Paediatrics (RCH) - Theses

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    Investigating the role of Insulin-like growth factor binding protein-2 (IGFBP-2) in childhood obesity and Type 2 Diabetes
    YAU, STEVEN ( 2014)
    Two thirds of adults and a quarter of children in Australia are overweight or obese. Central or visceral obesity is associated with numerous co-morbidities, including an increased risk of type 2 diabetes mellitus and heart disease. Insulin-like growth factor binding protein-2 (IGFBP-2) may represent a critical link between weight status and insulin sensitivity, as IGFBP-2 levels are reduced in obesity and type 2 diabetes mellitus. Precise mechanisms are unclear, although may involve specific domains of IGFBP-2 binding to cell-surface receptors. Recently, IGFBP-2 was shown to be regulated by leptin, a hormone secreted from white adipocytes primarily responsible for regulating energy balance and metabolism. However the regulatory mechanisms are not yet known. The overall hypothesis for this project is that IGFBP-2 plays a key role in the regulation of body weight and insulin sensitivity, and the leptin resistance which occurs in obesity decreases IGFBP-2 with deleterious effects on insulin sensitivity. The studies described in this thesis aim to, 1) explore the associations between circulating IGFBP-2 levels and clinical, environmental and metabolic parameters in overweight and obese children, 2) investigate the central (brain-mediated) and direct peripheral mechanisms by which leptin regulates IGFBP-2 expression in skeletal muscle, 3) delineate the molecular pathways by which IGFBP-2 enhances insulin sensitivity in skeletal muscle and 4) elucidate whether IGFBP-2 differentially affects adipogenesis and/or lipogenesis in subcutaneous and visceral pre/adipocytes. The clinical findings demonstrated that circulating IGFBP-2 levels in obese youth inversely correlate with obesity and its metabolic markers, whilst positive correlations were observed with insulin sensitivity. Furthermore, circulating IGFBP-2 levels appear to be regulated by hormonal rather than nutritional influences. These findings suggest that reduced levels of IGFBP-2 may play an important role in the pathogenesis of obesity and insulin resistance in early life. Furthermore, the in vitro studies revealed that leptin directly increases IGFBP-2 mRNA and protein (via both PI3K and STAT-3 signalling), as well as improving insulin signalling in human skeletal muscle. Subsequently, intracerebroventricular leptin infusion in sheep improved glucose tolerance, as well as increasing peripheral skeletal muscle IGFBP-2 and this effect is blocked by concurrent peripheral infusion of a β-adrenergic antagonist - indicating this centrally mediated regulation to be via the sympathetic nervous system. Silencing IGFBP-2 in cultured myotubes reduces insulin signalling and glucose uptake in response to both leptin and insulin treatment, and ‘add-back’ wild-type IGFBP-2 restores normal signalling and glucose uptake. Studies in cultured adipocytes demonstrated that IGFBP-2 inhibits adipogenesis and lipogenesis in visceral, but not subcutaneous, adipocytes. Further studies also revealed that these regulations are mediated via IGF-independent mechanisms involving cell-surface association and activation of integrins through the heparinbinding domain and integrin-binding domain of IGFBP-2. In summary, the studies described in this thesis have contributed to our steadily expanding knowledge regarding the role of IGFBP-2 in metabolism, and provide novel insights into the underlying molecular and cellular mechanisms of IGFBP-2 in insulin sensitivity and adipocyte development. These studies have also revealed possible IGF-independent metabolic actions of IGFBP-2 and that specific domains of IGFBP-2 may therefore be novel therapeutic targets for enhancing insulin sensitivity and reducing visceral obesity, therefore improving weight status and insulin sensitivity in those with clinical obesity and/or type 2 diabetes mellitus.
