The Hedgehog (Hh) family proteins and their signalling pathway are key mediators of, and important in, many mammalian developmental processes. Malfunction of the Hh signalling pathway contributes to developmental disorders and birth defects. This project aims to investigate the role of the Hh signalling pathway in murine haematopoiesis. In our study, we found that Dhh plays a negative regulatory role in normal erythropoiesis and under stress conditions. However, it is not required for regulating erythropoiesis in the fetal liver during embryo development. In contrast, analysis of conditional deletion of Smo in haematopoietic cells revealed that Smo controls early haematopoietic differentiation in the fetal liver but is dispensable for regulating haematopoiesis in adult bone marrow and spleen. Furthermore, pervious studies have demonstrated that Hh signalling is involved in T-cell development throughout maturation. We tested the hypothesis that Foxa2, a downstream target gene of Hh during pre-TCR signalling, is also required for late T-cell development and activation. Analysis of mice conditionally Foxa2-deficient in mature T-cells revealed that Foxa2 is important in the process of maturation in late thymocyte development. In addition, Foxa2 is also involved in regulation of T-cell activation, and the differentiation of T helper cells. Gene expression experiments confirmed that Foxa2 is also a Hh target gene in the thymus. Taken together, our findings revealed that the Hh signalling pathway and its target genes play critical roles in haematopoiesis during embryogenesis and in adult mice.

Amblyopia, a developmental disorder of the visual system, is widely known to cause a reduction in optotype acuity but it can also be associated with disrupted binocular vision, reduced contrast sensitivity and many other subtle high level visual processing deficits. The initial stages of the work presented in this thesis involved laboratory investigation of the functional visual deficit in global motion processing that has previously been reported abnormal in the presence of amblyopia. The key question is whether higher-levels of visual processing "inherit" abnormalities from lower levels, or whether additional developmental abnormalities arise in direct consequence of impoverished visual input. Overall, the results imply a far more complex perceptual change in amblyopia than would be predicted by the well -established losses in resolution and contrast sensitivity. The motivation behind Chapters 5 and 6 stems from the current observation that the recovery of visual function in amblyopia is contingent on even brief periods of correlated binocular vision, suggesting that amblyopia is intrinsically a binocular problem and that it is suppressive mechanisms that render the cortex, which is a structurally binocular system, functionally monocular. Research is now casting doubts on the idea that amblyopes do not possess cortical binocular connections, suggesting an active suppression rather than a deficit of cellular function. Interestingly, this is echoed in the clinical domain where, in cases of de-correlated visual input, strabismus clinical protocols have now established that the correction of refractive error alone can be sufficient to improve acuity, again implying incomplete inhibition mechanisms. The clinical investigations in this thesis have involved the validation of a series of psychophysical paradigms in cohorts of juvenile and adult amblyopes (as well as age-matched controls) to establish the degree of binocular interaction present and to explore the potential for treating amblyopia with prolonged viewing of a binocular stimulus adapted to correlate the visual imput from both eyes.

The underlying explanation for impairments in facial expression recognition in autism spectrum disorder (ASD) has been debated for the last over 50 years. Socially driven accounts examine whether a lack of attention to or aversion to the eye area is a primary factor that relates to impaired expression processing in ASD (Baron-Cohen et al., 1997a; Baron-Cohen et al., 1997b; Corden et al., 2008; Dalton et al., 2005; Klin et al., 2002; Kylliainen & Hietanen, 2006). A diminished reliance on configural information has dominated the field as an explanation for impairments in face processing in ASD (Happe & Frith, 2006; Mottron et al., 2006). The body of research carried out in this thesis draws on socially and perceptually driven explanations to investigate whether children with ASD can extract emotional cues from the eyes as well as TD controls. The four studies of this thesis examined two main research questions. One question was whether individuals with ASD attend to the eye area and can extract emotional cues from this region. Another question was what the perceptual strategies underpinning face processing were in individuals with ASD. This was examined by disrupting the configural and featural information within the face with the addition of sunglasses and masks to faces. The first two studies from this thesis found that there was a difficulty using or extracting emotional cues from the eyes in children with ASD and have atypical perceptual processing patterns. The third study of this thesis highlighted that the eye area can be used under certain task demands, as well as TD controls, to recognise expressions of emotion. The final experiment of this thesis examined the sensory components of face processing in ASD.

