Genetic kidney diseases are a heterogeneous group of disorders with varying phenotypes dependent on the affected nephron segment. Next generation sequencing has increased our appreciation of the breadth of gene variants associated with these diseases. It has also identified large numbers of variants of unknown significance (VUS), which require functional genomic validation. There is an unmet need for novel therapies for genetic kidney diseases as most invariably progress to dialysis or transplantation without any form of targeted treatment. Laboratory based research of genetic kidney disease requires the recapitulation of a disease phenotype in animal and/or in vitro cellular disease models. Interspecies variation in anatomy, physiology and gene function limits the translation of animal models to human disease and clinical care. Classical two dimensional cell cultures lack the complexity and intercellular cross-talk of in vivo three dimensional tissue. Kidney organoids are three dimensional, miniature, multicellular, human, in vitro micro-tissues, offering distinct disease modelling advantages over other models. Furthermore, kidney organoids can be regenerated from induced pluripotent stem cells (iPSC) reprogrammed from patients with genetic kidney disease, potentially providing outcomes with personalised clinical relevance. As a novel platform, the capabilities and limitations of kidney organoids as disease models are not well understood. By differentiating and characterising kidney organoids from the iPSC of patients with inherited kidney diseases, this thesis aims to explore the application of kidney organoids to disease modelling. As proof of concept, kidney organoids were first generated from iPSC reprogrammed from a patient with compound heterozygous variants in IFT140, an already validated nephronophthisis (NPHP) genotype. An isogenic control was generated by precision CRISPR-Cas9 gene editing. In this project, differential primary ciliary morphology within organoid tubules and transcriptional profiling of organoid epithelium validated the ability of the organoids to model genetic disease. Attempts were then made to validate novel, candidate variants for other pedigrees with unresolved trio whole exome sequencing. In a proband with clinically suspected NPHP, DNAH5 was selected as a candidate gene, despite previously association with a motile ciliary phenotype. In this project, kidney organoids were unable to validate the patient variant as pathogenic. In addition, a number of lessons were learned regarding the necessary variant curation process prior to making a commitment to modelling with kidney organoids. In the final chapter, kidney organoids validated a novel genotype for the glomerular disease steroid resistant nephrotic syndrome, via international collaboration with the laboratory of Prof Friedhelm Hildebrandt. Glomeruli within kidney organoids differentiated from iPSC expressing a patient-derived, homozygous variant in NOS1AP, displayed aberrant development, increased podocyte apoptosis and reduced expression of PAR polarity proteins. Together these projects demonstrate the strengths and challenges of using kidney organoids as models of inherited renal disease. Kidney organoids stand to complement animal and 2D unicellular disease models rather than replace them. We proposed that patient-derived kidney organoids are best placed to model paediatric onset kidney diseases with the future potential of providing personalised therapeutic screening.

Transient Receptor Potential Vanilloid 4 (TRPV4) is a non-selective calcium channel that plays an important role in the mechanotransduction system in chondrocytes. Heterozygous TRPV4 mutations cause skeletal disorders with varying severity. Heterologous cells such as fibroblasts and HEK-293 cells are commonly used to model TRPV4-inherited skeletal diseases in vitro. Studies using human chondrocytes are limited because cartilage is rarely available from patients and controls. Although heterologous cells cannot completely recapitulate the biological processes occurring in human chondrocytes, the studies show that two distinct disease phenotypes, TRPV4 skeletal dysplasia and arthropathy, might be caused by differences in the way the mutations change TRPV4 channel behaviour. TRPV4 skeletal dysplasia causing mutations show channel over-activity whereas arthropathy causing mutations show reduced channel activity upon channel stimulation. However, the downstream pathogenic mechanisms responsible for the distinct skeletal phenotypes remain undefined. Recognising the limitations of previous studies, human-induced pluripotent stem cells (hiPSCs) offer a new approach for inherited disease modeling. Their ability to differentiate into disease-relevant cells such as chondrocytes creates new opportunities for TRPV4-inherited skeletal disease modelling. Therefore, this PhD project aims to model the disorders caused by two TRPV4 mutations using hiPSCs and identify the pathogenic mechanisms underlying the two distinct TRPV4-inherited skeletal disease phenotypes. To obtain disease-relevant cells, establishing a robust and reproducible chondrocyte differentiation protocol is required. To do this, a reporter