Background The microcirculation makes up approximately three-quarters of the circulatory system but has attracted little attention as a target for cardiovascular disease (CVD) prevention, particularly in early life. Retinal microvascular parameters allow noninvasive assessment of the microcirculation, with adverse parameters (e.g., narrower retinal arterioles, wider venules) predicting CVD events in adults. In children, it is largely unknown when such microvascular changes become evident, their determinants and the underlying mechanisms. Aims In a large population-based sample of Australian children and mid-life adults (their parents), I aimed to determine whether: 1) adverse retinal vascular calibre and preclinical large arterial phenotypes covary; 2) body mass index (BMI) and waist-to-height ratio (WHtR; child only) over the preceding decade predict retinal vascular calibre; 3) inflammation mediates the association between obesity and retinal vascular calibre; 4) traditional CVD risk factors and large arterial phenotypes are related to retinal geometric parameters. Methods Participants: 1,288 11–12 year-olds (51% girls) and 1,264 parents (88% mothers). Cross-sectional measures: Retinal vascular calibre and (child only) geometric parameters; large arterial phenotypes (pulse wave velocity, PWV; carotid arterial elasticity; carotid intima-media thickness, CIMT); blood pressure (BP); BMI; WHtR; and metabolites (e.g., LDL, HDL cholesterol and the inflammation marker Glycoprotein Acetyls, GlycA). Biennial longitudinal measures: BMI and (child only) WHtR. Analysis: In all aims, linear regression models were used, and Aim 3 used causal mediation analysis. Results Aim 1: In children, narrower retinal arteriolar and wider venular calibre showed modest associations with faster PWV and lower elasticity but not with CIMT. In adults, results were stronger, and there was weak evidence of an association between wider venular calibre and higher CIMT. Aim 2: In children, higher BMI from age 4–5 years onwards was increasingly associated with adverse arteriolar calibre, but not venular calibre at 11–12 years. Effects were similar in adults. Less favourable BMI and WHtR trajectories predicted narrower arteriolar calibre. Aim 3: Compared to children with normal BMI, those with obesity had: (i) narrower arteriolar calibre, which was not mediated by inflammation; and (ii) wider venular calibre, which was partly mediated by inflammation. In adults with obesity, this association between obesity and wider venular calibre was fully mediated by inflammation. Findings were similar for WHtR. Aim 4 (in children only): BMI, systolic BP and PWV showed modest associations with lower arteriolar fractal dimensions, whereas only systolic BP was associated with arteriolar tortuosity. There was less evidence of associations with venular geometric parameters. Conclusion Preclinical phenotypes of large arteries and microcirculation have some shared, but mainly unique pathways to CVD, with shared pathways becoming more evident across the life course. The adverse impact of obesity on retinal microvasculature begins early in life, more markedly on arteriolar than venular parameters, with modest venular effects largely mediated by inflammation. In children, retinal geometric parameters did not add novel information about CVD risk beyond the vascular calibre. The microvascular parameter, retinal vascular calibre, may offer additional value to CVD prevention from childhood onwards, especially when combined with other risk assessment measures.

Immunisation reduces morbidity and mortality, and affects equity in health. This is evident between countries and regions, and within them, where there may be pockets of the population vulnerable to under-immunisation that experience more barriers to preventive health care. People with disability are one such group that are largely invisible in research and immunisation policy decisions. In particular, there is a paucity of data on adolescent immunisation in young people with disability in Australia. In Australia, students receive adolescent immunisations through the School-based Immunisation Program at 12 to 13 years of age. Vaccine uptake data for the majority of young people with disability attending specialist schools are not included in aggregate immunisation reports. Therefore, there is a clear need for coverage data, as well as qualitative research to clearly identify the barriers and enablers to immunisation for young people with disability in specialist schools. The aim of this thesis was to explore and describe acceptance and delivery of immunisation in specialist schools for young people with disability in Victoria, Australia. The research was designed as a mixed methods qualitative dominant and sequential explanatory study, with a quantitative phase followed by a qualitative phase. Phase One consisted of a prospective cohort study that aimed to measure the uptake of immunisations in specialist schools for young people with disability in Victoria, Australia. Data were collected on immunisation days in the 2017 school year from specialist schools in Victoria in order to determine uptake of diphtheria-tetanus-pertussis (dTPa) and human papillomavirus (HPV) immunisations in eligible students. Demographic data, motor and intellectual function of students, and reasons for non-receipt of dTPa and HPV vaccine were recorded using REDcapTM software and were analysed using descriptive statistics. Results from this study found that of the 28 specialist schools that participated, dTPa was received by 63% (237/374) of participating students, and HPV dose 1 (HPV1) was received by 66% (76/114) of female students and 67% (174/260) of male students. Three doses of HPV were received by only 41% (100/241) of students. The main reasons for missed immunisation were absence from school, lack of consent and inability to immunise due to the student’s behaviour and/or anxiety. Comparative data during the same time period for students in mainstream schools demonstrated significantly higher uptake, at 89% for dTPa and 75% for three doses of HPV, confirming under-immunisation of students in specialist schools. Phase Two consisted of an in-depth qualitative inquiry that utilised the Socio-Ecological Model (SEM) as a framework, which aimed to determine barriers and facilitators of immunisation for young people with disability in specialist schools in Australia. Data collection included 10 observations of specialist school immunisation sessions, 40 in-depth semi-structured interviews and two focus groups with key stakeholders, including representatives from state government, local government immunisation teams, specialist schools and parents of students. Data were coded and themed using Reflexive Thematic Analysis, as described by Braun and Clarke (2006; 2019). Five main themes were identified: an invisible population, searching for support, going the extra mile, competing priorities and trust takes time. The intersection of the themes across layers of the SEM varied, demonstrating the complex nature of the issue and the need for this multilayered approach. The integrated inferences from these two phases resulted in key recommendations. These recommendations include: ongoing rigorous coverage data recorded for ungraded schools; stronger central support for immunisation teams; a review of the immunisation funding model to reflect the extra work and resources required in some schools and to provide for increased follow-up and catch-up; clear guidelines for the use of restraint during immunisation in specialist schools; and a referral pathway for students with disability who cannot be immunised in the school setting. This thesis has generated new knowledge by establishing that young people with disability in specialist schools in Australia are missing their adolescent vaccinations, and that there are unique barriers to immunisation in this school setting. Phase One provided the first Victorian figures on coverage of adolescent immunisations of young people with disability and Phase Two constitutes the first qualitative research that has been conducted internationally on immunisation in children or young people with disability. Recommendations arising from the integration of findings from both phases have the potential to be translated into policy and practice, and thereby have a significant impact on the health and health equity of young people with disability.

