School of Biomedical, Biomolecular and Chemical Sciences HONOURS PROJECTS 2009
Biochemistry & Forensic Chemistry Molecular Biology
Genetics
Medical Science
Microbiology & Immunology Biomedical Science
Physiology Chemistry
Welcome to the Second Honours Exposition in the School of Biomedical, Biomolecular and Chemical Sciences.
2009 Honours
We hope that you will enjoy this event and that it will serve as a good introduction to the range of Honours projects offered in the School for 2009.
If you are interested in doing an Honours year at UWA, you maybe are already asking about the exciting prospects available within each of the Disciplines and sub-disciplines within the School. These are Biochemistry and Molecular Biology, Biomedical Science, Chemistry, Forensic Chemistry, Genetics, Medical Science, Microbiology and Immunology, Pharmacy, Physiology and Structural Biology. This Honours Projects book/CD will enable you to further explore the possibilities and talk to staff that will be on hand. If you intend to enrol in Honours in 2008, this booklet/CD will provide you with a comprehensive overview of the interests of each of the research groups within the School as well as outlining suitable Honours projects. The Honours Expo is designed to showcase the depth and diversity of research being undertaken in the School. Here, you will be able to talk to staff who will be available to explain their research in much detail.
Enjoy!
Professor GA Stewart Head of School
Honours Co-ordinators
Biochemistry and Molecular Biology Microbiology and Immunology Professor George Yeoh Assoc Professor Barbara Chang Phone: 6488 2986 Phone: 9346 2288 [email protected] [email protected]
Chemistry Physiology Professor Mark Spackman Dr Anthony Bakker Phone: 6488 3140 Phone: 6488 7859
[email protected] [email protected]
Genetics Forensic Science Professor Lawrie Abraham Professor John Watling Phone: 6488 1148 Phone: 6488 4488
[email protected] [email protected]
Table of Contents
Major Discipline Page
Biochemistry and Molecular Biology 1
Chemistry 33
Microbiology & Immunology 70
Physiology 96
End-On Honours Application Form 114
BIOCHEMISTRY AND MOLECULAR BIOLOGY PROFESSOR LAWRIE ABRAHAM Room 2.58, MCS building, Phone: 6488 3041 email: [email protected]
Human Molecular Biology Lab
Our group is interested in the transcriptional regulation of gene expression. We are also interested in the effects of genetic polymorphism (SNPs) on the expression of genes, particularly promoter and other regulatory variants. The focus is on genes that are involved in regulating inflammatory responses and understanding how genetically determined differences in expression contribute to diseases such as autoimmune disease, cancer and cardiovascular disease. To this end we are involved in the identification of transcription factors and upstream components of the signal transduction pathways that regulate these genes. Our long-term aim is to develop therapeutic strategies to modulate the activity of these genes through interference with such regulators in order to prevent disease. Prospective Honours students with a commitment to excellence and a background in Molecular Biology, Biochemistry, Genetics or Immunology 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, expression array analysis, siRNA knockdown, DNA binding assays (EMSA), protein analysis, DNase I Footprinting, Chromatin immunoprecipitation (ChIP) and FACS analysis.
PROJECTS
1. The Transcriptional control of the CD30 Gene in Anaplastic Large Cell Lymphoma
Anaplastic large cell lymphoma (ALCL) is a variant of immunoblastic lymphoma and tends to be clinically aggressive, resulting in the destruction of the involved lymph node structure, the infiltration of the lymph node sinuses by large transformed neoplastic cells with prominent nucleoli. The major diagnostic marker of ALCL is strong overexpression of the CD30 gene thought to result from a transforming event that leads to neoplasia. Fundamental to our understanding of the causes and treatment of ALCL is an understanding of the mechanism of overexpression of CD30. The CD30 gene promoter, including an ALCL-specific hypersensitive site we have discovered in the 1st intron, will be characterised with respect to transcriptional control elements by EMSAs, CD30 reporter gene analysis and CHART (chromatin accessibility by real-time PCR). The transcription factors binding to the promoter and the 1st intron will be identified by use of a 2-dimensional proteomics technique developed in our group. Once cloned, the identified proteins will be tested for the ability to repress endogenous expression and reporter constructs by overexpression in cell lines and by RNAi approaches. Chromatin immunoprecipitation (ChIP) assays will also be carried out to establish the in vivo relationship between the various cis-elements and trans-acting factors, including sites of histone modification. The long-term aim is to develop therapeutic strategies that interfere with the transcriptional regulation of CD30 and so block the deleterious effects resulting from overexpression of CD30.
2. Cell Signalling in Anaplastic Large Cell Lymphoma following CD30 Receptor Stimulation
As outlined above, one of our objectives is to understand the neoplastic phenotype in ALCL and develop targeted therapies. By understanding how CD30 gene regulation is aberrant in ALCL cells, one can develop appropriate therapies to reverse the neoplastic phenotype that results from CD30 receptor signalling. We have recently shown that when ALCL cells are stimulated via the CD30 receptor (using ligand CD153 - see Model above) some of the cells undergo apoptosis. However, many cells escape cell death and instead differentiate by becoming adherent and after extended culture begin to proliferate, thereby escaping the apoptotic signal. Due to the importance of adhesion in neoplasia and metastasis, the aim of this project is to define, initially at the gene level, the differentiation program by comparing the transcriptional profiles of apoptotic versus adherent cells
1 following ligand stimulation. Transcription profiles will be produced using Affymetrix U133A chips and comparisons made between control and CD153-treated cells induced for 0, 0.25, 4 and 24 hours; non-adherent and adherent cells will be arrayed separately. Expression changes will be compared using the clustering programs available in the Genesifter package. The objective is to identify uniquely expressed genes that correlate with the adhesion response. Following identification and confirmation of expression by quantitative PCR (qPCR) we will establish whether these potential facilitators show activation by coIP and immunoblotting. The results of these studies will be used to identify downstream effectors that are involved in the adhesion or apoptotic responses resulting from ligand-receptor engagement.
3. Post-transcriptional Control of the Cystinosis disease gene, Cystinosin.
Cystinosis is an inherited monogenic disease caused by the defective transport of cystine out of lysosomes. Mutations in the gene (CTNS) that codes for the lysosomal cystine transport protein, cystinosin, represent the known causes for this disease. A wide spectrum of causal mutations have been observed involving both complete elimination of the transport protein (in the most severe cases) and more subtle quantitative deficiencies of the protein seen in less severe cases. Recent genome-wide scans have identified a cluster of SNP's within the 3'UTR of the gene that may affect expression of CTNS. The project will involve characterising the polymorphic 3' regulatory elements and the cognate miRNAs that regulate CTNS expression. Knowledge of these effectors and their targets may elucidate new therapeutic options.
4. Characterisation of functional polymorphisms of Vanin 1, a QTL controlling HDL-C Levels
This collaborative project with the Southwest Foundation for Biomedical Research, Texas, USA involves the characterisation of the Vanin 1 gene, which has been shown to be genetically associated with low levels of High Density Lipoprotein-cholesterol ("good" cholesterol) levels in the blood. Low HDL levels are a strong risk factor for cardiovascular diseases such as arthrosclerosis and heart attack. Four promoter variants in the Vanin 1 gene (see Fig) show significant correlations with HDL-C as well as Vanin 1 mRNA expression levels. The most likely functional promoter variant at -137 exhibits a strong association with HDL-C levels (p = 0.002). The project aims are to characterise transcription factors that differentially bind to the -137 SNP region using EMSA/peptide mass fingerprinting, determine the effects of the -137 SNP on transcriptional activity using reporter gene analysis, and to identify modulators of VNN1 expression & determine their effects on allele-specific transcription of VNN1 using mRNA expression profiling. An understanding of how the gene is controlled will inform the development of therapeutic strategies and/or drugs to modulate the activity of the Vanin 1 gene with the objective of raising HDL-cholesterol levels in individuals at risk.
5. Mechanism of Action of Newly Synthesised Thalidomide Derivatives. (See co-supervised project with Dr Scott Stewart (Chemistry - Project 2)
In a collaborative project with Dr Stewart, newly synthesised thalidomide-based drugs will be screened for novel biological activities using TNF reporter gene assays. For those students interested in the functional aspects of thalidomide and newly synthesised derivatives, transcriptional profiling will be carried out, using Affymetrix microarrays to define novel cellular activities, with a focus on therapeutic application.
Also see co-supervised projects with Dr Daniela Ulgiati (Biochemistry & Molecular Biology).
2 Biochemistry and Molecular Biology DR PETER ARTHUR SENIOR LECTURER Room 2.41, MCS Building, Phone: 6488 1750 email: [email protected]
Reactive Oxygen Species as modulators of signal transduction pathways and biochemical systems
Where there is aerobic life, there are reactive oxygen species (ROS). ROS have the potential to disrupt cellular function but are usually held in check by an array of antioxidant systems. In many chronic diseases this balance is disturbed, and the resultant oxidative stress is thought to contribute to disease pathology. There is evidence of oxidative stress in a diverse range of diseases including atherosclerosis, diabetes, Parkinson's disease and Alzheimer's disease. There is also good evidence oxidative stress is an important contributor to dysfunction associated with aging.
There has been an intensive research effort into understanding how ROS disrupts cellular function by damaging cellular macromolecules such as DNA, protein and membrane lipids. An exciting development has be the realization that ROS could be interfering with a variety of biochemical and genetic systems involved maintaining cellular homeostasis. Of the various ROS, hydrogen peroxide (H2O2) has attracted interest because it may be involved in modifying key intracellular proteins (e.g. signal transduction proteins) to influence many aspects of cellular function (growth, metabolic rate, protein synthesis and catabolism, cell division to name a few). If this concept is correct, then it should be possible to develop drugs which can prevent or reverse the detrimental effects of this form of oxidative stress. One example serves to illustrate this point. Recently, over expression of catalase (an antioxidant enzyme) in the mitochondria was found to extend the maximum lifespan of mice by 20%. This exciting research also provides very strong support for the theory that oxidative stress is a cause of aging. We are following up on this research by developing our own transgenic mouse and we are keen to involve students in this research.
This research area is constantly developing, so I am happy to discuss the research area in general or work with you to develop a project that suit your interests. I am an experienced supervisor with a preference for collaborative projects so that you can gain the benefits of dual supervision. Please see below examples of current research projects to give you an idea of the type of work we do.
PROJECTS 1. Does a potential therapeutic cause oxidative stress? Collaborative with Dr Thea Shavlakadze & Professor Miranda Grounds, Anatomy
Insulin growth factor 1 (IGF-1) is widely investigated as a therapeutic agent for muscle ageing and muscular dystrophy. In both conditions oxidative stress plays a significant damaging role, but it is not certain how elevated IGF-1 affects ROS generation in skeletal muscle. Down-regulation of IGF-1/Insulin signalling has anti- ageing properties (i.e. extends lifespan), which has been attributed to reduced oxidative stress. On the other hand, IGF-1 has been shown to be protective against ROS induced apoptosis in cardiac muscle cells. The interaction between IGF-1 and oxidative stress in skeletal muscle has not been investigated. The objective of this project is to use proteomic technologies to establish the relationship between IGF-1 and oxidative stress in dystrophic muscle (mdx/IGF-1 mice (Fig 1)) through our oxidative profile measurements. This will be significant because if IGF-1 does not affect oxidative stress, then the protective effect of IGF-1 may be Mdx/IG enhanced by combination with appropriate antioxidant therapies. Additional techniques may include Immunohistochemistry, Western Blotting, quantitative PCR and EMSA. Figure 1. One year old male mdx/IGF-1 and mdx littermate mice. Mdx/IGF-1 transgenic mice are much Md bigger compared to their age matched litter mates and they have a pronounced skeletal muscle hypertrophy.
3 Biochemistry and Molecular Biology 2. Does oxidative stress cause muscle wasting? As skeletal muscle ages it loses strength and power leading to reduced mobility and deleterious changes in lifestyle. The relentless loss of muscle mass and function in elderly individuals impairs daily functions such as walking, using stairs and rising from chairs and results in an increased incidence of falls. Muscle wasting is also associated with immobility and diverse pathologies such as cancer, bacterial sepsis, AIDS, diabetes, and end- stage heart, kidney, and chronic obstructive pulmonary disease. We are using transgenic mouse models of muscular dystrophy (which we already have) and ageing (which we are developing) to investigate the role of oxidative stress in muscle wasting. Transgenic mouse models are particularly significant in biomedical research because they reflect the complexity of human disease processes. This work is supported by a grant from the National Health and Medical Research Council.
One signalling protein associated with muscle wasting is NFkB and there is evidence indicating that oxidative stress can activate NFkB. Muscle wasting has also been linked to increased rates of protein breakdown and increased oxidative stress. The objective of this project is to establish whether oxidative stress causes an increase in the rate of protein breakdown via a signalling pathway involving NFkB. For this work a muscle cell line (C2C12) will be used, as cell culture systems are particularly useful experimental systems to pin point the precise molecular mechanisms involved in disease processes. Techniques likely to be required (you will be trained) for this project include proteomic techniques, tissue culture, quantitative PCR for atrophy related genes and measurement of oxidative stress. This project is also related to our larger effort to understand the effects of mild oxidative stress (particularly ageing) by developing a transgenic mouse over-expressing catalase.
3. Oxidative stress in the heart Collaborative with Dr L. Hool, Physiology Mild oxidative stress is a feature of a variety cardiovascular disease states including ischemic heart disease, hypertension, and congestive heart failure. The L-type calcium channel plays a key role in contraction and is a likely target of mild oxidative stress. This year, an honours student established a method (immunoprecipitation) to isolate the L-type calcium channel from tissue. The objective of this project is to use this technique to evaluate whether the L-type calcium channel is oxidized in vivo. Establishing that the L-type calcium channel is oxidized would be an important step towards understanding the role of oxidative stress in the development of pathology. This work is supported by a grant from the National Health and Medical Research Council.
4. Developing proteomic technologies to identify proteins responding to mild oxidative stress in chronic diseases (collaborative with Proteomics International) Proteomics is an exciting and rapidly expanding field which seeks to unravel biochemical and physiological processes by focusing on proteins. Encoded proteins carry out most biological functions, and to understand how cells work, we need to study what proteins are present, how they interact with each other and what they do.
The West Australian-based biotechnology company Proteomics International and the University of Western Australia are associated with the “Diabesity Research Program” (DRP), which is flagship node of the comprehensive “Centre for Food and Genomic Medicine (CFGM)” based in Perth. Diabetes mellitus often referred to simply as diabetes, is a syndrome characterized by disordered metabolism leading to abnormally high blood sugar (hyperglycaemia). Diabetes is a common condition, on the rise worldwide. It has been estimated that 940,000 Australians have diabetes and that about half of these are not aware of it. If uncontrolled, diabetes can lead to a wide range of serious, long-term health complications which contribute to illness, disability and early death. Its onset is often associated with overweight. Diabesity is a relatively new term for diabetes caused by excessive weight, or the condition of having both diabetes and excessive weight.
As part of the DRP, we are undertaking identification of novel protein biomarkers for diabesity in human plasma. Such biomarkers are measurable proteins whose detection can be used clinically to enhance prediction of disease, diagnosis or prognosis.
The challenge is to identify valuable biomarkers from amongst the many thousands of proteins in human plasma. Oxidative stress has been implicated as a mechanism underlying hyperglycaemia-associated cellular damage and could play a role in the development of diabetes-related complications. This suggests that focussing on oxidised forms of particular plasma proteins may be the key to identifying valuable biomarkers for diabesity. This project will involve using proteomic technology including protein separation techniques (HPLC, 2D gel electrophoresis, antibody technology) and protein identification techniques (mass spectrometry).The effect of oxidative stress on proteins we will be evaluated using a patented technique developed by Proteomics International and Dr. Arthur.
4 Biochemistry and Molecular Biology A/PROFESSOR PAUL ATTWOOD
Room 3.69, MCS Building, Phone: 6488 3329 Email: [email protected]
The research focus of A/Prof. Attwood's laboratory is the structure and function of enzymes in general. However, there is a particular focus on two enzymes:
(i) pyruvate carboxylase, a biotin-dependent enzyme whose structure we have just determined and whose mechanism we are are investigation with a combination of site-directed mutagenesis, kinetic and physical methodologies;
(ii) mammalian histidine kinases which catalyse the phosphorylation of histidine residues in substrate proteins. This is a little understood form of phosphorylation in mammalian cells and its biological roles are not yet clear.
Honours projects for 2009 will be in these two areas of research.
5 Biochemistry and Molecular Biology
PROFESSOR CHARLIE BOND Room 4.16, MCS Building, Phone: 6488 4406 email: [email protected]
Structural Biology
Structural Biology research involves building a three-dimensional picture of biological molecules to shed light on the molecular interactions and events which drive many of the fundamental processes of life. Investigations in my lab address proteins of relevance to human health, including DNA repair enzymes and other nucleic acid processing proteins, and enzymes essential to the survival of life-threatening parasites, which may be drug targets.
Different aspects of this research can be tailored to students with strengths in Biochemistry, Chemistry, and Biophysics. Structural Biology research typically involves the opportunity to learn from a diverse set of useful techniques including molecular biology, protein purification and crystallisation, spectroscopy, X-ray crystallography, molecular modeling, bioinformatics, unix computing. The Structural Biology lab is equipped with state-of-the-art equipment including a crystallization robot and X-ray data collection facilities.
For further information, reprints of papers, a colour version of this page, or to find out about other research in the lab come and see me (MCS Lab 4.16) and look at http://xtal.uwa.edu.au/px/charlie .
PROJECTS NOTE: In addition to projects listed here, it may be possible to tailor a structural biology project to your specific interests.
1. HOW DO PROTEINS RECOGNISE RNA MOLECULES? ROP MUTANTS (collaboration with Dr Daniel Christ, Garvan Institute, Sydney)
Rop is a small alpha-helical protein which plays a critical role in bacteria where it regulates the number of copies of a DNA plasmid that the bacteria can accommodate. Rop does this by binding to a complex of two RNA hairpins – called the 'kissing' complex. By binding and stabilising this interaction, it stops the RNA being used to prime replication of the plasmids.
As it is a small protein and is known to crystallise, Rop makes an excellent target for research to understand the basis of protein:RNA interactions. We have a panel of mutant proteins, which were selected using in vitro evolution methods, that have higher affinity for the RNA than the wild-type protein. This project will involved expressing some of these mutant proteins, generating heterodimers of the mutants, measuring their affinity for RNA, attempting to crystallise them and solving their structures. Skills learned will include molecular biology (mutagenesis), protein expression and purification, protein:RNA interaction assays, crystallisation and protein crystallography.
A predicted model of the Rop:RNA complex.(from Christ, D and Winter, G, Proc Natl Acad Sci U S A. 2003 November 11; 100(23): 13202–13206)
6 Biochemistry and Molecular Biology
2. PROTEIN STRUCTURE PREDICTION: PPR PROTEINS (collaboration with Ian Small, CoE for Plant Energy Biology)
PPR proteins are modular proteins composed of tandem repeats of 35 amino acid sequences. A number of these proteins are known to bind and/or process RNA by recognising the RNA sequence. We want to understand how this sequence-specific recognition occurs. In similar protein families (TPR and ankyrin proteins) for which the structures are known, these repeats form alpha-helical hairpins which assemble to make a long ‘solenoid’. We have evidence from bioinformatics studies that PPR proteins have a similar, but different structure. Based on a specific type of bioinformatic (sequence covariation) data, we can predict both the secondary and tertiary structure of TPR and ankyrin proteins. We are interested in exploring which other protein families can be investigated with these Sequence covariation data can be used to methods, but our main aim is to produce plausible models of PPR protein structure which can be used to guide wet- predict the structure of helical repeat lab experiments into the function of these proteins. proteins, with a relatively high accuracy. This is a computation-based project which will involve learning about protein structure, molecular dynamics and bioinformatic analysis of proteins.
3 CHAPERONES AND CO-CHAPERONES OF THE MALARIA PATHOGEN, PLASMODIUM FALCIPARUM (WITH DR WILL STANLEY)
Schematic of the interactions in the multichaperone complex. Malaria is a widespread tropical disease killing about 2 million people annually, young children and pregnant women being especially vulnerable. It is a disease associated with poverty and classed as a neglected disease – no vaccine is available and prophylactic drugs are often too costly for those most at risk. The microbial pathogen, Plasmodium falciparum, is the major cause of life-threatening malaria amongst humans. This project explores a complex of P. falciparum chaperones – proteins essential for folding, stabilising and sorting of other proteins – which is critical to survival and proliferation of the pathogen, and thus a target for new kinds of antimalarial drugs. The complex consists of two housekeeping heat shock proteins, Hsp70 and Hsp90, and a Hsp organiser protein, HOP, which are involved in a complex set of intermolecular interactions to facilitate folding/sorting of a number of client proteins. Components of the complex can be recombinantly expressed and purified with the aim of detailed biochemical and biophysical studies of the assembly, structure and function of this multi-chaperone complex.
For other collaborative projects, please see entries for Dr Swaminatha Iyer and Prof Ian Small
7 Biochemistry and Molecular Biology PROFESSOR PETER HARTMANN Room 2.03, MCS Building, Phone 6488 3327 email: [email protected]
The long-term objective of the Human Lactation Research Group at The University of Western Australia is to facilitate successful breastfeeding, as defined by the WHO, by providing an evidence base for the clinical management of human lactation. Currently in Western Australia ~ 95% of mothers express a desire to breastfeed and ~ 93% leave hospital fully breastfeeding their babies. However, by 6 months postpartum only ~ 60% of these mothers are still breastfeeding. Thus, almost all mothers know that breastfeeding is best for their babies but many of these mothers do not achieve a successful lactation. Therefore, to achieve our objective a fundamental understanding of the physiology and biochemistry of milk synthesis, milk secretion, milk ejection, the mechanics of breastfeeding and infant appetite are required so that appropriate clinical assistance can be given to mothers who are not achieving the WHO recommendation to exclusively breastfeed babies for the first 6 months of life. These studies are particularly relevant to mothers who have delivered prematurely because the outcomes for premature babies who receive breastmilk are very much better than those who receive only infant formula.
Prof Hartmann’s research covers a broad spectrum of topics in the area of human lactation and infant nutrition. Honours candidates may participate in a variety of projects that fall within the following categories.
PROJECTS
1. Preserving the health benefits of donor breastmilk for pre-term babies Feeding human milk to infants provides nutritional, gastrointestinal, immunological and developmental benefits that impact on their long-term health and development. Human milk contains over 45 enzymes, growth factors and bioactive substances that enhance development and gut maturation and protect the infant from infection. In addition, the amino acid composition of human milk is ideally suited to the newborn and lipids are present in forms that are easily digested and absorbed. Many of these beneficial substances are not in artificial formula. These considerations are especially critical for pre-term infants who have been deprived of the full complement of developmental factors in utero. For example, pre-term infants have a six times greater risk of acquiring serious gastrointestinal infections such as necrotizing enterocolitis (NEC) if they are fed artificial formula. There are many challenges faced by mothers attempting to provide breastmilk for their pre-term infants and under these circumstances, mothers are often unable to produce enough milk to meet the full nutritional requirements of their baby. Currently in Australia the only option for these infants is artificial formula feeding. To overcome this problem, a donor milk bank has been established at King Edward Memorial Hospital. However to ensure that the donor milk does not introduce pathogens it first must be pasteurized before it is given to the pre-term babies. This project will focus on maintaining the quality of donor breastmilk in order to derive maximum benefits for pre-term babies. The research will involve determining the effect of the heat treatment involved in pasteurization on biologically important components of breastmilk, such as, bile salt stimulated lipase, sIgA, alpha-lactalbumin and lactoferrin. In addition, projects will also be available to investigate the retention of the nutritional and protective properties of breast milk.
2. The Stanford University Study A study is in progress, in collaboration with Stanford University, which has been designed to investigate the factors influencing the initiation and maintenance of lactation in women who deliver preterm. The method of breast expression and the changes in milk composition from day 1 to 60 postpartum will be assessed. These studies will provide a very comprehensive profile of milk composition of pre-term mothers and increase our understanding of the variation in milk composition both between pre-term mothers and the stage of lactation. These studies provide the basic rationale for the importance of measuring the composition of mother’s own milk prior to the fortification of breastmilk to achieve dietary intakes that are optimized for individual pre-term babies and hence increase their chance of survival.
8 Biochemistry and Molecular Biology
3. Milk intake and milk composition on gastro-intestinal tract physiology in breastfed infants Most babies in developed countries are breastfed to appetite (demand breastfeeding) and have relatively long breastfeeds and long intervals between breastfeeds, whereas babies in traditional societies have short but frequent breastfeeds (breastfeeds of a few minutes’ duration and more than one breastfeed per hour). Preliminary ultrasound studies have shown that it is possible to obtain a measurement of the volume of the baby’s stomach, determine whether the pyloric sphincter is either closed or open and determine if the gall bladder is empty or full. This study proposes to determine the relationship between the volume and composition of milk consumed by the baby at a breastfeed are related to the status of the pyloric sphincter and gall bladder. It is possible that age-related larger intakes of breastmilk may alter the dynamics of the flow of digesta through the gastro-intestinal tract and influence baby behaviour. In addition preliminary observations suggest that the development of the casein curd in the infant’s stomach can be observed using high definition ultrasound. Therefore the study also will examine the variation of clotting times both within and between babies. It is possible that differences in the clotting times and gastric emptying for different babies may be associated with either the nature of the mother’s caseins, the gastric digestive capacity of the baby or the fat content of the milk. These differences may be important in relation to optimizing breastfeeding frequencies for individual babies.
All of the above projects will provide information so that evidence based procedures can be developed that will directly impact on the care and health outcomes of both pre-term and term infants.
9 Biochemistry and Molecular Biology DR MARTHA LUDWIG SENIOR LECTURER Room 3.04, MCS Building, Phone: 6488 3744 email: [email protected]
Plant Nutrient Transport
The molecular evolution of photosynthetic pathways Terrestrial plants are typically grouped according to the biochemical pathway they use to fix atmospheric CO2 into carbohydrates – the so-called C3 plants, which include crop species such as rice and wheat as well as nearly all trees and herbs; the C4 plants, which include crop plants like corn and sugarcane, and some of the world’s worst weeds; and the Crassulacean Acid Metabolism (CAM) plants, which include cactuses, orchids and pineapple. C4 and CAM plants evolved from C3 plants, and some groups of plants have left “evolutionary footprints” that give us insights into how this process has occurred at the molecular level. Other plants are able to “switch” between pathways, depending on the environmental conditions and/or their developmental stage. Still other plants produce photosynthetic roots when flooded, allowing them to maintain oxygen levels in tissues that would otherwise become anoxic and die, and use dissolved CO2 in the flood waters to produce carbohydrates as an energy source. We are using tools of cell and molecular biology such as differential cDNA library construction and screening, quantitative reverse transcription PCR (qRT-PCR) and in situ hybridisation to identify key proteins involved in the above processes and examine the expression patterns of their genes. These studies will give insights into the evolution of photosynthesis, the process on which all life depends, and the plasticity of plants in obtaining nutrients from their environment. This information will open avenues for manipulating these pathways in economically valuable plants and will increase our knowledge of how plants may respond and cope with predicted future climate scenarios.
PROJECTS
1. Examining gene expression patterns of key enzymes in evolutionarily closely related plants that use the C3, C4 and intermediate C3-C4 photosynthetic pathways.
2. Identifying genes showing differential expression during photosynthetic pathway switching. This project is in collaboration with Dr Ed Barrett-Lennard ([email protected]), Department of Agriculture and Food (DAFWA). Students interested in this project will be eligible to apply for the DAFWA’s undergraduate studentship program, with the possibility of a bursary.
3. Identifying genes showing differential expression during the development of photosynthetic aquatic roots. This project is in collaboration with Assoc Prof Tim Colmer ([email protected]), School of Plant Biology.
Nutrient transporters The transport and exchange of ions and other nutrients across membranes is of fundamental importance to the survival of organisms. Intracellular symbiotic relationships are excellent systems with which to study these processes as both partners are typically separated from one another by at least one membrane and each partner is absolutely dependent on the coordination of solute transfer across these structures. Nitrogen is an essential nutrient for plants; however, it is often the factor that limits plant growth in many soils. Some plants, such as legumes, form intracellular symbiotic relationships with bacteria called rhizobia, which convert atmospheric nitrogen into forms of nitrogen that can be used by the plant. In exchange, the plant furnishes the rhizobia with carbon-containing compounds, typically dicarboxylates. Although agriculturalists have exploited this symbiotic relationship for centuries, in many cases, the proteins (transporters) and the mechanisms responsible for solute exchange between the bacterial symbionts and their host plants have not been characterised or even identified! We are focused on the identification of legume transport systems because of the great potential their manipulation offers not only to agriculture but also to animal and human biology as many of the transporters
10 Biochemistry and Molecular Biology characterised to date have homologues in humans. Understanding transport mechanisms will also contribute to the development of novel phytoremediation and phytofortification programs.
Projects include:
1. Examining gene expression patterns of several candidate legume dicarboxylate transporters in different tissues and during the development of the symbiosis with rhizobia.
2. Characterising candidate dicarboxylate transporters through complementation of plant dicarboxylate mutants.
Pollen Cell and Molecular Biology – Effects of Global Climate Change
Pollen-related allergy is a significant public health problem, which has increased in recent decades. Accumulating data indicate that this increase may be a consequence of climate change. Grasses are major contributors to airborne pollen levels and pollinosis worldwide; however, scant information exists on the effects of climate change on grass pollen biology, especially with regard to allergen content and dispersal. Ryegrass, a clinically important allergenic species, is being used as a model grass species to examine the impact of increased CO2 and temperature on the number, structure and allergen content of grass pollen. Outcomes of this work will include a greater understanding of the prevalence of grass pollen allergens and the mechanism of their dispersal under future climate change scenarios, which will contribute to the development of rational strategies for pollen-related allergy prevention, management, and therapies. We are producing recombinant ryegrass pollen allergens to use in cell culture assays and quantitative assays that will ultimately compare the levels of allergens in pollen from plants grown under climate scenarios differing in CO2 and/or temperature.
Projects include:
1. Cloning of recombinant ryegrass allergens and their expression in Escherichia coli, and the development of quantitative and functional cell culture assays. This project is in collaboration with Prof Geoff Stewart, School of Biomedical, Biomolecular and Chemical Sciences.
2. Examination of ryegrass pollen characteristics under increased temperature. Do plants produce more pollen or is the pollen structurally and/or biochemically modified under increased growth temperature relative to control plants?
11 Biochemistry and Molecular Biology
DR THOMAS MARTIN SENIOR LECTURER Room 3.47, MCS Building, Phone: 6488 3331 email: [email protected]
The Signalling Group Our group is interested in eukaryotic cellular signalling. As a model, we are investigating a gene family known as 14-3-3 genes. These proteins are important global regulators of many processes in all eukaryotes including the regulation of mammalian cell cycle and apoptosis as well as hormone responses and metabolism in plants.
In humans, epigenetic down-regulation of 14-3-3 gene expression was found in a great number of cancers. This indicates the importance of 14-3-3 proteins for correct cell function (for a review see Hermeking, 2003, Nature Reviews 3, 931-943). To learn more about the function of 14-3-3s, especially the role 14-3-3s play in correct cell cycle regulation, apoptosis and cancer development, we are collaborating with other researchers within Biochemistry and in Human Biology and projects are available to study the roles of 14-3-3 proteins in mammalian systems. We are also interested in the potential of 14-3-3s as targets for cancer therapy.
In plants, 14-3-3 proteins regulate metabolism, responses to hormones and to the environment. This has a great impact on the productivity of plants. For example the ability to assimilate nitrate is regulated by light, carbon dioxide, metabolite status, stress and developmental status. Many of these regulatory processes involve 14-3-3 proteins (for a review see: Comparot S, Lingiah G and Martin T, 2003, Journal of Experimental Botany 54, 595- 604). Our long term aim is to understand how plants manage to adjust metabolism to these manifold regulatory signals. Understanding these regulations may lead to generating improved crops or plants better adapted to extreme environmental conditions and poor soils.
My lab employs a combination of molecular and protein interaction approaches to investigate the roles of 14-3- 3s. Projects for students interested to study the signalling role of 14-3-3s in mammalian and plant systems are outlined below.
Please note, new projects may be available at the time you are choosing your Honours theme as the projects below are submitted a long time before you make your choice. So come and talk to me if you are interested in signalling and I will be happy to explain our work and the progress we are making in more detail.
PROJECTS
PLANT PROJECTS 1. Analysis of Protein Interactions in Living Plant Cells Protein interactions are involved in many regulatory processes. For example, 14-3-3 proteins bind to phosphorylated client proteins. By doing so, 14-3-3s determine the fate of such proteins which can be activated or inactivated, moved to a different cellular localisation or brought together with other proteins. 14-3-3 proteins act as dimmers. This project aims to investigate the formation of 14-3-3 dimers, the cellular localisation of dimer formation and the ways such interaction can be changed.
This figure shows the principle of the BiFC system. Two non-fluorescent parts of the YFP protein are fused to two proteins of interest (A and B). If these proteins do not interact (left) we will not observe fluorescence.
12 Biochemistry and Molecular Biology Interaction of A with B (right) brings the two YFP parts close enough together to fluoresce (from Bhat R.A. et al., Plant Methods 2006, 2:12). You will employ a system which will allow you to study protein interactions in living plant cells. The system you will employ is called Bimolecular Fluorescence complementation (BiFC, see figure above and Walter et al, 2004, Plant Journal 40, 428-38). It uses the yellow fluorescent protein (YFP) which can be split into two non- fluorescent parts. These two parts are fused on the gene level to two potentially interacting candidate proteins. Interaction of the candidate proteins will bring the YFP halves close enough together to fluoresce again. Thus fluorescence will indicate protein interaction the cellular localisation of such interaction and allows testing for conditions impacting on the protein interaction. The project evolved out of an ongoing PhD project. All technologies required are established and in use in my lab including transgenic plants to study interaction of 14- 3-3s. You will initially use these transgenic plants and study the interaction of 14-3-3s using fluorescent microscopy. You will further clone cDNA sequences into BiFC vectors of predicted 14-3-3 target proteins and test those in transient expression systems for interaction with 14-3-3s.
2. Analysis of 14-3-3 Gene Promoters 14-3-3 proteins are encoded by gene families. In Arabidopsis this family comprises of 15 genes. The encoded proteins act as dimers allowing for a great number of combinations. Understanding the regulation of 14-3-3 gene expression will allow for classification of these genes into groups with similar expression profiles. These profiles can be used to predict which of the 14-3-3 proteins act together in regulating cellular events. You will have access to a set of 14-3-3 promoter: reporter gene constructs and to promoter deletion constructs established in a previous Honours project and investigate their expression in transient expression systems. You will use molecular biology techniques to introduce mutations into 14-3-3 promoters and investigate the impact of those on gene expression to identify promoter elements shared between or unique to individual 14-3-3 genes. This analysis will be complemented by interrogation of promoter and microarray databases.
MAMMALIAN PROJECTS
3. Analysis of Tumour Suppressor Protein Interactions Mammalian Cells. We aim to understand the role a specific 14-3-3 isoform, 14-3-3 sigma, in cancer formation. This isoform is downregulated in a great number of cancers. This is significant as interaction of 14-3-3 sigma with the tumour suppressor p53 contributes to the prevention of cell division if DNA is damaged. You will transfer the BiFC protein interaction system (see project 1 above) which we are currently employing in plants to a mammalian cell system. You will clone 14-3-3 sigma into BiFC vectors, transfect mammalian cells and study the formation of sigma homodimers. You will further clone a tumour suppressor gene and investigate the interaction of the encoded protein with 14-3-3 sigma throughout the cell cycle and after induced DNA damage.
4. Investigating the role of 14-3-3s in liver tumour formation (collaboration with Prof Yeoh, Biochemistry) You will be continuing a current Honours project investigating the expression of 14-3-3 genes and proteins in liver cancer cells. You will work with two p53 mutant liver cell lines, one tumorigenic and one non-tumorigenic line. The ability of these lines to form tumours appears to be linked to the loss of 14-3-3 sigma expression. To find out which role this 14-3-3 protein plays in the tumour process, you will re-introduce 14-3-3 sigma into the tumorigenic cell line and inhibit its expression in the non-tumorigenic line. You will then investigate the impact these manipulations have on cell cycle, growth and apoptotic behaviour of the two cell lines to find out if sigma expression or the lack thereof is sufficient to cause the differences between these two cell lines.
5. 14-3-3 expression in cancer cells in response to chemotherapy (collaboration with Prof Dharmarajan, Human Biology) You will investigate whether absence or presence of 14-3-3 gene expression in cancer cells has an impact of these cells to resist chemotherapeutic treatments. A number of cell lines with sensitivity or resistance to single or multiple chemotherapeutic reagents as well as to androgenic treatments is available for this project. You will analyse the expression of 14-3-3s in such lines under control growth conditions and compare this to 14-3-3 expression in cells which received chemo or androgenic treatments. To achieve this you will use RT-PCR to compare RNA expression and cell based immunofluorescence assays and western blots to investigate 14-3-3 protein expression.
13 Biochemistry and Molecular Biology PROFESSOR HARVEY MILLAR PROFESSORIAL FELLOW ARC Centre of Excellence in Plant Energy Biology (http://www.plantenergy.uwa.edu.au/aboutus/scholarships_uwa.html) Room 4.74, Phone: 6488 7245 email: [email protected]
Using a combination of protein separation techniques, mass spectrometry and informatics my research group is seeking to understand the compartmentation of cellular functions in cellular organelles. The major organelles in green plant cells are the chloroplast (for photosynthesis), the mitochondrion (for respiration), and the peroxisome (for carbon and nitrogen metabolism). These three organelles divide and organize their energy conversion operations in a cooperative fashion. This cooperation is pivotal to directing energy capture and storage in the form of sugars, starch, oils, protein and fibre. The metabolism of plant organelles also underlies the growth and performance of plants including their ability to withstand environmental stresses. Further, the synthesis of key antioxidants, vitamins and cofactors is central to their development of products that are vital for human nutrition (e.g. vitamins A, C, and E, biotin, folic and lipoic acid, carotenoids). We are also studying honeybee biology and reproductive success through proteomics in order to understand the critical role of these social insects in plant pollination.
PROJECTS
1. Modification of Mitochondrial Proteins and Function by Oxidative Stress Aerobic organisms exploit the redox chemistry of oxygen to efficiently derive energy from oxidation of substrates. However, due to the tendency of molecular oxygen to gain single electrons and form reactive oxygen species (ROS) this energy production comes at a price. ROS, and the hydroxyl radical in particular, are highly reactive and can cause rapid and deleterious oxidation of biomolecules such as proteins, lipid and DNA. The mitochondrial electron transport chain produces significant quantities of ROS. However, despite this knowledge that plant mitochondria can produce ROS and contain mechanisms that may limit ROS accumulation, we are only beginning to understand the antioxidant system of plant mitochondria and the consequences for metabolism in this organelle if oxidative stress occurs. We have been studying specific proteins that are damaged by ROS, lipid peroxidation products and antioxidant defence enzymes (Chew et al 2003 ,Taylor et al 2005). This project will follow up on our past work using mitochondrial function assays, proteomics and transcript analysis to further unravel the damage done to mitochondria by oxidative stress and the response of mitochondria to protect themselves and repair damage. Publications 1. Chew O, Whelan J, Millar AH (2003) Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual-targeting of antioxidant defences in plants. The Journal of Biological Chemistry 278: 46869-46877. 2. Taylor NL, Heazlewood JL, Day DA, Millar AH (2005) Differential Impact of Environmental Stresses on the Pea Mitochondrial Proteome. Molecular & Cellular Proteomics 4:1122-1133. 3. Winger, AL, Taylor NL, Heazlewood JL, Day DA, Millar AH (2007) The cytotoxic lipid peroxidation product 4-hydroxy-2-nonenal covalently modifies a selective range of proteins linked to respiratory function in plant mitochondria. The Journal of Biological Chemistry 282:37436-47.
