Biomedical Research PhD projects for 2009 at the Murdoch Childrens Research Institute

With the completion of the Human Genome Project, advances in genetic technologies and increasing use of the internet, the availability of genetic tests has increased exponentially in recent years. Although the majority of tests for inherited conditions are offered through government funded clinical genetics services in Australia, direct-to-consumer (DTC) genetic tests are now offered by private companies, and are available via the internet. Such tests are often offered without genetic counselling or clinical input. DTC marketing not only includes tests for single gene conditions, which are often well-understood, but also include paternity tests and tests for susceptibility to complex conditions, pharmacogenomics and nutrigenomics, which are still considered to have questionable significance and interpretation. This project would examine the ethical, legal and social issues around DTC testing from an Australian perspective. There will also be an empirical study of community perspectives of DTC testing, using qualitative and quantitative approaches. Findings from this study could be helpful in informing policy makers.

2. Using children's genes in research

A/Professor Lynn Gillam
T 0417 536 785
E

Dr Merle Spriggs
T 90905237
E

Increasingly, genetic testing of children is becoming part of pediatric research. While a considerable amount of attention has been paid to the ethics of predictive testing in children for adult onset conditions there is little written about the ethical conduct of pediatric genetic research, especially that involving complex behavioural traits.

Questions that arise include:
• Is it ethically acceptable to enroll children and young people in gene-based prevention trials for traits such as obesity, addiction and ADHD?
• When is it ethically defensible for a parent to consent to their child taking part in such research?

Ethical issues include competence, proxy consent, autonomy and developing autonomy, best interests, discrimination, stigma, privacy, genetic determinism and the right not to know.

This project could be literature based, interview based or based on a questionnaire. The precise topic is negotiable and the methods are negotiable.

3. The ethics of surgically assigning sex to children

A/Professor Lynn Gillam
T 0417 536 785
E

Dr Merle Spriggs
T 90905237
E

Intersex conditions are variously referred to as ‘developmental anomalies of the external genitalia’, ‘atypical sexual differentiation’, and ‘ambiguous genitalia’. Controversy surrounding the surgical management of intersex conditions in newborns is an important ethical issue because it:
• Raises questions about the authority of parents and others to make irrevocable decisions for young children
• Tests the idea that surgery is only justified when it is for disease or malfunction
• Raises questions about what constitutes disease or malfunction
• Poses questions about what we should base treatment decisions on when there is little guidance in terms of evidence of outcomes
• Illustrates the shift from physician-centred medicine and paternalism to patient-centred medicine
• Highlights the need for evidence in the form of systematic outcome studies

This project could be literature based, interview based or based on a questionnaire. The precise topic is negotiable and the methods are negotiable.

4. Research, young people and the internet

Dr Merle Spriggs
T 90905237
E

A/Professor Lynn Gillam
T 0417 536 785
E

Internet communities and personal webspaces are a rich source of qualitative data for health and social researchers. It is a particularly fertile source for those whose research areas involve children and young people. Although there is some guidance for conducting research online, there are no detailed or universally accepted ethics guidelines for research of homepages, blogs or webspaces such as MySpace. Questions that arises are – “If MySpace is a public webspace, can research be done without consent?” “If it is thought that consent should be obtained, how should it be obtained in this context?”

Most teenagers use the internet without parental supervision and generally, researchers can use information that is in the public domain without obtaining consent. On the internet however, what is public and what is private is not so clear. Added to this, are the difficult issues around consent in research involving children and young people. In the context of research, children and young people are considered to be a vulnerable population requiring special protection such as consent from a parent or guardian. The immature judgment of some young people may also mean that a distinction between private and public is not meaningful.

This project will investigate and analyse the ethical issues, especially consent issues in research involving internet spaces in which children or young people participate. The precise topic and the methods used can be negotiated with the supervisors

5. Characterisation of a novel gene and mouse model of ciliary dyskinesia

Dr Paul Lockhart
T 61-3-8341 6322
E

Dr Martin Delatycki
T 83416284
E

Cilia are evolutionarily conserved microtubule-based hair-like organelles that project from nearly all mammalian cell types. Although they perform remarkably diverse functions they share a similar basic structure, consisting of a basal body, axoneme and ciliary membrane. Defects in cilial function have classically being associated with human phenotypes such as neural tube and patterning defects, male infertility and sinusitis. However, recent studies have broadened the range of associated syndromes and phenotypes to include obesity, diabetes and hypertension. There is limited understanding of the genes and proteins involved in the formation and function of cilia, but recent studies have taken advantage of mice models to study the pathogenesis of ciliary dysfunction in vivo. We have identified a novel mouse model characterised by male infertility and hydrocephalus (enlarged ventricles within the brain). We have identified the defective gene and have preliminary data suggesting it is a key protein required for the functioning of motile cilia. This project will utilise a range of molecular and cellular techniques to characterise the phenotype of the mouse and determine the molecular function of the gene involved.