hiPSC line, SOX9-T2A-tdTom, was generated from MCRIi001-A (PB001.1) (1) using CRISPR/Cas9 genome editing. Thus in vitro chondrocyte differentiation could be monitored in real-time. The T2A linker and tdTomato fluorescent reporter gene were inserted downstream of the SOX9 coding sequence through homology-directed repair. The targeted allele was designed to produce SOX9 with the T2A sequence at the C-terminal end and a separate tdTom fluorescent protein. Genomic DNA sequencing of the SOX9-T2A-tdTom hiPSC line confirmed that the hiPSC line had one SOX9 allele with the T2A tdTom gene fusion and one wild type allele. Pluripotency was maintained as indicated by expression of pluripotency markers OCT4 and NANOG (immunostaining); CD9, CD326, and SSEA-4 (flowcytometry); and the ability to form tissues derived from three germ layers. SNP array showed there were no aneuploidies. The SOX9-T2A-tdTom hiPSC line had a similar capability to the parental line, MCRIi001-A, to form sclerotome. Western blotting showed that SOX9 protein expression was similar between SOX9-2A-tdTom and its parental line suggesting that adding tdTom gene sequence downstream of SOX9 gene did not disrupt the SOX9 expression and stability. The chondrocyte differentiation protocol was established using the SOX9-T2A-tdTom hiPSC line. Two stages of differentiation were performed. First, sclerotome induction was achieved by culturing hiPSCs in a 6-day multiple-step chemically defined culture mimicking embryonic development with pellet culture format was established on day 4. Secondly, chondrocyte differentiation was performed by transferring day-6 pellets into chondrogenic media in swirling culture format up to 10 weeks. A 4-week course of FGF2 treatment followed by an optional TGFB3 and GDF5 treatment until week 10 was performed during chondrocyte differentiation. RNA was collected every day during a 6-day sclerotome induction and at different time points during chondrocyte differentiation. The optimised protocol that involved a multiple-step chemically-defined 3-dimensional (3D) culture with swirling in an extended culture that included a 4-week FGF2 supplementation and optional subsequent TGFB3 and GDF5 treatment was able to generate cartilage that closely resembles fetal cartilage. CRISPR/Cas9 genome editing was also used to introduce two human TRPV4 mutations, a TRPV4 c.819C>G (p.F273L) mutation causing familial digital arthropathy with brachydactyly (FDAB) and a TRPV4 c.2396C>T (p.P799L) mutation causing metatropic dysplasia, into the SOX9-T2A-tdTom hiPSC line. For in vitro disease modelling, the mutant and their isogenic wild-type control (SOX9-T2A-tdTom) hiPSC lines were differentiated towards chondrocytes using optimised chondrocyte differentiation. The phenotypic differences between mutants and wild-type were assessed using various techniques including gene (RNA sequencing) and protein expression analysis. The two mutant cell lines and their isogenic wild-type control (SOX9-T2A-tdTom) were able to form cartilage. The pellet cartilage histology did not show any striking differences between the two mutants and their isogenic control. COL2A1 and TRPV4 protein expression was similar between mutants and control and this was consistent with the RNA sequencing data. RNA sequencing suggested that the pathogenic mechanisms underlying the two distinct TRPV4-inherited skeletal diseases were different. Compared to the isogenic control, F273L mutant cartilage had 263 differentially expressed genes. F273L cartilage showed a slight reduction in cartilage related gene expression including COL2A1, CSPG4, BGN, and CILP2. The F273L cartilage tissue was also less mature than the wild-type as indicated by increased SHH expression. On the other hand, P799L cartilage had more differentially expressed genes (655 genes) than F273L. MEF2C, the main regulator of chondrocyte hypertrophy, was upregulated in P799L. The hypertrophic chondrocyte markers such as RUNX2, SPP1 or osteopontin, and PTH1R, were also upregulated in P799L suggesting increased chondrocyte hypertrophy of P799L chondrocytes. The other characteristics of hypertrophic chondrocytes such as a reduction in cell proliferation and increased apoptosis were also observed in P799L cartilage. In conclusion, this study is the first study that conducts global gene expression analysis using RNA sequencing to characterise gene expression changes downstream of TRPV4 mutations in hiPSC-derived chondrocytes. The pathogenic mechanisms underlying the two distinct TRPV4-inherited skeletal diseases are different. The fewer differentially expressed genes in the F273L cartilage than in P799L suggests a milder disease phenotype. The slight reduction in cartilage marker expression in F273L cartilage might cause the cartilage tissues less resilient to physical forces thus leading to FDAB. In contrast, accelerated chondrocyte hypertrophic maturation can be the pathogenic mechanism underlying TRPV4 skeletal dysplasia phenotype. Accelerated chondrocyte hypertrophic maturation can disrupt growth plate development and cause systemic skeletal defects seen in patients. This thesis demonstrates that hiPSCs are a powerful tool to model inherited skeletal disease in vitro.

