Behavioural emergencies due to high-risk behaviours are increasing globally in paediatric health care settings and place the child, their family, other inpatients and staff at high-risk of harm. High-risk behaviours have been reported in up to two thirds of children with autism spectrum disorder (ASD) and intellectual disability (ID). Admission to hospital can trigger high-risk behaviours in children, especially those with ASD or ID, and increase their intensity or frequency. Patient and staff safety can be compromised if staff are not confident or competent in managing aggressive outbursts. As well as the safety risks, these behaviours result in delayed treatment, prolonged procedure times, increased health care costs and poorer health outcomes. Having a critical mass of well-trained staff who are confident in preventing and managing behavioural emergencies is important. However, these skills may be beyond the usual scope of practice of the acute paediatric health care provider. Paediatric acute health care is a complex specialty encompassing children with a wide range of developmental levels who operate within an array of unique family-centred care systems. Staff need many and varied skills and strategies that can be adapted to effectively interact with the spectrum of ages, developmental stages, neurodiversity and parental/carer involvement experienced in the acute paediatric setting. Simulation-based education (SBE) has been found to be superior to traditional clinical medical education for clinical skill acquisition and is gaining acceptance as a training method for teaching skills in managing clinical aggression. Simulation training allows practice of skills without patient risk and may be more effective than traditional teaching formats for aggression management. The aim of this thesis is to better understand the cause of behavioural emergencies caused by children in hospital and to use this knowledge to develop, implement and evaluate a simulation-based training program for acute care paediatric staff on managing aggression in children with ASD or ID. The program of research was established with four objectives linked to four independent studies, each informing the next. First, we conducted a systematic review to assess the effectiveness of simulation-based training for increasing de-escalation knowledge, skills and behaviour of staff working in the acute care setting. Second, we conducted a one-year retrospective study of behavioural emergencies that triggered an emergency response team attendance in a quaternary paediatric hospital to understand their frequency, type, context and management. Third, we designed a simulation-based education session about prevention and management of aggression in a neurotypical adolescent. We used a proof-of-concept study design to evaluate the acceptability of this training and to gain an understanding of the impact on participants’ self-perceived confidence levels in managing clinical aggression. Fourth, we designed a more specific simulation training program about management of aggression and high-risk behaviours in adolescents with ASD and ID in the hospital setting. Our final study was a pilot and feasibility cluster randomised controlled trial (RCT) of this simulation-based education, with mixed method design. Understanding aggression and high-risk behaviours in paediatric health care settings is an emerging area of research. The findings of these four studies have increased our understanding of the prevention and management of aggression and high-risk behaviours in acute care paediatric settings. Simulation-based education could become an important part of a multi-faceted strategy that is now needed to reduce the prevalence of high-risk behaviours in acute care settings and paediatric hospitals, and to improve outcomes if behavioural emergencies occur. Further evaluation of this training format for managing high-risk behaviours (including aggression), in children with ASD and ID is required. As well as simulation education, features of provider and patient systems need to be considered when formulating solutions to this complex issue in the paediatric acute care setting.

