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Faculty of Medicine, Dentistry and Health Sciences
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2007 projects - PhD
1. Muscular dystrophies - understanding the molecular pathology of collagen VI mutations
Mutations in the extracellular matrix protein collagen VI cause Bethlem myopathy and Ullrich congenital muscular dystrophy, but we don't yet understand why mutations in such an abundant and widely distributed protein cause a muscle specific disease. One goal of this project is to use proteomic analyses (affinity chromatography, 2D electrophoresis and mass spectrometry) to identify the proteins that interact with collagen VI in muscle and examine the effect of collagen VI mutations on these interactions. Our research suggests that around half of Bethlem and Ullrich patients do not have collagen VI mutations. Collagen VI interacting proteins are the best disease gene candidates in these patients. The second goal of this project will be to screen the genes that encode collagen VI interacting proteins for mutations in muscular dystrophy patients who do not have collagen VI mutations.  2. The molecular genetics of skeletal disorders
Identification of novel genes responsible for human skeletal disorders will increase our understanding of the molecular cascades that regulate cartilage and bone formation as well as the pathways of degenerative diseases such as arthritis. Using whole genome linkage analysis we have mapped the genes for two dominantly inherited skeletal disorders. In the first disorder, familial digital arthropathy-brachydactyly (FDAB), affected individuals have progressive shortening of the fingers and toes and early onset arthritis of the hands and feet. The second disorder, Scheuermann disease, involves teenage onset of spine deformity and degenerative disc disease. The goal of this project is to identify the disease genes in these families. Selection of candidate genes for sequencing will involve analysis of tissue expression profiles using oligo microarrays, and bioinformatic approaches. Once the disease genes have been identified functional studies including tissue in situ hybridisations and immunostaining, and the generation of animal models will determine their role in skeletal development and degeneration. 3. Chromosome instability and human disease
Approximately 10 quadrillion cell divisions occur in the lifetime of a human. Each one of these divisional events requires the accurate distribution of the newly-replicated chromosomes to the daughter cells. Any faults occurring during this process may result in gains or losses of chromosomes. This imbalance can lead to human conditions such as Down syndrome, pregnancy loss, cancer, infertility, and a wide range of genetic disorders. The Chromosome and Chromatin Research Laboratory is currently investigating the cellular and molecular mechanisms that are responsible for chromosome number imbalances. To investigate these mechanisms, we will develop cutting-edge technologies and model systems, such as based on the use of embryonic stem cells, artificial chromosomes, mouse transgenics, identification of novel centromere and chromosome proteins, and sensitive screening methods for uncovering mammalian cell mutants showing chromosome missegregation defects. Our laboratory has a long-standing close association with the co-located Cytogenetics Laboratory of the Genetic Health Services Victoria that is able to provide patient materials for our research. Results gained from these studies will greatly assist in the understanding and ultimately the prevention of many common genetic diseases that have underlying chromosome instability aetiology. 4. Folate supplementation and disease prevention
Folate is a vitamin pivotal in DNA repair and synthesis, that can only be obtained from the diet. Folate deficiency can lead to increased DNA fragmentation and chromosome instability, observed in malignancies and developmental abnormalities. In 1995 the government has allowed the fortification of this vitamin in food. However, strong debate continues in the scientific community and the public concerning the risks and benefits of folate supplementation on the health of the general population. This project aims to study the possible ill-effects (for which we have preliminary data) of folate over or under supplementation. The project will investigate cellular, molecular, and chromosomal changes using cancer cell lines and primary cells, including stem cells. One aspect of the study will entail the use of mouse models to investigate relevance of dietary folate levels to developmental defects, with a longer-term view to extend the study to humans. Specific techniques used will include flow cytometry, immunofluorescence microscopy, FISH, real-time PCR, and gene-chip technology. An important outcome of the project is a much better informed understanding of what the safe levels of dietary folate supplement should be, and the cellular and molecular consequences of folate deficiency or excess. 5. Genetic and Epigenetic Regulation of the Structure and Function of Normal and Artificial Chromosomes
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 first discovered by us), and human artificial chromosomes to investigate how different constitutive and chromatin-modifying proteins and RNA components are organised at the centromere and non-centromeric regions. The organisation will be defined at the linear chromatin level using chromatin immunoprecipitation and microarray analysis, and at the 3-D level using electron microscopy. We also aim to study the protein complexes involved in the chromosome condensation process using advanced proteomic technology. The necessary technology and a number of promising novel RNA and protein components (such as the multi-subunit protein complex condensin that is key to chromosome condensation) for this project are already available. The knowledge gained will be fundamental to our understanding of how genetic and epigenetic factors regulate centromere assembly and function. The detailed analysis of the human artificial chromosomes, first engineered (and patent-protected) in our laboratory, will help us develop these artificial chromosomes as gene therapy tools. 6. Molecular genetics of deafness
