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Home  Study  Becoming a Student  Student Projects 2006

Student Projects 2006

Infectious Diseases & Immunology Division Therapeutic Development and Clinical Research Division Cancer & Cell Biology Division Population Studies & Human Genetics Division
How to apply:
Contact the Laboratory Head related to the project area of interest (see below).   Application and admission details in "Becoming a Student".

Legend:
P   = PhD, MPhil project
H   = Honours project
TU = Top-Up available

Infectious Deseases and Immunology Division

Jeffrey Gorman Protein Discovery Centre
Prof Jeffrey Gorman
Ph: 07-3845 3669
Email jeffG@qimr.edu.au
[ H,P]

Protein Discovery

The QIMR Protein Discovery Centre aims to discover the identities of proteins involved in or affected by physiological and disease processes and the ways in which these proteins function. This may involve a range of techniques such as sequencing proteins isolated from polyacrylamide gels through to identifying the ways in which the proteins are modified (post-translational modifications) in order to affect their specific functions.

In general the projects within the Protein Discovery Centre involve high-performance mass spectrometry.
A variety of projects within the Centre provide excellent opportunities for the involvement of post-graduate students. These projects range from the identification of the the complete complements of proteins (proteomes) secreted by parasites or present in viruses or neurosecretory vesicles, determination of the roles of post-translational modifications in regulating cellular systems and probing the structures and interactions of proteins at the molecular levels.

These projects are supported by the use of the latest equipment available for high sensitivity protein analysis, especially protein mass spectrometry. This equipment includes a high mass accuracy and high resolution Orbitrap mass spectrometer and ion-trap mass spectrometer with electron transfer dissociation which are the first instruments of their type in Australia. Other state-of-the-art mass spectrometers and ancillary equipment are also available in the Centre. Ancillary equipment includes the latest NanoHPLC equipment, robotic fractionators and a gel picking robot for processing 2D-gels.

Project 1.   Folding of Chaperonin 10 (Cpn10) and consequences of interactions with cellular receptors

Cpn10 has been identified as a protein with potential therapeutic uses for modulating human diseases. Rational exploitation of this small protein in disease modulation will benefit from gaining an understanding of its mode(s) of action. Accordingly the QIMR Protein Discovery Centre is collaborating with CBio as an industry partner to characterise the interactions of Cpn10 with cellular receptors.

An APA(I) scholarship is available as part of this project aimed at identifying structural features the protein in relation to functional activity and cellular pathways activated by the protein. This project has 2 possible streams available for a student to pursue either as independent objectives or in combination.
The scholarship is only available to Australian citizens, New Zealand citizens and Australian permanent residents and pays an annual tax-free stipend of $24148 (for 3 years). Suitable candidates should have a BSc (Hons) in chemistry or biochemistry.

One stream of research will involve hydroxyl radical footprinting to study the structure, folding and interactions of mammalian Cpn10. The second stream will involve analysis of post-translational modifications of cellular proteins induced by exposure of cells to Cpn10 as a means of identifying cellular pathways activated and/or repressed by the protein. Data emanating from the study will be shared in a collaboration with CBio scientists who will conduct functional studies.
The same techniques used in the Cpn10 project will be used for similar studies by more senior scientists within the QIMR protein Discovery Centre and ensure a supportive collegial environment.
Expertise in isolation of proteins and their analysis by mass spectrometry would be highly regarded.

Aims

Approaches

  1. Cpn10 will be irradiated with gamma-rays in a small animal incubator for various lengths of time and the protection from hydroxyl radical modification will be monitored by high-performance masss spectrometry. The effect of interacting molecules, such as antibodies, will also be analysed
  2. Mutations have already, or will be, introduced into Cpn10 and hydroxyl radical footprinting will be used to examine the stuctural effects of these modifications and their impacts on Cpn10 interactions
  3. Cells will be exposed to Cpn10 and disrupted fro 2D-SDS-PAGE analysis and/or analysis by 2D-HPLC methods. Cells without exposure to Cpn10 will be used as controls for comparative analyses. The method of Stable Isotope Labelling Analysis in Cell Culture (SILAC) will be used to determine quantitative differences in peptide profiles of digested proteins of exposed and unexposed cells. This will enable identification of sites of modifications and subsequent characterisation of specific modifications

Other Projects
Other projects, to be described at a later time, will be available within the Protein Discovery Centre
These will involve:

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Dave Kemp Katharine Trenholme Malaria parasites "coming unstuck"
Dr Katharine Trenholme
Ph: 07-3362 0432
Email kathT@qimr.edu.au
[ H,P]

Gametogenesis, Aminopeptidases, Clag genes and Malaria

There are an estimated 300-500 million clinical cases of malaria each year, resulting in between 1.5 and 2.7 million deaths annually. These are mainly caused by infection with the malaria parasite Plasmodium falciparum. The Malaria Biology Laboratory group at QIMR is involved in a number of projects that are designed to provide us with important information about Plasmodium falciparum and the way this organism functions. Many of the projects in our laboratory have both molecular and cellular components and involve determining the function of genes.

The molecular approaches often rely on transfection, a process that allows us to introduce foreign DNA into parasites so that we can determine function. We can alter genes, "knock" them out, tag them or simply permit them to be expressed in an environment where they may not have been previously expressed. We are also involved in assessing the ability of particular parasite proteins to protect the host from infection and recently we have become interested in assessing the activity of some novel anti-malarial agents as well as extending our molecular experience into the drug action and drug resistance arena.

Project 1   The Sexy Side of Malaria: Plasmodium falciparum and commitment to gametocytogenesis

The malaria parasite has a complex life cycle consisting of an asexual and a sexual phase and while significant advances have been made in understanding this disease much remains to be elucidated. The parasite interacts not only with the human host, but also with the mosquito vector which transmits this disease between people.

To allow the mosquito to carry this parasite it must undergo sexual differentiation in the human host to produce gametocytes which are taken up by the mosquito. This switch from asexual reproduction to sexual reproduction by the parasite has been shown to be sensitive to environmental stimuli yet we understand very little about how the malaria parasite recognises and processes these stimuli leading to sexual differentiation.