Cystic fibrosis (CF) is a multisystem disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Lung disease which is the major source of morbidity and mortality in CF, begins in infancy and persists and progresses in the first years of life. The most sensitive measure of CF lung disease is computed tomography (CT) and the majority of children show evidence of bronchiectasis, which represents irreversible structural lung disease, by five years of age. The rate with which lung disease develops and progresses varies greatly between people with CF. Accurate prediction of future lung disease severity in people with CF would facilitate significant improvements in clinical care, as well as provide helpful information to people with CF and their families, health authorities and CF researchers. It has been demonstrated that CF lung disease severity is determined by a combination of non-CFTR genetic and environmental factors. However, no robust predictive biomarkers have been developed. Epigenetic mechanisms are those which regulate gene expression but do not alter the DNA sequence itself. DNA methylation is the most widely studied epigenetic mechanism, and an individual’s DNA methylation profile is determined by their underlying genotype and environmental exposures (including in utero). DNA methylation has been studied as a predictive biomarker in other childhood diseases, and given it is determined by the same factors which determine CF lung disease severity, is well positioned to act as a biomarker in CF. Despite this, DNA methylation based biomarkers of future lung disease severity have not been explored in CF. The thesis presents a series of projects which aimed to identify DNA methylation based biomarkers of future CF lung disease severity. The projects in this thesis utilised biospecimens and clinical data from the world leading Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) cohort. In order to better understand previous attempts at identifying predictive biomarkers, Chapter Two and Three describe reviews of the evidence regarding non-CFTR genetic modifiers of CF lung disease and DNA methylation based biomarkers of future health outcomes in children. The reviews highlight the importance of appropriate study design when trying to identify predictive biomarkers, which then informed design of the subsequent epigenome wide association study (EWAS). Chapters Four and Five describe the development of methods that were necessary for subsequent EWAS. In Chapter Four protocols for cryopreservation, and flow cytometry based phenotyping and sorting of specific cell populations were developed and validated for use with paediatric bronchoalveolar lavage (BAL). These methods were then used in Chapter Five to obtain purified populations of the common cell types in BAL, which were then used to develop reference epigenomes for these cell types. These reference epigenomes allowed adjustment for cell composition in the subsequent EWAS, which is a crucial step in DNA methylation studies involving samples with multiple cell types. Chapters Six and Seven describe the EWAS studies that attempted to identify predictive biomarkers. Chapter Six involved genome wide DNA methylation profiling of BAL collected at six years of age, and assessed the ability to predict the presence or absence of bronchiectasis at nine years of age. No predictive biomarkers were identified. Chapter Seven involved genome wide DNA methylation profiling of BAL collected at one year of age, and assessed the ability to predict the presence or absence of bronchiectasis at five years of age. Seven predictive biomarkers were identified, which when assessed using area under the receiver operator curve analysis had extremely good performance as predictive biomarkers. Several of the predictive biomarkers were related to genes that are relevant to CF lung disease pathophysiology and hence may represent therapeutic targets. By following best practice for EWAS, and in particular using tissue specific samples, adjusting for cell composition and assessing outcome using the gold standard measure, this thesis succeeded in identifying DNA methylation based biomarkers of future lung disease severity in CF. If these biomarkers are validated in external cohorts they could dramatically improve the care of children with CF around the world. N.B. There are two files that have been submitted separate to this thesis document as supplementary files. They have been highlighted as appropriate in the thesis. In addition, the code used for statistical analysis is available at: https://atlassian.petermac.org.au/bitbucket/pages/OS/paed-cf- methylation/master/browse/docs/index.html

Abstract Introduction Kidney transplantation is the treatment of choice for eligible individuals with end-stage kidney disease, conferring superior quality and quantity of life compared to remaining on dialysis. However, transplant outcome for many patients remains suboptimal, with graft life years falling substantially short of the recipient’s life expectancy due to graft rejection and failure. At the same time, typical immunosuppressant doses to control rejection lead to cumulative morbidity and premature mortality from side-effects such as infections, malignancy, and cardiovascular disease. Objectives The overall objective of the research in this dissertation was to describe the clinical practice of immunosuppression in kidney transplantation and explore an approach to improve dosing to optimise safety and effectiveness. The thesis used time-to-event and pharmacoepidemiologic analysis of a large volume registry data set; systematic review and critical re-appraisal of pharmacokinetic-pharmacodynamic (PKPD) and randomised controlled trials (RCT) of immunosuppressants; and an investigator initiated, Australian multi-centre pharmacokinetic study with population pharmacokinetic modelling. 1. Optimal dose and dosing strategy of immunosuppressant agents over the long term have not been conclusively established. Using over 5 decades of immunosuppressant dosing and kidney transplant outcome data from the Australia and New Zealand Dialysis and Transplant Registry (ANZDATA) a time-to-event analysis and pharmacoepidemiologic data exploration was performed. This was used to describe immunosuppressant regimen and dose size evolution over time, and association with outcome (Chapter 3). 