There is now considerable evidence indicating that risk of many complex diseases in adulthood may be influenced by exposure to environmental exposures in utero. A growing number of studies suggest epigenetic markers, including DNA methylation, are involved in this process. Understanding how DNA methylation is impacted by pregnancy exposures, and related to later health, may both contribute to unravelling the aetiology of complex disease risk in later life and provide a potential early-life biomarker for risk prediction. However, current evidence is limited. There has been a predominance of small, poorly powered studies, failure to consider the effects of genetic variation, and limited replication of previous findings. In addition, previous studies investigating the relationship between DNA methylation and offspring health have been primarily cross-sectional. For these reasons, I investigated the associations between pregnancy exposures (in particular, maternal smoking, nutrition and metabolic health, psychosocial stress, and adverse pregnancy conditions), birth outcomes, and offspring blood DNA methylation of the insulin-like growth factor 2 (IGF2) and H19, hypoxia-inducible factor 3A (HIF3A), leptin (LEP) genes. I also considered how genetic variation impacted on these associations. I then investigated the longitudinal relationship between early life methylation and anthropometry, as well as the association between early life methylation and later childhood measures of weight, adiposity, and cardiovascular health. To do this, the large, population-based longitudinal Barwon Infant Study pre-birth cohort (n=1,074) was used, with clinical and questionnaire measures from 28 weeks pregnancy, birth, 12 months post-birth and 4 years post-birth time points. DNA methylation of candidate regions was measured using the Sequenom EpiTyper mass-spectrometry platform in cord (birth) and peripheral (12-month) blood. Infant genetic variation in and near the candidate genes was considered. Infant adiposity was assessed as sum of triceps and subscapular skinfold thicknesses in infancy, and with DEXA scanning at 4 years of age. We found evidence that exposure to maternal psychosocial stress, gestational diabetes, and pre-eclampsia was associated with differences in offspring methylation at the candidate regions, as was infant sex. Genetic variation showed strong effects on DNA methylation levels, with some evidence for the associations of pre-eclampsia and infant adiposity with LEP methylation differing by infant genotype. Early life methylation of HIF3A and LEP showed modest associations with four-year blood pressure and BMI, respectively. While these associations persisted with adjustment for potential confounding factors, they explained relatively little variance in the four-year phenotypes compared to traditional predictors, such as weight. These findings suggest that offspring DNA methylation of these candidate genes involved in regulation of growth and metabolism are sensitive to several environmental exposures and genetic factors. While there is modest evidence for methylation in infant blood associating with later phenotypes, methylation of these genes appears unlikely to have useful predictive utility in isolation. This study is the first to perform early life longitudinal analysis to investigate the association between anthropometry and methylation in infancy. It is also the first to report evidence of earlier methylation associating with later cardiovascular phenotypes. However, as gene expression data was not available, the functional consequences of the altered methylation observed in blood is unclear. Further work is required to replicate these findings in independent cohorts, to determine the nature of expression of these genes in blood, and to investigate if the relationship between early life methylation and later health persists into adulthood.

Kidneys are uniquely vulnerable to drug toxicity due to their role in filtering substances from the blood. Filtration and urine production takes places in specialised tubules called nephrons, which are divided into segments with functionally distinct cell types that express transporters and receptors for transporting water, solutes, or small molecules from the blood into the filtrate, or from the filtrate into the cells. Should a drug have an affinity for these transporters, it can accumulate within these cells and cause injury. As a result, there are many commonly-used pharmaceutical compounds which are nephrotoxic to some degree, which can lead to acute kidney injury in patients. The nephron segment called the proximal tubule is the most frequently affected, as proximal tubule cells express a wide array of transporters and receptors which can transport drugs, and have a high metabolic rate which is vulnerable to disruptions in cellular energy production. Developing new drugs which are less nephrotoxic has been challenging, as the in vitro assays and animal tests currently used to screen drugs in development for nephrotoxicity have decreased transporter and receptor expression compared to mature human proximal tubules in vivo. Recent advances in understanding kidney development in the embryo have led to protocols for differentiating human pluripotent stem cells to kidney organoids, which contain segmented nephrons that have proximal tubules. Hence, kidney organoids may be useful for in vitro nephrotoxicity screening during drug development if they show sufficient nephron maturation. Through immunofluorescence and qRT-PCR analysis, we found that while proximal tubules in organoids expressed some proximal tubule markers to a greater degree than primary proximal tubule cells cultured in 2D, expression was still low overall compared to foetal kidney, and some transporters were absent. We then performed a bioinformatics analysis of single-cell RNA sequencing datasets generated from foetal and adult mouse and human kidneys to find signalling pathways which changed during proximal tubule maturation. Among other changes, mature proximal tubule cells showed increased fatty acid oxidation, a decreased capacity for glycolysis, and decreased TGF-beta/BMP signalling, suggesting that inducing these changes in vitro might improve proximal tubule maturation in organoids. To aid our characterisation of proximal tubules in organoids, we developed two reporter lines, LRP2:mTagBFP2 and HNF4A:YFP, for genes which are highly expressed in the developing proximal tubule, and confirmed that these genes and their respective reporters were co-expressed in the proximal tubules of kidney organoids. We also developed a HAVCR1:mCherry reporter line for future use in detecting injury responses in kidney organoids. These reporter lines allowed live monitoring of proximal tubule development and function. Using the HNF4A:YFP reporter, we screened for culture conditions which improved proximal tubule maturity in organoids and determined that a low-glucose, insulin-free medium designed to induce fatty acid oxidation supplemented with a small molecule inhibitor of TGF-beta improved the expression of proximal tubule markers in organoids, consistent with our bioinformatics analysis. We also performed xenotransplantation of reporter organoids into immunocompromised mice to determine whether proximal tubules would mature in this environment, and found that while non-renal tissue would proliferate in the grafts, proximal tubules in transplanted organoids maintained reporter expression better than organoids maintained in vitro under standard conditions. Taken together, this thesis characterises proximal tubule maturation and has begun to optimise this process within kidney organoids using novel reporter tools. This represents a significant advance in facilitating improved screening of drug-induced nephrotoxicity using organoids in the future.