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    Predictors and phenotypes of IgE-mediated food allergy and the development of tolerance in children
    PETERS, RACHEL ( 2014)
    The prevalence of IgE-mediated food allergies has increased dramatically in recent decades. In clinical cohorts, food allergy resolves in the majority of children with egg allergy, but only 20% of those with peanut allergy. The relevance of these findings to the wider population is unknown since the natural history of challenge-confirmed food allergy has not been assessed in a population-based setting. Finally, the role that food allergy plays in the atopic march is yet to be fully elucidated. Although food allergy, eczema and wheeze commonly co-occur in infancy, the interplay between these conditions is not well understood. It is unclear whether different phenotypes of allergy and food allergy exist in infancy, and what the consequences of these are. The increase in food allergy has placed an unprecedented demand on the Australian health system with waiting times to see an allergy specialist now exceeding 12 months. Oral food challenges (OFC) remain the gold standard of food allergy diagnosis, however they are resource consuming and potentially dangerous. Tests of sensitisation, the skin prick test (SPT) or serum specific-IgE (sIgE), offer a more accessible approach to assess the risk of food allergy, although a positive test only confirms sensitisation, not necessarily a clinical reaction. The utilisation of these tests can be improved by identifying decision points with 95% positive predictive value (PPV), reducing the need for OFC. However, the predictive value of SPT and sIgE has not been assessed in a population-based setting nor in a large number of infants, the age most likely to present for diagnosis of food allergy. Although SPT and sIgE evolve with progression of food allergy, reliable indicators of when and in whom tolerance will develop are lacking. Presently, the only way of confirming the development of tolerance is through repeat OFC. Therefore this PhD has focussed on three key areas of paediatric food allergy, i) to identify thresholds for SPT and sIgE that have 95% PPV to the diagnosis of food allergy, ii) to describe the natural history of food allergy and identify predictors of persistence or tolerance, and iii) to identify phenotypes of allergy and food allergy. The HealthNuts study is the first population-based, longitudinal food allergy study using the gold standard OFC to confirm food allergy and is the ideal platform to address these aims. Over 5000 12-month-old infants were recruited and underwent SPT to four common food allergens, egg, peanut sesame and cow’s milk or shrimp. Any infant showing objective evidence of sensitisation was invited to undergo OFC (egg, peanut and sesame only). A subgroup of infants with egg allergy participated in a sub-study which examined tolerance to baked egg and the early resolution of egg allergy at age 2. All participants were invited to undergo follow-up at 4 years of age, which included repeat OFC to detect the development of tolerance, amongst those who were challenge-confirmed food allergic at age 1. In 12-month old infants, SPT wheal size ≥ 8 mm for peanut and sesame and ≥ 4 mm for egg, and sIgE levels ≥ 34 kU/L for peanut and ≥ 1.7 kU/L for egg have 95% PPV to challenge-confirmed food allergy. Only 20% of children with challenge-confirmed peanut allergy at 12 months-of-age, developed tolerance to peanut at 4 years of age. SPT and sIgE measured at age 1 were predictive of persistent of peanut allergy at age 4; 95% PPVs were SPT ≥ 13mm and sIgE ≥ 5.0 kU/L. Thresholds at age 4 with 95% PPV to persistent peanut allergy were SPT ≥ 8mm and sIgE ≥ 2.1 kU/L, while SPT ≤ 3mm had 90% negative predictive value (NPV) and SPT ≤ 2mm and sIgE ≤ 0.35 kU/L had 50% NPV to the development of tolerance. Among infants who were challenge-confirmed allergic to raw egg at 12-months, 80% could safely tolerate baked egg on challenge and nearly 50% outgrew their egg allergy by 2 years of age. Infants who were able to tolerate baked egg at age 1 were more likely to outgrow their raw egg allergy, compared to those who reacted to baked egg (aOR 5.27, 95% CI 1.36-20.50, p=0.02). Frequent ingestion of baked egg (≥ 5 times per month) was also associated with the resolution of egg allergy, (aOR 3.52, 95% CI 1.38 – 8.98, p = 0.009.) Finally, five phenotypes of food allergy in infancy where identified using latent class analysis: no allergic disease, non-food-sensitised eczema, single egg allergy, multiple food allergies (predominantly egg) and multiple food allergies (predominantly peanut). Shared and differential risk factors for these phenotypes were identified, suggesting that different mechanisms are involved in the development of food allergy phenotypes. In summary, the results presented in this thesis have identified important predictors and phenotypes of food allergy and the development of tolerance. This has important implications for the management of food allergy in the clinical setting and will improve our understanding of the development of food allergy and the role it plays in the atopic march.