2. Arabidopsis Mitochondrial Proteomics, Databases and Bioinformatics The mitochondrion is the organelle within the eukaryotic cell that is primarily concerned with the synthesis of ATP in the fundamental process known as respiration. It is predicted that mitochondria synthesise 3-5% of the proteins required for their function, with the remaining 95-98% of proteins required encoded by the nuclear genome and targeted back to the mitochondria as protein precursors using encrypted targeting information in the protein sequence. The detection of these encryptions and thus identification of the full set of these genes within the nuclear genome is a major challenge for biologists. We have established a database of these genes and proteins and incorporated bioinformatic targeting prediction tools and experimental data (see www.suba.bcs.uwa.edu.au ). This honours project will utilise the bioinformatic database of predicted mitochondrial proteins and experimental proteomics to further uncover the mitochondrial proteome. This will involve specifically targeting a particular group of proteins, such as RNA binding proteins, phosphoproteins, specific proteins in the intermembrane space, metal binding proteins or NADH binding proteins. Alternatively, strongly bioinformatic projects utilizing current data and exploring novel biological insights can be designed, for
14 Biochemistry and Molecular Biology this some interest and background in computer science, web interface design and script writing will likely be required.
Publications 1. Heazlewood JL, Verboom RE, Tonti-Filippini J, Small I, Millar AH. (2007) SUBA: the Arabidopsis Subcellular Database. Nucleic Acids Res. 35:D213-8. 2. Ito J, Heazlewood JL, Millar AH. (2006) Analysis of the soluble ATP-binding proteome of plant mitochondria identifies new proteins and nucleotide triphosphate interactions within the matrix. J Proteome Res. 2006 5:3459-69.
3. Rice Proteomics and Oxygen as a Trigger for Energy Metabolism The structure and functional status of mitochondria in the absence of O2 has intrigued researchers for decades. Mitochondrial structures appear to proliferate in rice seedlings even when they are grown under anoxic conditions from dry seed. We are using rice as a model system for studying the mitochondrial proteome in rice, the oxygen trigger in mitochondrial biogenesis, the establishment of aerobic metabolism, and the energy generating processes in rice during long-term anaerobic metabolism. An honours project in this area would involve proteomic analysis of rice tissues or isolated mitochondria under anoxia or during return to air and attempts to unravel specific aspects of the events and signalling process that regulate this process in rice cells.
Publications: 1. Millar AH, Trend AE, Heazlewood JL (2004) Changes in the rice mitochondrial proteome during the anoxia to air transition focus around cytochrome containing respiratory complexes. The Journal of Biological Chemistry 279:39471-39478 2. Howell KA, Cheng K, Murcha MW, Jenkin LE, Millar AH, Whelan J (2007) Oxygen initiation of respiration and mitochondrial biogenesis in rice. Journal of Biological Chemistry 282:15619-31 3. Huang S, Colmer TD, Millar AH (2008) Does anoxia-tolerance involve altering energy currency towards PPi? Trends in Plant Science 13:221-227.
4. Subcellular Proteomics in Arabidopsis Central metabolism, biosynthesis of high quality and high quantity products and cellular signalling pathways in defence from the physical environment and invading pathogens, are all essentially compartmented processes in plant cells, but only a small number of the proteins in plants have known location. The overall aim of my research in this area is to identify the intracellular location of proteins in the model plant Arabidopsis using proteomics in eight subcellular compartments isolated by cellular fractionation. This will allow a view of subcellular proteomes for plastids, peroxisomes, nuclei, plasma membrane, endoplasmic reticulum, golgi, tonoplast and cytosol. An honours project in this area would aim to biochemically purify one of these compartments based on modification of existing methods and to begin the mass spectrometry analysis of its constituent proteins using shot-gun proteomics (using LC-MS/MS analysis).
Publications: 1. Heazlewood JL, Tonti-Filippini J, Verboom RE, Millar AH (2005) Combining experimental and predicted datasets for determination of the subcellular location of proteins in Arabidopsis. Plant Physiology 139:598-
5. Honeybee Proteomics With Dr Boris Baer, ARC COE in Plant Energy Biology & School of Animal Biology (FNAS) Apart from being used for honey production, honeybees are the worldwide most important species for crop pollination. However, we currently face a dramatic and global decline in honeybee populations with severe expected consequences for agricultural yields. To counter the dramatic losses of honeybee colonies, detailed studies about honeybee reproduction will ultimately allow optimizing breeding and allowing to compensate for the current losses. Honeybee reproduction is quite spectacular, as queens only mate at the beginning of their lives, during one or very few mating flights. Afterwards they are able to store millions of sperm for years and use them in very economic ways to fertilise millions of eggs. We have very little information how queens are able to keep sperm alive of years, how active sperm remains during storage and how sperm potentially interacts with the female. This honours project will use proteomic tools to isolate and identify proteins that are relevant during sperm storage and reveal their possible effects on sperm survival and paternity success. The project is supported by Australian beekeepers and will use gel electrophoresis, mass spectrometry and the recently published bee genome sequence. Field work, using campus based honeybee colonies and artificial insemination techniques offer possibilities to test for effects of proteins in vivo.
15 Biochemistry and Molecular Biology PROFESSOR IAN SMALL ARC Centre of Excellence in Plant Energy Biology Room 4.03, MCS building, Phone: 6488 4499 email: [email protected]
Organelle Gene Expression Group
Our group is studying the regulation of gene expression inside mitochondria and chloroplasts and how this is coordinated with nuclear gene expression. Our focus is on genes that are involved in photosynthesis and respiration and include those that code for some of the most important and abundant proteins on the planet. These genes are mostly regulated post-transcriptionally but the control loops involved need to be linked to transcriptional control of nuclear genes via signalling pathways that are still to be discovered. Our aim is to understand how energy metabolism in plants is regulated, with the goal of generating discoveries relevant to optimal use of plants in agricultural and environmental applications such as biofuel production. Much of the research will build upon the discovery of the PPR protein family, a novel family of 450 sequence-specific RNA- binding proteins implicated in these processes. The experiments will be carried out on the model plant Arabidopsis thaliana to make full use of the existing data and resources. Prospective Honours students with a background in Molecular Biology, Biochemistry, Genetics or Computer Science are particularly encouraged to apply. The projects will benefit from the full-range of expertise and equipment on plant energy biology within the ARC Centre of Excellence and will be at the forefront of research in this field.
PROJECTS
1. Linking chloroplast and nuclear gene expression With Andeol Falcon de Longevialle and Dr. Etienne Delannoy
Several PPR proteins have been found to be needed for the correct activity of the chloroplast-encoded RNA polymerase (RPO) that transcribes almost all of the photosynthesis genes. These include pTAC2 and GUN1 (which contain DNA-binding domains in addition to their RNA-binding PPR motifs) as well as CLB19 and FLV, which are required for editing of the RPO transcripts. The phenotypes of mutants in these genes strongly suggest that some or all of them are involved in a retrograde signalling pathway from the chloroplast to the nucleus that controls the activation of nuclear photosynthesis genes. This project will try to elucidate of the role of these PPR proteins and the chloroplast RNA polymerase during chloroplast biogenesis in early seedling development. It will involve the detailed characterization of chloroplast and nuclear gene expression in wild-type and mutant plants using Affymetrix microarrays and quantitative RT- PCR. The assembly and activity of the RNA polymerase will be followed by immunodetection and transcription assays. The DNA and RNA-binding activities of the PPR proteins will be analysed using RIP- Chip or EMSA assays. Protein synthesis and accumulation inside chloroplasts will be followed by western blot with a suite of antibodies against the most important photosynthesis proteins.
1. Pfalz et al. PTAC2,-6, and-12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression (2006) Plant Cell 18(1): 176-197. 2. Koussevitzky et al. Signals from chloroplasts converge to regulate nuclear gene expression (2007) Science 316(5825): 715-719
16 Biochemistry and Molecular Biology 2. Discovering the mechanism and function of RNA editing in plant organelles With Dr Anne-Laure Chateigner-Boutin and Dr. Anne Bersoult
RNA editing is a site-specific modification of RNA molecules, occurring by nucleotide insertion/deletion, nucleotide substitution or nucleotide modification. Different types of editing have been described, generally involving a specificity factor (RNA or protein) that recognizes the editing site and an enzyme catalyzing the reaction. RNA editing alters the sequence of many different types of RNAs in many organisms including plants and animals and thus constitutes a form of epigenetic gene regulation. In many cases, RNA editing is essential for correct production of the protein encoded by the RNA, whilst in other cases, RNA editing changes the functional properties of the encoded protein. In higher plants, RNA editing consists of C to U changes and has been reported only in organelle transcripts, where over 500 different editing sites are now known. Four PPR proteins were found by our group and others to be essential for the editing of specific sites in chloroplast transcripts of Arabidopsis thaliana, raising the possibility that this large family of RNA-binding proteins could constitute the specificity factors recognizing the sequence around the target C. We have also identified a putative catalytic domain in some PPR proteins that phylogenetically correlates with RNA editing. The initial aim of this project is to identify further PPR proteins that are involved in RNA editing by screening knock-out mutants. The mutants will be characterized by analyzing the status of all editing sites using a novel high-throughput method we have developed that is based on real-time PCR and high-resolution melting analysis. Once a mutant is found to be impaired in editing, the consequence of the defect for the plant will be investigated using a full set of molecular techniques (global survey of the transcriptome and proteome, targeted analysis by qRT-PCR, western-blot, activity test…). A second aim of this project is to try and identify the proteins or protein domains that carry out the editing activity. Our group has several lines of evidence that indicate a particular motif associated with many PPR proteins as a strong candidate. By complementing editing mutants with proteins modified in this domain we can, for the first time, carry out experiments to test these candidates.
1. Kotera et al. (2005) A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature. 433, 326-30. 2. Okuda et al. (2007) Conserved domain structure of pentatricopeptide repeat proteins involved in chloroplast RNA editing. Proc Natl Acad Sci U S A 104, 8178-8183.
3. In vitro structural and functional analysis of Arabidopsis PPR proteins With Dr. Will Stanley and in collaboration with Prof. Charlie Bond, Chemistry Department
Little is known about the molecular mechanisms behind the functions of PPR proteins in processing of organelle encoded RNA transcripts. Some PPR proteins may specifically recognise RNA molecules and carry out the processing by themselves (route 1 in the figure) while others may recruit additional protein factors to catalyse processing (route 2 in the figure). Questions arise as to exactly how a given PPR interacts with a given RNA molecule – is there an RNA sequence-specific interaction, or does the RNA adopt a secondary structural arrangement amenable to PPR binding? Do some PPR proteins interact directly with both RNA and other protein factors? Could there be cooperativity in assembly of this type of multi-molecular complex?
Work is underway to study the atomic resolution structure and chemistry of RNA recognition for a selection of PPR proteins, using X-ray crystallography. Additional biophysical techniques, such as circular dichroism (CD) and fluorescence spectroscopy and isothermal titration calorimetry (ITC) are in use to dissect structural and thermodynamic parameters of multi-molecular complex assembly. Recombinant protein production and purification are prerequisites for biophysical analysis and specialised crystallisation techniques are required to make PPR crystals. Thus, an honours project covering basic molecular biology, protein biochemistry and high-fidelity macromolecular characterisation is available. The project would suit either a chemistry or biochemistry student.
17 Biochemistry and Molecular Biology PROFESSOR STEVE SMITH ARC FEDERATION FELLOW ARC Centre of Excellence in Plant Energy Biology and WA Centre of Excellence for Plant Metabolomics Room 4.05, MCS Building, Phone: 6488 4403 email: [email protected]
Using genomics approaches to discover genetic and metabolic function in Arabidopsis
The genome sequence of Arabidopsis thaliana is more accurate and better annotated than that of any other multi- cellular eukaryote. Similarly, the functional genomics resources available for analysis are better. Thus Arabidopsis provides the most powerful platform for modern genomics-based research in eukaryotes. Research using Arabidopsis can provide training in a range of disciplines including genomics, genetics, cell biology, biochemistry, and importantly, newly-emerging multi-disciplinary areas such as bioinformatics, systems biology and metabolomics. I aim to provide training in a specific area of Arabidopsis research, and an awareness of the future directions of research in biology. The following projects are proposed but there is plenty of room for flexibility and originality.
PROJECTS
1. Functional genomics approaches to plant energy metabolism
We are studying metabolism and sub-cellular distribution of enzymes of fatty acid beta-oxidation, the glyoxylate cycle and photorespiration. Surprisingly, our knowledge of these pathways is incomplete yet such metabolism underpins plant growth, responses to environmental stress and synthesis of valuable plant products. Genomic analysis in Arabidopsis has identified enzymes that could potentially function in these pathways. The roles of such enzymes can be addressed using reverse genetics to isolate a mutant lacking a specific enzyme. The metabolism and physiology of the mutant can then be studied. The project will involve identification of a putative enzyme of unknown physiological function, isolation of a mutant, and analysis of the mutant to understand enzyme function. The research may involve cell biology methods to study sub-cellular protein targeting, gene expression studies and metabolomics.
Metabolomics uses separation methods such as gas chromatography (GC) coupled to mass spectrometry (MS) to simultaneously identify a wide range of metabolites in a single experiment. This provides valuable information about the impact of a specific mutation on metabolism. It can be related to changes in gene expression to understand how the genome responds to perturbations in metabolism.
AT 780 Fo TAA
780 R T-DNA
References
1. Cornah JE et al. (2004) Lipid utilization, gluconeogenesis, and seedling growth in Arabidopsis mutants lacking the glyoxylate cycle enzyme malate synthase. J Biol Chem. 279:42916-23. 2. Pracharoenwattana et al (2005) Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination. Plant Cell 17: 2037-2048. 3. Bino RJ, et al. (2004) Potential of metabolomics as a functional genomics tool. Trends Plant Sci. 9:418-25. 4. Hirai MY, et al. (2004) Integration of transcriptomics and metabolomics for understanding of global responses to nutritional stresses in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 101:10205-10.
18 Biochemistry and Molecular Biology 2. Molecular mechanisms by which karrikins promote seed germination
To ensure their seeds germinate under the best conditions for survival of the emerging seedling, plants have adopted intricate mechanisms of germination control. For many species, their seeds can lie dormant in the soil for years until the appropriate set of stimuli trigger their growth. In the bush, a fire may wipe out all existing ground vegetation, but this is often quickly followed by a flush of seedlings eager to capitalize on the newly available space and resources. It has been known for many years now that the smoke from a fire is an important cue for breaking seed dormancy in many Australian native species.
Recently, a collaborative effort between Kings Park and the Chemistry department at UWA led to the discovery of the primary active compound in smoke, a butenolide (3-methyl-2H-furo[2,3-c]pyran-2- one). Several active analogues have since been discovered. We have adopted the family name karrikin (from ‘karrik’, the first recorded word for ‘smoke’ in Aboriginal Nyungar), to reflect the original source of these compounds, and karrikinolide (KAR1) to denote the parent butenolide molecule. It has been established that KAR1 can also promote germination and seedling vigor in species that do not normally encounter smoke, raising the possibility that karrikins represent a new class of plant growth-promoting substances of wide significance. The karrikins are expected to have major applications in weed management, land reclamation and plant conservation.
The goal of our research is to discover the molecular mode of action of karrikins in promoting seed germination and seedling vigour. We are using the model plant Arabidopsis thaliana to study the karrikin signaling pathway. The mechanisms of dormancy and seed germination have been best defined in Arabidopsis, although much remains unknown. The effects of karrikin on existing germination mutants, such as those affected in plant hormone homeostasis and signaling, will be investigated to uncover crosstalk with known genetic pathways. Interference or enhancement of exogenous hormone effects on plant development by karrikin will also provide useful clues. Global transcription profiling with microarray technology will be used to identify genes that respond to KAR1. This will provide insights about karrikin action as well as a set of genes that can be used to make reporter constructs. By fusing karrikin-responsive promoters to a visible marker such as firefly luciferase, we can generate transgenic plants that exhibit easily quantifiable responses to KAR1 treatment. Genetic approaches will be adopted to isolate karrikin response mutants, so that the genes required for karrikin action can be identified. This project offers scope for a range of different approaches including chemistry, metabolomics, molecular biology, genetics, and seed physiology.
References
Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD. (2004) A compound from smoke that promotes seed germination. Science. 305:977.
Bentsink L and Koornneef M (2002) Seed dormancy and germination. The Arabidopsis book. Eds Somerville CR, Meyerowitz EM. American Society of Plant Biologists, Rockville, MD.
Koornneef M et al (2002) Seed dormancy and germination. Current Opinion in Plant Biology 5:33-36.
19 Biochemistry and Molecular Biology DR NAOMI TRENGOVE LECTURER Room 2.04, MCS Building, Phone: 6488 4421 email: [email protected]
Wound Healing Research Group
The investigation of the causes of impaired wound healing in chronic wounds in humans represents a challenging area of medical research. Fortunately, with the emergence of new techniques in both cellular and molecular biology it is now possible to use the small tissue samples obtained from humans to understand the process more accurately. Venous leg ulceration is a debilitating chronic wound that occurs most often in the elderly and is the result of venous hypertension in the lower limb (venous disease). The pathogenesis of ulceration is not well understood, but there is evidence that elevated levels of inflammatory mediators (e.g. tumor necrosis factor-alpha) are involved (1,2). Susceptibility to ulceration in patients with venous disease varies. Professor Stacey’s team has identified a polymorphism in the tumor necrosis factor-alpha gene (TNFA-308A) that is associated with increased risk of ulceration (3). Further studies are required to determine whether carriage of this allele is part of the cause of venous leg ulceration or just a marker of another causal allele in close proximity on the chromosome.
PROJECT
With Professor Mike Stacey and Dr Hillary Wallace, School of Surgery and Pathology
This project will be undertaken in collaboration with the wound healing research group of the School of Surgery and Pathology (Faculty of Medicine and Dentistry), which undertakes clinical and laboratory studies into chronic venous leg ulceration.
The proposed Honours project will investigate whether the tumor necrosis factor-alpha (TNFA) genotype in humans with chronic venous ulceration is associated with differences in the TNF phenotype. That is, do individuals with different TNFA genotypes produce different amounts of TNF-alpha? The levels of TNF-alpha protein and mRNA will be assessed in the wound and in stimulated peripheral blood leukocytes of patients with leg ulcers who have been genotyped in a previous study. The working hypothesis is that TNF-alpha protein and mRNA levels will be increased in patients carrying the TNF-308A allele compared to the wild-type TNF-308G allele. Patients homozygous for the A and G alleles will be compared to minimise individual variation. This project will provide opportunities to interact with patients in a clinical setting, as well as giving a sound grounding in laboratory research techniques applicable to medical research.
Laboratory techniques for this study will include: Cell culture, RNA extraction from cells and tissue, ELISAs and real-time PCR and Immunohistochemistry.
References (1)Trengove NJ, Bielefeldt-Ohmann H, Stacey MC. Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers. Wound Repair Regen. 2000 Jan-Feb;8(1):13-25.
(2)Wallace HJ, Stacey MC. Levels of tumor necrosis factor-alpha (TNF-alpha) and soluble TNF receptors in chronic venous leg ulcers--correlations to healing status. J Invest Dermatol. 1998 Mar;110(3):292-6.
(3)Wallace HJ, Vandongen YK, Stacey MC. Tumor necrosis factor-alpha gene polymorphism associated with increased susceptibility to venous leg ulceration. J Invest Dermatol. 2006 Apr;126(4):921
20 Biochemistry and Molecular Biology DR ROBERT TUCKEY SENIOR LECTURER Room 3.71, MCS Building, Phone: 64883040, email: [email protected]
Molecular Steroidogenesis Group
Current research involves structure-function studies on cytochrome P450scc, synthesis and metabolism of vitamins D2 and D3 by cytochrome P450scc, and the activation and inactivation of vitamin D by other mitochondrial-type cytochromes P450.
Cytochrome P450scc Structure-Function Studies The conversion of cholesterol to pregnenolone by P450scc (CYP11A1), termed cholesterol side-chain cleavage, is the rate-limiting step in steroid hormone synthesis and occurs in the inner mitochondrial membrane of steroidogenic tissues (see Fig. 1). The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in the adrenal cortex, corpus luteum and testis. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P450scc converts this cholesterol into pregnenolone. This reaction involves three hydroxylations, all of which occur at a single active site on cytochrome P450scc. Electrons for the hydroxylation reactions are provided by NADPH via a short electron transport chain comprising adrenodoxin reductase and adrenodoxin. Using molecular modelling, cysteine mutagenesis and fluorescent labelling we have determined that the F-G loop of cytochrome P450scc anchors the cytochrome to the inner mitochondrial membrane.
Figure 1. Progesterone synthesis by steroidogenic mitochondria. Electrons flow from NADPH to cytochrome P450scc via adrenodoxin reductase (AR) and adrenodoxin (Adx). Pregnenolone is converted to progesterone by 3β-hydroxysteroid dehydrogenase (3βHSD). OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane.
Metabolism of Vitamins D2 and D3 and their Precursors by Cytochrome P450scc In collaboration with Professor Andrzej Slominski at the University of Tennessee, Memphis, we tested the ability of P450scc to metabolize vitamins D2 and D3 plus their precursors, ergosterol and 7-dehydrocholesterol. These potential substrates, structurally similar to cholesterol, were incubated with purified P450scc and in some cases were also incubated with P450scc in rat adrenal mitochondria. Products were purified by TLC or HPLC and identified by mass spectrometry and/or NMR. We found that human and bovine P450scc cleaves the side chain of the vitamin D3 precursor, 7-dehydrocholesterol, to produce 7-dehydropregnenolone at rates comparable to that seen for cholesterol metabolism. P450scc did not cleave the side chain of vitamin D3 but hydroxylated the side chain producing 20-hydroxyvitamin D3 and 20,23-dihydroxyhydroxyvitamin D3. P450scc converted vitamin D2 to 20-hydroxyvitamin D2 and 17,20-dihydroxvitamin D2, again with no cleavage of the side chain occurring. The major product of ergosterol metabolism was, 17,24-dihydroxyergosterol Thus, P450scc can metabolize vitamin D and its precursors producing novel hydroxylated metabolites with side chain cleavage
21 Biochemistry and Molecular Biology occurring only for 7-dehydrocholesterol. The cleavage of the side chain of 7-dehydrocholesterol by P450scc to produce 7-dehydropregnenolone provides an explanation for the accumulation of 7-dehydrosteroids in Smith- Lemli-Opitz syndrome where there is an excess of 7-dehydrocholesterol due to a 7-dehydrocholesterol reductase deficiency. We plan to directly test this hypothesis by examining the subsequent metabolism of 7- dehydropregnenolone by other steroidogenic enzymes such as P45017 (see Project 2).
Biological testing of these new metabolites of vitamin D is currently underway in collaboration with Professor Slominski. 20-Hydroxyvitamin D3 has been found to be as effective as the hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3, in inhibiting skin cell proliferation and promoting differentiation. The goal of this work is to identify new vitamin D3 derivatives that have antiproliferative activity but only minimal effects on calcium metabolism, to permit these compounds to be used in the treatment of hyperproliferative disorders including cancer.
Cytochromes P450 Involved in the Activation and Inactivation of Vitamin D. New research in my laboratory involves the characterization of 25-hydroxyvitamin D 1-hydroxylase (CYP27B1) and vitamin D 24-hydroxylase (CYP24). CYP27B1 catalyses the final step in the activation of vitamin D, 1- hydroxylation of 25-hydroxyvitamin D to produce 1,25-dihydroxyvitamin D3, which occurs primarily in the kidney. 1,25-Dihydroxyvitamin D3 is the hormonally active form of vitamin D that regulates calcium metabolism, but also has many other important effects including inhibiting proliferation and promoting differentiation of a range of cells, plus regulating the immune system. CYP24 acts on 1,25-dihydroxyvitamin D3, hydroxylating it at C24 which caused its inactivation. These cytochrome P450 enzymes are being expressed in E. coli, purified and incorporated into phospholipid vesicles to characterize their substrate specificity and kinetic properties.
PROJECTS
1. Metabolism of Vitamin D by P450scc Recent studies show that bovine and human P450scc act on vitamin D3 to produce 20-hydroxyvitamin D3 and 20,23-dihydroxyhydroxyvitamin D3, acts on vitamin D2 to produce 20-hydroxyvitamin D2 and 17,20- dihydroxvitamin D2 and acts on the vitamin D2 precursor, ergosterol, to produce 17,24-dihydroxyergosterol. Preliminary studies have shown that these products are biologically active and inhibit skin cell proliferation. The aim of this project is to determine the rate of production of these compounds from their vitamin D precursors by purified human P450scc. The project will involve bacterial expression of the enzyme, reconstitution into artificial membranes and a HPLC assay of the products of the reaction.
2. Can 7-Dehydropregnenolone be Metabolized by other Steroidogenic Enzymes and if so what are the Products?
We have shown that P450scc can convert the vitamin D3 precursor, 7-dehydrocholesterol, to 7- dehydropregnenolone. The observation that various 7-dehydrosteroids are produced in the Smith-Lemli-Opitz syndrome where there is an excess of 7-dehydrocholesterol due to a 7-dehydrocholesterol reductase deficiency, suggests that other steroidogenic enzymes can act on 7-dehydropregneolone to produce a number of novel steroids. Enzymes to be tested will include P45017, P45011β and 3β-hydroxysteroid dehydrogenase. Initial studies will involve incubating bovine adrenal microsomes which contain P45017 and 3β-hydroxysteroid dehydrogenase, with 7-dehydropregnenolone, isolating the products and analysing them by HPLC. Subsequent studies will involve purifying the enzymes (from adrenals or expressed in bacteria) and examining their ability to act on 7-dehydropregnenolone in a reconstituted system. Products of enzymatic metabolism of 7- dehydrocholesterol will be identified by mass spectrometry and/or NMR.
Note: other projects related to my field of study are possible and new projects arise as research progresses. I am happy to discuss other projects with students expressing an interest.
22 Biochemistry and Molecular Biology DR DANIELA ULGIATI LECTURER Room 3.03, MCS Building, Phone: 6488 4423 email: [email protected]
My research interest is in the role of complement in health and disease. My ambition is to clarify the roles of complement and B cell biology in autoimmune disease, using Systemic Lupus Erythematosus (SLE) as a model for this and other autoimmune diseases. Specifically, my research focuses on the control of complement receptor in health and disease. Students with a background in Molecular Biology, Biochemistry, Genetics or Immunology are able to apply. Students will be exposed to a range of techniques including Genotyping, Chomatin Assays, ChIP assays, DNA sequencing and cloning, cell culture, stable and transient transfection assays, PCR, DNA binding assays, proteomic analysis, and FACS analysis.
PROJECTS
1. Isolation of Transcription Factors Involved in Regulating Human Complement Receptor 2 (CR2/CD21) during B Cell Development.
Complement receptor 2 (CR2) plays an important role in the generation of normal B cell immune responses as demonstrated by CR2 knockout mice. As modest changes in levels of CR2 expression appear to effect B cell responses, understanding the transcriptional control of CR2 is critical. More recently, a role for this receptor has been established in the differentiation of normal B cells. Premature expression of CR2 resulted in marked reduction in peripheral B cell numbers as well as mature B cells that are defective in their antibody responses. This project involves the study of this gene during the B cell development process. Our analysis of the transcriptional control of human CR2 show that this gene is complexly regulated by the presence of both promoter and intronic silencer elements. Within these elements we have identified two regions critical for transcriptional regulation. The first is a CBF1 binding site within the intronic silencer and the second is a cell type specific repressor within the CR2 proximal promoter which binds E2A proteins as well as CBF1. Together with these known transcription factors, many as yet unidentified proteins bind the functionally relevant sites. This project involves studying the role of the identified factors during B cell development in vivo using chromatin immunoprecipitation assays (ChIPs) and B cells lines that represent different stages of B cell development. Isolation of and undentification of the unidentified binding factors will be achieved using 2D gel/proteomics based approaches.
2. The role of CR2 promoter polymorphisms in Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA).
Complement receptor 2 (CR2) is an important receptor that is required for a normal B cell immune response. It is expressed at a critical stage in B cell development and has been implicated in a number of autoimmune diseases. The significance of mechanisms that regulate CR2 expression is apparent by studies of human B cell CR2 expression in patients with SLE and RA. Both patient groups have abnormalities in the expression of CR2 on B cells (~50% of normal) and this decrease correlates with disease activity. With the recent advent of transgenic and knockout mice, several groups have examined the importance of CR2 in a lupus prone mouse model. Studies of these mice have also found an early decrease in CR2 expression that is initially detected prior to any major clinical manifestations. We have recently sequenced the CR2 promoter in a number of SLE patients and have found several single nucleotide polymorphisms (SNPs) within functional regions of the promoter. We are currently assessing the functional implications of these polymorphisms on the transcriptional regulation of CR2. This project involves determining the expression status of CR2 on patient B cells by correlating cell surface expression with mRNA levels and transcriptional activity. Furthermore, collating the expression and transcriptional data with the promoter phenotypes will ultimately determine whether these promoter polymorphisms are indeed having an effect on CR2 expression in patients with autoimmune diseases.
23 Biochemistry and Molecular Biology 3. Characterisation of the Upstream Repressor Element in the Complement C4 Gene and its control by Lupus-associated Factors. (Co-supervised with Prof Lawrie Abraham)
The fourth component of human complement (C4) is a serum protein involved in initiation of immune and inflammatory reponses. Previously, we have analysed the transcriptional regulation of the C4 gene. To determine the requirements for basal and regulated expression, we FIGURE 1 have analysed the promoter region of C4 in reporter gene assays, using deletion and mutant reporter constructs and in A. Induced C4 Expression EMSA analysis. We have mapped a number of promoter elements that are responsible for basal and interferon- gamma regulated expression. We also discovered a novel Initiation two-part regulatory element within the promoter which Upstream complex Repressor BKLF appears critical for C4 expression in hepatic cells. The element - Activation Sp1 reporter gene analysis results indicated the presence of repressor elements between –468 and –310 (which contain putative binding sites for GATA and Nkx2) that had the effect of decreasing promoter activity by more than 90%. In addition, these distal element/s appeared to be acting in B. Repressed C4 Expression concert with a complex of Sp1/3 and BKLF-binding GT box elements around –140. This interaction has the effect of masking the very strong negative effects due to the distal region. The mechanism for this masking effect is currently Upstream Dissociated unknown, but our hypothesis is that interaction with the – Repressor complex element BKLF 140 region prevents interaction of the upstream element Sp1 - no activation with the proximal basal elements (see Figure). We hypothesised that there would be an extracellular signal that regulated C4 expression via this repressor element. In Extra-cellular searching for such an agent we found an activity in serum signal from Luus nephritis NZW X NZB F1 mice that was able to repress C4 transcription via the two-part element in the C4 promoter. This project will involve the further characterisation of the repressor elements and the transcription factors that interact with them, and a subsequent investigation of the mechanism of repression. Also, the identity of the Lupus-associated factor will be investigated following purification.
4. Understanding the Role of Notch Signalling and associated Transcription Factors in Lineage Commitment. (Co-supervised with Prof Lawrie Abraham)
Notch signaling is an evolutionarily ancient mechanism which plays a critical role in dictating cellular fates. Signals transmitted via Notch receptors control how cells respond to developmental cues and in turn control lineage commitment. Notch signalling is intimately involved in lineage specification and differentiation of lymphocytes.
Commitment to the B-lineage requires inhibition of Notch signals in lymphoid progenitors. Notch signals in this context repress Pax5 expression thereby blocking B-cell differentiation. On the other hand, negative regulation of Notch signals by the inhibitory Notch modulator deltex1, skews commitment of lymphoid progenitors to the B-lineage. While, Notch1 signaling must be down-regulated to permit B-cell commitment, the involvement of
Notch signaling at subsequent stages of B-cell development in bone marrow have not been clearly defined. Notch signaling also has important consequences for T lymphocytes. Dysregulated Notch1 signaling leads to T cell leukemia in humans and mice. The ability of Notch to cause T cell neoplasia results from aberent expression during thymocyte development, where Notch receptor expression and signaling occur at distinct developmental stages. There is evidence that Notch expression at very early stages of lymphoid development commit progenitors to the T cell lineage. Recent evidence indicates that Notch may also influence mature T cell development.
We have recently developed an ex vivo model in which to study Notch signaling. Cells are co-cultured with stromal cell lines ectopically expressing the Notch ligand, delta-like-1 (OP9-DL). Cells attached to the stroma or in suspension following co-culture were harvested and can be analysed for differentiation and neoplastic markers and associated transcription factors. Since Notch signaling is known to upregulate the bHLH factor HES-1, we can also measure transcript abundance of this marker of Notch activation to ensure proper induction of Notch by dela-like-1 ligand in the co-cultures.
24 Biochemistry and Molecular Biology PROFESSOR ALICE VRIELINK Room 4.31, MCS Building, Phone: 6488 3162 email: [email protected]
Protein Structure by X-ray Crystallography The studies in my lab focus on crystallographic analysis of a variety of proteins with the aim of using structural analysis to better understand their biology. The structural biology laboratory is well equipped with state of the art robotic crystallization equipment, X-ray diffraction equipment and computational facilities for structure solution and analysis. Expression and purification resources are available in the laboratory in order to obtain sufficient quantities of protein for crystallographic studies. In addition we carry out kinetic and spectroscopic analyses to establish the quality of protein and pursue biochemical and biophysical studies to better correlate function with structure.
PROJECTS
1. Endotoxin Biosynthesis in Neisseria. The gram negative bacteria, Neisseria meningitidis, is the causative agent of meningitis and is responsible for significant mortality throughout the world. A characteristic feature of these bacteria is the presence of lipooligosaccharide (LOS) molecules on their outer membranes. These complex molecules, also called endotoxins, are structural components that play a role in the pathogenicity of the organism. Twelve different immunotypes are found depending on the structure of the LOS moiety. One aspect of complexity of the LOS group that plays a role in defining the specific immunotype is the presence and location of phosphoethanolamine (PEA) residues. The enzyme responsible for adding the PEA residue to the LOS group is phosphoethanolamine transferase. Different forms of PEA transferase are present depending on the precise location of the PEA moiety on the LOS molecule. Knowledge of the biosynthesis and regulation of meningococcal lipoooligosaccharides will provide a more detailed understanding of the role of this molecule in pathogenesis and disease. In collaboration with Dr. Charlene Kahler of the Department of Microbiology at UWA we have begun a study to determine the three dimensional structure of the enzyme LPTA, the phosphothanolamine transferase specific for phosphorylation of the lipid A core of LOS. This project will involve protein expression, purification, crystallization and structure determination using crystallographic techniques. The structural results will be correlated with functional studies carried out by Dr. Kahler and coworkers.
2. Transcription Elongation Factors Spt4/Spt5 RNA transcription is a dynamic process that produces an RNA transcript from a gene. It is the first step in gene expression and thus is Domains of Spt4 and Spt5 a focal point for cell regulation. In order for successful transcription to Zn finger domain occur, accessory proteins are needed that help initiate and promote elongation. In many eukaryotes, soon after initiation has occurred, Spt4 102 amino acids. blockage of the elongation process occurs resulting in abortive RNA 12kDa 1 40 transcripts. In yeast, a protein complex made up of Spt4 and Spt5 are 5 KOW domains required to circumvent these pauses. Studies have shown that this Acidic N-term CTR Region complex can regulate transcriptional elongation by modulating the Spt5 1063 a.a. processivity of RNA polymerase II. In collaboration with Professor 130kDa 1 244 283 840 Grant Hartzog at UC Santa Cruz we have undertaken crystallographic Spt4 binding region studies to establish the structural and functional relationships of NusG homology region Spt4/Spt5. A number of different constructs of the Spt4/Spt5 complex RNA Polymerase II Binding region have been designed, expressed and purified. This project involves protein expression, purification, crystallization and biophysical studies aimed at characterizing this complex in more detail.
3. Structural and Functional Studies of Substrate Channeling Multienzyme complexes are seen in many diverse metabolic pathways. Substrate channeling of intermediates between enzyme active sites within a multifunctional enzyme complex provides a mechanism to isolate labile and reactive intermediates from competing reactions as well as protect the cell from toxic intermediates. Our aim is to gain a better understanding of the role of protein-protein and protein-ligand interactions in intersubunit communication and synchronization of catalytic events with the channeling activity. This project will study
25 Biochemistry and Molecular Biology channeling activity in the bifunctional enzyme 4-hydroxy-2-ketovalerate aldolase-aldehyde dehydrogenase (DmpFG). Studies have shown that aldolase (DmpG) activity is not detectable in the absence of the dehydrogenase (DmpF) suggesting that acetaldehyde, the product of the aldolase reaction, may be channeled to the dehydrogenase active site.
We have determined the 1.7Å resolution structure of this enzyme. The presence of a 29Å long gated and sequestered tunnel in the structure, leads us to propose this as the channeling route taken by the acetaldehyde intermediate from the aldolase to the dehydrogenase active site. This Honours project involves using crystallographic and kinetic studies to probe the function of the observed channel. The structure of a complex of the acetaldehyde intermediate trapped within the channel will be determined. The physical nature of the residues that line the channel will be characterized by mutational, kinetic and structural studies. The mechanism by which access between the active sites and the channel is controlled and synchronized with enzyme activity at each of Secondary structure representation of the the active sites will be studied by a combination of site DmpFG heterodimer. directed mutagenesis, kinetic and crystallographic analyses.
4. Probing the Structure of Cholesterol Oxidase – A Novel Antibiotic Target. The flavoenzyme cholesterol oxidase constitutes an important virulent factor in immunocompromised patients prone to Rhodococcus equi lung infections. The longstanding problem with antibacterial drug resistance calls for a continued need to probe new targets for the design and development of novel antibiotic treatments. The design of novel antibacterial drugs is facilitated by a detailed knowledge of the architecture of active site, including the positions of hydrogen atoms, the ionization state of titratable groups and the precise conformational state of side chains and cofactors through the substrate binding and catalysis events. Electron density view of the isoalloxazine ring of cholesterol oxidase. The density clearly Our laboratory uses a combination of crystallographic, mutagenesis shows single electron differences for individual and kinetic methods to understand these events. We have crystals of atoms. cholesterol oxidase that diffract to sub-Ångstrom resolution providing an unprecedented view of the enzyme structure. Different redox states of the enzyme can be followed spectrophotometrically in the crystal and ligands bound to induce oxidation chemistry while maintaining sub-Ångstrom diffraction. This provides us with a unique opportunity to visualize transient states and establish structural changes as a function of the redox state. Honours projects, focused on testing hypotheses on redox activity and oxygen reactivity for the enzyme include:
(i). Investigating the structural and electronic differences between the oxidized and reduced enzyme and Difference electron density showing the (ii). Examining whether a tunnel is involved in oxygen access to the positions of hydrogen atoms in a region of the active site during the oxidative half reaction. structure.