We have previously identified a gene(Pfgig)within this region that when disrupted shows a 6 fold reduction in gametocyte production. A second gene PFI1770w located downstream of Pfgig is expressed solely in the gametocyte stages of the parasite. It is hypothesized that PFI1770w and Pfgig represent a point at which a connection between the asexual and sexual stages may exist. As little is known about the switch from asexual to sexual stages investigation into this area may lead to the identification of possible drug targets or to novel transmission blocking vaccines.

Project aim

Investigation of the gametocyte commitment pathway through the creation of a transgenic parasite line that expresses a Green Fluorescent Protein (GFP) tagged Pfs16 gene. Pfs16 is the earliest known gametocyte specific protein and allows for the identification of cells committed to the gametocyte pathway before they are morphologically distinguishable.

This will allow us to examine

Project 2   Aminopeptidases and haemoglobin degradation in Plasmodium falciparum infected erythrocytes

Malaria parasites live inside the host red blood cells for part of their lifecycle and digest host cell haemoglobin to obtain amino acids. In malaria parasites leucine aminopeptidase-like enzymes are believed to function in the final stages of haemoglobin digestion to generate free amino acids that are then used for either parasite protein synthesis, or exported out of the infected erythrocyte.
There are 4 aminopeptidase-like enzymes are expressed by malaria parasites and these are being studied as drug targets for the chemotherapy of malaria.

Project aim

To generate transgenic parasite lines which over express these genes so they can be further characterised.

This will be achieved by

Project 3   Characterisation of a clag gene located on Chromosome 7 of P falciparum

Our laboratory previously identified a gene called the Cytoadherence Linked Asexual Gene (clag 9) on chromosome 9 of P falciparum and demonstrated its involvement in cytoadherence to the cell adhesion molecule CD36.

Data generated by the malaria genome mapping project and previous work carried out in our laboratory has shown that clag 9 is in fact only one member of a multigene family with 4 other genes closely related to clag 9 being identified.

Two are located on chromosome 3, a third gene is located on chromosome 2 and a fourth gene on chromosome 7. Clag genes have also been identified in other malaria species, with every species examined to date containing at least one clag gene.

We are in the process of characterising these genes, recent work has indicated that some members of the clag gene family in both murine malarias and P. falciparum were associated with a protein assembly in rhoptries, but several members of this gene family remain to be characterised.

Project aim

To characterise clag 7 of P falciparum involving:

For Further Information please contact:

Dr Katharine Trenholme
Queensland Institute of Medical Research
Malaria and Scabies Unit
Post Office Royal Brisbane Hospital
Brisbane    4029
Australia.

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Peter Ryan Mosquito Control Laboratory
Dr Peter Ryan - Laboratory Head
07-3362 0351
Email peterR@qimr.edu.au
[ H, P]

Transmission of Ross River virus and Barmah Forest virus by backyard mosquitoes in south east Queensland

This project will determine whether Ochlerotatus notoscriptus (backyard container breeding mosquito species) from south east Queensland are important vectors of Ross River virus (RRV) and Barmah Forest virus (BFV). Although strains of Oc. notoscriptus mosquitoes from Sydney and Brisbane have been shown to be efficient vectors of BFV, the vector competence of Oc. notoscriptus from Brisbane and the Sunshine Coast has been shown to vary, at least for RRV.

Recent genetic analyses of Oc. notoscriptus from southern and central Q ueensland have identified three major genotypes. Although there does not appear to be any geographic clustering of these genotypes, we are interested in whether these genotypes of Oc. notoscriptus differ in their vector competence for RRV and BFV.

We propose to establish multiple colonies of Oc. notoscriptus here at QIMR. Isofemale lines would be established from wild caught material collected in adult mosquito traps or household surveys for immature stages. Individuals from each colony would be analysed to determine their genotype. Each pure colony would then be fed discriminating doses of RRV and BFV in blood via a membrane feeder, and infection and disseminated infection rates would be determined, and possibly extrinsic incubation period if time permits.

Aims

Approaches

References
Knox TB, Kay BH, Hall RA and Ryan PA. 2003. Enhanced vector competence of Aedes aegypti (Diptera: Culicidae) from the Torres Strait compared with mainland Australia for dengue 2 and 4 viruses.
J Med Entomol. 40: 950-6.
Ryan PA, Do KA and Kay BH. 2000. Definition of Ross River virus vectors at Maroochy Shire, Australia. J Med Entomol. 37:146-52.

This exciting project will provide broad experience in a range of different activities, including: field sampling to collect mosquitoes to be colonized at QIMR, mosquito infection experiments, insect dissections, basic genetic analysis, arbovirus assay, and statistical analysis. Motivated students interested in public health and arbovirology are encouraged to apply. A generous tax-free scholarship and maintenance funds will be available to the successful applicant.

This project is funded by the   Mosquito and Arbovirus Research Committee Inc.  an Australian based, independent body, consisting of representatives from Local Governments, Government Agencies, industry and scientific organisations. MARC is involved in mosquito control and the prevention of human diseases caused by mosquito-borne viruses.

Contacts:
Dr Peter Ryan, Deputy Head: Mosquito Control Laboratory, Queensland Institute of Medical Research [see above]
Assoc. Prof John Aaskov, School of Life Sciences, Queensland University of Technology, Ph 3864 2144

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Kadaba SriPrakash David McMillan Bacterial Pathogenesis Laboratory
Dr David McMillan
07-3845 3712
Email daveM@qimr.edu.au
[ H,P]

Group A Streptococcus infection and disease

Group A streptococcal(GAS) infection is associated with a wide range of diseases that range from relatively benign 'strep throat' to the more serious post-streptococcal glomerulonephritis (PSGN) and rheumatic heart disease. Streptococcal infection is endemic in Northern Australia. The Indigenous populations of these regions suffer from much greater incidences of disease than the Caucasian population.

Our research has shown that in addition to GAS, GGS is also highly prevalent in Northern Australia. This species has the potential to cause the same spectrum of disease as GAS. Our research his involved in identifying and characterising streptococcal genetic factors associated with disease. We are also interested in investigating how these factors may be transferred between streptococci.