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    The Fontan procedure in Australia and New Zealand: an outcomes-based registry to improve the lives of those born with a single ventricle
    IYENGAR, AJAY ( 2014)
    In the forty years since its introduction, the Fontan procedure has allowed a generation of children who would otherwise have died to survive into adulthood. These children, born with congenital heart disease at the worst end of the spectrum, must undergo a series of operations, ultimately resulting in bypass of the structures in their heart that were absent or poorly-formed. The resulting “Fontan circulation” consists of direct connection of the systemic venous return to the pulmonary circulation. As this population ages and grows, the long-term uncertainties of a procedure that was once considered palliative have remained unresolved. These uncertainties range from basic facts such as life expectancy, to complex considerations surrounding medication choices and family planning. Evolution of surgical techniques, patient selection and perioperative care have resulted in reductions in surgical mortality. However, patients remain at risk of long-term complications including arrhythmias, thromboembolism, heart failure and sudden death. Due to the small numbers of patients and heterogeneous range of conditions treated with the Fontan procedure, study of the long-term outcomes and risk factors in this population, beyond single large centres, has been difficult. For this reason, we created the Australia and New Zealand Fontan Registry. Now the largest registry of its kind in the world, it aims to (i) encourage population-based, longitudinal follow-up, (ii) understand the composition, outcomes and risk factors of the population, (iii) improve health service planning and provision and (iv) provide a base for recruiting patients to prospective studies. This thesis describes the fulfilment of these aims. Based on the data collected in creation of the registry, I present our current understanding of the population, their outcomes and risk factors for early- and late-postoperative adverse events and, finally, present initial data demonstrating the utility of the Registry as a platform for prospective hypothesis-generated research. Through the creation of the Registry, I have demonstrated that: (i) the Fontan population in Australia and New Zealand now numbers over 1000 individuals, half of whom are of adult age, (ii) there has been a recent rise in the proportion with hypoplastic left heart syndrome to 15% in the current era, (iii) early outcomes have improved, with contemporary hospital mortality falling to 1%, (iv) the earliest surgical techniques have been abandoned in favour of the contemporary extracardiac conduit technique, (v) long-term survival is over 76% at 25 years, which has far exceeded previous expectations, and (vi) there is a high prevalence of liver and renal dysfunction in long-term survivors. The journey involved in the creation of the registry began when I was a medical student, reviewing the outcomes of those operated at the Royal Children’s Hospital. The ensuing career-defining journey for me personally, as well as the whole research team, forms an ongoing labour of love for all involved.
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    Probiotics for treating infant colic
    SUNG, VALERIE ( 2014)
    Background: Infant colic is common and burdensome to families, health professionals and health services. Despite years of research, its aetiology remains unclear, and effective management options limited. Recent studies suggest a possible role of gut microbiota and inflammation in the pathogenesis of colic, and some randomised trials indicate probiotics may effectively treat colic in breastfed infants. Aims: Overall: to determine the role of probiotics in treating infant colic. Part 1: to investigate the role of probiotics in infant colic and identify the gaps in the literature. Part 2: to determine, through a randomised controlled trial of breastfed and formula-fed infants with colic, i) whether the probiotic Lactobacillus reuteri DSM 17938 is effective in reducing infant cry/fuss duration and episodes, and improving infant sleep duration, maternal mental health, parent quality of life, and family and infant functioning; ii) the probiotic’s effects on infant faecal microbial diversity, Escherichia coli load and calprotectin levels; and iii) if effective, whether the probiotic reduces healthcare utilisation. Part 3: to further clarify whether Lactobacillus reuteri DSM 17938 may be effective in treating certain subgroups of infants with colic through an individual participant data meta-analysis (IPDMA). Methods: Part 1: systematic review and meta-analysis of probiotics for infant colic treatment and prevention. Part 2: double-blind randomised trial of Lactobacillus reuteri DSM 17938 in breastfed and formula-fed infants with colic from Melbourne, Australia. Part 3: formation of an international collaboration to conduct an IPDMA. Results: Part 1: Five management trials and seven prevention trials were reviewed. There was insufficient evidence to support the use of probiotics to prevent infant colic. Meta-analysis from three small trials, all of which had methodological limitations, indicated Lactobacillus reuteri may be effective for treating breastfed infants with colic, but there was no evidence to support its use in formula-fed infants with colic. A larger, more rigorous randomised trial that includes both breastfed and formula-fed infants with colic was indicated. Part 2: Of 167 families randomised (85 probiotic, 82 placebo), 127 (76%) were analysed for primary outcome, 154 (92%) for secondary outcomes at one month, and 123 (74%) for secondary outcomes at six months. Lactobacillus reuteri DSM 17938 did not reduce infant crying/fussing, nor did it improve infant sleep, maternal mental health, family or infant functioning, or quality of life. It did not have any effects on infant gut microbial diversity, Escherichia coli load or calprotectin levels. Formula-fed infants in the probiotic group fussed more than those in the placebo group at all post-intervention time points, except at six months. Part 3: An international collaboration has been set up and published a protocol for an IPDMA to further evaluate whether Lactobacillus reuteri DSM 17938 may be effective in certain subgroups of infants with colic. Conclusion: Despite popular belief, Lactobacillus reuteri DSM 17938 cannot be recommended for all infants with colic, particularly those who are formula-fed. Its role in breastfed infants with colic is controversial. Results from the IPDMA will better inform whether certain subgroups of infants with colic may benefit from the probiotic.