26 Biochemistry and Molecular Biology ASSOCIATE PROFESSOR MICHAEL WISE Room 2.09, MCS Building, Phone: 6488 4410 email: [email protected]
Bioinformatics and Computational Biology
Research in the Bioinformatics and Computation Biology Lab. boils down to the application of computational techniques to investigate biological questions. Current application domains include: • Bioinformatics of anhydrobiosis (species’ ability to survive with minimal water) • Microbial bioinformatics • Low complexity/natively unfolded proteins • Simulation of metabolic pathways
PROJECTS
1. Viral Codons
You are no doubt aware that the "Universal" codon translation table in fact only applies to eukaryote genomes, and even then not to all of them; slime mold has a different table. The set of different tables can be found at: http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c If you look at that site you will notice that there is no mention of viruses. One may assume, however, that because viruses are dependent on the replication machinery of their hosts that their genes will be encoded like their hosts, i.e. use the same codon translation tables. So, for example, MUMPS will use the Universal table, while lambda phage will use a bacterial table.
The Codon Adaptation Index was developed some years ago and reflects the observation that some codons are far more used than other codons for a given amino acid, arguably reflecting greater numbers of the corresponding anti-codons. The authors also observed that highly expressed genes tend to use the most abundant codons. The Codon Adaptation Index was developed to reflect these observations.
The project is to examine viral genes in terms of their Codon Adaptation Index to gauge the extent to which the codon usage biases of a virus mirror that of its host. Is it possible to see significant differences between codon usage in the different isolates of the same virus which target different species, e.g. influenza virus affecting humans and birds.
2. Durability and Energy-Storage Genes – Under-Recognised Cofactors of Microbial Pathogenicity
The Sit-and-Wait hypothesis of microbial pathogenicity for non-vector-borne pathogens (Walther and Ewald 2004) suggests a correlation between the durability of a non-vector borne microorganism and its pathogenicity. (See also the review: Brown et al. (2006).) Under the hypothesis, durability – the ability to survive the stresses associated with existing for a period outside a host – is, in effect, a cofactor for pathogenicity, in concert with the necessary presence of conventionally understood virulence factors. That is, without an assortment of virulence factors, a microorganism is unable to colonise a host, but if the microorganism is labile, virulence will be tempered over time because an immobilised infective host is unable to move and thus unable to spread the infection. An extension of this proposal is to include long-term energy storage, particularly those used in stress situations, as a cofactor for pathogenicity because unless an energy store has been maintained the organism may have survived, but it will not have the energy to produce the range of invasion mechanisms it requires, such as pili. The overall aim of the project is to assemble a set of Hidden Markov Models (HMMs) that represent different abiotic stress tolerance protein domains, and another set that represents proteins involved long-term energy storage. The HMMs are then used to search the protein coding genes across a range of bacterial chromosomes to see which species use which mechanisms, which is then linked to published mortality data as a proxy measure pathogenicity.
27 Biochemistry and Molecular Biology
3. Computer Modelling and Simulation of Biochemical Pathways
DiMSim is a software system which embodies a novel methodology for simulating the flow of metabolites through networks of interacting metabolic reactions. Much as biochemists do, DiMSim views the reactions involved in metabolic processes as a graph and uses this as the basis of a simulation system. Each reaction is viewed as a simple automaton which "fires" when a complete set of inputs are available, producing outputs which are consumed by other reactions, and so on. A number of projects are available based on DiMSim.
3a. Modelling of a Cytochrome P450 Pathway With Dr Robert Tuckey, Discipline of Biochemistry In this project you will construct a detailed model of a cytochrome P450 medicated pathway, based on data from the BioCyc database in general, and relevant papers from the literature. The pathway involves the series of steps required to convert cholesterol to pregnenolone in the human placenta, which is essential for the maintenance of pregnancy during the second and third trimesters. The reactions are catalysed by cytochrome P450scc and electrons required for these reactions are supplied to the cytochrome via adrenodoxin and adrenodoxin reductase. Using the DiMSim software, what you will specifically be modelling is the interaction between adrenodoxin, adrenodoxin reductase and cytochrome P450scc to examine in impact of limiting the supply of adrenodoxin reductase.
4. A Novel Method for Building Phylogenetic Trees
Phylogeny is the study of the relatedness of species. The way this is done these days is through the computational analysis of genes in living organisms (after all, we can't go back in time to track speciation events as they happened). The phylogeny of organisms is often depicted as phylogenetic trees and there is a considerable literature on how best to create such trees. Most methods take as input data from a single gene or protein sequence across a range of taxa. That is, the same gene is found in all the species of interest and then compared to build the tree. The problem with this approach is that it assumes that the gene is "typical" and that evolutionary pressures have acted in the same way across all the species to shape that gene. A second problem is to find a gene that is both ubiquitous and conserved in its function, but with sufficient variability to differentiate the various species possessing that gene. In this project you will create an application which takes as its input data generated by an existing genome analysis application as it traverses whole bacterial chromosomes, and then, after normalising the elements of the data vectors, try different methods for building phylogenetic trees from the data. In other words, rather than trying to find the ideal gene around which to build a tree, this method will compare summaries of all the data available in chromosomes or, by extension, entire genomes.
28 Biochemistry and Molecular Biology PROFESSOR JIM WHELAN ARC Centre of Excellence in Plant Energy Biology Room 4.73, MCS Building, Phone: 6488 1749 email: [email protected]
Plant Molecular Genetics and Genomics
Our group uses a variety of ‘omic’ approaches to carry out discovery based investigations concerning the control of gene expression in cells, primarily using the plant models, rice and Arabidopsis, and to a lesser extent yeast. In addition to the characterisation of motifs and transcription factors that control gene expression, we use quantitative proteomic and metabolomic approaches, bio-informatic analyses, coupled with transgenic approaches to obtain a global view of the factors that control gene expression. Overall these approaches will lead to a greater fundamental understanding of Molecular Cell Biology.
This research is carried out in the ARC Centre of Excellence in Plant Energy Biology, which contains two additional State Centres of Excellence in Metabolomics and Systems Biology. This provides students with training and hands-on use of state-of-the-art equipment. Previous students have received international fellowships (EMBO, Human Frontiers) to carry out their own research project in Europe and the USA. National and International research agreements with the Australian National University, the University of Sydney, Zhejiang University (Shanghai, China), The Max-Plank Institute in Golm (Berlin, Germany), Ludwig-Maxmillians University (Munich, Germany) and Umea University (Sweden) provide students with the opportunity to study overseas, supported by a variety of grants or scholarships provided by the centre. In previous years this intellectual, technical and financial support for students has lead to many students receiving awards and prizes for their research and publication in international science journals. Research students will be supported in their studies by a number of post-doctoral and support staff in the centre.
PROJECTS
1. The role of WRKY transcription factors in controlling stress responsive gene expression
WRKY transcription factors are a family of plant specific transcription factors with 70 to 80 members in Arabidopsis. They are involved in a variety of processes in plants, most notably regulation of genes expression in response to stress The role in mediating stress response is mediated to some degree as the transcription of WRKY transcription factors is responsive to a variety of plant hormones that mediate stress responses in plants. The consensus- binding domain for WRKY transcription factors has been defined in a number of promoters. Mitochondria are both a target and play a central role in the stress response in plant cells. Apart from the fact that mitochondria play a central role in mediating programmed cell death, a variety of proteins are induced in mitochondria in response to oxidative stress. The role of many of these proteins is unknown. However the absence of some of these proteins, such as alternative oxidase lead to greater sensitivity to stresses and an altered response to stress. An analysis of the genes encoding mitochondrial proteins that are induced in response to stress reveal that they all contain at least one WRKY consensus binding site. This suggests that WRKY transcription factors play a role in regulating genes encoding mitochondrial stress responsive proteins. However given the variety of WRKY transcription factors and roles mediated by them, it is unclear which WRKY transcription factors are involved. Thus the overall aims of this projects are: i) to investigate if the WRKY consensus sequences in promoters of genes encoding mitochondrial proteins are active, i.e. are these domains necessary for stress induction; and, ii) to determine which WRKY transcription factors are bound to these domains. The approaches used will be to clone the promoters and determine the role of specific sequence elements in regulating expression in response to stress using reporter genes, site directed mutagenesis and transformation of plants. The binding properties of these sequences will be investigated using Yeast 1-hybrid analysis and electromobility shift assays. Additionally bio-informatic analysis of publicly available microarary data will be carried out, as a complete understanding of WRKY transcription factors and putative targets may yield insights into the role(s) of WRKY transcription factors in mediating the mitochondrial stress response.
29 Biochemistry and Molecular Biology
A simplified overview of stress signaling and the role of WRKY transcription factors in mediating stress responsive genes expression
2. Molecular dissection of the response of rice to anoxia
Rice is one of a handful of higher eukaryotic organisms that can withstand a prolonged period of anoxia. This is achieved by a variety of molecular responses that result in biochemical and morphological adaptations that results in survival under anoxia. We have used microarrays to investigate the changes in transcript abundance following alterations in oxygen and can identify a number of primary (within 1 hour) and secondary effects (after 1 hour). Analysis of the promoters of these genes reveal a number of conserved sequence motifs that putatively mediate response to oxygen. Thus the overall aim of this project will be to test the role of selected sequence elements in mediating oxygen responsive gene expression in rice.
This will be achieved as follows: 1) Cloning promoters in from of reporter genes and testing expression in the presence and absence of oxygen after transformation into rice. 2) Deletion analysis of predicted elements to determine the role they play in regulating gene expression
Summary of the number of changes in transcript abundance for genes in response to anoxia (N) in rice. The promoters regions of these genes contain common sequence elements that may play a role in mediating oxygen responsive gene expression.
Please refer to http://www.plantenergy.uwa.edu.au/ for recent publications and more details about lab personnel and scholarships.
30 Biochemistry and Molecular Biology PROFESSOR GEORGE YEOH Room 2.59, MCS building, Phone: 6488 2986 email: [email protected]
Liver Research Group Our research group focuses on the biology of the liver progenitor cell (LPC) called an “oval cell” which describes its shape. This has enormous potential as the vehicle for cell and gene therapy to treat liver disease. Liver disease has become a significant health issue because its causes which are mainly lifestyle related – alcohol, viral infection (HBV and HCV) and obesity are increasing at an alarming rate. All lead to chronic liver disease and liver cancer (HCC) is a common final outcome. Liver transplantation is the only option currently available for treating end-stage liver disease. This option is severely limited by the availability of livers for organ transplant and hence we are exploring the potential of the LPC for use in cell therapy. We contend it is superior to other cell types such as the differentiated hepatocyte or stem cells such as the embryonic stem cell (ESC) or adult stem cell (ASC) for many reasons. In particular, it is robust and simple to freeze and store, then thaw and grow by in vitro culture when required. It can be differentiated into either hepatocytes or cholangiocytes (bile duct cells) quite easily and rapidly when maintained under appropriate conditions, therefore it is more versatile than the hepatocyte. Accordingly, one of the objectives of our research is to identify cytokines that regulate LPCs and to understand their mechanism of action. This will underlie strategies to increase the contribution LPCs make to liver repair in vivo as well as to grow large numbers of functional liver cells; hepatocytes or cholangiocytes in culture for cell therapy applications. We are defining cytokine expression patterns in mice which are subject to chronic liver damage in which LPCs are induced and participate in liver repair. The aim of these studies is to identify factors which induce growth and the sebsequent differentiation of LPCs. We then test candidate cytokines in cell culture models. Our laboratory has developed cell culture models based on cells isolated from mice placed on a diet which induces fatty liver (primary cultures) as well as cell lines which we established which are now used internationally. There is also the potential to use our LPC lines in liver bioreactors and artificial organs. For this application they must be differentiated into hepatocytes which are functionally equivalent to mature adult hepatocytes so they are able to detoxify drugs, produce serum proteins and urea as well as perform the metabolic interconversion between lipids, carbohydrates and proteins. The LPC is also of interest to cancer researchers for it may be a target cell for transformation leading to the development of hepatocellular carcinoma (HCC). Our laboratory has established LPC lines from mice which have the p53 gene deleted. These are called p53 immortalised liver (PIL) cells. Interestingly we have PIL cell lines which are transformed and tumorigenic while others are normal (Ref 1). We are comparing these cell lines to identify important genes which are responsible for transformation of liver cells to hepatocellular carcinoma (HCC). Gene expression analyses have highlighted several dozen genes which are upregulated (oncogenes?) and as many that are down-regulated (tumour suppressor?). The overall aim of projects related to cancer is to identify genes which are causal in terms of HCC and also to reconcile the cancer phenotype with the altered pattern of gene expression.
1. Documenting the effect of cytokines on proliferation and differentiation of LPCs and determining their mechanism of action This project takes advantage of three resources available in our laboratory. First we have established a LPC line (BMOL TAT) which expresses beta-galactosidase when it differentiates into a hepatocyte (Fig 1). Second we have an instrument which measures cell growth continuously in cultured LPCs maintained in a 96-well format which allows for testing of many replicates under a variety of culture conditions such as the exposure to a variety of cytokines. We have published the application of this instrument called the Cellscreen to monitor growth of LPCs (Ref 1). Third we have identified several cytokines which are associated with liver inflammation induced by a choline deficient ethionine supplemented diet which are associated with the induction of LPCs in mice following liver damage (Ref 2 & 3). This project will focus on the role of TNF alpha, IL6 and IFN alpha in cultured BMOL TAT cells in terms of their respective effects on cell proliferation (using the Cellscreen instrument) and differentiation by quantifying the expression of beta-galactosidase. To confirm the effects of each cytokine on proliferation and/or differentiation, their ability to induce cyclin D (proliferation) and/or HNF4 alpha (differentiation transcription factor) will be determined by qPCR and Western blotting.
Fig 1. BMOL TAT cell line in growth conditions (top left) in which do not express beta-galactosidase (top right) Following differentiation induced by high density culture (bottom left) clusters of cells express beta-galactosidase (bottom right)
31 Biochemistry and Molecular Biology
References
1. Viebahn, C.S., J.E. Tirnitz-Parker, J.K. Olynyk, and G.C. Yeoh, Eur J Cell Biol, (2006). 2. Lim, R., B. Knight, K. Patel, J.G. McHutchison, G.C. Yeoh, and J.K. Olynyk, Hepatology, 43: 1074-83 (2006). 3. Knight, B., V.B. Matthews, B. Akhurst, E.J. Croager, E. Klinken, L.J. Abraham, J.K. Olynyk, and G. Yeoh, Immunol Cell Biol, 83: 364-74 (2005).
2. Assessing the drug metabolising capacity of LPCs during growth and after differentiation into hepaotcytes
This project will evaluate the drug metabolising ability of LPCs before and after differentiation. It will be addressed at two different levels. First the overall efficiency of drug metabolism will be measured by the conversion of a model substrate 7-Benzyloxyquinoline to its fluorescent product. This reflects the activity of CYP3A family – the major form of cytochrome p450 involved in drug metabolism. The CYP3A family comprises many isoforms and in the context of this project two are of special interest. CYP3A16 is the dominant form in fetal liver whereas CYP3A11 is the adult form (Ref 1). Besides documenting the overall change in CYP3A activity we wish to know if there is a switch from the fetal to adult form when LPCs differentiate. To accomplish this, the relative expression levels of mRNA coding for the respective isoforms of CYP3A will be measured by real time PCR.
References
1. Sakuma, T., M. Takai, Y. Endo, M. Kuroiwa, A. Ohara, K. Jarukamjorn, R. Honma, and N. Nemoto, Arch Biochem Biophys, 377: 153-62 (2000).
3. Identifying genes that are related to the transformation of liver progenitor cells into hepatocellular carcinoma. 7 Days 14 Days This project will exploit differences between a non- tumorigenic PIL4 cell line which does not grow in semi-solid medium in contrast to the tumorigenic PIL2 line which odes (see Fig 2). To identify genes that may be causal in the FRL transformation of LPCs we have profiled the pattern of gene 19 expression of PIL2 (transformed) and PIL4 (normal) cells. a b The list of genes which have been up-regulated (potential oncogenes) and down-regulated (potential tumour suppressor genes) is extensive. This project will adopt two approaches to PIL2 identify which are important genes. One is a bioinformatics approach to highlight genes which i) are associated with HCC c d and ii) are altered in LPCs as a result of deleting p53 (by comparison with wild type LPCs). The other approach is to follow up on recent findings which indicate that the lack of p53 in liver cells leads to up- PIL4 regulation of two intracellular molecules IAP and Yap which e f cooperate to induce tumorgenesis (Ref 2). Our array data indicates that expression of these genes is increased in PIL2 Fig. 2 PIL2 cells (c&d) are positive in the colony forming cells. This needs to be confirmed by qPCR, which can also assay whereas PIL4 cells (e&f) are negative. be used to investigate other genes highlighted by the first FRL19 = positive control (a&b) approach. Finally the differential expression of IAP and Yap needs to be confirmed at the protein level. Confirmation that these gene products are causative would be obtained by their ability to transform normal PIL4 cells by their over- expression.
References
1. Dumble, M.L., Croager, E.J., Yeoh, G.C.T., and Quail, E.A.,. Carcinogenesis. 23: 435-45 (2002). 2. Zender, L., M.S. Spector, W. Xue, P. Flemming, C. Cordon-Cardo, J. Silke, S.T. Fan, J.M. Luk, M. Wigler, G.J. Hannon, D. Mu, R. Lucito, S. Powers, and S.W. Lowe. Cell, 125: 1253-67 (2006).
32 Biochemistry and Molecular Biology CHEMISTRY ASSOCIATE PROFESSOR MURRAY BAKER Room 1.06, MCS building, Phone: 6488 2576. Email: [email protected]
My group's research interests are primarily in synthetic chemistry—we aim to apply our skills in synthesis to problems in areas such as catalysis, nanotechnology, surface science, biological chemistry/medicine, polymer science, molecular recognition, and sensors. The following project descriptions are a sample of current research projects in the group.
1. Cyclophane imidazolium salts and heterocyclic carbene complexes We are exploring the chemistry of a remarkable class of imidazolium salts (such as 1) in which the imidazolium units are part of a cyclophane macrocycle. These salts can easily be converted into fascinating heterocyclic carbene complexes, such as 2 and 3. Complex 2 is an outstanding, robust catalyst for industrially 6 important C-C bond-forming reactions (Dalton Trans. 2001, 111-20). Complex 3 contains an (η -arene)RuClL2 6 structure (with the L units in 3 being heterocyclic carbene groups). The (η -arene)RuClL2-type structure is found in a class of Ru complexes being investigated as anti-cancer agents, and related structures are found in a range of Ru complexes that promote useful isomerisation reactions under mild conditions. Complex 3 is especially interesting, however, because the η -arene and the two L units are part of the same ligand. The simultaneous binding of the η6-arene and the L units to the same Ru centre introduces considerable strain, so we expect 3 to exhibit unusual reactivity compared to the previously studied Ru complexes. Also, different analogues of the starting imidazolium N N N Cl cyclophane 1 are easy to synthesize (J. Org. N N N N N Pd Br Ru Chem. 2004, 69, 7640-52), which means that Br N N N N Cl we will be able to easily synthesize many 2 Br analogues of 3 to optimise any interesting catalytic or biological activity we discover. 2 1 3
2. Biodegradable and biocompatible materials for tissue engineering Collaboration with Prof Traian Chirila (Prevent Blindness Foundation, Qld) and Dr David Brown (Curtin). Biocompatible materials are materials that can be placed in contact with biological tissue without causing infection or other undesirable biological responses. A material is biodegradable if it will degrade under biological conditions, normally under the action of enzymes. Not all biocompatible materials are biodegradable (the prognosis for recovery from hip replacement surgery would be poor if enzymes in the body degraded the implanted hip joint) but biocompatible materials that are biodegradable have some fascinating applications. Slow release drugs, for example, are typically delivered from within a biocompatible, biodegradable matrix. Tissue engineering requires biocompatible, biodegradable materials. In a modern treatment for damaged knee cartilage, for example, a sponge made of poly(lactic acid) is cut into an appropriate shape and infused with a broth containing new cartilage cells, and is then used to replace the damaged cartilage in the knee. The poly(lactic acid) sponge holds the new cartilage cells in place until they grow into new cartilage material. Poly(lactic acid) is both biocompatible and biodegradable. Gradually, the poly(lactic acid) sponge dissolves (under the action of enzymes) and is replaced by the growing cartilage cells until the knee is repaired. Similar techniques based on poly(lactic acid) and closely related materials are being assessed for reconstructive surgeries that require cartilage in complex shapes (eg, ear, nose). A significant problem in tissue engineering is the difficulty in synthesizing the biodegradable material in a form that has appropriate morphology (porosity etc) to accommodate and encourage growth of replacement cartilage cells. One of the most important biocompatible polymers is poly(hydroxyethyl methacrylate) (PHEMA). PHEMA-based materials are made by co-polymerising O hydroxyethyl methacrylate with suitable crosslinking agents, and they are already used to OH O fabricate permanent medical implants, such as intra-ocular lenses. PHEMA materials have biocompatibility characteristics that would make them ideal for many applications in tissue engineering, and porous PHEMA can be easily produced. Unfortunately, hydroxyethyl methacrylate however, the PHEMA materials that have been synthesized to date are not biodegradable. We are investigating routes to biodegradable forms of PHEMA. Our approach uses new types of crosslinking agents that will form crosslinks susceptible to cleavage by enzymes. Research in this area will start with synthesis of new crosslinking agents and then explore copolymerisation of the crosslinking agents with
Chemistry 33 hydroxyethyl methacrylate to form new PHEMA-based materials. A good deal of synthetic organic chemistry will be involved, as well as NMR spectroscopy (for characterisation of crosslinking agents and polymers) and other polymer characterisation techniques.
3. Gold-carbene complexes as anti-cancer agents, catalysts, and luminescent materials Collaboration with Dr Huynh Han Vinh (NUS) and Prof Stephen Hashmi (Stuttgart). Cationic Au(I) carbene complexes such as 4 2 and 5 exhibit activity against certain cancer cell lines. These complexes appear to disrupt N N Cl NN Au NN mitochondrial function, and their selectivity for Au NNN N Au N N Au N N cancer cells over normal cells can be tuned by N N Cl variation of the hydrophilic-lipophilic character of the cation. The Au(I)-carbene complexes are 4 5 6 easy to synthesize and they offer the prospect of fewer toxic side-effects than their better-known Au(I)- phosphine counterparts. In addition, the gold carbene complexes are chemically interesting species in their own right. For example, in complexes such as 5 the ligand framework forces two Au atoms to be very close to one another, a trait that makes such complexes strongly luminescent. This luminescence may be useful for the development of sensors and for tracking the fate of complexes in cells (Angew. Chem. Int. Ed. 2006, 45, 5966- 70). Au(I) complexes such as 4 and 5 and Au(III) complexes such as 6 also have exciting prospects as robust catalysts for a range of oxidation and C-C bond forming reactions. In this project we are examining complexes such as 4 - 6 and additional new Au-carbene complexes, with a view toward delineating and enhancing their biological activity, catalytic activity, and luminescence properties.
4. QCM Sensors to detect Bacteria, Viruses, Proteins... This project seeks to use a quartz crystal microbalance (QCM) as a sensor for viruses and other particles. When stimulated by an appropriate electrical signal, a quartz crystal oscillates. If material is attached to the surface of the crystal, the effective mass of the crystal increases, and consequently the frequency of oscillation decreases. This phenomenon is the basis of operation of the quartz crystal microbalance—by analysing the change in oscillation frequency, the mass of the material attached to the QCM can be measured. If we can design a QCM with a crystal that selectively only adsorbs a specific type of virus, and nothing else, we can use the QCM as a sensor for that virus. One approach is to coat the surface of a QCM crystal with antibodies for the virus, and then expose the QCM crystal to the sample to be tested. If the virus is present, it will bind to the antibodies on the QCM surface, and the resulting mass change (just a few nanograms) will be detected by the QCM. This type of sensor has many potential advantages. It is inherently sensitive, since the QCM can detect very small mass changes. It is inherently specific, since the virus/antibody interaction is specific—tobacco mosaic virus will bind to tobacco mosaic virus antibodies, but influenza virus will not. It is also inherently flexible—we can easily target a different virus (or protein or bacterium) simply by changing the antibody that we attach to the surface of the QCM crystal. The QCM response is rapid (a few seconds), compared to the traditional assays (such as ELISA assays, which can require days of sample preparation time).
5. Functionalised triazacyclononane derivatives R R Collaboration with Dr David Brown (Curtin). N N N R N The coordination chemistry of 1,4,7-triazacyclononane and its M N N N derivatives is diverse. Many triazacyclononanes bearing pendant R N N R functional groups have been synthesized. In favourable cases, these R N functionalised triazacyclononanes can bind metals via both the 7 8 triazacyclononane unit and the pendant group. Triazacyclononanes functionalised with N-heterocyclic carbenes (as in 7) have not been reported to date, but are intriguing because they are likely to bind metals rapidly and strongly. This project will explore the synthesis of functionalised triazacyclononanes such as 8, and their conversion into metal complexes such as 7. Catalytic and biological activities of the metal complexes will be explored.
Chemistry 34 PROFESSOR SUE BERNERS-PRICE Room 4.09, MCS building, Phone: 6488 3258 Email: [email protected]
Medicinal Inorganic Chemistry Group Research at the interface between inorganic chemistry, biology and medicine is providing exciting new insight into biological processes as well as novel ideas for the design of therapeutic agents involving both novel targets and novel mechanisms. Our research is directed towards the design and mechanism of action of metal-based anticancer agents. All projects involve strong interdisciplinary collaboration, some internationally. Prospective Honours students with a background in Chemistry and interests in Biochemistry or Pharmacology are particularly encouraged to apply, but some collaborative projects are also suitable for students with a background in Molecular Biology and Biochemistry. Depending on interests, students will be exposed to a range of techniques including NMR spectroscopy, molecular modelling, cell culture, molecular biology and proteomics. Current research projects are encompassed under two broad areas funded by grants from the Australian Research Council.
PROJECTS
1. NMR and molecular modelling studies of the biological chemistry of platinum anticancer drugs
A major interest is in the exploitation of multinuclear NMR methods for the study of the chemistry of metal-based drugs under biologically relevant conditions. Recent [1H, 15N] H3NCl NMR studies have provided new insight into the kinetics and mechanism of binding of Pt cisplatin and other platinum drugs to DNA. H3N Cl Cisplatin Current projects focus on BBR3464, a trinuclear Pt drug currently in clinical trials. Whereas cisplatin binds to DNA to form a 1,2-GG intra- 4+ strand adduct, BBR3464 and analogs form long- Cl NH3 H3N NH2(CH2)6H2N NH3 range inter-strand DNA cross-links. In collaboration Pt Pt Pt with the inventor of BBR3464 (Prof Nick Farrell, H3N NH2(CH2)6H2NNH3 H3NCl Richmond USA) we are investigating the kinetics and mechanism of formation of these long-range BBR3464 inter-strand adducts, as well as studying other aspects of the biological chemistry aimed at understanding the unique antitumour activity of this novel clinical candidate and its second generation analogues. Our work uses a combination of multinuclear [1H,15N] NMR and molecular modelling. Projects in this area can be tailored for specific interests utilising these techniques.
2. Mitochondria as targets of novel metal–based antitumour agents With Dr Aleksandra Filiopvska, WAIMR
Recent developments in understanding the central place of mitochondria as a regulator of cell death have stimulated enormous interest in targeting mitochondria in new approaches to cancer chemotherapy. A major aim for this research is to overcome the two problems in cancer + R R' [Au(dppe)2] R = R' = Ph chemotherapy, drug resistance and the lack of selectivity of cancer PP + R R' [Au(dnpype)2] R = R' = drugs in differentiating between normal and tumour cells. In early Au R R' 2-, 3- or 4-pyridyl PP + work we identified a new class of metal phosphine antitumour agents R R' [Au(2-pyppe)2] R = Ph, R' = 2-pyridyl related to the Au(I) compound [Au(dppe)2]Cl. Clinical development of these compounds was abandoned when unacceptable levels of toxicity were identified in pre-clinical trials. However, more recent work has demonstrated that by fine-tuning the hydrophilic/hydrophobic balance of these types of compounds (by incorporating pyridyl substituents) it is possible to eliminate the toxicity and achieve selectivity for tumour cells over normal cells. These metal-based (Au(I), Ag(I) and Cu(I)) compounds appear to act by a mechanism of action that is different to all anticancer agents currently in clinical use. As they are lipophilic cations it is possible that the selectivity depends on selective uptake into tumour cell mitochondria and
Chemistry 35 recent work (in collaboration with Dr Aleksandra Filipovska) has identified critical protein targets responsible for the selective toxicity to cancer cells. This is an exciting new approach to cancer chemotherapy that involves targeting mitochondrial antioxidant proteins, which have altered function in cancer cells. Projects offered in this area can be tailored to suit students with backgrounds in either Chemistry or Biochemistry/Molecular Biology.
Recent References
Insight into the mechanism of action of platinum anticancer drugs from multinuclear NMR studies. S.J. Berners- Price, L. Ronconi and P.J. Sadler, Prog. Nucl. Magn. Reson. Spectrosc. 49 (2006) 65-98. Long range 1.4- and 1,6-interstrand cross-links formed by a trinuclear platinum complex. Minor groove pre- association affects kinetics and mechanism of cross-link formation as well as adduct structure. A. Hegmans, S.J. Berners-Price, M.S. Davies, D. Thomas, A.S. Humphreys, N. Farrell. J. Am. Chem. Soc.. 126 (2004) 2167-2180. Targeting the mitochondrial cell death pathway with gold compounds, P.J. Barnard and S.J. Berners-Price, Coord. Chem. Rev. (2007) 251, 1889-1902.
A gold(I) phosphine complex selectively induces apoptosis in breast cancer cells: implications for anti-cancer therapeutics targeted to mitochondria. O. Rackman, S.J. Nichols, P.J. Leedman, S.J. Berners-Price and A. Filipovska, Biochem Pharmacol. (2007), 74, 992-1002.
Gold(I) chloride adducts of 1,3-bis(di-2-pyridylphosphino)propane: synthesis, structural studies and antitumour activity. A.S. Humphreys, A. Filipovska, S.J. Berners-Price, G.A. Koutsantonis, B.W. Skelton and A.H. White. Dalton Trans. (2007), 4943 - 4950.
Effects of geometric isomerism and anion on the kinetics and mechanism of the stepwise formation of long range interstrand cross-links by dinuclear platinum antitumor complexes, J. Zhang, D. S. Thomas, S. J. Berners-Price and N. Farrell, Chem. Eur. J. (2008) 14, 6391-6405.
Mitochondria-targeted chemotherapeutics: The rational design of gold(I) N-heterocyclic carbene complexes that are selectively toxic to cancer cells and target protein selenols in preference to thiols. J. L. Hickey, R. A. Ruhayel, P. J. Barnard, M. V. Baker, S. J. Berners-Price and A. Filipovska, J. Am. Chem. Soc. (2008) DOI: 10.1021/ja804027j (Web Release date 26-Aug 2008)
The design of gold-based, mitochondria-targeted therapeutics, S. J. Berners-Price and A. Filipovska, Aus. J. Chem. (2008), 61, 661-668.
Chemistry 36
PROFESSOR CHARLIE BOND Room 4.16, MCS Building, Phone: 6488 4406 Email: [email protected]
Structural Biology Structural Biology research involves building a three-dimensional picture of biological molecules to shed light on the molecular interactions and events which drive many of the fundamental processes of life. Investigations in my lab address proteins of relevance to human health, including DNA repair enzymes and other nucleic acid processing proteins, and enzymes essential to the survival of life-threatening parasites, which may be drug targets.
Different aspects of this research can be tailored to students with strengths in Biochemistry, Chemistry, and Biophysics. Structural Biology research typically involves the opportunity to learn from a diverse set of useful techniques including molecular biology, protein purification and crystallisation, spectroscopy, X-ray crystallography, molecular modelling, bioinformatics, unix computing. The Structural Biology lab is equipped with state-of-the-art equipment including a crystallization robot and X-ray data collection facilities.
For further information, reprints of papers, a colour version of this page, or to find out about other research in the lab come and see me (MCS Lab 4.16) and look at http://xtal.uwa.edu.au/px/charlie .
PROJECTS
NOTE: In addition to projects listed here, it may be possible to tailor a structural biology project to your specific interests.
1. HOW DO PROTEINS RECOGNISE RNA MOLECULES? ROP MUTANTS (collaboration with Dr Daniel Christ, Garvan Institute, Sydney)
Rop is a small alpha-helical protein which plays a critical role in bacteria where it regulates the number of copies of a DNA plasmid that the bacteria can accommodate. Rop does this by binding to a complex of two RNA hairpins – called the 'kissing' complex. By binding and stabilising this interaction, it stops the RNA being used to prime replication of the plasmids.
As it is a small protein and is known to crystallise, Rop makes an excellent target for research to understand the basis of protein:RNA interactions. We have a panel of mutant proteins, which were selected using in vitro evolution methods, that have higher affinity for the RNA than the wild-type protein. This project will involved expressing some of these mutant proteins, generating heterodimers of the mutants, measuring their affinity for RNA, attempting to crystallise them and solving their structures. Skills learned will include molecular biology (mutagenesis), protein expression and purification, protein:RNA interaction assays, crystallisation and protein crystallography.
A predicted model of the Rop:RNA complex.(from Christ, D and Winter, G, Proc Natl Acad Sci U S A. 2003 November 11; 100(23): 13202–13206)
Chemistry 37
2. PROTEIN STRUCTURE PREDICTION: PPR PROTEINS (collaboration with Ian Small, CoE for Plant Energy Biology)
PPR proteins are modular proteins composed of tandem repeats of 35 amino acid sequences. A number of these proteins are known to bind and/or process RNA by recognising the RNA sequence. We want to understand how this sequence-specific recognition occurs. In similar protein families (TPR and ankyrin proteins) for which the structures are known, these repeats form alpha-helical hairpins which assemble to make a long ‘solenoid’. We have evidence from bioinformatics studies that PPR proteins have a similar, but different structure. Based on a specific type of bioinformatic (sequence covariation) data, we can predict both the secondary and tertiary structure of TPR and ankyrin proteins. We are interested in exploring which other protein families can be investigated with these Sequence covariation data can be used to methods, but our main aim is to produce plausible models of PPR protein structure which can be used to guide wet- predict the structure of helical repeat lab experiments into the function of these proteins. proteins, with a relatively high accuracy. This is a computation-based project which will involve learning about protein structure, molecular dynamics and bioinformatic analysis of proteins.
3 CHAPERONES AND CO-CHAPERONES OF THE MALARIA PATHOGEN, PLASMODIUM FALCIPARUM (WITH DR WILL STANLEY)
Schematic of the interactions in the multichaperone complex. Malaria is a widespread tropical disease killing about 2 million people annually, young children and pregnant women being especially vulnerable. It is a disease associated with poverty and classed as a neglected disease – no vaccine is available and prophylactic drugs are often too costly for those most at risk. The microbial pathogen, Plasmodium falciparum, is the major cause of life-threatening malaria amongst humans.This project explores a complex of P. falciparum chaperones – proteins essential for folding, stabilising and sorting of other proteins – which is critical to survival and proliferation of the pathogen, and thus a target for new kinds of antimalarial drugs. The complex consists of two housekeeping heat shock proteins, Hsp70 and Hsp90, and a Hsp organiser protein, HOP, which are involved in a complex set of intermolecular interactions to facilitate folding/sorting of a number of client proteins. Components of the complex can be recombinantly expressed and purified with the aim of detailed biochemical and biophysical studies of the assembly, structure and function of this multi-chaperone complex.
For other collaborative projects, please see entries for Dr Swaminatha Iyer and Prof Ian Small
Chemistry 38 Dr Craig Bullen Research Associate Centre for Strategic Nano-Fabrication (Incorporating Toxicology) Room 3.34, MCS building, Phone: 6488 4422 Email: [email protected] http://www.strategicnano.uwa.edu.au/
Current research projects cover: (i) process intensification using spinning disc and rotating tube processing for the fabrication of precisely tailored nanoparticles, including shaped controlled nanoparticles; (iii) investigations into kinetics and thermodynamic properties of metal and semiconductor nanocrystals systems; (iv) fabrication of nanoparticle based composite materials with polymer and sol gel hosts and processing of nanoparticle composites using electrospinning and related techniques. Projects involve the investigation of tailored nanoparticle synthesis, surface phenomena, application of continuous flow methods for nanocrystals processing, determination of size- dependent material properties, and development of materials for sensing and catalysis applications. These projects provide opportunities to gain experience and skill in materials chemistry, analytical techniques, NMR, optical spectroscopy, electron microscopy, x-ray diffraction, and other materials characterisation techniques. A brief description of projects is presented below. The research framework outline here does allow for a project to be tailored to the interests of the researcher. There are also may be opportunities for students to gain experience at the Australian synchrotron in Melbourne to characterise/study nanoparticle and nanocomposite materials.
PROJECTS
1. Shape directed metal nanoparticle fabrication using green chemical methods with Professor Colin Raston There are opportunities for interested researchers to produce nanoparticles with precisely controlled size and shapes using wet chemical methods using batch and continuous reactor methods. Current interest in the application of shape controlled nanoparticles for application in areas including cancer treatment, plasmonics, and optical data storage is stimulating a rapid development of the science required to make and process these unique materials. The Centre for Strategic Nanofabrication at the University of Western Australia is well equipped for the development of new anisotropic metal and semiconductor materials. The growing suite of spinning disc (SDP) and rotating tube reactors within the centre will be developed to produce commercially viable, continuous production methods for shape-controlled nanoparticles.
2. Studies of the kinetics of nanocrystal formation using batch and continuous systems with Professor Colin Raston and Dr Swaminatha Iyer The physical properties of materials can change dramatically as their dimensions are reduced below 100 nanometers. In order to control the specific size and shape of nanocrystals the chemical kinetics of nanoparticle formation need to be optimised. This research is aimed at the development of a quanitative framework covering the kinetic factors affecting nanocrystal nucleation and growth. A selection of metal and semiconductor systems are available for study using a combination of spectroscopic and electron microscopy techniques. One specific project is involved with identifying kinetic differences between different crystal facets of nascent nanocrystals, with a view to controlling particle shape.
Chemistry 39 3. Novel cavitand stabilized nanoparticle structures as photo-thermally activated molecular capsules with Dr Melissa Latter The development of functional coatings for nanoparticles is critical to most areas of application. The surface coating can have a number of roles in nanoparticles systems including: (i) regulating particle formation; (ii) stabilizing the particles against agglomeration within a host matrix; (iii) to sequester specific molecules close to the nanoparticle surface; (iv) be used to control charge transfer processes; and (v) act as a binding agent in nanoparticle based sensors. Projects in this cross-disciplinary research will involve the development of custom synthesized organic molecules and assessing their utility in the synthesis and surface tailoring of nanoparticles. One project involves tailoring a noble-metal nanoparticle surface with resorcarene-based molecular capsules (cavitands), and investigating the photo-thermal release of encapsulated target molecules. This system will be developed as a drug delivery method.