Hearing loss affects approximately 10% of Australians. Deafness is caused by genetic and environmental factors (incl. infections, noise exposure, and exposure to ototoxic drugs). It is also clear that genetic factors play a significant role in how susceptible we are to environmental deafness. Rapid progress has been made in identifying “deafness” genes. Despite the recent successes, many more genes important in normal hearing await identification and characterisation. Analysis of mouse models for deafness is a powerful approach to identifying human genes for hearing loss. The mouse inner ear is very similar to that of humans, and genetic and molecular studies can be easily be done using mice. We have identified more than 10 new and unique mouse strains with congenital or later-onset recessive deafness. We have discovered novel “deafness” genes in some of these strains, but others await identification. The project will involve identification and characterisation of new “deafness” genes using molecular and genetic techniques. The contribution of these genes to human deafness and the degree to which changes in the genes make us more susceptible to environmental deafness will also be investigated. 7. The relationship between airway inflammation, airway remodelling and airway hyperresponsiveness in asthma
Asthma is the commonest chronic disorder affecting Australian children. It is responsible for 1/250 deaths. Severe and persistent asthma involves airway hyperresponsiveness and irreversible loss of lung function. The relative contributions of airway inflammation and structural changes (airway remodelling) to these outcomes has not been elucidated. Airway remodelling is not addressed by current therapies. Our research interest is in developing treatments for airway remodelling and investigating its relationship with inflammation and hyperresponsiveness. Murine models of human asthma, cell culture and human biopsies are used in our studies of gene products and drug treatments. The current project aims to (1) determine the sequence of events in the pathogenesis of airway remodelling in asthma and their relationship to airway inflammation in vivo, (2) investigate the relationship between epithelium, fibroblasts and extracellular matrix in the development of airway remodelling using an in vitro culture model and (3) explore mechanisms of action and optimal delivery routes for potential therapies for airway remodeling and airway hyperresponsiveness in vivo in allergic airways disease models. 8. Liver cell transplantation for the treatment of metabolic liver disease
This project proposes to investigate the role of liver cell transplantation for the therapy of inherited metabolic liver disease using a methylmalonic aciduria mouse model developed within the CAGT group. Liver cell transplantation (LCT) is a new and developing field of therapy and provides an exciting alternative to the traditional whole organ liver transplantation. Currently the group has been investigating the use of syngeneic cells for transplantation. In clinical practice however, cells are obtained from one or multiple donors and the problem of rejection needs to be considered. This project aims to address this issue by using allogeneic cells for transplanattion. A number of antibodies have been identified that can prevent rejection and aid long term survival of transplanted cells. Engraftment and attrition of allogeneic liver cells will be examined following the co-administration of various antibodies. This will be achieved by either treating cells directly or alternatively via systemic administration of antibodies. 9. Pharmacological-based therapy for ß-thalassaemia: an insight into the epigenetic control of the ß-globin locus
Severe ß-thalassaemia (ß-thalassaemia major) is an inherited haemoglobinopathy arising from mutated ß-globin gene, resulting in reduced ß-globin chain synthesis. Much of the pathology of this disease is due to excess ß-globin chains forming toxic insoluble precipitates in erythroid cells resulting in severe anaemia. The ß-thalassaemias are one of a few medical conditions in which reactivation of a gene that is expressed during fetal life can ameliorate disease severity. We recently identified a number of novel pharmacological compounds that upregulated fetal globin expression. Many of these compounds were found to be pharmacological agents currently used in humans for conditions unrelated to thalassaemia. In this study, the therapeutic potential of these pharmacological agents will be investigated in our humanised ß-thalassaemia mice and human stem cells. Another aspect of this study will be to elucidate the molecular mechanisms of these agents at the epigenetic level by using ChIP assays, tissue-specific microarrays and bioinformatic strategies. The knowledge gained in this study will be essential for the development of safe and effective therapy for ß-thalassaemia patients. 10. Control of nerve cell migration: understanding Hirschprung's Disease
The nervous system of the intestine is crucial for peristalsis. Nerve precursor cells migrate from the brainstem into the intestine at 3-7 weeks post-conception in humans. A common and potentially fatal birth defect, Hirschsprung’s Disease (HD), is due to abnormalities in this migration. This is the clearest clinical example of a cell migration defect and this project focuses on understanding migration in normal and HD conditions. Mutations in at least 11 genes predispose to HD. Our group has made fundamental technical and intellectual advances in linking phenotype to genotype. In this project molecular and genetic control of intestinal neuron precursor cell migration will be explored in animal models, in the whole embryo and in organ culture, and using techniques to manipulate gene expression and function, including transgenic mice and in vivo gene transfer. This project meshes with mathematical systems analyses of cell migration with collaborators at Melbourne University, and San Antonio and U. Wisconsin, USA. For recent reviews (pdfs), contact above email. 11. How do cells become invasive in normal development and in malignant cancer?
