This review paper, the implications of the recently discovered syndrome of Developmental Amnesia (DA) for two opposing theories of medial temporal lobe (MTL) amnesia are evaluated: Some researchers describe both episodic and semantic memory as subsystems of declarative memory, dependent on medial temporal lobe and midline diencephalic structures, with episodic memory additionally depending on the frontal lobes (Unitary Account). They explain MTL amnesia in terms of an overall impairment in declarative memory functioning. Others describe episodic memory as a memory system that is additional to declarative/semantic memory (Dual-Process Account). They explain MTL amnesia in terms of various patterns of dissociation between episodic and semantic memory functioning. These two opposing accounts are used to interpret studies of the recently discovered syndrome of Developmental Amnesia, a type of childhood-onset amnesia that is characterised by selective damage to the hippocampus and selective episodic memory impairment, and which therefore presents an interesting context for evaluating the two theoretical accounts of MTL amnesia. It is argued that the Hierarchical Account, an elaboration of the Dual-Process Account, presents the most parsimonious explanation of Developmental Amnesia.

Infectious diseases are the current leading cause of human death and within this category nosocomial infections remain the most frequent complication of hospitalization. A range of infection prevention and control activities are employed to combat the selection and spread of these organisms. The principle components of which are: early identification of carriage/infection, patient isolation, improved hand hygiene, environmental control and good antimicrobial stewardship. In order to properly focus these interventions, it is essential to know how and when cross transmission has occurred. There is an ongoing debate about the role of the environment in the spread of healthcare associated infections and to what extent if any it acts as a potential vector for transmission. Within the healthcare setting patients spend a substantial amount of time surrounded by equipment and environmental surfaces that may be contaminated with microorganisms. In order to establish what role the environment could play, tracking the spread of organisms by molecular typing is key. The current methods used to do this are complex and often are only available at reference laboratories. This means that turnaround times are slow and only provide retrospective confirmation of cross-transmission events. Infection control interventions that can be used prior to receiving results play an important role. The selection and effectiveness of these interventions are often poorly supported by research studies, leading to problems with the introduction of evidence based practice and thus difficulty in selecting the most appropriate response to suspected cross transmission. This thesis aims to explore the role of the environment in cross transmission of infection by developing sampling methodologies to permit environmental surveillance, validating and developing typing techniques in order to establish epidemiological links between patients and environmental contamination and to evaluate infection control interventions to aid in prevention of cross transmission events.

Chromatin remodelling provides a key mechanism for the regulation of gene expression through dynamic alterations in nucleosome occupancy at promoters and enhancers. Haploinsufficiency for the ATP-dependent chromatin remodeller chromodomain-helicase-DNA-binding protein 7 (CHD7) causes human CHARGE syndrome. CHARGE is characterised by a distinct pattern of congenital anomalies, including cardiovascular malformations, and has traditionally been considered a neurocristopathy. However, a number of the congenital heart defects associated with CHARGE cannot be attributed to disruption to the neural crest alone. This thesis therefore addresses the tissue-specific requirements and roles for CHD7 during cardiogenesis. CHD7 protein is shown to be present throughout the developing heart until E13.5. Conditional ablation of Chd7 in the early cardiogenic mesoderm results in embryonic lethality due to severe cardiovascular defects. These include haemorrhaging and oedema, major venous and arterial pole malformations, and disruption to cardiac innervation and vascularisation. To further dissect tissue-specific requirements for CHD7, cardiomyocyte-, second heart field- and endothelial-specific knockdowns were also performed. Each cross results in a milder subset of the cardiac defects observed after mesodermal ablation, indicating that CHD7 is required in multiple lineages within the cardiogenic mesoderm. Microarray analysis and validation by in situ hybridisation were used to identify genes dysregulated in the heart following mesodermal Chd7 ablation. These included components of the Semaphorin and Slit-Robo signalling pathways, which have known roles in heart development. Furthermore, aberrant expression of genes involved in calcium handling within cardiomyocytes is seen. Excitation-contraction coupling is disrupted in mutant embryonic cardiomyocytes, demonstrating relevance of the gene expression changes at the cellular level. This work reveals a requirement for CHD7 in mesodermal cardiac progenitors for both inflow and outflow tract development. Novel pathways are identified downstream of CHD7 activity in the developing heart, including the extracellular Semaphorin and Slit-Robo pathways, as well as components of the excitation-contraction coupling machinery.

