faculty of Life and physical sciences Honours Projects 2012
Chemistry and biochemistry
Chemistry and Forensic Biochemistry and Genetics and Nanotechnology Science Molecular Biology Biomedical Science Biochemistry & Chemistry
2012 Honours
If you are interested in undertaking Honours at UWA, you may be already asking about the exciting prospects available within each of the Disciplines and sub-disciplines comprising the School. These include Biochemistry and Molecular Biology, Biomedical Science, Chemistry, Forensic Chemistry, Nanotechnology, Genetics, and Structural Biology.
This Honours Project book and the associated School Honours Expo are intended to help you explore the possibilities for 2012.
If you intend to enrol in Honours in 2012, this booklet will provide you with a comprehensive overview of the interests of our research groups as well as outlining specific Honours projects that are available.
The Honours Expo is designed to showcase the depth and diversity of research being undertaken in the School and will enable you to discuss particular projects or even discuss the design of new ones.
We hope that you will enjoy our Expo and that it will serve as a good introduction to the range of Honours projects available in the School for next year.
Professor M Spackman Head of School
Honours Co-ordinators
Biochemistry and Molecular Biology Chemistry Winthrop Professor Alice Vrielink Assoc Professor Sam Saunders Phone: 6488 3162 Phone: 6488 3153 [email protected] [email protected]
Genetics Forensic Chemistry Winthrop Professor George Yeoh Winthrop Professor John Watling Phone: 6488 2986 Phone: 6488 4488 [email protected] [email protected] or Nanotechnology Winthrop Professor Lawrie Abraham Dr Robert Woodward Phone: 6488 3041 Phone: 6488 2751 [email protected] [email protected]
Table of Contents
Research Expertise Table Page I - IV
Project Descriptions Page 1 - 66
How to Apply Page 68
Project Preference Form Page 69
Research Expertise Supervisor Research Area Discipline Page Genetics Biochemistry Genetics, Lawrie Abraham Biochemistry & 1 Biomedical Science Biomedical Science Molecular Biology
Oxidative stress Dystrophy Aging Peter Arthur Biochemistry 3 Muscle Diabetes Bioinformatics
Enzyme structure and function Enzyme kinetics Biochemistry & Paul Attwood Protein phosphorylation 5 Chemistry Histidine kinases Histidine phosphorylation.
Catalysis Nanotechnology Surface science Murray Baker Chemistry 7 Biological chemistry/medicine Polymer science Molecular recognition, and sensors
Structural Biology Protein Crystallography Biochemistry & Charlie Bond Protein:protein interactions 9 Chemistry Protein:nucleic acid interactions Gene regulation
Apoptosis Genetics & Bernard Callus 11 Cancer Signalling Biochemistry
Organometallic Chemistry Reto Dorta Chemistry 13 Catalysis
Nanotechnology Microalgae Ela Eroglu Nanotechnology 15 Wastewater treatment Biofertilizer
i Natural products Gavin Flematti Analytical Chemistry Chemistry 17 Separation Science
Crystallography Simon Grabowsky Electron Density Chemistry 18 Computational Chemistry
Physiology and biochemistry of milk Peter Hartmann Biochemistry 20 synthesis
Theoretical Dylan Jayatilaka Chemistry 22 Computational Chemistry
Organometallic Chemistry George Koutsantonis Inorganic Synthesis Chemistry 24 Molecular Electronics
Molecular evolution Molecular genetic Genetics, Martha Ludwig Biochemistry & 26 Molecular cell biology Molecular Biology Photosynthesis
Signalling Protein Interaction Bimolecular Fluorescence Complementation, 14-3-3 proteins Plant Histone Deacetylases Thomas Martin Plant nitrilases Biochemistry 28 Molecular Biology Sugar sensing in plants Nitrogen sensing in plants Sugar metabolism Nitrogen metabolism
Environmental Chemistry Physical Chemistry Allan McKinley Chemistry 30 Analytical Chemistry Medicinal Chemistry
Biochemistry plant mitochondria Oxidative stress and antioxidant defence Harvey Millar Biochemistry 32 Plant glutathione-S-transferases Protein mass spectrometry Proteome analysis
ii Synthetic Organic Chemistry Matthew Piggott Medicinal Chemistry Chemistry 34 Chemical Biology
Organic Synthesis Tissue Engineering Nano-chemistry Graphene Colin Raston Desalination Solar and Fuel Cell Nanotechnology 36 Technology Chemical Sensors Drug Delivery Microfluidics platforms
Atmospheric chemistry Gas phase chemical kinetics Sam Saunders Chemistry 38 Reaction mechanisms Computational chemistry
Genomics Ian Small RNA biology Biochemistry 40 Bioinformatics
Genomics Genetics Cell biology Steve Smith Biochemistry Biochemistry 42 Bioinformatics Systems biology Metabolomics.
Crystallography Mark Spackman Chemistry 44 Theoretical chemistry
Synthetic Organic Chemistry Natural Product Synthesis Scott Stewart Chemistry 46 Palladium Catalysed Reactions Domino Reactions
Carbohydrates Glycobiology Keith Stubbs Synthesis Chemistry 48 Inhibitors Enzyme kinetics
Swaminatha Iyer BioNanoChemistry Nanotechnology 50 iii Enzymes Cytochrome P450 Vitamin D Robert Tuckey Steroids Biochemistry 52 Hydroxylases, placenta Skin cancer Metabolism
Genetics Biochemistry Molecular Biology, Daniela Ulgiati Biochemistry, 54 Biomedical Science Genetics Molecular Biology
Protein structure Crystallography Biochemistry & Alice Vrielink Enzyme mechanism 56 Chemistry Structure-Function Relationships Rationale Drug Design
John Watling Forensic Chemistry Forensic Science 58
Mitochondrial biogenesis Gene regulation Biochemistry, Jim Whelan Phosphate metabolism Genetics, & 60 Biomedical Science Molecular cell biology Genetics
Physical Chemistry Laser Spectroscopy Duncan Wild Mass Spectrometry Chemistry 62 Van der Waals clusters ab initio calculations
Bioinformatics Microbial informatics Michael Wise Low complexity/natively unfolded Biochemistry 64 proteins Computational evolutionary biology
Liver stem cell Genetics, George Yeoh Cancer 66 Biochemistry Cell therapy
iv WINTHROP PROFESSOR LAWRIE ABRAHAM Room 2.58, Bayliss 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. 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 (Genetics, Biochemistry or Biomedical Science)
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. Characterisation of functional variants of Vanin 1, a QTL controlling HDL-C Levels (Genetics, Biochemistry or Biomedical Science)
This collaborative project with the Texas Biomedical Research Institute, 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. Twelve non-coding variants in the Vanin 1 gene were found that fall into 4 isocorrelated redundant variant sets (IRVS) 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
1 HDL-C levels (p = 0.002). The project aims are to characterise transcription factors that differentially bind to the IVRS variants using EMSA (see Fig) followed by peptide mass fingerprinting and also to determine the effects of the candidate functional SNPs on transcriptional activity using reporter gene analysis. A further aim is 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.
3. Mechanism of Action of Newly Synthesised Thalidomide Derivatives. (Biochemistry or Biomedical Science)
Thalidomide is a synthetic glutamic acid derivative used in the 1950‘s as a treatment for insomnia and as an antiemetic agent. Later investigations found that thalidomide had teratogenic properties. In a collaborative project with Dr Scott Stewart, newly synthesised and potentially safe 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 the newly synthesised derivatives, transcriptional profiling will be carried out, using Affymetrix microarrays to define novel cellular activities, with a focus on therapeutic application. The project also involves the identification of the cellular targets of thalidomide which will be informative in a more rational drug design. Photoactivatible biotin-derivatized thalidomide will be used to treat cells, followed by UV-catalysed cross-linking (see Fig). Proteins will be isolated and identified by biotin-streptavidin affinity chromatography and mass spectrometry. The proteins identified will be validated with respect to their interaction with thalidomide and by assessing functional aspects of the candidate proteins. Interactions will also be validated using confocal cell imaging.