The X chromosome and the Y chromosome are the sex chromosomes of mammals. While the X chromosome carries many genes with functions beyond sex, the Y chromosome is relatively gene poor. It serves as the trigger for development of a male embryo, with its genes having roles in testis determination and function. The sex chromosomes exist as a pair; in females, this pair are XX, and in males, XY. Two different mouse Y chromosomes were the focus of this project. The first was a transgenic fluorescent reporter Y chromosome, Ddx3y^mKate2, which was created to be used as a tool in chromosome instability research. The second was the naturally occurring Y chromosome from the A/HeJ inbred mouse strain, Y^A/HeJ. The work associated with these two Y chromosomes comprised the two studies in this project. The Ddx3y^mKate2 Y chromosome was generated by targeted insertion of a transgene cassette containing the red fluorescent protein gene, mKate2, into the mouse Y chromosome adjacent to the Ddx3y locus. This was selected as the relatively broad expression of Ddx3y indicated a permissive environment for expression of the mKate2 transgene. As male mice are XY, all male Ddx3y^mKate2 mice carried the fluorescent reporter. The mKate2 fluorescence in this strain has been previously assessed, revealing that the reporter was detectable from the preimplantation embryo, through to sexual maturity. To finalise the characterisation of the Ddx3y^mKate2 mouse, this study assessed the fluorescence in the Ddx3y^mKate2 male in advanced age. Additionally, this study characterised another transgenic reporter strain, Hprt^DsRed-Express, to serve as a comparison. To demonstrate the utility of the Ddx3y^mKate2 reporter, the Ddx3y^mKate2 Y chromosome was crossed onto two known chromosome instability backgrounds: the Trp53 knockout and Cenpagfp fusion knock-in backgrounds. Following in vivo and in vitro experiments, it was determined that the Cenpagfp background was the better background to model the Ddx3y^mKate2 reporter Y chromosome; however, more work using this genetic background in embryonic stem cells is advised for assessing the Ddx3y^mKate2 reporter Y chromosome. The Y^A/HeJ chromosome from the inbred A/HeJ strain was causally associated with a disturbance in the testis determination pathway. The Y^A/HeJ chromosome resulted in a subset of male mice having developed either ovotestes (gonads containing ovarian and testicular tissue) or abnormally small testes without epididymal sperm. It was confirmed that the Y^A/HeJ chromosome still carried the testis triggering gene, Sry. The gonadal and cytological abnormalities associated with this Y chromosome lead to the speculation that there was a structural change at or near its centromere. This study extended the characterisation of the A/HeJ phenotype, including identification of an age-related decline in male gonad mass, as well as assessment of the Y^A/HeJ chromosome. It was confirmed that the structural change to the Y^A/HeJ chromosome was a halving of the Rbmy tandem repeat array. This was predicted to adversely affect Sry gene expression during testis determination, resulting in the A/HeJ gonadal phenotype. Assessment of Sry regulation and gene expression from the Y^A/HeJ chromosome is recommended to complete the work associated with this Y chromosome.