6. Determining of the molecular basis of childhood dystonia

Dr Paul Lockhart
T 83416322
E

Dr Kirstee Martin
T 83416286
E

Dystonia is a common movement disorder which is characterised by involuntary sustained muscle contractions and abnormal movement. The condition affects approximately 1 in 5000 people, but remains poorly understood with limited treatment options.
Mutation of the TorsinA gene causes dominant early-onset general torsion dystonia, the most common heritable form of the disease. TorsinA is highly expressed in a subset of neurons within the brain, particularly the basal ganglia and associated motor circuits. Although the normal function of TorsinA is currently unknown we have preliminary data suggesting the protein functions as a chaperone and transports substrates within the neuron. We have performed a 2-hybrid screen and identified several proteins that interact with TorsinA. The aim of this project is to further characterise these interacting proteins and investigate their contribution to the disease phenotype. The project will involve a range of molecular and cellular techniques, initially utilising cellular models. In addition, we have established colonies of transgenic mice with specific defects in the TorsinA gene. These mice will allow us to identify the molecular pathways disrupted during disease and provide the means to develop and test novel therapeutic treatments.

7. Investigations into chromosome instability and human disease predisposition

Dr Paul Kalitsis
T 83416300
E

Professor KH Andy Choo
T 83416306
E

Approximately 10 quadrillion cell divisions occur in the lifetime of a human. Each divisional event requires the accurate distribution of the newly-replicated chromosomes to the daughter cells. Any faults occurring during this process can cause an imbalance in chromosome number and lead to clinical conditions such as Down syndrome, pregnancy loss, infertility, and cancer. Our laboratory investigates the cellular and molecular mechanisms that are responsible for such chromosome imbalances. One project involves the use of a fluorescent protein chromosome instability mutant screening assay in mouse embryonic stem cells to identify genes and environmental agents that contribute to chromosome missegregation events. Another project uses affinity purification and mass spec/proteomic techniques to identify novel chromatin protein components using known essential chromosome proteins as affinity baits. We anticipate finding many new genes/proteins whose functions will be further studied using techniques such as RNAi gene knockdown and gene knockout in cells and mice. These studies are expected to contribute important new insight into key mechanisms regulating chromosome stability and their potential role in causing the plethora of known chromosome-related human diseases.

8. Mental health problems in early childhood: Longitudinal population study

Dr Jordana Bayer
T 93457952
E

A/Professor Melissa Wake
T 93455937
E

The problem: Behaviour and emotional problems affect 1 in 7 Australian children aged 4-17 years, often with lasting negative impacts throughout life (such as family stress, academic and friendship problems, school dropout, substance misuse, depression, family violence, crime, and employment problems). The roots of these problems can often be found as early as toddlerhood.
Understanding at a population level how such problems develop can help develop effective primary prevention programs. The Toddlers Without Tears study has followed a population-based sample of 733 infants from 7 months to 3 years with excellent retention (80%). We have found that maternal stress and negative parenting practices are the primary, predictors of early behaviour and emotional difficulties at age 3.
PhD candidate (s): This project would examine child and family outcomes at school-entry (6 years) and early intervention service needs in the community. It would suit persons interested in health services research, and/or long term outcomes of early mental health problems.

9. Children’s Moods Fears and Worries: Clinical study of early anxious and depressive symptoms

Dr Jordana Bayer
T 93457952
E

Dr Lesley Bretherton
T 93455780
E

Emotional problems (anxiety, social withdrawal, depression) affect 1 in 7 Australian children and show continuity from preschool age onwards. Identifying symptoms of emotional difficulties in young children is a new field of study. The Children’s Moods Fears and Worries Questionnaire was newly developed as a sensitive measure of toddler-preschool age internalizing difficulties.
Masters candidate: This project would evaluate the new questionnaire in identifying internalising problems with 1-5 year old children attending outpatient clinics at the Royal Children’s Hospital Melbourne.

10. Novel mechanisms of chromosome and genome regulation and disease aetiology

Dr Damien Hudson
T 83416300
E

Professor KH Andy Choo
T 83416306
E

In order for our genetic material to be faithfully segregated into two daughter cells, the chromosomes must compact nearly 10,000 fold. Prior to this event chromosomes are an amorphous mass of DNA, but upon compaction they form visible X-shaped structures known as mitotic chromosomes.

A key component in this process is a multi subunit complex termed condensin. The aims of this project are to understand how condensin directs chromosome condensation and to find which components condensin interacts using gene knockout technology, and integrated proteomics and biochemistry. Furthermore we aim to look at the non-mitotic roles of condensin where a growing body of evidence suggests condensin has key roles in gene regulation and DNA repair. Critically malfunction of condensin is associated with human disease including cancer and immune deficiency. We expect to find novel interactors contributing to chromosome structure and to understand the mechanism of action of condensin and how it might contribute to disease.

11. Folate supplementation, neurodevelopment and epigenetics

Dr Richard Saffery
T 83416341
E

Dr David Godler
T 8341 6307
E

DNA methylation has been implicated in chromatin condensation, regulation of global transcriptional activity and nuclear organization. Folate is a principal methyl donor in the majority of biochemical reactions, and is an indirect substrate for S-adenosyl-L-methionine (SAM). Although folate deficiency can cause genome-wide DNA hypomethylation through depletion of SAM; limited folate intake has been also shown to induce silencing of many tumour suppressor genes, attributed to regional hypermethylation. To date the molecular relationship between these two paradoxical phenomena is unknown, however it indicates presence of a finely tuned regulatory mechanism of site-specific epigenetic changes affected by folic acid supplementation – a mechanism that is evident from our preliminary findings. Furthermore, there is ample evidence to suggest that these disturbances result in aberrant gene expression associated with abnormal neurodevelopment. This project aims to study the epigenetic bases behind the neurodevelopmental defects associated with folate over or under supplementation in a mouse model, with a longer-term view to extend the study to humans.