In normal development, sessile epithelial cells change to migratory mesenchyme cells in order to move as the embryo changes shape. This change is called the epithelio-mesenchymal transition (EMT). EMT-like changes also occur normally in adults during wound healing. Pathological EMT has recently been recognized when a benign carcinoma becomes invasive. Evidence is accumulating that in cancer an essentially normal EMT is re-activated, but at “the wrong time, wrong place”. This project uses a well characterised embryonic model of normal EMT and a recently described breast carcinoma cell line with inducible EMT, both systems developed by this group at MCRI/St. Vincent’s). These cells are used to probe the molecular and genetic controls of EMT in normal and cancerous contexts. This project emphasizes the roles of gene expression and signal transduction, and involves collaboration with the Cancer and Invasion Unit at St Vincent's Hospital. For recent EMT reviews (pdfs), contact above email. 12. Large-scale screen of genes controlling skeletal development
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. 13. Burden of genetic disease and paediatric services
The importance of genetics in public health and in paediatric medicine is increasingly recognised. The impact of genetic conditions on morbidity and health care resources can be measured relative to non-genetic conditions by examining hospital admissions. This will be done by reviewing medical records for evidence of the underlying condition and classification into categories depending on the presence or absence of a genetic condition. This health service research project will allow for quantification of the importance of genetics in health care and workforce planning by estimating the current prevalence of specific genetic conditions in the paediatric setting and associated lengths of hospital stay. This can be compared to data collected on this topic at the RCH in 1985. 14. Is Vitamin D deficiency a risk factor for birth defects?
Birth defects are a significant public health concern with an immense global toll affecting 6% of all births. In Australia 4% of births have a birth defect and they are the leading cause of infant deaths, and result in continuing mortality and morbidity in childhood. There is evidence from animal studies that vitamin D regulates cell differentiation and proliferation, for example in the heart and brain. Maternal vitamin D deficiency in pregnancy contributes to a number of adverse outcomes in children such as reduced bone-mineral accumulation or infantile rickets. However, the role of vitamin D in pregnancy in humans, in terms of fetal development is largely unexplored. This project will use a case-control design to examine whether a mother with markers of low vitamin D in pregnancy is at increased risk of having a baby with a birth defect. 15. How do Wilms tumor 1 (WT1) isoforms and Telomerase contribute to leukaemia?
WT1 and hTERT ( the catalytic subunit of the human telomerase complex) are frequently abnormally expressed in leukaemia and elevated expression has been correlated with poor outcome. Recent evidence suggests that aberrant expression of WT1 in the leukaemic stem cell contributes to transformation and is an early change in the progression pathway from a normal haematopoietic stem cell to a leukaemic cell. Clinical trials targeting WT1 protein expression in myeloid leukaemias are in progress overseas. In this project the roles of WT1 and hTERT will be explored in both normal and malignant haematopoiesis ( leukaemia ) using transgenic haematopoietic progenitor cell lines. Relationships between WT1 and hTERT expression and their involvement in proliferation, survival, clonogenic potential and differentiation will be ascertained with the aim of understanding how these proteins contribute to leukaemogenesis. 16. Roberts syndrome –when chromosome glue comes unstuck
Roberts syndrome involves limb reduction and craniofacial anomalies. It also results in separation of heterochromatin, which is found mainly in and around centromeres. The cause of Roberts syndrome has remained unknown until very recently when it was revealed that patients have mutations in a gene whose product interacts with cohesins: proteins that form a complex that joins sister chromatids together until the time of cell division. We have already made progress studying the cell biology in cells from a patient with Roberts syndrome and we expecting more patient samples soon. The main questions that this project aims to address are why does Roberts syndrome only affect centromeric cohesion and what is the role of the Roberts syndrome gene in limb and craniofacial development. Experiments in lower eukaryotes have implicated chromosome glues in the transcriptional regulation of developmental master genes. Techniques that will be used in this project include immunofluorescence, high resolution microscopy, Western blotting, and recombinant DNA technology. 17. Investigating the potential role of Tigger family transposons in cell division, reproductive fitness, and epilepsy in mammals
The highly conserved but poorly characterised Tigger family of transposon-related genes is interesting on many levels. We have identified several family members in both humans and mice but to date, only two have been studied in detail. Although ubiquitously expressed, gene knockout studies have revealed unexpected independent tissue specific functions for the CENP-B and JRK Tigger family members. These appear widely disparate (uterine integrity, neuronal functioning) and have clear implications for human health. Although such a role is not unprecedented for domesticated transposable elements, it is novel that members of such a family may have evolved two apparently separable functions within complex organisms; a possibly redundant role in cell division, in addition to a defined disparate tissue specific function. Preliminary studies of other family members have revealed a wide expression profile of these genes and nuclear localisation of all family members. Proposed studies will involve the further generation of knockout animals, characterisation of nucleic acid binding properties of Tigger family members, and the characterisation of functional domains of these proteins. 18. Environmental influences in the establishment of the epigenetic landscape in children