Premature infants represent 7% of all births in the UK and have higher rates of vaccine preventable infections. National recommendations for vaccine schedules focus predominantly on term infants. We aimed to assess the immunogenicity of the UK immunisation schedule in preterm infants randomising infants to 3 different PCV13 primary schedules and using data from the randomized controlled trial and a pooled data metaanalysis of preterm vaccine studies to identify factors affecting vaccine responses. Methods 210 infants «35 weeks gestation) received DTaP-IPV-Hib vaccine at 2, 3 and 4 months of age and meningococcal C conjugate vaccine at 2 and 3 months alongside 3 different PCV13 vaccine schedules (Group1: 2 and 4 months, Group 2: 2, 3 and 4 months, Group 3: 2,4 and 6 months). At 12 months of age participants received MMR, PCV13 and Hib-MenC-TI vaccines. Antibody concentrations and Iymphocyte subpopulations were measured before and one month after primary and booster vaccinations. Results The median birth gestation was 29+6 weeks (range 23+2-34+6). Younger gestation was associated with lower antibody concentrations at baseline but had limited effect on vaccine responses. For PCV13, group 3 had the highest antibody concentrations post-primary and prior to booster vaccination but the lowest antibody concentrations after the booster. There was no consistent effect of chronic lung disease, growth restriction or the receipt of blood products, antenatal or postnatal steroids on vaccine responses. For all antibodies a longer duration of vaccine course or an older age at final immunisation was associated with improved immunogenicity. Increased CD19+was associated with improved PCV13 immunogenicity Conclusion Premature infants can mount satisfactory responses to all routine vaccines but the schedule followed for PCV13 vaccine will significantly influence when optimal protection occurs. Disease epidemiology must be considered when deciding which schedule to follow.

The complex mechanisms by which early life factors can impact the developing mammalian system remain largely unclear. Animal studies have led to the identification of neurotransmitter systems that are influenced through manipulation of early life conditions. In addition, evidence suggests that early life events can influence signaling through the gut-brain axis to modulate brain function and behaviour. Specifically, the functions of the host gut microbiota and metabolism have been highlighted. Weaning and stress have both been shown to influence the development of central neurotransmitters implicated in emotional regulation. This thesis applied a multidisciplinary approach to investigate the effect of delayed weaning and its modulation by stress upon host development at multiple tiers of biological organisation. These included behavioural, neurochemical, gut microbial and metabonomic studies. Animal behavioural studies demonstrated that a lack of weaning at the standard age of 21 days in rats, leads to a marked 'depressive-like' phenotype. Furthermore, receptor autoradiography revealed non-weaned animals to exhibit altered oxytocin receptor binding in the amygdala and different stress coping mechanisms. Fluorescence in situ hybridisation studies exposed significant weaning and stress-induced effects upon gut microbiota composition. In addition, NMR-based metabonomic studies illuminated fluctuations in a number of endogenous and host-microbial co-metabolites known to be implicated in mood and stress-related disorders. Collectively, the results show that early life factors such as weaning and stress are able to have a marked influence on behavioural development. The findings highlight that signaling through the gut-brain axis may modulate behavioural development possibly through the production of neuroactive metabolites at critical stages of development and open new areas of investigation. The findings identify novel targets for interventions in the pathogenesis of paediatric neuropsychiatric disorders.