2. Mycophenolate mofetil (MMF) is used in the majority of kidney transplant recipients, yet there is conflicting opinion on whether dose should be individualised, along with substantial practice variability and ongoing prevalence of inefficacy and immunosuppressant toxicities. Through a comprehensive systematic review of both PKPD and RCT evidence the case for concentration-controlled dosing (CCD) of MMF was critically re-appraised, to refine understanding and guide best practice (Chapter 4). 3. Initial underexposure to mycophenolic acid (MPA, active metabolite of MMF) is seen in up to a third of kidney transplant recipients thus increasing risk of acute rejection. An investigator-initiated, Australian multi-centre pharmacokinetic (PK) study (ADOPT) was undertaken to describe the change in PK of total and unbound MPA in the pre-transplant and early post-transplant period. This was used to assess the ability of pre-transplant drug concentrations to predict post-transplant individual MMF dose requirement (Chapters 5 and 6), and to understand the change in total and unbound MPA drug concentrations in the early post-transplant weeks (Chapter 5). 4. Optimising MPA exposure is a practical burden in the initial phase post-transplant. Population PK (PopPK) modelling of the ADOPT data was performed to provide a deeper understanding of changing MPA PK peri-transplant and in the initial post-transplant weeks, in order to (a) re-assess ability to predict post-transplant MMF dose requirement after controlling for changing covariate factors and (b) understand the pattern of change in unbound MPA PK over the initial post-transplant weeks and (c) evaluate the potential value of Bayesian dosing to achieve an unbound MPA concentration target (Chapter 6). Key findings - There have been 4 key immunosuppressant regimen eras, with successive improvement in patient and graft outcomes by era. Multivariate analysis revealed more modest improvements in the maintenance phase in the period 2007 - 2015 compared with 1998 - 2006. Dose size for prednisolone and tacrolimus has decreased over the entirety of these 2 periods (1998 – 2015), in both initial and maintenance phases, with possible over-reduction of tacrolimus in the maintenance phase. The initial dose of MMF (combined with tacrolimus) has increased then decreased over this period. - The pharmacokinetic-pharmacodynamic (PKPD) and RCT evidence clearly favours CCD of MMF using Target Concentration Intervention. The contradictory RCT data can be objectively explained by accounting for the use of Therapeutic Drug Monitoring compared with Target Concentration Intervention CCD. - Pre-transplant concentrations were unable to improve prediction of post-transplant individual MMF dose requirement using the trapezoidal exposure calculation technique. Using a PopPK model and accounting for changing renal function, however, pre-transplant concentrations provided some information on post-transplant dose requirement. - There is a substantial increase in clearance of unbound mycophenolic acid (MPA, active metabolite of MMF) from pre- to post-transplant. Unbound MPA exposure stabilises by 2 weeks post-transplant, making it a more practical measure for CCD. A PopPK model has been developed that could be used for Bayesian dosing to an unbound MPA target.

Infection and inflammation are critical in the pathogenesis of cystic fibrosis (CF) lung disease. Detection of pathogenic bacteria and neutrophilic inflammation in the lower airways of infants with CF is associated with the earlier development of bronchiectasis and lower lung function at six years of age. The role of the airway microbiota, its bacterial community network, in the evolution of CF lung disease is not known, nor are the long-term sequelae of early life lower airway infection and inflammation. This thesis addresses these questions through analysis of a birth cohort of 100 infants, newly diagnosed with CF, predominantly by newborn screening, in Victoria, Australia, in the early 1990s, and managed in a single specialist CF centre where anti-staphylococcal prophylaxis was not prescribed. Participants in this birth cohort underwent serial bronchoscopy and bronchoalveolar lavage (BAL) from recruitment in infancy and semi-annually thereafter during early childhood. Quantitative microbiological culture was performed and inflammatory markers measured contemporaneously. 16S ribosomal RNA gene sequencing was subsequently performed on stored samples to describe the lower airway microbiota. Comparison of the lower airway microbiota of 13 newly diagnosed infants with CF to that of disease-controls, infants undergoing clinically indicated bronchoscopy but without evidence of respiratory infection, revealed profound differences in the CF airway microbiota within the first six months of life, driven by increased Staphylococcus and more pronounced in unwell infants. Longitudinal analysis of 95 BAL samples from 48 infants with CF, aged 6 weeks to 6.5 years, demonstrated a diverse and dynamic lower airway microbiota, in which reduced diversity was associated with dominance of proinflammatory pathogens and increased airway inflammation. Twenty-five year longitudinal follow-up of 79 of the 100 original birth cohort members (45.6% male, 67.1% P.Phe508del homozygous) was conducted to explore the associations between early lower airway infection with Staphylococcus aureus and Pseudomonas aeruginosa, as well as neutrophilic inflammation, and survival, rate of decline of pulmonary function and severity of structural lung disease in adulthood. Survival analyses were conducted using Kaplan Meier curves and Cox proportional hazard models. Annual rate of decline of forced expiratory volume in one second, percent predicted, calculated according to the Global Lung Initiative reference equations, was estimated using a linear mixed-effects model with random intercept and slope, and severity of structural lung disease was quantified using the CF-CT scoring system. There was no association between lower airway infection with S. aureus in the first two years of life and 25-year survival or development of functional or structural lung disease. Infection with the AUST-01 P. aeruginosa strain in the first five years of life was associated with increased mortality, while infection with other P. aeruginosa strains was associated with a trend towards reduced survival and poorer longitudinal lung function. Neutrophilic inflammation in the first two years was not associated with poorer long-term outcomes. Together, these findings highlight the need for careful evaluation of current approaches to anti-staphylococcal prophylaxis in newly diagnosed infants with CF, while suggesting a role for optimisation of the early lower airway microbiota.

