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.

Type I Diabetes Mellitus (TIDM) is one of the immune disorders and results from the loss of cells that make insulin (INS), a hormone that regulates the level and availability of glucose within the body. The most common treatment is exogenous insulin therapy which involves regular insulin injections or the use of an insulin pump. However, this treatment is imperfect and cumbersome, driving researchers to find more effective methods to control blood glucose levels. One potential treatment would involve making INS producing beta cells using pluripotent stem cells and then using these manufactured cells as a beta cell replacement therapy. Making insulin-producing cells in the laboratory involves several stages from stem cells to definitive endoderm, pancreatic endoderm, endocrine precursor, and the last step to mature beta cells. Any improvements in specific steps for the production of PSC derived pancreatic beta cells has the potential to lower the cost of an eventual treatment, making such therapy more widely available. In this project, we examined variations in the differentiation protocols with the ultimate aim of increasing the yield and functionality of the final cell product. One of the significant findings to emerge from this thesis is, in Chapter 4, we show that SCF addition has the potential to enhance cell proliferation during the stages of early differentiation. A surface receptor for Stem Cell Factor, cKIT, is widely applied to determine endoderm formation in the differentiation process of in vitro pluripotent stem cells. Despite its long-standing relationship with endoderm formation, a potential role for SCF itself during this process has not been systematically addressed. Using a suspension-based differentiation system, we show that the addition of SCF to differentiating cultures of PSCs enhanced the proliferation of early definitive endoderm, marked by the expression of CXCR4 and EPCAM. This effect of SCF was discernable within 2 days of differentiation initiation and coincided with the down-regulation of its receptor, cKIT. SCF treatment appeared not to affect the kinetics of differentiation, with key markers such as MIXL1, SOX17, CXCR4, and EPCAM showing identical expression patterns in untreated and treated cultures. Taken together, our results indicate that SCF addition at an early stage of differentiation will potentially reduce the number of input PSCs required to yield a given amount of endodermal products for the downstream application. This thesis is organized into six main chapters. Chapter 1 is a literature review concerning the introduction of pluripotent stem cells, Type I diabetes, pancreas development, normal molecules used in vivo differentiation protocol for beta cells generation. Chapter 2 describes the materials and methods of our experiments. Chapter 3 investigates the role of Activin A in definitive endoderm formation, which attaches great importance to adding Activin A from the beginning of differentiation. Chapter 4 shows that SCF promotes the proliferation during definitive endoderm differentiation. Chapter 5 presents images of the final cell product, which successfully show the signals of insulin. Chapter 6 is related to the limitations of previous research and the suggestions for future work.

Introduction: ADHD and ODD are two common developmental mental health conditions that can co-occur. There are associated functional impairments across clinical, biological, psychological, and social domains, which may be more severe when they co-occur. It is important to understand and recognise risk factors for young people with ADHD who are developing or maintaining ODD in order to target them early with appropriate and effective therapy interventions. To date, there are limited longitudinal datasets examining key risk factors associated with these comorbid conditions and more research is needed to investigate what places a young person with ADHD more at risk for ODD. This study examines the role of key child characteristics, psychosocial factors, ADHD subtypes and commodities. Methods: A three-year blinded evaluation of children with ADHD, investigating those with and without follow-up ODD. At baseline, relevant demographic, functional and clinical information was collected from clinically referred participants with ADHD (N=419). All participants were followed up three years later. Twenty-five percent (N=104), were unable to be reassessed. 150 participants were recruited with ADHD and follow-up ODD (120 males; 30 females) and 60 were recruited with ADHD alone (43 males; 17 females). Groups were compared for clinical presentations (ADHD subtype, comorbid disorders), key child characteristics (gender, IQ, temperament, Visual Spatial Working Memory, academic ability, emotional regulation, social problems), and psychosocial factors (parental level of education, family functioning, parental relationship functioning, parenting skill, parental psychopathology). Results: The two groups did not differ for age, gender, or SES at baseline. Young people with ADHD and follow-up ODD had increased temperament activity level, worse Verbal Comprehension, worse Visual Spatial Working Memory, increased aggression, and worse social problems in comparison with the ADHD alone group. In the psychosocial domain, lower parental level of education, overall higher parental psychopathology, and higher parental Obsessive Compulsive symptoms and Interpersonal Sensitivity significantly predicted ADHD and follow up ODD. ODD, CD and ADHD-C significantly predicted ADHD and follow up ODD group membership. In contrast, GAD acted as a protective factor, predicting membership in the ADHD alone group. Backwards stepwise logistical regressions were completed for all significant factors, finding that increased temperament activity, parent reported increased aggression and teacher reported increased social problems were the strongest predictors of ADHD and ODD follow up compared to the ADHD alone group. Discussion: The findings suggest that when working with young people with ADHD it is important to effectively manage their temperament activity levels, aggression and to teach skills that improve their social skills. Effectively treating ADHD-C symptoms, aiding Verbal Comprehension and Visual Spatial Working Memory difficulties, and supporting parents with furthering their education and mental health difficulties should be included in a comprehensive treatment plan. It is important for clinician’s to be aware of the bi-directional relationship between ADHD and parent behaviour and functioning, which highlights the importance of systemic intervention. Further understanding how to optimally manage the helpful aspects of a young person’s anxiety may assist in preventing behavioural problems. Study limitations, future clinical and research directions are discussed.