Sickle Cell Disease (SCD) is a collection of genetic haemoglobinopathies, the most common and severe being homozygous sickle cell anaemia. In the UK, it has been estimated that 1 in 2000 children are born with SCD. The disease is characterised by chronic anaemia, recurrent pain crises and vascular occlusion. Neurologically, there is a high incidence of stroke in childhood, as well as cognitive dysfunction. Newborn screening programmes and preventative treatments have allowed a much longer lifespan; however recently, neurological research has shifted to characterising subtler aspects of brain development and functioning that may be critically important to the individual’s quality of life. This thesis overviews the neurological and neurocognitive complications of SCD, and how magnetic resonance imaging (MRI) can provide biomarkers for severity of disease. During the PhD, retrospective and prospective cognitive and MRI data were collected and analysed. Diagnostic clinical MRI sequences and advanced MRI sequences were applied, as well as a neuropsychological test battery aimed at intelligence and executive function. First, this thesis reviews the intelligence literature in SCD and includes previously unreported data, finding patients, regardless of abnormality seen on conventional MRI, have lowered full-scale intelligence quotient than controls. Then, to determine imaging biomarkers, volumetric differences and diffusion characteristics were identified. Patients were found to have decreased volumes of subcortical structures compared to controls, in groups corresponding to disease severity. Results from a three-year longitudinal clinical trial suggest evidence of atrophy in paediatric patients, with no apparent protective effect of treatment. Diffusion tensor imaging revealed reduced white matter integrity across the brain, correlating with recognised markers of disease severity (i.e. oxygen saturation and haemoglobin from a full blood count). Overall, the four experiments bridge a gap in the cognitive and neuroimaging literature of the extent of neurological injury in children with SCD.

This project aimed to clinically characterise phenotypes with unknown genetic aetiology and proceed with customised gene identification strategies in selected cases/families. A systematic analysis of 116 medical records of undiagnosed cases observed at the Medical Genetics Department of the Coimbra Paediatric Hospital, corresponding to 52 families, was performed in order to identify phenotypes to better describe and identify their aetiologies. Thirty-three families were observed in the clinic to allow for careful examination and detailed phenotyping. In five of the observed families, likely previously clinically unrecognised syndromes were identified. Eighteen families with the clearest or distinct phenotype, evidence of consanguinity, or the most available family members, and therefore the highest chance of finding the mutated gene, were then selected for further research. In four of the selected cases, the chosen gene identification strategies were completed during this project. In one autosomal recessive family, no variants considered to be likely causative were identified and in the other three families, X¬exome sequencing revealed novel, likely pathogenic, variants in known X-linked intellectual disability genes: HUWE 1, SLC16A2 and PAK3. Whole-exome sequencing is ongoing on 6 families and the remaining 8 families were not possible to be studied. In addition to the familial cases, one sporadic case with the diagnosis of Lenz-Majewski syndrome was observed. Five other cases were subsequently recruited worldwide and a gene identification strategy, consisting of whole exome-sequencing and selecting variants according to the predicted genetic aetiology model, was implemented. We identified causative de novo missense heterozygous mutations in PTDSS1 gene, which encodes for phosphatidylserine synthase 1. We carried out further studies in patient fibroblasts and zebrafish embryos which led us to the conclusion that these mutations have a gain-of-function effect, associated with regulatory dysfunction. Another condition, for which I had a particular interest, was Nicolaides-Baraitser syndrome. Initial work was to clinically describe 23 cases, allowing the syndrome to be delineated and established as a discrete entity. Subsequently, work was to collect further data and DNA samples with the aim of identifying its causative gene. Using whole exome sequencing, dominant-negative de novo heterozygous mutations in SMARCA2, encoding one of two alternating catalytic subunits of the chromatin remodeller complex BAF, were identified in 37/44 cases. No definite genotype-phenotype correlations were established.

This review will consider the literature regarding the prevalence and impact of childhood physical abuse, and the potential link to the formation of delusions later in life. Childhood physical abuse is linked to a number of psychological difficulties both within childhood and later in the life cycle. As psychological models have increasingly been applied to psychosis (and delusional beliefs in particular) the role of early experience has increasingly been considered central. Given the high prevalence of past abusive experiences in individuals with psychosis, and the potentially far reaching effects of this abuse and resulting posttraumatic stress symptoms, further research is recommended to consider the role of abuse in order to improve clinical practice with these individuals.

This paper presents a review of the literature within the field of childhood onset disruptive behaviour disorders (DBD). Recent research in this area has focused on identifying risk factors, developmental pathways and causal mechanisms involved in DBD. Focus on these areas has led to somewhat of a neglect of intrinsic factors, which may also play a role in the development and maintenance of DBD and prove important to increase our understanding of how best to intervene with individuals with these difficulties. This review presents a brief introduction to the area and then considers the evidence for intrinsic factors being implicated in DBD. Initially cognitive factors are explored, in particular examining cognitive processes such as avoidance of responsibility, which has been proposed to play a role in maintaining disruptive behaviour. The review then moves on to explore emotional factors, including the distinction between children exhibiting callous-unemotional traits and those without this emotional profile. The review considers whether there is any evidence to consider any other emotional factors in a model of disruptive behaviour, focusing in particular on whether there is a role for shame. Finally a hypothetical model of the maintenance of disruptive behaviour is presented, integrating both emotional and cognitive factors. The implications of such a model on future research are discussed.