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    Neurodevelopmental outcomes of children with inherited metabolic disorders
    Brown, Amy ( 2014)
    Introduction: Inherited metabolic disorders (IMD) are caused by mutations that lead to deficiencies in enzymes or defects in transporters, channels or receptors etc. As a result there is a block in metabolic pathways, triggering toxicities and deficiencies that affect various organs. Their effect on the developing brain has the potential to disrupt neurodevelopment in childhood, and therefore impact upon cognition, behaviour and social skills. Background: An evaluation of the existing literature revealed that I comparison to research on Phenylketonuria, there was a lack of detailed research on neurodevelopmental outcomes of children with other IMD, especially in children diagnosed through newborn screening. More specifically, very few publications include follow up data, did not utilise standardised comprehensive measures and did not consider other environmental factors that may impact on outcomes. A distinct gap was evident in regards to social functioning in children. Aims: The aims of this thesis were (1) to investigate the neurodevelopmental outcomes of children with several inherited metabolic disorders; (2) to explore the impact of having a child with an IMD on parents and how this may affect child outcomes. Method: Children with a confirmed diagnosis of one of the following disorders; glutaric aciduria type I, maple syrup urine disorder, methylmalonic aciduria, propionic aciduria, isovaleric aciduria, glycogen storage disease, very long chain acyl-CoA dehydrogenase deficiency and ornithine transcarbamylase deficiency and their parents were invited to participate in the study. These disorders were targeted to represent a range of disorders (i.e, amino acid, carbohydrate, and fatty acid oxidation disorders) and based on the availability of patients within the hospital. Children underwent a neuropsychological assessment and parents completed questionnaires regarding their behaviour and social skills. Some parents completed questionnaires regarding their own coping, psychological distress and family management.   Results: The children in the glutaric aciduria type I follow-up study (all diagnosed through newborn screening) generally showed no evidence of decline in cognitive functioning, however all had impaired fine motor skills and some had problems with speech or language. Children with very long chain acyl-CoA dehydrogenase deficiency diagnosed through newborn screening had mostly normal motor and cognitive functioning, with some evidence for speech or language problems. As a cross sectional group, children demonstrated intact social perception and parents reported mostly average social skills, although some children were experiencing peer problems, hyperactivity and emotional symptoms. On reports of their own functioning, parents reported adequate coping although a small subset of parents reported at risk levels of psychological distress. Parent coping and family functioning were related to some child behaviour and social outcomes. Conclusion: Children with inherited metabolic disorders may be vulnerable to neurodevelopmental problems despite mode of diagnosis. Neuropsychological assessment is recommended and referrals made where necessary. Further consideration should be given to the child’s environment (parent stress and coping, family functioning) and how this may impact their outcomes. 
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    Structural and functional evaluation of gas-trapping in infants with cystic fibrosis
    Ebdon, Anne-Marie ( 2014)
    Air-trapping is frequently identified in infants with cystic fibrosis (CF) on chest CT scans. Aim was to examine the association between the measured functional residual capacity (FRC) using infant lung function under sedation, and air-trapping scores obtained from chest CT images, acquired under general anaesthesia. Thirty-eight lung function tests were analysed from CF infants aged 3-24 months. Twenty CT scans were performed on the infants. No statistical significance was found between the lung function and CT results when simple linear regression was applied. In conclusion, no associations were found between measured FRC and air-trapping CT scores in infants with CF.