The enteric nervous system (ENS) in the gastrointestinal tract is a complex nervous network. It is essential for gut secretion, absorption and peristalsis. Most of the ENS arises from vagal neural crest cells (NCCs) which migrate from the neural tube (level somite s1 to s7) into the foregut then colonize the rest of the intestine as a rostro-caudal wave and form the ENS. If this cell migration fails to be completed, the distal intestine lacks ENS, this results in Hirschsprung Disease (HSCR). My research project is going to answer: Do all levels of vagal NCC contribute to ENS equally? What is the enteric NCCs population expansion potential and how do individual initial enteric NCCs contribute to the final ENS? Does the ENS population retain its colonization capacity during development? How do enteric NC/glia cells and neurons interact in directing ENS cell invasion and axon extension? What is the mechanism that control ENS ganglion formation, especially the role of differential adhesion of enteric neurons and NCCs on gangliogenesis? This thesis using avian models combined with multiple techniques demonstrated that: The vagal NCCs of single somite levels origins migrate separately and converge at the foregut, where they all mixed. Along the whole vagal length, the mid-vagal region NC-derived cells -- that is s3 and s4 level -- arrive earliest at the foregut, and contribute the greatest numbers. During colonization along the gut, all levels of vagal NC-derived cells are mixed throughout the entire gut, but mid-vagal NC-derived cells contribute to all region of gut from foregut to distal hindgut in greatest numbers. Regarding the ENS forming potential, all levels of vagal NCC could form ENS, but mid-vagal NCCs (s3 and s4) have greatest competency. The temporal expansion of the enteric NC population in developing quail gut was investigated from E2.5 to E12, and the expansion potential was challenged by drastically reducing the starting enteric NCC numbers. By this means, the enteric NCCs showed an extremely high capacity to regulate their proliferation while forming the ENS. However, single cell lineage tracing technique developed in this thesis demonstrated that the contribution of individual enteric NCC to final ENS was unequal and unpredictabe, a few dominant ENS “superstar” cell clones emerge but most clones are small. These “superstar” clones are not predetermined, instead they achieve this status stochastically. The extremely high gut-colonization capacity of enteric NCCs from the early embryo gut was found to rapidly decline with embryonic age, and this declines was more rapid in the distal intestine-derived enteric NCCs than in more proximal enteric NCCs. This age-related loss in colonization capacity involves changes in the ENS cell population, rather than the maturity of the mesoderm. However, it is not caused by reducing the “undifferentiated” ENS cell number, since this capacity can neither be rescued with more time to catch up, nor mimicked by reducing ENS cell number from young donor. Actually, unlike their proportion in the ENS, the absolute number of apparently undifferentiated enteric NCCs does not decline with age. Therefore, this age-related loss in colonization capacity is caused by changes in qualitative aspects with age. The early development of the ENS shows two invasive events: rostral-to-caudal invasion by enteric NCCs and extension of ENS axons. Experimentally, the gut mesoderm supports these two invasive events for longer than the normal time-window. Both these events can occur bidirectionally when permitted, although normally they are unidirectional. However, a fresh invasion wave of enteric NCCs is prevent by pre-existing ENS cells. This prevention of invasion does not occur in gut with pre-existing ENS neurons and axons but without enteric NCCs. Therefore the conclusion is that this prevention is caused by the pre-existing enteric NCCs and not by other ENS components. In the other hand, pre-existing ENS neurons and axons inhibit neuronal differentiation of the invading enteric NCCs. In contrast, ENS axon invasion is not prevented by pre-existing ENS cells and axons. These invasive axons do not use pre-existing ENS axon tracts or ENS neurons to facilitate invasion of the gut, but have an absolute requirement for enteric NCC (either in situ or co-invading) in order to advance into gut mesoderm. Follow these colonizing aspects of ENS development, the ratio of enteric NCC to neuron is stabilized and ENS ganglia are formed with a core of neurons and a shell of enteric NC/glia cells. Using cell-cell aggregation assays this thesis revealed that during aggregation, both Ca+2-dependent and independent adhesion mechanisms are required. Neurons sorted to the core of aggregates, surrounded by outer enteric NCCs, showing that neurons had higher adhesion than enteric NCCs. The outer surface of aggregates became relatively non-adhesive, correlating with low levels of NCAM (Ca+2–independent) and N-cadherin (Ca+2–dependent) on this surface of the outer non-neuronal enteric NCCs. In addition, the ganglion size is intrinsically regulated by the ratio of enteric NCCs to neurons likely by generation of an outer non-adhesive surface. Overall, my research covers almost the entire process of the ENS development from initiating of NC on its origin site to the colonization of enteric NCC along the entire gut, and the final steps of ENS ganglion formation. The results from this thesis revealed many critical issues in understanding of the cellular mechanisms that control these processes. These basic researches have important implications for understanding not only ENS development but also enteric neuropathologies and for designing NC stem cell therapies.

Rett Syndrome (RTT) is a severe neurodevelopmental disorder (NDD) resulting in severe cognitive and physical impairments. Despite being predominantly caused by pathogenic variants in the methyl-CpG-binding (MECP2) gene, between 3 – 15% of classic and atypical RTT individuals do not have a genetic diagnosis. Classic RTT individuals exhibit an apparently normal development until 6 to 18 months of age after which developmental regression occurs. Atypical RTT individuals have many features of classic RTT but do not meet all the specific diagnostic criteria. Recently, the classification of RTT has been expanded to include individuals with clinical features overlapping RTT and other NDDs, often referred to as RTT-like individuals. The clinical and genetic diagnosis for RTT-like individuals is further complicated due to the complexity of NDDs and there is an unmet need to provide a precise genetic diagnosis for these individuals. Next generation sequencing (NGS) studies are continuously identifying hidden genetic links between relevant molecular pathways and RTT. Through whole genome sequencing (WGS), our lab had identified a de novo heterozygous missense variant [c.744C>A; (p.Asp248Glu)] in the motor domain of kinesin-3 family member 1A (KIF1A) in one classic RTT female. Single nucleotide heterozygous variants in KIF1A have been implicated in a number of severe neurological disorders, collectively known as KIF1A-associated neurological disorders (KANDs). KIF1A encodes a neuron-specific kinesin molecular motor protein essential for ATP-dependent anterograde axonal transport of synaptic cargos along microtubules. In order to determine additional RTT/RTT-like individuals with KIF1A variations, we collected further clinical and genetic information from our collaborators of 30 individuals with 18 different missense variants affecting the critical motor domain of KIF1A. After careful clinical assessment, we identified three additional individuals with different novel missense variants exhibiting overlapping RTT-like and KAND clinical features. In silico tools predicted all variants to affect proper protein folding and were predicted to be likely disease causing. In addition, in vitro functional analysis using the highly specific neurite tip accumulation and microtubule gliding assays, demonstrated all variants to have reduced microtubule based movement, suggesting these variants are indeed significantly pathogenic. Comparison of the clinical features of the remaining 27 KAND individuals with 16 variants in the KIF1A motor domain suggested that specific clinical features and phenotypic severity was largely dependent upon the location of the variant. Using an NGS approach, we identified pathogenic MECP2 variants, previously missed by mainstream genetic testing, in seven RTT individuals including a case of a mosaic male. In addition, we found variants in two genes that are known to be associated with NDDs and RTT-like syndromes; Structural Maintenance of Chromosomes 1A [SMC1A; c.3576delA; p.(Val1193Serfs*2)] and SH3 and multiple ankyrin repeat domains 3 (SHANK3; c.2265+1G>A). This work continues to expand the genetic basis of RTT. Through whole exome sequencing (WES), we have also identified an atypical RTT female with a heterozygous nonsense variant [c.3385C>T; p.(Arg1129*)] in Lysine Acetyltransferase 6A (KAT6A) that encodes a chromatin remodelling protein. Heterozygous protein truncating variants in this gene have been associated with KAT6A-related intellectual disability. Through various collaborations, we identified a further four individuals with KAT6A variants [c.3820G>T; p.(Glu1274*), c.3399_3400dup; p.(Lys1134Argfs*14), c.3377delC; p.(Ser1126Phefs*8) and c.3631_3632delGT; p.(Val1211*)] who had clinical symptoms overlapping with RTT/RTT-like individuals. Through systematic re-analysis of a reported cohort of 76 individuals with KAT6A-related intellectual disability we recognized two additional individuals exhibiting RTT-like clinical features with KAT6A variants [c.4254_4257del; p.(Glu1419Trpfs*12) and c.3661G>T; p.(Glu1221*)] . All the identified variants in the seven RTT-like individuals were clustered in the last exon of KAT6A and in silico analysis predicted the variants to cause a dominant-negative effect. These seven individuals exhibited clinical features overlapping between RTT and KAT6A-related intellectual disability that was previously unrecognized. Using singleton WGS, a novel heterozygous nonsense variant in another chromatin regulator gene, Chromodomain helicase DNA-binding protein 8 [CHD8; c.5017C>T; p.(Arg1673*)] was identified in an atypical RTT individual. Heterozygous protein truncating variants in CHD8 have been implicated in NDDs including Autism Spectrum Disorders (ASDs). Functional analysis confirmed reduction in CHD8 protein levels in her fibroblasts, confirming the pathogenicity of the identified variant. In another RTT-like female, a de novo heterozygous missense variant [c.271G>A; p.(Asp91Asn)] in the Eukaryotic Translation Elongation Factor 1 Alpha 2 (EEF1A2), involved in protein translation, was identified through singleton WES. This variant has been previously reported in a female with NDD. Interestingly, a single case with the same EEF1A2 variant [c.274G>A, p.(Ala92Thr)] has also been reported in a RTT-like female. Thus, our findings further established the casual association between EEF1A2 and a RTT-like phenotype. In a RTT-like individual, a de novo large deletion at chromosome 9q34.11 (hg19:131,455,942-131,743,585) resulted in the loss of 13 genes, including the 3’ end of the coding sequence of SET Nuclear Proto-Oncogene (SET) and the 5’ end of the coding sequence of Nucleoporin-188 kDa (NUP188). The truncation of SET resulted in the loss of a highly conserved critical nucleosome assembly protein (NAP) domain crucial for assembly of nucleosomes and chromatin fluidity. Subsequent WES in the same individual identified a missense variant in NUP188 [c.3922C>T; p.(Arg1308Cys)] on the other allele. Preliminary functional studies in individual’s fibroblasts showed reduced NUP188 protein levels and enlarged nuclei, suggestive of perturbed NUP188 function. In this individual two genes, SET and NUP188, may be contributing to the affected individual’s phenotype. Interestingly, a homozygous variant in a novel candidate disease gene, Potassium Channel Tetramerization Domain Containing 16 [KCTD16; c.937T>A; p.(Ser313Thr)], was also revealed in a classic RTT female. KCTD16 encodes an auxiliary subunit that associates with GABA-B receptor and regulates receptor response in an agonist concentration dependent manner. Although variants in the KCTD family of proteins have previously been reported in individuals with NDDs, defects specifically in KCTD16 are yet to be linked with any human disease. Our preliminary electrophysiological studies in Xenopus oocytes investigating perturbed GABA-B receptor kinetics in response to the KCTD16 variant [p.(Ser313Thr)] did not reveal any significant differences when compared to wild type, as well as a variant commonly found in the healthy population [p.(Asp160Asn)]. Despite this, we plan to continue these investigations in a mammalian Chinese Hamster Ovary (CHO) cell-based model to further evaluate the variant’s pathogenicity. Overall, this study has provided functional evidence of variations affecting the motor domain of KIF1A in the pathophysiology of RTT/RTT-like disorders. In addition to identifying pathogenic variations in four known RTT-related genes (MECP2, SHANK3, SMC1A and EEF1A2), in this project we have further expanded the genetic landscape of RTT/RTT-like disorders to include variations in five additional genes (KAT6A, CHD8, SET, NUP188 and KCTD16). We recommend that the testing of these genes should be considered routinely while analysing NGS data in mutation negative RTT/ RTT-like individuals. The identification of additional members of key molecular pathways perturbed in RTT has further expanded our understanding of the underlying biology behind RTT, and this may pave the way for future targeted therapeutic options for RTT.