Focal Cortical Dysplasia (FCD) is one of the most common brain malformations and a frequent cause of intractable epilepsy. Affected individuals often require surgical removal of the affected cortex for seizure control. The European Epilepsy Brain Bank estimates that different subtypes of FCD are the reason for up to 70.6% of epilepsy surgery related to brain malformations. Given the clinical importance of this disorder, there is an urgent need to further understand its biology and disease mechanism. Although FCD was first described 50 years ago, the genetic and pathogenic mechanisms of the disorder remain incompletely understood. We hypothesise that the delineation of disease mechanism underlying FCD will not only improve diagnosis and clinical management, but will also provide novel insights into the mechanisms underpinning human brain development and function. This project applied modern genomic technologies to comprehensively study a unique archive of surgical brain tissues derived from individuals affected by FCD. The first part of this study focussed on understanding the genetic basis of a subtype of FCD, known as bottom-of-sulcus dysplasia (BOSD). BOSD is a highly localised subtype of FCD with the dysplastic features being maximal at the bottom of sulcus. Using deep sequencing, we analysed blood and brain derived gDNA from 20 BOSD brain specimens. We found that brain-specific, pathogenic somatic MTOR variants accounted for 50% of the cases, with an additional two cases caused by heterozygous germline variant in DEPDC5 or NPRL3. These genes are all involved in the mTOR signalling pathway. Further characterisation of four somatic MTOR cases found that the somatic variants were more abundant in the bottom of sulcus compared to the nearby cortex, with a positive correlation with the severity of histopathology. Our study shows that BOSD belongs to an expanding group of “mTORopathies” and highlights that BOSD lesion is highly localised, with well-defined genetic and pathologic substrates. In the second part of this study, we tested the germline + somatic “two-hit” hypothesis in FCD. The “two-hit” hypothesis suggests that individuals with heterozygous germline variant acquired FCD lesion as a result of a somatic second “hit”. In a case study of an individual with FCD and a heterozygous germline DEPDC5 variant, we identified a somatic second “hit” in the same gene. By combining experimental and clinical findings, we showed in this individual that the brain region with the highest somatic variant allele frequency was also the region of seizure onset, suggesting a relationship between the somatic variant and epileptogenicity. We further characterised the brain specimens and found that somatic variant allele frequency positively correlated to the density of dysmorphic neurons. Furthermore, laser capture microdissection showed that the somatic variant was only detected in the dysmorphic neurons and not in the normal neurons. Collectively, we provided supporting evidence for the “two-hit” mechanism and demonstrated the molecular and cellular characteristics of FCD lesion. The third part of this study involved the application of single nucleus RNA-sequencing (snRNA-seq) to study 26 surgical brain specimens resected due to a range of brain malformations including FCD. These brain malformations are closely related and commonly characterised by the presence of abnormal cell types i.e. dysmorphic neurons and balloon cells. We generated 284,891 single nuclei transcriptomes, the largest snRNA-seq dataset for epilepsy brain specimens to date. Using a combination of bioinformatic analysis and molecular characterisation, we identified the probable transcriptional profiles of dysmorphic neurons and balloon cells. Furthermore, we identified commonly perturbed pathways in both cell types, namely calcium signalling pathway and glutamatergic synapse regulation, which may help explain the pathogenic mechanism underlying these different types of closely related brain malformations. In conclusion, this study has provided fundamental insights into the genetic and pathogenic mechanism underlying FCD and related brain malformations. In addition to improving genetic diagnosis and clinical management, these findings have the potential to pave the way for the identification of novel druggable targets by defining the genetic and transcriptomic profiles of FCD.