The t(8;21) translocation generates the aberrant transcription factor RUNX1-ETO and occurs in approximately 10% of all acute myeloid leukaemias. RUNX1-ETO transcripts can be detected in utero and in cells of patients in remission, but its sole expression is insufficient to cause overt leukaemia. Given that t(8;21) patient cells present additional mutations, the epigenetic reprogramming directly mediated by RUNX1-ETO remains unclear. To address this question, we generated human Embryonic Stem Cell lines carrying an inducible RUNX1-ETO transgene, which we subsequently differentiated into definitive haematopoietic progenitors. We show that induction of RUNX1-ETO in already formed progenitors (i) blocks differentiation at an immature stage, (ii) induces a cell-type specific and reversible cell cycle arrest, (iii) abrogates the RUNX1-mediated gene expression program by interfering with RUNX1 binding, resulting in downregulation of haematopoietic, cell cycle as well as DNA repair genes, (iv) closes down a large part of the chromatin accessibility pattern present in adult haematopoietic multipotent progenitors and (v) alters the differentiation of a defined sub-population of progenitors. Our data are consistent with the idea that RUNX1-ETO establishes a precondition for leukaemic transformation by maintaining a reservoir of quiescent pre-leukaemic multipotent progenitors with susceptibility to expand upon acquisition of additional oncogenic events.

BACKGROUND In low- and middle-income countries (LMIC) where most of the 15.1 million neonates who survive prematurity and serious illness every year reside, neurodevelopmental outcome data are scarce and needed to improve neonatal care including follow-up, developmental monitoring and early intervention. In Fiji, neonatal health policy has shifted from almost exclusive focus on neonatal survival to ensuring that children are also supported to reach their developmental potential. METHODS AIM To examine early childhood neurodevelopmental and health outcomes for neonatal intensive care unit (NICU) survivors in Fiji, to inform improvements in neonatal care and follow-up including developmental monitoring and early intervention. OBJECTIVES These were, to: 1. Systematically review existing neonatal outcomes studies in LMIC to understand knowledge gaps related to neurodevelopmental outcomes for high-risk neonates and inform research design and sample size estimates for our neonatal outcomes study 2. Assess early childhood neurodevelopmental and health outcomes for neonates discharged from the Colonial War Memorial Hospital (CWMH) NICU in Suva, compared with control, term neonates born in the same hospital 3. Evaluate accuracy of nurse-led developmental screening as a potential tool for early identification of developmental delay and impairment in high-risk neonates, compared with reference standard developmental assessment RESULTS Systematic review of 60 high-risk neonatal outcome studies in LMIC provided estimates of median prevalence of neurodevelopmental impairment (NDI) for survivors of prematurity/very low birth weight (VLBW) and ‘birth asphyxia’. This highlighted a need for studies that better describe multi-domain neurodevelopment, include hearing and vision and including control data. Our neonatal follow-up study in Fiji compared early childhood outcomes for high-risk NICU patients (n=149) with those of matched term, normal birth weight neonates (n=147) discharged from Colonial War Memorial Hospital between November 2008 and April 2010. NDI was defined as at least one of; cerebral palsy, moderate to severe hearing or visual impairment, or global developmental delay using Bayley Scales of Infant and Toddler Development Third Edition (i.e. score <70 in at least two of cognitive, language or motor domains). At median (IQR) age 36.1 (28.3, 38.0) months, prevalence of moderate to severe NDI % (95% CI, n) in high-risk and control groups was 12 (5 to 17, n=13) and 5 (2 to 12, n=5), respectively, an increased risk ratio (95% CI) of 2.7 (0.8 to 8.9. Risk factors for NDI were identified. Our nurse-led developmental screening study demonstrated that, while feasibility of a parent-report screening tool was attractive, sensitivity for detection of global developmental delay was poor compared with the reference standard. CONCLUSION Our research provided the first neonatal neurodevelopmental outcome data in Fiji, adding to limited international literature on neonatal neurodevelopmental outcomes in LMIC. Measurement challenges highlight a need for international collaboration to improve measurement to better understand neonatal outcomes beyond survival. Negative findings of our developmental screening study indicate a need for longitudinal research to establish developmental monitoring approaches which facilitate early identification of developmental delay and impairment in routine services. Implementation research is also needed to develop innovative models of early intervention which support high-risk neonates in LMIC to thrive.

