This review focuses on the nature of individual differences in Theory of Mind (ToM) and examines evidence concerning the links between ToM and children's emotional and behavioural problems. An introduction to ToM is given, followed by a brief synopsis of what is known about the influences on individual differences in ToM. The review considers ToM in relation to peer rejection, attention and behavioural difficulties, bullying and emotional difficulties. Research in these areas is in its infancy and the review describes the difficulties intrinsic to this type of research (for example, measuring ToM) as well as drawing the reader to the importance and potential implications of ToM research in child development and the development of psychological difficulties. Ideas for further research are proposed.

Background: Metabolic disorders are common in Saudi Arabia. Adherence to a special diet is essential to prevent developmental disability in phenylketonuria (PKU). Our aim was to identify the risk factors for non-compliance with treatment of aminoacidopathies and poor outcome in PKU patients at King Faisal Specialist Hospital & Research Centre in Saudi Arabia. Methods: A qualitative study assessed nutritional knowledge, attitudes and practices through interviews (n=5) and focus groups (2) with health care providers, and interviews with patients with aminoacidopathies (6) and families (17). A quantitative study assessed 40 PKU patients by anthropometric measurements, dietary records, phenylalanine blood levels, developmental assessments, and questionnaires with the patients and their mothers. The Vineland-II Adaptive Behaviour Scales were translated into Arabic and adapted to the Saudi culture to be used as the assessment tool to measure outcome. Regression analysis and independent t-tests were used to investigate relations. Results: Qualitative findings: Major themes identified: Lack of sufficient services, inadequate dietary knowledge, limited resources for families and dietitians, social and emotional attitudes towards diet, and compliance by the child and mother. Quantitative results: 1- Factors contributing to low Vineland-II Adaptive Behaviour Composite score (ABCs): a. Delayed diagnosis: Mean ABCs (}1SD) according to age at start of treatment (Significant difference p<0.0001): . Diagnosed .1 month: 92.2 (}11) . Diagnosed >1 month: 60.5 (}20.6) b. Disease severity: There was a significant difference (p=0.008) in the ABCs between patients with Mild PKU (diagnosis phenylalanine 600-1200ƒÊmol/l) and Classic PKU (diagnosis phenylalanine >1200ƒÊmol/l). 2- Factors leading to inadequate blood phenylalanine control: Delayed diagnosis, poor compliance with dietary phenylalanine restriction, and inadequate intake of the prescribed supplement. Conclusion: Newborn screening for PKU has been very successful in improving outcome but this could be further enhanced by targeted improvements in the education and support of families and in the metabolic care services.

Recurrent loss of imprinting at 11p15, paucity of recurrent genetic mutations and associated nephrogenic rests (NR; precursor lesions that resemble embryonic kidney (EK)) implicate aberrant DNA methylation in tumourigenesis of paediatric Wilms tumour (WT) and predict that interrogation of the methylome, rather than the genome, is more likely to reveal tumour-specific biomarkers To test if aberrant DNA methylation is implicated in tumourigenesis, methylome analysis was performed on 36 normal kidney (NK), 22 NR, 36 WT and 4 EK, including 20 matched trios and 34 matched NK-WT pairs, using Illumina 450k arrays. Findings were validated with bisulfite-sequencing and RNA sequencing. This thesis describes the successful identification of changes in methylation that distinguish between tissue types. Through analysis of DNA methylation, NR formation was associated with gain of methylation at developmental loci related to Polycomb target binding sites. Evolution to WT was associated with increase in methylation variability in a subset of WTs (group-1), which also showed common changes in methylation in comparison to their associated NR, including silencing of novel tumour suppressor genes. Group-1 WTs were significantly enriched for bilateral cases whereas those in group-2 showed no differences in methylation compared to their associated NR. Comparison between NK and WT identified three DMRs of genome-wide significance (P<5x10-8) for use as tumour-specific biomarkers. As proof of principle for clinical utility, DMR-2 was successfully used in a case study to monitor tumour burden during treatment in cell-free serum DNA. This thesis concludes that methylation levels vary during WT evolution. As group-1 WT included all bilateral cases, our data suggests that methylation analysis could aid treatment planning in bilateral disease and that some WT may be candidates for epigenetic-modifier therapy. These findings define the first cell-free epigenetic biomarker for WT with potential for clinical utility.