4. Composite nanoparticle processing Development of composite nanoparticles that combine the best optical, mechanical, or chemical properties of the constituent materials represent an important aspect of nanoparticle science. There are a number of systems that are available for interested researchers that are of fundamental scientific interest and others (for example gold-nickel oxide composites) that have potential in optical and biological applications. This work covers the fabrication and physical characterisation of uniform composite materials using batch and continuous processing.
5. Advanced Nanoparticle fibre generation with Dr Swaminatha Iyer
Using the newly acquired electrospinning equipment housed in the Centre for Strategic Nanofabrication, within the BCCS building, is able to produce nanofibres spun under a strong electric field. This work will be in association with researchers at the University of Padova in Italy, and will involve mechanical and gas sensing testing of spun fibres. One project involves the development of polarisation-dependent optical gas sensors produced by cross-weaving aligned nanocomposite fibres. This project will involve the development of composite materials, development of experimental methods for fibre electrospinning and physical characterisation of fibres.
Chemistry 40 Dr BEN CORRY
Room 4.29, MCS building. Phone: 6488 3166 Email: [email protected]
My research involves using a variety of theoretical and experimental techniques to understand the transport and binding of ions and water, and the proteins (known as ‘ion channels’) that regulate this in living organisms. These are large proteins responsible for signalling between and within cells that provide selective pathways for ions to pass across cell membranes. In addition, I am also interested in ion transport and binding in synthetic molecules. I use a variety of state of the art computational and fluorescence techniques, and these projects will provide a good introduction into the techniques of computational chemistry, macromolecular modelling or advanced optical imaging. Ultimately the understanding of ion selectivity in biological ion channels may aid the treatment of a range of neuromuscular diseases from epilepsy to muscular dystrophy. Other projects may be available after consultation
PROJECTS
1. Origins of selective ion binding in enzymes
Many biological proteins need to be able to discriminate between ion types in order to function. In most cases, however, it is not understood how this selectivity arises. We have been developing general models to understand the mechanisms that lead to ions selectivity, and can isolate the role that each possible effect has in creating ion differentiation. Now, we hope to test our theories on a number of real proteins. In this project you will conduct molecular dynamics simulations of an enzyme protein known to differentiate between Na+ and K+ to ascertain how this discrimination arises.
2. Quantum mechanical models of the origins of ion selective binding
As noted for project one, we have developed general models to explain the origins of ion selective binding. These models have involved the use of molecular dynamics calculations. In order to test the models, we are also interested to see if the same results can be achieved using a more detailed quantum mechanical system. In this project you will determine the magnitude and origin of ion selectivity in a range of model systems using quantum mechanical calculations to help create a general framework for both explaining ion selectivity and designing novel ion selective molecules.
3. Simulating interactions of organic foulants with carbon nanotube desalination membranes
Recent simulations within our group have shown that desalination of sea water can be achieved using much less energy than current technology by using reverse osmosis membranes made from carbon nanotubes. One thing that we have not examined is how susceptible such membranes would be to blockage from organic foulants. This project would involve examining the interaction of such foulants within simulations of desalination.
For an introduction to these research areas, see the following publications: z B. Corry. Understanding ion channel selectivity and gating and their role in cellular signalling. Molecular BioSystems, 2:527-535; 2006. z M. Thomas, D. Jayatilaka and B. Corry. The predominant role of coordination number in potassium channel selectivity. Biophys. J. 93:2635-2643, 2007 z B. Corry. Designing carbon nanotube membranes for efficient water desalination. J. Phys. Chem. B. 112:1427-1434. 2008
Chemistry 41 Dr. Swaminathan Iyer
Centre for Strategic Nano-Fabrication (Incorporating Toxicology) Room 3.32, MCS Building, Phone: 6488 4422 Email: [email protected] http://www.strategicnano.uwa.edu.au/
NanoBiotechnology: Interdisciplinary research encompassing Chemistry (Materials Science), Physics (Magnetism for targeted delivery, quantum dots) and Biology (Structural Biology [proteins]; Neuroscience/Animal Biology)
PROJECTS
Nanotechnology and Structural Biology 1. Nanoparticles and Biomacromolecules as applied to Efficient Protein Crystallization with Prof. Charles Bond and Dr. Will Stanley. A great deal of academic and industrial resource and effort is targeted worldwide to improve the success, quality and speed of protein crystallization (e.g. NASA’s Protein Crystal Growth (PCG) program). This need is becoming more and more crucial with the rapid advances in biotechnology, bioelectronics and molecular pharmacology that require understanding of biological processes at the molecular level. The prime importance of protein crystals in developing novel applications is centred on three major areas: (1) structural biology and drug design, (2) bioseparations, and (3) controlled drug delivery. The key to successful crystallisation is to ensure that the protein and precipitation reagents reach levels of local saturation appropriate for both nucleation and crystal growth to occur. The need to produce crystals more efficiently and rapidly is thus recognized world-over and has been a major undertaking in countless institutions. The project herein focuses on developing a novel protocol to crystallize proteins via controlled interactions with engineered nanoparticles. The project will result in hands on experience in nanotechnology, protein chemistry, crystallography and the use of Art- Robbins Phoenix robot.
2. Fullerene chaperones: carbon mediated refolding of thermally denatured proteins with Dr. Will Stanley, Prof. Charles Bond and Prof. C.L.Raston. Protein refolding is the process by which a denatured protein reconfigures to its characteristic functional native state. Denatured proteins are responsible for numerous diseases such as bovine spongiform encephalopathy (mad cow disease), and Alzheimer’s disease. Additionally, numerous medical and biotechnological applications require the rescue of misfolded proteins produced by in vitro or in vivo genetic expression. A practical solution to the issue of in vitro misfolding would be of great importance to biotechnology and would provide leads for the creation of in vivo therapeutics. Selective binding to the hydrophobic regions of proteins provides one route to protein refolding, with refolding dictated by complementary electrostatic interaction with the\ exposed charged residues of the denatured protein. The large surface area and surface tunability of nanoparticles make them excellent candidates for this approach. The project focuses on the use of fullerene: C60, C70 and carbon nanotubes as chaperones to interact with the charged residues on denatured protein
Nanotechnology and Neuroscience 3. Doped ZnSe quantum dots: “Green” alternatives as biomarkers for neuroscience with Prof. Sarah Dunlop, Dr. Lindy Fitzgerald and Dr. Martin Saunders. A quantum dot (QD) is a semiconductor whose excitons are confined in all three spatial dimensions. As a result, they have properties that are between those of bulk semiconductors and those of discrete molecules. Doping—the introduction of a small amount of ‘‘impurities’’ into the crystal lattice—is an interesting way to change the QDs physical properties. An important example is the
Chemistry 42 doping of II–VI semiconductors with paramagnetic Mn2+ ions, yielding materials denominated dilute magnetic semiconductors (DMS), which exhibit interesting magnetic and magneto-optical properties. The application of quantum dots as biomarkers for developing a recovery pathway following neurotrauma is an emerging field with great potential. Nanotechnology has been previously used to examine drug delivery across the blood brain barrier and to provide micropatterns to control neuronal growth and connectivity. The project will explore the synthesis of doped ZnSe nanocrystals, following which a pivotal emerging issue of importance: whether nanoparticles are innately toxic to, or biocompatible with, the central nervous system will be addressed : via in vitro analysis with retinal ganglion cells (RGCs).
Nanotechnology and Drug delivery 4. Multifunctional (Luminescent/Magnetic) Electrospun Scaffolds for targeted drug delivery with Prof Colin Raston and A/Prof Tim St. Pierre The natural extracellular matrix (ECM) is a complex structure that is built to meet the specific requirements of the tissue and organ, primarily consisting of nanometer diameter fibrils. ECM may contain other vital substances such as proteoglycans, glycosaminoglycan and various minerals. Current research in tissue engineering involves trying to replicate the ECM such that it provides the environment for tissue regeneration. Electrospinning uses an electrical charge to draw very fine (nano scale) fibres from a liquid. A variety of polymers and other substances have been incorporated into the artificial nanofibrous scaffold. The project will focus on fabricating mesoporous electrospun nano-hybrids of magnetite (iron oxide nanoparticles) and quantum dots with high magnetophoretic mobility. The resulting scaffolds potentially can be aligned under a magnetic field, for sustained release of drugs through the mesoporous channels.
5. Mechanical stimuli induced drug release nanosystems with Prof. Colin Raston. A potential limitation of most controlled drug-delivery systems is that they have been designed to operate under static conditions. However, mechanically dynamic environments are the norm for many locations in the body (e.g., compression in cartilage and bone, tension in muscle and tendon, and shear force in blood vessels). Many researchers have attempted to develop drug-delivery systems, which actively respond to external stimuli such as temperature, pH, ultrasound, electric or magnetic fields. However, mechanical signals have not been systematically exploited as an external stimulation for controlled drug delivery, regardless of its simple but critical function in the body. The use of self-healing materials was reported in Nature, vol 409, p794, 2001 for the release of a microencapsulated healing agent that is released upon cracking for repair of polymeric composites. The project will focus on developing nanocapsules for sustained drug release in response to mechanical signals. This approach would provide a useful means in the development of advanced materials for controlled drug delivery as well as for tissue engineering applications.
Chemistry 43 DR GEORGE KOUTSANTONIS
Room 3.11, MCS building, Phone: 6488 3177, Email: [email protected] Group Website: http://chem181.chem.uwa.edu.au/Koutsantonis_Group
Materials, Polymers and Nanochemistry Our group is interested in the role of metals in functional materials. While the role played by metals in materials is still evolving and there is a an increasing effort to incorporate redox–active centres into many materials, e.g. conducting polymers, in an effort to create highly efficient redox conductivity for sensor, catalytic, photochemical and photoelectronic applications. However, in conducting metallopolymers an understanding of the interactive roles that the metal centres and the organic polymer backbone play is in its early stages and thus much fundamental work remains to done to provide a firm background to applications. We are participating members of the WA Centre of Excellence in Nanochemistry.
PROJECTS 1. Redox-active Metallomicelles Metallosurfactants are an emerging class of materials which offer interesting alternatives to traditional “organic” surfactants due to the range of properties inherent to complexed metal ions Introduction of such a centre can impart the magnetic and electronic properties, as well as the redox and catalytic activity of the complex to the surfactant system, which of course can be concentrated at an interface, be it polar/apolar (e.g. micelles, vesicles), solid/liquid (e.g. monolayers) or liquid/gas (e.g. Langmuir-Blodgett films). Cationic surfactants have general applications such as biocidal agents, and there has been recent interest in their use as DNA delivery agents for gene therapy. We have shown that copper and cobalt metallosurfactants can form wormlike micelles in aqueous solution which may co-exist with, or easily interconvert with vesicle structures. The cylindrical micelle structures are of nanometer dimensions and these cylindrical structures are unusual for triple chain surfactants, not easily accounted for using geometrical packing arguments. The solution behaviour has been characterised by cryo-TEM and SAXS measurements. Both the Cu and Co compounds display viscoelastic solutions at 10 wt% which coupled with the wide variety of stable metal complexes formed by the cage head group augur exciting materials for possible application in the production of mesoporous silica structures loaded with metal aggregates for a variety of catalytic applications.
2. Redox-active Surfaces as Sensors and Devices With Heniz-Bernhard Kraatz (University of Western Ontario, Canada) Reagent immobilisation on surfaces is a sophisticated pathway to materials with a wide range of applications, heterogeneous catalysis being one obvious application, such applications depending on the functionality introduced with the bound reagent, as well as upon the ease and convenience of the immobilisation procedure and the stability of the final product. Given the remarkable stability and varied electronic, magnetic and redox properties of metal complexes of the macrobicyclic polyamines known as "sarcophagines" these are species of particular appeal as entities for attachment to surfaces and for various related applications. Reduction potentials for readily accessible species span a range of 2 V, subject to modification in an interfacial environment, and outer-sphere redox processes involving Co complexes, are, for example, known to be rapid steps in reactions leading to photoinduced hydrogen production and the reduction of oxygen to hydrogen peroxide. Of practical importance in relation to immobilisation of such complexes is the facile synthesis of the ligand in forms with reactive "external" functional groups R. Our immediate aim in the present work is to synthesize a series of disulfides bearing peptido-cage complex substituents, evaluate their solution and surface electrochemical characteristics and determine a suitable methods for their immobilisation onto gold electrodes. 3. Charge density analysis of fundamental host-guest supramolecular systems several projects, with Prof Mark Spackman, UWA
Chemistry 44 Although supramolecular chemistry is one of the most active fields of modern chemistry, very little seems to be known about the detailed nature of the host and guest systems that comprise these aggregates. Supramolecular systems – molecular aggregates – underpin the design and development of materials in areas as diverse as catalysis, targeted drug delivery, gas storage, chemical separation and nonlinear optics. They also serve as models for complex phenomena such as self-assembly and ligand-receptor binding. Projects in this area are part of a research program aimed at a greater understanding of intermolecular interactions and the properties of host- guest systems in the solid state, particularly organic clathrates and complexes formed by small molecules interacting with crown ethers, calixarenes, molecular tweezers and cages (some examples are given in the figure below). These projects will involve some synthesis, and measurement of highly accurate X-ray diffraction data, complementary neutron diffraction experiments, quantum chemical calculations and computer graphics. A particular focus of the charge density analyses will be the polarization and dipole moment of guest molecules as a function of the changing electrostatic nature of the host systems.
4. New Organometallic Materials In collaboration with Weiqiao Deng, Nanyang Technological University, Singapore. Photocatalysis and its application to solar energy conversion is an important research problem for the next century particularly in light of the peak oil problem that faces current energy generation strategies.
O O i HO C2SiPr 3 This project seeks to prepare new metallotectons with OHC KOH Pri SiCCLi + 3 the ability to potentially control energy and electron OHC O i O HO C2SiPr 3 transfer processes. One way in which to do this is to recruit pendant or bridging aromatic groups for this SnCl 2 purpose and a readily available moiety for this is the i SiPr 3 pentacene unit. Aromatic units of differing structure C
C will allow us to control the HOMO-LUMO and band gap. There is a significant synthetic component [M] C C C C [M] [M] C C C C [M] involved in this project the majority of which is C C supported by solid literature procedures. i SiPr 3 The molecule in blue will allow us to target additional allenylidene complexes with interesting properties and the molecule in red will allow a systematic investigation on metal-ligand combinations and their effect on the electronic properties of the complexes. 5. Ligand Scaffolds for Neutral Carbenes, Vinylidenes and Allenylidenes Most of our work in the chemistry of unsaturated ligands has involved the formation of cationic complexes whose isolation can be complicated by the formation of
NH2 N N mixtures, which can only be separated with difficulty or MX2 + N N N N N M O O not at all. Additionally, the construction of ditopic X X metallotectons (where there are two coordination sites for metal and the potential to form polymers) would lead to di-
N cations which have limited solubility. N N N N N N Alkynols N N NaBEt H 3 M Thus, the formation of neutral complexes containing M M N2 C X X unsaturated ligands is of interest and would allow us to use N2 N2 C these construct organometallic polymers without major C R2 R1 solubility issues interfering. Here, the synthesis of metal imine complex, pictured in blue, is available form literature procedures and would provide significant experience in synthetic chemistry coupled with complex formation. The complexes formed would be expected to have catalytic activity in a range of areas.
Chemistry 45 DR MELISSA LATTER Centre for Strategic Nano-Fabrication (Incorporating Toxicology) Room 3.32, MCS building, Phone: 6488 4422 Email: [email protected] http://www.strategicnano.uwa.edu.au/
Research at the Centre for Strategic Nano-fabrication is primarily involved in process intensification on rotating surfaces (PIRS) to fabricate nano-materials but more recently applications in supramolecular assembly and controlling chemical reactivity have also been demonstrated. Process intensification has significant advantages over more traditional batch processing such as continuous flow conditions, scalability, controllability and the use of green chemistry principles. To date spinning disc processing (see schematic below) has been the only available process intensification technology available at UWA but the recent arrival of electro-spinning and later in 2008 rotating tube processing will allow even more options to be explored. This provides a very exciting opportunity for students who wish to undertake novel research with technology unique in Australia to UWA. The projects offered are multi-disciplinary and offer students excellent opportunities to further skills in chemical synthesis, analytical and characterisation techniques and where relevant probing assembly modes. Projects will be undertaken in collaboration with other Centre researchers where relevant to take advantage of the vast skill base available within the Centre. Summaries of possible research projects are listed below but interested students are encouraged to discuss with me tailoring other options.
PROJECTS
1. Synthesis using process intensification on rotating surfaces (PIRS) The use of PIRS in chemical synthesis is relatively unexplored but early results from the Centre are encouraging for achieving selective, high yielding transformations. As it is a continuous flow process, all molecules experience the same reaction conditions allowing reactions to be easily scaled for production quantities. Overall PIRS is considered to be a more efficient process that produces less waste which are important factors in green chemistry principles. The purpose of this project is to use PIRS technology to investigate reactions like those shown below and improve the reaction conditions to increase the yield of product in a more benign manner than current batch processing.
2. Resorcinarene based host-guest chemistry Resorcinarenes are macrocycles that can adopt a bowl shaped geometry and arise from the condensation of resorcinol and an aldehyde. The curved framework provides a binding pocket that has been shown to be suitable for a variety of guests as well as being a popular building block for the assembly of larger, nano-sized containers that are able to completely enclose space. Many synthetic modifications of the core structure are possible, for example, introducing functionality for water solubility, incorporation of ligands for metal complexation and
Chemistry 46 producing larger and/or more rigid cavitand structures. The aim of this project is to prepare novel resorcinarene based hosts and investigate guest binding and possible assembly modes for potential nano-material applications.
3. Surface assembly of resorcinarene based systems for potential nano-material applications Modification of the feet of resorcinarene based hosts with surface binding (SB) groups provides ideal anchors to assemble molecules on a surface. Guest complexations with surface confined host molecules may be a useful avenue for delivery or sensor type devices. Further, self-assembly of nanoparticles with molecular capsules may produce ordered arrays which nano-material applications. The goal of the project is to synthesise suitably functionalised resorcinarene host systems to facilitate surface self-assembly and characterise the resulting arrays.
4. Investigating the packing of extended alkyl footed resorcinarenes towards potential tissue engineering applications Many resorcinarenes can be considered amphiphilic as they often possess hydrophobic long alkyl chain feet and a hydrophilic upper rim. Modifying the length of the alkyl feet alters the hydrophilic/hydrophobic balance, which is reflected in the packing of resorcinarenes units. In particular it has been shown that selective vesicle formation occurs when the alkyl chain length is extended to 18 carbons. In this project you will prepare extended alkyl footed resorcinarenes in an attempt to investigate the possible modes of packing and determine if any are suitable for applications in tissue engineering.
Chemistry 47 DR MOHAMED MAKHA Room 2.10, MCS building, Phone: 6488 1572, Email: [email protected]
Macromolecular and supramolecular Group
Research interests are wide ranging and are in the area of chemical sciences, biology and material sciences and these are some of the topics where research is underway: calixarenes, host-guest chemistry, crystal engineering, phase transfer catalysis, nanomaterials, biopolymers, drug masking and green chemistry. Special interest is in the area of organic synthesis associated with the preparation and application of macrocyclic bowl shaped molecules ‘cavitands’ and derivatives, which are key tectons in supramolecular chemistry and material sciences. Available projects for this year deals with the ‘green’ synthesis of new class calixarenes and the testing of their molecular recognition and catalytic activity in organic synthesis, in addition to a combinatorial approach to synthesize unique porous materials for selective binding, separation and storage. Other projects deal with the synthesis, application and cytotoxicity of modified biopolymer chitosan (eg. films, copolymers and composites). Some of these projects involve collaboration with Prof. Colin Raston, A/Prof. Lee Yong Lim and Prof. George Yeoh. The projects are excellent training in syntheses and analytical techniques and brief details for a few are given below.
PROJECTS
1. Synthesis of amphiphilic and zwetterionic calixarenes Minimisation of the use of organic solvents and the number of steps in chemical synthesis is of growing importance from both economical and environmental considerations. In this context, solventless reactions are important and can be used in the syntheses of a wide range of compounds, including cavitands (container calixarene molecules). The project looks at preparing water soluble calixarenes from para substituted phenols (e.g. p-sulfonato-, p-carboxy- and p-amino-phenol). Direct synthesis of large ring size and deep cavity are of interest in host-guest chemistry and inclusion studies involving the binding of large molecules and ions, and catalyzing organic reactions. The project will focus on preparing one of these classes of water soluble calixarenes.
R R R
MOH (M = Na, K) Δ + HCHO Δ/ 120 oC 200 oC 6 or 8 4, 5 OH OH OH
R = SO3Na, COONa, NH4Cl
Ph-SO3Na, Ph-COONa, Ph-NH4Cl
2. Calixarene folate conjugate as platform for drug delivery with A/Prof. Lee Yong Lim Cancer chemotherapy is often associated with unpleasant side effects due to the non-selective activity of many anticancer agents. Indiscriminate deposition of cytotoxic drugs in normal cells, particularly fast dividing cells in the bone marrow and hair follicles, can lead to serious and unpleasant side effects, such as myelosuppression and alopecia. To improve the selectivity of action of administered drugs, targeting delivery systems, which bypass normal tissues, have been developed for cancer chemotherapeutic agents. The aim of this project is to develop tuneable, nanoscale calixarene platforms for the targeted delivery of cancer chemotherapeutic agents. Water- soluble calixarenes with appropriate functionalities, topology and size will be synthesized for optimized drug delivery specifically to tumour tissues.
3. Nano-catalysis with Dr Christian Sandoval and Prof Colin Raston. Asymmetric hydrogenation of simple ketones catalyzed by molecular catalysts is a key transformation in modern organic synthesis. For this purpose and amongst the best catalysts for asymmetric hydrogenation of aromatic ketones are Noyori-type Ru catalysts. The project will look at fabricating nanoscale Noyori-type catalysts in order to effect rapid and efficient conversions. The test reaction will be the reduction of acetophenone to the (S)- isomer of 1-phenyethanol.
Chemistry 48 4. Nanoporosity in self-assembled organic ionic solids Nanoporosity in self-assembled molecular frameworks are gaining prominence and offering potential solutions to many current problems involving molecular separation, sensing, heterogeneous catalysis and stabilization or the ordering of nanoparticles. Molecular assemblies of organic ions contain large functionalised channels or voids as suitable environments for controlled chemical transformations, e.g. 'nanoreactors' or as porous materials for ion exchange chromatography. The project will focus on assessment of porosity in these self-assembled materials (see insert) targeting specific molecules entrapment and quantifying retention powers.
5. Synthesis of mercapto calicxarene as metal nanoparticles stabiliser Calixarenes are superb surfactants for enhancing the dispersion and self-assembly of metal nanoparticles into well-defined structures. Thiol functional groups containing ligands are renown for their binding to metals (Ag, Au) and this project is looking at the synthesis of water soluble mercapto calixarene derivative to stabilize and functionalise silver and gold nanoparticles.
SO3Na SO3Na SO3Na
(i) K2CO3/ClCH2CH2OH (i) KSCOCH3
(ii) NaOH/TsCl (ii) NaOCH3 4 4 4 OH O O
OTs SH CO2H
6. Extracting natural products by using calixarene carboxylates The project will involve the synthesis of p-methyl-calixarenes and explore the n oxidation of the methyl groups to carboxylic acids. The water soluble calixarene OH carbolyxates will be screened for their inclusion properties towards biologically n= 4-8 active molecules and ultimately testing their ability to complex specific compounds from various plant extracts (eg. Capsaicin). Capsaicin is found in O concentrated extract of hot peppers and it is believed to have antibacterial N H properties and other uses beyond pepper spay employed in rioting. HO OCH3 7. Tissue engineering using chitosan composites scaffold with Prof. George Yeoh Chitosan is a copolymer of glucosamine and N-acetylglucosamine obtained by N-deacetylation of chitin. The chitosan-based biomaterials have been noted for its wound-healing acceleration, cartilage repairing, and bone- forming ability in numerous studies. Polyanionic calixarenes sulfonates are found to have low cytotoxicity and tend to interact strongly with the ammonium functionality of the chitosans forming composites. Hence, it is believed that the combination of calixarene sulfonate and chitosan will be suitable for the preparation of artificial extracellular matrix for tissue engineering. The aim of this project is to investigate the physical and biological properties of the Calixarene-Chitosan composites and examine the in vitro proliferation and differentiation of liver progenitor cells into hepatocytes and bile duct cells in the presence of these composites.
8. Nano-materials for agriculture with Dr Thomas Martin The project is concerned broadly with the impact of nano-fabricated herbicides, pesticides, fungicides and fertilizers on plants. We are interested in looking at plants commercially interesting for Western Australia/Australia such as wheat, canola and common weeds occurring in this area. Initially the focus will be on herbicides as we can look at their action on plants at the physiological, biochemical and molecular level. The fabricated nano-particles may be delivered in liquid or solid form or contained in a film material. The study will target the impact of such substances on the model weed plant Arabidopsis thaliana, which is extensively studied on all biological and biochemical levels.
Chemistry 49 DR ALLAN McKINLEY SENIOR LECTURER Room 2.11, MCS Building, Phone: 6488 3165 Email: [email protected]
My research interests involve: applications of spectroscopy for the detection and characterization of reactive intermediates, theoretical modelling of the bonding in radicals, analysis and remediation of contaminated groundwater, and biological applications of Electron Spin Resonance spectroscopy.
PROJECTS
1. MATRIX ISOLATION STUDIES OF REACTIVE INTERMEDIATES We have built a state-of-the-art apparatus for measuring the ESR spectra of molecules trapped in solid neon at 4 K. There are less than half a dozen labs with this type of equipment in the world, no other in Australia. This is - cutting edge work and some of our recent successes CdCH3 [1], ZnCH3 [2], MgCH3 [3], Al2 [4] and HgCH3 [5] are published in top international chemistry journals. We have also completed the experimental phase for CdP, MgN, MgP, ZnN, ZnP, MgCH2 and MgCH radicals and articles on these molecules are in preparation. The results of our studies are important to improve understanding of models of chemical bonding as well as the chemical mechanisms involved in manufacturing computer chips, the wear-resistant coatings, and even the chemical processes occurring in circumstellar dust clouds.
2. Radicals of Environmental or Astrochemical Relevance We have been studying radical adducts formed between OH radical or O2 molecule and a water molecule. To date we have published three papers in this area [6-8]. We want to extend this work and investigate nitrogen containing radical adducts with water. Such species could be intermediates in the chemical reactions occurring in the atmosphere of, or on the surface of Titan. The atmosphere of Titan is mainly nitrogen with traces of water and organic compounds. Such studies are particularly relevant at the moment as the NASA Cassini-Huygen space probe has reached Saturn and one of the targets of this mission is to gather information about the chemistry of the atmosphere of one of Saturn's moons Titan. One of our collaborators on this project is Professor R. E. Johnson from the University of Virginia. Professor Johnson is a team leader on the Cassini-Huygen programme and will be able to supply us with spectroscopic data from the mission. We intend to perform ESR and optical spectroscopic measurements of the nitrogen-containing radicals mainly in argon matrices but some experiments are likely to be performed in ice matrices.
3. Environmental Chemistry of Contaminated Groundwater. For some years now we have had a collaboration with Drs Greg Davis and Brad Patterson at the Land and Water division of CSIRO at Floreat. In Australia, water is a key resource. In WA much of our water reserves are underground and very vulnerable to pollution. We have studied the degradation in groundwater of BTEX hydrocarbons (from leaking petrol stations), the mobility of pesticides such as atrazine and fenamiphos in soils and we are evaluating the possibility of employing a new method for remediation of contaminated groundwater using polymer-mats to introduce reagents into groundwater to promote microbial consumption of the pollutants. As well as remediation of groundwater contaminated by BTEX and other volatile organics [10] we have studied denitrification of ammonium nitrate contaminated groundwater[9]. There is a plume of ammonium nitrate flowing into Cockburn Sound and we have tested this remediation technique on this plume [11]. In this field study oxygen was introduced first to oxidize the ammonium ions to nitrate, and then ethanol was introduced downstream to reduce the nitrate ions directly to nitrogen gas. While this study was reasonably successful there are still many issues to clarify here and we want to extend this work to other pollutants. Projects in this area would involve either the analysis of the chemistry occurring in, or the mathematical modeling of the mass transport phenomena involved with, pilot scale test-rigs for groundwater remediation which are set up at CSIRO in Floreat.
Biological Applications of ESR Spectroscopy: 4. Detection of Reactive Oxygen Species. - Radicals play a very important role in biology. Reactive oxygen-containing radicals e. g. OH and O2 are believed to be involved in many degenerative processes for example aging, atherosclerosis and renal failure. Some of the complications arising from diseases such as diabetes are thought to involve reactive oxygen species. ESR spectroscopy combined with the spin trapping technique has been used extensively as an extremely
Chemistry 50 sensitive and selective technique for quantifying and identifying oxygen containing radicals in biological samples [12]. This project would involve collaboration with Associate Professor Kevin Croft at Royal Perth Hospital to develop the methodology to perform these types of ESR experiments at UWA.
5. Site Directed Mutagensis and Spin Labeling for the Determination of Protein Structure We are collaborating with Professor Boris Martinac from The University of Queensland to use site directed mutagenesis combined with spin labeling to interrogate the conformation of ion channel proteins. We have developed a new apparatus to measure the ESR spectra of proteins that have been labeled with a nitroxide radical [13]. The label is introduced onto a specific position of the protein backbone by incorporating a cysteine residue at that position using site-directed mutagenesis then binding the nitroxide label selectively to the introduced cysteine residues. By analyzing the ESR signals of the spin label and by assessing the effects of adding a relaxation agents to the aqueous or lipid phase we can infer the secondary structure of the protein. With this approach we are examining functional ion channels and it is hoped we can characterize the open as well as closed forms. This work is only performed by a handful of groups in the world [14].
6. Development of New Antibiotics. Multidrug-resistance in pathogenic strains of bacteria has in the last decade presented an increasing problem in treatment of bacterial infections and diseases. The re-emergence of tuberculosis (TB), for instance, is one of the serious threats, which is spreading rapidly throughout the world. Furthermore, many strains of enterococci have acquired resistance to vancomycin, the last antibiotic that was still able to fight them successfully for example last year many wards at Royal Perth Hospital were plagued by VRE (vancomycin resistant enterococci). Professors Boris Martinac (UQ), Matthew Wilce (Monash), Professor Tom Riley (Microsbiology, UWA) and I have received NHMRC funding for a joint project to exploit a novel target for anti-bacterial activity and we hope to develop a new generation of antibiotics. This project would involve characterization of the target using ESR spin labeling methodology, although molecular modeling approaches are possible as are microbiological studies with Professor Riley for appropriately qualified students.
References: Copies are available from Dr Allan McKinley.
1.Karakyriakos, E.; Davis, J. R.; Wilson, C. J.; Yates, S. A.; McKinley, A. J.; Knight, L. B. Jr.; Babb R.; Tyler, D. J. “Neon and 12 12 13 12 111 12 113 argon matrix ESR and theoretical studies of the CH3Cd, CD3Cd, CH3Cd, CH3 Cd, and CH3 Cd Radicals” J. Chem. Phys. 1999, 110, 3398-3410. 2.McKinley, A. J.; Karakyriakos, E.; Knight, L. B. Jr.; Babb, R.; Williams, A. “Matrix isolation ESR studies of the various isotopomers of the CH3Zn and ZnH radicals; comparisons with ab initio theoretical calculations” J. Phys. Chem. A 2000, 104, 3528-3536. 3.McKinley, A. J.; Karakyriakos, E. “Neon matrix isolation ESR and theoretical studies of the various isotopomers of the CH3Mg radical” J. Phys. Chem. A 2000, 104, 8872-881. 4.Stowe, A. C.; Kaup, J. G.; Knight, L. B. Jr.; Davis , J. R.; McKinley, A. J. “Matrix-isolation investigation of the diatomic - - anion radicals of aluminium and gallium (Al2 and Ga2 ): An electron resonance (ESR) and ab initio theoretical study.” J. Chem. Phys. 2001, 115, 4632-4639 5.Karakyriakos, E.; McKinley, A. J. “The Matrix Isolated HgCH3 Radical: An ESR Investigation” J. Phys. Chem. A. 2004, 108, 4619-4626. 6.Langford, V. S.; McKinley, A. J.; Quickenden, T. I. "Identification of OH·H2O in argon matrices." J. Am. Chem. Soc. 2000, 122, 12859-12863. 7.Cooper, P. D.; Kjaergaard, H. G.; McKinley, A. J.; Quickenden, T.I.; Schofield, D. P. "Infrared measurements and calculations on H2O·HO" J. Am. Chem. Soc. 2003, 125, 6048-6049. 8.Cooper, P. D.; Kjaergaard, H. G.; Langford, V. S.; McKinley, A. J.; Quickenden, T. I.; Robinson, T. W.; Schofield, D. P. "Infrared Identification of Matrix Isolated H2O·O2" J. Phys. Chem. A. 2005, 109, 4274-4279. 9.Patterson, B. M.; Grassi, M. E.; Davis, G. B.; Robertson, B.; McKinley, A. J. “The use of polymer mats in series for sequential reactive barrier remediation of ammonium-contaminated groundwater: laboratory column evaluation.” Environ. Sci. Technol. 2002, 36, 3439-3445. 10.Patterson, B. M.; Davis, G. B.; McKinley, A. J. “Polymer mats to remove selected VOCs, PAHs and pesticides from groundwater: laboratory column experiments” Ground Water Monit. Rem. 2002, 22, 99-106. 11.Patterson, B. M.; Grassi, M. E.; Robertson, B. S.; Davis, G. B.; Smith, A. J.; McKinley, A. J.; “The Use of Polymer Mats in Series for Sequential Reactive Barrier Remediation of Ammonium-contaminated Groundwater: Field Evaluation.” Environ. Sci. Technol. 2004, 38, 6846-6854. 12.Rosen, G. M.; Britigan, B. E., Halpern, H. J.; Pou, S. Free Radicals: Biology and Detection by Spin Trapping; Oxford University Press: New York, 1999. 13.Tsai, I.; Liu, Z.; Rayment, J. Norman, C.; McKinley, A. J.; Martinac, B. M.; “ The Role of the Periplasmic Loop Residue Glutamine 65 for MscL Mechanosensitivity” Euro. Biophys. J. 2005, 34, 403-412. 14.Perozo, E.; Marien Cortes, D.; Sompornpisut, P.; Kloda, A.; Martinac, B. “Open channel structure of MscL and the gating mechanism of mechanosensitive channels.” Nature 2002, 418, 942-948.
Chemistry 51 DR MATTHEW PIGGOTT LECTURER Room 3.29, MCS Building, Phone: 6488 3170 Email: [email protected]
Medicinal and Synthetic Organic Chemistry
Our expertise in organic and medicinal chemistry is applied to the design and synthesis of therapeutic drugs and small molecule probes to help investigate complex biological systems. We have active collaborations with neurobiologists, biochemists, cell biologists, immunologists, microbiologists and molecular biologists. The synthesis of biologically active natural products and compounds with novel, aesthetically pleasing architecture, are other areas of interest.
PROJECTS
1. “Urinomics” With Professors Barry Marshal and Kevin Croft
Helicobacter pylori is the bacterium that causes most gastric ulcers. Its discovery won UWA’s Professor Barry Marshall and Dr Robin Warren the Nobel Prize in Physiology or Medicine in 2005. This project will involve the use of gas and liquid chromatography coupled with mass spectrometry to search for diagnostic metabolites in the urine of infected patients. Once identified, a simple chemical test for the metabolite will be investigated.
2. Redesigning the designer drug, ‘ecstasy’
Recently, MDMA has been shown to induce apoptosis in Burkitt's lymphoma and related cancer cell lines. Burkitt’s lymphoma is rare in the western world, but is endemic in H Africa where it primarily affects O N children. The disease manifests as horrific facial tumours, which can O double in size in 1 day, and is fatal if left untreated. In collaboration with MDMA Professor John Gordon (Institute of Biomedical Research, Birmingham) we have discovered an analogue that is 300 times more potent than the lead compound, MDMA, and is selectively toxic to cancer cell lines. Our search for even more potent and selective compounds is ongoing.
Chemistry 52
3. Total Synthesis of Biologically Active Natural Products
Inspiration for synthetic targets comes from biologically active natural products that present an opportunity to investigate new methodology or strategy which improves on literature precedents. An overriding aim is to develop concise syntheses so that useful quantities of end-products, intermediates and analogues can be provided for biological testing. A sample of the synthetic targets in our sights is shown below.
O HO O OH OH O O N N O N N O N N N O
N N N O
sebastianine B arnoamine A alpkinidine Sch 538415 human colon tumour cytotoxin cytotoxic cytotoxic antibiotic/anticancer
4. Cool molecules
Some of these beautiful molecules may be easier to make than you think. Want to be the first?
Chemistry 53 PROFESSOR COLIN RASTON Director, Centre for Strategic Nano-Fabrication (Incorporating Toxicology) Fledgling Centre for Green Chemistry and Molecular Discovery Room 3.09, MCS Building, Phone: 6488 3045 Email: [email protected] http://www.strategicnano.uwa.edu.au/
Nano-chemistry, Nano-technology, Organic Synthesis, and Green Chemistry and Molecular Discover Current research projects cover: (i) Inter-related process intensification using spinning disc processing and rotating tube processing, fabrication of nano-materials, nano-chemistry, supramolecular chemistry, and crystal engineering. (ii) Benign process technology – process intensification in organic synthesis, green sustainable chemistry, and drug discovery and drug delivery. Integration of these areas has led to novel chemistry and applications. Projects for 2009 deal with organic synthesis, process intensification, drug discovery, structure, theory and applications in new materials, separation science, device nano-technology, controlling chemical reactivity and selectivity, molecular recognition, catalysis, and drug delivery. The projects are excellent training in a wide range of techniques, including chemical engineering, nano-technology, inorganic and organic synthesis, X-ray diffraction, NMR, electron microscopy, analytical techniques, and other characterisation techniques. Brief details of some projects available are given below. Other projects are also available depending on the interests of the researcher. Some projects are in collaboration with colleagues in other disciplines including engineering, pharmacy, medicine and biochemistry. Our recent findings on the application of process intensification has important implications in organic synthesis with the ability to control chemical reactivity and selectivity, and in controlling the size, shape, agglomeration, phases and defects in nano-particles while under continuous flow, and thus the chemistry is scalable with the research reaction potentially the production reactor. There are summer vacation scholarships available, including one linked to an Honours Scholarship sponsored by AGR Matthey.