Juvenile idiopathic arthritis (JIA) is a complex autoimmune disease affecting children aged between 6 months and 16 years. JIA represents a group of 7 subtypes of disease, with the most common being oligoarticular JIA (oJIA). Despite a prevalence of up to 1 in 400, rates similar to those in T1D, JIA research is relatively sparse. Research into disease pathogenesis has largely focussed on genetic risk factors, and has also identified CD4+ T-cells as likely to mediate the autoimmune process. However, research is particularly needed regarding diagnosis and prognosis of disease and its outcomes. Currently, diagnosis is almost entirely dependent on clinical observation and history, with little in the way of biomarkers to classify patients or to guide clinical management. Epigenetics represent biological modifications to DNA and chromatin that control gene expression and chromatin structure. DNA methylation is perhaps the most accessible modification available for study, and is known to modulate immune cell function particularly amongst CD4+ T-cell subsets. A number of autoimmune diseases have reported significant DNAm associations, and have also provided intriguing data on the potential of DNAm to predict clinical outcomes. This study hypothesised that DNAm is important in oJIA pathogenesis, and potentially provides a biological basis for the diagnosis and prognosis of disease. This study utilised CD4+ T-cells and a case-control study design to analyse the associations between DNAm and oJIA, with data generated from the Illumina Infinium HumanMethylation450 BeadChip array. Cases were matched with controls according to age and sex. Further, cases were subtyped according to current diagnostic criteria and had active disease, both of which attempted to ensure all cases were clinically homogeneous. The first aim was to profile DNAm in oJIA cases compared to controls. Processing of data through analysis pipelines resulted in high quality data. Differential methylation analysis suggested that oJIA cases and controls could be segregated in cluster analysis using DNAm data, despite no genome wide significant hits being produced. Immune system pathways analysis suggested the top hits were relevant to disease, being enriched for receptor binding of cytokines such as IL6, IL17 as well as MHC class II. In addition, a number of top ranking probes were enriched within cell death and survival functions. Indeed, gene expression data suggested genes within those pathways were also correlated with DNAm. Technical validation of a selection of probes was highly successful, with all probes validating. A small replication study, however, was not able to reproduce these findings. Of particular note, a wide distribution of DNAm values was observed for many of the validated probes. Since technical validation was so successful, this DNAm heterogeneity potentially derived from sample group heterogeneity, which may well have played a part in difficulties replicating data. Therefore, biological sources of heterogeneity were explored in chapter 5, focussing primarily on the genetic associations with DNAm. Probes utilised for technical validation were analysed for genetic associations associating with either mean or variable DNAm. Both analyses suggested that the most robust associations were for known mQTLs and enhancer SNPs. Indeed, DNAm differences according to genotype were up to 13% and 27% for 2 probes analysed, representing a many-fold difference over case-control differences (typically approximately 5%). Combined with an intermediate level of minor allele frequency for many of these robustly associated SNPs, these mQTLs represent a likely source of biological variation contributing to oJIA DNAm variation. These minor allele frequencies increase the likelihood of inadvertent sampling bias, potentially resulting in difficulties in replicating DNAm data. Deeper analysis provided some initial indication that these mQTLs may also be potential oJIA risk loci, with the most significant associations again coming from known mQTL or enhancer SNPs. This also suggested DNAm data may well identify regions of interest for genetic risk loci discovery. The final chapter hypothesised that sources of potential clinical heterogeneity not captured within current classification criteria may well lead to DNAm heterogeneity, as could recognised subgroups within oJIA. Of primary focus, age of disease diagnosis was assessed for associations with DNAm. This study found that case-control analyses of older diagnosed samples (greater than or equal to 6 years) resulted in case-control clustering using far fewer probes. Indeed, the reduction of probes required for clustering was more pronounced in the analysis of younger diagnosed samples (less than 6 years of age), and also resulted in a genome wide significant hit. These subgroups represented 2 highly divergent populations, since top ranking probes from each subgroup had virtually no overlapping probes. This data suggested that age subgroups in oJIA represent sources of sample heterogeneity, leading to DNAm heterogeneity. Technical validation for a large majority of the select probes from the younger-diagnosed analysis was also successful. However, a small replication study could not reproduce these initial findings. In light of the potential for mQTLs to have pronounced effects on DNAm, as explored in chapter 5, larger replication groups (or, indeed, discovery groups) will likely be needed to mitigate the risk of sampling error to enable reproduction of findings. OJIA heterogeneity was also explored by looking at known subgroups, Persistent vs Extended disease. A number of oJIA cases would go on to develop extended disease, and the possibility existed for DNAm signatures to identify these cases prior to disease extension. This was indeed the case, with an exploratory analysis suggesting a number of probes can cluster persistent cases from extended-to-be cases. Further, these probes were able to produce a highly sensitive and specific test to predict disease extension, thereby providing a proof of principle for a prediction test using DNAm data. This study is the largest case-control analysis of JIA DNAm to date, and provided insights into the potential for DNAm to identify pathogenic pathways, identify sources of oJIA heterogeneity, and opened the possibility for biological markers of disease to be used in clinical management. The findings regarding the pronounced effect of mQTLs on DNAm also suggest that genetics is a large source of DNAm variability, far larger than group differences typically found in a complex diseases (such as oJIA). The identification of subgroup specific differences, even with a clinically homogeneous subtype, warrants further investigation to explore potential differences in pathogenesis between age groups and the use of DNAm as biomarkers for classification or disease management.