Identifying early signs of lung disease reliably in asymptomatic infants with Cystic Fibrosis (CF) diagnosed by newborn screening (NBS) is a challenge. Very little is known about the origin and progression of lung disease in these infants hence there is uncertainty on best interventions to protect the lungs of these infants from functional and structural decline. This collaborative observational study aims to assess lung function and structure in CF NBS infants. Lung function tests (LFT) were performed in contemporaneous healthy controls and CF infants at 3 months and a year of age. In addition, CF infants underwent chest computed tomography (CT) at 1 year under general anaesthesia within 2 weeks of the LFTs. At a year, CF NBS infants had impaired lung function compared to contemporaneous healthy controls. However the percentage of CF NBS infants demonstrating what was considered abnormal lung function had reduced at a year of age compared to those with abnormal result at 3 months. Although some improvements were observed at 1- year LFTs, this was not universal using the different techniques. Some measurements improved, others remained stable and certainly none deteriorated. In terms of CT structural changes in CF NBS infants, fewer abnormalities were detected in our cohort compared to other reported studies and changes seen were mild. There was poor correlation between lung function and structure in this group of CF infants. To accurately detect CF infants with impaired lung function or structural abnormalities using important physiological and clinical determinants would play an important role in the management of these infants. This study could inform power calculation for future interventional studies using relevant clinical trial endpoints related to lung function and structure.

Neural tube defects (NTDs) are amongst the commonest birth defects, affecting 1 in 1000 pregnancies. During neurulation, failure of neural tube closure in the low spinal region (at the posterior neuropore, PNP) results in spina bifida. Mechanical force relationships in the normally closing neural tube and in the NTD-developing neural tube were investigated through incision of the most recently closed neural tube roof, in order to eliminate tension in the closed region. After incision in wild-type embryos, immediate springing apart (re-opening) of the elevating neural folds was observed, associated with partial relaxation of the bending degree of the Dorsolateral Hinge Points (DLHP) and the Median Hinge Point (MHP) in transverse sections. When incision was performed on Zic2 mutant embryos developing spina bifida, a larger re-opening of neural folds was observed than in wild-type controls. These findings coincided with measurements of the elastic modulus of the mutant NPs using Atomic Force Microscopy, which revealed that the dorsal NP of the mutant embryos is stiffer than wild-type. Finite Element Method (FEM) modelling also showed that a larger closing force is required for a stiffer NP. Moreover, in unincised embryos, nuclei were found to be more elongated in the wild-type NP, where DLHPs are present, than in the mutant NP, where DLHPs are absent, suggesting possible roles of cell packing in DLHP formation. In conclusion, apposition of the NFs is an elastic process exogenously driven by the force field that originates from the most recently closed point. DLHP formation, however, is a partially plastic process, closely related to cell packing within the dorsal NP as an endogenous event. In Zic2 mutant embryos, a stiffer NP and the absence of cell-packing in the dorsal NP region are perhaps the main mechanical causes of spinal neurulation failure.

Magnetic resonance imaging (MRI) is widely used both in the clinic and as a research tool in the management of brain tumours. While most studies focus on adult tumours, which have a higher incidence than those in children, paediatric brain tumours differ widely in terms of biology and treatment management. Furthermore, as a non-invasive and non-ionising imaging tool, MRI is used in the diagnosis, prognosis and assessment of treatment response of such tumours. This work focuses on diffusion MRI to study childhood brain tumours. The thesis is divided into three main parts: a reproducibility study of diffusion MRI parameters in order to answer the question of whether clinical imaging may be used interchangeably across multiple-centres to combine data from different institutions; a study on a tumour border diffusion measure as a prognostic biomarker in children with embryonal brain tumours – the apparent transient coefficient in tumour (ATCT); and a study analysing the functional diffusion map (fDM) as a tool for assessing treatment response in paediatric brain tumours. Diffusion MRI has been shown to have a good reproducibility and thus data from multiple centres and scanners can be combined in order to analyse clinical data for patients treated at different institutions; particularly where data for specific tumour types would otherwise be limited. In addition, ATCT has been shown to be a useful prognostic biomarker in children with embryonal brain tumours. Finally, while the fDM may be beneficial in assessing treatment response, the underlying biology of both tumour and healthy tissue needs to be carefully considered, and in particular, areas of necrosis, tumour grade and change in tumour size need to be taken into account. In conclusion, diffusion MRI is a valuable tool in the management of childhood brain tumours, with multi-centre studies paving the way for further research and validation of biomarkers.