Background: Children raised in families experiencing adversity (e.g. unemployment, financial hardship, family violence, parent mental health difficulties) are at greater risk of poor health outcomes. Physiological stress is one mechanism thought to explain the effects of early adversity on children's health. Understanding this role of physiological stress and how best to measure it, particularly in population cohorts of young children, is limited. My PhD used child hair cortisol as a measure of physiological stress to address this evidence gap by examining associations between adversity, physiological stress and health in a community-based cohort of young children experiencing adversity. Aims: To investigate 1) whether indicators of adversity are associated with children's physiological stress at 2 years; 2) the role that maternal physiological stress and parenting behaviours play in explaining children's physiological stress response to adversity at 2 years; and 3) whether social adversity is associated with children's health at 3 years, and whether physiological stress is a mechanism which mediates these effects. Methods: This was a prospective longitudinal cohort study nested within the ‘right@home’ randomised controlled trial of nurse home visiting, comprising 722 Australian women recruited for their experience of adversity while pregnant and their subsequent children. Child hair cortisol was collected at ages 2 and 3 years. Social adversity was measured as maternal reported indicators of adversity according to sociodemographic and psychosocial characteristics, collected at pregnancy and child ages 1, 2 and 3 years. Maternal physiological stress (hair cortisol) and parenting were measured at 2 years. At 3 years, child health outcomes of mental health, wellbeing and weight status were assessed. Analyses for Aims 1 and 3 used linear regression models; for Aim 2 used structural equation modelling. Results: Hair cortisol data were available for 319/603 (53%) participating children at 2 years and 297/500 (59%) at 3 years. Aim 1 showed limited evidence of associations between 18 indicators of adversity and child hair cortisol at 2 years across different types, timing and persistence of adversity. Aim 2 showed that maternal and child hair cortisol were associated with one another at 2 years. There was no evidence that adversity or maternal parenting were associated with child hair cortisol; as such there were no pathways by which maternal stress or parenting explained effects of adversity on children’s physiological stress. Aim 3 showed children experiencing greater adversity had higher externalising behaviour problems and poorer physical wellbeing at 3 years, and higher hair cortisol was associated with higher externalising problems; however, there was no evidence that hair cortisol mediated any effects of adversity on health. Conclusions: At ages 2 and 3 years, social adversity was not consistently associated with children’s physiological stress, measured using hair cortisol, nor was there any evidence that physiological stress mediated the effects of adversity on young children’s health. These findings suggest that hair cortisol may be limited as a measure of stress in young children or that measuring one aspect of the physiological stress pathway at the population level provides limited insight into the complex mechanisms underlying children’s health inequalities.