Recruiting a representative sample of participants is becoming increasingly difficult in large-scale population health and epidemiological surveys, even for studies using a well-documented sampling frame and a sound sampling process. In view of this and with the increasing appeal of online recruitment, due to significantly lower cost and rapid accrual, many researchers undertaking health surveys are faced with the challenge of analysing data obtained from a sample that is not representative of the target population of interest. Statistical methods for appropriately addressing this selection or participation bias are therefore critical to ensuring reliable and accurate population inference from large-scale complex health surveys. This is particularly important as results of such surveys often influence health care decision making and policy development. Historically, inverse-probability weighting using survey sampling weights has been the standard method for adjusting for known or expected discrepancies between sample and population when estimating descriptive population parameters in complex health surveys. A recently developed model-based approach is multilevel regression and poststratification (MRP). MRP was first described in the context of political polling and social research in the US. MRP first uses multilevel regression to model individual survey responses for the outcome measure of interest as a function of individual-level demographic and area-level geographic covariates. The resulting estimates of the target parameter for each demographic-geographic respondent subtype are then combined using a weighted average across the subtypes (poststratification cells), weighting by the proportions of each subtype in the actual population, to produce an overall population-level estimate. The research of this PhD investigated the use of MRP for producing valid and accurate inference for descriptive population parameters in large-scale population health and epidemiological surveys where samples may not be representative of the target population of interest. This approach was evaluated in comparison to inverse-probability weighting using a combination of simulation experiments and a case-study analysis of data from the baseline wave of Ten to Men: The Australian Longitudinal Study on Male Health. MRP was consistently found to achieve greatly superior precision as well as increased uniformity of estimates across population subsets relative to inverse-probability weighting. In simulation studies, while sampling weights produced estimates with smaller bias on average, the reduced variance associated with MRP was shown to result in estimates that were more often closer to the true population parameter values. As well as establishing MRP as a valuable analytic approach for large-scale health surveys where samples may not be representative of the target population of interest, this research explored the practical challenges associated with the application of MRP to real survey data and provided a number of recommendations to support future applications of MRP to health-related outcomes.