PROJECTS 1. Nano-chemistry using process intensification on rotating surfaces (PIRS) with Dr Swaminathan Iyer, Dr Craig Bullen & Assoc Prof Tim St Pierre (Physics). Several projects are available and several students can work in this area. We have recently established that spinning disc processing (SDP) can be used to prepare nano- particles in a controlled way, for silver nano-particles (medical and chemical catalysis applications), magnetite (medical imaging), gold (medical technology), and drug molecules (drug delivery). Related to this is new technology based on rotating tube process (RTP), which allows controlling the residence time of the reaction mixture on the rotating tube. This is in collaboration with the company NanoDynamics. We now plan to develop PIRS chemistry to make a range of different shapes and arrays like those shown in the cartoon. New materials will have potential in synthesis and medical applications, for example as multi-functional imaging and drug release reagents, as well as in fuel cell and solar cell technology. The PIRS facility at UWA is one of a few such facilities in the world, to be further enhanced with the RTP and a larger SDP with accessible temperatures ≥ 600oC. Gold nano-particle chemistry is supported by AGR Matthey, with other projects in collaboration with industry, including drug delivery (with iCeutica), and solar cell technology (with Dyesol).
2. Controlling chemical reactivity and regio-selectivity in organic synthesis using process intensification on rotating surfaces (PIRS) with Keith Stubbs and Melissa Latter. We have established the remarkable utility of PIRS in preparing organic compounds, and projects here will focus on further applications in organic synthesis targeting molecules with biological activity. There are two noteworthy effects of PIRS: (i) Plug flow conditions which control chemo-selectivity without the need for protection and de-protection.
Chemistry 54 (ii) The ability to control kinetic and thermodynamic outcome of chemical reactions which is not possible using classical stirred flask reaction vessels. All this is under continuous flow conditions. In consequence of these findings we are mapping out the plethora of organic reactions to establish the versatility of PIRS in organic synthesis in general, and then to use the technology to prepare molecules with particular function for biological applications. In the first instance we have used PIRS to prepare new classes of pyridine compounds which have application in medicine, including diabetes inhibitors, and anti-cancer and anti-inflammatory activity (in collaboration with Prof Geoff Stewart). Other applications of PIRS in organic synthesis include: (i) The use of tailed nano-particles in catalysis. (ii) Generation of nano-particles of organic compounds for photolysis for high yield, high stereo- and enantio-selective solid state conversion, such as C-C bond formation with elimination of CO. 3. Probing molecular structure using process intensification with Dr Swaminathan Iyer and Dr Solvent/SDP
Melissa Latter Molecular capsules based on six C- - H 2 alkylpyrogallol[4]arenes held together by a seam of 72 H-bonds, can be disassembled/assembled using SDP (dynamic light scattering). A flow of H2 gas over the disc at ambient pressure and temperature results in high mass transfer of H2 into solution. Instantaneous re- assembly of the capsules post SDP results in Solvent/H2 confinement of H2 in the capsules (NMR). [(Solvent/H 2) ⊂(Calixarene)6] The internal capsule concentration of hydrogen is representative of the concentration of hydrogen in chloroform under SDP – nano-sampler. This breakthrough technology can be used to probe the structure of matter, and to disassemble DNA, large assemblies of protein molecules in the form of ferritin and plant viruses, and liposomes, for drug delivery, and more. 4. New device technology for sensors and energy with Dr Swaminathan Iyer and Craig Bullen Recently Lee Hubble (PhD applicant in the research group) developed technology for detecting hydrogen gas and detecting and discriminating organic molecules in the gas phase. This has exciting possibilities in sensor technology for fuel cells (including hydrogen gas), forensics (explosives and their breakdown products) and solar cell technology. The core of the device is based on carbon nano-tubes which can be decorated with selected nano- materials to tailor specific applications. 5. Composite nano-materials for multi-purpose medical applications with Dr Swaminathan Iyer. Recently we have Mesoporous silica shown that it is possible ot construct nano-particles comprised of superparamagentic particles coated with a therapeutic material (curcumin), encapsulated with mesoporous silica. This has exciting possibilities for targeted drug delivery using a magnetic field, as well as us as contrasting agents in magnetic resonance imaging. The proposed research will focus on constructing particles binding other drug molecules, as well as studying the all important release profile of the drug under physiological pH. 6. Applications of phosphonated calixarenes in tissue engineering same time able to bind drug RO OR P(O)(OH)molecules in t h eir cavities (see with Prof Fiona Wood and Prof Sarah Dunlop Relatively unexplored 1 2 OR OR phosphonated calixarenes, 1, R = H, n = 4, 5, 6 and 8, have been insert). nano-textured features suitable for application in tissue prepared. This allows access to derivatives with alkyl chains attached n PO H PO H engineeriH2O3P ng. In3 2additio3 2 n, the to the phenolic O-centres, and various functionalised moieties in the OR PO3H2 Molecular calixarenn+ e s c an actn+ as same position, including unsaturated chains (for photolytic cross M encapsulation M linking) and groups with specific binding prowess (towards metal surfactants in stabilising nano- H2O3P PO3H2 particles (for imagingPO3H2 and centres and organic molecules). Long alkyl chain (R) derivatives H2O3P assemble into intertwining nano-fibres with the overall material having manipulation using a magnetic nano-textured features suitable for application in tissue engineering. In fields), at the same time able to bind drug OmoleO O Ocules in their addition, the calixarenes can act as surfactants in stabilising nano- Molecular Interaction particles (for imaging and manipulation using a magnetic fields), at the cavcapsuleities (see insert)Complementarity . same time able to bind drug molecules in their cavities (see insert). Nano-particle
Chemistry 55 Dr. SAM SAUNDERS Room 3.10, MCS building, Phone: 6488 3153, Email: [email protected]
ACER – Atmospheric and Environmental Chemistry Research Group My research interests have wide environmental implications. One of my keen interests is to measure anthropogenic impacts, to develop practical tools for environmental impact assessment. The research projects range from laboratory based chromatographic analysis of environmental samples, field campaign measurements, to chemical simulations of the atmosphere using established software tools. Study regions extend from local WA to wider Australia, Hong Kong and as far a field as the UK.
PROJECTS
Atmospheric Science and Air Quality Issues a) Integrated Air Monitoring Campaign Field measurements and analysis. Health impacts attributed to industrial air emissions has become a major social and environmental issue especially in Western Australia, which has a large mining and refining industry. In order to reliably identify the environmental signature of specific sources, (to assess their impact on air quality) requires a comprehensive, multi-dimensional investigation involving highly accurate and detailed measurements supported by computer modelling studies, over an extended time period, to cover both seasonal and background variability. The DEC is actively supporting and promoting the development of technical expertise within the state, towards understanding and characterising the unique and complex atmospheric processes of various regional air masses in WA. To advance current air contaminant measurement capabilities a Proton Transfer Reaction Mass Spectrometer (PTRMS) instrument was commissioned in 2006. For atmospheric trace gas analysis PTRMS is the state-of-the-art method, having resolution down to sub-ppb concentrations (100 to 500 ppt depending on compound) and the ability to cyclically sweep for many compounds every few minutes. The latter capability is vital for capturing short term events and is unique to the PTRMS.
Further project developments include the main objectives: • optimise the PTRMS utilization; • development of the method of ambient air monitoring; • develop efficient data collection and analysis; and • generate validation and processing protocols
Ambient air inlet
+ H3O ion source and drift tube
PTR-MS side view PTR-MS top view
Chemistry 56 The projects will provide dedicated personnel for PTRMS method development within a comprehensive research program and results generate new baseline measurement data within the State by investigating both seasonal and background variability, in biogenic and anthropogenic VOC sources. b) Simulating the experimental data from the CSIRO smog chamber facility An important area in the development of air quality policy is in the use of experimental data from smog chambers. Simulation of the experiments is used to help validate reaction mechanisms used in air quality assessment. For this project data from several experiments conducted at the CSIRO smog chamber facility are available. The project aims to accurately simulate the experimental data, and investigate the impact of mechanistic details. Initial work has focussed on 3 different VOC (toluene, 1,3-butadiene and isoprene) under different [VOC] and [NOx] experimental conditions. c) Assessment of Hong Kong emissions, monitoring and meteorological data to develop a regional chemical mechanism for simulating observed ozone formation and ambient VOC measurements in Hong Kong Collaborating with the Hong Kong EPD, and Hong Kong Polytechnic University this project gives the opportunity to make a significant contribution in developing a comprehensive tropospheric chemical degradation mechanism, to provide simulations of the extensive sets of VOC and ozone measurements from the Hong Kong monitoring network. During a recent visit to Hong Kong in June 2004, the daily ozone measurement exceeded 200ppb, almost twice the level recognised as having detrimental impacts on human health. Only through developing an understanding of the chemistry occurring in this airshed, will it be possible to work towards viable remediation strategies and reduce the air pollution episodes in the region.
Chemistry 57 PROFESSOR MARK SPACKMAN ARC PROFESSORIAL FELLOW Room 4.11, MCS Building, Phone: 6488 3140 Email: [email protected]
Crystallography and theoretical chemistry
Our research investigates in detail the structure of crystals, in particular the electron distribution and properties related to it, such as electric moments of molecules (dipole, quadrupole, etc.), electrostatic potential and electric field, and also measures of its response to external perturbations, including polarizability and hyperpolarizability. All research projects in this area incorporate different aspects of physical and theoretical chemistry. They utilise ab initio computational methods along with some computer programming and computer graphics and, where applicable, measurement and detailed analysis of high-resolution, low-temperature X-ray diffraction data. The Honours projects listed below will provide valuable practical experience with the techniques of modern computational chemistry and a familiarity with state-of-the-art ab initio quantum chemical calculations, as well as some practical experience in the use and applications of X-ray crystallography. The amount of hands-on experience with computer programming and graphics on the one hand, and experimental measurement of X-ray diffraction data on the other hand, can be tailored to suit the project and the candidate.
PROJECTS
1. Electrostatic complementarity as a guiding principle in molecular crystals with A/Prof Dylan Jayatilaka
In recent years much of our research has focused on a detailed exploration of the attributes and uses of Hirshfeld surfaces, which are now making a substantial contribution to the improved understanding of intermolecular interactions in bulk materials, and especially crystal engineering (the understanding of intermolecular interactions in the context of crystal packing and exploiting that understanding in the design of new solids with desirable physical and chemical properties). Details and examples of this exciting work can be found at the web site associated with this project: http://www.hirshfeldsurface.net/, and CrystalExplorer, developed in collaboration with Dylan Jayatilaka's group is described at http://hirshfeldsurface.net/CrystalExplorer. This Honours project will explore in more detail the electrostatic potential mapped on these surfaces, especially the way in which the electropositive part of one molecule coincides with the electronegative region of an adjacent molecule (an example is given in the figure). This qualitative picture of intermolecular interactions will be compared with the more quantitative results obtained with ab initio calculations of intermolecular interaction energies, and for a range of molecular crystals incorporating hydrogen bonds, halogen bonds and other interactions.
Chemistry 58 2. Charge density analysis of fundamental host-guest supramolecular systems several projects, with Dr George Koutsantonis
Although supramolecular chemistry is one of the most active fields of modern chemistry, very little seems to be known about the detailed nature of the host and guest systems that comprise these aggregates. Supramolecular systems – molecular aggregates – underpin the design and development of materials in areas as diverse as catalysis, targeted drug delivery, gas storage, chemical separation and nonlinear optics. They also serve as models for complex phenomena such as self-assembly and ligand-receptor binding. Projects in this area are part of a research program aimed at a greater understanding of intermolecular interactions and the properties of host- guest systems in the solid state, particularly organic clathrates and complexes formed by small molecules interacting with crown ethers, calixarenes, molecular tweezers and cages (some examples are given in the figure below). These projects will involve some synthesis, and measurement of highly accurate X-ray diffraction data, complementary neutron diffraction experiments, quantum chemical calculations and computer graphics. A particular focus of the charge density analyses will be the polarization and dipole moment of guest molecules as a function of the changing electrostatic nature of the host systems.
3. Reactivity in crystals and its relationship to voids and cavities with Dr Ben Corry and A/Prof Dylan Jayatilaka
Reactivity in crystals has been the focus of increased activity in recent years, in particular the recent kinetic studies of E/Z photoisomerizations occurring in co-crystals, [2+2] photodimerizations in organic crystals (for example, (a) to (b) in the adjacent figure) and single-crystal to single crystal transformations in molecular framework materials. Many studies such as these use concepts of "reaction cavity" and "void space" to rationalize the observed reaction products, and in particular the differences between solution and solid state products. The Hirshfeld surface (see Project 1, above) is a measure of the space occupied by a molecule in a crystal, and hence it should be able to provide a considerable amount of relevant information. This project will explore the correlation of void locations and volumes with experimental information on crystal reactivity of various kinds. This information will be used to rationalize the way in which the void space influences reactivity, and if possible relate these conclusions to the results of MD simulations being undertaken by Alexandra Nemkevich.
Chemistry 59 DR SCOTT STEWART Room 3:30, MCS building, Phone: 6488 3180 Email: [email protected]
Research Interests Research interests include the construction of biologically active natural products utilising modern organic synthetic methods. Many these syntheses are designed using palladium catalysed cross coupling reactions as the key step transformation. Two natural products the hexahydroanthracene mensacarin (1),1,2 and arboflorine (2)3 have been targeted within this group because OMe OMe OH O of their interesting molecular architecture OH O A and biological activity. Related to this field, B C N methodological studies involving the A B O N H improvement various reactions including, R H Suzuki, Buchwald-Hartwig and O O OH N intramolecular Heck reactions through the 1 2 H modification of nickel(0) and palladium(0) catalytic conditions are currently being explored. Also, associated with this area projects which deliberately target domino transformations mediated by palladium are in the initial stages. Medicinal chemistry interests include the synthesis of libraries of small molecules for the treatment of cancer. Recently, a library of new thalidomide analogues have been synthesised for the inhibition of tumour necrosis factor (TNF) expression.
Project 1: The Synthesis of Amphibian Alkaloids through the Tsuji-Trost Reaction
Several classes of compounds can be found in the skin of Amphibians displaying a wide range of biological activity. One such class of compound includes the pumiliotoxins of the general indolizidine structure 3, where R is an alkyl side chain.4 Several compounds in the pumiliotoxins subclass have cardiotonic and myotonic activity through interactions with unique binding sites on the voltage dependent ion channel. This project will focus on the synthesis of the indolizidine ring system starting with L-proline (4) with the appropriate stereochemsistry at the -carbon. The key step for this proposed synthesis is an intramolecular Tsuji-Trost cross coupling reaction. Such palladium mediated cross coupling reactions have regularly been applied in complex natural product syntheses.5
O OH H H HO
N R N H
4 3
Project 2: The Synthesis of Pyrrolosesquiterpene Natural Products
The pyrrolosesquiterpene 5 has recently been isolated from a Streptomyces sp. (NPSOO8187) found in a marine sediment collected in Alaska.6 This compound belongs to a polyene pyrrole family of compounds which in the past have drawn much attention due to their biological activity and unique structure. This project involves the synthesis of 5 through pathways witch will involve the initial preparation of the iodinated pyrrole ring precursor 6. The key transformation in this proposed total synthesis will involve the cross coupling of fragment 6 and derivatives of geraniol (7). OH I HO OH O O 7 NH NBs 5 6
Chemistry 60 Project 3: Exploring Domino Transformations (with Dr. M. Połomska and C. Heath)
Domino transformations are defined as the execution of two or more bond-forming transformations under identical reaction conditions, in which the latter transformations take place at the functionalities formed by preceding transformations.7 Recently within our group we have discovered a palladium mediated domino process which involves firstly a Tsuji-Trost transformation followed by a Heck reaction. With this reaction sequence we can construct ring systems such as the as 3-benzazepines and azepino[4,5-b]indole ring systems in a single pot. This project will focus on exploring similar transformations involving ally ethers among others to explore the utility of this domino reaction sequence.
NHR () () Br OH Br O N R "Pd(0)" I I "Pd(0)" 918 011
Project 4: Synthesis of Thalidomide Derivatives (with A Prof L. Abraham)
Since its discovery, thalidomide [(R,S)-2-(2,6-Dioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione (R,S)-2 has had a tumultuous history as a medicinal agent. Administered in the 1950’s as a treatment for insomnia and as an antiemetic agent, the racemic compound (R,S)-12 was assumed to be non-toxic. Later investigations found that while the R-isomer (R)-12 was responsible for the sedative effect the S-isomer (S)-2 had teratogenic properties (causing foetus deformities). As a result, in 1962 this popular drug O O H (R,S)-12, as prescribed for morning sickness, was withdrawn. 1 N 1' Currently, thalidomide (R,S)-12 use is undergoing a N 3' O resurgence in the medical world. Attention has been focused on a 5 widespread application in the research for treatment of various types 3 H 5' 4 of cancer including, multiple myeloma (MM), an as yet incurable O form of bone marrow cancer. It is the aim of this project, through the 12 invention of new synthetic pathways, to provide more active O O H analogues of thalidomide through the inhibition of the expression of 1 N 1' tumour necrosis factor (TNF). TNF is a cytokine which is described 3' N O as the central regulator of the inflammatory cascade the main being anti-angiogenic, anti-inflammatory and immunomodulatory. 3 H 5' 4 O Compound 13, has recently been produced within the group, and has shown to have THF inhibitory activity twice that of thalidomide 12.8 BuO 13 This project will involve the synthesis of new related thalidomide analogues through modern synthetic methodology.
References 1. L. F. Tietze.; S. G. Stewart.; M. E. Polomska.; A. Modi.; A. Zeeck, Chem, Eur. J. 2004, 10, 5233. 2. L. F. Tietze.; S. G. Stewart.; M. E. Polomska, Eur. J. Org. Chem. 2005, 1752. 3. Lim, K-H.; Kam, T-S. Org. Lett., 2006, 8, 1733. 4. Daly, J. W.; Spande, T. F.; Garraffo, H. M. J. Nat. Prod. 2005, 68, 1556. 5. Nicolaou, K. C.; Bulger, P. G.; Sarlah, D. Angew. Chem. Int. Ed. 2005, 44, 4442. 6. Macherla, Venkat R.; Liu, Jehnan; Bellows, Christopher; Teisan, Sy; Nicholson, Benjamin; Lam, Kin S.; Potts, Barbara C. M. J. Nat. Prod, 2005, 68(5), 780 7. Tietze, L. F.; Brasche, G.; Gericke, K. M. Domino Reactions in Organic Synthesis, Wiley-VCH 2006. 8. Stewart, S. G.;Spagnolo, D.; Polomska, M. E.; Sin, M, Karimi, M.; Abraham, L. Bioorg. Med. Chem Letts. 2007, 17, 5819.
Chemistry 61 DR KEITH STUBBS ARC AUSTRALIAN POSTDOCTORAL RESEARCH FELLOW Room 4.14/Lab 4.22, MCS Building, Phone: 6488 1755 E-mail: [email protected]
Research Interests Carbohydrates are present in every living system from prokaryotes to eukaryotes and traditionally, have been known for their role in the structural integrity of plants and as energy sources. Recently, however, carbohydrates have been shown to be involved in a variety of fundamental biological processes such as protein folding and trafficking, as well as cellular signaling and recognition. As we gain greater understanding into the roles that carbohydrates play at the cellular level, scientists are faced with new challenges. On the chemistry side, unique carbohydrate-based tools need to be developed and in turn used to investigate the specific roles that a single mono- or polysaccharide plays in the dynamics of the cell in order to keep up with the biochemical discoveries of new glycan structures and the enzymes that regulate them. My research aims are to address the development of such tools.
All the projects outlined below will initially involve the synthesis of compounds using both new and literature methods. Once the compounds have been prepared, investigation(s) using biochemical and microbiological assays will be conducted.
PROJECTS
1. Developing inhibitors of an enzyme critical to antibiotic resistant bacteria. Everyone in society benefits from the use of antibiotics for the treatment of bacterial infections. One problem associated with the widspread use of antibiotics is the ability of bacteria to develop what is called antibiotic resistance. One class of antibiotics used today is the -lactams, which constitute nearly 50% of clinically used antibiotics. The use of -lactams is currently under serious threat because of the increasing prevalence of bacteria with enzymes ( - lactamases) that destroy them. The bacteria in question produce these enzymes in such a manner that is greatly dependent on the activity of two proteins: AmpR and a carbohydrate processing enzyme, NagZ.
Research into the preparation and testing of potent inhibitors of NagZ will be done to determine their effectiveness in reversing -lactam antibiotic resistance in Gram-negative bacteria. This research will lead to the development of alternative eradication therapies.
Students with interests in synthetic chemistry or both synthetic chemistry and microbiology are very well suited for this project.
2. Using affinity-based proteomics probes (ABPP) to investigate proteomes.
Glycoside hydrolases are an extremely large class of enzymes that act to cleave OH carbohydrate residues. They are found throughout nature in organisms ranging from HO O bacteria to humans and have been classified into over 100 families on the basis of HO F HN structural similarity. In light of their biological significance, the rapid detection of F proteins involved in processing glycoconjugates is of considerable interest. For example, O the study of NagZ (Topic 1) was greatly facilitated when the development of an ABPP, N3 2AA5FGF, was achieved to detect the NagZ protein at nanogram levels in bacteria. 2AA5FGF
Chemistry 62 One project involves the profiling of proteomes of Gram-negative bacterium, such as H. influenzae, S. marcesens, B. cepacia, P. mirabilis and E. cloacae, to identify the presence of NagZ.
Another project involves using chemical synthesis to design new ABPPs to look for different carbohydrate processing enzymes in proteomes. This research will give leads for the development of new strategies to treat antibiotic resistant bacterial infections.
Students with interests in synthetic chemistry or both synthetic chemistry and biochemistry/microbiology are very well suited for this project.
3. Development of new scaffolds to inhibit carbohydrate processing enzymes such as O-GlcNAcase and OGTase.
The enzymes that regulate the structures of glycans are extremely important and have been implicated in a wide variety of diseases. For example, the post-translational modification of serine and threonine residues of nucleocytoplasmic proteins with N-acetylglucosamine (GlcNAc) is a reversible and dynamic process implicated in multiple cellular processes in eukaryotes. The enzyme O-GlcNAcase catalyzes the cleavage of O-GlcNAc from modified proteins whereas the enzyme OGTase catalyzes the addition of these monosaccharide units. Dysregulation of this modification has been linked to Type II Diabetes, Alzheimer’s disease and cancer.
Recently, compounds have been prepared that are very potent inhibitors of O-GlcNAcase and have been shown to be excellent candidates for a prevention of Alzheimer’s disease.
One project described here would involve expanding the repertoire of compounds that are inhibitors of O- GlcNAcase and OGTase. The prepared compounds will be tested for their potency against the human enzymes and they will also be tested in vitro to determine their effectiveness at the cellular level. These compounds will provide new leads for the treatment of Alzheimer’s disease.
Another project under this heading will be the design and synthesis of new inhibitor scaffolds that can be used to investigate the role that carbohydrate processing enzymes play in other diseases.
Students with interests in synthetic chemistry or both synthetic chemistry and biochemistry are very well suited for this project.
4. Investigations into the glycobiology of Helicobacter pylori. (with Prof. Barry Marshall and A/Prof. Mohammed Benghezal)
Helicobacter pylori is a Gram-negative, microaerophilic bacterium that infects the stomach and duodenum. It has been shown that many cases of peptic ulcers, gastritis, duodenitis, and stomach cancers are caused by H. pylori infections. Whilst a lot of information has been gathered on the genetics and pathology of H. pylori infection, the role that carbohydrates play in this bacterium’s life cycle and in mediating host-pathogen interactions is lagging.
The projects here will investigate, through chemical synthesis and molecular biology, what roles carbohydrates and larger glycan structures play in the pathogenesis of H. pylori.
Students with interests in synthetic chemistry or both synthetic chemistry and microbiology are very well suited for this project.
Please feel free to come and chat with me about any one of these projects if you are interested.
Chemistry 63 PROFESSOR ALICE VRIELINK Room 4.31, MCS Building, Phone: 6488 3162 Email: alice.vrielink@ uwa.edu.au
Protein Structure by X-ray Crystallography The studies in my lab focus on crystallographic analysis of a variety of proteins with the aim of using structural analysis to better understand their biology. The structural biology laboratory is well equipped with state of the art robotic crystallization equipment, X-ray diffraction equipment and computational facilities for structure solution and analysis. Expression and purification resources are available in the laboratory in order to obtain sufficient quantities of protein for crystallographic studies. In addition we carry out kinetic and spectroscopic analyses to establish the quality of protein and pursue biochemical and biophysical studies to better correlate function with structure.
PROJECTS
1. Endotoxin Biosynthesis in Neisseria. The gram negative bacteria, Neisseria meningitidis, is the causative agent of meningitis and is responsible for significant mortality throughout the world. A characteristic feature of these bacteria is the presence of lipooligosaccharide (LOS) molecules on their outer membranes. These complex molecules, also called endotoxins, are structural components that play a role in the pathogenicity of the organism. Twelve different immunotypes are found depending on the structure of the LOS moiety. One aspect of complexity of the LOS group that plays a role in defining the specific immunotype is the presence and location of phosphoethanolamine (PEA) residues. The enzyme responsible for adding the PEA residue to the LOS group is phosphoethanolamine transferase. Different forms of PEA transferase are present depending on the precise location of the PEA moiety on the LOS molecule. Knowledge of the biosynthesis and regulation of meningococcal lipoooligosaccharides will provide a more detailed understanding of the role of this molecule in pathogenesis and disease. In collaboration with Dr. Charlene Kahler of the Department of Microbiology at UWA we have begun a study to determine the three dimensional structure of the enzyme LPTA, the phosphothanolamine transferase specific for phosphorylation of the lipid A core of LOS. This project will involve protein expression, purification, crystallization and structure determination using crystallographic techniques. The structural results will be correlated with functional studies carried out by Dr. Kahler and coworkers.
2. Transcription Elongation Factors Spt4/Spt5 RNA transcription is a dynamic process that produces an RNA transcript from a gene. It is the first step in gene expression and thus is Domains of Spt4 and Spt5 a focal point for cell regulation. In order for successful transcription to Zn finger domain occur, accessory proteins are needed that help initiate and promote elongation. In many eukaryotes, soon after initiation has occurred, Spt4 102 amino acids. blockage of the elongation process occurs resulting in abortive RNA 12kDa 1 40 transcripts. In yeast, a protein complex made up of Spt4 and Spt5 are 5 KOW domains required to circumvent these pauses. Studies have shown that this Acidic N-term CTR Region complex can regulate transcriptional elongation by modulating the Spt5 1063 a.a. processivity of RNA polymerase II. In collaboration with Professor 130kDa 1 244 283 840 Grant Hartzog at UC Santa Cruz we have undertaken crystallographic Spt4 binding region studies to establish the structural and functional relationships of NusG homology region Spt4/Spt5. A number of different constructs of the Spt4/Spt5 complex RNA Polymerase II Binding region have been designed, expressed and purified. This project involves protein expression, purification, crystallization and biophysical studies aimed at characterizing this complex in more detail.
3. Structural and Functional Studies of Substrate Channeling Multienzyme complexes are seen in many diverse metabolic pathways. Substrate channeling of intermediates between enzyme active sites within a multifunctional enzyme complex provides a mechanism to isolate labile and reactive intermediates from competing reactions as well as protect the cell from toxic intermediates. Our
Chemistry 64 aim is to gain a better understanding of the role of protein-protein and protein-ligand interactions in intersubunit communication and synchronization of catalytic events with the channeling activity. This project will study channeling activity in the bifunctional enzyme 4-hydroxy-2-ketovalerate aldolase-aldehyde dehydrogenase (DmpFG). Studies have shown that aldolase (DmpG) activity is not detectable in the absence of the dehydrogenase (DmpF) suggesting that acetaldehyde, the product of the aldolase reaction, may be channeled to the dehydrogenase active site.
We have determined the 1.7Å resolution structure of this enzyme. The presence of a 29Å long gated and sequestered tunnel in the structure, leads us to propose this as the channeling route taken by the acetaldehyde intermediate from the aldolase to the dehydrogenase active site. This Honours project involves using crystallographic and kinetic studies to probe the function of the observed channel. The structure of a complex of the acetaldehyde intermediate trapped within the channel will be determined. The physical nature of the residues that line the channel will be characterized by mutational, kinetic and structural studies. The mechanism by which access between the active sites and the channel is controlled and synchronized with enzyme activity at each of the active sites will be studied by a combination of site Secondary structure representation of the directed mutagenesis, kinetic and crystallographic analyses. DmpFG heterodimer.
4. Probing the Structure of Cholesterol Oxidase – A Novel Antibiotic Target. The flavoenzyme cholesterol oxidase constitutes an important virulent factor in immunocompromised patients prone to Rhodococcus equi lung infections. The longstanding problem with antibacterial drug resistance calls for a continued need to probe new targets for the design and development of novel antibiotic treatments. The design of novel antibacterial drugs is facilitated by a detailed knowledge of the architecture of active site, including the positions of hydrogen atoms, the ionization state of titratable groups and the precise conformational state of side chains and cofactors through the substrate binding and catalysis events. Electron density view of the isoalloxazine ring Our laboratory uses a combination of crystallographic, mutagenesis of cholesterol oxidase. The density clearly and kinetic methods to understand these events. We have crystals of shows single electron differences for individual cholesterol oxidase that diffract to sub-Ångstrom resolution providing atoms. an unprecedented view of the enzyme structure. Different redox states of the enzyme can be followed spectrophotometrically in the crystal and ligands bound to induce oxidation chemistry while maintaining sub-Ångstrom diffraction. This provides us with a unique opportunity to visualize transient states and establish structural changes as a function of the redox state. Honours projects, focused on testing hypotheses on redox activity and oxygen reactivity for the enzyme include:
(i). Investigating the structural and electronic differences between the oxidized and reduced enzyme and
Difference electron density showing the (ii). Examining whether a tunnel is involved in oxygen access to the positions of hydrogen atoms in a region of the active site during the oxidative half reaction. structure.
Chemistry 65 R John Watling
Professor, Forensic Chemistry Room 3:31, MCS Building, Phone: 6488 4488 E-mail [email protected]
BSc (Hons) (Lond), DIC, PhD (Lond) FRSC, FRACI, CChem, CSci, Registered Analytical Chemist
Forensic Chemistry Research Group Expertise and Interests: The Group has two main research initiatives. Firstly, spectral fingerprinting of crime scene evidence and provenancing metals, projectiles, gemstones, glass, oriental ceramics, paintings, foodstuffs, explosives, plastics, drugs and environmental materials, and secondly, nano-forensics - a completely new area of forensic science associated with the development of nano-sensors for real-time crime scene and terrorist activity investigations by determining the presence of explosive gases, biological agents and residues.
Group Activities: It is impossible to discuss in detail the diversity of projects being undertaken by the Forensic Chemistry Research Group at UWA, however, any student wishing to obtain information should contact John Watling for a CD of the group’s activities.
Introduction: With the increase in both sophistication and frequency of crime and the continuous decrease in Governmental funding of police and law enforcement authorities it has become necessary for forensic chemists to be aware of, to develop and to apply, relevant new analytical technology to assist them in "fast tracking" forensic investigations. Furthermore, as criminals become more careful about leaving "debris" at a crime scene the amount of evidentiary material is becoming smaller and increasingly more difficult to analyze using conventional analytical methodology. A significant setback for criminals occurred with the advent of ICP-MS. This technique provides an improvement in detection limits for most elements in the Periodic Table of often more than three orders of magnitude over conventional absorption and emission techniques. Consequently it has now become more possible to obtain analytical information for a wide range of elements on much smaller samples. Incorporation of laser ablation with ICP-MS has the potential to solve many of the existing problems associated with provenance establishment of scene of crime evidence as even the initial Nd-YAG lasers were capable of volatilization of relatively small craters (<100μm in diameter) thereby removing often only a relatively tiny amount of the evidentiary material. The recent advent of UV and Excimer lasers decreased the sampling volume to crater sizes of <10μm and thereby decreased the size of potentially analyzable debris. The current research group in the application of lasers to forensic investigations in a world leader in this technology and is a founder member of the international NITECRIME Network of forensic mass spectrometric CSI laboratories.
Chemistry 66 The science of “Spectral Fingerprinting” is in its infancy and although recorded in case law in five countries, researchers have only scratched the surface of the technology. Consequently, application of this technology is suited to Honours, masters and PhD projects as well as considerable post doctoral research initiatives. Therefore, while some overview project types are discussed in this document, rather than identify specific projects in detail to students, the student is encouraged to use their imagination to identify areas where the application of this technology is relevant and to suggest these to members of the Forensic Chemistry Group. In this way it will be possible to tailor specific projects of particular relevance to the student to suit student interest and commitment. Suggestions such as the spectral fingerprinting of Tapes and ties used in rape and drug transport, pencils and inks used in forgeries, glass, pollen, plants, plastic rope, metals from crime scenes, fibres, abrasive minerals, paper and canvass used in art forgery, statues, clays, guns and projectiles are all relevant for consideration. Give it a thought yourselves and come and see us. Current Honours students are investigation the provenance establishment of diamonds, gold and identifying the provenance of oil at ram raids and hit and run events.
Some Possible Suggestions for Projects in Environmental Forensics:
1) The recent recognition of a lead problem in Esperance has resulted in an increase in interest in the distribution of lead in the environment. Of particular risk are young children and babies. We propose to develop a method of teeth analysis (lead is sequestered in teeth) to plot the history of lead intoxication by humans and to look at methods of determining changes in the lead pollution of the environment with time. In addition we will look at an Ibex tooth from the last European Ice Age ad determine if we can see the reflection of pasture changes from summer to winter and tell how old the animal was when it died some 20,000 years ago.
2) The international requirement to provenance foodstuffs has led to the Forensic Chemistry Group at UWA pioneering the inception of PROOF (The Australian and New Zealand Proof of Origin of Foodstuffs) programme. This programme interfaces with the European equivalent programme (TRACE). We have projects on developing methodology for the elemental fingerprinting of Milk Powder, Mineral Waters and Wine. We even have some research dollars to buy some of the necessary ingredients! These projects will lay the foundation of our involvement with the European programmes in these products and will compliment our existing projects for tea and drugs.
Please remember that these are not the only projects on offer, they only from a basis for discussion towards a relevant equivalent which can be mutually developed.
Chemistry 67 Duncan A. Wild Room 1.28, MCS building, Phone: 6488 3178, Email: [email protected]
B.Sc. (Hons), PhD. (Melbourne) Alexander von Humboldt Fellow (2004) Research fellow, MPI für biophysikalische Chemie (2004-06) UWA lecturer (2007- )
Laser Spectroscopy
My research interests include: Spectroscopic investigations of small gas phase clusters, ab initio calculations to predict infrared and photoelectron spectra, and utilising graphical programming techniques in physical chemistry. PROJECTS
PHOTOELECTRON SPECTROSCOPY AND AB INITIO CALCULATIONS OF ANION CLUSTERS Two projects are offered for prospective students. The first is concerned with spectroscopic interrogation of important gas phase species using the recently built TOF-PES apparatus. The second project involves modeling photoelectron and infrared spectra via ab initio methods.
1. PHOTOELECTRON SPECTROSCOPY OF GAS PHASE CLUSTERS AND PARTICLES
Our group is in the final stages of the construction of gas phase photoelectron spectrometer. This machine combines a time of flight (TOF) mass spectrometer with a 1.6 metre photoelectron flight tube, see figure for simple schematic, or visit the website for more details; http://www.chem.biomedchem.uwa.edu.au/staff/homepages/dr_duncan_wild You will be involved with some of the very first experiments to be conducted with the apparatus (glory days!). These will involve probing the solvation of small anions. The idea behind the experiments is to use the mass spectrometer to select certain sized clusters, i.e. a gas phase anion cluster such as Cl-…HCCH and then record its photoelectron spectrum. If one follows the photoelectron spectra as the size of the cluster is changed (by increasing the number of solvent molecules bound to the anion, easily followed via mass spectrometry), then this provides us with information on the electron affinities of the clusters, and also the structures and sizes of the solvation shells around the anions (by discontinuities in the size dependant spectra). This project is ideal for those who are interested in spectroscopy, intermolecular interactions, and probing neutral reaction chemistry. The project also offers the exciting opportunity to extend your knowledge into areas outside of chemistry; for example into the realms of i) basic electronics, ii) high vacuum technology, iii) data acquisition programming, and much more!
Chemistry 68 2. MODELLING PHOTOELECTRON AND INFRARED SPECTRA OF SMALL DIMER (1:1) COMPLEXES
Ab initio methods (calculations from first principles, i.e. no experimental input) are used routinely to predict structures and energetics of molecules and clusters (for some examples see references 3 and 4 and citations within). In this project it is planned to model photoelectron spectra of small dimer clusters (1 solvent molecule bound to an anion). In particular, the project will involve simulating the photoelectron spectra of the gas phase - - - complexes formed between the halide anions F , Cl , Br and various solvent molecules, such as PH3 and HCCH. You will aim to predict the photoelectron spectra that will guide the experiments that are planned in the future, thereby testing the predicted cluster structures and energetics. In fact, this project will path the way for many of the experiments planned for the TOF-PES.
References:
15. W. C. Wiley and I. H. McLaren, Rev. Sci. Instrum. 26, 1150 (1955) 16. see http://www.agilent.com/find/vee for information about VEE graphical programming 17. D.A. Wild and T. Lenzer, Phys. Chem. Chem. Phys., 2005, 7, 3793-3804 18. D.A. Wild and T. Lenzer, Phys. Chem. Chem. Phys., 2004, 6, 5122-5132
Chemistry 69 MICROBIOLOGY AND IMMUNOLOGY DR MANFRED BEILHARZ SENIOR LECTURER Laboratory 1.4, Microbiology and Immunology, L Block, QEII Medical Centre Phone: 9346 2663 Email: [email protected]
Research Interests
Regulatory T cells Research conducted in the Beilharz laboratories focuses primarily upon Regulatory T cell biology and the ability of these cells to modulate the immune response to tumours, viral infection, transplantation and low dose oral interferon therapy. Projects running in the laboratory utilise both in vivo and in vitro techniques to characterise and manipulate the Regulatory T cell response in each of these models of human pathology
Fermentation in Wine Production A more recent addition to our research team is the wine research group. This group is exploring microbial and molecular aspects to stuck and sluggish ferments which place a significant economic burden on the productivity of Australia’s burgeoning wine industry.
PROJECTS
1. Immunomodulation to improve Myoblast Transfer Therapy, a Treatment for Duchenne Muscular Dystrophy Supervisors: Dr Manfred Beilharz and Mr Clayton Fragall
Description:
Of the 700 neuromuscular diseases currently described in man Duchenne Muscular Dystrophy (DMD) is both the most common and most severe of those diseases affecting primary muscle function. Mutations in the dystrophin gene result in the loss of dystrophin or production of a non-functional dystrophin protein, an essential element in the membrane stabilising machinery of muscle fibres.
Myoblast Transfer Therapy (MTT) is aimed at utilising the muscles’ natural repair mechanism via the direct injection of donor myoblasts (muscle precursor cells), which express a functional dystrophin molecule, into the dystrophic tissue to provide a cellular based gene rescue. Whilst numerous animal model studies and some human clinical trials have indicated the MTT approach to be feasible in principle, the innate and acquired immune rejection of the donor myoblasts appears to limit the practicality of this potential therapy (Sammels et al., 2004). The precise nature of the donor cells is also being investigated.