This thesis addresses the question of what is the best evidence-based management for children with ASD (autism spectrum disorder) coming under anaesthetic care in the hospital setting. The increasing prevalence of ASD (1) has meant that most anaesthetists need to become proficient in understanding and managing children with ASD. Children with ASD have higher hospital contact than their neurotypical peers.(2) Behavioural problems, sensory sensitivities, language deficit, and inflexibility with change contribute to the difficulties experienced by children with ASD in the hospital setting. (3)Hospitals may be inherently challenging to children with ASD: being inflexible places, with the sound of crying children, with invasive monitoring techniques and bright lights.(4) One unpleasant anaesthetic experience can lead to heightened anxiety and future refusal to attend hospital. In order to find the context for best anaesthetic care, we have reviewed the existing literature about ASD and its management in chapter one. The first part of chapter two is a review of anxiety and premedication in general terms. The evidence for current best practices in managing children with ASD in the perioperative period is outlined in the second part of chapter two. To further understand the family and staff perspective of optimal care, we conducted a qualitative study of 29 individuals including 15 parents of children with ASD who had had a recent anaesthetic and 14 staff members that had looked after them in different capacities at two hospitals in Melbourne, Australia in chapter three. Chapter four contains discussion and conclusion. It includes discussion about the discontinued preparation/premedication trial (CLOMID). The flaws in the design and obstacles in its execution are examined. Our data showed important organisational, educational and resource matters. Problems such as prolong waiting for an operation date, lack of training of staff including anaesthetists and nurses, lack of availability of simple equipment and private spaces in the recovery rooms- were to be addressed. Good communication, clear explanation, and friendly attitudes as well as flexibility and individualised care of patients were considered useful. The supplementary material includes a protocol for a preparation /premedication study that has not been concluded as well as two social stories that I have designed.

Neurodevelopmental disorders such as cerebral palsy (CP) and epilepsy are some of the most prevalent childhood neurological disorders caused by damage to the growth and development of the brain. Early life environments predispose children to later health outcomes evidenced by the developmental origins of health and disease (DOHaD) phenomenon. Epigenetics, which refers to modifications of DNA without change in DNA sequence, is one way by which environmental exposures may contribute to development of disease. DNA methylation, arguably the most highly studied epigenetic mark, has been correlated with early life environmental exposures and have implications in both disease mechanisms as well as clinical biomarkers of neurodevelopmental diseases. These modifications most likely originate in utero, in line with the DOHaD hypothesis. The study of monozygotic (MZ) twins, in which genetics, age, sex, parental factors and shared environment are controlled for, helps in distinguishing the extent of effect of genetics and environment. Discordance for neurodevelopmental disorders has been recorded in MZ twins indicating a potential role of non-shared factors in disease risk. The aim of this PhD was to utilise the discordant MZ twin model to understand epigenetic changes associated with neurodevelopmental disorders. Genome-wide DNA methylation was measured within MZ twin cohorts discordant for CP or epilepsy using Illumina’s Infinium HumanMethylation450 and EPIC arrays. Statistical and bioinformatics pipelines were applied to evaluate the association of DNA methylation data to disease phenotypes. As detailed in Chapter Three of this thesis, DNA methylation analysis of CP-discordant twin pairs provides the first evidence that environmentally mediated differential methylation in genes involved in known processes such as hypoxia and inflammation, and processes such as cell adhesion, may contribute to the development of CP. As detailed in Chapter Four, an epigenome-wide analysis of epilepsy discordant MZ twin pairs revealed distinct patterns of DNA methylation within subtypes of epilepsies of unknown cause. Differentially methylated genes within epilepsy subtypes included those with a role in metabolic pathways, voltage-gated channel signalling and neurotransmitter processes. This research paves the way for future larger studies, as understanding DNA methylation profiles associated with neurodevelopmental disorders, may facilitate biomarkers for earlier diagnosis. This could lead to possible intervention strategies for patients suffering from a broad spectrum of disorders. Analysing epigenetic data from disease discordant twins provides an elegant study design and has the power to explore non-shared environmental factors that further refine models of disease mechanisms and biomarkers. The findings of this thesis suggest that epigenetic factors may play a role regulating biological pathways that underlie neurodevelopmental disorders, some of which arise as early as the prenatal period. Replication in other larger and similar cohorts of discordant twin pairs may provide novel targets for biomarker development, thereby allowing for early interventions and helping the health of children.

Scabies is a parasitic disease and a global health problem that predominantly affects disadvantaged communities in low-resource settings. Scabies significantly impacts the health and quality of life of those with the disease. To accurately assess the global burden of disease and to compare data across regions, standardised diagnosis with consistent disease definitions is necessary. In low-resource settings, diagnosis by clinical assessment is the principal diagnostic method. In the absence of available experts, non-expert health workers are likely to play critical roles in diagnosis, including for prevalence mapping. Currently, standardised processes for clinical diagnosis for scabies do not exist. This thesis explores the diagnosis of scabies in low-resource settings and the role and utility of non-expert health workers in the diagnosis of scabies. Chapter 3 describes the evaluation of non-expert health workers in the diagnosis of scabies and impetigo using clinical criteria. The diagnosis of four briefly-trained nurses was compared to the consensus diagnosis of two experienced doctors. The sensitivity of the nurses’ diagnosis compared to the reference standard was 55.3% for scabies with a specificity of 89.9% Sensitivity for moderate to severe scabies was 93.5% with a specificity of 74%. The accuracy of diagnosis by non-expert health workers is promising and may be acceptable for scabies and impetigo disease mapping in low-resource settings. Chapter 4 describes the development and evaluation of a training protocol for the diagnosis of scabies and impetigo for non-expert health workers. The aim of this study was to measure the change in knowledge and confidence of the participants and explore their experience and perceptions of the training. Training was evaluated using a case-based test, a questionnaire and semi-structured interviews. The overall results of the case-based test were 90% for scabies and 75.5% for impetigo. The mean score for both self-reported knowledge and confidence increased from 2.5 to 4.5 following training and scores increased for all nurses (mean difference 2, 95%CI 1.1-2.9, P=0.005). The study showed that training local health staff in scabies diagnosis was enjoyable for participants and led to improvements in self-reported knowledge and confidence. Chapter 5 investigates the prevalence of scabies and impetigo using a cross-sectional study in a primary school in Gizo in the Solomon Islands. Using the International Alliance for the Control of Scabies (IACS) diagnostic criteria the classified the diagnosis of scabies. The prevalence of scabies was 54.3% and prevalence of impetigo was 32.1%. 63.5% of those with impetigo had scabies, corresponding to a population attributable risk of 11.8%. The study highlighted the extremely high burden of these diseases supporting the need for interventions for scabies in this community. Chapter 6 evaluates the methods of data collection, analysis and display for describing in detail the distribution of scabies lesions in a pilot study. The study used a novel technique of representation of dermatological lesions in the form of a choropleth map. The study found that the methods used were feasible for a larger population and would describe valuable detailed information on specific lesion location in scabies. The study will provide information on lesions at specific body sites to determine if simplified examinations are appropriate for prevalence surveys. The public health control of scabies requires identification of high-prevalence communities to target interventions, as well as methods to monitor the effectiveness of interventions. Such programs would be dependent on accurate and standardised diagnosis for population mapping. This thesis suggests methods to improve the diagnosis of scabies in low-resource settings. Modifications to training and diagnostic methods are likely to improve diagnostic accuracy. Improvements to scabies diagnosis will contribute to efficient collection methods and reliability of prevalence data.