The requirement to produce complex, therapeutic proteins that possess human-like post-translational modifications for treatment of disease has accelerated the use of mammalian cell lines, in particular Chinese hamster ovary (CHO) cells, in the biopharmaceutical industry. However, producing stable, high expressing cell lines quickly, reproducibly and with relative simplicity whilst achieving a desired product quality profile proves challenging. Conventional methods involve random integration of the expression vector carrying the gene of interest (GOI) into the host genome, often resulting in clonal lines with highly variable protein expression. In order to overcome some of these challenges the artificial chromosome expression (ACE) system, comprising a functional artificial chromosome (AC) present in a CHO-K1 cell line, ACE targeting vector (ATV) with GOI, and a mutated λ integrase which catalyses site-specific integration of the ATV into the AC, has been developed. This targeted integration negates ‘position effects’ and reduces phenotypic variation. In this study this novel, minimally characterised system was investigated; the number of AC recombination sites was determined and optimal selection pressure and vectorology was established for the generation of highly productive cell lines. In an attempt to further improve protein production, two consecutive rounds of gene delivery were performed and its potential evaluated. Further, stability of the AC over long-term culture and its uncompromised structure over 50 cell passages was proven. In addition, a series of cell lines expressing recombinant proteins have consequently been investigated for stability over long-term culture, with the results underlining the need for optimal metabolite support for the cells to ensure consistent protein production. Another key feature of the system is the ability to isolate and transfer the AC into alternative host cell lines. To test this portability, the AC was isolated by FACS and an attempt to transfect into alternate CHO and HEK293 cell lines was made. Work presented in this study demonstrates the potential of the ACE system to become a predominant commercial platform for the production of recombinant proteins, owing to its high efficiency, competitive timelines for mammalian cell line generation, and low clonal variability.

Background: Any clinical decision made has an effect on both the patient and the outcome of their treatment. There is little known about how dentists weight up factors before they make a clinical decision. Aim and Objective: A pilot qualitative investigation of the factors influencing dentists allocating children needing operative treatment to local anaesthetics only, Local anaesthetic with a form of sedation or general anaesthetic. Method: 14 dentist invited to take part in an in-depth interview with a trained interviewer. Dentists were asked about their dental training background, how they assessed children, treatment planning, dental anxiety, parental influence and how they decide by which pharmacological behaviour management technique the child should be treated. Interviews were recorded, transcribed then themes developed using a framework analysis approach. Result: Dentists were found to be the main influencing individuals in clinical decision making. Parents and young children in particular having a limited input. They are also happy with current methods and have a poor opinion about anxiety scales. Pharmacological behaviour techniques are selected mainly based on the magnitude and type of treatment to be conducted. Dentists tend to seek alternative behaviour techniques or change their initial treatment plan only if parents disagreed with them; especially if general anaesthesia was considered. An audit of a 1000 patient file treated on the department also found that treatment to be conducted is the main influencing factor; especially the number of teeth to be extracted. Dentists were found to be poor in predicting patient whom would benefit from treatment on the dental chair, and were better in identifying patients who needed general anaesthesia. Additional clinical decision making influential factors were identified; history of pain and swelling, child’s behaviour, parental opinion and previous dental experience. Conclusion: In conclusion, clinical decision making in this sample appeared to be subjective. A more systematic approach to behaviour and anxiety assessment is required.