12. Food Allergy

A/Professor Katie Allen
T 93455060
E

Dr Nick Osborne
T 99366506
E

Peanut allergy has been reported to be increasing in industrialised countries. Children with IgE-mediated food allergies such as peanut allergy are at risk of life-threatening episodes of anaphylaxis. Along with obesity, atopy has been touted as the new epidemic of the 21st century. This project aims to examine the epidemiology of food allergy in a large cohort of Melbourne children. The primary goal will be to catalogue the prevalence of peanut allergy in the community and measure if there are modifiable factors that can alter this prevalence. As up to 20% of individuals develop tolerance to food allergy, a secondary focus of this project will be to elucidate what mechanisms promote tolerance to foods. Infants who are 12-month-old and pre-peanut ingestion will be recruited from the community at vaccination sessions in and across metropolitan Melbourne via council-led immunisation clinics. Skin prick test for food allergens will be administered and a questionnaire completed. Children with positive skin prick tests will be invited to attend the Royal Children’s Hospital to be offered an inpatient-based food challenge to confirm whether the infant has true peanut allergy. Other genetic and biochemical analyses will be undertaken as potential markers to predict allergy or development of tolerance.

13. Epigenetics of mammalian germ and stem cell development

Dr Jeffrey Mann
T 9936 6516
E

The health of reproductive stem cells can be measured by genetic integrity, that is, are there mutations in the DNA code which can lead to congenital abnormalities? Also, their health can be measured by epigenetic integrity, epigenetics being the overlying system of regulatory controls of genes or DNA sequence. Germ cells are unique in undergoing a large scale rebuilding of their epigenetic architecture, in preparation for their own unique developmental programme, and for the developmental programme of the fertilised egg. In doing so, mistakes can be made (epimutations), and these can be as severe in consequence as genetic mutations. The aim of our research is to find out more about how and why reproductive stem cells undergo this large scale rebuilding of epigenetic systems. Also, how mistakes are made in this process, and how these may affect the development of germ cells and embryos. Please contact Jeff Mann for further information on specific projects.

14. Classification of regulatory elements involved in gonadal differentiation

Professor Andrew Sinclair T 83416424 E	Dr Stefan White T 83416426 E
Dr Thomas Ohnesorg T 83416426 E

Intersex disorders, ranging in severity from genital abnormalities to complete sex reversal, are surprisingly common and as such represent a major paediatric concern. The cause of these problems is most often the failure of the complex network of genes that regulate development of testes or ovaries. However, we understand relatively little of this regulatory network. Our research seeks to understand the molecular basis of testis and ovary development and how key genes are regulated. We are currently studying regulatory elements controlling a range of genes involved in gonadal differentiation. Methods that are being applied include DNaseI hypersensitivity analysis, Chromatin Immunoprecipitation and reporter assays. Other methods involved include cell culture, (quantitative) PCR and expression analysis.

15. Cardiac molecular signaling mechanisms during the progression of heart failure

Dr Salvatore Pepe T 93454114 E	A/Professor Joe Smolich T 93454571 E
Professor Dan Penny T 93455922 E

Congenital and acquired myocardial disorders, despite diverse etiology, commonly involve a reduced capacity to manage oxygen and nitrogen free radical metabolism. Chronic augmented oxidative stress, particularly in fetal and neonatal development, not only leads to post-translational structural modification of proteins, but also impacts gene transcription, ultimately with consequences to structural, metabolic and functional remodeling during adaptive and maladaptive heart failure. These pathological changes remain to be well defined in the developing heart at molecular and cellular level in order for potential therapeutic targets to be identified. Students ideally should have a background in at least one of the following: biochemistry, immunology or pharmacology. Studies will explore novel molecular signaling pathways (intracellular/mitochondrial/nuclear) using unique in vitro and ex vivo models and a range of immunohistochemical, biochemical, molecular, genetic and cell biology methods.

16. Offering population carrier screening for fragile X syndrome

A/Professor Sylvia Metcalfe
T 83416309
E

Dr Melissa Hill
T 83416415
E

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability, with other medical, behavioural and emotional features of varying expression. The gene involved in FXS is located on the X chromosome, and expansions of a CGG repeat in the promoter region of the gene lead to the phenotype, such that the risk of being affected relates to the length of this repeat. Carriers of FXS are at risk of developing a late-onset tremor ataxia and female carriers have an additional risk of premature ovarian failure, as well a risk of passing on an expanded allele to their children (unlike male carriers). Carrier frequency in females is about 1 in 150. We conducted a recent study in which carrier screening was offered to non-pregnant women in a Victorian family planning clinic, and found that, overall, offering testing in such a healthcare setting was acceptable and feasible to women and staff, although various barriers to uptake were identified. The proposed project will examine acceptability and feasibility of offering carrier screening to women in other healthcare care settings, eg GP and obstetric clinics.