The Ventx genes are a family of non-clustered homeobox transcription factors that confer a ventral phenotype on mesodermal cells in the developing embryo. Ventx genes are conserved in vertebrates but have been lost in rodents. Studies in Xenopus have proposed Ventx as a transcriptional repressor during early mammalian embryo development. Although not much is known about the expression of this gene in humans, studies have shown that VENTX promotes myeloid differentiation in the human haematopoietic system and it is expressed in some acute myeloid leukaemias, including those with a RUNX1-ETO translocation. To further explore its function during mesoderm patterning and differentiation, we used a human pluripotent stem cell (hPSC) reporter line in which a GFP reporter marked the expression of VENTX. Surprisingly, differentiation studies revealed that early expression of VENTX identified a subset of early primordial germ cells (PGCs). Following these results, we used a double reporter hPSC line in which a mCHERRY reporter marked the pluripotency gene OCT4 and a third line in which the homeodomain of VENTX was deleted, creating a VENTX functional knockout. Human PSCs from the double reporter line and VENTX knockout line were differentiated into VENTX expressing PGC-like cells (PGCLCs). Transcriptional profiling of GFP-expressing cells from both cell lines confirmed expression of early PGC associated genes, including BLIMP1, PRDM14, SOX17 and TFA2PC. Examination of the RNA-sequencing data confirmed that VENTX is expressed in a subset of human PGCLCs, however it did not reveal significant transcriptional consequences when VENTX was deleted. These results suggest that VENTX is not required for early human PGC ontogeny, but does not exclude a role later in human PGC development. We also used a doxycycline (DOX) inducible VENTX expression system to investigate the role of VENTX during haematopoietic development. We found that VENTX overexpression in hPSC impaired mesoderm formation, but enforced VENTX expression after mesoderm commitment resulted in the emergence of blood cells that co-expressed the cell surface markers CD90, CD34 and CD43, identifying them phenotypically as immature haematopoietic cells. These cells displayed high clonogenic capacity in methylcellulose, but only after DOX was removed from the medium. This suggested that VENTX expression held cells in an immature but non-proliferative state. Transcriptional profiling revealed increased expression of HOXA genes in haematopoietic cells following VENTX induction, consistent with their immature phenotype. Conversely, genes involved in myeloid differentiation were downregulated during VENTX overexpression, as were genes involved in proliferation, such as MYC and MYB. Chromatin accessibility examination using ATAC-sequencing demonstrated that VENTX closed selected chromatin loci, in particular areas targeted by homeobox genes like HOXB13 and CDX transcription factors. Overexpression of VENTX also closed areas of chromatin found in promoter regions of homeobox genes. We hypothesise that VENTX acts as a transcriptional repressor during differentiation, preventing haematopoietic proliferation and differentiation into myeloid lineages, and leading to the retention of quiescent immature blood cells. We have initiated experiments to investigate possible genomic targets of VENTX, combining ATAC and ChIP-sequencing with transcriptional profiling. Our preliminary findings have revealed few VENTX DNA targets to date. Finally, we have initiated studies to explore the possible synergy between VENTX and RUNX1-ETO by building a cell line expressing inducible versions of both genes.

Immunisation is the most cost-effective life-saving medical intervention and is estimated to save at least 2.5 million lives each year. However, there is substantial variation between individuals in the immune response to immunisations. This has consequences for both protective efficacy and duration of protection. My PhD research project focused on evaluating factors that influence antibody responses to routine immunisations given in infancy. The first section of my thesis focuses on intrinsic, perinatal and external factors that might influence responses to immunisation in infancy. Within this, I investigated the effect of maternal immunisation during pregnancy. Antenatal diphtheria-tetanus-acellular pertussis (dTpa) was associated with significantly reduced infant antibody responses to both specific (diphtheria and pertussis) and heterologous (polio and pneumococcal) vaccines. In contrast, maternal influenza immunisation had minimal effect on infant vaccine responses. I also investigated the effect of early-life factors, namely sex, delivery mode, breastfeeding and antibiotic exposure on antibody responses to routine immunisations given in the first year of life. While there were some differences between males and females in antibody responses to routine immunisations in the first year of life, delivery mode, breastfeeding and antibiotic exposure did not exert a substantial influence. The second section of my thesis focuses on the ‘non-specific’ or immunomodulatory effects of the Bacillus Calmette-Guerin (BCG) vaccine, one of the most widely used vaccines worldwide, and specifically, how it influences vaccine responses to heterologous (routine) immunisations. It comprises a systematic review of studies that investigated the effect of previous or co-administered BCG on heterologous vaccine responses in infants and adults, and an original study in which I investigated the effect of BCG immunisation at birth on antibody responses to routine infant vaccines. In this study, although not statistically significant, antibody responses in BCG-vaccinated infants were consistently higher against diphtheria, tetanus, pneumococcal, measles and mumps antigens, but lower against Haemophilus influenzae type b compared to BCG-naive infants. These findings add to the evidence that BCG immunisation at birth has broad heterologous effects on the infant immune system. The third section of my thesis focuses on the correlation between the response to different vaccines, for which there was previously no data available. I found that correlation between antibody responses to similar antigens in the same vaccine (such as different serotypes of a bacterium or virus), as well as responses to antigens conjugated to similar carrier proteins are strong. In contrast, correlation between responses to other vaccines are weak and there is a negative correlation between responses to measles-mumps-rubella vaccine antigens and non-live vaccine antigens. There was also a weak correlation between antibody responses to vaccines of the same type (e.g. polysaccharide or toxoid vaccines). This has important clinical implications as it means the current practice of measuring antibody responses to one or a few vaccine antigens does not offer a reliable surrogate marker of responses to unrelated vaccines. Recognising factors which might influence vaccine antibody responses, is important for the design of vaccine studies and decisions on vaccine schedules. It also offers ways to optimise vaccine immunogenicity and effectiveness, as well as the possibility to individualise immunisation. The complex interplay between different components of the immune system in vaccine responses is not yet fully understood, and future studies should focus on additional factors, including the innate and cellular immune system.