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    The decision that would give me the most peace of mind: exploring informed choice in non-pregnant women offered population genetic carrier screening for fragile X syndrome
    Ames, Alice ( 2014)
    Advances in genetic screening capabilities drive the need to evaluate the social and ethical implications of new screening programmes. One ethical aspect to evaluate is informed choice, as recommended in genetic screening guidelines. An informed choice can be facilitated by providing relevant, up-to-date information and offering screening in a way that allows individuals to make a free choice. Population carrier screening for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, can provide personal and reproductive risk information to individuals. Female carriers of FXS are at risk of having children with FXS and may develop premature menopause. Screening before pregnancy allows women the choice of learning their carrier status, and to inform family planning. FXS population carrier screening provides a useful model for exploring informed choice because it has implications for both reproductive and personal health. Women may value the benefits and harms of FXS screening differently, therefore it is the individual’s choice to decide whether or not to have screening. This PhD research aimed to explore informed choice in a programme offering population carrier screening for FXS to non-pregnant women. Using a mixed methods approach to respond to the research aim, qualitative and quantitative datasets were used to explore informed choice from different perspectives. A systematic review of informed choice measures used in population-based reproductive genetic screening programmes identified the multidimensional measure of informed choice (MMIC) as one key validated measure. The MMIC requires a condition specific knowledge scale, and measures of attitudes and uptake (i.e., value-consistency) to evaluate informed choice. Deliberation and decisional conflict were also identified as measures important to the evaluation of informed choice. These measures were applied in the quantitative datasets of this PhD study, and explored further using qualitative and quantitative methods. The findings of this mixed methods PhD study indicate that for non-pregnant women offered FXS carrier screening, informed choice, and their decision-making processes are indeed complex. When measured quantitatively, the majority of women in the study appeared to make informed choices. There were various factors affecting and contributing to informed choice when measured with the MMIC. Informed choice was affected by perceived susceptibility, family experience, decisional conflict and how participants were approached to participate in the study. Stepping outside of the MMIC paradigm to draw on the in-depth qualitative interview findings, we see that women described a number of different ways of coming to a decision about FXS carrier screening. Yet, despite these diverse processes, the majority of women indicated that how a choice feels is an important outcome when making their choices. This feeling was described as the choice ‘feeling right’, or comfortable, or being ‘at peace’ with the choice. This finding adds another dimension to how informed choice is generally understood. Women’s perceptions of their choices, such as whether the choice ‘feels right’, could be incorporated into the evaluation of informed choice as this aspect is not included in the MMIC. Therefore, an informed decision-making approach using a combination of objective and subjective measures such as knowledge, decisional conflict and ‘feeling right’ are suggested to better evaluate informed choice in FXS carrier screening programmes in non-pregnancy settings. The key findings of this PhD research suggest that: - an important decision outcome for women when offered FXS carrier screening is whether the decision ‘feels right’ - informed choice is affected by an individual’s perceived susceptibility, family experience, decisional conflict and the way individuals were approached to participate in the study - the active approach of individuals to participate in the study has implications for how we approach potential participants in future research and how we offer individuals screening The recommendations arising from this work have implications for the design of screening programmes and decision interventions such as those performed by genetic counsellors. The revised model of evaluating informed choice can be used to evaluate the decision quality and outcome by incorporating an objective measure of knowledge and the individual’s perspective of their decision using measures of decisional conflict and whether the choice ‘felt right’
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    Transabdominal Electrical Stimulation to treat constipation: developing a new medical device
    TAN, ANDRE ( 2014)