17. Chord Blood and Cardiac Stems Cells for Repair of Congenital Myocardial Dysfunction

Dr Salvatore Pepe T 93454114 E	Dr Christian Brizard T 93455200 E
Professor Dan Penny T 93455922 E

As the heart has recently been found to be capable of self renewal, the current work is aimed at acquiring a basic understanding of the growth and differentiation of cardiac progenitor cells. Related genetic transcriptional signaling, immune and endocrine regulatory factors will be studied to develop models of myocardial cell recruitment for potential treatment of congenital heart disorders such as hypoplastic left heart syndrome which currently only have palliative treatment options. Students ideally should have a background in immunology and biochemistry or pharmacology, and will predominantly utilise genetic, molecular and cell biology techniques.

18. Dissecting Survival And Apoptosis Pathways In Myeloid Cells

A/Professor Paul Ekert
T 93455823
E

Dr Anissa Jabbour
T 93455835
E

Growth and survival of haemopoietic cells is regulated by growth factors such as Interleukin-3 (IL-3). When IL-3 is removed, dependent cells kill themselves by a mechanism that can be regulated by the Bcl-2 family of apoptosis regulators. The genes that regulate this process are likely to be important in the development of leukaemia since leukemic cells acquire the ability to survive and proliferate independently of growth factors. We have identified members of the Bcl-2 family that are required for apoptosis to occur in the absence of IL-3 and also genes that couple growth factor signalling to the survival of haemopoietic cells. Some of these genes do, as predicted, have important roles in myeloid leukaemia. The projects in the laboratory seek to determine how growth factors promote cell survival, and how the genes we have identified are regulated by growth factor signalling. The projects will provide students with opportunities to master a wide range of cell biological and molecular biological techniques, including PCR, cloning, tissue culture, flow cytometry, confocal microscopy, Western blotting and co-immunoprecipitation. In addition, students will learn about gene expression techniques and gene discovery, as they address the importance of various genes in the biology of tumours such as leukaemia.

19. Growth Factor CheckPoints in Neuroblastoma Cells Tumorigenesis: Cellular and Molecular Mechanisms

Dr Vincenzo Russo
T 93457931
E

Professor George Werther
T 93455951
E

Neuroblastoma is a common and devastating childhood cancer, highly invasive and resistant to treatment. Cells derived from these tumours are undifferentiated aggressive and disseminating. By modulating the growth factor environment of these cells we have discovered a number of critical genes involved in arrested differentiation, proliferation and motility of neuroblasts. The products of these genes control key cell cycle checkpoints, intracellular signaling and the activation of modulators of the extracellular matrix. We aim to dissect further these cellular and molecular mechanisms, particularly those related to the cell cycle and growth factor / cytokine signaling and reorganization of the extracellular matrix.
The methodological approach includes: cell cycle analysis by FACS; RNA extraction & molecular hybridisation (real time PCR and gene-array): functional gene analysis with validated siRNA: nuclear extracts and gel shift assay (EMSA); cell extracts, receptor activation and signaling pathway analysis (immunoblotting, phospho- protein, pathway inhibitors, etc): signaling-protein interactions using wild type and mutated signaling molecules.

20. Major or minor birth defects? Hospitalisations as a measure of morbidity.

A/Professor Jane Halliday
T 83416260
E

Dr Vijaya Sundararajan
T 9096 1306
E

The project will utilise an administrative dataset to identify cases with one of two selected birth defects and follow their hospitalisation patterns over time. This will give us the opportunity to monitor the impact of these conditions over the course of the children’s lives. The results of this study will provide us with important information to inform health policy, better plan for services and direct future research on the clinical management of these children.
Two examples of birth defects to be studied:
• Hypospadias are among the most common birth defects of the male genitalia, involving an abnormally placed urinary opening. While hypospadias is generally considered a ‘mild’ birth defect, there are degrees of severity and the impact of this condition on the course of a child’s life is not well documented. The prevalence of hypospadias in male babies is 40 per10,000.
• Tetralogy of Fallot (TOF) is a congenital heart defect, involving three to four anatomical abnormalities. TOF is the most common cause of the cyanotic ‘blue baby’ syndrome. Total surgical repair is possible, but surgical success and long-term outcome vary and lifetime follow-up is necessary. TOF occurs in approximately 3-6 births per 10,000.

21. Experiences of a genetic counselling intervention to facilitate communication within families

A/Professor Sylvia Metcalfe T 83416309 E	Dr Jan Hodgson T 83416308 E
A/Professor Clara Gaff T 83416241 E

We are conducting an NHMRC funded project of a randomised controlled trial of a genetic counselling intervention to facilitate communication to at risk family members after diagnosis and genetic testing of an inherited condition.
Utilising a qualitative approach the PhD project will aim to explore the experiences of the trial participants and health professionals carrying out the intervention.The findings will form part of the process evaluation for the study.

22. Large-scale screen of genes controlling skeletal development

Dr Peter Farlie
T 8341 6409
E

Dr Christopher Gordon
T 8341 6418
E

Congenital defects of the skeleton are common and have a major impact on health and well being of affected children. Microarray RNA expression analysis of skeletal development is a powerful genome scale screening technology that is beginning to reveal essential pathways in skeletogenesis. However, a major limitation in this process is the functional analysis of identified candidate genes. To address this limitation in the analysis of our microarray data, we have developed a high-throughput screen to analyse the function of candidate genes in early skeletal development using avian retroviral delivery of expression and knockdown constructs. This screen will allow the student to rapidly analyse gene function in a whole animal model and will facilitate large-scale functional analysis of the genes controlling skeletal development and causing human skeletal defects and disease.