The MCRI Epigenetics laboratory is collaborating with experts in genetics, nutrition, obstetrics, public health, epidemiology, biostatistics and bioinformatics to identify the nature and causes of epigenetic differences in twins at birth. More importantly, we are searching for the factors in the intrauterine milieu that, during pregnancy, influence the epigenetic profile of the conceptus and with this, the health of the individual at birth and in later life. Furthermore, we also aim to study the rate of epigenetic change and the effect of postnatal environment in twins by 18 months of age. Our ultimate aim is to empower individuals to influence the health of their offspring though the optimization of their environment before and during pregnancy. We have started collections of blood and tissue from the umbilical cords of newborn twins from three major Melbourne hospitals that will form the starting materials for epigenetic analysis. This project will involve tissue culture, expression analysis and a number of techniques to look at DNA methylation and histone modification, including microarray analysis. 19. Mental health across generations: pre- and post-conception Predictors of early life risks
There is increasing evidence that adolescent onset mental and behavioural disorders predict similar disorders the next generation. A range of mechanisms other than simple genetic mechanisms have been postulated including the post-natal maternal infant relationship, genetic, and epigenetic inheritance. To date, few studies have had the capacity to prospectively examine the impact of pre-conception and antenatal exposures on the health and wellbeing of the next generation. We are using the Centre for Adolescent Health prospective study of 2,000 young people surveyed on 9 occasions from around the time of puberty to their late twenties in order to achieve this aim. The offspring from this cohort have begun to arrive and we aim to investigate the effects of pre-conception and antenatal parental mental health and behaviour on perinatal psychosocial and biological risk factors for offspring mental disorder. The epigenetic components of this study will involve the investigation of the role of parental mood and behaviour before and during pregnancy on patterns of DNA methylation in the offspring, both globally and within specific candidate genes (implicated in mental health), and to relate patterns of DNA methylation in offspring to fetal growth in-utero and various infant behavioural measures. 20. Experimental arthritis in genetically-modified mice
Arthritis is a debilitating disease that affects babies, children and teenagers, as well as adults. A common feature of all forms of arthritis is the gradual loss of cartilage from the surface of bones, leading to long-term joint dysfunction. In arthritic cartilage, the major structural molecules, collagen and aggrecan, are destroyed by metal-dependent proteinases, or metalloproteinase. We have generated several new strains of genetically-modified mice, with knock-in and knock-out mutations designed to help elucidate the role of specific metalloproteinases in arthritis. A number of biochemistry, cell biology and molecular biology-based projects are available to characterise these genetically-modified mice in vivo and in vitro. Some projects will involve mouse models of experimental arthritis. 21. Food Allergy in Children: Identifying modifiable risk factors
Peanut allergy has been reported to be increasing in Western-industrialised countries. Why? 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 allow this. Infants who are 12-month-old will be recruited from the community across metropolitan Melbourne via Maternal and Child and Health 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 a formal peanut challenge and appropriate prescription of an Epipen. Other genetic and biochemical analyses will be undertaken as potential markers to predict allergy, anaphylaxis and tolerance. 22. Intestinal adaptation in children with short bowel syndrome
Short bowel syndrome (SBS) is the state of malabsorption that follows massive small bowel resection. Parenteral nutrition therapy can provide fluid and nutritional requirements, however, long-term PN therapy is associated with a ~5% annual mortality due to complications such as sepsis and liver failure. Intestinal transplantation may offer hope for some individuals, however, may be unnecessary if therapeutic strategies could be developed that enable the small intestine to adapt and compensate for the loss in bowel length. This project focuses on mechanisms associated with intestinal adaptation in a preclinical model of SBS and aims to translate promising interventions aimed at improving the adaptive response onto clinical trials in children with short bowel syndrome. 23. Mouse models for neurodegenerative disease
