Research has demonstrated that the human brain undergoes significant change in both structure and function during adolescence, but little is known about the role of puberty in this developmental process. The aim of this thesis is to investigate the relationship between puberty and brain development during adolescence. The first two chapters of this thesis summarise the current understanding of the behavioural and brain changes associated with both adolescence and puberty, and review the methods employed to assess puberty in research. Chapters 3 and 4 focus on the relationship between puberty and changes in brain structure. In Chapter 3, the influence of puberty on subcortical structural development is investigated in a large longitudinal MRI dataset, using a mixed effects modelling analysis method. Chapter 4 investigates the relationship between pubertal status, as measured by physical pubertal stage and levels of salivary sex steroid hormones, and white matter structural development in a cross-sectional sample of 12-16 year old boys, using diffusion tensor imaging. In Chapters 5-7, functional brain changes with puberty are explored. Chapters 5 and 6 focus on social emotion processing, where social emotions (e.g. embarrassment) are defined as emotions that require an awareness of other people’s mental states, while basic emotions (e.g. fear) are those which do not. In Chapter 5, the neural correlates of social and basic emotion processing are investigated in relation to pubertal status. In Chapter 6, the fMRI data are reanalysed using psycho-physiological interaction (PPI) analysis to investigate puberty-related changes in functional connectivity during the same task. Chapter 7 explores, in males, how developmental changes in brain function when performing a risk-taking task are related to puberty, independently of chronological age. Finally, in Chapter 8, the results of the empirical studies are summarised and the findings and implications of the thesis are discussed.

Congenital hyperinsulinism (CHI) is the commonest cause of recurrent and persistent hypoglycaemia in infants. Histologically there are two distinct types: focal and diffuse. The medical therapy for diffuse CHI involves diazoxide, glucagon and octreotide. The patients who are unresponsive to medical therapy require a near total pancreatectomy. However, this often fails to provide the best outcome as some patients continue to have recurrent hypoglycaemia whilst others develop diabetes mellitus in the long term. This study aimed to understand the gene expression pattern in the pancreatic tissues of the patients with diffuse CHI so as to identify novel mechanism(s) and therapeutic options. Gene expression microarray using RNA extracted from fresh frozen pancreatic tissue samples (obtained from children who underwent pancreatectomy) revealed significant overexpression of mammalian target of rapamycin (mTOR) and insulin-like growth factors in the diffuse CHI patients in comparison with normal controls. Immunostaining suggested an activation of mTOR pathway (which regulates cellular proliferation) in diffuse CHI and transdifferentiation of exocrine pancreatic elements into insulin producing cells, contributing to β-cell hyperplasia. Further clinical study on the role of the mTOR inhibitor sirolimus in patients with medically unresponsive diffuse CHI (who would have required pancreatectomy otherwise) revealed a good glycaemic response to sirolimus and the infants were able to come off intravenous fluids, glucagon and octreotide infusions, thereby preventing the need for a major surgery. Subsequent follow up to 12 months revealed that the patients were normoglycaemic on sirolimus therapy without any major side effects. Hence treatment with mTOR inhibitors offers an alternative therapeutic option for the patients with severe forms of diffuse CHI. Further studies involving larger group of patients to assess the long term safety and efficacy of mTOR inhibitors in the management of diffuse CHI are needed.