23. ENU mutagenesis screen for genes controlling craniofacial and limb development

Dr Peter Farlie
T 8341 6409
E

Professor John Bateman
T 8341 6422
E

ENU mutagenesis is a forward genetics approach that allows phenotypes of interest to be identified without any prior knowledge of specific genes involved in the development of the organ system of interest. We have begun an ENU mutagenesis screen for genes involved in the development of the craniofacial and limb skeletons and have a number of mouse strains with phenotypes that are of great interest. All strains identified so far have both craniofacial and limb anomalies that appear similar to human syndromes. Identification of the mutated genes harboured within these strains may facilitate identification of disease genes in humans. This project will involve the phenotypic analysis of one or more strains of mice with developmental anomalies of the skeleton and identification of the genetic pathways associated with the malformation. The techniques to be employed in this project include wholemount and section in situ hybridisation analysis of embryos, skeletal preparations and histology.

24. Neuropathogenic mechanisms of mitochondrial dysfunction

Dr Denise Kirby
T 83416234
E

A/Professor David Thorburn
T 83416235
E

Mitochondrial dysfunction causes a range of early-onset neurological conditions and contributes to neurodegenerative conditions such as Parkinson Disease. The mechanisms of neuronal damage are unknown, and study of these at a cellular level may lead to improved treatment and greater understanding of the role of both nuclear- and mitochondrial-DNA mutations in both rare and common conditions. This project will focus on complex I deficiency, the most common respiratory chain defect. It will study the effects of mutations on mitochondrial membrane potential, reactive oxygen species, ATP production and cellular calcium dynamics, mostly using fluorescent imaging techniques. We already have data on most of these parameters for patient fibroblasts with characterised mutations in complex I subunits and assembly factors. Three other cell culture models that more closely reflect neuronal function will be used to study possible mechanisms:
1. Mouse embryonic stem cells with mitochondrial DNA mutations, which can be differentiated into neurons and glia.
2. Neural cell cultures from two mouse models with complex I deficiency due to mutations in two different nuclear complex I subunits.
3. Olfactory stem cell cultures derived from nasal epithelial biopsies from patients with mtDNA mutations affecting complex I activity or with Parkinson disease, which can be grown as neurospheres and differentiated into neurons and glia.

25. Understanding the role of infectious agents in children with early onset Crohn's disease.

Dr Carl Kirkwood T 83416439 E	Dr Josef Wagner T 83416450 E
Professor Ruth Bishop T 93455062 E

Crohn’s disease is a major cause of morbidity throughout the world. It is an incurable condition associated with chronic inflammation of the gastrointestinal tract of genetically susceptible individuals. Crohn’s disease usually begins in early adulthood and is treated with potent immunosuppressive medications, but often requires surgery. We have conducted preliminary studies designed to identify an infectious agent in gut biopsy tissue obtained from children with suspected Crohn’s disease. Using microarray and subtractive hybridisation techniques we have identified a candidate viral agent that we propose could initiate the gut damage and ongoing immune activation. This project will continue to utilise molecular techniques to genetically characterise the viral agent in children with Crohn’s disease and develop specific PCR detection assays to determine the prevalence of the virus in clinical specimens collected from children at disease onset.

26. Characterisation of novel viral agents responsible for acute diarrhoea in children.

Dr Carl Kirkwood
T 83416439
E carl.kirkwood

Professor Ruth Bishop
T 93455062
E

Diarrhoea is responsible for over 2 million deaths worldwide, primarily in children under 5 years of age. A diverse group of pathogens including viruses, bacteria and parasites can cause diarrhoea, with rotavirus, adenovirus and astrovirus the chief viral agents identified. However, up to 30% of diarrhoeal cases are of unknown etiology, suggesting a role for novel or unknown agents. We have conducted preliminary studies using a mass-mini sequencing approach to explore the viral communities present in children with undiagnosed diarrhoea. Using this approach we have detected known enteric viruses as well as multiple sequences from novel viruses including bocavirus and picobirnaviruses.
In this research project we propose to further explore to the role of novel viruses using a mini-mass sequencing approach and genetically characterise these novel viruses to allow classification. RT-PCR assays will be developed and used to determine the prevalence of these agents in clinical specimens collected from children with diarrhoea.

27. Hearing loss: why?

A/Professor Henrik Dahl T 83416253 E	Dr Shehnaaz Manji T 83416254 E
Kerry Miller T 83416254 E	Dr Louise Williams T 83416254 E

Ten percent of the Australian population has a significant hearing loss. The major objective of our research is to identify and understand the causes of deafness in children and adults so that deafness can be prevented or new therapies developed. We have identified several mouse strains with inherited hearing loss. These mice are a new, unique and valuable resource for studying the contribution of genetic and environmental factors to hearing loss. We are identifying and characterising new “deafness” genes in these mouse strains. These genes will be identified through database mining and DNA sequencing. We will then study the function and expression of these novel “deafness” genes to expand our understanding of how the ear develops and functions. Expression of “deafness” genes will be analysed by in-situ and immunohistochemical techniques using isolated mouse inner ears, as well as by biochemical methods in tissue cultures. These investigations will be complemented by analysis of human DNAs and provide much needed insights into hearing loss in humans.
This project provides an opportunity to work with and develop skills in a wide variety of techniques, encompassing the biology of hearing loss at both the DNA and protein levels.

