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.

When an infant is admitted to the neonatal intensive care unit (NICU), parents are confronted with the challenge of developing relationships with their very sick infant while trying to comprehend the medical situation and their own emotional reactions. Although Parental Reflective Functioning (PRF) is important for early parent-infant relationships, its role in mental health and attachment relationship outcomes of infants admitted to NICU and their parents has not been evaluated. The prospective “Reflecting on Babies in NICU” (ROBIN) study aimed to determine whether PRF was measurable in NICU and predicted subsequent mental health problems. Parents of infants who were at least 3 weeks of age and admitted to a quaternary NICU at the Royal Children’s Hospital Melbourne without ever having been home were prospectively enrolled. PRF was evaluated using the Parental Reflective Functioning Questionnaire (PRFQ) and, in a nested substudy, a specifically developed NICU version of the Parent Development Interview (PDI:NICU). Acute stress disorder (ASD) and post-traumatic stress disorder (PTSD) were classified according to the Acute Stress Disorder Scale (ASDS) and Posttraumatic Stress Disorder Checklist (PCL-5) respectively. The association between baseline PRF and parent self-reported feelings of attachment, measured by the Maternal, and Paternal, Postnatal Attachment Scales (MPAS/PPAS), and parental emotional functioning, was evaluated both while the infant remained in NICU and at 10 months of infant-age. Evaluable data were available for 69 infants (67 mothers and 38 fathers) enrolled between July 2014 and April 2016. Baseline PRFQ subscale scores were similar to previously published studies. Baseline PRF scores from the PDI:NICU were available for 19 mothers. The mean overall score was 5.4 (SD 1.1, range 4-7), and the pattern of individual demand question scores was similar to previously published profiles. This indicates that PRF is measurable in the NICU-setting. Paired PRFQ results at baseline and at 10 months were available for 57 parents. For mothers, the PRFQ subscales correlated closely over time, whereas for fathers only the Pre-Mentalizing Mode subscale was correlated. This suggests that the PRF being measured in NICU may be an enduring trait, rather than a state influenced by the acute condition, more for mothers than fathers. PRF at baseline was highly correlated with self-reported attachment-quality at 10 months. Specifically, high PRFQ Pre-Mentalizing Mode (PM) subscale scores in NICU predicted dyads with subsequent “at risk” attachment, implying that parents with poorer PRF in NICU are more likely have parent-infant relationship difficulties as their child grows. Acute stress disorder affected 38% of mothers and 41% of fathers at baseline. Higher baseline PRF, especially the PRFQ Interest and Curiosity about Mental States (IC) subscale, was associated with risk of concurrent ASD. Higher baseline IC scores were also associated with risk of PTSD at 10 months in mothers. These findings present a dilemma. Lower capacity for PRF in NICU may jeopardise the developing parent-infant relationship, but higher PRF may create emotional trauma for parents. Infant Mental Health resources and a “nested mentalization” approach to care are vital for infants and their at-risk parents in NICU, so they can be held “in mind” in supported ways.

Introduction: Crohn’s Disease (CD) is a chronic, relapsing condition of the human gastrointestinal system with several significant extraintestinal manifestations. It can affect any age group; 10% of patients being diagnosed prior to their 18th birthday. Pediatric CD is known to have a more severe and difficult to manage phenotype. A rising incidence has been observed in recent years although the disease phenotype has remained largely unchanged. There is a growing body of literature describing dysbiosis in patients with CD. Despite extensive research, the role and behaviour of the gut microbiome in pediatric CD is not well understood. Aims: We aimed to characterize the microbiome in patients with CD longitudinally and compare it with non-IBD patients. We also sought to explore if there were any microbiome differences between ASCA positive and ASCA negative patients. Methods: We analysed the microbiome of 345 biopsies from 204 patients, including 88 CD first diagnosis (CDFD) patients, 38 patients in relapse (CDRL), 12 patients in remission (CDRM), and 66 controls. Species identification was conducted using oligotyping in combination with ARB/SILVA taxonomic annotation. Comparison was also made between ASCA status, microbial diversity and clinical characteristics. Results: We observed 45 bacteria to be statistically different between CDFD samples and controls, with Fusobacterium being the most implicated species in CDFD patients. We also identified gender specific differences in CD. Five species showed a strong association with patients with CD in relapse and 10 species in patients with CD in remission. Three taxa showed a positive co-occurrence across the two groups. Hespellia porcina (closest taxonomic neighbor to Clostridium oroticum) had the strongest association with samples from patients with CD in relapse. Interestingly, Fusobacterium was not part of the CD relapse associated taxa group. Faecalibacterium prausnitzii was equally present in CDFD and in control samples. ASCA was highly specific but poorly sensitive for the diagnosis of CD. In patients with CD, ASCA positivity was more likely to be present in patients older than 10 years, and associated with increased likelihood of ileocolonic disease distribution and long-term risk of surgery. Microbial alpha and beta diversity were similar in patients with CD with or without ASCA, but significantly less when compared to non-IBD controls. 14 bacterial species were statistically associated with ASCA positive patients with CD and 14 species with ASCA negative patients (p< 0.05). By using a false discovery rate corrected P value, two species remained statistically associated with both the groups. Ruminococcus torques and bacterium Yersinia enterocolitica were statistically associated with CD ASCA positive patients (p = 0.0178). Enterobacter cloacae and Faecalibacterium prausnitzii were statistically associated with CD ASCA negative patients (p = 0.0178 and 0.0342, respectively). Conclusions: This is the first study to investigate gut mucosal microbiome in a pediatric CD cohort with longitudinal sampling. Significant differences in microbiome were observed between treatment naïve patients with CD, patients with CD in relapse, patients with CD in remission and non-IBD patients. We also identified differences in the gut microbiome between patients with CD depending on presence of ASCA.

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.