This portfolio presents the work completed towards partial fulfillment for the degree of Practitioner Doctorate in Psychotherapeutic and Counselling Psychology. My professional development as a counseling psychologist is represented across the Academic Dossier, Therapeutic Practice Dossier and Research Dossier. The Academic Dossier includes three essays and three pUblications. In the first essay, the psychodynamic appl~oach was considered in relation to therapeutic work with a fictitious client affected by combat related PTSD. In the second essay, trauma focused cognitive behavioural therapy and its therapeutic relationship was evaluated in relation to militalY veterans and combat related PTSD. In the third essay, the conceptualisation of parasuicidal behaviours and how this is approached in Dialectical Behavioural Therapy was explored. The three publications were written in my second year of training. The first explains a counseling psychology approach towards combat related PTSD. The second is a report on a conference I attended titled 'DSM: The HistOlY, TheOlY and Politics of Diagnosis'. Both were published in the Wessex Psychological Bulletin. The third is a copy of the essay, which explored psychodynamic approach to combat related PTSD. This essay won the Division of Counselling Psychology RUlmer up for Annual Trainee prize 2013 and was subsequently published in the Counselling Psychology Review. The Therapeutic Practice Dossier includes a brief summmy of all four of my clinical placement and the final clinical paper. This includes the setting, brief summmy of client work, and other placement activities. My therapeutic practice was assessed through in-depth client studies and process repOlis, log books and supervisors' reports. To preserve the anonymity of the placement settings and the clients presented in the in-depth client studies and process repOlis; these documents were included in the attachment and appendix for the pemsal of examiners. Also included in this dossier is the final clinical paper, which explicates my process in becoming a counselling psychologist. This paper draws from and integrates my personal therapy, research and clinical development. The Research Dos'sier includes three research repOlis and tlu'ee conference presentations. Drawing from the NICE (2005) guidelines for PTSD, the literature review aimed to provide a comprehensive review of the various psychotherapies that are available. The second research project used Qualitative Content Analysis to examine practitioners' understanding of trauma work with militalY veterans. The third research project used Interpretative Phenomenological Analysis to examine military veterans' experience of therapy.

Anxiety disorders and social anxiety disorder in particular, are common in childhood and associated with negative short and long term consequences. Psychophysiological theories suggest that anxiety disorders are associated with chronic dysregulation of the autonomic nervous system, as indexed by respiratory sinus arrhythmia (RSA) and heart rate (HR). Findings to date regarding autonomic arousal at rest and in response to stress, among children with (social) anxiety disorder(s) have been inconsistent however, and/or subject to a range of methodological limitations. The first study presented in this thesis aimed to investigate whether children with social anxiety disorder, other anxiety disorders and nonanxious children differ in terms of their autonomic arousal at rest and in response to stress, taking account of various methodological shortcomings of previous studies. Results showed limited evidence for autonomic dysregulation in anxious versus nonanxious children. These findings were contrary to expectations on the basis of psychophysiological models. This may suggest however, that it is how children interpret their physical symptoms that is particularly important in relation to childhood anxiety disorders, and social anxiety disorder in particular. Past research has suggested that negative interpretation of physical symptoms and/or inferring threat from a situation on the basis of one's own physical experience, is associated with high levels of anxiety in children. Furthermore, theories of social anxiety disorder suggest that socially anxious individuals in particular interpret their internal physical symptoms in a negative way which influences their view of themselves as a social object. The second study presented in this thesis investigated these hypotheses among children with social anxiety disorder, other anxiety disorders and nonanxious children, again taking into account methodological limitations of previous studies. Children with social anxiety disorder were more likely than both other groups to assume that physical symptoms are associated with negative consequences, and to view ambiguous situations as anxiety provoking, whether physical information was present or not. The findings highlight that cognitive characteristics may be particular pertinent in the context of social anxiety disorder in childhood, and may be a potential target for treatment.

Identification of bacterial pathogens in paediatric and neonatal bloodstream Infection by blood culture is associated with significant delays. Rapid species level diagnosis in bloodstream infection may allow early introduction of targeted antibiotic therapy, and improve outcomes. Molecular tests can detect bacterial DNA directly from blood and are rapid, but not sufficiently sensitive for adoption as routine tests. Aims: The aim of the project was to test the hypothesis that a shortened culture step prior to molecular testing would improve the sensitivity of molecular testing, and reduce the time to species-level diagnosis in neonatal and paediatric bloodstream infection. Methods: A panel of real time polymerase chain reaction assays was assembled, specifically targeting the common bacterial pathogens in neonatal sepsis and neutropenic sepsis. A novel method incorporating whole genome analysis was developed to identify target sequences for bacterial speciation, and an assay detecting Enterobacter cloacae designed and validated. A suitable DNA extraction protocol was identified. In a group of 88 episodes of suspected sepsis, in preterm infants and neutropenic children, clinical samples were cultured in blood culture media for 6 hours before DNA extraction and molecular testing. The results were compared with matched blood culture samples. Results: 11 out of 88 blood culture samples were positive by blood culture, and the qPCR panel detected five of these. The remaining six isolates were coagulase-negative staphylococci. The culture-molecular approach adopted here had an overall sensitivity of 45% (17 -76%) and a specificity of 97% (91- 99%). The processing time for the molecular test was 9 hours in total. Conclusion : The incorporation of a culture step into a molecular testing strategy did not improve sensitivity of molecular tests in children, but the panel developed was highly specific. Further refinement of this approach may impact favourably on diagnostic accuracy.