This thesis represents a compilation of peer-reviewed papers addressing the care for children presenting to emergency departments with bronchiolitis. After an introductory chapter, papers related to one of several themes are briefly introduced and placed in context with each other. The papers, and their compilation in this thesis, aim to provide guidance for paediatricians and emergency physicians caring for children on how to bronchiolitis in hospital, with specific attention paid to hydration methods, reducing low value care and high flow nasal cannula use. Many aspects of this work, including certain research findings, are being used by, or have been implemented in, the Emergency Department at Royal Children’s Hospital and other emergency departments across Victoria, Australia, as well as interstate and overseas. I take primary responsibility for the initial concept, design, protocol development and successful execution of all projects used for this thesis. To my knowledge, no other co-author has presented papers from this compilation for submission as part of a degree. Exceptions are the use of parts of the projects in the fulfilment of Advanced Trainee requirements for the Royal Australasian College of Physicians or the Australasian College for Emergency Medicine, or for advanced medical students in the fulfilment of a Bachelor of Science through the University of Melbourne.

Introduction: Reference intervals (RI) are an important clinical assessment tool used by physicians and laboratory professionals to interpret test results. A RI is commonly defined as a range between the 2.5th and 97.5th percentile and represents the middle 95% of the reference population. The estimation of RIs is a complex process which involves defining a reference population, selecting reference individuals, collecting, and testing reference samples, and applying appropriate statistical methods. Estimation of RIs in children is challenging for a number of reasons. There is a need for adequate representation of the general population in the reference population. Analytes are influenced by the nutrition, development, and growth of a child. Hence, RIs should reflect these age-dependent changes in the analytes as children grow older. Most laboratory tests are instrument dependent and variation between different analysers has been documented in several studies. In addition, the best statistical methods for estimating and reporting RIs varying with continuous age are still not widely agreed upon. Finally, there are no available guidelines for laboratories to validate continuous age-specific RIs for their use. Hence, this project addressed the following four gaps: 1) the lack of appropriate and reliable continuous age-specific paediatric RIs, 2) an absence of head-to-head comparison of laboratory results on different analysers, 3) the lack of clear evidence and accompanying guidelines regarding the best statistical methods for constructing continuous age-specific paediatric RIs, and 4) the lack of guidelines for validating continuous age-specific RIs. Methods: This research project consists of four studies. Data collected as part of the HAPPI Kids study, from children 30 days to < 18 years, were used to compare laboratory test results on different analysers and to establish and validate continuous age-specific RIs. Differences in mean test results of analytes by analyser types were investigated using mixed-effects regression analysis and by comparing maximum variation between analysers with analyte-specific allowable total error reported in the Westgard QC database. Continuous age-specific paediatric RIs were estimated using quantile regression where power variables in age were selected based on fractional polynomial regression for the mean, with modification by sex when appropriate. The continuous age-specific paediatric RIs were considered valid if more than 90% of test results from a routine laboratory were within the intervals. A systematic review was conducted to examine the range of statistical methods used over the past 25 years for the estimation of age-specific RIs and to identify trends in usage and reporting. A simulation study was conducted to evaluate and compare statistical methods for constructing continuous age-specific RIs in children under different scenarios and for different sample sizes. Results: The variation in the mean test results across five analysers was not clinically significant for 24 out of 30 biochemistry analytes examined and common continuous age-specific RIs were established for children 30 days to 18 years. The continuous age-specific paediatric RIs were validated in a routine laboratory after initial analysis for most analytes and after secondary analysis for a few analytes. According to the results of the systematic literature review, a wide variety of statistical methods have been used for estimating age-specific RIs in children over the past 25 years. However, there has been insufficient uptake of modern statistical methods in estimating continuous age-specific paediatric RIs for analytes that change with age. The results of the simulation study show that in comparison to the commonly used methods for estimating continuous age-specific paediatric RIs, the applied novel method that we applied performed better in most scenarios for sample sizes of 400 and over. Discussion: The comparison of test results for common biochemistry analytes should provide evidence of lack of differences between analysers and in turn improve clinical interpretation. It is recommended that continuous age-specific RIs are used as a gold standard for interpreting test results. Laboratories should estimate or validate continuous age-specific paediatric RIs for their use by applying appropriate statistical methods based on the evidence provided in this research.