Mitochondrial diseases are the most common metabolic disease, caused by pathogenic variants in both nuclear and mitochondrial genomes. The molecular diagnosis can be challenging, sometimes taking years and requiring multiple invasive and risky diagnostic techniques. Genome sequencing (GS) is an emerging technology that allows the analysis of nuclear and mitochondrial genomes simultaneously. However, the interpretation of data can be complex, and the use of this technology in mitochondrial diseases is limited. This PhD thesis explores the diagnostic utility of GS by investigating retrospective and prospective cohorts of patients with suspected (but molecularly undiagnosed) mitochondrial disease. The thesis is divided into eight chapters. Chapter 1 begins with a general introduction to mitochondrial diseases and associated diagnostic challenges. Chapter 2 describes the subjects and methods used throughout the PhD. Chapters 3 to 6 use trio genome sequencing to analyse a retrospective cohort of patients with paediatric onset mitochondrial disease. In Chapter 3, trio GS uncovers TEFM and COX11 as new candidate mitochondrial disease genes. In Chapter 4, trio GS was useful to identify a deep intronic variant in NBAS, enabling diagnosis of a patient who showed clinical overlap with mitochondrial disease. In Chapter 5, trio GS was used to identify a novel apparently synonymous variant in PNPT1 where in silico and functional studies revealed a splicing defect instead. Clinical information and functional evidence from other patients with PNPT1 variants were studied through an international collaboration to gain a better molecular and clinical understanding of PNPT1-related mitochondrial disease. In Chapter 6, trio GS data was interrogated to study mtDNA inheritance, showing an absence of biparental mitochondrial DNA transmission in the cohort. In Chapter 7, the use of singleton GS data was studied in a prospective cohort of patients with suspected mitochondrial disease. A molecular diagnosis was made in 37% of cases. Examples that show the utility of GS include the identification of a mtDNA variant with low levels of heteroplasmy in blood (NC_012920.1(MT-TL1):m.3243A>T with 2% heteroplasmy), deletions in mtDNA (9kb deletion with 50% heteroplasmy) and nDNA (heterozygous 4.1kb intragenic deletion in AARS2), and the use of GS data to aid in paternity confirmation and variant phasing in a patient with suspected Perrault syndrome and PEX6 variants. Finally, Chapter 8 concludes that GS is an effective tool for the diagnosis of mitochondrial diseases. The diagnostic potential is increased by combining GS with other techniques such as RNA sequencing. Further research is being conducted to assess the cost-benefit of this technology.

The overall cure rates for paediatric cancer have improved dramatically over the last 60 years. There is, however, a subset of paediatric embryonal tumours that carry a poor prognosis. In particular, metastatic neuroblastoma, in association with MYCN amplification, remains difficult to cure. The phosphatidylinositol-3-kinase (PI3K)/Protein Kinase B (AKT)/mammalian target of rapamycin (mTOR) cell signalling pathway, as well as epigenetic factors, play a key role in numerous cellular functions including cell growth, survival and angiogenesis. Activation of the PI3K/AKT/mTOR pathway is common in a variety of tumours including those associated with the MYC family of proto-oncogenes. As a result of its multiple cellular functions it is not surprising that deregulation of this pathway is frequently observed in cancer. The importance of MYCN as a therapeutic target, and the impact of PI3K inhibitors in MYCN amplified neuroblastoma has been well documented. Rapamycin and its analogues everolimus (RAD001) and temsirolimus (CCI-779) exert selective cytostatic/cytotoxic effects on by targeting mTORC1. PF-502 is a combined PI3K/mTOR inhibitor that serially inhibits multiple targets within this pathway. Accordingly, interest in both mTOR inhibitors and combined PI3K/mTOR inhibitors as anticancer drugs, with particular applications to neuroblastoma, has recently surged. When used in the aggressive TH-MYCN murine model of neuroblastoma, PF-502 produced a significant survival benefit by both apoptotic and anti-angiogenesis effects whereas temsirolimus worked primarily by an anti-angiogenic effect. Epigenetic modulation of tumours has recently received increasing attention recently. Histone deacetylase inhibitors have shown pre-clinical promise in a variety of paediatric tumours. Specifically, they have been shown to promote both tumour apoptosis and terminal differentiation in other paediatric embryonal tumour pre-clinical models. Accordingly, the role of agents targeting these pathways was studied in the aggressive TH-MYCN mouse model of neuroblastoma. Panobinostat, when given as a continuous, low dose, resulted in a significant survival benefit as a result of both apoptosis and differentiation, with terminal differentiation achieved by prolonged exposure to the drug. Following on from these pre-clinical studies, an open label, Phase I (3+3 design), multi-centre study evaluating panobinostat in pediatric patients with refractory solid tumours, including neuroblastoma, was conducted. Primary endpoints were to establish MTD, define and describe associated toxicities, including dose limiting toxicities (DLT) and to characterise its pharmacokinetics (PK). Secondary endpoints included assessing the anti-tumour activity of panobinostat, and its biologic activity, by measuring acetylation of histones in peripheral blood mononuclear cells (PBMNC). Panobinostat significantly induced acetylation of histone H3 and H4 at all time points measured when compared to pre-treatment samples. A significant biological effect of panobinostat, measured by acetylation status of histone H3 and H4, was achieved at a dose of 15mg/m2 and was well tolerated. PK data and drug tolerability at 15mg/m2 was similar to that previously published. In summary, pre-clinical experiments support serial inhibition of the PI3K/AKT/mTOR pathway and the use of histone deacetylase inhibitors as treatment approaches in neuroblastoma.