Project 1  Characterisation of genes involved in post-streptococcal glomerulonephritis

A major contributor to glomerulonephritis is infection by the group A streptococcus(GAS). How GAS causes post-streptococcal glomerulonephritis (PSGN) is not yet understood. Importantly, it appears that only a subset of GAS strains is associated with the onset of glomerulonephritis. By identifying and characterising genes that are present in PSGN-associated strains but not PSNG-non-associated strains, we hope to gain a greater understanding of the pathogenic mechanisms associated with this disease.

In preliminary work, we identified 22 gene fragments present in an PSGN associated GAS isolates that were not present in an PSGN non-associated GAS isolate. These gene fragments may therefore encode proteins involved in PSGN pathogenesis. Seven of these DNA fragments have streptococcal homologues in the Genbank database. However, fifteen of the DNA fragments are novel genes without homologues in the Genbank database, despite the presence of five complete genomic GAS sequences. The aims of this study are to begin the characterisation of these genes.

Aims

Approaches

  1. A lambda phage library containing M55 GAS DNA will be constructed. The phage library will be probed with labelled DNA probes corresponding to the gene fragments identified in preliminary work. Reactive phagemids will be isolated and the DNA subcloned into manageable sizes.
  2. DNA sequencing of the subclones will be undertaken to generate complete nucleotide sequence data for each of the genes. BioInformatical approaches will then be utilised to predict structure/function/cellular locations of the putative proteins encoded by the genes.
  3. Based on the above gene sequences, diagnostic PCR primer will be designed to investigate the presence/absence of these genes within a panel of PSGN-associated and non-associated GAS isolates
Project 2   Characterisation of group A streptococcal biofilms

Biofilm formation is an ancient and integral characteristic of prokaryotes that evolved as a response to the fluctuating and harsh conditions of primitive earth. For pathogenic bacteria, a biofilm may provide the organism with greater antibiotic tolerance, and resistance to host innate immunity. Biofilm formation is well documented in oral streptococci. However, little work has been done on biofilm formation by group A streptococcus (GAS).

Preliminary work by our group and others has shown that the majority of GAS isolates form biofilms We have also shown that addition of glucose increases biofilm formation in GAS. The exopolysaccharide matrix, the lower growth rate and changes in gene expression are all attributes of biofilms that could contribute to the process of antibiotic insensitivity. Biofilm formation by GAS may be one mechanism to explain the antibiotic treatment failure encountered by many pharyngitis cases.

Aims

Approaches

  1. A crystal violet assay has been developed and validated in our lab to measure biofilm formation in GAS strains. We will utilise this assay to measure biofilm formation in GAS isolates
  2. Minimum Inhibitory Concentrations (MIC) of penicillin and erythromycin required to kill GAS strains when in planktonic or sessile (biofilm) phase will be determined
  3. Using genetic and proteomics approaches we will investigate differences in expression profiles of bacteria in biofilm and planktonic growth phase
Project 3  Genetic transfer between streptococci

The transfer of DNA between bacterial isolates of the same species is mediated by bacteriophage and transposons. These transfer events are important as they represent a mechanism for super-charged evolution, potentially converting a non-pathogenic to pathogenic strains. As an example, GAS bacteriophages often carry genes for superantigenic virulence factors. Bacteriophages not only enable the intra-species DNA transfer, they may also promote inter-species transfer.

Our previous work has identified a bacteriophage (315.1G) in one GGS strain associated with bacteraemia that is highly homologous to a GAS bacteriophage. In the same strain we have also identified a transposon (GGSt.1) with a high degree of homology to a Group B streptococcus (GBS) transposon. We have completed the genetic sequences of these mobile elements and are now studying their functional attributes. The aim of this project will be to demonstrate the transfer of these mobile elements between bacterial species.

Aims

Approaches

  1. In separate isolates DNA encoding 315.1G and GGSt.1 will be labelled by the incorporation of a kanamycin (Km) resistance cassette into their chromosomes. Recipient strains will be chromosomally marked with a streptomycin (Strr) resistance cassette
  2. The donor GGS Kmr strain and recipient Strr strains will be incubated together. Evidence of transfer will be demonstrated by growth of bacteria in the presence of Kanamycin and Streptomycin
  3. Chromosomal mapping techniques will be utilised to determine where the bacteriophage and transposon integrate into the recipient chromosome
Students who undertake an Honours program in the Bacterial Pathogenesis Laboratory will have the opportunity to make valuable contributions to our ongoing projects, and will be exposed to a wide range of laboratory techniques.

For further enquires please contact Dr Sri Sriprakash or Dr David McMillan.

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Chris Engwerda Immunology and Infection Laboratory
Dr Christian Engwerda
07-3362 0428
Email chrisE@qimr.edu.au
[ H, P]

Immunology and Infection

Cerebral malaria (CM) and visceral leishmaniasis (VL) are significant parasitic diseases in the developing world. We work with experimental models of CM caused by Plasmodium berghei and VL caused by Leishmania donovani. Both protozoan parasites induce strong inflammatory immune responses by their hosts that contribute to the development of tissue pathology.

One of the major aims of the Immunology and Infection laboratory is to understand how these inflammatory responses are initiated, identify the immune cells that cause pathology and to devise strategies to protect the host from disease, without impeding the development of protective immunity. We focus on two stages of infection. First, the priming of parasite-specific T cells that produce inflammatory mediators, and second, the migration of activated T cells to sites of infection and their interaction in local tissue micro-environments.

Many of our studies are performed in situ with infected tissue so that we avoid changes to cell behaviour once they have been removed from local tissue micro-environments. Therefore, these studies require the use of unique reagents and specialised techniques such as immunohistochemistry, confocal microscopy, laser micro-dissection and whole-tissue imaging.

Students in our laboratory would become familiar with all of these techniques. There will also be opportunities for successful Honours students to enrol for PhD studies and continue work in the Immunology and Infection laboratory.