Congenital anomalies of the pulmonary veins and arteries are rare congenital cardiac defects. Without surgery, mortality during infancy is high with few children surviving to adulthood. Prognosis after surgery is improving with many children now surviving the initial operative period. Our understanding of the outcomes after surgery is evolving as emerging information from longitudinal studies have shed light on longer term survival of these patients. There is now a growing population of these patients surviving into adulthood. This project aimed at expanding our understanding of the prognosis and long-term outcomes of children who underwent surgery for partial and total anomalous pulmonary venous drainage (PAPVD and TAPVD). It has explored the entire spectrum of these patients and shows that excellent long-term survival and freedom from reoperation can be achieved in these patients into early adulthood. To better understand the long-term disease perceptions and impact, we conducted quality of life assessments on adult PAPVD and TAPVD patients. These patients were found to have similar quality of life outcomes compared to age-matched Australian population data. Furthermore, we examined the outcomes of surgery amongst TAPVD patients with a univentricular circulation. This group of patients have been rarely reported in the literature due to the extremely poor prognosis and limited experience even amongst the largest paediatric centres. We showed that these patients have a poor prognosis with high mortality. Future research into improving outcomes amongst these patients are needed. With the aid of a bi-national registry, we have reported a large cohort of TAPVD patients with a univentricular circulation who have reached Fontan completion. This has enabled better understanding of the composition, outcomes and risk factors for mortality of the population. Finally, to complete our study we addressed the long-term outcomes of the rare pulmonary artery sling condition. These patients often present in respiratory failure due to concomitant airway compromise and the need for airway surgery is associated with a higher mortality and morbidity. As part of a multi-centre study, we found that the overall results have improved in patients requiring concomitant airway surgery. Importantly, we found that at late follow-up, many of these patients had evidence of obstructive airway defects on lung function testing. Though currently asymptomatic, the long-term impact on functional status and respiratory function is unknown. As the early survival outcomes of these patients improve, our focus is now shifting towards understanding the long-term prognosis, functional impacts and quality of life. Enhanced knowledge in these areas will allow us to better inform families and develop strategies to, ultimately, improve the lives of the children and young adults with these diseases.

Neonatal infectious diseases result in an estimated 40% of neonatal deaths worldwide and contribute significantly to chronic morbidity. Childhood disease-specific immunisation is irrefutably linked to the decline in deaths from targeted infections over the last century. However, neonatal immunisation strategies are limited, in part, due to impairments in their adaptive immune function. Vaccine-induced protection from severe forms of tuberculosis (TB) with the Bacille Calmette-Guerin vaccine (BCG) and perinatally-acquired hepatitis B infection with the hepatitis B vaccine (HBV) are two exceptions, with these vaccines commonly administered to neonates worldwide. Evidence for heterologous (‘non-specific’) effects (NSEs) of various vaccines used in childhood, most notably for BCG, is increasing. This refers to the immunomodulatory capabilities of vaccines to influence immune outcomes beyond inducing protective immunity against the vaccine’s specific targeted disease. There is limited evidence for these effects in neonates, particularly for HBV. The research reported in this thesis aimed to explore the influence of BCG, HBV and concurrent administration of both vaccines at birth on the neonatal immune responses to unrelated antigens compared with unvaccinated babies in a randomised control trial (RCT): The Early Life Vaccine and Immunity Study (ELVIS). Neonatal blood samples from 128 participants, collected seven days after randomisation, were stimulated with various unrelated antigens for 20 hours. Cytokine responses, measured in the supernatants by an enzyme-linked immunosorbent assay (ELISA) method, were analysed using non-parametric statistical tests to determine differences in median responses between the four groups: BCG alone, HBV alone, concurrent BCG and HBV and the unvaccinated control group. I found minimal differences in the median in vitro cytokine responses to all stimulants between the four groups. However, both vaccines independently influenced cytokine-stimulant responses. Effects on responses were strongest for BCG-vaccinated babies, but only decreased interferon gamma (IFN-gamma) responses to the Toll-like receptor (TLR) ligand resiquimod (R848) and monocyte chemoattractant protein-1 (MCP-1) responses to heat-inactivated E. coli were significantly different from controls. Combined vaccines tended to induce similar cytokine-stimulant response patterns as BCG alone, although for some cytokine-stimulant pairs, the BCG-induced effects were mitigated by HBV and vice versa. This study adds to the evidence for NSEs of vaccines in neonates. It is the first study to investigate the influence of HBV immunisation on immune responses to unrelated antigens, finding no statistically significant differences in median cytokine responses compared with controls. The finding that concurrent vaccination with HBV and BCG at birth induced the same cytokine-stimulant response pattern as BCG alone, suggested that cytokine responses to unrelated stimulants are driven by BCG. This is also the first study to show that in neonates concurrent HBV and BCG vaccination at birth weakens the NSEs of BCG for certain cytokine-stimulant pairs. Further research into neonatal vaccine NSEs are warranted. Future studies should explore and further investigate the clinical relevance of certain cytokine-stimulant response signatures identified in my thesis and the mechanism for these observations in neonates. These results will direct research on how we could potentially exploit any beneficial vaccine NSEs to provide protection against infection in the very young.