4. Identification of Genetic Variation in Preeclampsia by Whole-Genome Exome Sequencing (Genetics or Biomedical Science)
The genetic analysis of preeclampsia continues to be one of the most critically important and unresolved areas of obstetric medicine. There is currently no known cure for preeclampsia other than delivery of the baby. Like many other common human diseases there is a large genetic component underlying susceptibility to developing preeclampsia but the genetics are complex and not yet fully understood. This project is a collaboration with W/Prof Eric Moses and involves the identification of functional genetic variants associated with preeclampsia. The emphasis is on whole-genome exome sequencing in families and represents the current state-of-the-science for genetic dissection of complex traits. The goal is to identify the specific genetic polymorphisms responsible for susceptibility to preeclampsia with the view to informing the development of much-needed diagnostic reagents and therapeutic strategies. This approach has been made possible by recent technological advances and efficiencies in high-throughput next generation DNA sequencing. This project involves a multidisciplinary team of investigators who have led the field in the recruitment and genetic analysis of preeclampsia and cardiovascular disease in families. The collection of 72 preeclampsia families from Australia/New Zealand, Finland, Iceland and Norway are the best available worldwide, making this a time of unprecedented opportunity for finding the most likely functional variants influencing susceptibility to preeclampsia.
2 ASSOCIATE9B PROFESSOR PETER ARTHUR 10B Room 2.41, Bayliss Building, Phone: 6488 1750 Email: [email protected]
Reactive Oxygen Species as modulators of signal transduction pathways and biochemical systems
Oxidative stress is caused by reactive oxygen species (ROS) and is thought to exacerbate pathology associated with many chronic diseases and conditions. Examples include Alzheimer‘s disease, atherosclerosis, dementia, diabetes, emphysema, heart disease, HIV/AIDS, kidney disease, liver disease, muscular dystrophy, Parkinson's disease, Rheumatoid arthritis, some cancers and aging. However, preventing the harmful effects of oxidative stress is not a simple matter, as antioxidant treatments have generally been ineffective in the treatment of these conditions.
One challenge has been the lack of understanding of the various molecular mechanisms by which oxidative stress causes pathology. We have established that cysteine residues on proteins are particularly sensitive to oxidative stress and our laboratory is playing a leading role in identifying proteins sensitive to oxidative stress. Our work, and the work of others, has established that multiple proteins are sensitive to oxidative stress, which means oxidative stress could have a widespread impact on many cellular processes (metabolic pathways, ion transport, protein synthesis, protein degradation, gene expression, signal transduction pathways). Our work into how oxidative stress affects cellular processes will offer new opportunities to treat oxidative stress.
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. How does oxidative stress affect cell signaling pathways? Collaborative with Dr Thea Shavlakadze & Prof. Miranda Grounds, School of Anatomy and Human Biology Insulin growth factor 1 (IGF-1) is a potential therapeutic agent for muscle ageing and muscular dystrophy. In both conditions oxidative stress plays a significant damaging role, and has the potential to block the actions of IGF-1. The objective of this project is to use a cell culture model to examine the effect of ROS on the function of signal transduction proteins. This project will involve using proteomic technology including protein separation techniques (HPLC, 2D gel electrophoresis, antibody technology) and protein identification techniques (mass spectrometry). Additional techniques may include Immunohistochemistry, Western Blotting, quantitative PCR and EMSA.
2. Does oxidative stress cause muscle wasting? Collaborative with Dr Thea Shavlakadze & Prof. Miranda Grounds, School of Anatomy and Human Biology 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.
The objective of this project is to establish whether oxidative stress causes changes in protein turnover in muscle, since decreased protein will lead to muscle wasting. 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 for this project include proteomic techniques, tissue culture, quantitative PCR for atrophy related genes and measurement of oxidative stress. This
3 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. Mdx/IG 3. Oxidative stress in ageing mice Collaborative with Dr Thea Shavlakadze & Prof. Miranda F-1-1 Grounds, School of Anatomy and Human Biology The trend of ageing populations in many countries has become a significant concern because age itself is a key risk factor for Md many chronic degenerative diseases. Examples include sarcopenia and neurodegenerative diseases such as Alzheimer's x and Parkinson's disease. Many of the age-dependent pathologies been linked to oxidative stress, so targeted interventions aimed at treatment or prevention of oxidative stress have the potential Figure 1. One year old male mdx/IGF-1 to alleviate ageing pathologies. In this context, it is interesting and mdx littermate mice. Mdx/IGF-1 to note that both cardiac pathologies and cataract formation were transgenic mice are much bigger delayed in mitochondrial catalase knock-in mice. compared to their age matched littermates and they have a pronounced One strategy for addressing the challenges posed by ageing skeletal muscle hypertrophy. populations is to understand the molecular mechanisms underlying the ageing process. We have hypothesized that oxidative stress affects susceptible proteins which disrupt cellular homeostatic mechanisms and lead to pathological consequences including increased oxidative stress sufficient to cause irreversible damage to cellular macromolecules. To develop evidence for these hypotheses, a range of markers of oxidative stress will be used to assess how oxidative stress develops in ageing mice. One experimental approach will involve using proteomics to identify proteins susceptible to developing oxidative stress. A second experimental approach will test whether peroxiredoxins can be used as sensitive indicators of oxidative stress. Peroxiredoxins are thought to be significant contributors to cellular removal of hydrogen peroxide, yet are readily inactivated by oxidation of susceptible thiol groups. Inactivation of peroxiredoxins may also have significant biological consequences by exacerbating oxidative stress.
4. Oxidative stress in Diabetes Collaborative with Prof. Paul Fournier, School of Sport Science, Exercise and Health Type 2 diabetes mellitus (T2DM) is a complex disorder that has reached epidemic proportions in Australia, affecting nearly one sixth of its adult population above 40 years old. This is a source of much concern because intensive personal and medical attention is required to manage and treat this condition. In addition, there is the large burden of the many long-term microvascular and macrovascular complications associated with diabetes. These include diabetic retinopathy which may lead to blindness, diabetic neuropathy with associated increased risk of amputation and early death, diabetic nephropathy leading to end-stage renal disease, and macrovascular complications such as stroke, coronary artery disease, and myocardial infarction.
Diabetes is characterised by impaired insulin secretion and a marked resistance to the action of insulin, particularly in skeletal muscles. We have hypothesized that protein thiol oxidation is contributing to insulin resistance. The objective this project is to establish whether oxidative stress is interfering in the function of key proteins involved in the insulin signaling pathway (eg IRS1, AKT) in an animal model of insulin resistance (high fat fed rats). 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 Dr. Arthur.
5. Systems Approaches to Oxidative Stress Collaborative with Professor Michael Wise The objective of this project is to develop and use bioinformatic methods to identify the cellular processes and organelles that are particularly sensitive to oxidative stress. This will involve categorizing the involvement of proteins (those identified as sensitive to oxidative stress) in different cellular processes. You will be using pathway analysis software such as IPA (www.ingenuity.com), keyword clustering software (Protein Annotators Assistant) and databases such as BioCyc, Reactome and Kegg to look for common themes/processes. Protein- protein interaction data and data about predicted location may also be useful.
4 PROFESSOR PAUL ATTWOOD Room 3.69, Bayliss Building, Phone: 6488 3329 Email: [email protected]
The research focus of Prof. Attwood's laboratory is the structure and function of enzymes in general. However, there is a particular focus on two enzymes:
1. Pyruvate carboxylase is a key biotin-dependent enzyme that provides oxaloacetate for the TCA cycle, gluconeogenesis and neurotransmitter synthesis, whose structure we have just determined. There is also a strong correlation between the activity of this enzyme and insulin secretion and thus an association with Type II diabetes. We have determined the first structure of a biotin-dependent carboxylase holoenzyme, the pyruvate carboxylase from Rhizobium etli:
St. Maurice et al. (2007) Science 317, 1076-1079.
We are investigating the structure-function relationships in this enzyme, with a combination of site-directed mutagenesis, kinetic and physical methodologies. The ultimate aims of this project are to understand the mechanism of action of the enzyme and design drugs that will act either as inhibitors (anti-fungals and anti- bacterials) or stimulate the activity of the enzyme (diabetes treatment). We are currently working on the mechanism of allosteric regulation of the enzyme by acetyl CoA and the mechanism of catalysis with respect to half-of-the sites reactivity in the enzymic tetramer.
Suitable for students with a biochemistry or biochemistry/chemistry background.
5
2. Mammalian histidine kinases 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, although we have established a link between enhanced histone H4 histidine kinase activity and hepatocellular carcinoma in human liver and shown it to be a possible oncodevelopmental marker of hepatocellular carcinoma (see below). This discovery offers a potential target for treatment or diagnosis of liver cancer.