28. Gene expression in ear hair cells.

A/Professor Henrik Dahl
T 83416253
E

Dr Shehnaaz Manji
T 83416254
E

A goal of our laboratory is to develop cell-based therapies for hearing loss using progenitor or terminally differentiated hair cells generated in vitro. Hearing loss affects more than six hundred million people worldwide. The financial, social and personal costs of deafness to affected people, their families and the society are significant. Management options include learning sign language or fitting hearing aids or cochlear implants. However, none of these options are fully satisfactory. The availability of stem cells has given us the opportunity to develop novel therapies based on replacing or regenerating cells in the inner ear. For such cell-based therapies to work we need to understand the differentiation pathways that the inner ear hair cells and their supporting cells follow during the development of the auditory system. We are able to isolate inner ear hair cells from mice with green fluorescent hair cells at different developmental stages by fluorescence-activated cell-sorting (FACS). RNA will be isolated from these cells and analysed on microarrays. This project will focus on analysing the microarray data and studying selected developmentally regulated genes using techniques
commonly used to study gene expression.

29. Stem cell transplantation for the treatment of MMA

Dr Heidi Peters
T 83416257
E

Dr Nicole Buck
T 83416236
E

Methylmalonic aciduria is an autosomal recessive inborn error of organic acid metabolism, affecting approximately 1/140,000 children. The condition results from a functional defect in the enzyme methylmalonyl CoA mutase.
This project aims to investigate the degree to which a transplanted immature liver cell line can reduce disease and biochemical phenotypes observed in a mouse model with an intermediate phenotype for the human disorder MMA.
We have established colonies of transgenic mice which will be transplanted and then characterised biochemically and phenotypically. Tissues will be examined for the presence and level of EGFP expression using fluorescent microscopy, RT-PCR of mRNA, western blot and flow cytometry. The activity of the transplanted cells will also be confirmed by specific liver enzyme assays and measurement of metabolite levels by mass-spectrometry.
This project will lead on to extending the work to examine methods of increasing the level of production of MMA enzyme from the cell line. This would involve the use of both viral gene therapy techniques and non viral methods prior to transplantation.

30. Epigenetic factors that regulate the chromatin at the telomeres in pluripotent embryonic stem cells.

Dr Lee Wong
T 83416240
E

Professor KH Andy Choo
T 83416306
E

There is currently a huge interest in understanding how embryonic stem (ES) cell pluripotency is controlled, and what (epigenetic) changes occur at the chromatin level during differentiation. However, until the present study, no one has investigated the chromatin status of the telomere in pluripotent and differentiating ES cells. In our study, we have found that the telomere chromatin of the pluripotent ES cells is unique compared to that of the differentiating ES cells. Specifically we show enrichment of histone variant H3.3 at the telomeric chromatin in mouse ES cells. The study involving RNAi-depletion of H3.3 that results in impairment of the telomere structure also confirms that H3.3 is essential for the maintenance of telomeric integrity in these cells. This project involves the investigation of the roles of H3.3 and its interacting partner as a ‘reprogramming cue’ for the maintenance of prolonged telomere-self renewal in pluripotent ES cells, and in other adult stem cells.

31. Genetic and Epigenetic Regulation of the Structure and Function of Human Chromosomes

Professor KH Andy Choo
T 83416306
E

Dr Lee Wong
T 83416240
E

This project aims to study the role of genetic and epigenetic factors in regulating the structural and functional integrity of chromosomes and chromatin. We will use normal centromeres, neocentromeres (a new class of centromere devoid of alpha-satellite repetitive DNA first discovered by us), and human artificial chromosomes to investigate how different chromatin-modifying proteins (including constitutive centromere proteins, histone variants, boundary element insulator and DNA repair checkpoint proteins) and non-coding RNA components (including centromeric alpha-satellite and retrotransposon transcripts) are organised at the centromere regions. The organisation will be defined at the linear chromatin level using molecular biology analysis, and at the 3-D level using electron microscopy. The knowledge gained will be fundamental to our understanding of how genetic and epigenetic factors regulate centromere hierarchical assembly and its function in the maintenance of mitotic activity.