Research indicates inconsistent associations between early attachment patterns and later developmental outcomes. In light of these discrepancies, this study aimed to further investigate the matter by considering both mothers' attachment representations and early infant-mother attachment patterns, as antecedents of children's behaviour problems in early childhood, preadolescence, and adolescence. Specifically, this study tests the hypothesis that children's psychosocial development may be better predicted by mothers' attachment representations, assuming stability in this variable to be greater, than infant-mother attachment patterns. The study consisted of a sample of 51 children and their mothers. Child-mother attachment patterns were measured by the Strange Situation procedure at 12 months and by the MacArthur Coding System at 5 years of age. Maternal attachment representations were evaluated by the Adult Attachment Interview when mothers were pregnant (in their last trimester) with their first-born child, and five years after. Children's mental health was assessed by maternal reports (Child Behavior Checklist) at 5 and 11 years, and by maternal and children's reports (Child Behavior Checklist and Youth Self-Report) at 16 years. The results indicated that mother-infant attachment patterns were not stable from infancy to the preschool years. However, high stability of mothers' adult attachment representations was found. In prospective analyses, while children's attachment patterns were not predictive of later outcome, mothers' dismissing attachment representations were associated with children's externalising difficulties at 16 years. The results are discussed in relation to developmental challenges to parenting posed by the transition to adolescence and the possible connections to attachment representations. Limitations and suggestions for future research are also discussed.

Advances in pharmacological and interventional strategies for the treatment of ischaemic heart disease and acute myocardial infarction (MI), have paradoxically increased the number of patients living with heart failure. Re-vascularisation of the infarcted myocardium can improve cardiac repair; and thus minimise morbidity and mortality rates in heart failure patients. While a number of re-vascularisation strategies are currently being explored, a major hindrance to successful neovascularisation has been concomitant smooth muscle (SM) support. The epicardium, the epithelial tissue that surrounds the heart, is a developmental source of SM cells (SMCs). Previous work in our lab demonstrated a role for Thymosin Beta 4 (Tβ4) in epicardial activation and systemic SM progenitor differentiation. The aim of this project was to investigate the role of Tβ4 in the endogenous response to MI. I hypothesised that endogenous Tβ4 recapitulates its role during development and activates the adult epicardium post-MI, providing SM support for new vasculature. My data provide evidence that Tβ4 does indeed have a role in the endogenous response post-MI. Tβ4 was found to be upregulated post-MI in the epicardium, and Tβ4 deficiency resulted in a significant reduction in epicardial activation and SM contribution to neovascularisation. A yeast-two-hybrid screen identified Low density lipoprotein receptor-related protein 1 (LRP1) as a potential Tβ4 binding partner. Evidence is further provided of epicardial expression of LRP1 and of its role in epicardial function and neovascularisation during development. Further studies are required to confirm a direct interaction between Tβ4 and LRP1 and to elucidate the precise mechanisms through with the proposed Tβ4-LRP1 interaction may impact epicardial activation and SM differentiation and together contribute to myocardial revascularisation and repair.

During development, cells of the epicardium undergo epithelial-mesenchymal transition (EMT) to generate multipotent progenitor cells (epicardium-derived cells, EPDCs) that contribute to multiple lineages of the cardiovascular system. Critical to this process is the epicardially-expressed Wilms’ tumour 1 (Wt1) gene. In the adult, the epicardium is largely quiescent and no longer expresses Wt1. Upon myocardial infarction (MI), however, the adult epicardium is reactivated; it re-expresses Wt1, reverts to its embryonic potency, and contributes to cardiac repair. Previous work showed that this reactivation is enhanced by ‘priming’ mice with the G-actin-binding protein thymosin β4 (Tβ4), although the molecular mechanisms were not defined. Here we investigated Wt1 regulation in the epicardium during development and post-MI and found Wt1 to be regulated by the SWI/SNF chromatin remodelling complex in combination with Tβ4. During development, Wt1-expressing cells in the epicardium expressed the SWI/SNF ATPases, Brg1 and Brm. Both Wt1 and SWI/SNF were largely absent from the uninjured adult epicardium, but the re-expression of Wt1 post-MI was accompanied by substantial upregulation of both Brg1 and Brm. Exogenous Tβ4 in the form of priming increased the post-MI expression of Wt1, Brg1 and Brm, whilst endogenous Tβ4 was found to be required for maximal Wt1 reactivation. Furthermore, we found that Tβ4 physically interacts with the SWI/SNF complex and that it potentiates SWI/SNF-mediated activation of specific regulatory regions of the Wt1 promoter and intron 1, suggesting that this interaction is functional. Loss of Brg1 in the epicardium using Gata5Cre;Brg1flox embryos showed that Brg1 is not required for Wt1 expression, which we postulate is due to compensation from Brm, whilst loss of Brg1 in Wt1-expressing lineages (using the tamoxifen-inducible Wt1CreERT2) caused no obvious phenotype. Further understanding of the mechanisms underlying Wt1 expression, during both development and epicardial reactivation post-MI, is essential in our quest to utilise resident-cell-based therapy in cardiac repair.