HCCT = HEPATOCELLULAR CARCINOMA TISSUE HCCN = NORMAL TISSUE SURROUNDING HEPATOCELLULAR CARCINOMA NORMAL = NORMAL ADULT LIVER
Tan et al. (2004) Carcinogenesis 25, 1-6.
However, we really need to know more about the cellular role of histidine phosphorylation in general and particlularly in histone H4. One of the difficulties in the investigation of histidine phosphorylation is the detection of proteins containing phosphohistidine in cells and tissues, partly due to the lability of the P-N bond and also because there are two isomers of phosphohistidine N1 and N3 (se below). To address this problem I am currently collaborating with Dr. Matthew Piggott to develop pan-phosphohistone antibodies for the detection of histidine-phosphorylated proteins, by synthesizing and using non-hydrolysable analogues of phosphohistidine as immunogenic haptens (see triazole analogues below). This would be part of the Honours project which would be jointly supervised by Prof. Attwood and Dr. Piggott and the chemistry content could be adjusted to suit either a Chemistry major student or a biochemistry major student. Other components could include some purification and characterization of histidine kinases.
O O O H H H N protein histidine N protein N protein protein N kinase protein N protein N H H OR 1 H 3 ATP N HN 3 N N 1 N O N 1 P O 3 O P O O O histidine residue N1-phosphohistidine N3-phosphohistidine residue residue
O O
H3N H2N O O N N1 N N 3 N N 1 O P O 3 P O O O O stable triazole analogue stable triazole analogue
Suitable for students with a biochemistry or chemistry background.
6 PROFESSOR MURRAY BAKER Room 4.09, Bayliss 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. Honours projects are currently available in the following areas.
1. Biodegradable and biocompatible materials for tissue engineering Collaboration with Prof Traian Chirila (Prevent Blindness Foundation, Queensland), Prof Kathy Luo (Nanyang Technological University (Singapore), Dr Keith Stubbs (UWA), 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. One of the most important biocompatible polymers is poly(hydroxyethyl methacrylate) (PHEMA). PHEMA-based materials are made by co-polymerising hydroxyethyl methacrylate (HEMA) with suitable crosslinking agents. PHEMA is O already used to fabricate permanent medical implants, such as the artificial cornea OH developed by Prof Traian Chirila. An important feature of PHEMA is its ability to be O easily fabricated in a porous form that is suitable for hosting cell growth. The pictures here show a scanning electron microscopy image of a sample of porous PHEMA (left) hydroxyethyl methacrylate and an optical microscope image of a similar sample of PHEMA after implantation into a mouse (right). In the latter image, blood vessels and regenerating tissue growing into the pores in the PHEMA are clearly visible. In collaboration with Prof Chirila, we are now developing biodegradable forms of PHEMA, for new applications in tissue engineering. This research includes study of: (1) new biodegradable crosslinking agents based on peptides and (with Dr Stubbs) carbohydrates); (2) new methods of controlled polymerisation of HEMA; (3) incorporation cell adhesion factors and cell-growth factors into PHEMA; and (4) new forms of biodegradable PHEMA (e.g., powders and thin films on surfaces) as substrates for tissue growth in the laboratory. In collaboration with Prof Luo we are investigating PHEMA as a component of polymer-cell conjugates to build artificial tumours for use in cancer research. This work includes: (1) development of polymerisation methods using non-toxic reagents and catalysts and (2) polymerisation of HEMA and HEMA oligomers in the presence of live cells.
2. Chemical and Biological Applications of N-Heterocyclic Carbene Complexes N-Heterocyclic carbenes (NHCs) are analogues of phosphines, but they have some significant advantages, including ease of synthesis and strong N donor ability. NHC complexes are easily accessible via azolium ions (eg N N N Br 1). We are exploring the synthesis and properties of interesting azolium N Pd N N Br ions and transition metal NHC complexes. We have found that N complexes such as 2 are excellent catalysts for certain C-C bond forming 1 2 reactions. The unique ruthenium complex 3 is of interest as a potential anti-cancer agent, since it is an analogue of a well-known class of anti- cancer compounds such as 4. + R + Cationic Au(I) NHC complexes such as 5 and 6 exhibit activity N Ru against certain cancer cell lines. This activity appears to be a consequence N N Ru Cl H2N Cl of Au binding to an enzyme in mitochondria, and the selectivity for N NH2 3 4 7 killing cancer cells over normal cells can easily be tuned by variation of the hydrophilic-hydrophobic character of the NHC ligands.
2
N N Cl N N Au N N Au Au N N N N N N Au N N Cl N N
5 6 7 The Au(I)-NHC complexes are easy to synthesize and they offer the prospect of fewer toxic side-effects than their better-known Au(I)-phosphine counterparts. Au(I) complexes such as 5 and 6 and Au(III) complexes such as 7 also have exciting prospects as robust catalysts for a range of oxidation and C-C bond forming reactions. An exciting goal in the Au-NHC area is to replace one of the NHC ligands with other ligands that have their + own innate biological activity. Thus, complexes of form [(NHC)-Au-L] have two potential modes of action: deactivation of an enzyme by Au, and separate anti-cancer activity exhibited by L. Another area of interest is the use of NHC-metal complexes as antibacterial agents to treat drug-resistant infections. We have found that Au-NHC complexes such as 6 tend to concentrate in lysosomes of some cells, and one way in which some bacteria resist drug treatments is by sequestering themselves in lysosomes. Thus, any Au-NHC complexes (or Ag-NHC complexes, which are structurally similar to the Au complexes) that show anti-bacterial activity have the potential to be therapeutic agents for some bacterial infections that are difficult to treat using existing drugs.
3. Azamacrocycles and Catalysis of Organic Reactions by Iron Compounds Triazacyclononanes (TACNs) are excellent ligands for transition metals. Numerous TACN complexes are known, many have demonstrated interesting catalytic and biological activity, and some have served as model systems for the active sites in metalloenzymes. The main disadvantage of TACN ligands is that their syntheses
are long and tedious. TACH TACN ADACH
Triazacyclohexanes (TACHs) are smaller analogues of TACN. N N N N N N The chemistry of TACHs is relatively undeveloped, but TACHs N N are easy to make (just one step from formaldehyde and a primary NH2 amine) and they form many compounds analogous to TACN N N N N N complexes. Because the TACH ring is small, however, bonding N N N NH Mo 2 Mo in TACH-metal complexes is much more strained than in Mo C C C O C C C TACN-metal complexes, and so TACH complexes are quite O C C O C O O O O labile. O O Recently, aminodiazacycloheptanes (ADACHs) have been proposed as analogues of TACN. ADACHs may be a "happy medium" between TACHs and TACNs, since ADACHs are easy to synthesize and they offer a coordination geometry similar to that of TACN. We have already used complexes such as the molybdenum tricarbonyl adducts shown above to compare the chemistry of TACH, TACN, and ADACH systems. Now we are starting a new project in this area, to examine new classes of iron complexes as potential catalysts. Iron is the most abundant transition metal, it is very cheap, and it is non-toxic. Over the last few years, iron compounds ranging from ferric nitrate through to tetrahedral iron phosphine complexes have been found to catalyse organic transformations that previously had been achieved only by catalysts based on much more expensive metals such as palladium. Catalysis by iron complexes is still in its infancy and is not well understood, and selectivity is still poor in most cases. There are great opportunities for the development of useful, cheap, and non-toxic catalysts based on iron. One way to address the problem of selectivity in iron-catalysed reactions may be to bind the iron in a favourable coordination N environment, such as the environments provided by the facially-coordinating TACH and N N Fe ADACH ligands. These environments would bind the iron and so inhibit certain Cl unfavourable processes (eg formation of iron oxides) but leave three mutually cis Cl Cl coordination sites for catalytic reactions to occur. A few Fe-TACH compounds are known. 8 The Fe(III) complex 8 would serve as a convenient starting point for this study.
8
PROFESSOR CHARLIE BOND
Room23B 4.32, Bayliss Building, Phone: 6488 4406
Email: [email protected] U
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 nucleic acid processing proteins involved in regulating gene expression, 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 or to find out about other research in the lab come and see me (MCS
Rm 4.32) and look at http://www.crystal.uwa.edu.au/px/charlieHU UH .