In 2002, Kingsley et al. discovered that CD4+CD25+ T cells (Tregs) were generated using a tolerance induction protocol before allograft transplantation while in 2003, Bushell et al. showed that following adoptive transfer from a tolerised host, these cells could confer antigen specific tolerance to naïve mice. IL-10 and TGF-β have been shown to be critical mediators of Treg suppressor function in numerous studies of peripheral tolerance while type I IFN have been implicated in the induction of Tregs, possibly via secretion by DCs.
70 Microbiology and Immunology 2. Interferon and Influenza Supervisors: Dr Manfred Beilharz and Mr Clayton Fragall
Description: The persistence of highly pathogenic avian influenza within wild bird populations has forged interest in control measures to limit a possible human pandemic. We therefore investigated the efficacy of low dose oral administration of IFN-α as a potential therapy against influenza infection in a murine model. We have identified an optimal low oral dose of IFN-α that when delivered daily as prophylactic therapy protects C57BL/6J mice from a lethal challenge with mouse adapted human influenza virus A/PR/8/34 H1N1. These results provide strong support for the application of low dose type 1 IFN pretreatment to human influenza control. These results have been i) published in BBRC (Beilharz et al., 2007 ) and ii) internationally verified by researchers in the Trudeau Institute (USA and the Friedrich Loeffler Institute (Germany). In 2009 a human clinical trial will be conducted in Perth with WA health department funding.
3. Immuno and Complementary Therapy for the Treatment of Cancer Supervisors: Dr Manfred Beilharz, Dr Sara Greay and Dr Demelza Ireland
Description: In the year 2000, 6 million people died from cancer world-wide, and current estimates indicate that one third of people will suffer from some form of the disease by the age of 75. Current cancer treatments such as chemotherapy and radiotherapy have a wide range of negative side effects that seriously reduce the patients’ quality of life. Furthermore, many forms of cancer are unresponsive to traditional treatments. The goal of immunotherapy is to use the host immune system to fight the cancer. This approach has had a mostly unsuccessful history that can largely be attributed to the lack of understanding of the interaction of tumours and the immune system. Recent advances in our understanding of this field have resulted in new immunotherapies with increasing rates of success. One of these advances is the discovery of regulatory T-cells (Tregs) and the immuno-suppressive role that they play in tumour immune evasion. Our lab has shown that removal of Tregs from tumours results in activation of the host immune response and inhibition of tumour growth (Needham et al., 2006). One limitation of this treatment is that Treg cell populations eventually return to the tumour which correlates with the resumption of tumour growth. Our lab is currently investigating the role of dysfunctional dendritic cells in the expansion of Treg cell populations in tumour bearing hosts and developing an immunotherapy that aims to reverse dendritic cell dysfunction to prevent the return of Tregs to tumour bearing hosts following Treg depletion. The activity of Tea Tree Oil (TTO) in effecting immune mediated tumour clearance is also being investigated.
4. Stuck and Sluggish Wine Fermentations Supervisors: Dr Manfred Beilharz and Mr Clayton Fragall
Description:
Hexose transporters play a critical role in the wine fermentation process by importing the hexoses glucose and fructose into the yeast cell for fermentation (Luyten et al., 2002). It has been observed that the process of hexose transport is the rate limiting step in the fermentation pathway (Kruckeberg et al., 1993) thus implicating expression of the genes encoding the hexose transporter proteins as a potential cause of problem ferments. Research conducted in our laboratory comparing yeast gene expression in both a normal and a sluggish commercial wine ferment has shown different expression patterns of two hexose transporter genes (HXT3 and HXT6/7).
This project will further characterize the expression of these genes during stuck and sluggish fermentations and attempt to associate these gene expression profiles with specific environmental conditions and the presence of competing micro-organisms.
71 Microbiology and Immunology ASSOCIATE PROFESSOR BARBARA CHANG Room 1.8, Microbiology & Immunology, L Block, QEII Medical Centre. Phone: 9346 2288 Email: [email protected]
Molecular Bacteriology
The first research area in my laboratory concerns the biology and genetics of the bacterial viruses known as bacteriophages (phages), and their use as biocontrol agents in human disease and in aquaculture. Research has included the isolation of Vibrio harveyi phages and an examination of their use in aquaculture (in collaboration with Dr David Sutton), genome sequencing of a phage of Clostridium difficile (with Prof Tom Riley) and a study of the role of phages in virulence of Aeromonas (with Dr Harry Sakellaris). The second general area is in molecular studies of virulence mechanisms of bacterial pathogens including Aeromonas spp., Burkholderia spp., Clostridium difficile, Moraxella catarrhalis, S. maltophilia and Vibrio spp.
PROJECTS 1. Virulence determinants of Western Australian Aeromonas pathovars With Dr Tim Inglis, PathWest
Aeromonads are ubiquitous aquatic Gram-negative bacteria that cause disease in amphibians and fish. Three species of Aeromonas, A. hydrophila, A. caviae and A. veronii biovar sobria, are human pathogens. They are associated with gastrointestinal infections and a variety of extra-intestinal infections such as septicaemia, wound infections, soft tissue infections, and occasionally meningitis, peritonitis and haemolytic- uraemic syndrome. A large number of putative virulence factors have been identified in Aeromonas, including the production of pili and other adhesins, O-antigens and capsules, lateral flagella, exotoxins such as haemolysins and enterotoxins, and extracellular enzymes such as proteases, amylases and lipases. A type III secretion system gene cluster has been identified in A. hydrophila, although the role of proteins secreted by this system and how they interfere with host cellular processes is not yet known. In addition, a phage-associated genomic island has been reported, although no role in virulence is yet proven. Some Aeromonas isolates have the ability to invade human epithelial cells in a process which has not been completely characterised, but which has been shown to involve actin polymerisation.
There have been several clusters of invasive Aeromonas sp. infection in Western Australia in recent years. The reason for this has not been fully elucidated, but a comprehensive cataloguing of Aeromonas spp. in WA is now under way in our laboratories. Carefully characterised Aeromonas isolates will be available for virulence factor analysis using nucleic acid amplification to probe key gene sequences, DNA sequencing to identify potential mutations, an expression PCR to analyse possible variability in virulence expression. If rapid progress is made, fluorescent hybridisation will be used to explore gene expression in a cellular model of infection. Together with the molecular work, a phenotypic analysis of selected virulence factors will be performed. In particular, adhesion to and invasion of human epithelial cell lines will be examined. This project will lead to a better understanding of the multifactorial nature of Aeromonas pathogenicity, and a determination of the association of putative virulence genes with clinical invasiveness in WA isolates.
References: 1. Yu HB et al. (2005) Identification and characterisation of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91. Appl Environ Microbiol 71: 4469-77. 2. Snowden L et al. (2006) Prevalence of environmental Aeromonas in South East Queensland, Australia: a study of their interactions with human monolayer Caco-2 cells. J Appl Microbiol 101: 964-75. 3. Gavin R et al. (2003) Lateral flagella are required for increased cell adherence, invasion and biofilm formation by Aeromonas spp. FEMS Microbiol Lett 224: 77-83. 4. Chang BJ and Janda JM (2005) Aeromonas. In: Topley and Wilson’s Microbiology and Microbial Infections.
72 Microbiology and Immunology 2. Aeromonas phages and their role in Aeromonas virulence and biology With Dr Harry Sakellaris
There is currently renewed scientific interest and rapid development world-wide in the study of phages. This has been partly fuelled by the emergence of multiple antibiotic resistance in bacterial pathogens and the development of alternative antibacterial therapies, including phage therapy. However it is also becoming increasingly clear that phages play a major role in bacterial evolution and specifically in the virulence of many bacterial pathogens. For example, phages often encode toxins and other virulence factors of bacterial pathogens, such as the ctx phage of Vibrio cholerae which encodes cholera toxin. Little is known about Aeromonas phages and whether they have any role in disease. Thus the aim of this project is to determine the role of selected temperate phages in virulence of Aeromonas and in the biology of their host strains.
We have a collection of 19 Aeromonas phages, and have been able to construct a number of strains lysogenic for some of these phages. The role of phages in virulence will be studied by assaying lysogens (harbouring phage genomes or prophages) and their parental non-lysogenic strains using in vitro assays such as haemolysis, haemagglutination, adhesion to and invasion of human cell lines such as HEp-2 cells, cytotoxicity and serum resistance. Other phenotypes examined will include growth characteristics and metabolic functions. Phage DNA isolation and pulsed-field gel electrophoresis, in order to determine the size of the phage genomes, may be undertaken in order to further characterise the phages. Southern hybridisation using DIG-labelled phage DNA probes will be used to determine the number and location of phage genomes within lysogens, and wild-type Aeromonas isolates will be screened to determine the presence of related phages; correlation with source and virulence of strains can then be determined. Another molecular approach will be to use PCR to amplify regions of known virulence genes of Aeromonas using as templates the isolated phage genomes, as well as DNA from lysogens and parental strains. This project will increase our understanding of the contributions made by Aeromonas phages to their hosts.
References: 1. Janda JM and Abbott SL (1998) Evolving concepts regarding the genus Aeromonas: an expanding panorama of species, disease presentations, and unanswered questions. Clin Infect Dis 27: 332-344. 2. Boyd EF and Brussow H (2002) Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Trends in Microbiology 10: 521-529. 3. Goh S, Riley TV and Chang BJ (2005) Isolation and characterization of temperate bacteriophages of Clostridium difficile. Appl Environ Microbiol 71: 1079-1083. 4. Wagner PL and Waldor MK (2002) Bacteriophage control of bacterial virulence. Infect Immun 70: 3985- 3993. 5. Brussow H et al. (2004) Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol Molec Biol Revs 68: 560-602.
NB Also see project co-supervised with Dr David Sutton
73 Microbiology and Immunology Dr Tim Inglis, Clinical Associate Professor PathWest Laboratory Medicine WA, Room G13, K block, QEII Medical Centre, Phone: 9346 3461 A Levy E-mail: [email protected]
Clinical Microbiology
Recent research by my laboratory group has focused on bacteria that cause human infections including the Burkholderias, Legionella, Mycobacteria, Listeria and other facultative intracellular species. Current work spans bacterial survival strategies, the role of secondary metabolism in mechanisms of disease, bacterial taxonomy, molecular diagnostic and molecular epidemiological methods and the cell biology of bacterial-eukaryote interactions. Recent highlights include participation in an international whole genome sequencing project, the discovery of bacterial species that are new to Australia, the development of new molecular epidemiological methods and work on direct molecular detection of bacteria in early bloodstream infections.
PROJECTS
1. The laboratory Diagnosis of Legionella longbeachae infection With Drs Charlene Kahler and Gerry Harnett
In most of the world, including much of Australia, the majority of cases of Legionnaire’s Disease are caused by Legionella pneumophila serotype 1. However, in Western Australia L. pneumophila is an unusual cause of infection. Much more common is Legionella longbeachae, which is associated with exposure to potting mix and other garden products. It is not clear whether this apparent difference is due to real differences in the epidemiology of Legionella infection in WA, or due to differences in the way laboratory diagnostic tests are used and interpreted. Legionella spp are quite difficult to grow in the diagnostic laboratory, requiring the use of special selective agar and experienced staff. In recent years, L. pneumophila infections have benefited from PCR-based molecular diagnostic methods and an easily performed urinary antigen test (UAT). A UAT is not available for L. longbeachae and PCR methods are of variable specificity. There is a pressing need to improve the currently available laboratory tests for L. longbeachae infection.
The project will combine a review of currently used laboratory methods for L. longbeachae infections, and seek to improve the PCR-based method by designing a new set of PCR primers from sequence data that has recently been uploaded to GenBank. Sera from patients with confirmed Legionella infection will be tested by Western blot to help differentiate antibody responses to L. pneumophila and L. longbeachae antigens. Suitable candidate antigens for differentiation of L. longbeachae infections from other causes of Legionnaires’ Disease will be characterized and preliminary purification attempted. If time permits, a polyclonal antibody preparation will be raised in laboratory animals for use in a prototype urinary antigen test, and evaluated on clinical samples from patients with suspected Legionella infection. References. 1 Nazarian EJ et al. Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples. Diagn Microbiol Infect Dis 2008 Jul 11 [e-pub ahead of print] 2 Dominguez JA et al. Comparison of the Binax Legionella urinary antigen enzyme immunoassay (EIA) with the Biotest Legionella Urin antigen EIA for detection of Legionella antigen in both concentrated and unconcentrated urine samples. J Clin Microbiol 1998; 36: 2718-22.
Methods used: • Selective culture of Legionella species • Nucleic acid amplification by PCR • Serodiagnostic methods for Legionella infection • Western blot • Polyclonal antibody development in laboratory animals • Antigen characterisation, purification and detection in clinical specimens
74 Microbiology and Immunology
2. The intracellular survival of Burkholderia species With Drs Avram Levy and Gerry Harnett
The Burkholderias are a group of soil-dwelling environmental Gram negative bacteria that include human pathogens such as B. mallei, B. pseudomallei and B. cepacia as well as a large range of non-pathogenic near-neighbour organisms that inhabit the upper layers of the soil. Disease-causing members of this genus have a capacity to invade eukaryotic cells such as tissue macrophages and can remain dormant there for a very long time. We have shown that these bacteria can persist in naturally occurring eukaryotic inhabitants of the upper soil layer such as free-living amoebae and mycorrhizal fungi and have proposed that the ability to invade human cells is as a result of an intracellular lifestyle in the wider environment. It is possible that this environmental niche is also a means of exposure to infection by disease-causing species such as B. pseudomallei. We have collected a library of soil samples from the north of Western Australia and isolated Burkholderia species from these. A previous Honours project measured the biodiversity of Burkholderias from these samples but stopped short of recovering them from amoebic cysts or mycorrhizal fungal spores in the soil sample collection. The proposed project will build on that earlier work, specifically looking for evidence of intracellular Burkholderia species in our collection of soil samples. Further fresh samples will be obtained from northern WA during the course of the project to ensure further Burkholderia-positive cultures and viable eukaryotic microbiota to analyse for intracellular bacteria. A positive result will strengthen evidence for a new mode of transmission of Burkholderia infection.
References: 1 Levy A et al. Expanded range of Burkholderia species in Australia. Am J Trop Med Hyg. 2008 Apr;78(4):599-604. 2 Levy A et al. Invasion of spores of the arbuscular mycorrhizal fungus Gigaspora decipiens by Burkholderia spp. Appl Environ Microbiol. 2003 Oct;69(10):6250-6. 3 Inglis TJJ et al. Interaction between Burkholderia pseudomallei and Acanthamoeba species results in coiling phagocytosis, endamebic bacterial survival, and escape. Infect Immun. 2000 Mar;68(3):1681-6.
Methods used • Selective culture using specialized solid and liquid media • Targeted nucleic acid amplification by PCR and sequencing of amplicons • Recovery of mycorrhizal fungi and amoebic cysts • Electron microscopy techniques • Fluorescent in-situ hybridization
75 Microbiology and Immunology DR CHERYL JOHANSEN RESEARCH FELLOW Room 1.29, Microbiology and Immunology, L Block, QEII Medical Centre, Phone: 9346 4656 Email: [email protected]
Arbovirus Surveillance and Research Group
The Arbovirus Surveillance and Research Laboratory (ASRL) monitors activity of mosquito-borne viruses including the flaviviruses Murray Valley encephalitis virus (MVEV), Kunjin virus (KUNV), and the alphaviruses Ross River virus (RRV) and Barmah Forest virus (BFV) in Western Australia (WA). In addition, the program aims to detect incursions of medically important exotic mosquito-borne viruses, such as Japanese encephalitis virus, West Nile virus and chikungunya virus. These viruses have caused large outbreaks of potentially fatal disease in other countries, often including neighbouring countries in southeast Asia. Monitoring of mosquito fauna and arbovirus activity is undertaken at key locations, particularly in the southwest of WA and the Kimberley and Pilbara regions, but also other parts of the state on an opportunistic basis. Year-round flavivirus surveillance is conducted in northern WA using sentinel chicken flocks. Environmental conditions and predisposing factors, and the incidence of human disease (provided by the DOH) are also monitored. In addition, research initiatives aim to improve the speed/specificity/sensitivity of detection of viruses and their infections, to improve the detection of flaviviruses that are serologically cross-reactive and difficult to distinguish, and to research the epidemiology and ecology of arboviruses and mosquito vectors in WA.
PROJECTS
1. Is the infection rate and titre of Ross River and Barmah Forest viruses in vector mosquitoes informative? With Dr Michael Lindsay (Mosquito Borne Disease Control Branch, Western Australian Department of Health, 9385 6001, [email protected])
RRV and BFV are two medically important mosquito-borne alphaviruses that cause outbreaks of debilitating arthritis, rash and fever in people in WA. The ASRL has monitored activity of these viruses in mosquitoes along the Swan Coastal Plain in the southwest of WA since 1987. A large number of mosquito pool homogenates have been confirmed to be infected with RRV and/or BFV since surveillance of these viruses in mosquitoes commenced. Although the minimum infection rate (MIR) of RRV and BFV in mosquito populations can be extremely high preceding and during outbreaks of disease, the infection rate is variable. Preliminary analyses in the early 1990s showed that high MIRs were often linked to large outbreaks of RRV disease, yet not always, and no detailed analysis has been undertaken to formally investigate the relationship between infection rate in the mosquito population and the likelihood of outbreaks of disease. In addition, determination of the titre of virus in mosquito pools has been shown to enhance mosquito-based arbovirus surveillance programs elsewhere. This project aims to investigate whether MIRs in mosquito populations and mosquito abundance are of predictive value in relation to outbreaks of RRV and BFV disease in WA, and to investigate the benefit of determination of virus titre in infected mosquito pools, particularly pertaining to potential vector incrimination. MIRs in mosquito populations in the Swan Coastal Plain will be determined using several published methods and compared to the number of human cases of disease and mosquito abundance. Virus titre will be determined by virus titration using TCID50s and tissue culture enzyme immunoassays (TCEIA). Given that this method can be cumbersome, time consuming and not practical in the long term, real-time reverse transcription (RT)-PCRs specific for RRV and BFV will also be developed to rapidly quantify RRV and BFV in mosquito pools, and the relationship between titration using TCID50/TCEIA and quantification of RRV and BFV RNA by real-time RT-PCR will be analysed. It is envisaged that these results will enhance the ability of the ASRL to predict future outbreaks of RRV and BFV disease and improve understanding of vector mosquito species in WA.
76 Microbiology and Immunology 2. Development of virus-specific real-time reverse transcription PCRs for arbovirus surveillance in WA
Flaviviruses commonly isolated from mosquitoes collected in northern WA include the potentially fatal MVEV and the closely related KUNV. These viruses are endemic in northern WA, and can spread further south and east when environmental conditions are suitable. Surveillance of MVEV and KUNV activity currently relies on detection of seroconversions in sentinel chicken flocks at approximately 30 locations in WA, primarily in northern, remote areas. Sentinel chickens are bled regularly, and the blood samples are transported to the laboratory where they are tested for the presence of antibodies to these viruses. Detection of flavivirus activity in sentinel chickens enables early warning of flavivirus activity. Annual adult mosquito collecting field trips are undertaken in the Kimberley region to investigate vector ecology and mosquito infection rates, and to monitor incursions of new arboviruses and mosquitoes into the region. Additional field trips to the Pilbara region are also currently being undertaken, to investigate the possibility that MVE virus has become established in enzootic transmission cycles in the region.
Surveillance of human cases of RRV disease and BFV disease is currently the only mechanism for monitoring activity of these viruses in northern WA, although these viruses are isolated from mosquitoes collected during annual audits of mosquito populations and arboviruses during the late wet season each year.
Given the remoteness of many parts of WA, we are currently investigating the possibility of using mosquito traps to collect adult mosquitoes continuously for surveillance in remote areas. This would require detection of arboviruses in large pools of mosquitoes using real-time reverse transcription (RT) PCRs. This method would not only enable surveillance in remote areas, but also longitudinal studies into vector ecology, mosquito infection rates with indigenous arboviruses, as well as detection of incursions of foreign mosquito species and arboviruses new to the region.
However in addition to MVEV and KUNV, several other flaviviruses are also regularly isolated from mosquitoes collected in WA, including Alfuy virus (ALFV), Kokobera virus (KOKV), Stratford virus (STRV) and Edge Hill virus (EHV). Real-time RT-PCRs have been developed for detection of MVEV and KUNV, however virus-specific real-time RT-PCRs for other flaviviruses, as well as the alphaviruses RRV and BFV, are not yet available. Furthermore, the MVEV and KUNV assays are not specific, as they also detect the closely related ALFV and West Nile virus (WNV), respectively. The aim of this project will is to design and develop virus-specific real-time RT-PCRs for detection and identification of flaviviruses in mosquitoes from WA.
77 Microbiology and Immunology DR CHARLENE KAHLER SENIOR LECTURER Room 2.03, L Block, QE II Medical Center. Phone: 9346 2058, Email: [email protected]
Bacterial Pathogens Causing Sepsis
Neisseria meningitidis and N. gonorrhoeae are two closely related obligate human pathogens. N. meningitidis is the causative agent of epidemic meningococcal meningitis and septic shock. It colonizes mucosal surfaces of the nasopharynx and in susceptible individuals, particularly children under the age of two years, the bacterium becomes systemic resulting in fatal bacteremia. Despite the continued sensitivity of meningococcus to multiple widely available antibiotics, including penicillin, the case-fatality ratio for meningococcal disease remains around 10%–14% (CDC, unpublished data, 2004). Vaccines have been developed based on the polysaccharide capsules to prevent community spread and therefore have become an effective means of reducing meningococcal disease. However, in the West Australian community, type B remains prevalent and there is no vaccine against this organism since the type B polysaccharide is a poor immunogen. Neisseria gonorrhoeae on the other hand, is the causative agent of the sexually transmitted disease (STD) gonorrhoea. Comparatively, the rate of disease in developing nations is approximately ten times that of developed countries and globally approximately 20-60 million new cases are reported per annum (WHO). In males, gonococcal infection is generally acute and resolves rapidly with treatment. However, higher morbidity is seen in women as the infection remains asymptomatic and without treatment progresses to pelvic inflammatory disease (PID) resulting in infertility in approximately one third of patients. Unlike meningococci, this organism is increasingly resistant to antibiotics with a recent report of the emergence of a “superbug” resistant to all antibiotics. To date no successful vaccine strategies have been developed for this organism, primarily because the cell surface proteins expressed by this organism are highly antigenically variable, thus eliciting limited immunological protection against other strains. As a result individuals can contract the disease multiple times throughout their lifetime. My group is interested in three different facets of these important human pathogens: a. Endotoxin is the primary toxin that results in septic shock and death of the patient. We are interested in understanding the biosynthesis pathway and regulatory networks controlling the production of this important toxin. b. The regulatory networks within these pathogens that are triggered during attachment to the human nasopharynx. c. Understanding the invasive mechanisms used by these organisms.
Prospective Honours students with a background in Molecular Biology, Biochemistry, and Microbiology are particularly encouraged to apply. Students will be exposed to a range of techniques including DNA sequencing, DNA cloning, cell culture, RT-PCR, protein analysis, and FACS analysis.
PROJECTS
1. Endotoxin of Neisseria meningitidis
This work is being conducted in collaboration with Professor David Stephens (Emory University, Atlanta, USA) and Dr Russell Carlson (Complex Carbohydrate Research Center, Athens, USA).
Neisserial endotoxin (or lipopoligosaccharide [LOS]) is a glycolipid related to E. coli lipopolysaccharide and contains lipid A attached to a conserved outer core of sugars. Although the complete biosynthetic pathway of this structure has been determined, very little is known about transportation of this structure across the inner and outer membranes of Gram – negative bacteria. In an effort to understand the LOS transport pathway, we will examine whether the LOS biosynthesis enzymes form an interactome for efficient biosynthesis and transport of LOS. To do this we will clone a number of LOS biosynthesis enzymes and tag them with known epitopes which
78 Microbiology and Immunology will allow us to specifically detect each protein. The location of each tagged protein in the cell will be determined by cell fractionation and Western Immunoblot. Candidate proteins that may interact intracellularly will be assessed using a protein two-hybrid system.
AIMS:
1. To epitope-tag known LOS biosynthesis enzymes and assess their localization within the bacterial cell by cell fractionation and Western Immunoblot. 2. Bacterial two hybrid system for detection of protein:: protein interactions
2. Sigma factors in Neisseria gonorrhoeae
This work is being performed in collaboration with Professor John Davies (Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Victoria) and Dr Paul Rigby (BIAF, UWA).. Bacterial sigma factors are essential components of the RNA polymerase holoenzyme and determine promoter selectivity and specificity. Analysis of the Neisseria gonorrhoeae genome indicates that apart from the rpoD and rpoH genes there was only one other gene that encodes a potentially functional sigma factor. This gene, ecf, appears to encode a member of the extracytoplamic function (ECF) alternative sigma factor family. Microarray analysis suggested that 8 genes (including ecf) were up-regulated when Ecf levels are increased. Five of the Ecf- regulated genes are clustered with ecf on the genome, and appear to form a single transcriptional unit. The first two open reading frames in this cluster, NGO1947 and NGO1948, appear to encode putative anti-sigma factors which regulate the amount of free ECF factor in the cytoplasm. To examine their direct role in regulating ECF, we will clone each gene and add epitope tags for protein purification and the generation of monoclonal anti-sera. These epitope tagged proteins will be over-expressed in N. gonorrhoeae and the location of the proteins determined by cell fractionation and Western immunoblot. To examine the interaction of the proteins with one another, the genes will be cloned into a bacterial protein two-hybrid system for analysis of protein::protein interactions.
AIMS:
1. To epitope-tag ECF, NG01947 and NGO1948 and assess their localization within the bacterial cell by cell fractionation and Western Immunoblot. 2. Protein purification for anti-sera production.
3. Cellular biology of the human nasopharynx
This work is being performed in collaboration with Professor David Stephens (Emory University), Dr Steve Webb (Royal Perth Hospital), Dr Bastiaan DeBoer (PathWest) and Dr Paul Rigby (BIAF, UWA). Neisseria meningitidis naturally inhabits the nasopharynx of humans, and in some instances, causes invasive infections culminating in rapidly fatal sepsis. Early studies showed that meningococci bound to non-ciliated epithelial cells in the nasopharyngeal organ culture model (NPOC). Upon attachment by meningococci, microvilli on the surface of these cells became elongated, eventually forming lamellipodia which engulf the bacterium as it is internalized. Transformed or primary epithelial cell cultures have provided the simplest model to analyze bacterial adherence and invasion, and has allowed for the identification of a number of neisserial adhesins (i.e. pili, Opa, Opc) and additional putative virulence determinants which affect bacterial adherence and invasion into host cells (i.e. lipooligosaccharide [LOS], capsule, PorB). To date, this data has been obtained using transformed cell lines growing in vitro, and attempts to translate these observations to the original NPOC model have yet to be attempted.
AIMS:
1. Characterize human nasopharyngeal epithelium for expression of cellular antigens required by meningococci for invasion. 2. Examine the co-localisation of meningococci with known cell types in human nasopharyngeal epithelium. 3. Examine the role of lipopolysaccharide in attachment of meningococci to nasopharyngeal epithelium.
79 Microbiology and Immunology DR THELMA KOPPI
Room 2.29, Microbiology and Immunology, L Block, QEII Medical Centre, Phone: 9346 2215 Email: [email protected]
Dendritic Cell Biology Research in my laboratory involves the regulation of dendritic cells in humans. Research has centred on how cells die and are replaced and the influence of inflammatory proteases on cell migration.
PROJECTS
1. To determine whether lineage negative DC (plasmacytoid and myeloid DC) undergo CD95-mediated apoptosis as has been found for monocyte-derived DC Dendritic cells (DC) are potent activators of primary immune responses during their interaction with naïve T cells in secondary lymphoid organs. Activation of DC and their subsequent migration from non-lymphoid tissues to regional lymph nodes have been shown to be early steps during inflammatory processes and crucial events in the generation of cell-mediated immune responses against various pathogens. DC have been isolated from adherent blood mononuclear cells which have been cultured for seven days with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 which give rise to cells with the phenotypic and functional properties of DC (Monocyte-derived DC or MoDC). CD95 (Fas/APO-1) and CD40 are members of a family of cell surface proteins that include the two TNF receptors, the nerve growth factor receptor, CD27, CD30, CD40, OX40, 4-1BB and the TNFR related protein (LTβ receptor). Incubation of Mo-DC expressing CD95 with either agonistic CD95-specific antibodies or CD95 ligand (CD95L) leads to the death of the cells by apoptosis. CD40 ligand (CD40L, T-BAm, TRAP, or gp39) is a transmembrane protein of 32-39 kDa that is expressed on the surface of T cells and mast calls and has homology to TNF and other members of the TNF ligand family. Cross linking CD40 with either CD40L or anti-CD40 mAbs rescues Mo-DC from apoptotic death. Since Mo-DC are 7- day-cultured immature cells it is important to compare these results with immature DC obtained by direct immunodepletion from blood (lineage negative DC). Both plasmacytoid and myeloid DC will be prepared by immunodepletion. Many detectors of apoptotic death stain dead or dying cells, which may be washed away after staining. The cell-permeant nucleic acid stain LDS 751 has been used to discriminate intact nucleated cells from nonnucleated cells and cells with damaged nuclei, as well as to differentiate apoptotic cells from nonapoptotic cells. It is a vital, nucleic acid stain and can be added to living cells. There is no washing process and dead cells may be stained with propidium iodide and the results assayed by flow cytometry. It is proposed to use this novel method to measure apoptosis and compare with other methods of measuring cell death.
2. Enhanced survivability of dendritic cells: potential role of protease-activated receptors The failure of host antigen presenting cells (APC) to present tumour antigen to the immune system is a means by which tumours escape detection. However, APC taken from cancer patients may be loaded in vitro with tumour antigens prepared from host tumour cells. These APC may then present these antigens, upon injection back into the patient, to host T cells. These T cells, once activated can recognize and destroy the tumour. It has also been shown that human blood-derived immature DCs are potent anticancer cytotoxic cells capable of inducing selective apoptotic death in a variety of human cancer cell lines both in vitro and in vivo. The generation of large numbers of APC (DC) for scientific study in recent years has led to the utilisation of these DC in immunotherapy However, DC after injection into patients, do not survive and migrate to the tumour site and many die at the site of injection. Koppi et al., 1997 have shown that Mo-DC easily undergo programmed cell death, but also that DC can be protected by the addition of cytokines or by blocking receptors for initiating apoptosis. It is also known that after stimulation with antigen, DC upregulate chemokine receptors which aid in their movement. Dead and dying DC are not functional and so cannot be stimulated with antigen to upregulate maturation or chemokine markers, secrete chemokines, or release the cytokines that initiate and regulate responses. Enhancement of the survival and migration of DC to the tumour site is crucial in immunotherapy and it is important to discover suitable adjuvants which can bring this about. Our preliminary data have shown us that PAR on DC may be activated by PAR agonist peptides with consequent improvement in DC viability and induction of cluster formation. We will discover what this/these factor(s) are by HPLC and mass spectrometry and use them directly on DC to improve DC survival and migration.
80 Microbiology and Immunology
CLINICAL PROFESSOR BARRY MARSHALL The Marshall Centre for Infectious Diseases Research and Training, Room 2.14, Microbiology and Immunology, L Block, QEII Medical Centre, Phone: 9346 4815 Email: [email protected]
Helicobacter Research Laboratory and Ondek
Our group is interested in the bacterium, Helicobacter pylori, a ubiquitous gastrointestinal pathogen which infects more than half the population of the world and is the aetiological agent of gastritis and peptic ulcers. Generally, research has focused on the eradication of this organism. However, Ondek Pty Ltd. (Prof. Barry Marshall’s vaccine development company) is taking the novel approach of using this bacterium as a vaccine delivery system. Ondek Pty Ltd. aims to use genetically modified H. pylori to present parts of other pathogens to the immune system. It is anticipated that infection of the host with these genetically modified H. pylori will result in immunization against these other pathogens. Specifically, Ondek Pty Ltd. is aiming to produce an inexpensive, stable and easily administered vaccine against influenza, which would greatly benefit both developed and developing countries. Ondek is based in the new state of the art Marshall Centre for Infectious Diseases. Prospective Honours students with a background in Molecular Biology, Microbiology, Biochemistry, Genetics or Immunology are encouraged to apply. Students will be involved in genetic engineering of H. pylori, and the evaluation of immune responses to these genetically modified bacteria. Students will be exposed to a number of techniques including DNA cloning, DNA sequencing, bacterial transformation, protein analysis, immunoassays, confocal microscopy, FACS analysis and animal infection models.
PROJECTS
1. Systems for the Presentation of antigens by Helicobacter pylori for Vaccine Development With Assoc Prof Mohammed Benghezal ([email protected]) and Dr Tobias Schoep ([email protected])
The development of live attenuated vaccines requires a thorough understanding of the host-pathogen interaction and the availability of tools to engineer safe genetically modified organisms (GMOs). Molecular genetics of H. pylori will be used to present proteins of pathogens to the immune system, in order to elicit a specific immune response. Strategies for presenting antigens include cell surface display, secretion or cytoplasmic expression. Aims include: fusing antigens to various carrier proteins or macromolecules, examining their presentation in vitro, and identifying which of these result in an immune response in mice.
81 Microbiology and Immunology
2. Quantitative detection of Helicobacter pylori in experimentally infected mice by real-time PCR. With Dr Helen Windsor ([email protected])
Accurate detection of the level of infection of H. pylori in experimentally infected mice is critical for determining success in this animal model. At present this is routinely measured by bacterial culture but the use of real-time PCR will allow the measurement of the intensity of the fluorescently labelled amplified DNA and should enable the detection of as few as 10 bacterial cells. This new technique will need to be optimized with the new Roche LC 480 Light cycler and validated in our laboratory using biopsies from H. pylori- infected mice. If wanted hands-on animal work could be done by the honours student or else close collaboration will be needed with the researchers in our team who are responsible for mouse handling. This technique, once established in our laboratory, will also be used to determine the levels of infection in human patients.
82 Microbiology and Immunology DR JASON PLUMB ADJUNCT SENIOR LECTURER Room W4.10, CSIRO, Floreat, Phone: 9333 6253 Email: [email protected]
Urban and Industrial Water Microbiology Group
Our group is interested in the use of microorganisms and biotechnological processes for environmental and industrial applications, such as in the mining, water supply and waste treatment industries. Our work investigates the microbial ecology of natural and industrial microbial processes and provides an assessment of the capabilities and limitations of these processes for beneficial applications. We work with a diverse range of Bacteria and Archaea including extremophilic acidophiles, thermophiles, oligotrophs, and halophiles and have interests in measuring rates of microbial growth and activity towards the development of bioreactor or other processes for industrial application. We are also interesting in discovering novel microorganisms and characterizing their role in natural or industrial processes e.g. mineral sulfide bioleaching, drinking water treatment and distribution and waste treatment. Prospective Honours students with an interest in environmental microbiology, biotechnology and microbial physiology are encouraged to apply. Our laboratories are equipped with facilities to perform microbial growth and activity studies using bioreactors, respirometry, aerobic and anaerobic growth conditions and also to conduct molecular biology-based analyses including, DGGE, real-time PCR, DNA cloning, flow cytometry and fluorescence microscopy.
PROJECTS
1. Biofilm Growth and Activity in Water Distribution Systems Supervisors: Dr Jason Plumb and Dr Maneesha Ginige, CSIRO, Floreat
The influence of the treatment and distribution systems on water quality has recently been given considerable attention. Changes in water quality can occur that lead to considerable problems due to lack of compliance with drinking water guidelines, and through elevated potential risks to human health. Of particular concern is the deterioration of water quality due to ineffective treatment and disinfection of water in long pipelines. Increased water age is correlated with increased disinfectant demand usually due to biofilm regrowth. Biofilm regrowth can lead to the proliferation of pathogenic microorganisms and cause water quality problems related to decreased water aesthetics and other issues. Avoiding problems associated with biofilm regrowth involves determining ways to prevent microbial growth in water distribution systems. Improved water treatment processes that remove bioavailable and biodegradable organic matter have the potential to reduce biofilm regrowth. Other important factors include the choice of disinfectant and also the effect of temperature on biofilm growth and water quality.
Complex interactions between natural organic matter, inorganic chemical species, microorganisms and disinfectants determine the potential for the formation of biofilms in water distrubution systems. The scope of this project is to develop and apply advanced microbiological techniques to enable better characterisation of biofilms, and the effect of prevailing physico-chemical and disinfection regime conditions on the biofilm growth and activity in order to inform the better management of water distribution systems for delivery of safe high quality drinking water to consumers.
References Piriou, P., Dukan, S., Kiene, L. 1998. Modelling bacteriological water quality in drinking water distribution systems. Water Science and Technology 38, 299-307. Rittmann, B.E., Snoeyink, V.L. 1984. Achieving biologically stable drinking water. Journal AWWA 76, 106-114. van der Kooij, D., Visser, A., Hijen, W.A.M. 1982. Determining the concentration of assimilable organic carbon in drinking water. Journal AWWA 74, 540-545.
83 Microbiology and Immunology
2. Determining Sources of Faecal Contamination in Western Australian Aquatic Environments Supervisors: Dr Jason Plumb and Dr Geoff Puzon, CSIRO, Floreat
Waterborne pathogenic microorganisms are a potential threat to public health. Maintenance of good microbiological quality in aquatic environments used for drinking water supply or for recreational activities is essential to limiting the risk to public health. Perhaps the biggest challenge to the maintenance of good microbiological quality in aquatic environments is faecal contamination either from human or animal sources. In order to manage and minimise the risks associated with faecal contamination of aquatic environments, an understanding of the source of faecal contamination is required. Knowledge of the source of faecal contamination provides an insight into the extent of the associated health risks and an opportunity to eliminate the contamination.
This project seeks to apply established and novel molecular typing methods to profile selected pathogenic microorganisms from aquatic environments in WA. Possible sources of faecal contamination will be sampled and profiled for comparison with waterborne faecal indicators. An assessment of the effectiveness of profiling methods for application to WA aquatic environments will be made. The project will provide an insight into key issues facing the environment and water resources sector and the challenges associated with protecting water resources.
Aims 1. To survey possible sources of faecal contamination and profile selected indicator microorganisms 2. To assess the suitability of established and novel methods for profiling faecal contamination sources in WA aquatic environments
Techniques to be used include 1. Molecular typing techniques (e.g. real-time PCR, PCR-DGGE, ribotyping) 2. Analysis of chemotaxonomic markers (e.g. signature lipid analysis) 3. Microbial culturing and identification 4. Water and environmental sample collection 5. Data manipulation, and analysis (e.g. nucleic acid analysis) 6. Literature interrogation
References: Blanch, A.R., Belanche-Muňoz, L., Bonjoch, X., Ebdon, J et al. 2006. Integrated analysis of established and novel microbial and chemical methods for microbial source tracking. Applied and Environmental Microbiology, 72, 5915-5926. Carson, C.A., Shear, B. L., Ellersieck, M.R. and Schnell, J.D. 2003. Comparison of ribotyping and repetitive extragenic palindromic-PCR for identification of fecal Escherichia coli from humans and animals. Applied and Environmental Microbiology, 69, 1836-1839. Scott, T.M, Rose, J.B., Jenkins, T.M., Farrah, S.R. and Lukasik, J. 2002. Microbial source tracking: current methodology and future directions. Applied and Environmental Microbiology, 68, 5796-5803.