Disorders of mitochondrial energy generation cause a wide range of diseases. Severe mitochondrial defects affect ~1/5000 individuals, often causing childhood neurodegenerative diseases. At least 1 in 10 people carry nuclear or mitochondrial DNA genetic variants that cause milder mitochondrial dysfunction, which appear to contribute to common conditions such as Parkinson disease and diabetes. We have a very incomplete understanding of the pathogenic mechanisms linking genotype to phenotype in mitochondrial disorders and at present there are few useful animal models. We identified a novel gene causing a lethal mitochondrial disease of childhood (NDUFS6) and have very recently generated a knockout mouse model using GeneTrap embryonic stem cells. This project will characterise the phenotype using a range of physiological, molecular, immunochemical and cell biological approaches. Homozygous mice will be used in studies of therapeutic approaches. Heterozygous mice will be used to investigate the role of mild mitochondrial dysfunction as a risk factor for common diseases. This will involve testing their susceptibility to toxins used to induce diabetes and Parkinson disease and by breeding with other mouse models to investigate digenic inheritance. 24. Neonatal rotavirus vaccine: clinical trials and surveillance systems
RV3 vaccine has been developed from a strain of rotavirus (G3 P6) isolated from healthy newborn infants in Melbourne. Natural infection was 100% protective against severe rotavirus disease and 65% protective against moderate disease through the first 3 years of life. Thus it was considered a potential vaccine candidate. With collaboration with the WHO, Q-Gen and the Indonesian vaccine manufacturer BioFarma, we are developing RV3 vaccine. The goal is an effective rotavirus vaccine affordable for the global community. Clinical trials will be conducted in Melbourne, Indonesia and possibly other developing countries. Opportunities also exist for involvement in development of adverse surveillance projects for rotavirus vaccines. 25. Identification of novel human disease genes using integrative genomics
Mitochondrial energy generation disorders are the most common inborn errors of metabolism. They cause a wide range of severe childhood diseases and contribute to common conditions such as Parkinson disease and diabetes. Over 40 nuclear genes have so far been shown to cause mitochondrial disorders in children, but the genetic basis in most cases remains to be discovered. We have recently identified two novel (nuclear) genes causing severe mitochondrial diseases of childhood using an approach that combined genomic approaches (linkage analysis and microarray expression analysis) with cell biological studies (complementation analysis and phenotypic rescue by microcell-mediated chromosome transfer), bioinformatics and candidate gene analysis. This project will use similar integrative genomic approaches to study other potential novel chromosomal loci for genes causing mitochondrial disease. 26. Mapping neural control of the gut and changes following bowel removal
Movement of food along the gut is controlled and coordinated by neurons located in the walls of the gut tube. More than 20 different neurotransmitters are involved in contraction and relaxation pathways. Each transmitter is released from nerves and binds to receptors. How does the gut respond if a large segment has to be removed? Some babies have blockage of the blood supply of their intestine resulting in death of the segment and removal of a large segment. There are many problems to overcome to adjust to loss of a major part of the organ responsible for nutrient absorption. This project will determine the distribution of transmitters and receptors in animal and human colon using fluorescence immunohistochemistry and confocal microscopy in gut from guinea pigs, rats and pigs and in biopsies from children. This study will also look at changes in the muscle and nerves following bowel removal. This project will suit science students with neuroscience, physiology or anatomy backgrounds or medical/surgery graduates. 27. New treatments for chronic idiopathic constipation in children
Over 200 children at the Royal Childrens hospital have chronic constipation that does not respond to treatments such as laxatives, diet and enemas. We are testing transcutaneous electrical stimulation to overcome constipation. We are testing different types of electrical stimulation and assessing if there is improvement in bowel activity and quality of life. This study will look for defects in hormones, neurons, muscle and pacemaker cells (interstitial cells of Cajal) in slow colonic transit using fluorescence histochemistry and confocal microscopy. This project will suit science students with neuroscience, physiology or anatomy backgrounds or medical/surgery graduates. 28. Role of HoxB8 in myeloid cell differentiation
HoxB8 is a homeobox gene that can regulate myeloid cell differentiation and contribute to the development of leukaemia. We have used HoxB8 expression to generate novel cell lines, which are dependent on the growth factor Interleukin-3, from gene-deleted mice. We now wish to determine the mechanism by which HoxB8 regulates differentiation of myeloid cells. This project will employ an inducible expression system we have developed to determine the effects of inducing or withdrawing enforced HoxB8 expression has on cell differentiation in our novel cell lines and to screen for the genes (both novel and known) regulated by HoxB8 using expression array. These studies will provide insight into myeloid cell differentiation and gene mutations contributing to leukaemia. This project is laboratory-based and will expose the student to a wide range of cell and molecular biological techniques. Our laboratory has established links with groups at the Walter and Eliza Hall Institute and LaTrobe University. 29. Regulation of growth-factor withdrawal-induced apoptosis