In Australia, state-based statutory Child Protection (CP) systems intervene when children are deemed to be at risk of harm from exposure to neglect and/or abuse. On 30th June 2019, 44,906 (8.0 per 1,000) Australian children were subject to court orders placing them with a relative (kinship) or foster carer in an out-of-home care (OOHC) placement. These children have often experienced socio-economic disadvantage in addition to adverse experiences such as neglect or abuse, with resultant high health needs. Australian and international child health experts uniformly recommend that health is routinely assessed upon entry to OOHC. Australia has national standards that stipulate an initial assessment within 30 days and a comprehensive assessment (paediatrician-led, with dental, audiology, and optometry assessments) within three months of entering OOHC. Despite this, Victorian state policy is less clear. Some routine assessments (medical, dental, optical, and auditory) are advised within one month of entry to care. While some areas have access to dedicated multi-disciplinary assessment clinics, there is no state-wide recommendation for routine comprehensive assessment. The majority of Victoria has no model of adequate service provision for health assessments. There are no data that monitor health assessments at either a state or national level, and little is known in Australia about the barriers to healthcare for children in OOHC. This research therefore aimed to determine the extent to which Victorian children entering OOHC were having their health needs assessed and addressed in a timely way, and to identify barriers and enablers to timely health assessment. This was a convergent parallel mixed-methods study with 3 sub-studies: Study 1, an audit of children seen at a multidisciplinary OOHC comprehensive assessment clinic, confirmed high rates of need and identified that only 25% of children attended within timeliness recommendations. Study 2 included a survey of 290 foster and kinship carers and interviews with 19 survey participants. Barriers encountered when accessing health services for children in OOHC included time-consuming consent processes, insufficient publicly funded health services, long wait times, poor communication of health information and delays in receiving key identifying information such as the child’s Medicare number. Increased health navigation support, greater prioritisation of health within CP, greater prioritisation of children in OOHC by health services, and flexible delivery of healthcare were identified as potential solutions. Study 3, a retrospective data linkage cohort study, examined the health service use of 6,201 children in the first year after entry to OOHC between 2010 and 2015, through analysis of administrative health data from one federal (Medicare) and four Victorian datasets. Only 41 children had attended all recommended services (GP, paediatrician, dentist, audiologist, and optometrist). While 90% attended a GP, fewer than 40% attended other health services, and a minority attended within recommended timeframes. This research has demonstrated that health assessment for Victorian children upon entry to OOHC is neither routine nor timely, and potentially modifiable barriers exist within the health and child protection systems. The findings have implications for cross-sector service and policy development, and for monitoring of compliance with national standards.