Project 1  Defining early immune events following Plasmodium berghei infection

The activation of parasite-specific T cell responses is a key event in the pathogenesis of cerebral malaria (CM) in mice caused by Plasmodium berghei. Dendritic cells (DCs) play an important role in the activation of T cells by presenting parasite antigens, providing co-stimulatory signals and producing pro- and anti-inflammatory cytokines that influence the phenotype of activated T cells. We will isolate splenic DC's from mice at various times after infection with P. berghei to determine the co-stimulatory molecules and cytokines being expressed by these cells.

Initially, we will study the important co-stimulatory molecules CD80, CD86 and CD40, as well as the pro-inflammatory cytokine IL-12 and anti-inflammatory cytokine IL-10. Blocking antibodies raised against these molecules will be administered to mice prior to infection and the effects of DC function and T cell activation will be analysed, as well as effects on the development of CM. This work will be the first step in identifying the key molecules that activate T cells involved in the development of pathology during CM. Once identified, strategies to modulate their expression can be devised with the aim of preventing the development of cerebral malaria in humans.

Techniques used in this project will include cell biology, molecular biology, histology, microscopy and analysis of blood parasite levels. Resources are available to continue this work as a PhD project.

Reference: Good M. F., H. Xu, M. Wykes and C. R. Engwerda. 2005. Ann Rev Immunol 23:69.

Project 2  Characterising changes in splenic structure during visceral leishmaniasis

Visceral leishmaniasis (VL) is characterised by an organ-specific immune response, whereby the liver is a site of an acute, but resolving infection, and the spleen becomes chronically infected. We have previously shown that TNF produced by host cells in response to infection causes many changes in the structure of the spleen during Leishmania donovani infection.

However, we recently showed that production of the closely related molecule lymphotoxin alpha (LTα) is dramatically decreased in the spleen during VL. This cytokine plays an important role in maintaining tissue structure, and decreases in the production of this molecule might also contribute to tissue re-modelling following L. donovani infection.

We recently showed that by providing lymphotoxin signals to L. donovani infected mice using an agonistic anti-lymphotoxin receptor antibody, we were able to cause a significant reduction in parasite burden in the spleen.

In this project, the mechanism of action of the anti-lymphotoxin receptor antibody will be defined. We will analyse changes in the structure of the spleen following treatment with the antibody, as well as the production of anti-microbial products. In particular, we will investigate changes to the composition of the marginal zone, a region of the spleen that is critical for capturing blood-borne antigen and mediating lymphocyte migration into the specialised areas of T and B cell accumulation.

In addition, levels of pro- and anti-inflammatory cytokines will be measured. The use of antibodies against TNF receptor molecules to compensate for decreased cytokine activity is a unique and promising way to alter the course of diseases such as VL.

This study will involve the use of histology, microscopy, and molecular and cell biology. Resources are available to continue this work as a PhD project.

Reference: Kaye P. M., M. Svensson, M. Ato, A. Maroof, R. Polley, S. Stager, S. Zubairi and C. R. Engwerda. 2004. Immunol Rev 201:239.

Project 3  Defining the biological activities of plant-derived proteases

We have discovered several proteases from pineapple stems with potent immunomodulatory properties. These molecules are able to inhibit a major cell signalling pathway involving Erk2 (MAP kinase), thereby suppressing cell activation and making them potential anti-cancer agents.

We have also found stimulatory activities for some proteases, including the activation of macrophages and NK cells. In order to further define the biological properties of these molecules, we have begun a program to produce recombinant cysteine proteases from the pineapple stem. In this project, specific proteases will be cloned from mRNA extracted from pineapple stems and then expressed in yeast (Pichia pastoris). Recombinant proteases will then undergo extensive biochemical analysis, including assays for substrate specificity and enzyme kinetics. They will then be tested in vitro for biological function, including the ability to inhibit Erk2 activation, stimulate macrophages and NK cells, and suppress T cell activation.

Depending on the outcome of these experiments, the recombinant material will then be tested in an appropriate in vivo disease model, such as cancer, graft versus host disease, cerebral malaria or visceral leishmaniasis.

Reference: Mynott, T. L., A. Ladhams, P. Scarmato, and C. R. Engwerda. 1999. J Immunol 163:2568.

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Qin Cheng Darren Krause
Malaria Chemotherapy & Resistance Laboratory
Dr Darren Krause
Dr Qin-Cheng
Phone: 3332-4929
Email: darren.krause@defence.gov.au; Darren.Krause@qimr.edu.au [ H, P]

Analysis of human antibody specificity to PfEMP1 and their effect on the parasites ability to bind to cellular ligands.

Malaria is a disease in over 90 countries and infecting approximately 300 million people worldwide, causing over one million deaths each year. The majority of these deaths are in children and pregnant women in tropical countries. Resistance of malaria to drugs has increased in many areas in the last few years, resulting in increased morbidity.

Plasmodium falciparum parasites remodel the surface of human blood cells on invasion by the insertion of parasite-derived proteins in knob-like protrusions. We have been looking at one such protein, PfEMP1, which is coded by a set of genes called Var genes. Var genes are switched to different variants by the parasite after infection of human red blood cells. It does this switch to evade the host immune response and to survive longer in the host. PfEMP1 is also involved in pathogenesis caused by severe malaria.

We have cloned regions of the PfEMP1 gene and have expressed them in E.coli to analyse immune responses from malaria-infected volunteers. We have also made antibodies in mice to analyse switching of PfEMP1 variants on the red blood cell surface.
We hypothesise that various domains of PfEMP1 trigger antibody production and that some of these antibodies will block the binding of parasites to various ligands.

This project aims to investigate what region of PfEMP1 triggers the human immune response and what ligand binding is blocked by these antibodies.
The project will use molecular biology techniques (such as cloning and sequencing), protein technology (such as purifying expressed proteins) and antibody production work to investigate variants of expressed PfEMP1 and ligand binding.

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Qin Cheng Michelle Gatton
Malaria & Chemotherapy Laboratory
Dr Michelle Gatton
07-3362 0416
Email michelleG@qimr.edu.au
[P, MPhil, H]

Investigating Plasmodium falciparum resistance to LapDap using mathematical simulation models

Malaria is a disease occurring in over 90 countries and infecting approximately 300 million people worldwide, causing more than one million deaths per year. The majority of these deaths occur in children and pregnant women in tropical countries. In recent years, there is a resurgence and increased morbidity of malaria in many areas that is partially attributed to the development and spread of drug-resistant malaria parasites.