Gaps in the quality of hospital care in low- and middle-income countries contribute to neonatal death and morbidity. Most neonatal deaths occur in the first few days of life, many from preventable or treatable causes. Essential newborn care consists of low-cost interventions, such as basic resuscitation, early breastfeeding and skin-to-skin contact, which have been shown to improve outcomes. Successful essential newborn care implementation requires understanding of the setting in which it is being delivered and the contextual factors that enable healthcare workers to provide quality care for newborns. Solomon Islands is a low-resource country in the Western Pacific and a Small Island Developing State with high neonatal mortality rates, increasing births per capita and limited healthcare resources and personnel. The Ministry of Health and Medical Services (MHMS) and stakeholders implemented an intervention to improve newborn quality of care in Solomon Islands. This thesis evaluates the quality of care in hospitals of Solomon Islands and describes contextual factors that enabled successful implementation of a multifaceted intervention to improve essential newborn care. This thesis used a mixed methods design comprising the following sequential studies: (1) Quality of hospital care for newborns was described through a cross-sectional study using a structured assessment tool to identify strengths and limitations in structure and process components of existing care. (2) Three years of perinatal outcomes were audited to determine stillbirth, perinatal and neonatal mortality rates and the main causes of neonatal morbidity and mortality. (3) The impact of the World Health Organization Early Essential Newborn Care training program on knowledge and skills of healthcare workers was assessed, using a before-and-after study that identified the factors associated with improved retention of knowledge and skills. (4) The implementation process was described through interviews of healthcare workers, and interview data were triangulated with quantitative results to describe features of the intervention that supported implementation. This thesis demonstrated gaps in structure and process elements in quality of newborn care, which limited provision of appropriate, timely clinical care in the hospitals. Very high perinatal mortality rates, mainly owing to stillbirths, were identified. The provinces had higher rates of perinatal mortality than the National Referral Hospital. The main causes of neonatal mortality were complications of prematurity, birth asphyxia and infection. Knowledge and skills of healthcare workers significantly increased following the coaching program. At the time of evaluation, some loss of skills over time had occurred, particularly in cadres of healthcare workers that did not routinely use relevant skills. The evaluation of the implementation process reflected strengths of the intervention, specifically the training methods (content, short duration and practical approach) and the impact of a small training team of MHMS midwives and nurses who delivered the program independently. The challenges and sense of anxiety faced by healthcare workers in remote, isolated settings with infrequent exposure to resuscitation were highlighted. The barriers to establishing independent, ongoing quality improvement activities reflect the challenge of sustaining action across a large geographical area that has a dispersed health workforce when there is little capacity for regular oversight and support. This thesis highlights the potential impact from a multifaceted intervention to improve essential newborn care. Together with efforts to address stillbirths and improve intrapartum quality of care and quality of care in the neonatal period, essential newborn care may form an important part of a strategy to improve outcomes for newborns.