Acute lymphoblastic leukaemia (ALL) is the most common form of paediatric malignancy and while the prognosis for patients has dramatically improved, specific molecular subtypes are still associated with inferior outcomes. Comprehensive sequencing studies of large cohorts have identified an expanding number of recurrent drivers associated with B-cell ALL (B-ALL), notably gene fusions, and has resulted in the characterisation of 23 subtypes. Associated risk of relapse has been established for many of these molecular subtypes and is incorporated into clinical risk stratification algorithms. However, molecular features of T-cell ALL (T-ALL) are yet to be incorporated into these algorithms. For a subset of B-ALL patients and the majority of T-ALL patients there is a great need to expand of molecular diagnostic testing to identify recurrent molecular features or potentially actionable lesions. In this thesis, we aimed to identify novel and rare fusion genes in paediatric ALL using RNA sequencing (RNA-seq) and understand the mechanisms by which these fusions function to drive leukaemia. We performed RNA-seq on 126 ALL patients diagnosed at the Royal Children’s Hospital (RCH) to test the utility of implementing RNA-seq into standard of care diagnostic pipelines. We showed that RNA-seq reliably detected gene fusions identified by clinical diagnostics but has limitation to detect gene fusions in samples with low tumour burden, lowly expressed fusion transcripts (KMT2A rearrangements), and rearrangements involving promotor or enhancer regions (IGH rearrangements). We additionally developed analysis tools to identify IKZF1 deletions and a gene expression classifier to predict Ph-like, ETV6-RUNX1+, and ERG-deleted/DUX4 rearranged ALL, and showed that we could use RNA-seq data to molecularly classify patients that did not express standard lesions. This chapter of the thesis is accompanied by supporting material contained in "Supplementary_table_1.xlsx", which details transcript information for fusion genes identified by the fusion-finding algorithm, JAFFA, in this patient cohort. Using RNA-seq data, we identified a number of rare and novel tyrosine-kinase activating fusion genes, which are potentially therapeutically targetable with tyrosine kinase inhibitors (TKIs). We cloned and validated the transforming capacity of these fusions in cytokine-dependent cell lines and tested response to therapies. These included a rare CNTRL-FGFR1 fusion that we identified in two patients with biphenotypic leukaemia, and two rare ABL1 fusions, SFPQ-ABL1 and SNX2-ABL1, identified in two patients with B-ALL. We showed that the structure of the CNTRL-FGFR1 fusion differed from what was previously described, and that the fusion is transcribed from exon one of CNTRL and undergoes alternative splicing. We confirmed that cells expressing the full-length form of CNTRL-FGFR1 were sensitive to TKIs that targeted FGFR1, notably ponatinib. In addition, we designed a Droplet Digital PCR (ddPCR) assay that detects CNTRL-FGFR1, using transcript-specific primers and probes, down to approximately 1 cell in 100,000 (0.001%). This level of sensitivity suggests that this assay could be utilised clinically for minimal residual disease (MRD) monitoring. Given little is known about the biological function of rare ABL1 fusions, including SFPQ-ABL1 and SNX2-ABL1, we utilised cell viability and proliferation assays to compare the function of these fusions to BCR-ABL1. Cells expressing SFPQ-ABL1 and SNX2-ABL1 have reduced proliferative capacity, reduced sensitivity to TKIs targeting ABL1, and different subcellular localisation, compared to BCR-ABL1. We performed phosphoproteomics to understand signalling differences between SFPQ-ABL1 and BCR-ABL1. We showed that proteins involved in transcriptional regulation and spliceosome were enriched in SFPQ-ABL1, suggesting that this fusion may in part function by altering gene transcription and RNA-splicing. In this thesis we show that RNA-seq is a valuable tool for the identification of molecular drivers of ALL including gene fusions and IKZF1 deletions. Our data suggests that RNA-seq is most valuable for the molecular classification of patients that do not harbour standard lesions. Our functional studies show that the 5’ fusion partner of tyrosine kinase fusion genes may play a role beyond facilitating dimerisation and activation of the kinase and may mediate subcellular localisation and contribute to signal transduction. In all, the fusion identification and biology pipeline we have developed has the potential to inform clinical management of patients, to both tailor treatment and design molecular assays to track MRD.