Globally, the population of vertically HIV-infected young adults is increasing. The effect of vertically acquired HIV infection on B cell development and adaptive immunity is relatively unexplored. HIV infection is known to result in perturbations in B cell turnover and signalling, reflecting an accelerated drive to terminal differentiation. Whilst control of HIV load with ART is generally reported to result in recovery of normal B cell dynamics, persistent damage to memory B cell populations is well described and generation of new memory responses may remain impaired. This thesis explores how the development of B cell memory and the immune response to pneumococcus is altered when ‘immune education’ in early life occurs in the context of vertically acquired HIV infection. Young adults with vertically acquired infection were compared with those infected horizontally in early adulthood and with healthy adults. HIV infection was associated with expanded populations of abnormally activated and immature B cells compared to healthy controls. Vertically infected patients showed decreased marginal zone and switched memory populations compared to the horizontally infected group, especially in those patients with controlled HIV viral load. HIV-infected patients showed impaired baseline anti-pneumococcal immunity and diminished humoral responses to immunisation with the pneumococcal polysaccharide vaccine, with a trend to lower antibody concentrations in the vertically compared to the horizontally infected population. There was some suggestion of benefit of early sustained viral control in the vertically infected group. In those patients infected with HIV from early childhood, damage to B cell memory populations and impairment of generation of humoral immunity to pneumococcus is evident in early adult life. It appears likely that viral control in early childhood may help to limit this damage.

During an immune response, dendritic cells (DCs) capture and process antigen, upregulate co-stimulatory molecules and migrate to lymphoid organs to maximise antigen recognition by rare T cell clones. A crucial step for successful T cell activation is cell-cell interaction at the immunological synapse (IS), the organised contact interface which allows optimal communication. IS formation requires the dynamic remodelling of the actin cytoskeleton to spatially distribute membrane areas with distinct protein compositions. While evidence exists for a role of the T cell cytoskeleton in this process, the driving force behind the specific organisation on the DC side is unknown. One important actin regulator is Wiskott-Aldrich Syndrome protein (WASp), expressed exclusively in immune cells. Using a murine model, we investigated the role of WASp in DC-mediated actin-dependent synapse formation. Utilising both confocal and electron microscopy (EM), we observed a disorganised, asymmetric interface between T cells and WASp-deficient DCs. Immunofluorescent staining shows reduced polarisation of certain synapse markers in WAS knock-out conjugates; while high resolution 3D EM reconstructions show severely impaired cell spreading and a reduced contact area. We have used fluorescence recovery after photobleaching (FRAP) to show reduced stability of the actin network in WASp DCs. Our experiments on supported planar bilayers have shown an unexpected interface organisation and highlight a novel role of the DC cytoskeleton in this process. Crucially, we have demonstrated reduced T cell activating capacity of WASp-deficient DCs, as measured by T cell proliferation, cytokine production and transcription factor expression. The results highlight the critical role of cytoskeletal regulation in DC-mediated IS formation. Further, a second murine immunodeficiency model, Dock8 deficiency, and two novel human primary immunodeficiencies were briefly investigated to examine the role of other actin regulators in normal DC function.