Truncus arteriosus is a rare congenital cardiac defect which results in a single common arterial trunk exiting the heart which supplies the systemic, pulmonary, and coronary circulations. The truncus itself is guarded by a single, often large common valve – the truncal valve – which separates the truncus from both the left and right ventricle. Truncus arteriosus has an incidence of 3 to 10 per 100,000 live births. Although only 0.7% – 3% of all congenital cardiac anomalies are due to truncus arteriosus, it accounts for 4% of all critical congenital cardiac anomalies. Patients typically present early in life with symptoms of cyanosis and congestive cardiac failure. Nowadays, surgery is undertaken early in life prior to the development of irreversible pulmonary hypertension. Improvements in surgical techniques and perioperative management has drastically reduced early mortality to 3 – 20%. Therefore, many children who have undergone truncus arteriosus repair are living well into adulthood. Despite this, there are few large studies addressing the long-term outcomes of truncus arteriosus repair. Furthermore, the impact of concomitant anomalies and the truncal valve are insufficiently described. This Doctor of Philosophy focuses on the long-term outcomes of truncus arteriosus repair in order to determine the current results and risk factors for poor outcomes. This research constitutes the largest single-institutional and multi-institutional experience assessing the long-term outcomes of truncus arteriosus repair with the longest follow-up time. I demonstrated that the majority of mortality following truncus arteriosus repair occurs within the first year after repair, and survival beyond the first year is excellent. I found that the presence of a coronary artery anomaly was associated with both early and late mortality and suggest that the coronary anatomy be clearly identified intraoperatively. Furthermore, patients with DiGeorge syndrome are at risk of late mortality, most commonly due to infection. Interestingly, I found that patients with mild or less truncal valve insufficiency are free from truncal valve surgery for up to 25 years, despite their truncal valve anatomy. In contrast, most patients with moderate or greater truncal valve insufficiency – particularly those with a quadricuspid truncal valve – will require truncal valve surgery at some stage in their lifetime. Of note however, the durability of truncal valve repair as a whole is poor, with most patients requiring reoperation on the truncal valve. In those with a quadricuspid truncal valve, repair by tricuspidization appears to be the most durable option with good long-term outcomes. Tricuspidization provided better long-term outcomes even if the non-tricuspidization group included younger children (less than 6 years of age), in whom truncal valve replacement was performed. This is an important finding as it suggests that younger children may benefit from truncal valve repair rather than a replacement with a smaller (non-adult sized) mechanical prosthesis which may require repeat replacement. Furthermore, if repair of the truncal valve is possible, this would avoid life-long anti-coagulation and the associated risks. In the long-term, patients had an excellent functional status following truncus arteriosus repair but had a high rate of reoperation due to the use of a conduit for reconstruction of the right ventricular outflow tract. Despite the high reoperation rate, patients have similar quality of life compared to age-matched Australian controls. This Doctor of Philosophy has redefined our understanding of the long-term outcomes of truncus arteriosus repair. The findings presented will impact clinical decision making, and I envision an improvement in the outcomes of these rare and complex patients.

Background: Epidemiological evidence suggests that Bacillus Calmette-Guerin (BCG) vaccine exerts non-specific (heterologous) effects in infants; decreasing neonatal mortality in high-mortality settings and preventing allergy and morbidity from infection in developed countries. New tuberculosis (TB) vaccines could potentially lack these beneficial effects. Immune mechanisms underlying the non-specific effects of BCG vaccine have been linked to ‘trained immunity’ or innate immune memory. Aims: Part 1: To investigate whether neonatal BCG vaccination alters the immune response to heterologous pathogens and Toll-like receptor (TLR) ligands in (i) the neonatal period and (ii) infancy. Part 2: To use a systems vaccinology approach to identify innate immune signatures underlying the non-specific effects of BCG vaccine. Methods: Part 1: In the Melbourne Infant Study: BCG for allergy and infection reduction (MIS BAIR), 1272 infants were randomised to receive BCG vaccine or no BCG vaccine within the first 10 days of life. A subset of participants was recruited to an immunological sub-study. A whole blood stimulation assay of ‘specific’ mycobacterial antigens, heterologous bacterial or fungal antigens and TLR ligands was used to interrogate cytokine responses at 7 days (n=212) and 7 months (n=167) post randomisation. Part 2: In the BabyBAIR study, 44 infants who were BCG vaccinated prior to travel to a TB-endemic area were recruited. Blood was collected from participants prior to BCG vaccination, and at 7 days and 3 months post vaccination. Cytokine responses and cell populations were analysed following in vitro stimulation of whole blood as above. RNASeq was also done on whole blood and the transcriptome was analysed for differentially expressed genes. Pathway analysis was done using functional gene set enrichment analysis (fGSEA) at each time point compared to baseline.   Results: In the MIS BAIR study, infants who were BCG-vaccinated had significant differences in their heterologous cytokine responses at both 7 days and 7 months post randomisation compared to BCG-naive infants. At 7 days post randomisation, compared to BCG-naive neonates, BCG-vaccinated neonates had evidence of a pro-inflammatory bias in RPMI-stimulated (nil) samples. Following heterologous stimulation, BCG-vaccinated neonates had decreased chemokine (MCP-1, MIP-1alpha, MIP-1beta, IL-1RA, IL-6 and IL-10 responses following stimulation of TLR2 (PEPG) and TLR7/8 (R848). At 7 months post randomisation, compared to BCG-naive infants, BCG-vaccinated infants, had decreased IFN-gamma responses to stimulation with heterologous pathogens. Decreased IFN-γ responses in the BCG-vaccinated group were attributable to a reduction in the proportion of individuals mounting an IFN-gamma response. Heterologous cytokine responses were modified by sex and maternal BCG vaccination status in both neonates and older infants. In the BabyBAIR study, longitudinal cytokine analysis showed BCG vaccination to be associated with a pro-inflammatory bias at baseline (prior to BCG vaccination), a robust pro-inflammatory heterologous response at 7 days post BCG vaccination and downregulation of pro-inflammatory cytokines 3 months post BCG vaccination compared to baseline. Flow cytometry suggested that both myeloid and monocyte derived dendritic cells were associated with the observed heterologous cytokine responses. RNASeq analysis of the whole blood transcriptome following BCG vaccination indicated widespread changes in innate immune signalling pathways and identified several potential mechanisms by which BCG vaccine could mediate its beneficial heterologous effects. Conclusions: Neonatal BCG vaccination leads to significant changes in the immune phenotype of vaccinated individuals. Following in vitro stimulation with heterologous pathogens, BCG-vaccinated infants have altered immune responses which might improve regulation of the inflammatory response during acute infection and a subsequent reduction in all-cause mortality. These results support the paradigm of BCG-induced trained immunity and provide additional information regarding the nature of the response in neonates and between different classes of pathogens.