Background ASD is a neurodevelopmental condition that affects an individual’s ability to communicate and interact with others. There has been an increase in the prevalence of ASD diagnosis, with the most considerable increase in adolescents who are intellectually able. These adolescents have difficulties with adaptive behaviour and struggle to participate in society, even if their intelligence is in the average or superior range. A better understanding of the relationship between impairments associated with ASD such as social impairments, executive function, adaptive behaviour, and participation and quality of life (QoL) is needed in intellectually able adolescents with autism. Aims Using the International Classification of Functioning, Disability and Health (ICF), with subjective functioning added, as a framework, this thesis aimed to study the association between 1) executive function (EF) and adaptive behaviour, over and above the contribution to adaptive behaviour of social impairment and intelligence; 2) EF and participation and the mediating role of adaptive behaviour in their association; 3) participation and self-reported QoL, over and above the contribution to self-reported QoL of social impairment and symptoms of anxiety and depression, in adolescents with ASD and without ID. Methods To address these aims, a cross-sectional study of 39 adolescents with ASD, aged from 10 to 16 years and 11 months (M = 13 years 4 months; SD = 3 months) and with Full Scale Intelligence Quotient (FSIQ) ranging between 78 and 144 (M = 107.97; SD = 18.25) was conducted. Adolescents completed a battery of tests to assess the ICF domains of Health Condition (social impairment, anxiety and depressive symptoms), Body Functions/impairments (EF, such as cognitive flexibility, inhibition, generativity, planning), Activity (adaptive behaviour), Participation (participation in extra-curricular activities) as well as the domain of subjective functioning (self-reported QoL). The domains of Environmental Factors (family social advantage) and Personal Factors (sex, age and intelligence) were also considered for each participant. Measures were chosen based on their use in clinical practice and research with young people with ASD, with the exception of participation, as there was no clear understanding of how to measure this domain for young people with ASD. To identify how participation has been measured in young people with ASD a systematic review of the literature was performed, with consideration of the measurement properties of the tools identified. As published, nine studies out of the 2539 screened investigated the measurement properties of the tool to assess participation in young people with ASD. These nine studies included seven tests, namely Adolescent and Young Adult Activity Card Sort, the Children’s Assessment of Participation and Enjoyment/Preference for Activities of Children (CAPE/PAC); the Experience Sampling Method; the Pediatric Evaluation of Disability Inventory Computer Adaptive Test (PEDI-CAT) and the School Function Assessment. The tests’ measurement properties were investigated in studies with high risk of bias and therefore, the quality of the evidence for these tools is limited. The PEDI-CAT demonstrated adequate measurement properties but lacked the assessment of the subjective dimension of participation. For this reason, the CAPE was chosen in this study. Preliminary analysis of the data from the cross-sectional study comprised a description of the sample, univariate analysis of the association between independent and dependent variables by hypothesis, and explorative univariate analysis of the association between covariates and dependent variables. To address the three aims of this thesis, three hierarchical regressions were performed with covariates entered in the first steps, and the independent variables by hypothesis entered last. Results The descriptive statistics fit well with the conceptualisation of autism as a spectrum condition with scores on measures of the constructs mentioned earlier varying between participants. Hierarchical regression relative to aim 1 showed that inhibition was positively associated with adaptive behaviour, above and beyond social abilities (reversed social impairments) and intelligence. Inhibition and social abilities were positively associated with adaptive behaviour and explained 50% of its variance. There was no association between cognitive flexibility, generativity, planning and adaptive behaviour over and above the contribution of social abilities and intelligence. Hierarchical regression relative to aim 2 showed that there was no association between EF abilities and participation. Furthermore, there was no association between adaptive behaviour and participation and, as such, adaptive behaviour was not found to be a mediator in the association between EF and participation. Hierarchical regression relative to aim 3 showed that there was no association between participation and self-reported QoL, above and beyond reversed anxiety and depressive symptoms. Reversed depressive symptoms were positively associated with self-reported QoL and explained 49% of the variance in self-reported QoL. Discussion Assessment of EF abilities, together with the assessment of social abilities, should become routine in the care of adolescents with ASD who have difficulties with adaptive behaviour. However, there is a need to investigate other factors in addition to EF abilities and adaptive behaviour which may influence participation in extra-curricular activities as impairments in the former are not associated with those in participation. Treating comorbid mental health symptoms of young people with ASD, in particular depressive symptoms, may result in an improvement of these young people’s self-reported QoL.

Food allergen oral immunotherapy (OIT) is emerging as a potential treatment for food allergy. Probiotic and Peanut Oral Immunotherapy (PPOIT) showed effectiveness inducing non-responsiveness to peanut in the immediate post-treatment period. This doctoral thesis investigates the long-term clinical and immunologic effects of PPOIT in children with peanut allergy and the efficacy and tolerability of a modified PPOIT dose escalation schedule designed to improve treatment feasibility.