RPTPs have striking patterns of expression within the neural tube; raising interesting questions as to their role in the development of this structure. PTPγ expression is initially observed in the first born neurons and this expression domain progressively expands to the lateral motor columns and intermediate zone of differentiating neurons. Short hairpin encoding RNAi constructs were generated against PTPγ, which effectively knocked down this gene in vitro and in ovo. Analysis of silenced embryos through neuronal markers presented a range of phenotypes. The most striking was a dorsoventral truncation of the neural tube arising from a loss of LIM-HD expressing cells not observed among controls. During the onset of neurogenesis, a loss of Lim1/2/3, Islet1/2, and Mnr2/Hb9 positive cells was observed. At later stages, Islet1/2 cells showed no such sustained effect essentially recovering. A further heterotopic phenotype was observed with mislocated neurons located ectopically in the ventricular lumen, that were Islet1/2 and Nkx2.2 positive but never Lim1/2/3. The targeted regions showed a significant decrease in apoptosis suggesting newly born neurons were dying before reaching their pool specific domains. A reduction in the proliferative capacity in the ependymal zone demonstrated by Histone3 activity. Over expression of PTPγ showed a similar reduction in LIM-HD expression however no overall change in S-Phase was observed yet cells at M Phase were reduced with no apoptosis detected through Caspase3 activity. The data suggests PTPγ silencing may result in a failure of Lim1/2/3 cells to reach their final destinations and acquire LIM-HD identities, implicating this gene in the development of inter and motor neuron populations. Preliminary RNAi experiments against PTPσ show a reduction in LIM-HD fated cells and a triggering of the apoptosis programme along with a reduction in number of mitotically active cells. This is consistent with a potential role for PTPs during neurogenesis and the birth of the first neuronal populations in the neural tube.

Primary speech and language impairment (PSLI) is a term used to describe children with a range of significant speech and language impairments, in the context of normal cognitive abilities. The aims of this thesis were firstly to explore identity construction in children with PSLI, and secondly to explore how these children made sense of their experiences. Narrative inquiry was used to conduct this study. The participants were 11 children aged 9-12 years-old, presenting with PSLI. The aim was to generate storied accounts of events and happenings in their lives using interviews, supplemented with visual methods. An innovative analytical framework was designed, drawing on a range of narrative analytical methods, including an analysis of verbal and nonverbal evaluation markers, cohesion markers, as well as an analysis of agency and identities presented in the children’s narratives. The key findings were four interrelated themes, which were conceptualised in a working model comprising facilitators and potential barriers to well-being and belonging. The four themes which contributed to well-being and belonging included: relationships; autonomy, agency, and competence; identities of belonging and difference; and hope and concern for the future. Facilitators of well-being and belonging were life events and experiences that the children evaluated in positive ways, whereas potential barriers were life events and experiences which they evaluated in negative ways. Although some children evaluated their experiences in mixed and sometimes contradictory ways, their evaluations were predominantly positive. This thesis contributes to the field in four ways. Firstly, it provides new insights into identity construction in children with PSLI. Secondly, it adds to understandings of ways in which children conceptualise communication impairment. Thirdly, it deepens understandings of the determinants of well-being and belonging in children with PSLI. Finally, this thesis highlights the value of narrative inquiry as a means for listening to the voices of children with communication impairments.

