Supervising Staff

MOLECULAR BIOLOGY OF GENE EXPRESSION

Phone: 6488 3041 (Biochemistry, UWA); or 9224 0363 (Royal Perth Hospital)
Email: labraham@cyllene.uwa.edu.au

Our group is interested in the transcriptional regulation of gene expression. Our focus is on genes that are involved in regulating the immune and inflammatory responses and the identification of transcription factors that regulate these genes. Our long-term aim is to develop therapeutic strategies to modulate the activity of these genes in order to prevent disease. Prospective Honours students with a background in Molecular Biology, Biochemistry, Molecular Genetics or Immunology/Microbiology are particularly encouraged to apply. Students will be exposed to a range of techniques including DNA sequencing, DNA cloning, cell culture, transfection assays, RT-PCR, Northern and RNase Protection analysis, EMSA, protein analysis, DNase I Footprinting and FACS analysis. Projects will be carried out in both Biochemistry, UWA and the Western Australian Institute for Medical Research laboratories at Royal Perth Hospital.

MEDICAL RESEARCH - OXIDATIVE STRESS, SIGNALING AND CELL DEATH

Phone: 6488 1750
Email: parthur@cyllene.uwa.edu.au

Partially reduced oxygen in the form of reactive oxygen species (ROS) are generated as a by-product of normal cellular metabolism and in normally functioning cells a variety of antioxidant defenses protect against injury from these potent oxidants. Nevertheless, ROS can affect cell function (replication, growth, protein synthesis, ion transport etc) and is an ongoing threat to cell viability. For example, ROS are thought to contribute to the complications of many chronic diseases including blindness, Alzheimer’s, emphysema, Parkinson’s disease, diabetes, renal failure and aging. Consequently there is intensive research into understanding the molecular mechanisms by which ROS exert their effects with view to developing appropriate therapies to prevent damage.

Research into ROS has historically focused on the damage ROS causes to cellular components (eg DNA, membranes and proteins). An exciting development has been the realization that ROS can act as signaling molecules to activate a wide variety of biochemical cascades. This is particularly interesting when examining how ROS cause cell death (necrosis or programmed cell death) because it may possible to prevent cell death by targeting one or more of the processes (likely proteins) involved. A major challenge is to identify proteins involved in the pathways. The development of new fluorescent techniques and technologies to assess ROS have also contributed to the exploding interest in ROS.

A MAMMALIAN HISTONE H4 HISTIDINE KINASE INVOLVED IN THE REGULATION OF CELL PROLIFERATION AND CANCER

Phone: 6488 7007
Email: Paul.Besant@uwa.edu.au

Protein kinases have been found to be major components of cellular signalling systems that regulate almost all aspects of cellular function. The most well-known protein kinases in mammalian cells are serine/threonine kinases and tyrosine kinases which phosphorylate their substrate proteins on these hydroxyamino acid residues. There are however protein histidine kinases that phosphorylate proteins on histidine residues and their importance to cell regulation in bacteria, yeast and plants has long been recognised. It is now becoming evident that such protein kinases exist in mammalian cells and may also play important roles in the regulation of cell function. We have been studying a histone H4 histidine kinase activity that is induced in regenerating hepatocytes following partial hepatectomy and in liver oval cells during liver regeneration following chronic chemical damage. In addition, we have found that the levels of the activity of this protein kinase are greatly increased in transformed tumorigenic oval cells compared to normal proliferating oval cells. The time course of increase in the activity of the protein kinase in the regenerating livers just precedes the increase in DNA synthesis corresponding to cell division. This evidence strongly suggests that the histone H4 histidine kinase plays a role in the regulation of cell division. The aims of this project are to characterise the histidine kinase and investigate its role in the regulation of cellular proliferation and cancer.

EVALUATING SIGNAL TRANSDUCTION NETWORKS AND THEIR CELLULAR EFFECTS

Phone: 6488 1348
Email: marieb@cyllene.uwa.edu.au

The Cell Signalling Lab has a broad interest in signal transduction events. In addition, we are interested in signalling events in the muscle cells (myocytes) of the heart. Why? Recent statistics from the Australian Institutes of Health clearly show that diseases of the heart and circulation are the greatest causes of death in Australia. Prominent amongst the causes of death is injury to the heart in the form of heart attack (infarction) or high blood pressure (hypertension), as well as failure of the heart itself.

Additional Projects:

We are actively pursuing a number of other areas in collaboration with members of the local, national and international research communities. Students with specific interests in any of the following project areas should see Dr Bogoyevitch as early as possible in 2004 to discuss the development of projects of interest to start in 2005.

