beta-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2) and GDP-mannose pyrophosphorylase B (GMPPB) to validate them as causative of dystroglycanopathy. The muscle biopsies and fibroblasts of patients with variants in these enzymes had reduced α-DG glycosylation compared to healthy controls. In some patient fibroblasts, complementation with the wild-type gene partially restored α-DG glycosylation. Additionally, several of the identified missense mutations in both genes resulted in a change in the subcellular localisation and/or aggregation of the recombinant enzyme compared to wild-type, suggestive of possible altered function. The results of this study provide insight into the complexity of α-DG glycosylation and pose questions that should help lay the foundation for future studies.

Childhood onset epilepsy can have a profound effect on cognitive, emotional and behavioural development. As such, early intervention is crucial. Approximately 25-50% of children with epilepsy show resistance to medication however. For these children, neurosurgical intervention may be considered. The decision for surgery is a multi-disciplinary process, including functional Magnetic Resonance Imaging (fMRI) to assess the risk posed by surgery to language. Based on feasibility and behavioural pilot studies, I developed an fMRI task panel optimised for pre-surgical investigations of language in children. This task panel maps different language systems (word retrieval, sentence generation, auditory comprehension and reading comprehension) and localises critical language functions (semantic and syntactic processing). I validated this task panel in healthy children (N=43, 5-16 years). This included assessments of scan quality, comparison of methods for artefact repair, and definition of typical activation patterns. I also piloted the new task panel in a representative sample of children with epilepsy, who were being considered for surgery (N=13, aged 5-16 years). Patient case studies are reported to highlight methodological challenges associated with localisation of critical language regions on an individual basis. Finally, I present experimental analyses which highlight the importance of the ventral system to semantic processing. Activation in this network was reduced in children with epilepsy and predicted language outcome. Further investigation showed prolonged development of specific nodes within this system, supporting multimodal semantic processing (independent of effort and performance accuracy). These regions included ventral occipito-temporal cortex, whose role in semantic processing has so far been underappreciated in the developmental literature. These analyses provide evidence for a core language system, which may be crucial for post-surgical language outcome. The findings from this thesis contribute towards extending and improving the role of fMRI in the surgical decision-making process, with the potential for improving long term outcome. They also contribute to models of typical and atypical language development.

BACKGROUND We identified patients from consanguineous families from within a genetic isolate who presented with an unknown combination of renal tubular proteinuria and sensorineural hearing loss. METHODS Patients were carefully characterised concerning kidney function including 99m-Tc-DMSA scan and renal biopsy. We applied multipoint parametric whole-genome linkage analysis and haplotype reconstruction in four families with six affected children. We performed exome capture and next generation sequencing in five affected children. Newly identified mutations in a gene with presumed intracellular trafficking functions were evaluated with the use of different cell models. Protein expression within the kidney and intracellular trafficking were further investigated by immunocytochemical analysis. RESULTS All patients investigated had normal glomerular filtration rate, a pathological 99m-Tc-DMSA renal scan with no tracer uptake and abnormal electron microscopy finding within the microvilli of the proximal tubule. Linkage studies identified a single significant genome-wide locus with a LOD score of 7.2 on chromosome 11. This region of 1.5 million bases, contained a total of 48 annotated genes. Analysing the sequencing results of those genes within this linked region revealed a homozygous missense mutation in the EHD-1 gene in all affected individuals. The mutation segregated in an autosomal recessive fashion within the families and was absent from 196 healthy ethnically matched control alleles. This mutation, when expressed in HeLa and renal proximal tubular LLC-PK1 cells caused significant and specific changes in the endosomal recycling compartment, an intracellular compartment related to processing and recycling of cellular substrates. CONCLUSION We identified a founder mutation in a gene causing a previously unrecognised and undiagnosed rare autosomal recessive Mendelian disorder, showing clinically signs and symptoms of renal tubular low molecular weight proteinuria and hearing deficit. Our findings indicate that this gene plays a major role in renal tubular low molecular protein handling and inner ear function.

This thesis is concerned with an exploration of low-intensity treatments for common mental health problems in childhood and adolescence. Part one is a narrative literature review of trials of low-intensity interventions for children and adolescents with depression or anxiety, including bibliotherapy, computerised CBT and attention-bias modification. Part two is a long term follow-up study examining the effects of age, symptom severity and comorbidity at initial assessment on outcomes for children who had received guided CBT bibliotherapy via their parents four years previously. Part three is a critical appraisal of the thesis, considering the wider methodological and conceptual challenges associated with research across the developmental period.