Nephronophthisis (NPHP) is a major cause of pediatric renal failure. Currently there is little understanding of the aetiology of the disease. In order to identify the molecular events leading to NPHP, we have created a novel mutant mouse strain containing a truncating mutation in the Cep290 gene. Patients with mutations in CEP290 present with a ciliopathy phenotype that includes retinal dystrophy, cerebellum defects and NPHP. Characterisation of Cep290LacZ/LacZ mice confirms that they display all the features of the human condition. Microarray analysis of newborn kidney tissue was used to explore initiating events leading to NPHP. Ciliopathies have recently been associated with either disrupted Wingless integrated (Wnt) or sonic hedgehog (Shh) signaling. We show that mutant kidneys display abnormal Shh signaling in the absence of Wnt signaling abnormalities. Primary cell cultures of collecting duct (CDT) cells (isolated from Cep290LacZ/LacZ mice and wild-type litter mates crossed with the “immorto” mouse) were established and characterised. CDT cells expressed the mineral corticoid receptor (MR) and the epithelial sodium channel (ENaC) alpha subunit. The CDT cell lines formed epithelial layers and formed tubules when maintained in 3D culture media. Cep290LacZ/LacZ CDT cells displayed ciliogenesis abnormalities as well as abnormal spheroids with loss of lumen when grown in 3D culture. Pharmacological activation of Shh signaling (purmorphamine) partially rescues the spheroid and ciliogenesis defects in Cep290LacZ/LacZ CDT cells. This implicates abnormal Shh signaling in the onset of NPHP and suggests that targeted treatment of Shh antagonists have therapeutic potential.

The social profiles seen in Williams syndrome (WS) and autism (ASD) have often been cited as mirror opposites of one another, with hyper-sociable behaviours seen in Williams syndrome and a disinterest in social engagement evidenced in autism. Studies investigating the social-perceptual abilities of individuals with these neurodevelopmental disorders have found overlapping profiles, with a tendency towards using more featural processing strategies when interpreting information from faces, and deficits in recognising and interpreting the various facial cues that provide social information. It is therefore likely that differences in social approach behaviours in the two groups are driven by a more social-cognitive mechanism. The focus of this thesis was on answering the overarching question: What meaning do faces and socially relevant stimuli have for children with Williams syndrome and autism? Six experiments examined the recognition, attribution, description and understanding of emotions and social cues from faces and socially relevant scenes, amongst WS and ASD individuals relative to their typically developing peers. It was found that the social-perceptual profiles of individuals with the neurodevelopmental disorders were markedly similar, with accuracy for identifying emotions being at nonverbal mental (but not chronological) age level. A tendency towards differences emerged in terms of the types of attribution and descriptions that individuals made, with those with ASD focusing more on physical aspects of social and non-social stimuli whilst individuals with WS showed more of an atypicality in the understanding of emotions and social contexts. The lack of any clear differentiation between individuals with ASD and WS in both the social-perceptual and social-cognitive domains is in line with recent research pointing to the extreme heterogeneity seen in these groups. The issue of overlaps and dissociations within such heterogeneous groups provides the theoretical framework for this thesis.

A large amount of resource is expended attempting to change clinical practice; however the effects of these interventions are seldom assessed. A systematic literature review revealed that existing outcome measures to examine the effects of a ‘practice changing intervention’ (patient video cases as the instructional element in an e-learning package) lacked validity. As existing frameworks for the evaluation of practice changing interventions may not adequately determine an interventions’ true effectiveness a novel “7Is framework” was developed. This contains 7 domains: Interaction, Interface, Instruction, Ideation, Integration, Implementation and Improvement. To produce bespoke outcome measures for the “7Is Framework” domains the following studies were performed: i) Focus groups with junior doctors demonstrated poor differentiation between concepts of competence and confidence. A new indicator of self-awareness, the perception of “safeness”, was generated (Ideation). ii) Paediatricians rated quality and acuity of a selection of clips at differing technical qualities to produce gold standard responses for video case questions (Integration). iii) A novel matrix determining how experience and tacit knowledge may impact on patient outcomes was validated as a measure of behaviour change (Integration). iv) A time series of presentation and admission rates of feverish children provided outcome data for the evaluation (Implementation / Improvement). An audio-visual intervention in paediatric fever was designed, delivered and tested against the new system (studying 202 UK doctors). Interaction with the intervention was variable, only 28.7% completed the post learning section and issues were identified with accessing the video cases. Although measures of ideation increased and there was a trend towards behaviour change, full implementation of the guidance did not occur and overall admission rates increased. The 7Is Framework allows the various effects of an intervention to be conceptualised, promoting the development of a set of valid and specific outcome measures, ultimately leading to more robust evaluation.