PROJECTS13B (The exact scope of each project will vary depending on the interests and experience of the student). 1. Structure of the paraspeckle interactome (suitable for more than one student) Collaborative with Dr Archa Fox and DrSven Hennig (WAIMR) An emerging and exciting research area is the role of noncoding RNAs in controlling gene expression. ‗Noncoding‘ RNAs are molecules that are functional as RNAs, and do not encode for proteins. Paraspeckles are the first sub-nuclear structure known to form around a long noncoding RNA (lncRNA), making them an important model system within lnRNA research. This is particularly relevant when it comes to cancer, as several lncRNA have been shown to act as molecular scaffolds, recruiting proteins to form oncogenic complexes that drastically alter gene expression leading to metastasis and ultimately poorer outcome for patients. Paraspeckles contain a number of different proteins that are either (1) responsible for paraspeckle formation (2) required for paraspeckle function, or (3) are regulated by sequestration within paraspeckles. The Bond lab has recently solved the 3D structure of a number of homo- and heterodimers of paraspeckle proteins (see figure 2). In an effort to determine the roles of the other known paraspeckle proteins in paraspeckle formation and function, we are undertaking a large-scale interactome analysis of paraspeckle components. This project involves investigating interactions of key paraspeckle proteins PSP2 and Matrin3 with other paraspeckle proteins. It will involve mapping the domains in each protein responsible for protein:protein interactions. A number of techniques will be applied, including molecular biology, yeast-two-hybrid assays, protein Figure 1. Intermolecular interactions in expression in bacteria, purification and in vitro interaction paraspeckles assays. The ultimate goal is to crystallise and solve the structure of protein complexes. In many cases sophisticated expression strategies are used such as co-expression of interacting proteins, in an effort to stabilise interaction partners, leading to large-scale protein production. This project will provide important building blocks for understanding how nuclear proteins together build up a lncRNA-structure, and how their sequestration affects function.
9 2. Structural studies of DBHS proteins – Key factors in gene regulation Collaborative with Dr Mihwa Lee
Key paraspeckle proteins include the „Drosophila behavior/Human Splicing‟ (DBHS) family of proteins have been implicated as important regulators of gene regulation in mammals. Here they are involved in a regulation mechanism whereby mRNA molecules containing a particular structural motif are stockpiled in the nucleus so they cannot be translated into protein. On a specific signal, the stockpiled mRNA is released and a burst of protein production takes place. In mammals this process is controlled by three highly-conserved related DBHS proteins which can form heterodimers which then form into large nuclear bodies called paraspeckles. The Bond lab has recently solved the 3D structure of a number of homo- and heterodimers of DBHS proteins (see Figure 2). Figure 2. Crystal structure of a DBHS protein heterodimer
from our lab This project will build on this exising structural knowledge of DBHS proteins to investigate the propensity of DBHS to for larger aggregates via a coiled-coil interaction motif. Protein samples will be cloned, expressed in bacteria, purified and studied by a panel of biophysical techniques including crystallization and X-ray diffraction, dynamic light scattering and analytical ultracentrifugation.
The student will learn the principles of basic molecular biology, protein expression and purification, X-ray crystallography and complementary biophysical techniques.
10 DR BERNARD CALLUS Senior Research Fellow Room 3.49, Bayliss building, Phone: 6488 1107 Email: [email protected]
Apoptosis and Cancer Signalling Our research group focuses on the mechanisms of apoptosis (programmed cell death) as well as the signalling pathways that regulate cell death pathways. Particular focus is given to how the abnormal regulation of these signalling pathways can contribute to the development of cancer. Typically, cancer cells are profoundly resistant to apoptotic stimuli, e.g. chemotherapeutic drugs, radiation, and this apoptotic resistance is considered to be an essential component in the development of tumours. Often this is due to amplification of oncogenes, e.g. Bcl-2, or the loss of tumour suppressors, e.g. p53, or a combination of both which impart apoptotic resistance in cells. Our research incorporates molecular biology and cellular based assays to examine the impact of increased oncogene expression or loss of tumour suppressor gene expression in cells to examine how they impact on apoptotic mechanisms as well as the signalling pathways that are regulated by them that ultimately contribute to the development of cancer. Our research aims to identify novel regulators involved in apoptosis and cancer as candidates for rational drug design leading to the development of new therapies to kill cancer cells. Feel free to come and discuss your research interests and to learn more about our ongoing projects in the lab. PROJECTS 1. The role of the Arf/Ink4a tumour suppressors on the proliferation, differentiation and transformation of liver-progenitor cells. With Professor George Yeoh, Biochemistry and Molecular Biology. We have previously shown that the expression of the Arf tumour suppressor is significantly down-regulated to undetectable levels during the process of tumorigenic transformation of liver progenitor cells (LPCs) (see Fig 1). We hypothesise that the loss of Arf is a critical early step in the transformation of LPCs. The observation that Arf is expressed in LPCs is significant as Arf is not expressed in many adult and has also led us to hypothesise that Arf may be a marker of non-transformed LPCs. We have generated LPCs from embryos of Arf null (-/-) mice and these cells show an increased propensity to transform (see Fig 2 below). Consistent with this, karyotype analysis of the Arf-/- LPCs indicates the cells already display signs of chromosomal abnormalities and instability. Also the culture of Arf-/- LPCs to high density has revealed that the lack of Arf sensitises the cells to apoptosis and cellular destruction leading us to hypothesise that Arf plays a significant role in the survival and proliferation of LPCs especially at high density. This project will involve numerous follow-up experiments aimed at characterising the role of Arf in these processes. This will include elucidating the role of Arf in regulating the function of key transcription factors such as c-Myc and FoxM1 to prevent LPC transformation. We are also developing a system for reducing Arf expression utilising lentiviral shRNAs that will allow us to demonstrate a direct role Arf plays in these cellular processes.
2. The role of the p19Arf tumour suppressor on chromosomal stability in liver-progenitor cells. With Professor George Yeoh, Biochemistry and Molecular Biology. We have previously found that the expression of the Arf tumour suppressor is significantly down-regulated to undetectable levels during the process of tumorigenic transformation of liver progenitor cells (LPCs). We hypothesise that the loss of Arf is a critical early step in the transformation of LPCs. We have generated LPCs from embryos of Arf null (-/-) mice and these cells show an increased propensity to transform. Consistent with this, we have also performed karyotype analysis of the Arf-/- LPCs and the results indicate that the cells already display signs of chromosomal abnormalities and instability, a key trait of cancer cells. This project will involve follow-up experiments aimed at determining the role of Arf in preventing chromosomal instability. We hypothesise that routine culture of LPCs results in chromosomal instability and that loss of Arf and/or culture under normoxic conditions will hasten this process. Therefore Arf-/- and Arf +/- cells will be serially passaged under both normoxic and anoxic conditions and cells will be systematically examined with increasing passage for chromosomal instability. Cells will also be treated with Arf shRNA to reduce Arf levels and together with parental cells (controls) these will be passaged under normoxic and anoxic conditions and systematically examined with increasing passage for chromosomal instability. In addition, an inducible system will be developed to knockdown Arf levels using shRNA. These studies will allow us to determine the role Arf plays in preventing chromosomal instability and will provide the framework for developing chromosomal molecular probes for fluorescent in situ hybridisation (FISH) to detect transformed liver stem cells in mouse and ultimately human liver pathologies.
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C A B
Figure 1: Chromosomal changes seen during transformation of LPC (BMEL) cells at passage 5 (A), 10 (B) and 15 (C). Large chromosomal abnormalities (upper panels) including loss (red arrowheads) and gain (blue arrowheads) of genomic material are seen concomitant with transformation of the cells indicated by the ability of the cells to grow in soft-agar (lower panels). Cells used for growth in soft-agar are the same cells used for karyotyping at passage 6 (A), 11 (B) and 16 (C). Note the increase in size and number of colonies with increasing passage number.
Figure 2: p19Arf expression is lost during LPC transformation. A) p19 Arf expression in non-transformed (NT) and transformed (T) BMEL and BMOL LPC lines showing Arf expression is lost during transformation. B) BMEL p19Arf-/- LPCs have the propensity to form colonies when grown in soft-agar. Note the increase size and number of colonies compared with the non-tranformed BMEL A-EGFP (p12) control cells. Approximately 21% of BMEL Arf null (-/-) LPCs form colonies in soft-agar at p20.