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 (apoptosis) that can be regulated by the Bcl-2 family of apoptosis regulators. Our goal is to connect, molecule by molecule, the pathways that links growth-factor signalling with apoptosis mechanisms, since these pathways are important in normal development and the development of malignancies. We have generated a comprehensive panel of IL-3 dependent cell lines from gene-deleted mice lacking components of the apoptosis mechanism to test the requirement for each gene in the IL-3 withdrawal-induced apoptosis pathway. This project involves the generation and testing of new IL-3-dependent cell lines for growth-factor withdrawal responses and biochemical aspects of IL-3 signalling and screening a library for novel genes that co-operate with mutations in apoptosis pathways in the development of leukaemias or growth-factor independence. This project is laboratory-based and will expose the student to a wide range of cell and molecular biological techniques. Our laboratory has established links with groups at the Walter and Eliza Hall Institute and LaTrobe University. 30. mRNA surveillance in human disease: How cells detect and degrade deleterious mutant mRNA (nonsense-mediated mRNA decay)
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 diseases as well as normal development [see Hum Mol Genet. 8:1893 (1999)]. Our studies will explore the molecular basis of NMD by the production of mutations in reporter gene constructs we have developed to measure NMD in vitro by transfection of cells and determination of mRNA levels by PCR and primer extension assays. The project will characterise the mRNA sequences that specify NMD in different cell types and determine role of known and novel RNA binding proteins using electromobility shift assays. These trans-acting proteins will be identified by proteomics (electrophoresis and mass spectrometry) and their role in NMD tested by suppression of expression using RNA interference. 31. Molecular Regulation of Gubernacular Migration – the missing link in sexual development
The gubernaculum or “genito inguinal ligament”, was once thought to be a static or shortening ligament, pulling the testis down to the scrotum. We now know that it elongates out from the abdominal wall with many anatomical characteristics of an embryonic limb bud. This laboratory has played a leading role in identifying the anatomical processes and hormonal regulation of testicular descent, but little is known of the molecular regulation of gubernacular growth. The first goal is to test the hypothesis that molecular signalling in the gubernaculum is similar to that in an embryonic limb bud. The project will then examine the genes expressed in the gubernacular tip during growth and subsequent differentiation in cremaster muscle, with the ultimate aim of determining whether mutations in these genes lead to cryptorchidism. 32. The use of proteomics to investigate the mechanisms of cartilage degeneration in osteoarthritis
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. Our hypothesis is that by determining the proteome (“the dynamic protein complement”) of articular cartilage, and the changes in the proteome during the initiation and progression of cartilage degeneration in OA, we will gain key insights into the molecular mechanisms of OA and will identify new proteins and protein fragments as biomarkers for diagnosis of disease onset and progression. The studies will use proteomic methods for cartilage analysis, including micro-dissection, protein extraction, 2-D gel resolution (mapping) of cartilage proteins, and protein and protein fragment identification my mass spectrometry developed by us [Proteomics, In press]. We will also use the cutting-edge proteomic technology, 2-D Fluorescence Difference Gel Electrophoresis (DIGE), to allow the precise determination of the differential proteome of healthy and OA cartilage during the initiation and progression of arthritis. With these experimental tools we are in a unique position to identify the key protein changes and underlying mechanisms of cartilage degeneration in OA. 33. Role of a new cartilage matrix protein, WARP, in arthritis
We have recently identified and partially characterised WARP, a novel extracellular matrix molecule that has a unique expression pattern in joint cartilage. WARP forms an important structural network in cartilage during development [see J. Biol. Chem. 281(11):7341-9 (2006)] and in adult cartilages and it’s location at the articular surface suggests that it could be degraded early in the arthritic process, and the detection of WARP release and/or fragmentation during cartilage degeneration would provide important new diagnostic biomarkers and insight into the molecular mechanisms of arthritis. This project will study WARP synthesis, breakdown and release from cartilage in both in vitro and in vivo models of cartilage degeneration. We have developed a WARP knock-out mouse and analysis of cartilage development and degeneration in this will be of crucial importance in further defining the role of WARP in development and disease. The study will use a range of immunohistochemical, biochemical and molecular methods and proteomic analysis (2D-electrophoresis and mass spectrometry) skills. 34. A qualitative study of genetic screening for haemochromatosis in high schools
ironXS is a project which will offer screening to 15,000 year 10 and 11 students for genetic predisposition to haemochromatosis. The PhD student will be part of a multi-disciplinary team working on this large project. The qualitative component of the study will form the basis of the PhD. In depth qualitative data will be collected from students and members of the school community, via semi-structured interviews, about their experiences of having the program run in their school. These will be undertaken pre- and post- screening. The student will receive training in qualitative methodology. 35. GeneScreen: Mapping Australian attitudes to population genetic screening
