B.Sc. HONOURS
DISCIPLINE OF BIOCHEMISTRY AND MOLECULAR BIOLOGY

Molecular Sciences Bldg 21, James Cook University,
Townsville, 4811, Queensland, Australia
Telephone: 61-7-4781 6265 Fax:  61-7-4781 6078


EXAMPLES OF PROJECTS ON OFFER:

Supervisor: Jim Burnell.
Purification of and sequence determination of pyruvate, orthophosphate dikinase regulatory protein.
        Pyruvate, orthophosphate dikinase regulatory protein (PDRP) catalyses the dark-dependent inactivation and the light-dependent activation of pyruvate, orthophosphate dikinase (PPDK) in plants. It is a unique enzyme for a number of reasons and, to date, it has eluded scientist's attempts to determine the nucleotide  (and amino acid) sequence.  This project will involve site-directed mutagenesis, bacterial expression of proteins, affinity column chromatography, isolation of chloroplasts, spectrophotometric assays, and molecular biology techniques including library screening, subcloning and DNA sequencing.

Characterisation and expression of maize leaf carbonic anhydrase
        Carbonic anhydrase is present in all plants however its intercellular and intracellular localization differs between C3 and C4 plants.  In C3 plants CA is mainly found in the chloroplasts while in C4 plants most of the CA activity is located in the mesophyll cytosol. In maize and sugar cane (NADP-malic enzyme type C4 plants) there appear to be three CA isozymes and the nucleotide and amino acid sequences have been determined. In contrast to other CAs the three maize CA isozymes have repeat sequences which do not match up with the molecular weights of the mature proteins found in plants. This project is designed to investigate and answer the questions raised relating to the protein and nucleotide sequence data available.
This project will involve molecular biology techniques as well as basic biochemistry techniques.

Supervisor: Alan Baxter.
Identification of the role of NKT cells in the aetiology of type 1 diabetes
        A relative deficiency in NKT cell numbers exists in NOD mice, a model of type 1 diabetes. Adoptive transfer of NKT cells into these mice at an young age can completely prevent the progress of disease. Genetic linkage analyses have unidentified a region on mouse chromosome 2 that encodes both susceptibility to diabetes and control of NKT cell numbers.  Mice congenic for genes controlling these phenotypes have been bred on the NOD mouse background. This project seeks to characterise the genetic, immunological and pathological features of this strain with a view to determining the role NKT cells play in controlling susceptibility to diabetes. The project will involve cellular immunological techniques, molecular genetics and clinical assays.

Characterising the role of CD3 zeta polymorphisms in autoimmune disease
 Both lupus and genetic control o NKT cell numbers have been mapped to chromosomal region containing the CD3 zeta gene on chromosome 1. This project aims to test the hypothesis that CD3z polymorphism plays a role in controlling susceptibility to lupus as well as NKT cell numbers. The project will involve cellular immunological techniques, molecular genetics and clinical assays.

Fine mapping of the gastritis susceptibility locus Gasa1
       In previous studies, we have mapped the genes conferring susceptibility to gastritis in BALB/c mice to two genes on distal mouse chromosome 4. This project will apply a positional cloning strategy to narrow the region of interest to a size that can be studies using large insert (BAC) transgenesis. This is will achieved by performing a specific series of crosses between congenic mouse stocks,  fine mapping of the region of interest using cutting edge robotics and automated genotyping technologies, phenotyping mice with recombinations in the region of interest and deriving new subcongenic stocks. The project will involve molecular genetics and clinical assays.

The role of Toll-like receptors in autoimmune disease
       Toll-like receptors (TLR) mediate the state of activation of multiple elements of the innate immune system and are believed to play a critical role in determining the nature and strength of adaptive immune responses. This project tests the hypothesis that TLR ligation can also modulate the severity of ongoing autoimmune responses. The project will involve mouse handling, cellular immunological techniques, and clinical assays. The student may have the opportunity to help generate a transgenic mouse line expressing a dominant negative TLR mutant.