3. Examining the role of YAP in transformation of liver progenitor cells. Previous research has established that over-expressing the YAP oncogene in cells results in increased cell growth and acquired resistance to certain forms of apoptosis, two key traits of cancer cells. We have also observed that YAP localizes to the nucleolus in non-transformed liver progenitor cells (LPCs) but not in transformed LPCs. We hypothesise that loss of nucleolus localized YAP results in its activation leading to cellular transformation. Furthermore, the expression of the p19Arf tumour suppressor is reduced to undetectable levels in transformed LPCs. Interestingly YAP nucleolus localization is abolished in Arf-/- LPCs suggesting that Arf is involved in this process. We also hypothesise that Arf and YAP interact biochemically and that expression of YAP in Arf-/- LPCs will result in an enhanced rate of cellular transformation. This project will (i) determine whether Arf and YAP interact by performing co-immunopreciptitation (co-IP) experiments; (ii) examine and compare the effect of YAP over-expression in Arf+/+ and Arf-/- LPCs on the rate of cell growth and transformation by examining the ability of the cells to grow in low serum and in soft-agar, a key indicator of transformation and (iii) examine the effect of reactive oxygen species (ROS) on YAP-induced cellular transformation by culturing LPCs under normoxic and anoxic conditions or by culturing LPCs in the presence of anti-oxidants, e.g. L-ascorbic acid (vit C) to determine whether ROS contributes to the transforming ability of YAP.
12 ASSOCIATE PROFESSOR RETO DORTA Room and Phone available soon (December 2011) Email: [email protected] Group webpage: http://www.oci.uzh.ch/group.pages/dorta/home.html
Organometallic Chemistry and Catalysis
Our research is directed toward the preparation of reactive transition metal complexes for stoichiometric and catalytic applications. We focus our attention on the development of new chiral and non-chiral auxiliary ligand systems which are able to bind, activate and functionalize the substrates at the metal center. The ultimate goal of the research program is to identify new ligand families and their corresponding metal complexes for new, more selective or more widely applicable catalytic transformations. Projects for honours students offer a unique opportunity for getting hands-on experience in modern organic and inorganic chemistry. State-of-the-art routine lab equipment will be made available and includes synthetic aspects of the project (Schlenk-line techniques, Glovebox techniques) as well as analytical aspects (GC-MS, GC‟s and HPLC instruments with chiral stationary phases within the laboratory, NMR and X-ray analysis and other necessary equipment within the department). The projects will be such as to provide real insights into new developments in the field of catalyst development and organic synthesis within the timeframe of the honours degree. Additional related projects will be made available upon request.
PROJECTS
1. Ligand Systems Based on Chiral Sulfoxides and Their Use in Late-Metal Chemistry and Catalysis several possible projects
Expanding the ligand families capable of acting as Possible disulfoxide ligand structures: successful entities in metal-mediated reactivity and catalysis R' O R R'' is crucial for future discoveries in this field and will lead to R R'' S S S S R systems that show unprecedented reactivity patterns. One of R' O O O S R' O O O O our recent research goals is to identify and apply chiral S S S Fe,Ru R R chelating sulfoxides as sulfur-based ligands in late-transition R' R metal chemistry. First results show that these ligands indeed R,R'' = alkyl, aryl; R' = hydrogen, alkyl, aryl are able to perform well in a conjugate addition reaction catalyzed by Rhodium. The honours projects available in Possible catalytic application: this area of our research will focus on novel ligand systems of this family and will expand catalytic reactivity to other R chiral Rh/Ir/Pd/Pt cat. * R reactions catalyzed by late-transition metals. For additional EWG + M–Nu EWG R' R' H+/electrophile Nu information on our research, please consult the following
R = H, Alkyl, aryl, OR", NR"2 publications: R. Mariz et al., J. Am. Chem Soc. 2008, 130, R' = Alkyl, aryl, OR", NR"2 M = B, Al, Zn, Si, Ti 2172; J. J. Bürgi et al., Angew. Chem. Int. Ed. 2009, 48, 2768; R. Mariz et al., Chem. Eur. J. 2010, 16, 14335.
13 2. New Chiral N-Heterocyclic Carbene Ligands in Asymmetric Catalysis several projects available
Reactions incorporating NHC metal complexes represent some of the most Successful NHC ligands significant advances in homogeneous catalysis during the last decade, particularly for alkene metathesis and for coupling reactions. Nevertheless, there is a very R1 R restricted architectural choice for these ligand system and this is particularly N N 1 hindering development of chiral monodentate NHCs. In the last few years, we R R2 2 R1 R1 have therefore initiated a research program that proposes the synthesis of new classes of monodentate, chiral NHCs that incorporate substituted naphthyl Proposed Chiral NHC ligands sidechains on the nitrogen atoms. In doing so, we are indirectly relying on a very successful design motif in chiral ligand synthesis that goes back to Noyori‟s bis-
R2 phosphine ligand BINAP. These new types of ligand systems will allow for the R1 synthesis of new transition metal complexes, where our focus will particularly lie N N on the isolation of highly unsaturated precatalysts. Special emphasis in subsequent R1 applications will be put on the identification of more active chiral rhodium and R2 iridium NHC compounds in catalysis, development of better asymmetric nickel, C2-symmetry palladium and ruthenium mediated transformations and the development of unknown NHC-Ag catalysis. For preliminary data from our group on this project, see: X. Luan et al., Org. Lett. 2008, 10, 5569; X. Luan et al., Org. Lett. 2010, 12, 1912.
3. New Catalysts and New Substrates in Ruthenium-catalyzed Metathesis Reactions
Olefin metathesis has experienced a significant evolution in the last R7 decades and is becoming one of the most useful synthetic transformations R2 for generating carbon-carbon double bonds. The reaction can be applied in N N a great variety of synthetically useful permutations that include ring- Cl R2 7 closing metathesis (RCM), cross metathesis (CM) and enyne metathesis. Catalyst: R Ru Cl Among the catalysts that have been developed, ruthenium alkylidene O complexes incorporating an N-heterocyclic carbene (NHC) ancillary ligand i Pr (Grubbs‟ second-generation catalyst) have become the most widely used in Ph organic synthesis. The goal of this project is twofold; we have already been able to show that modifying the NHC ligand (see project 2) can bring about Ring-closing metathesis a clear increase in catalyst performance; further fine-tuning is therefore a n n Ru cat. worthwhile target and is also expected to lead to new reactivity in difficult Y Y + or novel applications of the metathesis reaction. Indeed, metathetical X X reactivity of relatively electron-rich double bonds is still very challenging, presumably due to the fact that stable, Fischer-carbene type complexes are Cross metathesis generated upon reaction with the catalyst‟s metal center. Here, new results
R Ru cat. R in our group have shown that modification of the substrates themselves + CR2 + CR2 might lead the way to exploiting metathesis reactions that have previously X X not been known. For recent results from our group, see: X. Luan et al., J.
X = Halide, OR, NR2 etc. Am. Chem. Soc. 2008, 130, 6848; M. Gatti et al., J. Am. Chem. Soc. 2009, 131, 9498; M. Gatti et al., J. Am. Chem. Soc. 2010, 132, 15179.
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DR ELA EROGLU RESEARCH ASSISTANT PROFESSOR Centre for Strategic Nano-Fabrication and ARC Centre of Excellence in Plant Energy Biology Room 3.41, Bayliss Building, Phone: 6488 2558 Email: [email protected]
My research projects involve photosynthetic microorganisms (such as microalgae and photosynthetic bacteria) and their applications to nanotechnology. The following topics cover several ―green‖ bioprocesses while combining several interdisciplinary fields including Biotechnology, Nanotechnology, Agricultural and Environmental Sciences
PROJECTS
1. Wastewater treatment processes with Immobilized Algae with Prof. Steve Smith (CoE in Plant Energy Biology), Prof. Colin L. Raston, and Dr. Swaminathan Iyer
Wastewater treatment is the process of eliminating unwanted chemicals, or biological contaminants from the impure water. It mainly includes liquid wastes released by houses, industrial properties, and/or agricultural processes; while having a wide range of contaminants at various concentrations (Metcalf and Eddy 2003). As a relatively recent bioprocess, microalgal cultivation in wastewaters has a combination of several advantages such as integrated wastewater treatment and simultaneous algal biomass production, which can be further exploited for biofuel production (in the form of biodiesel, biohydrogen, or biogas), food additives, fertilizers and soil conditioners, cosmetics, pharmaceuticals, and many other valuable chemicals (Mallick 2002). Microalgae are the recent organism of choice for the renewable generation of hydrocarbon-based biofuels, with high biofuel yields in comparison with plant-oils (Eroglu and Melis 2009). Microalgae have several other advantages as it can grow within short time intervals, does not require many resources to produce, and can be utilized for the reduction of CO2 emissions by using carbon dioxide for biomass and/or energy production. In addition to the utilization of the wastewater contents for algal biomass formation, the dissolved oxygen released by the algae is also useful to oxidize waste organic matter.
