Paediatrics (RCH) - Theses
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ItemMicroRNA expression and genome-wide epigenetic analysis of paediatric acute myeloid leukaemia( 2014)Introduction: Paediatric Acute Myeloid Leukaemia (AML) is the third most prevalent cancer in Australian children under the age of 14. Chromosomal and genetic lesions are commonly found within specific subtypes of paediatric AML and these help to direct treatments and prognostic predictions. However, there are no common genetic lesions across subtypes of paediatric AML and subtype-associated genetic changes may fail to induce leukaemogenesis. DNA methylation is the most commonly studied molecular alteration in cancer. Strong evidence indicates that methylation variations exist within paediatric cancer known to modify gene expression. Similarly, microRNA are small non-coding RNA that also regulate gene expression and whose dysregulation within paediatric cancer is now beginning to be appreciated. Therefore altered methylation and miRNA expression may contribute to malignancy through activating oncogenes or inactivating tumour suppressor genes, similar to genetic mutations. Furthermore, both DNA methylation and microRNA expression represent viable epigenetic mechanisms for application to clinical biomarkers of disease diagnosis, prognosis and disease tracking. At the beginning of this study, limited interrogations of genome-scale DNA methylation and microRNA expression had been conducted within paediatric AML with appropriate non-leukaemic controls. Furthermore, there has been few extensive studies evaluating the utility of current epigenetic techniques on primary clinical specimens. Therefore I chose to study genome-wide DNA methylation and large-scale miRNA expression changes in paediatric AML, as well as interrogating techniques for using archived primary patient samples. Materials and Methods: Two approaches were used to interrogate large-scale DNA methylation and microRNA expression; TaqMan OpenArray Human MicroRNA Panel (Life Technologies) was used to interrogate 5 primary paediatric AML patients with matched controls; and Illumina Infinium HumanMethylation450 BeadChip array (HM450) was used to interrogate 18 primary AML alongside matched non-leukaemic samples. Validation of AML DNA methylation alterations was conducted using the SEQUENOM MassARRAY EpiTYPER with an expanded cohort of 28 patients, in conjunction with validation of microRNA expression utilising quantitative real-time PCR (qRT-PCR) and TaqMan singleplex assays on 32 primary paediatric AML, together with matched control groups. This study also developed extensive extraction techniques for the analysis of microRNA expression within a range of samples, including archived bone marrow aspirate smear slides. Results: Genome-scale DNA methylation disruptions were characterised within paediatric AML, and identified hundreds of genes associated with disease compared to matched control samples. Interrogation of a refined subset of target genes also identified gene expression alterations within these regions, which were further associated with patient disease onset and predicted outcome. Conversely, large-scale microRNA expression disruptions were characterised within paediatric AML, whereby a small number of reliable targets were identified. Such microRNA disruption was found to be associated with DNA methylation regulation on the microRNA gene, and could also be used as reliable biomarkers to predict disease onset and patient outcome in connection with identified patient cytogenetic abnormalities. Integration of data from genome-scale DNA methylation and combined gene and microRNA expression analysis, identified common epigenetic disruptions within paediatric AML affecting known tumour suppressor genes, cytoskeletal organisation, cellular proliferation and immune function. Conclusions: The findings of this study reveal that DNA methylation alterations within paediatric AML can associate with gene disruptions with the potential to initiate and perpetuate malignant phenotypes. Likewise, microRNA deregulation can establish widespread gene disruptions as a catalyst for leukaemogenesis. Interestingly we establish that combined deregulation of epigenetic mechanisms, and the occurrence of one epigenetic mechanism working to deregulate another, may be a common feature of paediatric AML. Lastly this study identifies targets and techniques to extend such studies into the clinic and provides attractive targets for therapeutic intervention.
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ItemLong-term psychosocial outcomes following childhood traumatic brain injury( 2014)Introduction: Childhood traumatic brain injury (CTBI) is one of the most common causes of mortality and impairments in children, impacting on the development of neuropsychological, social and psychological functioning. A disruption in these areas often results in long-term impairments with interpersonal relationships, participation in leisure and social activities and quality of life. Background: Available literature has shown that children and adolescents with a CTBI demonstrate greater social and psychological dysfunction than typically developing children and adolescents. They often show lower self-esteem, have fewer friends, express more maladaptive, aggressive and antisocial behaviour in a social environment and have poorer emotional and behavioural regulation. These symptoms may become more severe and develop into a clinical disorder, with children and adolescents with a CTBI having a 49% risk of developing any kind of psychiatric disorder compared to 13% risk for healthy developing children and adolescents. Most research in this field has investigated these outcomes up to 5 years post-injury, although less is known about the long-term outcomes after CTBI into adulthood. Aim: The aim of this thesis was to investigate the development of long-term psychosocial outcomes over a period of 16 years following CTBI. The participants were recruited from a prospective, longitudinal sample ascertained between 1993-1997 and aged 1-7 years at time of injury. Children were included in the study if there was sufficient information regarding their CTBI to classify illness severity, if they were able to speak English and complete the protocol. They were excluded in cases of a non-accidental, penetrating or previous brain injury or if there was evidence of a pre-existing neurological or developmental disorder. A healthy control group was also recruited during the same period, matched for age, gender and socio-economic status (SES) with the CTBI sample. Both groups were followed up several times, with this thesis utilising data from the acute, 6 month, 5 year and 16 year post injury time points. Results: The results showed that young adult survivors of CTBI reported more depressive, anxious and withdrawn symptoms then their healthy peers. Ratings on psychosocial measures rated by the young adults with CTBI were compared with the ratings of their significant other, with analyses indicating fair to excellent agreement on the obvious and noticeable behaviours, such as communication, and alcohol and drug use. In contrast, there was poor agreement on the more internalising symptoms such as depressed/anxious and withdrawn symptoms. With regard to the developmental trajectory of social skills, and internalising and externalising symptoms, the CTBI and control group did not differ. However, there was a trend for the CTBI group to be rated higher on internalising symptoms by the parent at every time point. Conclusion: In summary, young adults with a CTBI experience more internalising symptoms than their healthy peers, and are at higher risk of developing more severe symptoms and even clinical disorders. Appropriate screening by health professionals and early intervention should be considered to prevent the development of any significant social or psychological symptoms later in life.