There are hundreds of genetic conditions that could be screened for and there are several screening programs already established in Australia. We believe that research involving all stakeholders is critical prior to the implementation of genetic screening programs. Hence it is important to determine community attitudes to genetic screening, with an emphasis on current programs as well as potential programs. This study will involve participants from several communities including, health professionals involved in providing services, schools, couples with and without children, expectant parents and adults at risk of late-onset disorders. The aim of the study is to: Describe the attitudes of lay and health professional communities in Australia regarding genetic screening. Specifically to focus on the following: • when genetic screening should be offered and for which conditions • what setting is best • what factors influence attitudes to screening; including, health, ethical, social, cultural, privacy, gender and familial issues 36. Investigation of the novel gene, Parkin Co-Regulated Gene (PACRG)
Parkinson's disease is a common neurodegenerative disorder that primarily affects elderly individuals. However, familial forms of the disease with early onset have been identified. Recently, we identified PACRG, a novel gene that is physically and functionally linked to the early-onset Parkinson's disease-linked gene, Parkin. The functional role of PACRG has not been well characterised, although evidence suggests PACRG may play a role in diverse cellular processes, for example mediating neuronal cell death and spermatogenesis, via interaction with microtubules. The overall aim of this project will be to investigate the cellular expression and function of PACRG, with a particular focus on its role in the brain. The project will utilise a range of molecular and genetic techniques including tissue culture, immunohistochemistry, gene expression studies and protein analysis. In addition, we are generating several strains of genetically-modified mice to help investigate the specific role of PACRG in neuronal function. This project will involve the generation and analysis of some of these animal models. 37. An assessment of prenatal and pre-pregnancy screening for cystic fibrosis
Cystic fibrosis is the commonest severe inherited condition of childhood, affecting approximately 1:2500 Caucasians with 1:25 being carriers. A carrier screening program is offered through private obstetricians in Victoria. We wish to study this program using qualitative and quantitative methodologies. Participants and potential participants will be interviewed and, based on the outcomes of these, questionnaires will be developed and administered. The outcomes will be used to inform all aspects of the screening program, as well as to lobby governments for funding for CF screening in the public sector. The student undertaking this project will receive formal training in qualitative and quantitative research methodologies. 38. An investigation of neural re-organisation and recovery associated with language impairment in children following cerebrovascular accident
Cerebrovascular accident (CVA) acquired during childhood is a significant cause of lifelong morbidity, with common sequelae including severe communication and learning problems (Steinlin et al., 2006). Despite these well-known issues, there has been an absence of studies investigating language outcome in this population. In addition, little data exists relating the neuroanatomical site of lesion to behavioural outcome. Furthermore, few studies have examined the functional reorganisation or recovery of neural mechanisms subserving language function in this group of children. Knowledge of language outcome related to the neurological profile is crucial for informing diagnosis and prognosis of language outcome in this group. Therefore the present PhD proposal aims to systematically address the following research questions: 1. What are the medical and neurological predictors for language outcome in children post-CVA? 2. Which structural brain regions and white matter fibre connections are associated with language impairment in children post-CVA (using modern structural neuroimaging techniques such as Voxel-Based Morphometry; Diffusion Tensor Imaging). 3. Which functional brain regions are associated with language impairment in children acutely post-CVA, and how does the brain recover or re-organise language following CVA over time? 39. Understanding the contribution of gene reassortment to the diversity of human rotaviruses, and potential impact on vaccine efficacy.
Rotavirus is the major viral cause of acute gastroenteritis in children worldwide. The virus is characterised by a genome of 11 segments of RNA. Reassortment is a process whereby viruses with segmented genomes can rapidly undergo genetic changes, however little is known about the extent of reassortment in wild type rotavirus populations. The overall aim is to investigate the contribution of genetic reassortment (intra- and inter-species) to the evolution and genetic diversity of human rotaviruses and to assess its importance in the generation of new rotavirus strains that arise during successive epidemic seasons. A greater understanding of the evolution of wild type virus populations is required to predict and evaluate the long-term efficacy and impact of rotavirus vaccines. These studies will utilise molecular biology techniques such as RT-PCR, Northern hybridisation and sequence analysis, in addition to cell culture techniques. 40. Does the rotavirus RNA polymerase influence viral virulence?