Drug resistance is a major problem for the effective treatment of malaria. P. falciparum parasites have demonstrated the ability to become resistant to most commonly used anti-malarial drugs, and there is little doubt that this trend will continue as new drugs become available. Understanding the processes leading to the development of resistance, and the factors which promote or inhibit the spread of resistant parasites is fundamental to combating drug resistance in the future.

This project aims to investigate the role that treatment dosage, host immunity and parasite genotype have in the development and spread of parasites resistant to LapDap, a newly formularised anti-malaria combination.

The project will utilise an existing stochastic simulation model of the within-host dynamics of P. falciparum infections to mimic infection outcomes after treatment with LapDap. Simulation of biologically relevant treatment parameters in malaria naïve and semi-immune individuals will be conducted to allow identification of factors important in the development and spread of resistance to this drug.

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Mathieu Taveau Human Progressive Kidney Disease:  molecular aspects of proteinuria induced tubular damage
Dr Mathieu Taveau
Ph: 07-3362 0488
Email Mathieu.Taveau@qimr.edu.au
[ H ]

Molecular aspects of proteinuria induced tubular damage in human progressive kidney disease

Proteinuria, which refers to an abnormal large amount of proteins in the urine, is a hallmark of progressive renal disease, independently of the type of initial insult. Proteinuria has been classically used as a marker of the severity of the renal damage, however, it is now accepted that protein species present in the urine directly damage tubule cells and therein participate in the progression of renal failure.

The Kidney Research Laboratory has specialised in the investigation of the molecular mechanisms leading to proteinuria-induced renal damage with 2 main orientations:

Identification of the urinary protein specie(s) responsible for proteinuria induced renal damage

Our laboratory has set up and extensively used an in vitro model of proteinuria using human proximal tubule epithelial cells (PTECs), the renal cells mainly involved in the reabsorption of urinary protein species. Using plasma protein fractionation, we recently reported that deleterious effects on PTECs are induced by a high molecular weight (HMW) plasma protein fraction (Morais C et al., 2005).
These effects were not induced by the albumin-rich low molecular weight fraction in our model, despite albumin is considered as the key protein inducing proteinuria related tubular damage.

We are currently extensively comparing the effects of the HMW protein fraction to different forms of albumin to confirm and extend our previous results. We are also performing experiments aiming at identifying the critical protein(s) in the HMW fraction inducing PTECs damage.

Identification of the early molecular pathways induced by proteinuria in proximal tubule epithelial cells

A number of molecules have been identified to be induced by proteinuria in PTECs using in vitro models (MCP-1, RANTES, ICAM-1, TGF-beta1, FAS…). Several of these pathways have been investigated and validated in animal models of proteinuric renal diseases. However, there is a paucity of mechanistic information available in the human disease. The few described pathways have been usually investigated in only one particular human renal proteinuric disease, raising the question of the reproducibility of these results in the general context of proteinuria. In addition, no integrated view of the complexity of the human PTECs molecular response to proteinuria has been reported yet.

We are currently deciphering the early molecular pathways induced in PTECs in the human disease, by comparing the transcriptomes of PTECs extracted from patients with proteinuric renal diseases from various origins. Access to a substantial biopsies archive through collaboration with the Anatomical Pathology Department of Queensland Health Pathology Service and to technologies available at the Queensland Institute of Molecular Research (laser capture microscopy, Illumina gene expression profiling system), allow us to perform experiments on limited human biological material. We also compare the results obtained from the human disease situation to our in vitro model profiled at early stages following PTECs stimulation to more specifically identify the early molecular pathways induced by proteinuria.

Our long term goal is to use RNA interference to silence the key early genes induced by proteinuria in PTECs to ultimately slow the progression of chronic renal disease.
Techniques used in the Kidney Research Laboratory:

Key publication
Morais C, Westhuyzen J, Metharom P, Healy H.  High molecular weight plasma proteins induce apoptosis and Fas/FasL expression in human proximal tubular cells. Nephrol Dial Transplant. 2005; 20(1):50-8.

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Therapeutic Development and Clinical Research Division

Alejandro Lopez Dendritic cells and Cancer Laboratory
Dr Alejandro Lopez
07-3845 3794
Email alejL@qimr.edu.au
[ P, H]

Dendritic cells and Cancer therapy

Recent developments in the generation of dendritic cell (DC)-based therapy has yielded promising clinical results(1). Around 12% of patients with stage IV melanoma were rendered into complete clinical responses lasting for over 5 years. While the mechanisms involved in the protective responses elicited remain to be established, it appears that the use of complete tumour cells in this immunotherapy approach is definitive.
In contrary to other clinical trials reported in the literature, the results described were obtained upon immunization with irradiated autologous melanoma cells and DC(2).

Our laboratory is interested in pursuing this encouraging lead in three main directions:

DC-based immunotherapy for breast cancer
Despite the increase in early detection, there is still a great the need for improved therapies for patients with advanced breast cancer. We have extensively studied the function of DC in breast cancer (3) and established that they are significantly dysfunctional, contributing to explain the evasion of the immune surveillance (4). While circulating functional DC appear reduced in number we have develop in vitro methods to render them functional in vitro (5). This opens the possibility to develop DC-based immunotherapy in patients with breast cancer(6).
Together with an interactive team at the RB&WH, we are currently completing the pre-clinical evaluation for a novel approach in DC-based therapy in breast cancer.

The interactions between DC and tumour cells
We have established that DC interact with tumour cells forming conjugates and followed their fate in vitro; however, it is not clear how these conjugates elicit immune responses in patients immunised. We are studying models mimicking the clinical in vivo to further examine the mechanisms responsible for protection.

Evaluation of a closed system to generate DC for immunotherapy
In the process of translating the results described above to a wider clinical application, there is the need to produce DC within closed culture conditions. We have recently compared two methods for generating DC and produced a large supply of material that needs to be evaluated under various culture conditions. This project would suit to a student interested in the applied edge of research combined with basic science.