• Genetic screening to identify inhibitors of the kinase, Aurora-A, and the ultimate development of new drugs for the treatment of breast cancer.

• Proteomic and microarray analysis to identify proteins involved in cardiac protection downstream of the ERK MAPK pathway.

• Evaluation of cardioprotection in a transgenic mouse line overexpressing insulin-like-growth factor.

• The role of the beta-splice form of the transcription factor STAT3 as a repressor of transcription, and its implications in the development of heart failure.
  

Note, at the end of each Project description, specific references relating to each project are given. Further information on specific techniques and approaches employed can also be found in our recent papers:

Phone: 6488 1107
Email: mdcregan@cyllene.uwa.edu.au

Human milk contains a myriad of different cell types, including secretory epithelial cells (lactocytes), immune cells and progenitor cells that offer a unique potential of non-invasive source of tissue. The overall goal of the research in my laboratory is to isolate, characterise and utilise these cells for studies into the synthesis of human breastmilk and the consequent benefits for the mother and breastfed infant. The presence of lactocytes in breastmilk is believed to be due to them having exfoliated from the breast due to the physical pressures associated with the continued filling and emptying of the breast, whereas the immune cells are believed to be present to protect the mammary gland from infection and to provide protection for the infant from disease. We have also recently identified a third cell-type in human breastmilk – progenitor cells that may be adult stem cells.

CONTROL OF THE SYNTHESIS AND REMOVAL OF BREASTMILK

Phone: 6488 3327
Email: hartmanp@cyllene.uwa.edu.au

Breast development during pregnancy and the initiation of milk production after birth (lactogenesis 2) are under endocrine control, whereas during established lactation local (autocrine) negative feedback mechanisms match milk synthesis to the infant’s appetite. It is possible that imbalances in these physiological changes are largely responsible for mothers concerns about their ability to produce enough milk for their babies during the first six to eight weeks of lactation.

Specifically we aim to gain a greater understanding of the local control of milk synthesis. To identify the mechanisms controlling milk synthesis, it is important to identify the factors that influence the removal of milk from the breast and to understand how milk synthesis responds to breast emptying. Recent studies conducted in collaboration with the Texas Tech Medical Centre, USA, have shown constant rates of milk synthesis over 4-5 hour periods, provided the breast is drained hourly. Therefore, these studies have provided a methodology for investigating milk synthesis and the rates of synthesis of specific components in the milk.

The projects offer students the unique ability to conduct research with a multidisciplinary approach and the rare opportunity to gain both laboratory and clinical experience. In this regard, my laboratory is part of the Women & Infants Research Foundation at King Edward Memorial Hospital for Women (KEMH) and part of the research will be carried out in the Breast Feeding Centre of WA, KEMH.

It is hoped that these studies, will lead to a greater understanding of the basic mechanisms controlling milk synthesis, providing evidence-based procedures to discriminate between normal and abnormal function of the lactating breast and thereby provide a scientific basis for the nourishment of pre-term babies.

Phone: 6488 3331
Email: tmartin@cyllene.uwa.edu.au

The production and allocation of metabolic resources and hence plant productivity is greatly influenced by environmental challenges such as nutrient availability, light conditions, abiotic and biotic stresses. Plants strongly rely on and use manifold sensing, signalling and response mechanisms enabling a flexible response to ever changing conditions. A class of small acidic soluble proteins named 14-3-3 proteins play crucial roles in many of these regulatory pathways (for a recent review see 1). They bind to and regulate key enzymes in carbohydrate and nitrogen metabolism such as nitrate reductase and sucrose phosphate synthase (see ref. 2-4). Furthermore they mediate hormone related gene expression in plants, control hormone biosynthesis and interact with other signalling components such as protein kinases (CDPKs and SNF1-like kinases, reviewed in 1 and see ref. 5 and 6). These manifold functions have led to the hypothesis that 14-3-3s play key roles in bringing various sensing, signalling and response pathways together. In plants, up to 15 genes encode for 14-3-3 proteins. These proteins act as homo and hetero dimers, thus allowing for a great number of putative dimer combinations. It is possible that each 14-3-3 dimer has specific function or that several dimers have overlapping functions and can substitute for each other. Two projects are on offer which should help to progress our understanding of 14-3-3 specificity and to analyse the roles of individual 14-3-3 proteins.