32. RNA interference therapy: Applications in ß-thalassaemia

Dr Jim Vadolas
T 8341 6232
E

Dr Heidi Peters
T 8341 6233
E

Severe ß-thalassaemia (ß-thalassaemia major) is an inherited haemoglobinopathy arising from mutated ß-globin genes, resulting in reduced ß-globin chain synthesis. Much of the pathology of this disease is due to excess a-globin chains forming toxic insoluble precipitates in erythroid cells resulting in cell death, ineffective erythropoiesis and severe anaemia. Decreased a-globin chain synthesis leads to milder symptoms, exemplified by individuals who co-inherit a- and ß-thalassaemia. Therefore, a possible therapeutic strategy in the treatment of ß-thalassemia could include targeted reduction of a-globin chains to mimic co-inheritance of a/ß-thalassemia. RNA interference (RNAi) is an innovative new strategy for modulating gene expression and this pathway can potentially be exploited to mediate reductions in a-globin. Our group has identified key regions in the a-globin mRNA sequence which can be targeted with high efficiency using short-interfering RNA (siRNA) to mediate significant reductions in a-globin expression. We have also successfully demonstrated that RNAi-mediated reduction of a-globin results in phenotypic improvements in ß-thalassaemic cells. This project aims to develop strategies for targeted delivery of siRNA into erythroid progenitor cells. Initial studies will be conducted in vitro and will involve culture of both cell lines and primary cells. Further studies will also be conducted in vivo using our unique humanised ß-thalassaemia mouse models and patient-derived cells.

33. Stem cell and gene therapy: Targeted integration of functional genomic loci

Dr Jim Vadolas
T 8341 6232
E

Dr Heidi Peters
T 8341 6233
E

One of the major obstacles to successful gene therapy is the random integration of the therapeutic transgene, which is associated with insertional mutagenesis and oncogenesis. Using specific elements derived from adeno-associated virus (AAV) our research group has developed a novel strategy to enhance the delivery, and site-specific integration of large DNA molecules into the human genome. We have recently shown that we can enhance the delivery, and facilitate the site-specific integration of the entire human ß-globin locus. This project will investigate the site-specific integration of functional genomic loci into stem cells. Reporter gene expression and fluorescence in situ hybridisation will be used to monitor targeted integration and tissue-specific expression. In vitro differentiation will be used to assess the capacity of modified stem cells to differentiate along multiple lineages. The transplantation potential of modified stem cells will be investigated using the immunodeficient NOD/SCID mouse model. We propose that this non-viral gene therapy strategy may be used in conjunction with patient-derived stem cells to facilitate persistent and stable transgene expression while avoiding the risks associated with random integration.

34. Mouse models for mitochondrial disease: Mendelian genetics and synergistic heterozygosity

A/Professor David Thorburn T 8341 6235 E	Dr Jasper Komen T 8341 6287 E
Dr Bi-Xia Ke T 8341 6287 E

Disorders of mitochondrial energy generation cause a wide range of diseases. Severe mitochondrial defects affect ~1/5000 individuals, often causing childhood neurodegenerative diseases. Increasing evidence suggests that some patients have digenic or multigenic disorders rather than simple single gene defects of one mtDNA or nuclear gene. In the general population at least 1 in 10 people carry nuclear or mtDNA genetic variants that cause milder mitochondrial dysfunction, which may contribute to common conditions such as Parkinson disease and diabetes. We have generated two mouse models of Complex I deficiency, the most common type of mitochondrial disease. Ndufs4-/- mice have a systemic Complex I defect and neurodegenerative disease while Ndufs6 GeneTrap mice have a Complex I defect primarily affecting heart and develop cardiomyopathy. This project will complete characterization of the phenotype of heterozygous and homozygous mice using a range of physiological, molecular, immunochemical and neuropathological approaches. Homozygous mice will be used in studies of therapeutic approaches such as high fat diet or upregulation of mitochondrial biogenesis using bezafibrate or resveratrol. Heterozygous mice will be used to investigate the role of mild mitochondrial dysfunction as a risk factor for common diseases. In addition, doubly heterozygous mice (ie, heterozygous for both Ndufs6 and Ndufs4 mutations) will be studied to investigate possible effects of synergistic heterozygosity.

35. Discovering novel genes that cause childhood mitochondrial disorders

A/Professor David Thorburn
T 8341 6235
E

Dr Alison Compton
T 8341 6287
E

Mitochondria are the powerhouses of the cell, generating cellular energy through the oxidative phosphorylation (OXPHOS) system. Pathogenic mutations in genes required for correct assembly of the OXPHOS protein complexes (I-V) result in a variety of neurodegenerative disorders collectively known as mitochondrial disorders. Over 90 (nuclear and mitochondrial) genes are known causes of mitochondrial disease, however there are still many more novel disease genes awaiting discovery. We recently identified four novel (nuclear) disease genes in which mutations cause severe childhood-onset OXPHOS complex I deficiency, the most common mitochondrial enzyme defect in humans. These are NDUFS6 (Kirby et al., 2004 J Clin Invest 114: 837-45), NDUFAF1 (Dunning et al., 2007 EMBO J 26: 3227-37), C8ORF38 (Pagliarini et al., 2008 Cell 134:112–123) and C20ORF7 (manuscript under review). Recently, our collaborators at the Broad Institute, Harvard have compiled a compendium of 1098 ‘mitochondrial’ genes called Mitocarta, including 19 novel genes proposed to be involved in OXPHOS complex I biogenesis (Pagliarini et al., 2008 Cell 134:112–123). We are currently performing SOLEXA high throughput sequencing of 88 ‘mitochondrial’ genes in 110 of our patients with defined complex I defects. This project will follow up on sequence variants identified in that study to identify novel OXPHOS disease genes and determine their normal function and disease pathogenesis using a combination of cell biology, molecular biology and biochemical approaches.