    Chronic constipation has significant health, social and economic impacts, and standard medical interventions may be ineffective. Non-invasive, pain-free electrical stimulation applied daily to the abdomen (Transabdominal Electrical Stimulation (TES)) has been shown to overcome slow-transit constipation in children at our centre. Historically, electrical therapies have been associated with quackery and charlatans, and their medical use has been contentious and controversial. It is therefore important to determine how this novel therapy can be controlled and optimised in the home environment, and what its physiological mechanism of action may be. The aim of this thesis is to examine how TES applied at home may be applied incorrectly and how it may be optimised, and investigate how a large animal (pig) model may be developed to better understand the physiological aspects of TES. First, the issues raised by patients and clinicians in previous studies were listed. Prescribed stimulation systems were difficult to use, and placed a lot of burden on patients and their families, and so 24 potential causes of failure to apply TES correctly at home were identified (Chapter 2), and these were examined and analysed using a Failure-Mode and Effects Analysis (FMEA). Issues were ranked, and risk scores assigned; this identified the most severe hazards, and options for mitigation. Many application hazards could be mitigated by designing a custom stimulation system to deliver TES to the abdomen. The analysis also identified the electrode-hydrogel-skin interface as a hazard that required further investigation (Chapter 3). The skin has a large electrical impedance; the electrodes were tested to characterise frequency response and effect of reducing electrode contact. The best-designed skin-applied electrodes resulted in comparable stimulation to current delivered using needle electrodes at stimulation frequencies at or above 4kHz. Well-designed skin-applied electrodes can deliver more current, however, poor contact greatly increased electrode-hydrogel-skin interface impedance (i.e., reduced current flow). A computer model was developed to understand the flow of current in the abdomen (Chapter 4). To understand the physiological mechanisms of TES, a pig model was developed. Methods were developed to measure direct colonic contractions of anaesthetised pigs (Chapter 5), water content (Chapter 6) and oral-rectal transit of markers in conscious pigs to measure transit rate (Chapters 7 and 8). One hour of TES did not change amplitude and frequency of colonic contractions (Chapter 5), 30 minutes of daily TES for up to 2 weeks did not change stool water content (Chapter 6), and 30 minutes of daily TES for one week did not change oral-rectal transit of 4mm-diameter plastic markers (Chapter 8). Next, to allow measures of the effect of TES on a constipated pig, experiments were performed to develop a constipated pig (Chapter 9). Opiates produce constipation, and a peripherally active opiate, loperamide, was tested. However, making pigs constipated with loperamide was difficult. Orally-fed loperamide powder did not slow-down oral-rectal transit of plastic markers, but may have decreased stool water content. Alternative methods to make pigs constipated should be investigated. In the final Chapter (10), overall conclusions are drawn. Future studies should investigate longer daily TES, over more weeks in pigs. Also, imaging the flow of electrical current through the abdomen may provide insight to where TES may exert its physiological effects. TES is a promising treatment for overcoming constipation, and the work presented in this thesis opens the door to further understanding and optimising this novel therapy.
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    Investigation of the functional role of disease genes in Parkinson's disease
    Stephenson, Sarah Elizabeth Marie ( 2014)
    Mutations in parkin (PARK2) can result in Parkinson’s disease (PD). PARK2 shares a bidirectional promoter with Parkin Co-Regulated (PACRG). Together PARK2 and PACRG span almost 2 Mb of the human genome because of super-expanded introns. However, the transcriptional start sites are separated by only ~200 bp. Bidirectionally regulated genes have been shown to function in common biological pathways. Parkin is an ubiquitin ligase that mediates protein and organelle turnover via the proteasome and autophagy systems. PACRG is a highly conserved protein of unknown function that is involved in cilia, aggresome formation and autophagy in response to proteasomal impairment. We hypothesise that the function of PACRG is coupled to parkin by its co-regulation through the shared bidirectional promoter. The overall aim of this study is to test this hypothesis utilising several distinct but complementary approaches; molecular evolutionary analysis and the generation of novel in vivo models. Aim 1: To determine the evolutionary and expression evidence that suggests that PARK2 and PACRG function together. The molecular evolution of PARK2 and PACRG orthologues from 47 metazoan species were verified by phylogenetic analysis. Conserved synteny and gene structure of orthologues was used to determine the origin of the bidirectional PARK2-PACRG promoter and super-expanded intron structure. A duplication of PARK2 appears to have occurred in an ancestor of the chordate genome, which co-localised PARK2 antisense to PACRG. There was evidence that the promoter driving PACRG in the ancestral chordate genome may have already had capacity for bidirectional expression. However, the modern human bidirectional PARK2-PACRG promoter originated as a result of the gain of a new first exon of PARK2 within the regulatory region of PACRG. Furthermore, the bidirectional promoter and super-expanded intron structure of PARK2-PACRG appears to have originated simultaneously, and intrinsic features of the locus appear to be responsible for the maintenance of this organisation. To investigate the regulation of PARK2 and PACRG, conserved transcription factor binding sites were identified and the expression of PARK2 and PACRG in 41 human tissues including 22 unique brain regions was interrogated by qRT-PCR to determine the expression profile of both genes. The sequence of the bidirectional PARK2-PACRG promoter was found to be highly conserved, which suggests that the regulation of the genes is likely to be conserved across species. The expression of PARK2 and PACRG in human tissues