Phone: 6488 7245
Email: hmillar@cyllene.uwa.edu.au

Phone: 6488 3040
Email: rtuckey@cyllene.uwa.edu.au

Research being undertaken concerns the mechanism and regulation of steroid hormone synthesis. Steroid hormones are produced by the adrenal cortex, gonads and placenta and play essential roles in glucose and salt homeostasis as well as in reproduction. The enzymes catalysing steroid synthesis are either steroid dehydrogenases or members of the cytochrome P450 family of enzymes. Of particular interest is cytochrome P450scc, which catalyzes the first step of steroid synthesis, the conversion of cholesterol to pregnenolone (termed the cholesterol sidechain cleavage reaction). Cytochrome P450scc is located in the inner-mitochondrial membrane of steroid producing cells and uses cholesterol from the membrane as its substrate. The conversion of cholesterol to pregnenolone by cytochrome P450scc involves three hydroxylations which all occur at the same active site on the cytochrome. Electrons for the hydroxylation reactions are supplied by NADPH via the electron transport proteins adrenodoxin and adrenodoxin reductase. Adrenodoxin is a one electron carrier with an iron sulphur centre and adrenodoxin reductase is a flavoprotein that can donate two electrons. These proteins serve as electron donors for all mitochondrial cytochromes P450 (see Fig. 1).

In the adrenal cortex, ovary and testis the delivery of cholesterol to P450scc in the inner mitochondrial membrane is rate-limiting for steroid synthesis. This step is regulated by trophic hormones (ACTH of LH) and is mediated by the steroidogenic acute regulatory protein (StAR protein). A deficiency in the StAR protein occurs in the disease Congenital Lipoid Adrenal Hyperplasia where adrenal cells die due to accumulation of excessive cholesterol. In collaboration with Prof. Walter Miller at the University of California, San Francisco (1,2) we are investigating the mechanism of action of the StAR Protein (see Project 1 below).

The mechanism of regulation of the progesterone synthesis in the human placenta is also under investigation. Progesterone is a hormone essential for human pregnancy. It is made from pregnenolone, the product of cytochrome P450scc action on cholesterol. Its rate of production during pregnancy is dependent upon the amount of cytochrome P450scc activity in the placenta. Unlike the adrenal cortex, the placenta does not express the StAR protein but rather uses a related protein, MLN64, to transfer cholesterol to P450scc for progesterone synthesis (2). Recent work with intact and disrupted placental mitochondria has shown that cytochrome P450scc activity is limited by the rate of electron delivery to cytochrome P450scc, and not cholesterol delivery (3-7). In particular, the concentration of adrenodoxin reductase has been found to be limiting. Thus, the slowest and therefore rate-limiting step in progesterone synthesis is the reduction of adrenodoxin by adrenodoxin reductase (see Fig 1). The mechanism of regulation of adrenodoxin reductase levels is poorly understood. This regulation is of fundamental importance to our understanding of the control of progesterone synthesis during pregnancy and Project 2 (below) will explore this.

Figure 1. Progesterone synthesis by placental mitochondria. Electrons flow from NADPH to cytochrome P450scc via adrenodoxin reductase (AR) and adrenodoxin (Adx). Pregnenolone is converted to progesterone by 3²-hydroxysteroid dehydrogenase (3²HSD). OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane.

A new area of interest is steroid synthesis by the skin. Prof. Slominski's group at the University of Tennessee has reported that the skin expresses a number of P450 enzymes involved in steroid synthesis including P450scc (6). In collaboration with Prof. Slominski we have recently shown that P450scc, adrenodoxin, adrenodoxin reductase and MLN64 are all present at low levels in human skin and the system is functional in the cleavage of the side chain of cholesterol. Skin is also the site of vitamin D3 synthesis from 7-dehydrocholesterol in a reaction that requires UV light. We have shown that cytochrome P450scc can act on both 7-dehydrocholesterol and vitamin D3 providing strong evidence that the skin can make a new class of steroid-like molecules not previously described. The third project (next page) aims to further characterize steroid synthesis by P450scc in skin.

References

Tuckey, R.C. Headlam, M. J., Bose, H. and Miller, W. L. (2002) Transfer of cholesterol between phospholipid vesicles mediated by the Steroidogenic Acute regulatory Protein (StAR). J. Biol. Chem. 277, 47123-47128.

Tuckey, R. C., Bose, H. S., Czerwionka, I. and Miller, W. L. (2004) Molten globule structure and steroidogenic activity of N-218 MLN64 in human placental mitochondria. Endocrinology 145, 1700-1707.

Tuckey, RC (2004) Progesterone synthesis by the human placenta.Placenta, in press

Woods, S. T., Sadleir, J., Downs, T., Triantopoulos, T., Headlam, M. J. and Tuckey, R. C. (1998) Expression of catalytically active human cytochrome P-450scc in Escherichia coli and mutagenesis of isoleucine-462. Arch. Biochem. Biophys 353, 109-115.