36. Investigating the role of altered methylation in schizophrenia

Dr Jeff Craig
T 83416346
E

Dr Richard Saffery
T 83416341
E

Despite the relatively high prevalence (approaching 1%) and devastating social and financial impact, there is presently no “objective” biological test to screen for Schizophrenia risk. The identification of SZ biomarkers is a crucial step towards improving current diagnosis, developing new presymptomatic treatments, identifying high-risk individuals and disease subgroups, and assessing the efficacy of preventative interventions at a rate that is not currently possible. In addition, the identification of brain-specific biomarkers of SZ has the potential to reveal valuable insights into the development of this disorder.
We have data demonstrating that, SZ cases have lower serum folate level than controls with altered epigenetic modification of specific genes in the prefrontal cortex compared to disease free brains.
We now aim to further characterise specific epigenetic biomarkers of SZ in post-mortem brain samples, to identify peripheral epigenetic biomarkers of SZ in blood; and to characterise the downstream functional consequences of this altered methylation profile.

37. Epigenetics and the interaction between folate and vitamin D metabolism at the fetomaternal interface.

Dr Jeff Craig
T 83416346
E

Dr Richard Saffery
T 83416341
E

The fetomaternal interface (placenta: decidua) represents a major site of accumulation of active vitamin D. However the exact role of this remains to be elucidated but may involve immune modulation, regulation of cell division, and/or maximal transfer of VitD to the developing fetus.
Recent data have demonstrated a role of epigenetic modification generally (and DNA methylation specifically) in the regulation of genes involved vitamin D metabolic enzymes that control vitamin D bioavailability and action. As one-carbon donors derived from maternal dietary folate are critical for the establishment of DNA methylation, we believe that sub-optimal circulating levels of vitamin D and folate may act cooperatively to alter vitamin D bioavailability in the developing pregnancy.

38. The role of altered epigenetics in Paediatric Leukaemia development and outcome?

Dr Richard Saffery T 83416341 E	Dr Nicholas Wong T 83416205 E
Dr Jeff Craig T 83416346 E

Leukaemia is the most common form of cancer in children, accounting for over 30% of newly diagnosed cases. Most cases involved specific genomic rearrangements (translocations). However, these are neither sufficient nor absolutely necessary for disease development. Despite the fact that ~80% of cases are successfully treated by chemotherapy, the underlying causes of childhood leukaemia remain unclear and cannot be explained by genetic or environmental factors alone. Epigenetics is an emerging field examining the modulation of gene expression in the absence of underlying genetic change. We believe disruption of epigenetic profile could play a major role in the aetiology of paediatric leukaemia in conjunction chromosome translocations. This project will catalogue epigenetic changes at gene promoters from archived matched leukaemia and remission bone marrow samples in an attempt to identify changes in associated with development or outcome of specific leukaemia subtypes.

39. What is the role of Epigenetics in Paediatric Leukaemia?

Dr Richard Saffery T 83416341 E	A/Professor Jane Halliday T 83416260 E
Dr Jeff Craig T 83416346 E

40. Unravelling molecular mechanisms and developing treatments for inherited musculoskeletal disease

Professor John Bateman
T 83416422
E

Dr Richard Wilson
T 93456601
E

Inherited musculoskeletal disorders are a significant disease burden and although many mutations have been defined, our knowledge on the molecular mechanisms that cause them, and ultimately how these mechanisms could be manipulated, is only just beginning to be explored. Many of the gene mutations result in the production of mutant protein that is compromised in its ability to form the correct 3-D folded functional structures. Recent research has shown that these unfolded proteins can cause cellular stress and activate intracellular signalling pathways that have profound effects on cell gene expression that may contribute to cellular pathology. The proposed studies will explore the molecular signalling pathways using in vitro and transgenic mouse models and a range of immunohistochemical, biochemical, molecular methods and proteomic analysis (2D-electrophoresis and mass spectrometry) skills. In addition, our studies will explore the use of new therapeutic agents to overcome protein misfolding and cell stress, as a proof-of-principle that some of these diseases can be effectively treated.

41. What causes cartilage degeneration in osteoarthritis?

Professor John Bateman
T 83416422
E

Dr Richard Wilson
T 93456601
E

Degeneration of articular cartilage is the central pathological feature of osteoarthritis (OA) and it is this progressive erosion of cartilage that leads to joint failure and necessitates joint replacement surgery. We have a major research program determining the molecular events in the initiation and progression of cartilage breakdown. Using microarray analysis to look at gene expression changes and proteomic approaches we have determined new arthritis candidate genes. Several PhD projects are available in this program explore the detailed biology of these genes and the biological pathways that result in the onset and progression of OA. These studies will involve the use of a wide range of molecular biology, biochemical, cell biology and proteomic techniques in both in vitro experiments and the analysis of mouse osteoarthritic tissues.

42. mRNA surveillance in human disease: How cells detect and degrade deleterious mutant mRNA (nonsense-mediated mRNA decay)

Professor John Bateman
T 83416422
E

Dr Jacqueline Tan
T (03) 9345 6601
E

Cells have several critical quality control processes to reduce the impact of mutations on cell function. We are studying nonsense-mediated decay (NMD), a mRNA quality mechanism that degrades mRNA containing premature stop codons. Since mutations that introduce premature stop codons account for one-third of inherited disorders, NMD is of immense importance in many disea