The projects are offered within the DC & Cancer Laboratory and in close collaboration with the Cancer Immunotherapy Laboratory, lead by Dr. Chris Schmidt.
All projects involve the mastering of sterile culture techniques, basic immunological evaluations including ELISA, ELISPOT, Cytotoxicity and Flow Cytometry.

References

  1. O'Rourke, M. G., M. Johnson, C. Lanagan, J. See, J. Yang, J. R. Bell, G. J. Slater, B. M. Kerr, B. Crowe, D. M. Purdie, S. L. Elliott, K. A. Ellem, and C. W. Schmidt. 2003. Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine. Cancer Immunol Immunother 52:387.
  2. Lopez, J. A., and D. N. Hart. 2002. Current issues in dendritic cell cancer immunotherapy. Curr Opin Mol Ther 4:54.
  3. Pinzon-Charry, A., T. Maxwell, and J. A. Lopez. 2005. Dendritic Cell Dysfunction in Cancer: A Mechanism for Immunosuppression. Immunology and Cell Biology 85:451.
  4. Pinzon-Charry, A., T. Maxwell, M. A. McGuckin, C. W. Schmidt, C. Furnival, and J. A. Lopez. 2006. Spontaneous apoptosis of blood dendritic cells in patients with breast cancer. Breast Cancer Res 8:R5.
  5. Pinzon-Charry, A., C. Ho, R. Laherty, T. Maxwell, D. Walker, R. Gardiner, L. O'Connor, C. Pyke, C. W. Schmidt, C. Furnival, and J. A. Lopez. 2005. A population of HLA-DR+ Immature Cells Accumulate in the Blood Dendritic Cell Compartment of Patients with Different Types of Cancer. Neoplasia 7:1123.
  6. Pinzon-Charry, A., T. Maxwell, S. Prato, C. Furnival, C. W. Schmidt, and J. A. Lopez. 2005. HLA-DR+ Immature Cells Exhibit Reduced Antigen Presenting Cell Function but Respond to CD40 Stimulation. Neoplasia 7:1112.

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Cancer and Cell Biology Division

Barbara Leggett Kevin Spring Vicki Whitehall Gasteroenterology Laboratory
Dr Vicki Whitehall
07-3362 0170
Email Vicki.Whitehall@qimr.edu.au

Dr Kevin Spring
07-3362 0487
Email kevinS@qimr.edu.au
[ H, P ]

Candidate genes in the development of colorectal cancer

Characterisation of the genetic changes underlying the progression of a pre-cancerous colonic polyp to colon cancer will increase our understanding of this disease and improve treatment options

We are studying both polyps and cancers in an effort to identify genetic markers for progression, prognosis and response to therapy.

Colorectal cancer continues to be one of the most common internal malignancies occurring in the Australian population. One in twenty-three of our population will develop it during their lifetimes and half of these cases will not survive beyond five years. However, colorectal cancer has a great potential for prevention as most of these malignancies develop within pre-cancerous growths called polyps which can be removed at colonoscopy.

Colorectal cancer is a somewhat heterogeneous disorder. Across the spectrum of colorectal cancer types, there is a gradient of genetic causation tempered by the effects of environment such as smoking, diet and the use of anti-inflammatory drugs.

Over the last decade our laboratory has contributed to the growing understanding that these different influences result in a number of distinct subtypes of colorectal cancer. These develop along different molecular genetic pathways and have characteristic somatic changes.

This work has important implications for improving prevention and therapy of the disease.

Honours project

Potential honours projects will examine candidate genes for a role in the development of colorectal cancer. Putative tumour suppressor genes can be evaluted by a number of techniques to examine expression changes, altered methylation, mutation and gene deletion. This would then be compared with important molecular events characteristic of specific colon cancer subgroups. Gene candidates can then be expressed in cell lines for functional analysis. Finally, we have detailed patient records for further comparison of genetic alterations with tumour pathology and clinical outcome.

PhD project

Potential PhD projects will examine candidate genes for a role in the development of colorectal cancer. Such genes will be selected either by a traditional candidate gene approach, or as a result of expression or methylation profiling experiments. Putative tumour suppressor genes can be evaluted by a number of techniques to examine expression changes, altered methylation, mutation and gene deletion.
This would then be compared with important molecular events characteristic of specific colon cancer subgroups. Gene candidates can then be expressed in cell lines for functional analysis. Correlation can then be sought between the observed genetic alterations, tumour pathology and clinical outcomes based on our detailed patient records.

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Nathan Subramaniam Membrane Transport Laboratory
Dr Nathan Subramaniam
07-3362 0179
Email nathanS@qimr.edu.au
[ P, H]

Genes of the iron storage disease Haemochromatosis

Project 1  Functional consequences of mutations in hepcidin and hemojuvelin causing juvenile haemochromatosis

Hereditary haemochromatosis is one of the most common inherited disorders in humans. It is associated with an increase in iron absorption and iron deposition in the body. Untreated, this accumulation of iron leads to tissue damage including cirrhosis, diabetes mellitus, arthropathy, cardiomyopathy, endocrine abnormalities and hepatocellular carcinoma. The most common form of haemochromatosis (type 1) is caused by mutations in the HFE gene.

Mutations in the recently identified hepcidin and hemojuvelin (HJV) genes are associated with juvenile or type 2 haemochromatosis. The mechanisms of action of these proteins have yet to be completely defined. The characterisation of these proteins will lead to a better understanding of the complex mechanisms involved in the control of body iron homeostasis and may lead to novel therapies for the treatment of disorders of iron overload and deficiency.

Aims

To study the consequences of juvenile haemochromatosis causing mutations on trafficking and localisation.