Tuckey, R. C. and Sadleir, J. (1999) The concentration of adrenodoxin reductase limits cytochrome P450scc activity in the human placenta. Eur. J. Biochem. 263, 319-325.

Tuckey, R. C., McKinley, A. J. and Headlam, M. J. (2001) Oxidized adrenodoxin acts as a competitive inhibitor of cytochrome P450scc in mitochondria from the human placenta. Eur. J. Biochem. 268, 2338-2343.

Tuckey and Headlam, M. J. (2002) Placental Cytochrome P450scc (CYP11A1): Comparison of catalytic properties between conditions of limiting and saturating adrenodoxin reductase. J. Steroid Biochem. Molec. Biol. 81, 153-158.

Slominski, A., Ermak, G and Mihm, M. (1996) ACTH receptor, CYP11A1, CYP17 and CYP21A2 genes are expressed in skin. J. Clin. Endocr. Metab. 81, 2746-2749.

ORGANELLE BIOGENESIS, GENE EXPRESSION AND PROMOTER CHARACTERISATION

Phone: 6488 3325
Email: seamus@cyllene.uwa.edu.au

The evolution of eukaryotic cells was triggered by the endosymbiotic event that led to the formation of mitochondria. As such mitochondrial biogenesis and function is vital to many important functions in the cell. In fact it is often stated that mitochondria control the “life and death” cycle of cells due to their role in energy production, synthesis of essential compounds and the role they play in cell death. Thus the study of mitochondria and the roles they play in cellular function is linked to many aspect of cellular function. Thus signal transduction pathways controlling energy production, reactive oxygen species (ROS) signalling, synthesis of essential metabolic compounds (amino acids etc) and cell death itself all use mitochondria as an important hub or site through which these pathways can be controlled, received and integrated to achieve an appropriate cellular response. The overall aim of research in my laboratory is to study and understand these responses.

We study the various aspects of mitochondrial function using a variety of post-genomic or functional genomic responses. By this we mean that we use model organisms to study particular processes, and do not limit our investigations to single genes, proteins or metabolites – rather we use all the available resources provided by model systems to answer fundamental biological questions. Thus approaches employed in the projects below range from Biochemical approaches (activity assays, Western blots, organelle isolation and protein uptake experiments), Molecular approaches (gene expression analysis, over-expression of proteins, protein tagging etc, site-directed mutagenesis, gene cloning) to Genomic approaches (Microarray analysis, transgenics using knock-out or overexpression and Bio-informatics). Overall this yields a powerful, integrated and team approach to investigations with national and international collaborations.

A. Harvey Millar, David. A. Day, and James Whelan (2004) Mitochondrial Biogenesis and Function in Arabidopsis In 'The Arabidopsis Book'. pp. doi/10.1199/. (Ed. C.R. Somerville and E.M. Meyerowitz). http://www.aspb.org/publications/arabidopsis/toc.cfm

A number of projects are outlined below with some references. These give an overview of the types of projects available. Visit http://www.mitoz.bcs.uwa.edu.au for web page. A number of references are listed for each project, which gives an idea of the approaches used, and output from these projects. Specific references will be provided after discussion of individual projects.

STRUCTURAL AND COMPUTATIONAL BIOLOGY

Phone: 6488 3337
Email: wilceja@cyllene.uwa.edu.au

IN VIVO AND IN VITRO DIFFERENTIATION OF LIVER PROGENITOR CELLS DERIVED FROM A TRANSGENIC MOUSE

Phone: 6488 2986
Email: yeoh@cyllene.uwa.edu.au

BACKGROUND :

The TAT GRE lacZ transgenic mouse offers a means of easily identifying cells, which have differentiated into hepatocytes during development. The transgene is activated in hepatocytes, which have assumed the adult phenotype so such cells stain blue with the chromogen x-gal. In the last 6 months, liver progenitor cells (LPC- lacZ) have been isolated from these mice. These cell lines represent a powerful tool to identify factors and for clarifying molecular mechanisms which are responsible for determination of the hepatic lineage of liver progenitor cells. In culture, under growth conditions they proliferate rapidly and do not express the transgene (Figure 1&2). Hence they can be expanded very quickly. Preliminary experiments show that under specific conditions of culture e.g. when they are allowed to reach a density which induces the formation of clusters (Figure 3), or when cultured in matrigel where they form large 3-dimensional spheroids, these cells can be induced to become hepatocytes and thus express the transgene and be marked using x-gal which stains them blue.

1. LPC-lacZ cell line in growth medium (confluent culture phase contrast)		2. LPC-lacZ in growth medium (no blue cells after x-gal staining)
3. Cell clusters which stain positively (differentiation medium)		4. Spheroids which stain postively (matrigel medium)