Primary ciliary dyskinesia is a genetic disorder where patients develop lung disease as they are unable to clear airway infections effectively. There is currently no treatment for the underlying genetic defect. This thesis describes advances towards the development of a gene therapy targeting the airway disease in PCD patients with DNAH5 mutations, the most common cause of PCD. Little work has been done in the field so many challenges remain. DNAH5 is a large gene and the cDNA has not been cloned. In addition PCD models are currently inadequate. Finally, non-viral vectors for airway gene delivery produce sub-optimal levels of transgene expression and more efficient viral vectors cannot package the large DNAH5 cDNA. To address these issues functional DNAH5 cDNA was cloned from healthy airway cells and the cDNA validated by sequencing and expression studies. Lentiviral shRNA transduction of healthy cells was used to knock down DNAH5 in airway cells semi-immortalised with BMI-1, a proto-oncogene that allows extended growth capacity of cells whilst retaining their mucociliary differentiation potential. The novel cell lines lacked cilia motility as is seen in patient cells. Minicircle DNA, in the context of airway gene delivery, was found to enhance gene expression in vitro and in vivo. A non-viral vector was optimised, characterised and used to deliver DNAH5 coding minicircle DNA to PCD models but poor transfection efficiency of DNAH5 prevented functional correction. Transfection of smaller genes was efficient so the vector in its current form could be useful for gene therapy treatment of the majority of PCD causing genes. Studies are needed to determine and overcome the bottlenecks in the efficient transfection of large transgenes to help advance PCD gene therapy.

γδ T cells are a rare subset of T cells present in human blood and lymphoid tissues. Functionally, they stand at the interface of the innate and the acquired branch of the immune system as they express somatically rearranged antigen receptors but they also share many characteristics with NK cells. One of those is the ability to express CD16. CD16 is a cell-surface receptor that binds the Fc portion of IgG. In the isoform found on tissue macrophages and NK cells it is coupled to an intracellular signalling domain that allows it to initiate cellular processes leading to the internalisation of IgG-coated particles or cytotoxicity against IgG-coated cells. Therefore, it has a role in the clearance of IgG-coated complexes and the destruction of infected or malignant cells. In this thesis, I show that a population of γδ T cells present in human blood from healthy donors express the CD16 receptor and implicate a role for this receptor in phagocytosis of IgG-coated bacteria and synthetic beads coated with recombinant influenza M1 protein. These cells were subsequently shown to activate influenza-specific T cells through MHC II presentation. Importantly, neither αβ T cells nor NK cells were capable of significant phagocytosis. In addition, once CD11c+ cells had been removed from the NK fraction, neither αβ T cells or NK cells were capable of MHC II antigen presentation of influenza M1 antigen. The CD16+ γδ T cells displayed a phenotype of effector memory and late effector memory T cells suggesting a role for these cells in patrolling the blood and rapidly migrating to sites of inflammation for a combination of rapid effector and antigen presentation function.

The lymphatic vasculature is a blind-ended network covering most tissues and organs of the body, crucial for tissue fluid homeostasis, immune surveillance and lipid adsorption from the gut. Recent evidence has proposed an entirely venous-derived mammalian lymphatic system. The major focus in the field has been on the systemic lymphatic vasculature, whilst organ-based lymphatics have been largely overlooked. In particular, the cardiac lymphatic vasculature has not been studied in great detail and the cellular origin of these vessels is yet to be determined. In contrast to the current dogma, the work presented in this thesis revealed that cardiac lymphatic vessels have a heterogeneous cellular origin. By utilizing multiple Cre transgenic mouse lines for lineage tracing studies and conditional Prox1 and Vegfr3 knockout mice, the data showed that the cardiac lymphatic vasculature is derived from both the blood and hemogenic endothelium during development. We hypothesized that the mechanisms underlying the development of the cardiac lymphatics might be reactivated following adult heart injury, therefore the innate cellular and molecular response of the cardiac lymphatic system to myocardial infarction (‘heart attack’) was investigated. A significant upregulation of lymphatic genes as well as a physical expansion of the lymphatic network via lymphangiogenesis was observed. These results prompt the re-evaluation of a century-long debate on the origin of lymphatic vessels and emphasize the significance of focusing on organ-based lymphatics during development and under pathological conditions. Specifically in the heart, this will enable better understanding of how to modulate the innate inflammatory response to promote cardiac wound healing and repair following myocardial infarction.