Developmental amnesia (DA) is a selective episodic memory disorder associated with hypoxia-induced bilateral hippocampal volume reduction. Despite the systemic impact of hypoxia-ischaemia, the resulting brain damage has been previously reported to be limited to the hippocampus. This thesis explores damage outside of the hippocampus in patients with DA and in those with less severe memory impairment (MI), to provide a full anatomical characterisation of a hypoxia-induced memory disorder. Seventeen patients with DA and fifteen patients with MI participated alongside an equal number of controls. The extent of brain injury was assessed in structural magnetic resonance images using whole-brain (voxel-based morphometry and tract-based spatial statistics) and region-of-interest approaches (automatic and manual measurement of structure volumes). Results showed that in addition to damage to the hippocampus, patients with DA had severe atrophy of the mammillary bodies, mild volume reduction of the thalamus and widespread abnormalities in the white matter. There was only limited evidence of damage in the medial temporal lobe neocortices. By comparison, brain injury in patients with MI was much less extensive. A series of correlations examined the integrity of brain structures in relation to patients’ cognitive outcome. Global abnormalities in the white matter were related to patients’ intelligence, but not to their memory, whereas regional abnormalities in the ventral cingulum predicted performance on tests of verbal recognition. In addition, volumes of a thalamic segment were related to patients’ face recognition ability. Together, these results show that extra-hippocampal abnormalities contribute to the cognitive outcome of patients with DA, independent of the hippocampus, offering support for dual-process models of memory. The findings could lead to new magnetic resonance imaging-based diagnostic measures of developmental amnesia.

Enteropathogenic Escherichia coli (EPEC) is a non-invasive, gram-negative bacterium that causes acute and persistent diarrhoea in infants under the age of five. Infantile diarrhoea is the second leading cause of mortality in children recorded by the World Health Organization (WHO). At present the role of innate and adaptive immunity in providing protection or mediating pathology in response to EPEC remains less well studied. Herein, multiple facets of EPEC interaction with the immune system were explored in co-cultures utilising wild-type and bacterial isogenic mutants in murine and human model systems. EPEC-mediated bone-marrow derived dendritic cell (BMDC) activation was investigated. The flagellin moiety and the type three secretion system (T3SS) inhibit DC surface marker and IL-10 expression, thus modulating the innate IL-12/IL-10 axis. The T3SS of Enterohemorrhagic E.coli (EHEC) and Salmonella typhimurium (STM) exerted similar effects on the cytokine milieu, supporting the hypothesis that enteropathogens may promote inflammation by inhibiting IL-10 expression. Infection led to potent Th1 and Th-17 immunity, the latter response was generated in the absence of any detectable IL-23. Evidence suggests a central role for IL-1 signalling in Citrobacter rodentium (murine model for EPEC) infection and inflammation. We investigated EPECmediated inflammasome activation, a protein-complex involved in the release of bioactive IL-1 . The present study is the first to implicate NLRP3 in EPEC-mediated BMDC inflammasome activation. The T3SS was crucial for NLRC4 but not for NLRP3-driven inflammasome assembly. K+ ion influx was identified as an important mediator of EPEC-driven NLRP3 function. EPEC sabotages intestinal epithelial actin cytoskeleton in multiple ways, cellular events that promote disease on-set. Wiskott-Aldrich syndrome (WAS) is a rare immunodeficiency where the WAS protein, an important mediator of actin polymerisation is defunct. The role of actin in EPEC-BMDC crosstalk was studied in WT and WASP-deficient murine and human innate immune cells. WASP deficiency resulted in increased bacterial load, impaired autophagy and deregulated inflammasome activity. Similar effects were also observed in response to infection with the intracytosolic enteropathogen Shigella flexneri. Our observations suggest for the first time that WASP is a pivotal rheostat between bacterial Autophagy and the Inflammasome axis.

Photoreceptor cells are the sensory cells of the retina, responsible for detecting light and conducting the signals to secondary neurons. Because they cannot be regenerated, loss of photoreceptor cells leads to irreversible blindness. Cell transplantation with postmitotic photoreceptor precursor cells has been shown as a feasible approach to rescue vision in animal models, but the molecular properties of these transplantation-competent cells are not understood. The aims of this thesis are to 1) determine the properties of photoreceptor precursor cells by transcriptome and proteome analysis; 2) identify cell surface molecules that can be used to isolate these cells from cell mixtures and/or that are important for their migration and correct integration in development and in a transplantation context. Nrl/CrxGFP transgene-labelled photoreceptor precursor cells were separated from other retinal cells by flow cytometry and subjected to microarray and mass spectrometry analysis. Bioinformatics analysis showed that the photoreceptor precursor cells were enriched in expression of genes encoding cell projection proteins. Over 200 cell surface molecule candidates were identified and 32 genes encoding confirmed extracellular domains were expressed > 5-fold higher in photoreceptor precursors than other retinal cells. These included the stem cell marker Prom1 (CD133), which was specifically expressed in photoreceptor cells (particularly on their cilia) throughout development as well as on transplanted photoreceptors. Together with CD73 and CD24, it serves as a specific marker to isolate photoreceptor cells for transplantation. An axon guidance molecule Sema7a was shown to be highly expressed in photoreceptor cells. It co-labels with PlxnC1, rather than the expected receptor Itgb1, in developing retina, as well as transplanted migrating photoreceptor cells. Knockout of Sema7a resulted in retinal holes and abnormal photoreceptor synapse projection indicating a role of Sema7a in outer retina lamination. This study sets the foundation for future work on photoreceptor cell surface molecules in development and retinal repair.