Approaches


References

  1. Wallace DF, Dixon JL, Ramm GA, Anderson GJ, Powell LW, Subramaniam N. Hemojuvelin (HJV)-associated hemochromatosis: analysis of HJV and HFE mutations and iron overload in three families. Haematologica. 2005 Feb;90(2):254-5.
  2. Papanikolaou G, et al. Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis. Nat Genet. 2004 Jan;36(1):77-82.
  3. Heeney MM, Andrews NC.Iron homeostasis and inherited iron overload disorders: an overview. Hematol Oncol Clin North Am. 2004 Dec;18(6):1379-403.
Project 2  Molecular and cellular analysis of novel ubiquitin-like domain proteins

Ubiquitin is a 76 amino acid protein used by the cell to tag proteins for degradation by the proteasome. However recent studies have shown that ubiquitylation is also implicated in protein trafficking, DNA repair, etc. Ubiquitin-like proteins have been shown to interact with the proteasome, endocytosis and regulate DNA repair and transcription.

We have identified and cloned two novel proteins with ubiquitin-like domains. Current studies are aimed a thorough molecular and cellular characterisation of these molecules.

Aims

  1. To analyse the expression of two novel ubiquitin-like proteins in various tissues and cell types
  2. To study their interaction with the proteasome and identify other binding partners

Approaches

References

  1. Welchman RL, Gordon C, Mayer RJ. Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nat Rev Mol Cell Biol. 2005 Aug;6(8):599-609
    2. .
  2. Mani A, Gelmann EP. The ubiquitin-proteasome pathway and its role in cancer. J Clin Oncol. 2005 Jul 20;23(21):4776-89.

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Martin Lavin Radiation Biology and Oncology Laboratory
Prof Martin Lavin
07-3362 0341
Email martinL@qimr.edu.au
[H, P]

Radiation Signaling and Disease

The major emphasis of our research is the recognition of damage in DNA using human and animal models to investigate genome instability, cancer predisposition and neurodegeneration.
Research is also directed to isolating novel therapeutic compounds from snake venom, early detection of prostate cancer and identification of genes in development.

Much of our work focuses on the gene defective in the human genetic disorder ataxia-telangiectasia (A-T), ATM, that plays a central role in recognising double-strand breaks in DNA, and signalling of these breaks to slow the passage of cells through the cell cycle so that the DNA damage can be repaired. Failure to recognise and repair this damage in A-T patients results in increased frequency of cancer and a progressive neurodegeneration. Being able to slow or halt the progress of this neurodegeneration would be of great benefit to A-T patients.

More recently we have extended this work to include investigations on other ataxia syndromes where a defect in DNA repair is also implicated in the phenotype.

Student projects:


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KumKum Khanna Signal Transduction Laboratory
Dr Kumkum Khanna
07-3362 0338
Email kumkumK@qimr.edu.au
[ H]

Signaling in Cancer cells

DNA damage and cancer susceptibility

DNA damage is an extremely common event in cells life-time and cells activate pathways that help them repair damage to DNA. Diseases with diminished repair capacity are associated with high rate of cancer. It is now thought that cancer is due to inability of our cells to repair DNA damage. Accumulating evidence also suggests that familial breast cancer is due to an inherited insufficiency in DNA repair. We have recently identified novel factors involved in protecting cells from DNA damage and we propose to study their mechanism of action and link with cancer susceptibility.

Understanding molecular pathways controlling cell division cycle

Cell division is a highly regulated process involving many components to produce two daughter cells, which contain equal amount of DNA. We have recently identified factors involved in regulating final stages of cell division, which involves the cleavage of cell membrane to produce two daughter cells.

Small interfering RNA (siRNA)- mediated depletion of these factors leads to several defects including multinucleation and cells connected by cytoplasmic bridges.
We propose to study how these factors regulate final stages of cell division to produce daughter cells, which have a stable genome.

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Population Studies and Human Genetics Division

Greg Anderson Iron Metabolism Laboratory
Dr Greg Anderson
07-3362 0187
Email gregA@qimr.edu.au
[ H, P]

Genes that regulate Iron metabolism

Project 1  Transcriptional regulation of the hepcidin gene by interleukin-11

Iron is an essential trace element that participates in a wide range of biological processes. Despite this essentiality, excess iron can be extremely hazardous as it can catalyze the formation of toxic oxygen radicals. Consequently, iron loading disorders such as haemochromatosis can have significant clinical sequelae. The amount of iron in the body is determined by how much is absorbed by the diet and this in turn is regulated by a liver-derived peptide called hepcidin.

We know that hepcidin represses iron absorption and can be regulated by a number of factors, such as the body iron level, hypoxia and cytokines. It appears that most of this regulation is at the level of transcription of the hepcidin gene, but few details of the regulatory mechanism are known.

One cytokine that has been shown to stimulate iron absorption is interleukin 11 (IL-11).

Aims

The goal of this study is to investigate the role of IL-11 in the regulation of the hepcidin gene. The hypothesis under investigation is that IL-11 exerts its effects on iron absorption by decreasing the expression of hepcidin through specific cis-acting elements in the hepcidin promoter.

Approaches

This project will be based on the analysis of the hepcidin promoter in the liver cell line HuH7. Initial studies will involve treatment of HuH7 cells with IL-11 to confirm that the cytokine alters transcription of the hepcidin gene. In subsequent work, existing hepcidin promoter luciferase reporter gene constructs will be transfected into the cells, which will then be stimulated by IL-11 and luciferase activity determined. To localize the nucleotide sequences responsible for the regulation, a series of nested deletions of the promoter will be generated and tested for activity in the reporter assay.

Any regulatory regions identified will be studied by generating further deletions and point mutations. These studies will be supplemented with DNA mobility shift assays to verify that the regulatory sequences are able to bind nuclear proteins.

If the regulatory elements and their cognate transcription factors are know, supershift assays with antibodies to the transcription factor(s) will be carried out to verify their involvement. These studies will identify factors involved in the regulation of hepcidin in response to IL-11 and will provide the basis for a more detailed analysis of the signal transduction pathways leading to the activation of the gene.

References

  1. Frazer DM and Anderson GJ. 2003. The orchestration of body iron intake. How and where do enterocytes receive their cues. Blood Cells Molec Dis 30:288-297.
  2. Ganz T. 2003. Hepcidin A key regulator of iron metabolism and mediators of anemia of inflammation. Blood. 102:783-788.