One of the main problems to obtain a productive algal water treatment and bioenergy systems is the harvesting, dewatering and processing of algal biomass. In this project novel algal immobilization approaches will be adopted by employing various nanotechnological strategies, such as electrospinning. This work has great prospects to combine interdisciplinary fields in the national and international collaborations. Several industrial waste treatment plants, and the Governmental or private water-cooperation are potential end-users of the developed-technology. Image: http://www.westernenvirosolutions.net/
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2. Nutrient Recovery for the Generation of Sustainable Biofertilizers with Dr.Sasha Jenkins (from the School of Earth and Environment)
Several effluent wastewaters (i.e., agricultural, municipal, industrial) contain high amounts of nitrate and phosphates that needs to be removed from their effluents before discharging into their environment. The exposure of surrounding system and groundwater to pollution brings severe environmental regulations to be imposed on these industries. Removal of these nutrients is very essential especially to avoid eutrophication of the surrounding water sources that can result several environmental impacts. Nitrate and phosphate uptake can be achieved via algal pond systems, while the algal biomass is harvested and can be recycled as a fertilizer.
In this study, we‟ll be investigating the treatment of effluent wastes with high nitrate and phosphate loadings by immobilized algal cultures. Then the algal cultures will be harvested and the immobilized algal biomass will be recycled on land as “slow-release” fertilizer which will be highly beneficial for organic farming. Goal of this research is to develop and operate a sustainable nutrient recovery system from various wastewaters by using immobilized algal systems and mixing these algae-hydrogel combinations with the soil as a moisture-rich fertilizer and soil enhancer. Nitrate and phosphate recovered from the effluent wastewater will be recycled back to soil by algae, whereas hydrogel matrix can also be beneficial for providing moisture to the soil. Image: http://www.biosynherb.com/bio-fertilizer.html
3. Biosynthesis of nanoparticles with Prof. Steve Smith (CoE in Plant Energy Biology), Prof. Colin L. Raston, Dr. Swaminathan Iyer, and Dr. Jeremy Shaw (from Centre for Microscopy, Characterisation and Analysis)
Metal nanoparticles have recently been receiving significant interest, due to their distinctive chemical, magnetic, electronic, and optical properties. As a result of their high surface-to volume ratio, they have been used for various applications such as catalysis, biological labelling, electronics, and optical devices (Lengke et al. 2007). Rather than following the conventional chemical pathways, biological materials can be used for the synthesis of metal nano-particles as ecological stabilisers.
For this context, several microorganisms will be investigated for their nanoparticle (such as Palladium nanoparticles) production capability. Transmission Electron Microscopy (TEM) techniques will be developed and applied for the imaging and the characterization of nanoparticles.
Image: Gillian Walters and Ivan P. Parkin (2009) J. Mater. Chem., 19, 574
References Lengke et al. (2007). Langmuir, 23, 8982-8987 Eroglu and Melis (2009) Biotechnology and Bioengineering, 102(5): 1406-1415 Mallick (2002) Biometals, 15: 377–90 Metcalf and Eddy, Inc (2003) Wastewater engineering: treatment and reuse. 4th ed., McGraw-Hill, New York.
16 DR GAVIN R FLEMATTI ARC Postdoctoral Fellow Room 4.17, Bayliss Building, Phone: 6488 4461 E-mail: [email protected]
Research Interests
My main research interest is in the field of bioactive natural products. I work closely with A/Prof Emilio Ghisalberti in this regard and together with collaborators from other disciplines we are interested in the detection, isolation and identification of natural products that demonstrate some form of biological activity. Some possible honours projects are summarised below which I am happy to discuss further.
PROJECTS
1. Isolation of bioactive compounds that reduce methane emission in ruminants. Collaboration with A/Prof Phil Vercoe, School of Animal Biology, UWA
In Australia, 90% of the total greenhouse gas emissions from agriculture stem from gases produced as a natural end-product of the digestion in ruminants (sheep and cattle), including methane as the most potent greenhouse gas. One way to reduce methane emissions from animals is to feed them plants that contain naturally occurring secondary compounds with antimicrobial properties that can inhibit methanogenic microorganisms in the rumen. A/Prof Phil Vercoe‟s research group at UWA Animal Biology has identified several Australian native plants with these antimethanogenic properties in the rumen. However, the chemistry, the metabolism in the rumen and the mode of action of these compounds is unclear.
This project aims to isolate and identify the major secondary metabolites from selected Australian native plants and investigate their role in reducing methane production by the rumen microbes.
2. Investigation of volatile organic compounds emitted from Australian truffles. Collaboration with Prof Garry Lee, Centre for Forensic Science, UWA
Truffles are subterranean edible fungi that traditionally grow in various parts of Europe, particularly in Italy and France. They are highly appreciated due to their characteristic aroma and are used mainly uncooked in French and Italian cuisine, particularly the black perigord truffle (Tuber melanosporum). Previous research has identified over 200 volatile compounds that are emitted from truffles, including many alcohols, ketones, aldehydes, aromatics and sulphur compounds.1
Studies show that the geographical location plays a significant role on the composition of the truffle volatiles.2 To date, there have been no reports on the composition of volatiles from Australian grown truffles. This project will investigate the volatiles emitted by black truffles (T. melanosporum) at various stages of maturity from at least three different locations in Australia (Western Australia, New South Whales and Tasmania). Commercially available truffle oils will also be analysed and compared with fresh samples using solid phase micro-extraction (SPME) and GC/MS. The purpose of this work will be to identify volatiles that allow differentiation of truffles grown from different regions and at different stages of maturity.
Refs: 1 Cullere, L., Food Chemistry, 122, 300-306 (2010). 2 Gioacchini, M. A., Rapid Communications in Mass Spectrometry, 22, 3147-3153, (2008).
17 DR SIMON GRABOWSKY Room 4.29, Bayliss building, Phone: 6488 3515, Email: [email protected]
The aim of our research is to find the exact locations of electrons within molecules and make them visible. We can use the electron density, which tells us about electron concentration and depletion, and we can use electron localisation functions, which tell us where electron pairs are localised. If we know the exact distribution of electrons within molecules and where they preferably pair up, we can derive information about chemical bonding and reactivity. We use two ways to extract the desired information: an experimental one and a theoretical one. X-ray diffraction experiments on single crystals at very high, i.e. sub-atomic, resolution and at ultra-low temperatures (down to 8K) allow us to obtain the electron-density distribution of the scrutinised compound. New techniques go even beyond this and allow to derive an experimental wavefunction from the X-ray diffraction data, which can be used to calculate electron localisation functions additionally to the electron density. The theoretical way uses quantum-mechanical ab-initio calculations on the computer to derive all necessary information from a theoretical wavefunction. Applications of these techniques are widespread: On the one hand, we are interested in compounds suitable for drug design and study potential active centres for interactions with enzymes; on the other hand, we are interested to shed light on unusual bonding situations in organic and inorganic compounds.
PROJECTS
1. Comparing the electronic nature of different potential protease inhibitors to facilitate drug design
Proteases are enzymes that catalyse the hydrolysis of peptide bonds. They are essential for any organism in many different ways. But they also play an important role in the dissemination of cancer. Tumour cells release pathogenic forms of autologous proteases like cathepsin or collagenases. Therefore, drugmakers search for compounds which can irreversibly inhibit specific proteases. Our collaboration partners (University Wuerzburg, Germany) synthesise compounds which attack cysteine or asparagine groups in proteases via an electrophilic attack at the mercapto (SH) group in the case of cysteine or at the carboxyl group (COOH) in the case of asparagine. We have already measured high-resolution X-ray diffraction data sets of three of these compounds with different active electrophilic centres (an epoxide group, an electron- deficient double bond, a sulphur-containing five- membered ring). See the figure for the electrostatics around an epoxide model compound. In this honours project, we aim to measure a fourth high-resolution data set at the in-house X-ray diffractometer and evaluate the crystal electron density as well as electron-pair localizability within these four protease-inhibitor model compounds. A detailed comparison of the different active centres at the electronic level will give insights into the inhibition mechanism and thus the usefulness for drug design.