Rotavirus is the major viral cause of acute gastroenteritis in children worldwide. The virus is characterised by a genome of 11 segments of RNA. Gene segment 1 encodes the RNA polymerase protein, which transcribes the dsRNA segments into mRNA prior to translation into viral proteins. It is well documented that rotavirus cause both symptomatic and asymptomatic infections. This projects aims to address whether differences in activity of the RNA polymerase contribute to virus attenuation. Functional assays utilising the CAT reporter gene replication system and tissue culture techniques will be applied to answer this question. 41. Can managment of sleep problems in preschool children improve child behaviour, quality of life and learning? - A randomised controlled trial
Nearly 20% of Australian parents report a problem with their preschool child's sleep (eg frequent night waking, difficulty falling to sleep and tiredness upon waking in the morning). Such problems are associated with an increased risk of child behaviour problems, poorer quality of life, and an increased likelihood of a child being diagnosed with ADHD or sustaining an injury requiring medical attention. Brief behavioural interventions are known to be clinically effective in reducing child sleep problems. This PhD will determine whether community-level surveillance and intervention for sleep problems improves children's behaviour, learning and quality of life in the school entry year. It will be in two parts: 1) a screening questionnaire at school entry for child sleep problems and 2) a randomised controlled trial involving only children whose parents report a sleep problem in their child, with the intervention arm receiving a brief sleep intervention at their school. Outcomes will include child sleep, behaviour, quality of life, parent mental health and children's learning over a six month period. 42. Intersex patient analysis using high density SNP microarrays
Intersex disorders, ranging in severity from genital abnormalities to complete sex reversal, are surprisingly common. Uncertainty about a child’s gender is extremely traumatic for the individual and other family members. There are profound psychological and reproductive consequences in later life for the affected patient. The cause of these problems is most often the failure of the complex network of genes that regulate development of testes or ovaries. Our research seeks to understand the molecular basis of testis and ovary development and how mutations in key genes can lead to abnormalities. We have identified several major genes that play critical roles in testis development. Mutations in these genes explain 20% of intersex cases but we have no explanation for the remaining 80%. To identify these other genes we will use gene chips with 500,000 SNP markers covering the human genome. DNA from intersex patients will be used to interrogate these chips for micro-deletions and micro-duplications in the human genome. Data from the chips will be subjected to detailed bioinformatic analysis to pinpoint novel genes responsible for gonad development. These genes will be subsequently analysed in mouse models to determine their precise molecular function in testis /ovary development. 43. The molecular genetics of gonad development and dysfunction.
We have used a variety of screens in mouse to isolate genes that are differentially expressed from either the male or female developing gonad. Such differential expression suggests a potential role in either testis or ovary development. We are currently conducting a range of functional assays on these genes in mouse embryos to determine the role of these genes in developing gonads. Null mice have been created for several genes and thes are being examined in detail for phenotypic changes in gonad development. However, many other genes are being analysed using more rapid assays. These rapid approaches include siRNA knockdown and overexpression in embryonic mouse gonad cultures. The effects of knockdown or overexpression are being monitored by changes in histology of the gonad and by assessing perturbations in the expression profile of known genes using real-time quantitative PCR. These more rapid functional assays will help assign a number of genes to the testis or ovary determining regulatory network. This work is closely integrated with project 1 as many of the transgenic or knock-out mouse models of gonad development will find correlates in human intersex patients. 44. Analysis of genes responsible for infertilty and germ cell cancer.
Germ cells are unique in that they contribute to the next generation. To reiterate the program of embryonic development germ cells must retain underlying totipotency. In this quality germ cells appear similar to embryonic stem cells. Using molecular and computer based screens we have identified genes expressed in embryonic stem cells and during embryonic germ line development. These genes may function in establishing/maintaining totipotency in ES cells and early germ cells or may have other functions during later germ line development. We have characterised the germ line expression of these pluripotency genes using in situ hybridization and immunohistochemical techniques and found that their expression is downregulated as germ cells exit the cell cycle and differentiate, indicating that these genes have important germ line roles. The current project aims to functionally examine the role of these pluripotency genes in the developing germ line by using genetic techniques to overexpress and/or mutate these genes in the developing mouse germ cells. This work will address the function of genes expressed in the normally developing mouse germ line and their possible roles in human infertility and germ line cancer. |
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