Acute lymphoblastic leukaemia (ALL) takes up the highest percentage of paediatric cancer. The treatment requires intensive chemotherapy for two to three years, as well as haematopoietic stem cell transplantation for poor-prognosis cases. Compared to B cell lineage ALL (B-ALL), T cell lineage ALL (T-ALL) has a lower 5-year event free rate, higher rate of relapse, and a worse outcome for relapsed cases. Individualised therapy, targeting at oncogenic changes in each patient, can make treatment more effective and less harmful. This requires understanding of the oncogenic biology of each individual leukaemia. We have attempted to develop a T-ALL model based on hiPSC-derived T cells, which will be in human origin, maintain normal genetic pattern, mimic in vivo T cell development, and can be massively produced for high throughput lab work. This model may make up for the shortcomings of conventional leukaemia cell lines and mouse models. This project investigates the biology of T-ALL by focusing on two novel fusion genes – TCF7-CSF1R and ETV6-CRX – identified by RNA sequencing of paediatric T-ALL patient samples. We have shown that TCF7-CSF1R is sufficient to immortalise mIL-3 dependent Ba/F3 cells. The ETV6-CRX fusion gene is anticipated to block differentiation. Establishing consistent expression of this fusion will require further optimization. The feasibility of setting up a hiPSC-derived T-ALL model was also assessed, with respect to protein expression in human T-ALL/lymphoma cell lines, hiPSC differentiation efficiency, hiPSC-derived T cell lentiviral infection rate, and cytokine withdrawal during differentiation. This project provides potential directions for improvement of methods for exogenous gene expression, such as the usage of CRISPR-Cas9 based techniques to introduce gene modifications for fusion genes such as ETV6-CRX that are difficult to express, particularly in hiPSC-derived T cells that have a low viral infection level. The T cell differentiation protocols also need to be optimised to make the T cell production easier and efficient. Detailed functional assay during T cell differentiation needs to be conducted in the future. In this thesis, Chapter 1 presents the background of this project; a literature review introducing human haematopoietic system, in vivo thymocyte development, paediatric TALL, novel oncogenic fusion-related genes, and in vitro T cell generation; the aims and hypothesis. Chapter 2 introduces the methods and materials used in this project. Chapter 3 presents the identification, cloning, and expression of novel fusion genes. Chapter 4 investigates the ability of these novel fusion genes to support cell survival and proliferation in conventional Ba/F3 cell line. Chapter 5 assesses the feasibility of setting up the hiPSC-derived T-ALL model. Chapter 6 makes a discussion on the results and concludes the whole project.

Background: The Fontan procedure was developed approximately 50 years ago and has become the definitive treatment for children born with single ventricle physiology. Surgical refinement of the Fontan technique and improvements to critical care have led to a dramatic reduction in mortality, with the vast majority of patients expected to live well into adulthood. The reduction in mortality has led to a shift in research efforts in the Fontan population towards defining the long-term expectations and identifying modifiable factors that can reduce Fontan-associated morbidity. Thrombosis and stroke represent important post-Fontan complications. As such, thromboprophylaxis with warfarin or aspirin is accepted as standard of care post-Fontan. However, there is a notable lack of evidence to inform best practice in the setting of thromboprophylaxis for Fontan patients hence, selection of thromboprophylaxis has been largely dependent on institute practice and clinician preference. Objectives: This thesis explores the long-term outcomes and the impact of current thromboprophylaxis practice in the Australian and New Zealand (ANZ) Fontan population. This study compared cerebrovascular injury, bleeding, bone mineral density (BMD) and quality of life (QoL) in Fontan patients receiving warfarin as compared to aspirin. Methods: This was a cross-sectional, multicentre study of the Australian and New-Zealand Fontan (ANZ) population. Participants underwent cerebral magnetic resonance imaging (MRI) to detect the presence of cerebrovascular injury, dual-energy x-ray absorptiometry (DXA) to assess bone mineral density (BMD). Bleeding and quality of life were assessed using validated questionaries (WA-BAT and PedsQL, respectively). Results: 84 participants underwent brain MRI. Stroke was detected in 33 (39%) participants, with only 5 (15%) of those being clinically symptomatic. There was no association between stroke and Fontan type (p value 0.38, CI 0.39- 1.43) or thromboprophylaxis type. Micro- haemorrhage and white matter injury were detected in almost all participants (96% and 86% respectively), regardless of thromboprophylaxis type. BMD was reduced in our cohort (n=120) compared to the general population however, this reduction was greater in participants receiving warfarin than those on aspirin. High rates of bleeding were reported in both warfarin and aspirin groups however, participants on warfarin experienced more epistaxis and ceased anticoagulation more commonly prior to a medical procedure than those on aspirin. QoL was similar between warfarin and aspirin groups; however, higher QoL scores were reported in patients on warfarin therapy with access to home-INR monitoring compared to those on warfarin therapy without home-INR monitoring. Conclusions: This study adds significantly to the current evidence with regard to the potential consequences of warfarin and aspirin therapy, post-Fontan surgery. Specifically, cerebrovascular injury are frequent occurrences in the ANZ Fontan population, regardless of thromboprophylaxis type. No benefit of long-term warfarin prophylaxis could be demonstrated over aspirin; however, this finding needs to be considered in relation to important clinical features such as cardiac function and lung function. Furthermore, the association of reduced bone health in children on warfarin warrants further mechanistic studies.