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2. Transferability of sub-molecular properties in the electron density
The electron-density distribution for any molecule can be subdivided into functional group or even atomic regions. This is part of Richard Bader‘s Quantum Theory of Atoms in Molecules. Physics predicts that these sub-molecular fragments are transferable between different molecules. The figure shows a cut-plane through the electron density of two fused rings. Regions in the electron density belonging to the individual atoms can be identified. You can imagine how these regions could be extracted from the picture using a scalpel and could be glued together in a different arrangement to construct the electron density of a different compound. In fact, the total electron density of a compound, e.g. a large one like a protein which cannot be measured to high resolution, can be built up from atomic fragments like a three-dimensional puzzle.
This concept is extremely useful and has led to the development of electron-density data banks which store atomic electron-density building blocks. However, this has only been tested for and has been applied within the so-called multipole expansion of electron-density modelling. But we want to test this in a more general way and for more functions than only the electron density. We have measured the high-resolution X-ray diffraction data sets of six different tripeptides of the type L- alanyl-X-L-alanine in the past where X is a variable amino acid. The aim of this honours project is to use these data sets to extract experimental wavefunctions and to compare derived properties with respect to transferability of sub-molecular fragments.
Other projects may be available after consultation
For an introduction to these research areas, see the following publications:
P. Luger, Fast electron density methods in the life sciences – a routine application in the future? Org. Biomol. Chem. 2007, 5, 2529-2540. T. Koritsanszky, P. Coppens, Chemical Applications of X-ray Charge Density Analysis. Chem Rev. 2001, 101, 1583-1627 Watch the lecture on his website http://www.chemistry.mcmaster.ca/bader/ A. Savin, R. Nesper, S. Wengert, T. F. Faessler, ELF: The Electron Localization Function. Angew. Chem. Int. Ed. Engl. 1997, 36, 1808-1832. S. Grabowsky, T. Pfeuffer, W. Morgenroth, C. Paulmann, T. Schirmeister, P. Luger, A comparative study on the experimentally derived electron densities of three protease inhibitor model compounds. Org. Biomol. Chem. 2008, 6, 2295-2307. S. Grabowsky, T. Schirmeister, C. Paulmann, T. Pfeuffer, P. Luger, Effect of Electron-Withdrawing Substituents on the Epoxide Ring: An Experimental and Theoretical Electron Density Analysis of a Series of Epoxide Derivatives. J. Org. Chem. 2011, 76, 1305-1318. S. Grabowsky, R. Kalinowski, M. Weber, D. Foerster, C. Paulmann, P. Luger, Transferability and reproducibility in electron-density studies – bond-topological and atomic properties of tripeptides of the type L-alanyl-X-L-alanine. Acta Cryst. B 2009, 65, 488-501.
19 WINTHROP PROFESSOR PETER HARTMANN Room 2.03, MCS Building, Phone 6488 3327 Email: [email protected]
Human Lactation
Winthrop Professor Peter Hartmann leads a large research group that carries out both basic and applied lactation research with women and infants. Despite a plethora of evidence showing breast milk is the best nutrition many women fail to sustain exclusive breastfeeding for 6 months as recommended by WHO. The aim of this group is to provide an evidence base for clinical protocols and management of lactation difficulties. To achieve this objective a fundamental research into the physiology and biochemistry of milk synthesis milk secretion, cell in milk (immune and stem), milk ejection, the mechanics of breastfeeding and the control of infant appetite is carried out.
The following projects will increase the knowledge base of lactation substantially and are available to honors students
PROJECTS
1. Reactive oxygen species in human milk with Dr James Lui and RA/Prof Ching Tat Lai
Reactive oxygen species (ROS) have received much attention due to their high reactivity and ability to modify other biomolecules. These modifications may potentially be so devastating that they precipitate damage to tissue and subsequently cause disease. ROS can be generated at the cellular level as well as during environmental stress (e.g. ultraviolet irradiation, ultrasound or heat exposure). Although the human lactating breast produces high quantities of antioxidant proteins and molecules that scavenge these ROS, recent evidence suggests that ROS may function as antimicrobial agents. We have tested several assays to detect ROS in human milk and preliminary results have identified ROS. This project will extend this work with the aim of extensively documenting ROS and determining their role in human milk.
2. Peptide profile of human milk with Dr James Lui
Certain classes of peptides in human milk have been shown to have bioactive functions such as providing infant immunity and stimulating infant growth. In addition antimicrobial properties have been demonstrated in in vitro experiments. Data from previous studies have only considered either one or several specific groups of peptides derived from protease-digested proteins found in human milk. It is well known that large variations in the components of human milk exist between lactating mothers therefore it is impossible to draw firm conclusions about the bioactive functions of these peptides. This project will endeavor to characterize the natural peptide profile of human milk at different stages of lactation thus providing a fundamental understanding of the involvement and significance of peptides in human milk with regard to both the mother and the infant.
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Mass spectrometry data and PCA analysis
3. Bacteriostatic properties of human milk with RA/Prof Ching Tat Lai
While breastfeeding is recommended first and foremost by WHO there are many situations where the mother needs to express her milk to be fed to the infant for instance premature infants are too ill and weak to breastfeed. Since breastmilk improves preterm infants short and long term health outcomes it is imperative that they receive this milk safely. Human milk is unique in that it possesses bacteriostatic properties that are apparent when milk is stored over time. These properties are diminished when the milk is pasteurized prior to feeding. Despite the importance of this property to the infant little investigation has been carried out in this area. This project is designed to further investigate the bacteriostatic effects of milk and to determine which components are responsible for these effects.
4. Cellular biochemistry of human milk with Dr James Lui and RA/Prof Ching Tat Lai
Recent research indicates the existence of cell population, intact cellular organelles and bacteria in expressed breast milk. Although current studies are beginning to characterize the different cell types and bacteria existing in breast milk, our understanding of the functional significance of the cells, organelles and bacterial populations in breast milk are still unclear. Biochemical contribution of these populations to the milk could be a window of opportunity to observe physiological changes in the lactating breast. This study will take a focus approach to explore cellular biomolecules in breast milk to determine the functional relationship between these populations in breast milk. This may lead to a way of monitoring any changes in the health of the mother, which may inadvertently affect the health of the newborn infant.
These projects provide exciting insights into the components of breast milk and their functions. They provide important knowledge that will contribute to the development and refining of optimal storage conditions for the milk. Ultimately it may be possible to tailor components in the milk to benefit ill and premature infants.
21 ASSOCIATE PROFESSOR DYLAN JAYATILAKA Room 4.30, Bayliss building, Phone: 6488 3138, Email: [email protected]
Theoretical and Computational Chemistry
I am interested in a number of areas, including:
Quantum chemistry: using quantum mechanics to calculate molecular properties e.g. shapes, dipole moments, polarisabilities. We use existing computer programs and we write our own too. Chemical concepts from quantum mechanics. Although quantum mechanics can produce properties, by following the rules, it is often difficult to understand and interpret these properties in terms of “atoms” and “bonds” and all the usual terms that chemists use. I'm interested in developing theories and methods to do this. Crystallography and diffraction: I'm interested in using diffraction experiments to improve quantum chemistry calculations, and vice versa, using quantum chemistry to improve measurements from X-ray and polarized neutron diffraction experiments. Development of reusable software. I have written a program library called Tonto which makes developing new quantum chemistry and crystallography methods easier than normal. Visualisation of complex chemical data. I have helped developed a program called Crystal Explorer to visualize crystal structure packing information in high quality 3D graphics.
What do I need to know to do a computational project?
You need to be familiar with computers (who isn't) and if not, you need to be good at maths. You will develop skills in dealing with Unix computers in the projects to run existing programs. For more specialist projects, you will need to be interested in writing programs to solve problems. A general facility with numbers helps.
PROJECTS
1. Why do crystals have the shape they do? What about the spaces between molecules in crystals? With: Prof. Mark Spackman, Dr Mike Turner
The physical properties of a crystal are directly connected to the way the molecules pack to form a crystal. Why do molecules pack in one way, and not another? Even though we know the underlying laws, this is a basic question that is still largely unanswered. A unique “fingerprint” can be made of this surface (shown left, for urea). The fingerprint is easier to see and understand since it is two dimensional. In this project, we want you to systematically examine the fingerprints for many structures and to try and compare them to see if we can at least classify all the different types of crystal structure. You could also develop a new kind of fingerprint which characterises the void spaces between molecules. The voids between molecules are useful in themselves, because other molecules (such as hydrogen) can be fitted into them for storage.