Supervisor: Gerald Münch.
Thiamine (Vitamin B1) analogues ? are they potential inhibitors of thiamine pyrophosphate synthesis?
       Thiamine pyrophosphate is an essential cofactor for thiamine diphosphate-dependent enzymes such as pyruvate dehydrogenase (E1 subunit), alpha-ketoglutarate dehydrogenase (alpha KGDH) and transketolase.  Thiamine is only the pro-vitamine, it has to be converted to the active cofactor, thiamine pyrophosphate (TTP), by the action of thiamine dikinase. ( other names: Thiamine pyrophosphokinase (E.C.2.7.6.2)).
Thiamine analogues are currently tested in clinical trials as AGE Crosslink Breakers that function unlike any previously developed pharmaceutical agent to reverse the disease states associated with age (or diabetes)-related stiffening of vessels, tissues and organs.
In this project, we want to investigate the hypothesis that such AGE-inhibitors / thiamine analogues are competitive inhibitors of Thiamine pyrophosphokinase. A  subsequent depletion of Thiaminepyrophosphate (TTP) may result in serious clinical consequences after long-term use.
 

Activation of microglia by ß-amyloid peptide and advanced glycation endproducts (AGEs) - therapeutic inhibition by natural antioxidants from medicinal plants
       The overall aim of this project is to inhibit pro-inflammatory signal transduction pathways involved in the induction of oxidative stress and cytokine release by activated microglia in a cell culture model of Alzheimer’s disease.  Activation of murine microglia will be achieved by a combination of ß-amyloid peptide and AGEs. These cells will then be used to study the effects and actions of a range of natural antioxidants (from tropical fruits, vegatables and herbs) on cellular activation and cytokine release. Our project is intended to provide “natural” therapeutic options to treat the chronic inflammatory process in AD.
 

Effect of carnosine on pro-inflammatory signalling and toxicity of advanced glycation endproducts
       Advanced glycation end products (AGEs), formed by the reaction of reducing sugars or dicarbonyl compounds on long-lived proteins (such as amyloid) or under conditions of high sugar concentration (such as in diabetic tissue), play an important role in Alzheimer’s disease and diabetic complications including diabetic retinopathy and nephropathy. AGEs inhibit proliferation and cause hypertrophy in tubulus cells. In addition they induce the expression of cytokines such as TGF-ß, IL-1 and IL-6 and inducible nitric oxide synthase. Furthermore, AGEs are toxic to a variety of cells including neurons. The human body has evolved a variety of mechanisms to lower AGE levels including physiological production of millimolar amounts of the carbonyl scavenger carnosine. We want to investigate the hypothesis that the application of carnosine during AGE formation will produce less neurotoxic and pro-inflammatory AGEs.

Supervisor: Heiner Körner.
Generation of TLR-2, 4, 9 transfected HEK-293 cells and analysis of the TLR ligands LPS and CpG
       Toll-like receptors (TLRs) are transmembrane proteins and represent a newly recognized family of vertebrate pattern recognition receptors. As a consequence of an infection and subsequent pathogen-associated molecular pattern engagement, TLRs activate downstream mediators such as NF-kB. TLR2 in combination with CD14 bind to bacterial lipoproteins, TLR4 and CD14+MD2 bind to lipopolysaccaride and TLR9 recognizes conserved bacterial DNA motives (CpG). Changing patterns of TLRs are epressed on most cells but TLR2, 4 and 9 are of particular interest for the activation/maturation of dendritic cells. In the proposed project a cell line negative for these TLRs will be transfected with TLR 2, 4 and 9 together with the necessary auxillary molecules. This will give us the possibility to test the purity of the different TLR ligands and will be essential for our further work with the role of TLRs in dendritic cells. Experimental techniques used: flow cytometry, PCR, tissue culture, transfection.

EAE in CCR6-negative mice
       The migration of autoimmune cells to the central nervous system is a fundamental event in the establishment of the autoimmune disease Multiple Sclerosis (MS). The mechanisms governing how these cells find their way to the brain have not yet been resolved. This project will be carried out in the model disease experimental autoimmune encephalomyelitis (EAE), which is a tissue specific, cell-mediated autoimmune disease that resembles the early stages of MS. EAE will be induced and analysed in mice negative for CCR6. This model will be used to analyse the role of one chemokine receptor molecule, CCR6, which potentially helps autoimmune cells to navigate to the brain. Experimental techniques used: immunohistology, flow cytometry, PCR, tissue culture.