was showed a dynamic pattern across tissues, which suggested that tissue of origin cues may play a role in mediating expression of both genes. Furthermore, PARK2 and PACRG displayed convergent expression in the brain that suggested co-regulation of the genes. Collectively, the evolutionary and expression analysis provided evidence that suggests that PARK2 and PACRG function together in the brain. Aim 2: To generate two novel mouse models to facilitate the investigation of the functional roles of parkin and PACRG in vivo. Traditional homologous recombination mediated modification of mouse embryonic stem cells was used to generate a single Pacrg knockout mouse line and a double Park2-Pacrg knockout mouse line. Successful modification of the targeted allele was confirmed at the genomic, transcriptional and steady state protein levels for both genes. In addition, two independent phenotypic evaluations – infertility and hydrocephalus – were used to verify loss of PACRG function in both lines. Unexpectedly, the steady state levels of parkin were increased in the single Pacrg knockout. Introduction of transgenic Pacrg did not alter parkin steady state levels, suggesting a PACRG independent mechanism. At 12 months of age, neuropathological examination suggested there was no gross abnormality of the dopaminergic system in the substantia nigra and no significant difference in the number of dopaminergic neurons in either knockout model. However, in the single Pacrg knockout there was a trend of a ~15% reduction in the number of dopaminergic neurons (P=0.07, n=6). Given that PD is an age dependent syndrome, analysis of an increased number of mice at older time points is ongoing to fully characterise the phenotype of these mouse models.
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    Diagnostic methods for checking multiple imputation models
    Nguyen, Cattram ( 2014)
    Multiple imputation is an increasingly popular method for handling missing data. A key task in the imputation process is the specification of a model for generating imputations. The validity of imputation-based inferences depends on the adequacy of this imputation model. Constructing imputation models is not straightforward and requires careful decision-making. The imputer must decide, for example, which variables to include in the imputation model and what functional form these variables should take. In many cases, there is no consensus in the literature to inform the modelling decisions. If the imputation model is poorly specified, such as through the omission of important variables, this can lead to biased results. It is therefore important that researchers check the goodness-of-fit of their imputation models. Despite the popularity of multiple imputation, the checking of imputation models is not widespread. This may primarily be due to the scarcity of guidelines and computational tools for performing imputation diagnostics. Although some diagnostic methods have been proposed in the literature, very few studies have formally evaluated whether the proposed techniques are useful for identifying problems with imputation models. Thus, we have found ourselves in an environment where the wide availability of multiple imputation is coupled with a lack of software and guidelines for assessing the adequacy of the models used in this process. The current research addressed this knowledge gap by evaluating diagnostic methods for checking imputation models. This included an examination of proposed methods including graphical diagnostics, the Kolmogorov-Smirnov test and posterior predictive checking. These techniques were evaluated using simulation experiments and they were illustrated using data from the Longitudinal Study of Australian Children. The investigations in this thesis revealed both advantages and disadvantages of all evaluated diagnostics. The graphical checks were useful for exploring the imputed values, but it was challenging to apply them routinely to all imputed variables, especially when working on large-scale datasets. The Kolmogorov-Smirnov diagnostic was straightforward to implement, but it had limited usefulness when the data were missing at random, an assumption which is commonly made when performing multiple imputation. Posterior predictive checking was preferable to methods that focus on the plausibility of imputations, because it checks the fit of the model with respect to quantities of scientific interest. Posterior predictive checking was able to successfully identify model misspecifications such as the omission of the outcome variable from the imputation model. However, users of posterior predictive checking need to be aware of the shortcomings of this approach, particularly its reduced usefulness in the presence of large amounts of missing data. Given that all of the evaluated methods were imperfect, there is the need for further development and evaluation of diagnostic techniques for checking imputation models. In addition, rather than expecting any individual diagnostic to provide a complete solution, it might be preferable to treat each of these techniques as separate elements of a diagnostic toolkit. When applied together, these diagnostic methods can be used to check different aspects of the imputation model. Finally, to encourage the practice of model checking, it is crucial that guidelines for imputation diagnostics are developed and communicated to the research community. It is also important that tools for diagnostic checking are made available in statistical packages that support multiple imputation. This will become increasingly important as multiple imputation becomes further established as a standard missing data method into the future.