Refeeding adolescents with anorexia nervosa (AN) carries risks, the extent of which have been much debated but subject to little research. As such, the optimal nutritional management of such patients is unknown, and the lack of evidence from interventional studies has led to worldwide disparities in clinical management recommendations. In this first randomised controlled trial in this area, we tested the hypothesis that refeeding with a higher energy intake than that currently recommended in Europe, improves outcomes in low weight adolescents with AN. The aim of this study was to investigate the association between total energy intake on QTc interval, heart rate and hypophosphataemia. The primary outcome was QTc interval (ms). Secondary outcomes were heart rate, electrolytes (phosphate, magnesium and potassium) and anthropometry (weight [kg] and %BMI). Participants were 38 adolescents’ aged 10-16years with a DSM IV diagnosis of AN recruited from six acute paediatric services around the UK and were randomly allocated to commence refeeding at 1200kcal/ day (intervention) or at 500kcal/ day (control). Energy intake was incrementally increased by 200kcal day up to 80% of estimated energy requirements. The results showed that compared to controls, adolescents randomised to the higher calorie group had a greater weight gain. However, randomised groups did not differ statistically in QTc interval or heart rate. Refeeding hypophosphataemia (serum phosphate <0.9mmol/l) developed in a proportion of patients. However, there was no statistical difference in the incidence or severity of refeeding hypophosphataemia between the two groups. Participants that were below 68%BMI and those with low WBC’s (WBC <3.8 x 109/l) had a greater reduction in post refeeding phosphate. Together, these findings suggest that oral refeeding at 1200kcal/ day (38kcal/ kg/ day) which increases incrementally to 1900kcal/ day (58kcal/ kg/ day) is more beneficial for the majority of patients than commencing refeeding at 500kcal/ day in low weight adolescents with AN. The findings from this study challenge current European and UK recommendations.

Brushite cements have potential as drug carriers and bone filling materials. They can also act as a reservoir for calcium and phosphate ions in remineralisation of hard tissues. Aim and objectives: To optimize brushite cement properties and assess the effect of incorporation of a novel antimicrobial ε-polylysine (PLS) into brushite. Materials and Methods: Powders were mixed with aqueous solutions at a powder to liquid ratio of 3.3:1 or 4:1 to produce cement pastes and start the setting reaction. The powder consisted of 1g of monocalcium phosphate monohydrate (MCPM) and 1.23 g of β -tricalcium phosphate (β-TCP). Two different types of MCPM and different β-TCP particle size range from 4 micron to 34 micron were used. The liquid phase was prepared by dissolving PLS powder in aqueous citric acid 800 mM in incrementally percentages. In control formulations, only citric acid 800 without the PLS was employed. Biaxial flexural strength and modulus were determined using a ball on ring jig. Setting kinetics and chemistry were examined using Fourier transforms infrared spectroscopy (FTIR). Microstructure of brushite cements were examined with scanning electron microscopy (SEM). Results: The largest particle size of â-TCP (34 micron) produced the highest flexural strength of 30 MPa. The handling of brushite cements was better with MCPM 2 (Sigma). This was observed to have much larger flat crystals rather than the more powdered MCPM 1 from Himed. Powder to liquid ratio 4:1 overall increased the strength 5 MPa – 7 MPa compared to powder to liquid ratio 3.3:1. High levels of PLS could be added with only a minor reduction in the strength. Setting time however was delayed and an alternative anhydrous dicalcium phosphate complex formed rather than all brushite which is hydrated dicalcium phosphate. Conclusion: The findings of this research demonstrated that very high levels of PLS could be introduced into brushite cements without serious detrimental effects on mechanical properties. PLS however, did delay the setting time and altered the final chemistry and microstructure of the dicalcium phosphate product.

The early onset epileptic encephalopathy of infantile spasms is frequently associated with acute cognitive regression, long-term learning disability and autistic spectrum disorder. Although there may be a structural basis to the epilepsy, it appears that seizure activity is directly implicated in the process associated with the above disabilities. There are strong indications of the crucial role of temporal lobe dysfunction in children within this and related epileptic regressions including the site of lesions in tuberous sclerosis and the EEG localisation in a later onset epileptic encephalopathy, the Landau-Kleffner-syndrome. Thus the hypothesis for this study was that the temporal lobe is functionally abnormal in children with infantile spasms. This was tested by recording event related potentials, the electrical indicators of the brain's perception and processing of auditory stimuli. The aims of the current study were to describe the normal developmental changes of mismatch-negativity (MMN) and novelty P3 in the first year of life and to identify whether these ERPs are abnormal in children with infantile spasms. The developmental status of infants with infantile spasms was assessed at presentation. The MMN was only shown in a group mean average in control infants. All obligatory and the endogenous P250, P500, Ncl and Nc2 ERP components of the control infants showed age dependent latencies and differed in latency between wakefulness and stage 2 sleep. Using nonparametric calculations infants with infantile spasms had prolonged latencies of the obligatory and endogenous components during both wakefulness and sleep compared to controls. The results of this study support the hypothesis that the auditory processing is interrupted in infants with infantile spasms. As the auditory cortex is very immature during the first year of life it is therefore suggested that infantile spasms may interfere with crucial maturational processes during the first year of life.