84 Microbiology and Immunology Dr Alec Redwood Dr Lee Smith Dr Megan Lloyd Professor Geoffrey Shellam
The Marshall Centre for Infectious Diseases Research and Training, Room 2.04, Microbiology and Immunology, L block, QEII Medical Centre, Phone: 9346 2512 Email: [email protected]
Murine cytomegalovirus (MCMV) is a mouse-specific herpesvirus that is commonly used as a model for human cytomegalovirus infection. MCMV has similar growth characteristics to HCMV and induces similar disease states. Our group is interested in the natural genetic variation found in strains of MCMV that have been isolated from wild mice and the effect of genetic variability on the pathogenicity and tissue tropisms of the virus. Several genetically different strains of MCMV have been isolated from a single mouse, and we are interested in the relationship between these different strains and the resulting disease in the host animal. Additionally, we have demonstrated over many years that MCMV is a remarkably effective vector for the delivery of foreign antigen to vaccinated animals. A number of immunogenic antigens such as ovalbumin, haemagglutinin, and various reproductive antigens have been incorporated into the viral genome, and rapid and long-lived immune responses have been induced. The Cytomegalovirus Research Group is located in state of the art laboratories within the new Marshall Centre for Infectious Disease Research and Training in the Discipline of Microbiology and Immunology.
Cytomegalovirus Research Group
Projects
1. Requirements for MCMV endothelial cell tropism
Dr Alec Redwood and Dr Lee Smith
MCMV replicates in most cells in the body, however its replication in host endothelial cells is of particular interest because these cells play a role in the transmission of the virus to macrophages, which then enable the virus to spread to distal sites. In addition, infection of endothelial cells is likely to play a role in the development of atherosclerosis, which has been associated with human cytomegalovirus infection. We have recently identified two strains of MCMV that demonstrate different capacities for endothelial cell replication. One strain C4C replicates to high titres in endothelial cells and the other, C4B replicates poorly in these cells. This project will investigate the causes of poor endothelial cell replication by C4B, specifically the role of apoptosis, cell entry and cell-to-cell spread in this defect. It is anticipated that these viruses will be used to develop models of atherosclerosis in mice. As such this project would suit a student interested in molecular biology, virology and basic cellular biology and will involve cell culture, molecular biology and flow cytometry.
85 Microbiology and Immunology
2. Molecular mechanisms of cytomegalovirus immune evasion
Dr Lee Smith and Dr Alec Redwood
MCMV encodes multiple genes which are non-essential for viral growth in tissue culture. These genes interact with components of the host immune system, allowing the virus to modulate numerous defence mechanisms and to therefore establish persistent infection. MCMV is able to affect host natural killer (NK) cell responses, to regulate cell surface major histocompatibility complex (MHC) class I expression, and to modulate apoptosis of infected cells.
We have noted that low passage wild-derived strains of MCMV affect some of these host defense mechanisms in different manners. This project will extend previous findings, and assess the capacity of these low passage isolates to inhibit MHC I and class II on the surface of infected cells. MHC class I and class II molecules are responsible for presentation of viral peptides to host CD8+ and CD4+ T cells respectively, and are therefore are critical in control of viral infection. As most mice are naturally infected with multiple strains of MCMV, we will also assess the capacity of variant MCMV strains to complement the function of each other by co-infection of cells. As such this project would suit a student interested in virology, and will involve cell culture, flow cytometry, and molecular biology. This project builds on previous work and will be part of an on-going research project.
3. The effect of MCMV on pregnancy
Dr Lee Smith, Dr Megan Lloyd, Professor Geoff Shellam
Human cytomegalovirus (HCMV) has replaced rubella virus as the most important viral infection of the fetus in utero, and is responsible for significant morbidity and mortality worldwide. Because of the strict species specificity of HCMV, experimental studies of CMV infection employ the mouse model using murine CMV (MCMV). However, although most characteristics of MCMV-induced disease mimic HCMV infection, it is generally thought that MCMV does not readily cross the placenta and infect fetal mice. However, preliminary data obtained in our laboratory suggests that this is not the case, and that fetal infection with MCMV is possible.
This project therefore seeks to define the effect of both laboratory and wild-derived MCMV strains on mouse pregnancy by investigating both fetal and maternal health. Female mice at different gestational stages will be infected with different MCMV strains (either one strain, or a mixture of several strains) and the effect of this infection on fetal and maternal health will be determined by measuring fetal and placental weights and looking for the presence of MCMV in fetal and maternal tissues. Virus detection will be carried out using a variety of methods such as quantitative PCR, in-situ hybridization and ELISA. By characterising the effect of these strains on pregnancy, we will be better able to model congenital HCMV infection. This would significantly contribute to the understanding of cytomegalovirus disease.
86 Microbiology and Immunology PROFESSOR THOMAS RILEY DR CHRISTINE CARSON DR KATE HAMMER K Block, QEII Medical Centre. Phone: 9346 3690/3288/1986 [email protected] [email protected] [email protected]
(www.tto.bcs.uwa.edu.au)
Antimicrobial and Natural Products Research (Tea Tree Oil Research Group)
Our group is located within the PathWest Laboratory Medicine building at the Queen Elizabeth II Medical Centre and we conduct, in the main, applied research. This research covers a wide range of areas, with a broad focus on antimicrobial agents and some specific micro-organisms. As such a variety of projects is always available covering any aspect of clinical microbiology.
This laboratory has a long history of projects investigating the antimicrobial activity of antimicrobials, natural products and other compounds. The major natural product investigated so far is tea tree oil, the essential oil obtained from the Australian native plant Melaleuca alternifolia. This essential oil has been the subject of research by the group for over 10 years. The group has investigated other natural products such as essential oils from other native Australian plants and the honey derived from Australian native bees. We have been contracted by several pharmaceutical and biotechnology companies to investigate new antimicrobials.
1. Formation and characterisation of the cytoplasmic inclusion bodies seen in Pseudomonas aeruginosa after exposure to terpinen-4-ol Dr Christine Carson and Dr Kate Hammer
The essential oil of Melaleuca alternifolia, commonly known as tea tree oil (TTO), has indisputable antimicrobial activity against bacteria, fungi and viruses and is becoming increasingly well-accepted as an alternative topical antimicrobial agent. Pseudomonas aeruginosa is more resistant to TTO and its components than most other bacteria. Previous work in our laboratory has suggested that P. aeruginosa may be made even more resistant to TTO and terpinen-4-ol, one of its main antimicrobial components, and that inclusions form in the cytoplasm when it is exposed to terpinen-4-ol (see figure). This project will involve characterising the formation and content of these inclusions using methods similar to those described previously for other bacteria. Numerous mutants resistant to TTO and/or terpinen-4-ol previously generated in the laboratory will also be examined. The formation of inclusions in the presence and absence of energy uncouplers such as CCCP or sodium azide will be observed to determine if accumulation is an energy dependent process. Growth of P. aeruginosa on minimal media containing terpinen-4-ol as a sole carbon source will also be attempted. If time permits, the effects of exposure to other components of TTO will be evaluated. Results from this study will contribute to our appreciation of the mechanisms that P. aeruginosa uses to withstand normally toxic levels of these terpenes, and hydrocarbons in general, and to a broader understanding of the potential for resistance to develop in other bacteria.
2. Effects of bacterial exposure to tea tree oil on antimicrobial susceptibility profile Dr Christine Carson, Dr Kate Hammer, Dr Kerry Carson and Barbara Henderson, PathWest Laboratory Medicine WA
The use of TTO as a topical antimicrobial agent is supported by a growing number of studies, both in vitro and in vivo. However, recently published data suggests that the exposure of bacteria to tea tree oil reduces their subsequent susceptibility to clinically important antibiotics. This phenomenon requires much further investigation. Bacteria are known to have a range of adaptive mechanisms that they employ to counter harsh environments or adverse conditions. There is also evidence that bacteria that have their inbuilt stress responses induced are subsequently less susceptible to TTO than non-stressed cells. However, since little is known about how bacteria respond to TTO or what kind of protective mechanisms they may use, very little can be done to counter or reduce their protective efforts. It is critical to investigate the manner in which microorganisms
87 Microbiology and Immunology respond or adapt to TTO and whether this influences the subsequent effectiveness of conventional antibiotics and our ability to effectively treat infectious disease. The primary aim of this project is to investigate if the adaptive changes caused by TTO habituation that seemingly offer cross-protection for antimicrobial compounds, are due to a stress response in microorganisms. Initially the work of McMahon et al. will be replicated and we will also look for reversion to initial antimicrobial susceptibility profiles after a period of growth without TTO. We will also examine whether the adapted responses seen are the same for whole oil as for individual TTO components such as terpinen-4-ol. Comparison of the responses shown previously for other compounds such as organic solvents will also be undertaken to determine whether any of the responses are unique to TTO. Finally, the mechanisms by which the stress responses protect cells against TTO will be investigated. This is a collaborative project with PathWest Laboratory Medicine WA.
3. Characterisation of antimicrobial activity of honey produced by Australian native stingless bees Dr Kate Hammer, Dr Christine Carson and Dr Kerry Carson (PathWest Laboratory Medicine WA)
Interest in the use of honey for medicinal purposes has increased in recent years, in parallel with rising interest in other alternative or natural therapies. The major therapeutic uses of honey take advantage of its antimicrobial properties. The main factors that account for the antimicrobial activity of honey are the high sugar content, pH and hydrogen peroxide activity. Most of the recent honey research has focussed on manuka honey, which is honey produced by the European honeybee Apis mellifera. European honeybees are not the only bees to produce honey. Stingless bees from genera such as Trigona and Melipona also produce honey, known as sugarbag. Sugarbag differs chemically from European honeybee honey in several important ways, including sugar and water content. Since most honey is known to have antimicrobial activity it would seem reasonable to assume that sugarbag also possesses activity. However, very few investigations into the antimicrobial activity of sugarbag have been conducted and these studies have not focussed on sugarbag from Australian native stingless bees. This project will investigate the antimicrobial activity of sugarbag from Australian stingless bees and explore its potential as an alternative topical medicinal product. The specific aims are to (1) investigate the antimicrobial activity of several sugarbag samples, (2) compare the activity of sugarbag with a control sugar solution and several commercially produced honeys and (3) investigate which factors within the sugarbag are most critical for activity.
4. Clostridium difficile Professor Tom Riley
There is great concern world-wide about a new infectious diseases threat following the recent emergence in Canada, the USA, and now Europe, of a highly virulent strain of Clostridium difficile (called PCR ribotype 027 in Europe and NAP1 in the USA). Rates of detection of C. difficile have risen dramatically, C. difficile disease has been more severe, and attributable mortality was >10% in those aged >60 years. C. difficile is the most commonly diagnosed cause of infectious hospital-acquired diarrhoea in developed countries. The majority of patients with C. difficile-associated diarrhoea (CDAD) have been exposed to antimicrobials that reduce ‘colonisation resistance’ of the large intestine allowing subsequent infection with C. difficile. Acquisition of C. difficile is facilitated by its ability to form spores that are resistant to many disinfectants allowing it to remain viable in the hospital environment for long periods of time. Toxigenic isolates of C. difficile usually produce two toxins, toxin A and toxin B, and these are thought of as the major virulence factors. Some strains of C. difficile produce an additional toxin, binary toxin (actin-specific ADP-ribosyltransferase, CDT), first reported in 1988 but not considered important until now. Binary toxin producers make up the majority of strains isolated in the large outbreaks of disease overseas, however, the significance of binary toxin needs further investigation. A second important feature of this “new” organism is that it produces more toxin A and B than other strains. The third important feature of these strains is that they are resistant to fluoroquinolone antibiotics, and excessive fluoroquinolone use appears to be a contributing factor in the recent outbreaks. We are undertaking surveillance to see if PCR ribotype 027 is in Australia and collaborating with Monash University on an investigation of antimicrobial resistance, virulence factors and genetics.
References 1. Carson, C.F., Hammer, K.A., and Riley, T.V. 2006. Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties (Review). Clin Microbiol Rev 19: 50-62. 2. Riley, T.V. 2006. Epidemic Clostridium difficile (Editorial). Med J Aust 185:133-4. 3. McMahon M.A. et al. 2007. Habituation to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia) is associated with reduced susceptibility to antibiotics in human pathogens. J Antimicrob Chemother 59:125-7.
88 Microbiology and Immunology DR HARRY SAKELLARIS LECTURER Room 2.02A, L Block QE2 Medical Centre, Phone: 9346 2286, Email: [email protected]
Bacterial Pathogenesis Group
Our research is focused on understanding the pathogenesis of diarrhoeal diseases caused by Enterotoxigenic E. coli (ETEC), a Gram-negative bacterium which is responsible for a cholera-like illness that causes massive water loss from the intestine. An estimated 650 million people suffer from ETEC infections and 650,000 – 800,000 people, mainly children less than five years of age, die from ETEC infections annually (1). Despite the heavy toll on human life, there is no broadly effective vaccine to prevent infections and our understanding of the pathogenesis of ETEC infections is incomplete. ETEC is transmitted to humans via the consumption of contaminated water and the bacterium subsequently attaches to and colonises the small intestine via pili (fimbriae). Once the pathogen has established a foothold in its new host, it secretes enterotoxins that induce diarrhoea. Therefore the Recombinant E. coli K12 expressing binding of pili to intestinal cells represents the critical first step in the CS1 pili pathogenesis of ETEC diarrhoea. We have cloned the four structural genes for CS1 pili and can express pili in recombinant E. coli strains that are otherwise “bald.” This has allowed us to manipulate the genes to study the functions of the proteins they encode and to determine how pili are assembled on the bacterial cell surface (2-4).
The expression of CS1 pili on the surface of ETEC cells is directly under the control of a transcriptional regulator called Rns (5, 6). Work in our laboratory has established that Rns also activates the expression + - of genes other than those involved in CS1 pilus production. These CS1 CS1 include at least two known virulence proteins, suggesting that, rather than being a specific regulator of CS1 expression, Rns may be a master regulator of virulence genes in ETEC. Therefore, we propose that identifying the full complement of genes controlled by Rns may reveal new ETEC virulence genes and therefore increase our understanding of the disease process.
PROJECT AIMS In the Honours project proposed here, the full complement of genes regulated by Rns will be determined by constructing an rns mutation in ETEC and, by proteomic analysis of the mutant and wild-type strains, determine which proteins in the cell are regulated by Rns. The significance of regulated genes will be investigated by applying a combination of bioinformatics, cloning, expression and functional analysis. This approach will define the complete Rns regulon and has the potential to identify new virulence factors in ETEC. Techniques that will be used in this project include PCR, cloning, DNA sequencing, recombinational mutagenesis, gene expression analysis by quantitative RT PCR, proteomic analysis and routine microbiological techniques.
REFERENCES 1. Gaastra, W., and A.-M. Svennerholm. 1996. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends in Microbiology 4:444-452. 2. Sakellaris, H., D. P. Balding, and J. R. Scott. 1996. Assembly proteins of CS1 pili of enterotoxigenic Escherichia coli. Mol Microbiol 21:529-41. 3. Sakellaris, H., G. P. Munson, and J. R. Scott. 1999. A conserved residue in the tip proteins of CS1 and CFA/I pili of enterotoxigenic Escherichia coli that is essential for adherence. Proc Natl Acad Sci U S A 96:12828-32. 4. Voegele, K., H. Sakellaris, and J. R. Scott. 1997. CooB plays a chaperone-like role for the proteins involved in formation of CS1 pili of enterotoxigenic Escherichia coli. Proc Natl Acad Sci U S A 94:13257- 61. 5. Murphree, D., B. Froehlich, and J. R. Scott. 1997. Transcriptional control of genes encoding CS1 pili: negative regulation by a silencer and positive regulation by Rns. J Bacteriol 179:5736-43. 6. Froehlich, B., L. Husmann, J. Caron, and J. R. Scott. 1994. Regulation of rns, a positive regulatory factor for pili of enterotoxigenic Escherichia coli. J Bacteriol 176:5385-92.
89 Microbiology and Immunology PROFESSOR GEOFFREY A STEWART Room G02, MCS Building, Phone: 6488 4699 Email: [email protected]
Inflammation and Infectious Diseases Group
Our group is interested in inflammatory and atopic lung diseases such as asthma and allergic rhinitis as well as respiratory and prostate infections. Our long-term aim is to elucidate the immunomodulatory effects of the allergens and pathogen associated molecular patterns responsible for these diseases and to develop novel therapeutic strategies for their intervention. We have isolated and characterised several house dust mite, pollen, and cockroach allergens. Prospective Honours students with a background in Immunology, Microbiology, Molecular Biology, Biochemistry or Molecular Genetics are particularly encouraged to apply. Students will be exposed to a range of techniques including DNA cloning gone array, cell culture, transfection assays, cytokine ELISA, RAST and RAST-inhibition assays, RT- and Q-PCR, protein expression and analysis, bacterial characterisation, enzymology, confocal and epifluorescence microscopy, immunohistochemistry and FACS analysis.
PROJECTS
1. Anti-Microbial Peptides and Pro-Phenyloxidase Activity in the House dust Mite With Dr Leslie Mathaba ( [email protected])
Invertebrates produce a variety of cationic antimicrobial peptides (e.g., defensins, cecropins and attacins) and enzymes involved in melanisation in response to potential pathogens. Most of the work has been performed in insects and little information exists with regard to their presence in other arthropods such as arachnids (spiders and mites). Similarly, the enzyme pro-phenyloxidase is involved in producing melanin which results from the production of quinones and plays an important antibacterial role. Whether dust mites, which are associated with asthma, produce anti-microbial peptides or possess the pro-phenyloxidase system is unknown. Our research on dust mites, has resulted in the isolation of 2-3 bacteriolytic enzymes, which could play a role in defending mites against bacterial pathogens. Students will undertake an analysis of such antimicrobial products in mites using homology and enzymatic based approaches. Our preliminary studies have revealed the existence of such peptides using a mite cDNA library and primers based on the amino acid sequences of androctonin and gomesin, hairpin-like beta sheet structures from Androsctonus australis and Acanthoscurria gomesiana in a low stringency PCR analysis.
2. Modulatory Effects of Rye Grass Pollen Allergens on Respiratory Epithelial Cell Function With Dr Martha Ludwig ([email protected]) and Professor Alice Vrielink ([email protected])
Aeroallergens are considered to be the most common cause of allergic disease and are derived from a variety of complex particulate sources present in the environment. They include pollens, fungal spores, insect and mite faeces, animal danders and dusts, and exposure may be perennial or seasonal. Allergens from the pollens of several grass species have been described at the molecular level and include species belonging to the clinically important subfamilies Pooideae and Panicoideae. There is significant sequence similarity and, hence, immunological cross-reactivity between allergens from botanically related pollens which has made it possible to group allergens from related species together. This has resulted in the description of more than 13 distinct groups of proteins with diverse biochemical properties. To cause disease, an allergen must make contact with the respiratory mucosa and, in previous studies, we have shown that respiratory epithelium responds to exposure to allergens in this way by producing a range of pro-inflammatory mediators that are able to contribute to allergic disease. However, whether pollen allergens do this is unclear but recent exciting preliminary data from our laboratories suggest that they do. These novel findings indicate that further studies are warranted and in this project, students will continue and extend these studies. In doing so, students will be exposed to state-of-the-art cloning and protein expression techniques, gene array studies, cytokine measurements and protein structure determination by crystallographic methods.
90 Microbiology and Immunology
3. Cloning, expression and characterisation of mite proteases and bacteriolytic enzymes. With Dr Leslie Mathaba ( [email protected])
Domestic mites are medically and economically important pests responsible for the development of allergic diseases and for the destruction of stored grain and seed resulting in poor yields. Our laboratory has been involved in the isolation and characterisation of mite allergens. Recently, we isolated a new cysteine protease from the mite Dermatophagoides pteronyssinus designated Der p 9/LM-1. Der p 9/LM-1 has been partially characterised and shown to be a very important allergen recognised by over 70% of mite allergic individuals. We also observed that mites produced enzymes possessing bacteriolytic activity. Bacteriolytic enzymes have never been reported in mites and this was exciting for us given that some bacteriolytic enzymes have been implicated in allergies. We have isolated and sequenced a cDNA encoding a 14 kDa bacteriolytic enzyme and the sequence data showed homology with bacterial rather than eukaryotic proteins suggesting that it could be of bacterial origin. Expression of these cDNA is essential to enable a detailed physicochemical and immunochemical characterisation of Der p 9/LM-1 and the 14 kDa bacteriolytic enzyme. In addition, we have shown that these proteins are produced by other mites (e.g., Blomia tropicalis). Using the PCR and other techniques the genes/cDNA coding for these proteins in other mites will be isolated for comparison with the D. pteronyssinus cDNA sequences.
4. Protection from Complement-Mediated Damage on Respiratory Epithelium. With Dr Asokananthan ([email protected])
Complement comprises groups of proteins that play an important role in both innate and adaptive immunity. They act as opsonins to facilitate phagocytosis and cause lysis of cells via the membrane attack complex (MAC). So potent are they that a number of regulatory proteins exist to moderate their activities once activated. In this regard, CD55 and CD59 are important because they accelerate the decay of membrane bound convertases and inhibit the MAC activity. We have recently shown the upregulation of the genes encoding these proteins by pro- inflammatory stimuli. These are novel findings in the context of the lung and we wish to further explore the biology of these proteins. In addition, these proteins exist as isoforms which are produced by alternate splicing mechanisms and may be secreted. In this study, students will determine whether their secretion is modulated by pro-inflammatory stimuli and whether different isoforms are differentially modulated. Stimuli will include microbial pathogen associated molecular patterns, proinflammatory proteases and allergens. In doing so, students will be exposed to cell physiology techniques, ELISA, real time PCR and protein characterisation studies. Findings from such studies may help us understand the mechanisms involved in protecting the lung from pro-inflammatory insults.
91 Microbiology and Immunology DR DAVID SUTTON
SENIOR LECTURER Room 1.16, Microbiology & Immunology, L Block, QEII Medical Centre. Phone: 9346 4873 Email: [email protected]
Applied and Environmental Microbiology Group
Our group is interested in using an understanding of the diversity and roles of microbes in natural (particularly marine) systems to identify potential applications, particularly in the area of disease control and industrial applications. Our focus is on microbes with the potential to act as biological or probiotic control agents against bacterial agents of human and aquaculture disease, and on natural products (of microbial or marine invertegrate origin) that have antimicrobial activities which may have application in clinical and industrial settings. We also study marine microbial diversity and its interactions with host organisms (from mutualistic symbiosis to disease), as study of these is central to detecting interactions mediated by chemicals with potential commercial applications. To this end we have developed appropriate isolation, identification and bioassay techniques for use in an established model disease situation. Our long-term aim is to develop biological control agents and antimicrobials for use in disease prevention and control strategies and in control of biofouling. Prospective Honours students with a background in Microbiology and Molecular Biology, Biochemistry or Chemistry are particularly encouraged to apply. Students will be exposed to a range of microbiology and molecular biology techniques.
PROJECTS
1. Quorum sensing inhibitors – an exciting new weapon in bacterial disease control? Supervisors: Dr David Sutton (UWA) Email: [email protected] Ph: 9346 4873 A/Professor Barbara Chang Email: [email protected] Ph: 9346 2288
Aims of the project are to seek quorum sensing inhibitors of Vibrio harveyi and investigate their nature and modes of action, and to assess their potential application in bacterial disease control. Quorum sensing is a bacterial cell communication process essential in many disease-associated processes including biofilm formation, virulence mechanisms or avoidance of host immune responses, and is mediated by ‘autoinducer’ molecules via pathways which have been extensively studied. Quorum sensing inhibitors (termed ‘quorum quenching compounds’) offer exciting applications in control of bacterial disease, as there is a growing body of evidence that such chemicals can reduce virulence in some pathogenic bacteria. As quorum quenching compounds only inhibit communication and do not kill bacteria, development of bacterial resistance, as occurs with antibiotics, may be reduced or not occur. A model system for detecting and investigating quorum quenching compounds, their mode of action and effect on virulence has been developed in Microbiology at UWA. The model is based on Vibrio harveyi, a bioluminescent bacterium and devastating aquaculture pathogen causing ‘luminous vibriosis’ of cultured finfish, crustaceans and molluscs. Bioluminescence in V. harveyi is a quorum sensing process which appears to be related to disease-causing ability, as mutants in quorum sensing genes lose virulence. The V. harveyi isolate used in our system is highly pathogenic to the crustacean Artemia, the disease host used in the established model. Quorum quenching compounds are produced by a number of bacteria (e.g. some Bacillus spp.) and certain plants and algae. Secondary metabolites of the marine alga Delisea pulchra have been reported to both inhibit bioluminescence of V. harveyi and reduce its virulence to prawns. In recent studies we have found strong evidence that other marine organisms, including sponges, also contain compounds with quorum quenching activity against V. harveyi. The production of such compounds as part of evolved defense mechanisms in marine organisms is not surprising, as they live permanently bathed in a medium (seawater) containing very large bacterial populations, typically in excess of 106/ml. In this project quorum quenching compounds which inhibit V. harveyi quorum sensing will be sought from a diversity of marine bacteria. Active compounds will be partially or fully purified, and the bacteria producing
92 Microbiology and Immunology them will be identified using molecular methods. The extracted compounds and the bacteria that produce them will be tested for their ability to inhibit quorum sensing processes (eg bioluminescence, biofilm formation, virulence factor expression) and to protect Artemia from infection by V. harveyi. The discovery of these types of compounds may well provide new opportunities for drug development as well as enhancing knowledge of defense mechanisms in marine organisms. Techniques to be used include PCR and sequencing, screening of natural products for inhibition of Vibrio harveyi or its quorum sensing system; chemical extraction and purification of bioactive compounds; bioassays (bioluminescence, biofilm, virulence factors); and Artemia bioassays for assessment of control of disease caused by Vibrio harveyi. References: 1. Chytanya, R. et al. 2002. Aquaculture, 208: 1-10. 2. Defoirdt, T. et al. 2005. Environmental Microbiology, 7: 1239-1247. 3. Lavilla-Pitogo, C.R. Leonon, E.M. and Paner, M.G. 1998. Aquaculture, 164, 337-349. 4. Manefield, M. et al. 2000. Applied Environmental Microbiology, 66: 2079-2084. 5. Moriarty, D. 1998. Aquaculture, 164: 351-358. 6. Rasch, M. et al. 2005. Systematic & Applied Microbiology, 27(3) 350-359. 7. Sullivan, A. and Nord, C.E. 2005. Journal of International Medicine, 257: 78-92.
93 Microbiology and Immunology
DR MARK WATSON
ADJUNCT SENIOR LECTURER HCV Research Group, Royal Perth Hospital Phone: 9224 1175 Email: [email protected]
Hepatitis C Virus Research Group Hepatitis C virus (HCV) is a major health problem in Australia with over 200,000 people currently infected, around 9,000 new cases annually and in 1998 estimated treatment and care costs were in excess of 40 million dollars. Primary HCV infection is only resolved by about 20% of individuals with the remaining 80% developing varying severity chronic infection that may persist for decades if untreated. During the chronic stage of disease, HCV can cause progressive liver damage and is the commonest cause of liver transplantation in all developed countries. It is also associated with increased risk of hepatocellular carcinoma. HCV is currently treated with pegylated interferon-α ( PEG-IFN ) and ribavirin to increase the response rates to between 50 and 80% depending on the viral genotype. Despite current advances, many patients are still refractory to treatment; the reasons for this are not completely understood. Some patients are able to carry HCV at significant viral loads with little or no associated pathology, whilst others with very low viral loads may have substantial liver damage. This dichotomy is not without probable cause as the immune responses that may assist with virus eradication may also play a role in the destruction of liver tissue in kind of double edged sword. For untreated or treatment failure patients progression to end stage liver disease is the commonest outcome. With poor prognosis liver transplant is the only treatment option. Transplant patients are immune suppressed after transplant to aid organ engraftment. Unfortunately the immune suppression also allows HCV still within the patient’s blood to infect the new organ and replicate unchecked by the immune system. This can cause serious damage to the new organ and rejection. Current therapy for HCV infection is contraindicated in transplantation due to immune suppression and thus there are no therapies available to reduce the viral burden. With HBV, anti HBV immunoglobulin is used and maintains viral load at a low level improving transplant survival and minimising damage. In HCV infection this is not yet available, largely due to the lack of suitable immunoglobulin that is in turn related to our inability to generate good antibodies against HCV. Despite the apparent lack of B cell mediated immunity directed at HCV in infected patients, there are infected individuals that have strong antibody responses to the virus and maintain low or undetectable viral loads. This indicates that in these patients virus replication may be suppressed by circulating antibody. These patients are a primary target for cloning B cells producing infection neutralizing antibodies. Production of monoclonal antibodies (MAbs) is not new and was first described in 1975 by Kohler and Milstein and is now commonplace in both mouse and rat systems. The production of human antibodies is also possible with the availability of suitable human myeloma fusion partners. Fig 1.X marks sites of targets for antiviral agents These systems are now quite efficient but in the case of HCV to date, there have been some inherent problems associated with the production of effective (infection- neutralizing) monoclonal antibodies.
Monoclonal antibody production involves several critical steps. These are 1) vaccination or infection of the target organism from which the B cells will be cloned; 2) obtaining B cells from the organism (usually spleen or lymph nodes used) and fusion with an immortalised cell line to immortalise the B cells and 3) screening of cloned B cells for the production of antibody directed to the target antigens or organisms. In the case of the hepatitis C virus all of these steps are problematic and prohibitive to the production of infection neutralizing antibodies. Firstly HCV cannot be produced in small animal models or grown in culture preventing the whole virus being used to vaccinate or infect mice or rats, the usual animals used to produce monoclonal antibodies. Secondly, Fig 2. Cultured cells infected with HCV virus
94 Microbiology and Immunology most HCV antigens have been expressed in E. coli often resulting in loss of native protein conformation and glycosylation, both of which are particularly important for the HCV surface glycoproteins E1 and E2. Some researchers have thus attempted to express HCV proteins in cell culture systems to promote native protein structure. Unfortunately only small amounts of antigens are available from these systems making their use as immunogens problematic. Also, without a cell culture system or small animal model for HCV infection, identification of infection neutralizing antibodies is near impossible. Most monoclonal antibodies produced and characterised to date against HCV recognise linear unglycosylated epitopes, a consequence of screening with bacterially expressed and denatured antigens, often in a Western blot format. We have developed one of the few HCV culture replicative models for HCV and are using this model to screen candidate antibodies produced from a human hybridoma line. We also have access to a more efficient myeloma fusion partner that we will use to generate more antibody lines. Our long term goal is to use our model system to study antibody and drug inhibitors of HCV infection with an end goal to developing therapeutic agents to control HCV infection. Our projects ar concerned with advancing our antiviral antibody projects in terms of identification of the antibody targets and developing new assays to identify cross reactivity with other HCV genotypes. We hope that this will identify new therapeutic agents to combat this serious infectious disease. For further explanation of projects described below it is recommended that interested students contact Dr Watson or Dr Lee directly.
PROJECTS
1. Development of alternative culture methods for HCV (Supervisors Dr Mark Watson, Dr Silvia Lee ([email protected]) and Associate Professor Jim Flexman)
We already have a replicative model for HCV in culture however there are significant drawbacks to this system. HCV exists as many genotype variants and even within genotypes the variation can be high. HCV’s inherent low fidelity replication results in a high mutation rate that results in a large genetic diversity. A single model system therefore is not ideal and one would like to be able to culture virus from any patient sample. This has not been possible with HCV until recently. Traditional culture techniques use serum from bovine sources but it seems clear that HCV prefers human serum supplements furthermore HCV does not replicate well in culture- adapted hepatocytes. It does appear that HCV isolates will replicate readily in cultured foetal liver cells and possibly hepatocyte stem cells. In this project we will use a variety of molecular (Real time RT PCR), flow cytometric and immunohistochemistry techniques to identify replicating HCV under an array of growth conditions to identify the most efficient replication system for primary viral isolations.
2. HCV Inhibitor target identification and assay development (Supervisors Dr Mark Watson, Dr Silvia Lee ([email protected]) and Associate Professor Jim Flexman)
This project will investigate a set of monoclonal antibodies we have isolated that appear to have antiviral activity against our existing HCV model. These antibodies along with several others under production may offer an immediate therapeutic treatment to HCV infected patients. Antibodies will be characterized and their target ligands determined using a variety of molecular techniques. These include immunoreactive staining using immunogold and immunohistochemistry (IHC) techniques, flow cytometry and western blotting. The immunogold and IHC assays will need development as new assays within the laboratory however, standard protocols as starting points already exist for these techniques in the literature. Should alternative models become available these will be used to test cross genotype reactivity to target antigens.
95 Microbiology and Immunology PHYSIOLOGY
DR TONY BAKKER SENIOR LECTURER Room 1.20, Physiology Building, Phone: 6488 7859 email: [email protected]
Skeletal Muscle Damage & Disease
Skeletal muscle function can be severely compromised by injury and disease, leading to loss of mobility and decreased quality of life. In the case of inherited muscle wasting diseases such the muscular dystrophies, damage to respiratory muscles (diaphragm) can lead to death of patients in their early twenties.
Ca2+is a universal second messenger that is essential for the normal function of almost all cells. Changes in intracellular Ca2+ can activate a diverse array of processes such as muscle contraction, cell secretion and cell to cell communication. However, if Ca2+ levels remain high within a cell for too long, cell death can occur. In Duchenne muscular dystrophy, a pathalogical rise in intracellular Ca2+ is thought to be the ultimate cause of muscle cell death in these patients. The high Ca2+ is thought to kill the muscle cells by over-simulating cellular 2+ Ca -activated enzymes such as the calpains and phospholipase A2 (PLA2).
Sporting and workplace muscle injuries can also important as they result in decreased the quality of life for the affected individual and result in increased costs to the community in terms of working days lost and increased health care costs. Loss of control of intracellular Ca2+ is also the major cause of muscle cell death after injury.
In this laboratory, we are interested in determining the cellular mechanisms responsible for causing muscle cell death after skeletal muscle damage and during disease, and uncovering of strategies to inhibit these pathways in order to provide therapies to affected individuals.
PROJECTS
1. The Role of Phospholipase A2 in Skeletal Muscle Ca2+ Homeostasis and Fatigue
Phospholipase A2 (PLA2) is an important enzyme system in all cells. The products PLA2 activation, archidonic acid and lysophospholipids, act as second messengers at low concentrations, but are cytotoxic and important mediators of disease and injury at high levels. PLA2 is highly expressed in skeletal muscle and in this laboratory we have recently published important findings showing that PLA2 has marked effects on the control of 2+ intracellular Ca in skeletal muscle cells. These findings indicate that PLA2 could be an important cellular mechanism that contributes to fatigue and exercise-induced muscle damage in skeletal muscle. Furthermore, it has been shown that the skeletal muscle of patients with Duchenne muscular dystrophy exhibit markedly elevated (≈ 600%) PLA2 activity, indicating that inhibition of PLA2 could reduces injury –induced muscle damage in dystrophic muscle, and possibly provide a future therapy against this devastating disease.
Aim of Study: To determine the ability effect of PLA2 inhibitors to prevent eccentric-contraction-induced damage in dystrophic skeletal muscle. Successful prevention of muscle damage by PLA2 inhibitors could lead to the use of these drugs in the treatment of Duchenne muscular dystrophy.
Supervisors: Dr Tony Bakker & Dr Gavin Pinniger.
96 Physiology 2. The Role of Protease-Activated Receptors (PARs) in Muscle Injury
In skeletal muscle, inflammation results from muscle injury. This inflammation is thought contribute to the skeletal muscle weakness seen in these conditions, although it is unclear how this occurs. PARs are a newly identified family of G protein-coupled receptors that are activated by serine proteases such as thrombin and tryptase. PARs have been shown to mediate an extensive range of cellular activities, particularly during inflammation. In this laboratory, preliminary results in isolated skeletal muscle cells indicate that PAR-1 activation results in a large (≈50%) decrease in sarcoplasmic reticulum Ca2+ release, which should induce significant muscle weakness. Therefore, PARs could act as an important link between inflammation and muscle weakness. However, the effect of PAR activation on force production in intact skeletal muscle is unknown.
Aim of Study: To investigate the effect of PAR activation on skeletal muscle contractile performance under normal and inflammatory conditions.
The results of this study could have important ramifications for the treatment of sports injuries, and diseases where inflammation and skeletal muscle weakness are associated. These include the muscle weakness associated with muscular dystrophy, cancer, chronic heart failure, chronic obstructive pulmonary disease and repetitive strain injury.
Supervisors: Dr Tony Bakker & Dr Gavin Pinniger.
3. The Ability of Antisense Oigonucleotides to Improve the Function of Dystrophic Skeletal Muscle
In collaboration with Prof Steve Wilton & Dr Sue Fletcher, Australian Neuromuscular Research Institute. Duchenne muscular dystrophy is a lethal muscle wasting disease that results from a mutation in the gene coding for dystrophin. This protein is important for maintenance of the structural integrity of the cell. Patients with Duchenne muscular dystrophy do not have a functional form of dystrophin, and therefore, they become prone to damage during normal muscle activity. Muscle cells die as a result, and the muscles eventually waste away. Boys with Duchenne muscular dystrophy show signs of muscle weakness as infants, are restricted to a wheelchair by the age of 12 years and 90% will die before their 20th birthday from cardiac and respiratory problems.
In this project a genetic manipulation technique called ‘exon skipping’ will be used to try and restore a functional form of dystrophin in mdx mice (a mouse model of human
Aim of Study: To test whether treatment with antisense oligonucleotides leads to functional improvement in muscle function in dystrophic ‘mdx’ mice.
This project will provide important functional evidence about the ability of antisense oligonucleotide treatment to reverse the dystrophic phenotype. A positive outcome in this study could help pave the way for the use of this treatment as a future therapy for human Duchenne muscular dystrophy.
Supervisors: Dr Tony Bakker, Dr Gavin Pinniger & Dr Sue Fletcher.
97 Physiology
ASSOCIATE PROFESSOR ALAN EVERETT Room 1.18, Physiology Building, Phone: 6488 3317 Email: [email protected]
PROJECTS
The broad aim of the work is to identify key cellular pathways in airway motoneurons that control acetylcholine release from nerve terminals (boutons) and subsequent airway narrowing. In particular, experiments will investigate the role of Rho-kinase in the mobilization of transmitter-containing vesicles in nerve terminals. The mobilization of these vesicles from a reserve pool within terminals determines the number of vesicles available for releasing transmitter. Understanding what controls how much transmitter is released, and therefore synaptic strength, could lead to dramatically improved outcomes in the pharmacological treatment of asthma.