Project 2  The analysis of intestinal iron transport: defining the mechanism by which phenobarbital alters iron absorption

The amount of iron in the body is determined at the point of absorption in the proximal small intestine since humans have a very limited capacity to excrete the metal. When iron absorption is perturbed, as in diseases such as haemochromatosis and -thalassaemia, the body accumulates excessive amounts of iron and organ damage can result. Understanding the mechanism by which iron absorption is regulated will place us in a much better position to define the pathogenesis of disorders such these and to treat them more effectively.

In recent years considerable advances in this area have come from the analysis of how various chemicals that are known to alter absorption exert their effects. One chemical that has not been examined in detail is phenobarbital. A series of studies in the 1970s showed that phenobarbital is able to stimulate iron absorption, but at that time our knowledge of how iron moved across the intestinal epithelium was rudimentary and the precise mechanism of phenobarbital action proved elusive.

Aims

The goal of this study is to investigate the mechanism by which phenobarbital stimulates intestinal iron absorption. The hypothesis under investigation is that phenobarbital exerts its effects by enhancing the cell surface expression of the brush border and basolateral membrane iron transporters in intestinal enterocytes.

Approaches

This project will use the mouse as an experimental model and will employ a range of analytical techniques to determine the effects of phenobarbital on iron absorption. In initial studies wild-type mice will be treated with phenobarbital and iron absorption will be investigated by whole body counting. These experiments will confirm the earlier observations and establish the treatment regimen for further experiments. In subsequent studies, the effect of phenobarbital on the expression of a range of intestinal iron transport molecules will be investigated.

Expression will be studied at the mRNA level by quantitative PCR and at the protein level by western blotting and immunohistochemistry. In recent years it has become apparent that a series of proteins in the liver play critical roles in the regulation of iron absorption and the expression of these will also be investigated. One of these regulatory proteins is hfe and, since hfe knockout mice are available in the laboratory, the effects of phenobarbital on iron absorption in these mice will also be determined.

Finally, the effects of phenobarbital will be studied in an in vitro system using the intestinal cell line Caco-2. Cells will be treated with the compound and both the expression of iron transport molecules and capacity of the cells to transport iron will be investigated

References

  1. Frazer DM and Anderson GJ. 2003. The orchestration of body iron intake. How and where do enterocytes receive their cues. Blood Cells Molec Dis 30:288-297.
  2. Andrews NC. 1999. Disorders of iron metabolism. N Engl J Med. 341:1986-95.

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Grant Ramm Hepatic Fibrosis Laboratory
Dr Grant Ramm and Dr Richard Ruddell
07-3362 0177 and 07-3362 0175
Email grantR@qimr.edu.au or richardR@qimr.edu.au
[ H, P]

PKCζ Signalling and Hepatic Fibrosis

Liver disease associated with viral infection, obesity (fatty liver disease), alcoholism, biliary obstruction and iron overload is a considerable cause of morbidity and mortality in today's modern society. Once thought to be irreversible, liver fibrosis is now the subject of intensive study, with more and more data proving that the condition can be resolved.

Our group is particularly interested in the role of the hepatic stellate cell(HSC), which is now known to be critical in disease progression and maintenance. Upon liver injury the quiescent HSC undergoes a miraculous transformation from Dr Jekyll to Mr Hyde resulting in cellular proliferation, recruitment to the site of injury and the deposition of persistent scar tissue.

An exciting opportunity exists in our laboratory to help contribute to the understanding of the role the hepatic stellate cell in liver disease. The project is very much cell biology based and the candidate will learn a number of prerequisite techniques that can be used in many different fields of biological research.

Primary hepatic stellate cells isolated from rat liver will be culture activated on plastic for up to 10 days. At specific time points (Days 1, 2, 3, 4, 5, 7 and 10) stellate cells will be harvested and whole cell RNA and protein isolated. RNA can then be reverse transcribed into cDNA and then subjected to real time RT-PCR analysis, looking at the expression patterns of PKCζ and other PKC isotypes.

Western blotting will be used to confirm PKCζ protein expression and phosphorylation, indicating relative levels of PKCζ activity. Use of specific PKC ζ inhibitors at various time points of culture activation will be used to determine the relative importance of PKC ζ in the HSC activation process.

Specifically, will inhibition of PKCζ alter HSC proliferation and expression of key markers of HSC activation e.g. the contractile microfilament, a-SMA and scar tissue fibril, Collagen a1(I)?
If effects are observed on activated HSC phenotype will they result in the eventual apoptosis or quiescence of the HSC?

Key techniques to be employed:

Isolation and purification of HSC from rat liver
Cell culture techniques
Generation of whole cell RNA and protein
Western Blotting
Real time polymerase chain reaction using SYBR green technology
Bromodeoxy-Uridine incorporation assay
Caspase-3 assay
DNA fragmentation assay
TUNEL staining

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Nick Martin Genetic Epidemiology Laboratory
Prof Nick Martin
07-3362 0278
Email nickM@qimr.edu.au
[ P, H]

Psychiatric Genetics

The Genetic Epidemiology Unit at QIMR conducts a number of large twin-family studies of psychiatric conditions, including anxiety and depression and addictions to alcohol, nicotine, cannabis, opioids and gambling. Large numbers of genetic markers are also being typed for these subjects.

A range of interesting honours and PhD topics are available. The work is mainly statistical genetic analysis of existing datasets so good statistics and computing skills and a knowledge of genetics are an advantage.
Please contact Professor Nick Martin [as above] for more details.

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David Duffy Genetic Epidemiology Laboratory
Dr David Duffy
07-3362 0217
Email davidD@qimr.edu.au
[ P, H]

Asthma studies using twins

Asthma is a major and still increasing public health problem. There are both genetic and environmental contributions to the disease, but the exact nature of these is still far from clear.

Dr David Duffy in QIMR's Genetic Epidemiology group is looking for bright student (Hons or PhD) to work on a large twin-family study of asthma in which extensive environmental and genetic data have been collected. The work primarily involves statistical genetic analysis of existing data, so good statistical and computing skills, as well as a knowledge of genetics would be an advantage.


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How to apply: Contact the Laboratory Head related to the project area of interest (see above) in the first instance.

Application details here.

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