The consensus is that children born following ART are delivered earlier and at a lower birthweight that those naturally conceived and have a higher likelihood of requiring admission to a neonatal intensive care unit (NICU). The longer term prospects for artificially conceived children seem comparable to the naturally conceived population for the majority of medical conditions. This study examined NICU admission registry data of children born 111/2001 to 31/12/2007. Odds ratios (OR) for NICU admission were produced. All paediatric hospital admissions in Northern Ireland 1/7/1996 through 3016/2009 were also studied. Standardised admission ratios (SAR's) for children up to 13 years old born following ART were calculated. Unadjusted OR for NICU admission was 0.92 (95% CI 0.71, 1.18) for singleton~ and 0.83 (95% CI 0.69, 0.99) for twins. Adjustment for hospital, gestation and year showed OR for NICU admission to be 0.68 (95% CI 0.49, 0.93) for singletons and 0.76 (95% CI 0.60, 0.97) for twins. SAR for all- and first hospital admissions in ART children were 80 (95% CI 76, 84) and 74 (95% CI 68, 80) respectively. Children born fOllowing IVF had increased all-hospital SAR compared to those born following ICSI (SAR 85, 95% CI 80, 90 vs. SAR 71,95% C164, 77; p=0.001). Those born following fresh embryo had increased all-hospital SAR compared to those born fallowing frozen embryo transfer. All-admission analysis showed Significant increases in SAR with diagnoses of cerebral palsy and inguinal hernia. Non-significant trend for increased SAR for all-hospital admission and first-hospital admission was found for some diagnoses. This study adds some reassurance to the neonatal and long-term child health of those born following ART. IVF children and those born following fresh embryo transfer may not be as healthy throughout childhood as their ICSI and frozen embryo transfer peers. Published concerns regarding neurological conditions and congenital abnormalities in children conceived using ART are supported by this study.

The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are inherited neurodegenerative diseases of children. In all types of NCL glial activation, the innate immune response of the brain, precedes neurodegeneration. However, it is unclear whether adaptive immune responses are also involved in these diseases, or if they directly contribute to disease pathogenesis. Therefore, we examined the role of adaptive immune cells in mouse models of Infantile NCL (Ppt1-/- mice) and Juvenile NCL (Cln3-/- mice) by identifying, localising, and subsequently genetically removing immune components. Characterising the adaptive immune response within Ppt1-/- and Cln3-/- mice, we revealed evidence for progressive CNS infiltration by different classes of T-cells in both forms of NCL. To analyse the pathogenic impact of these lymphocytes, we crossbred PptP/- mutants with mice deficient in Rag-1, which lack T- and B-lymphocytes. Disease progression was significantly delayed in immune deficient PptP/ /Rag-1-/- mice, which displayed ameliorated neuroinflammation and neuron loss. We also crossed our Infantile and Juvenile NCL mouse models with mice deficient in sialoadhesin (Sn), a binding protein found on macrophages and microglia, and is involved in pro-inflammatory T-cell regulation. Disease progression was also slowed down, with significantly increased neuron survival in both Sn deficient NCL mice. However, contrasting effects on glial activation were observed between Ppt1~/-/ ’ Sn~/-and Cln3-/-/Sn-/- mice. Whereas glial activation was only marginally attenuated in Cln3-/-/Sir/- mice, significantly decreased microglial activation and enhanced astrocytosis were observed in Ppt1-/-/Str/- mice. These latter findings suggest an unexpected link between macrophage-expressed Sn and astrocytosis in Taken together, our findings prove that adaptive immune cells and sialoadhesin each appear to contribute to disease progression in Infantile and Juvenile NCL mice. Therefore, both immune components could prove to be suitable therapeutic targets for these fatal disorders.