The understanding of synaptic function and vesicle pool dynamics in combination with recent work on the potential role of the Rho-kinase enzyme in the regulation of cholinergic transmitter release has lead to our working hypothesis. This enzyme normally promotes the phosphorylation of myosin light change kinase and increases intracellular calcium levels, at least in smooth muscle. Our own work (Fernandes et al., 2006 Eur J Pharmacol 550, 155-161) has shown that Y-27632 suppresses cholinergic nerve-mediated contractile activity in murine airways and increases acetylcholine release from murine tracheal preparations. Rho-kinase has been shown to phosphorylate endophilin. Endophilin, in turn, is involved in clathrin-mediated endocytosis of synaptic vesicles.
Our working hypothesis is that the Rho-kinase pathway controls the phosphorylation level of endophilin in synaptic boutons of autonomic nerves. Endophilin is involved in clathrin-mediated endocytosis of synaptic vesicles and thus regulates the number of synaptic vesicles available for recycling and neurotransmitter release. A change in the activity of Rho kinase will therefore impact on cholinergic nerve-mediated contraction of airway smooth muscle.
Supervisors: Assoc Professor Alan Everett and Dr Lynette Fernandes (Pharmacology, 9346-4517)
98 Physiology
DR LIVIA HOOL RESEARCH FELLOW Room 1.96, Physiology Building, Phone: 6488 3307 email: [email protected]
Ion Channels in Heart Muscle
Currently, cardiovascular disease accounts for 41% of all deaths in Australia. This is a staggering proportion when compared with the 22% from all cancers and 4% from road deaths. A number of the deaths in the cardiovascular group are due to arrhythmia or disturbances in the electrical activity in the heart.
The normal electrical activity in the heart is controlled by the movement of ions through specialised channels in the membranes of cardiac cells. The autonomic nervous system plays an essential role in regulating cardiac function and many of its effects are mediated via sympathetic neurotransmitters that regulate the activity of these ion channels.
Certain pathophysiological conditions contribute to arrhythmias such as hypoxia and oxidative stress. Under these conditions there is a reduction in blood flow to the muscle in the heart resulting in a reduction in available oxygen and reactive oxygen species production. This is then followed by an increase in generation of reactive oxygen species. There is increased sympathetic drive and the heart has a greater vulnerability to sudden cardiac death. Understanding how cardiac ion channels are regulated under these conditions is crucial to understanding the ionic mechanisms involved in the triggering of ischemic arrhythmias and the mechanisms that lead to chronic pathology such as cardiac failure.
The laboratory uses molecular biology techniques for expression and purification of ion channel protein and biochemical techniques for assay of generation of reactive oxygen species and cell viability. The method that will be used to study membrane currents is the patch-clamp technique. This technique is an extremely powerful method for studying the electrophysiological properties of biological membranes and its contribution to the advancement of research was justly recognised with the awarding of the Nobel Prize in Physiology or Medicine in 1991 to its developers Erwin Neher and Bert Sakmann. The technique can be used to study ion channels both at a whole-cell level or at the level of a single channel. In addition, since the intracellular composition of the cell can be controlled, this can be exploited to determine any second messengers involved.
PROJECTS
1. How does the L-type calcium channel regulate mitochondrial function in pathology where actin filaments are disrupted? (in collaboration with Dr Peter Arthur-Biochemistry and Molecular Biology UWA, Professor Nigel Laing, WAIMR).
Mitochondrial respiration is abnormal in hearts where actin or cytoskeletal proteins are disrupted and it is not understood why. This project follows from data generated by a PhD student in the lab, Helena Viola. We have evidence to suggest that the L-type calcium channel can regulate mitochondrial function via the actin cytoskeleton. The project involves the use of patch clamp technique to study L-type calcium channel currents in mouse myocytes isolated from hearts of Duchenne Muscular Dystrophy mice and fluorescent detection of changes in mitochondrial membrane potential, NADH and superoxide production after activation of the channel.
99 Physiology 2. How does oxidative stress alter the sensitivity of the L-type calcium channel to isoproterenol? What effect does this have on the action potential? (in collaboration with Professor Yoram Rudy, University of Washington, St Louis, Missouri, USA).
This question seeks to understand how arrhythmias occur during ischemia/reperfusion in the heart (after a heart attack). Isoproterenol is a beta- adrenergic agonist (and mimics the effects of catecholamines such as adrenaline in the heart). This project will use patch clamp technique to study the effect of oxidative stress and isoproterenol on L-type calcium channel currents in addition to K and Na channel currents and record changes in action potentials. Information gained from patch-clamp studies are incorporated into the Rudy-Luo model. Changes in action potential configuration are modelled and the relative risk of arrhythmia is determined.
3. How does a brief oxidative stress (eg 5 min exposure to 30 uM hydrogen peroxide) alter protein synthesis in cardiac myocytes? (in collaboration with Dr Evan Ingley, WAIMR, Dr Richard Lipscombe, Proteomics International)
This question looks at how hypertrophy (enlarged heart) is triggered after ischemia/reperfusion. Hypertrophy leads to heart failure and increases risk of arrhythmia and mortality. It is based on studies currently being undertaken in the laboratory that have identified increased protein synthesis measured as 3H-Leucine uptake in cardiac myocytes after 5 min exposure to 30 uM hydrogen peroxide. A proteomics approach will be used to identify alterations in protein synthesis in conjunction with mass spectrometry.
100 Physiology
ASSOCIATE PROFESSOR LEE-YONG LIM Room 2.06, Pharmacy Building, Phone: 6488 4413 email: [email protected]
Laboratory for Drug Delivery
Our research focuses on understanding the mechanisms and constraints of drug delivery across biological barriers, and developing methodologies to overcome these barriers. Nanotechnology is the enabling technique for the development of drug delivery systems in our laboratory. Delivery platforms based on polymer nanoparticles, liposomes and plant viral protein cages have been manufactured for drugs and genes. In addition, targeting ligands, such as plant lectins and folic acid, have been applied to enhance the tumour uptake of anticancer drugs. To address current concerns over the impact of nanotechnology on human health, we have also initiated safety assessments of biomedical nanomaterials, with emphasis on hepatotoxicity. Our laboratory is affiliated with the UWA Centre for Strategic Nanofabrication, incorporating Nanotoxicology.
We have 2 projects in Year 2008 for prospective Honours students. Students with a background in Chemistry, Biomedical Science or Biochemistry are encouraged to apply.
PROJECTS
1. Production of drug nanoparticles by spinning disc processing technology
With Prof Colin Raston, Dr Mohamed Makha
Drugs with poor water solubility cannot be effectively absorbed if they fail to achieve an adequate degree of dissolution in the gastrointestinal tract following oral administration. Many approaches have been developed to overcome the solubility problem of such drugs, including solubilization, the formation of inclusion compounds and complexes.
101 Physiology More recently, the reduction of drug crystals to the nanoscale range has been found to be highly effective in increasing drug dissolution and absorption in vivo. Drug nanoparticles have been produced by milling, high speed homogenization and supercritical fluid technology. This project proposes to harness the unique advantages of the spinning disc processing technology to produce drug nanoparticles of tuneable size, shape and agglomeration status under continuous flow conditions. The aim is to develop generic protocols for producing nanoparticles of superior dissolution profile for drugs with inherently poor water solubility. As a starting point, griseofulvin, a classical example of a poorly water soluble drug, will be used as a model drug to develop the protocols. The student will use the spinning disc processor to produce the nanoparticles and a variety of techniques, including laser light scattering, X-ray crystallography and transmission electron microscope, to characterize the drug nanoparticles. There is scope for patenting the manufacturing process.
2. Production of liposomes by spinning disc processing technology
With Prof Colin Raston, Dr Mohamed Makha
Liposomes are widely applied in the food and cosmetic industries. They are also widely investigated as delivery vehicles for drugs. A successful example is Doxil®, in which the anticancer drug, doxorubicin, is encapsulated within liposomes to minimize its debilitating toxic effects. Liposomes are conventionally prepared by swelling previously dried lipids with water, followed by size reduction by homogenization, extrusion or sonication to produce small unilamellar vesicles. The purpose of this project is to evaluate the feasibility of manufacturing small unilamellar liposomes by a single pass on the spinning disc processor. This would offer significant advantage over the conventional and even other patented methods of liposome manufacture. The incorporation of hydrophilic and lipophilic cargoes within the liposomes will also be evaluated in this project. The student will be using the spinning disc processor for liposome manufacture, and a variety of techniques, including laser light scattering and transmission electron microscope, to characterize the liposomes. There is scope for patenting the manufacturing process.
102 Physiology DR SHANE MALONEY SENIOR LECTURER Room 1.02, Physiology Building, Phone: 6488 3394 email: [email protected]
Comparative Physiology of Adaptation
Our group is interested in the physiological mechanisms whereby animal species (including humans) adapt to environmental stressors. We Mainly focus on thermal and osmotic stress, but exercise, inanition (starvation), and infection are also studied. Most experimental work is on systems level adaptations, but organ level adaptations are also studied. Our long-term aim is to identify specific adaptations that allow animals to survive and reproduce in challenging environments, and to identify how “homeostasis” handles the trade-offs when simultaneous challenges are presented to an organism, (such as combined thermal and osmotic stress, or combined inanition [starvation] and infection stress). Prospective Honours students with a background in General Systems Physiology, Exercise Physiology, Applied Animal Physiology, or Comparative Physiology are encouraged to apply. Depending on the project chosen a background in Cell Physiology could be an advantage.
Students will be exposed to a range of approaches and techniques including (in non-human animals) recovery anesthesia and surgery, implantation of physiological recording equipment, blood sampling for hormone measurement, and husbandry techniques for various species. In human research we use state of the art equipment to record physiological parameters in ambulatory subjects, including core and skin temperature transmitters, ambulatory blood pressure recording equipment, laser Doppler skin blood flow techniques, and infra-red thermography for surface temperature measurement.
PROJECTS
1. Is Brain Temperature a Signal for Brain Vascular Reactivity? With Dr Peter McFawn
Brain blood flow is autoregulated over a wide range of arterial blood pressure, and is thought to be influenced mainly by changes in local PCO2. The vasodilator effect of PCO2 couples increases in brain metabolism to increases in brain blood flow, and should result in tight coupling of brain temperature to cerebral arterial blood temperature. But that is not what has been observed in several species. One explanation is a “thermal vasodilator” signal in the brain. To ascertain if such a signal exists, fresh cerebral blood vessels from rats or rabbit or sheep will be extracted and suspended in an organ bath under a dissecting microscope. Changes in tension developed by the vessels (smooth muscle contraction and relaxation) visualised as changes in vessel diameter, will be recorded while the PCO2 and temperature are varied systematically. The measurements will answer whether there is an effect of temperature on brain blood vessel contractility and / or whether temperature alters the vasodilator response to PCO2. A cornerstone of cerebral physiology is the autoregulation of brain blood flow mediated in part by the vasomotor actions of PCO2. The identification of another mediator of brain blood flow would be an important advance to our knowledge and has implications for brain circulation and recovery from stroke.
2. The Physiology of Avian Fever. With Prof Dave Gray (Physiology, University of the Witwatersrand)
During fever following infection, the body acts as if an internal thermostat has been turned up; thermoregulation during fever is normal, but defends an elevated temperature. The presence of a fever response in birds surprises many people because normal body temperature in birds is hotter than febrile temperature in mammals. The presence of the febrile response in birds points to an early phylogenetic origin for the fever response. Over the last few years we have studied the physiological responses of a model bird (the Pekin Duck) to bacterial infection. With the current interest in Bird Flu we are interested to study the physiology of viral fever in birds, particularly the severity of anorexia and inhibition of activity (sickness behaviour). Small microprocessor / loggers will be implanted into Pekin Ducks to measure core body temperature and activity (accelerometer). Food intake will also be measured. Viral fever will be simulated with injection of Poly (I:C), and the effects of standard antipyretics (aspirin like drugs) and steroids will be studied. Whether birds develop fever and sickness behaviour in response to viral infection, and identification of the mediators of those responses, will increase our
103 Physiology knowledge of the evolution of fever. Knowledge of the sickness behaviour during viral infection in birds also has implications for models of disease spread in birds infected with avian influenza.
3. Can we use Vibration to Enhance Cooling from Hyperthermia? With Prof Brian Dawson (Human Movement and Exercise Science)
Industrial workers habitually exposed to hand vibration (such as jackhammer operators) often develop a condition called “whitefinger” where vasoconstriction of the hands is exaggerated. It turns out that there is a range of frequencies where vasoconstriction is exaggerated, but another where it is inhibited. When a hyperthermic human places their arms in cold water, it feels good, but not much heat is transferred to the water because the cold stimulus causes skin vasoconstriction. If we can determine the optimum vibration stimulus for inhibition of vasoconstriction to a cold stimulus during hyperthermia, we would have an ideal method for treating hyperthermia. Laser Doppler flowmetry will be used to measure skin blood flow, and thermocouples to measure skin temperature, of the arm and hand while the arm is vibrated at various frequencies. Initially the flow and temperature responses in euthermia will be investigated to determine the optimum frequency (frequencies?) for vasomotor effects in the skin. Then the study will turn to effects in hyperthermia and with cold stimuli applied to the skin. A more efficient, non-pharmacological method of cooling will be useful in any endeavour where hyperthemia impacts on health or performance, such as competitive sport, military activities, or outdoor events.
4. A Role for Prostaglandins in the Vasodilator Skin Blood Flow Response to Heat Exposure and Exercise? With Prof Brian Dawson (Human Movement and Exercise Science)
When humans are placed in situations where enhanced heat loss is required to maintain thermal balance, skin blood flow increases. An elevation in skin blood flow is achieved by reduction of vascular resistance in the skin. It is clear that central thermal input is important and leads to a reduction in vasoconstrictor tone and activation of a vasodilator system. For many years it was thought that the vasodilator system was mediated by Nitric Oxide, but subsequent study in humans has offered little support for NO mediation. The system seems to involve sympathetic cholinergic nerves, but the mediator is not Acetyl Choline. Attention has thus turned to Non- Adrenergic-Non-Cholinergic (NANC) mediators released from these nerves. In skeletal muscle prostagladins and histamine have been implicated in ACh induced vasodilation, and so an involvement of these mediators in skin blood flow is possible. The project will involve exposing subjects to high ambient temperature (37°C) and light exercise, while core body temperature, skin blood flow, and skin temperatures are measured. Blood pressure and heart rate will also be recorded. Three experiments will be performed in random order 1) Control, 2) prostaglandin blockade, 3) histamine blockade. The results will have implications for sport and general medicine, because drugs that inhibit prostaglandins and histamine are freely available and they may be important in the etiology of heat illness.
5. Why Does Exposure to High Ambient Temperature Inhibit Reproductive Efficiency? With Dr Dominique Blache (Animal Biology) and Drs Anne Barnes and David Beatty (Murdoch Vet School)
In cattle the reproductive efficiency (pregnancies per insemination) decreases from about 50-60% in winter to 10-15% in summer. The effect is similar, but less well studied, in other species. The mechanism of the inefficiency will be studied by measuring changes in hormones released from the pituitary (that stimulate the ovaries) and those from the ovaries (that feedback to the pituitary) to determine if the effect of heat exposure on reproduction is mediated at the brain or gonad level. Measurements of body temperature, food intake, reproductive hormones (analysed from blood samples drawn at regular intervals from a catheter implanted in the jugular vein), and reproductive behaviour (onset of oestrus, length of oestrus, presentation behaviours) will be made on Merino ewes exposed to thermoneutral and hot conditions. Several different protocols will be covered in different projects, including the response to cycle synchronisation within the normal breeding season and the response to the “male effect” outside of the normal breeding season. “Pair fed” ewes exposed to thermoneutral conditions and fed the same amount as the intake observed in the heat exposed ewes will allow distinction of the effect of food intake from the effect of heat per se. The projections for global warming sets a worrying scene for production species that we rely on for food and fibre, as well as endangered species already facing extinction. Identification of the mechanisms underlying the changes in reproductive function are a first step to finding ways to improve reproductive efficiency.
104 Physiology DR DANIELLE MEYRICK LECTURER Room 2.01C, Old Biochemistry Building, Phone: 6488 2498 email: [email protected]
Radiopharmaceutical Chemistry Group
Our research focuses primarily on the development and application of novel radiopharmaceuticals for imaging, therapy and management of a range of conditions in nuclear medicine. With the installation of a cyclotron for producing radioisotopes at Sir Charles Gairdner Hospital, applications of radioisotopes to medicine for positron emission tomography (PET) and therapy is an area attracting increasing interest locally. This interest parallels the world-wide growth of this field of medicinal chemistry. Radioisotopes of particular interest to this group are copper-64, useful for PET, and Re-188, a therapeutic beta emitter. The research aims to develop methods for targeted delivery of radioactivity to, for example, neoplasms. The targeting entities may incorporate biomolecules, chelating agents or micro/nanosystems.
The laboratory also focuses on elucidating the in vivo physical/solution chemistry of radiopharmaceuticals and other drugs incorporating metals. There is a broad interest in bioinorganic chemistry, including biomineralisation, and the thermodynamic behaviour of metal ions in biological systems (including, for example, the chemical interaction of the medically-important metal zirconium with human biochemicals).
Students with an interest in the applications of chemistry to biological systems (including those discussed above) should note that there are also joint Pharmacy/Chemistry projects available. These are to be completed under the joint supervision of Chemistry and Pharmacy. PROJECTS
1. Reducing the renal radiation burden during peptide receptor radiation therapy for neuroendocrine tumours.
When a radiopharmaceutical is administered for internal radiotherapy, the injected activity distributes through the body with varying degrees of uptake in each of the organs. In the case of radiopeptide therapies, the total radiation dose that can be administered to a patient is limited by the maximum dose that can be tolerated by the kidney without causing irreversible injury. If the radiation dose to the kidneys can be selectively reduced whilst at the same time allowing delivery of a lethal radiation dose to the tumour, the clinical efficacy of radiopeptide cancer therapy can be greatly increased. Importantly, a larger total radiation activity, and therefore dose to tumour, can be administered.
This project involves the development of chemical strategies for reducing the uptake and retention of radioactivity by the kidneys. The potential outcomes of this project have great clinical relevance.
105 Physiology 2. Direct labelling of peptides with Re-188 for treatment of neoplasms.
Peptide receptor radiation therapy exploits the over expression of peptide receptors by some types of cancer as a molecular basis for targeted delivery of radioactivity to the disease site. The chemical challenge lies in finding methods for binding the radionuclide (usually a metal) to the peptide of interest without changing the targeting properties of the peptide.
Rhenium-188 is a therapeutic beta-emitting radionuclide with a half-life of approximately 17 hours. Fortuitously, rhenium-188 also has a gamma emission, allowing it to be imaged by gamma camera. This combination of emissions allows the delivery of therapy to the tumour sites and simultaneous imaging of treatment success/disease progression. Rhenium displays varied redox and coordination chemistry, providing scope for the development of methods to bind radioactive rhenium to clinically important peptides. This project will focus in particular on the labelling of rhenium to bombesin and somatostatin analogues with the long-term view of application to the treatment of prostate and neuroendocrine malignancies.
3. Assessing the bactericidal and bacteriostatic properties of positron-emitting and gamma-emitting radioisotopes and radiopharmaceuticals.
The quality control of radiopharmaceuticals can often present challenges in the clinical setting, primarily due to the half-life of these agents. Although in most cases aseptic technique is employed in the preparation of these pharmaceuticals, sterility testing is undertaken retrospectively. It is routinely assumed that bacteriocide or bacteriostasis will be induced by the radiation.
Whilst radiation-induced cell-death is a well-studied process, the effect of different forms of emission is not well-characterised. Furthermore, the different response to radiation of different strains of bacteria remains to be fully elucidated, as does the effect of the pharmaceutical on the bacteria. Some widely used PET agents – e.g fluorodeoxyglucose (18FDG) - may in fact be a food source for the bacteria. The impact of ionising radiation on bacteria during growth vs lag phase is not well-understood.
This study will investigate the above effects, and establish a dose-response relationship of bacteria to a number of radiopharmaceuticals. Comparisons will be made between the capacity of different radiopharmaceuticals to induce bacterial death or terminate growth.
4. Development of Cu-64 labelled peptides for combined PET imaging and therapy of neuroendocrine tumours.
Positron-emitting copper radioisotopes are attracting interested as important positron emission agents. They may be bound to peptides or antibodies for diagnosis and staging of a range of conditions. This study will investigate the optimum chemical conditions for labelling a number of peptides with Cu-64. Importantly, the biodistribution in tumour-bearing animal models of these agents will be determined, with the aim of progressing to application in the clinic.
5. Thermodynamic modelling of zirconium in blood plasma.
Zirconium is a metal with widespread applications in nuclear medicine, prosthetic technology, health care and environmental fields, yet important aspects of its aqueous and biological chemistry are yet to be determined. By generating important thermodynamic data in this study and critically reviewing data already reported in the literature, a computational model of the metal-ion binding to low molecular weight ligands in blood plasma can be developed. This will facilitate a better understanding of the biochemistry of the metal ion, and allowing a degree of prediction of drug, chelating agent and other interactions, together with uptake and excretion behaviour.
106 Physiology Respiratory Group HOWARD MITCHELL, PETER MCFAWN and PETER NOBLE Room 2.25 & 2.26, Preclinical Link, Phone: 6488 3314, 6488 3341, 6488 3310 email: [email protected] [email protected] [email protected] L to R: Prof Howard Mitchell, Dr Peter McFawn, Thom Ansell, Dr Peter Noble, Respiratory Physiology Group Dr Adrian West.
The respiratory group in Physiology has had a long-standing interest in the control and function of conducting bronchi. The trachea, bronchi and other airways conduct air into and out of the lung. During an asthma attack contraction of airway smooth muscle (ASM) narrows the conducting bronchi and obstructs airflow. Airway obstruction also occurs in several other respiratory disease including Chronic Obstructive Pulmonary Disease (COPD), chronic bronchitis and emphysema. The focus of our research has been in understanding the detailed mechanisms involved in the control of airway diameter and airway obstruction.
PROJECTS
1. Impact of simulated breathing movements on the response to bronchodilators. With Lin Fernandez, Pharmacology (School of Medicine and Pharmacology)
Inflation of the lung dilates airways and stretches ASM so that the length of ASM changes during breathing. Deep inhalations or sighs are now known to provide a powerful break on airway narrowing in healthy people, an inhibitory system that fails in asthma. The response of isolated bronchi to deep inhalation and simulated breathing movements is a current focus of our work, which studies the mechanism of deep inhalation induced bronchodilatation. Projects to investigate the actions of breathing on control of smooth muscle by bronchial nerves or on interactions between bronchodilator drugs and deep inhalation can be undertaken. Do the drugs used to treat asthma help or inhibit the normal bronchodilatation from deep breaths? This project will use isolated bronchi Pig bronchi stained with smooth muscle specific (red) and dissected from pig lung that are oscillated using a nerve specific (green) anti-bodies. servo control system to simulate respiratory movements. Bronchi will be contracted with cholinergic stimuli (acetylcholine or synthetic analogues) and the response to inhibitory nerve stimulation and bronchodilator drugs measured with and without simulated breathing movements.
2. Relative importance of radial and longitudinal stretch to the bronchodilatation produced by deep inflation. With Lin Fernandez, Pharmacology (School of Medicine and Pharmacology)
In a human or whole animal deep inflation of the lung both dilates bronchi and stretches them longitudinally, producing bi-directional strain in the smooth muscle. The relative impact of these two different directions of stretch, have not been studies and projects to characterise the importance of radial and longitudinal stretch maybe undertaken. This project will use isolated pig bronchi and measure the suppression in contraction produced by directional stretch. Sections of bronchi subjected to radial or longitudinal stretch would then be studied using histochemistry to identify changes in cytoskeleton structure produced by different stretch directions.
107 Physiology
3. Is bronchial hyperresponsiveness of immature animals due to reduce deep inflation bronchodilatation?
Deep inhalation is the most important bronchodilatation mechanism in adult humans but the actions of deep breaths on immature airways are not known. Some studies suggest that the bronchodilator response to deep inhalation is not present in immature animals. Projects in this area would investigate maturational changes in the response to deep inhalation and the development of normal bronchodilatation using isolated animal bronchi.
4. Maturational changes in calcium release during ASM contraction. With Tony Bakker, Physiology
Developmental changes in airway smooth muscle have long been an interest for the respiratory laboratory. Significant changes in responsiveness of airway smooth muscle occur in many species after birth, including humans. Excitation contraction signalling in smooth muscle can occur through both calcium dependent and calcium independent pathways that control myosin phosphorylation. This project would investigate signal transduction and force production in isolated smooth muscle from immature and mature pigs. Currently we know immature airway smooth muscle shows a greater rise in intracellular calcium during contraction than in mature muscle. The primary aim of this project would be to use simultaneous measurements of force and intracellular calcium to identify the mechanism for the increased calcium rise.
5. Cellular basis for deep inflation produced bronchodilatation. With Prof Geoff Stewart, Microbiology
The biochemical changes in smooth muscle produced by stretch have been suggested as the mechanism behind deep inhalation produced relaxation of airway tone. In isolated smooth muscle cells stretch can produce depolymerisation of actin filaments and remodelling of the contractile proteins. Contraction of smooth muscle also promotes myosin filament formation so that in smooth muscle the contractile machinery is assembled as the cell moves. How these cellular processes change during maturation is unknown and even the importance of these cellular events to the whole animal is not well defined. Projects in this area would use cell biology techniques to study the regulation of contractile proteins and assembly of contractile filaments.
6. Response of isolated human bronchi to deep inflation. With Alan James, Medicine (School of Medicine and Pharmacology)
Animal and cell studies are important in understanding normal physiology and the mechanisms underlying pathology and treatment. However, nothing beats human tissue for studying pathophysiology of disease. A new direction for the respiratory group is the study of human airways obtained from lung cancer patients, and the study of deep inflation in patients with respiratory disease. Projects in this area will run in collaboration with Dr Alan James from the School of Medicine and Pharmacology (Pulmonary Physiology) and will include significant work with patients as well as tissue. The aim of the initial project is to show bronchodilatation response to inflation of isolated human bronchi and attempt to identify differences between normal bronchi and those from patients with COPD. Students will be required to liaise with hospital staff, work with the patients before surgery to obtain base line lung function and allergy data as well as perform isolated tissue studies using lung removed following surgery for lung cancer. As a caution the availability of human tissue is highly variable and exciting new areas tend to have development problems that can make these projects more challenging.
7. Breathing pattern and rate of spontaneous sighs in obstructive airway disease With Alan James, Medicine (School of Medicine and Pharmacology)
The failure of deep inhalation to produce bronchodilatation in both asthma and COPD is well characterised. Anecdotal evidence and limited studies on asthmatics suggest that the failure of deep inflation to give bronchodilatation does not change the pattern of breathing and that asthmatics sigh as often as normal people. Unfortunately the studies so far have been limited in both the number of subjects investigated and the characterisation of the extent and severity of disease. This project will run in collaboration with Dr Alan James and aims to measure breathing pattern in healthy, asthmatic and COPD patients.
108 Physiology DR GAVIN PINNIGER LECTURER Room 1.10, Physiology Building, Phone: 6488 3380 email: [email protected]
Muscle Physiology Group
Muscle contraction involves the cyclic interaction between myosin heads (crossbridges) on the thick filaments with binding sites on the thin (actin) filaments, a process that is driven by ATP hydrolysis. The original Huxley (1957) model for crossbridge cycling provides the foundation for current theories of muscle contraction and can account for various aspects of skeletal muscle function such as the force-length relationship and the force- velocity relationship during muscle shortening (concentric contraction). However, current models of muscle contraction fail to fully account for the force response when an active muscle is lengthening (eccentric contraction).
Repeated lengthening of activated muscle causes muscle damage, loss of force producing capacity, and pain, and has been associated with disruption of structural proteins. This exercise-induced muscle damage (EIMD) also triggers and inflammatory response that is characterized by the slow development of tenderness, swelling and stiffness, in the several days after the initial exercise (often referred to as delayed onset muscle soreness or DOMS). These debilitating effects are enhanced if the eccentric exercise is excessive or unaccustomed, but may be reduced through an adaptation process brought about by frequent exposure to eccentric exercise. The increased susceptibility of dystrophic muscle to EIMD is considered a major contributor to the progressive muscle wasting that is characteristic of patients with muscular dystrophy.
This research is aimed at unravelling the complex molecular events occurring during the lengthening of an active muscle with the aim of better understanding the mechanisms of EIMD and the subsequent inflammatory response. Further research is aimed at understanding the adaptation process brought about by repeated exposure to eccentric contractions and investigating various therapeutic interventions aimed at reducing the susceptibility of dystrophic patients to EIMD. PROJECTS
1. MOLECULAR MECHANISM OF STRETCH-INDUCED FORCE ENHANCEMENT With Dr Tony Bakker Experiments on isolated muscle preparations have shown that stretch of an active muscle causes a transient increase in force arising from the strain of both contractile (crossbridges) and non-contractile (structural) components of the sarcomere. The relative contributions of these components can be determined from their force-velocity characteristics and by the use of specific myosin inhibitors (Pinniger et al., J Physiol, 2006). Structural proteins such as titin, act to stabilize the sarcomere allowing the transmission of force within and between muscle fibres and disruption to these proteins is associated with the development of exercise-induced muscle damage (see Figure above). Although the contribution of the structural proteins (titin) to stretch-induced force enhancement is unknown, there is evidence that titin stiffness increases upon activation in a calcium- dependent manner. This study aims to determine the contribution of titin filaments to stretch-induced force enhancement. Experiments will be performed on single skinned muscle fibres to determine the calcium sensitivity of stretch-induced force enhancement. This research is focused on unravelling the complex molecular mechanisms of tension development during active muscle lengthening. The outcomes of this research will provide valuable insight into the mechanisms of exercise induced muscle damage and help to identify key features of the adaptation process brought about by repeated exposure to eccentric exercise.
2. The role of circulating cytokines in skeletal muscle weakness With Dr Tony Bakker & Dr Paul Fournier(Human Movement) Low-level systemic inflammation has been implicated in numerous pathological conditions such as cardiovascular disease, and chronic obstructive pulmonary disease and may contribute to the muscle weakens that is characteristic of Duchenne muscular dystrophy and in the aged population. Acute muscle injury can also trigger an inflammatory response leading to further loss of force and morphological damage. The precise pathways linking inflammation to muscle weakness are unknown. Our laboratory is currently investigating various aspects of the inflammatory pathway using isolated animal muscle preparations (see Dr Tony Bakker).
109 Physiology This study will further our understanding of the role of systemic inflammation in muscle weakness by examining the effect of circulating inflammatory cytokines on skeletal muscle function. These experiments will be performed on a new, state of the art, the HSE-HA Universal Perfusion System (UNIPER UP-100). This system will allow for the administration of precise levels of cytokines into the hind limb circulation of rats or mice while maintaining an intact muscle vasculature. This perfusion system has distinct advantages over isolated muscle systems as the actions of most hormones and cytokines affecting muscle function are mediated to a significant extent by their effects on muscle blood flow. The effects of specific levels of inflammatory cytokines on skeletal muscle function will be determined while recording force output, and other critical variable such as blood pressure, pH, pO2 and temperature.
3. The role of the inflammatory cytokine TNF-α (Tumour Necrosis Factor-alpha) in muscle damage With Dr Tony Bakker & Prof. Miranda Grounds (Anatomy and Human Biology) Regardless of the initial injury mechanism, the days following EIMD are characterised by the development of typical DOMS symptoms including pain and inflammation. The time-frame of these symptoms correspond to a secondary decline in force producing capacity and extensive morphological damage. The localised muscle damage and sarcolemmal lesions allow the infiltration of inflammatory cells and key cytokines such as TNF-α which may play a critical role in the development of secondary muscle damage. The cytokine TNF-α promotes an excessive inflammatory response causing direct damage to muscle fibres. This damage may be potentiated in patients with muscular dystrophy as the loss of dystrophin is reasoned to cause sarcolemmal instability through a vulnerability to physical stress. It has recently been shown that blockade of TNF-α activity (using Remicade a specific antibody to TNF-α) results in a striking reduction of myofibre necrosis in dystrophic mdx mice. Clinically, the TNF-α neutralising drugs Remicade and Enbrel, are highly effective at reducing symptoms of inflammatory diseases such as Rheumatoid Arthritis and Crohn’s Disease. The established clinical success of these drugs makes them very attractive as potential treatments for muscular dystrophies. cV1q, is a mouse specific version of the antibody Remicade® that has been shown to neutralise TNF-α.
This study aims to determine the role of TNF-α in the secondary decline in muscle force associated with DOMS development after exercise-induced muscle damage. It is hypothesised the cV1q will reduce the extent of myofibre necrosis and enhance muscle regeneration following EIMD. Experiments will be carried out on normal healthy mice and dystrophic, mdx mice using a combination of in vivo eccentric muscle testing as well as isolated, intact muscle fibre experiments.
4. Ventilation induced diaphragmatic dysfunction With Dr Tony Bakker & A/Prof Jane Pillow (School of Women’s and Infants’ Health). The diaphragm is the major muscle of the respiratory pump hence a functional diaphragm is fundamental to respiratory well-being. Studies over the last 6-8 years have shown that in adults, diaphragm dysfunction may contribute to difficulty weaning from the ventilator which has given rise to the term Ventilator Induced Diaphragmatic Dysfunction (VIDD). Mechanical ventilation unloads the diaphragm which becomes inactive leading to the development of VIDD. In adult models of prolonged ventilation VIDD is characterized by loss of diaphragmatic force-generating capacity and endurance with evidence of muscle fibre atrophy, structural injury and myofibre cellular dysfunction. Experiments have shown that the preservation of spontaneous breathing activity may blunt the severity of VIDD. Whilst VIDD has been well characterized in the adult, there is very little information about the relevance of this disorder in the immature newborn infant. The extremely premature neonate may be at increased risk of developing VIDD due to the need for extended periods of ventilation, immature myofibre composition, rapid respiratory rates, high mechanical loads, and exposure to steroids, infection, poor nutrition and sedation. Pilot studies in the preterm lamb suggest that the onset of VIDD in the newborn may be more rapid than in the more mature adult model, and have highlighted the already reduced forced generating capacity and increased susceptibility of the preterm diaphragm to injury. Future studies will investigate the time course of ventilation injury in preterm versus more mature lamb, the impact of antenatal steroid treatment on susceptibility to VIDD, and the effect of different ventilation strategies on the development of VIDD.
110 Physiology PROFESSOR DON ROBERTSON & Dr HELMY MULDERS Room 2.02, Physiology Building, Phone: 6488 3291 email: [email protected]
The Auditory Laboratory (See also projects by Dr R Patuzzi)
Deafness and other hearing disorders such as tinnitus are among the most common forms of sensory impairment with profound consequences for the individual and society. Normal hearing depends on the proper function of the many component parts of the inner ear and the brain pathways to which it is connected. Our laboratory seeks an integrated understanding of the normal operation of this sense organ and its associated neural pathways and to describe the mechanisms underlying various hearing pathologies. Visit the lab’s website for additional information on our history, funding and publication record. (http://www.auditory.uwa.edu.au)
Projects
1. Regulation of inner ear function by ATP receptors Hearing sensitivity is determined by a number of variables in the peripheral receptor organ. One of these is the voltage in the scala media, the fluid compartment into which the stereocilia of the hair cells project. This large extracellular voltage is a vital element in the total driving force on ions through the hair cell transduction channels. Disturbances of this voltage could underly a variety of hearing pathologies including tinnitus and hence it is interesting to study the mechanism by which it is regulated. We have consistently found that perfusion of the cochlea with agonists of receptors for ATP (so-called purinergic receptors) causes a marked rise in the voltage in the scala media (endocochlear potential). Three possible hypotheses of the mechanism of this increase are 1) that there is a change in activity in a neural efferent feedback loop regulating the voltage, 2) that there is activation of P2 receptors in the stria vascularis (the transporting epithelium that is responsible for generating the voltage) and 3) that there is a reduction in the current drain on the scala media voltage by closure of ion channels in hair cells and/or supporting cells lining the scala media. These hypotheses will be tested by a series of experiments using intracochlear perfusion, intracochlear electrical measurements and surgical interventions in the brainstem.
2. Effects of loud sound on gene expression in the cerebellum (With Dr Jenny Rodger) Loud sound exposure is known to provoke changes in gene expression in central auditory pathways and these changes are of interest for understanding the molecular and cellular basis of phenomena such as hyperacusis and tinnitus. It has recently been reported that human tinnitus patients have elevated neuronal activity in part of the cerebellum, a structure with multisensory roles but not normally associated with auditory processing. This project will use experimental animals to investigate changes in gene expression in the cerebellum that may be associated with this hyperactivity. Primers for a number of candidate guinea pig genes have already been constructed and qPCR will be used to measure changes in mRNA expression after loud sound exposure.
Note: this project would be suitable for a student with level 3 knowledge of molecular biological techniques (either PHYL3300 orPHYL3340 or GENE3300 together with SCIE3325).
111 Physiology 3. Mapping connections of an efferent control system in the auditory nervous system The auditory nervous system consists of ascending (afferent) components that carry information from the periphery to the higher centres, and descending (or efferent) pathways by which the brain exerts synaptic control over the flow of incoming information. The efferent system is thought to play a number of roles in auditory processing, but its detailed connectivity with afferent subsystems is poorly understood. In this project, we will label selected populations of afferent neurons using retrograde tracers injected into the brainstem of experimental animals and then anatomically map the synaptic connections that the efferent systems make with these labeled neurons by using anterograde tracers injected into the sites of origin of the efferent pathways.
4. Plasticity in the auditory midbrain after partial unilateral deafness We have now established that after a partial unilateral deafness induced by loud sound exposure, there is a time- dependent rise in the background neuronal firing levels in the contralateral auditory midbrain (the inferior colliculus). This neuronal hyperactivity is of interest because it is a likely neuronal correlate of tinnitus (phantom auditory sensation) that is an increasing problem in today’s society. We have studied the inferior colliculus contralateral to the sound-exposed ear because the principal projection from cochlea to midbrain is crossed. However, there is also a substantial projection from the damaged ear to the ipsilateral midbrain. This altered ipsilateral input must interact with the intact contralateral projection from the unexposed ear. It is of interest to know whether hyperactivity is observed here as well, or whether the intact contralateral input acts as a brake on plastic alterations. The project will involve recording single neuron activity in anaesthetized guinea pigs. Students will acquire skills in small animal handling, anaesthesia and surgery, single neuron electrophysiology , digital data acquisition and analysis and histological verification of recording sites.
5. Binaural effects on responses of auditory midbrain neurons In the auditory midbrain of mammals, neurons are known to have a diversity of responses to sound when controlled acoustic stimuli are presented to the ear that supplies the dominant synaptic drive to those neurons. However, there is no good investigation in the literature of how these diverse “monaural” responses patterns may be shaped by two possible factors 1) spontaneous afferent input from the opposite ear converging onto the same neurons and 2) acoustically-driven input from the opposite ear when sound presented to one ear is loud enough to physically leak across the head. Such information is vital to a proper interpretation of the responses seen in the auditory midbrain, both in normal animals and in animals with various peripheral auditory pathologies. In this project, students will use metal microelectrodes to record the activity of single neurons in the auditory midbrain of anaesthetized animals and will study the effects on response patterns caused by experimental elimination of inputs from the non-dominant ear.
112 Physiology
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