TNF-dependent “tissue conditioning”
       Microbial products and inflammatory cytokines stimulate dendritic cells (DC) and DC precursors, such as monocytes or immature DCs, which have been recruited from the blood, to pass through a regulated sequence of maturation and migration steps. Immature DCs express receptors for inflammatory chemokines and, once maturation has been induced, up-regulate CCR7, a chemokine receptor that drives their migration to the local lymphoid node. The proinflammatory cytokine TNF plays a central role in the attraction of DC precursor cells from the peripheral blood and in the subsequent accumulation of those cells in the tissue. It has been shown that the addition of TNF to the site of an immune reaction significantly increases the number of mature DCs that present antigen. This project explores the activity of TNF further using TNF-negative mice. We will induce immune responses in TNF-knockout mice with the artificial antigen Ovalbumin (Ova) and add TNF to induce and modulate the response. The number of DC presenting Ova in the draining LN will be analysed. Experimental techniques used: immunohistology, flow cytometry, PCR, tissue culture.
xperimental cutaneous leishmaniosis is one of the best-characterised mouse models of infection by a parasite. The analysis of this model has contributed significantly to our understanding of the host response to an infection by complex pathogens. We demonstrated that CD8 alpha-negative inflammatory dendritic cells that infiltrated the lesion early after infection and transported antigen to the local lymph node. In genetically modified B6.TNF-/- mice inflammatory infiltration and antigen transport were severely diminished. The project aims at an analysis of the size and the composition (CD4+ and CD8+ T cells, B cells, NK cells, NKT cells) of the cutaneous inflammatory infiltrate at the site of infection in B6.WT and B6.TNF-/- mice. Furthermore, we want to learn more about the expression of the cytokines Il-4, Il-10, Il-12 (p40, p35), Il-23 (p40, p19) and IFN-gamma in the infected tissue. In this project methods of cellular immunology and molecular biology will be used.

Supervisor: Bill Warren.
Analysis of Novel Regulators of Sister-Chromatid Cohesion
       The objective of this study is to better understand how chromosomes are accurately partitioned during cell division.  'Cohesin' is an evolutionarily conserved multi-protein complex thought to be the primary effector of sister-chromatid cohesion in all eukaryotes. In yeast, cohesin is loaded onto chromosomes in S-phase and maintains chromatid cohesion until the metaphase-anaphase transition. Sister-chromatid separation is then triggered by the site-specific cleavage of the Rad21 cohesin subunit. We have used the Drosophila system to identify a number of regulators of rad21 that alter the dynamics of sister-chromatid separation.  Using a range of genetic and molecular techniques, this project will work toward determining the molecular identity of these novel regulator genes. These studies will lay the groundwork for future genetic, cytogenetic and biochemical analyses of genes and proteins that regulate these processes in human cells.

The role of Glipr2  in innate immunity
       In a genome screen of coral, a highly conserved gene, Glipr2, was identified. This gene has strong homology with the PR1 family of plant antifungal defensins, the human glioma pathogenesis-related protein (GliPR) and multiple proteins identified through whole genome analyses of a broad range of organisms, including Drosophila, C. elegans, and S. cerevisiae.  This project will utilize the Drosophila system to determine whether Glipr2 plays a role in immune function in metazoan species. RNA interference will be used to knockdown the function of the endogenous Glipr2 in adult Drosophila by microinjecting double-stranded RNA corresponding to Glipr2.  Real-time quantitative PCR will be used to monitor the level of RNAi-induced transcriptional suppression. Glipr2 knock-down animals will then be challenged with bacteria or fungi using by septic injury.  This involves pricking the thorax of adult with a tungsten needle dipped in a concentrated microbial culture with the number of insects succumbing to infection being indicative of innate immune function.

A Structure/Function Study of the Drosophila deflated Gene
       Proper regulation of the cell cycle is essential to coordinate proliferation with differentiation and to prevent disastrous outcomes such as cancer. Using a comparative genomics approach, we have identified an evolutionary conserved gene, which we have named deflated, as a putative regulator of cell proliferation in multicellular eukaryotes. This project will generate a number of in vitro plasmid constructs to express the deflated protein product in both bacterial cells and in transgenic Drosophila. These studies are aimed at determining the structure function relationship of the various deflated domains to better determine the role that this gene plays in cell cycle regulation both biochemically and in a developmental context.

Supervisor: David Miller
Origins of eyes and vision ? evolution of sine oculis and related genes.
        Pax-6 is often referred to as the “master control gene” for eye specification, but does not operate on its own. Key regulators of Pax-6 genes are also conserved between fly and mouse (and possibly much further), and one of these is a homeobox gene known as sine oculis in Drosophila and Six-3 in vertebrates. Vertebrates have several genes closely related to fly sine oculis; these are known as the Six family, and of them Six-3 seems to correspond most closely to sine oculis. We have recently identified coral homologs of several Six famly genes, and aim to characterise these at the level of expression (virtual northerns; in situ hybridisation) and function (band shift analysis; transgenic expression). Key questions that we would hope to answer include whether coral Six genes are expressed in neurons (or their precursors), and whether genetic interactions like those seen in Drosophila could occur in “lower” animals. These experiments should enable a better understanding of the evolution of vision in the animal kingdom.

Characterisation and functional analysis of novel Pax genes
        A novel mechanism has been proposed by which homedomain transcription factors, such as Pax proteins, may be secreted from cells and regulate the activities of genes in adjacent cells. One aim of this project would be to study aspects of this process by studying the localisation of GFP-tagged Pax constructs in cultured mammalian cells (this will be done in collaboration with Prof David Jans, at Monash University). The other aim of this project would be to better understand how Pax proteins recognise their binding sites. Mammals have at least nine Pax genes, and fly a comparable number ? these encode closely related paired domains (PDs) that are somehow able to recognise different binding sites. We have identified two Pax proteins whose binding specificity we wish to study (by band-shift assays and site-directed mutagenesis) and expression patterns we also need to determine (by virtual northern and in situ hybridisation).

Sex determination mechanisms ? the Doublesex genes.
        Genes related to Drosophila Doublesex (Dsx) are known as the DM family; these genes encode a type of transcription factor which is thought to play a common role in sex specification across the animal kingdom. Analysis of the entire DM gene family allowed us to recently identify a novel conserved domain, the DDM (Downstream of DM) domain, that is present in some (but not all) DM genes in vertebrates and other animals (Miller et al., 2003). The precise roles of most of these DDM genes are unknown. However, the expression patterns of at least one of the human DDM genes are consistent with roles in sexual development, and the 9p region to which one of the human DDM genes maps is associated with XY sex reversal (Ottolenghi et al., 2000). The major aim of this project is to initiate functional analysis of the DDM domain. We propose two approaches: use of the yeast 2-hybrid system to identify protein interacting with the DDM domain and generation of Drosophila strains in which the activities of the DDM gene dmrt93B can be selectively disabled (ultimately enabling mutant phenotypes to be characterised). Together these approaches are likely to provide novel perspectives into the evolution of function in the DM gene family, and into the evolution of sex-specification in animals.

Characterisation of the Xoom/ARM1 family, conserved regulators of gastrulation.
        Gastrulation has been called the single most important time in your life ? it is the process in the early development of animals when the multilayered body plan is established. Typically it involves dramatic and highly coordinated cell movements, and results in the formation of the three body layers (ectoderm, endoderm and mesoderm). In man and the fly, gastrulation is initiated by mesoderm-inducing molecules, but the downstream implementation of these signals is much less clear. One good candidate is the Xoom/ARM1 (Adhesion regulating protein 1) protein family, which appear to be key regulators of the gastrulation process in vertebrates. Normally the activity of the Xoom/ARM1 gene is tightly regulated, and its misexpression is characteristic of specific cancer classes in man. We have identified uncharacterised genes related to Xoom/ARM1 in Drosophila and in Acropora. The aim of this project is to characterise these genes in order to better understand the function of the Xoom/ARM1 family in development and cancer.

Conservation of nervous system patterning processes
        In the developing Drosophila nerve cord, patterning along the dorsoventral (DV) axis is achieved largely by three homeobox genes that interact directly ? ventral neuroblasts defective (vnd), intermediate neuroblasts defective (ind) and muscle segmentation homeobox (msh). Very similar genes are expressed in very similar ways in vertebrates, so it seems that this system may be universal in higher animals. We think it may also pattern the most ‘primitive’ extant nervous systems ? those of anthozoan cnidarians such as Acropora. We have shown that the Acropora ind gene is expressed in a very similar pattern to its fly and human counterparts, and are carrying out mutant rescue experiments in the fly to examine conservation of function. We want to extend these studies to the cnidarian vnd and msh genes, which we have recently identified. The aims of this project are to characterise the expression patterns of the two cnidarian genes, to carry out yeast one-hybrid work and initiate Drosophila experiments aimed at understanding the function and interactions of the cnidarian vnd, ind and msh genes.

Other possibilities:

 

If a student has a specific project in which they are interested they are encouraged to speak with relevant potential supervisors to investigate the possibility (and feasibility) of conducting research to investigate the specific research of interest.


OTHER LINKS: Comparative Genomics Centre, James Cook University, Key words: Autoimmune diabetes, Type 1 diabetes mellitus, childhood diabetes, lupus, systemic lupus erythematosus, haemolytic anaemia, hemolytic anemia, Coombs' test, antinuclear antibodies, renal failure, glomerulonephritis, gastritis, type A gastritis, pernicious anemia.