Further reading: Spackman MA, Jayatilaka D (2009) Hirshfeld surface analysis. Cryst. Eng. Comm. 11:19-32.
22 2. What defines a bond? Bond indices from quantum mechanics.
Have you ever wondered when you are allowed to draw two lines between an atom in a molecule and call it a bond? Recently we published a paper that allows to calculate the bond index between two atoms in a molecule from the wavefunction of the molecule. Furthermore, we showed how to calculate an ionic bond index and a covalent bond index. We've tested the method on a few systems, and the results are mostly very good, but there are a few anomalies, especially for group II elements. In this project you will use our programs and calculate bond indices for a range of interesting systems. We are particularly interested in performing calculations on a recently reported compounds which is claimed to have a quintuple bond. You will try to discover the reason for the anomalous results.
Further reading: Gould MD, Taylor C, Wolff SK, Chandler GS (2008) A definition for the covalent and ionic bond index in a molecule An approach based on Roby's atomic projection operators. Theor. Chem. Acc.:275- 290.
3. Visualising energy densities in molecules
The energy of a molecule is a crucial property. However, the energy is a property of the whole molecule: so where exactly is the energy located in the molecule? In this project we are interested in obtaining properties such as the energy density in the molecule. Prof. Gibbs and I have found new expressions for a number of property densities (kinetic energy density, potential energy density, and so on) obtained from wavefunctions. None of these properties has ever been plotted before. In this project you will investigate these new energy densities (there are 48) for a range of simple molecules. Shown to the right is the plot of the electron localisation function for urea (this is not one of the ones you will look at). This will be a colourful project.
Further reading: Grimwood DJ, Bytheway I, Jayatilaka D (2003) Wave functions derived from experiment. V. Investigation of electron densities, electrostatic potentials, and electron localization functions for noncentrosymmetric crystals. J. Comp. Chem. 24:470-83.
4. 3D and dome visualisation with Crystal Explorer With: Prof. Paul Bourke, Dr. Mike Turner
The ability to visualise and interpret crystal structures is an important aspect of structural science. We have developed a program called Crystal Explorer used to visualise crystal structures, Hirshfeld surfaces, and Hirshfeld surface fingerpints (see project 1). Crystal Explorer is popular and widely used. In this project you will extend Crystal Explorer to display on a dome projector to aid visualisation (shown left). You will also try to convert the program to display in 3D, using 3D glasses. This project will require some background in programming or a strong desire to develop skills in this area. It will be co-supervised with Paul Bourke from the WA supercomputing facility (WASP), which has dome and 3D projection facilities.
23 PROFESSOR GEORGE KOUTSANTONIS Room 3.11, Bayliss building, Phone: 6488 3177, Email: [email protected]
Metals in Chemistry 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. We are participating members of the WA Centre of Excellence in Nanochemistry. PROJECTS
1. Biomimetic Complexes of the Mg/Ca oxide cluster of Photosystem II The inorganic cubane complex known as the manganese-calcium oxide cluster, commonly referred to as the "Oxygen Evolving Complex" or OEC (also referred to as a photosynthetic water oxidase). The OEC is located on the oxidizing side of Photosystem II (PSII), and isolated within chloroplasts, a plastid found in all plants and algae. The OEC is also found in one group of bacteria, the Cyanobacteria. It is believed that the Cyanobacteria are the endosymbiotic ancestors of modern day chloroplasts. At the active site water oxidation procceds at a pentanuclear Mn4Ca oxide particle with an “organic sheath” protecting the core. An attractive method for the formation of nanoparticles derived from metal compounds is the use of a particular ligand to excise clusters from the lattice of a simple species. More commonly, however, the "excision" is a formal process, in that while the cluster may be recognisable as a portion of an extended lattice, it is not, in fact, formed by direct fragmentation of that lattice.. In this project we will utilise polyphenolic compounds, called calixarenes, as a template to build Mn/Ca clusters upon and to introduce the geometric constraints required for the enzyme function to be mimicked.
2. Molecular Computing: Dihydropyrene-based organometallic molecular switch with Assoc. Prof. Matthew Piggott) Dimethyldihydropyrenes are fascinating molecules with a planar 14- -electron periphery, making them aromatic. They are easily converted to their valence tautomers, cyclophanedienes, by irradiation with certain wavelengths of light. This project will involve the synthesis of a novel diethynyl-substituted dihydropyrene that will be used to prepare organometallic complexes. Switching between the valence tautomers is expected to drastically change the conductivity of the organic ligand, which in turn will affect properties such as colour, crystallinity and redox potential. We will prepare metal complexes of dimethyldihydropyrenes that will have the potential to fine tune the physical properties of these materials for application in new computing technologies.
3. 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
24 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.
4. Redox-active Metal Complex Oligiothiophenes as Sensors and Devices with Dr Gavin Collis, CSIRO Material Science and Engineering The drive for new devices that have utility in electrochemical sensing applications or for clean electrocatalysis has seen considerable effort expended in the modification of electrodes. Two intensely studied approaches to construct of such electrodes has been the formation of self-assembled monolayers or by the deposition of a funtionalised polymer on the electrode surface. In this latter case the most widely studied class of monomers for the production of polymer modified electrodes are functionalised thiophenes and oligothiophenes, as they can be readily electropolymerized directly onto the electrode. We have S recently shown S that S S S S H H oligiothiophenes that have metal complexes directly N N attached to the polymerisable unit have difficulty in M O M beingpolymerised. Thus this project will strive to prepare M = metal complex new monomers for polymerisation that have variable linkers for attachment to metal complexes. The targets in the first instance are shown adjacent.
5. Charge density analysis of fundamental host-guest supramolecular systems several projects, with Prof Mark Spackman and Dr Alex Sobolev, UWA 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.
6. New Organometallic Materials with Assoc Prof Matthew Piggott) Photocatalysis and its application to solar energy conversion is an important research problem for the next O O i HO C2SiPr 3 OHC KOH Pri SiCCLi century particularly in light of the peak oil problem that faces + 3 OHC O i current energy generation strategies. O HO C2SiPr 3 This project seeks to prepare new metallotectons with the ability to SnCl2
i potentially control energy and electron transfer processes. One SiPr 3 C way in which to do this is to recruit pendant or bridging aromatic C groups for this purpose and a readily available moiety for this is the [M] C C C C [M] [M] C C C C [M] C pentacene unit. Aromatic units of differing structure will allow us C i to control the HOMO-LUMO and band gap. There is a significant SiPr 3 synthetic component involved in this project the majority of which is supported by solid literature procedures. 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.
25 ASSOCIATE PROFESSOR MARTHA LUDWIG On study leave July-December 2012 Room 3.05, Bayliss Building, Phone: 6488 3744 Email: [email protected]
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; 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. Many CAM plants are able to “switch” between pathways, depending on the environmental conditions and/or their developmental stage.
Harnessing the photosynthetic biochemistry of C4 plants for increased food, fodder and fuel – supercharging C3 plants The global demand for cereals, which are major food sources for animals including humans and are important in the biofuels industry, has been forecasted to increase by 60% for 2050, and with consumption being greater than production in seven of the last nine years, and 2008 stockpiles at 70 days of global consumption, major challenges face agricultural sectors and governments with respect to food, feed and fuel securities. Increasing productivity is unlikely to be accomplished only by conventional breeding methods. A second “green revolution” that includes biotechnology is inevitable for some crops and regions. The higher photosynthetic rates, greater efficiency in the use of water and nitrogen of C4 plants relative to C3 plants in arid and saline environments – environments that are expanding in many parts of the world due to global climate change – are desirable traits, which if introduced into C3 plants, have the potential to increase yield. In other words, we are looking to “supercharge” C3 crops like rice and wheat by giving them a C4 pathway. Toward this objective, a major aim of the work in the lab is to understand the molecular biology, biochemistry and cell biology of the enzymes in the C4 photosynthetic pathway. This includes the identification of the control regions of the genes coding for these enzymes. Such information will be used to make informed and strategic decisions regarding the transfer of particular C4 enzymes, or an entire C4 pathway, into C3 plants to increase yield while restricting negative impacts on the environment.
We are using tools of cell and molecular biology and molecular genetics such as differential cDNA library construction and screening, quantitative reverse transcription PCR (qRT-PCR), transcriptome sequencing, and immunocytochemistry to identify key proteins involved in the above processes and examine the expression patterns of their genes. These studies will give insight into the evolution of photosynthesis, the process on which all life depends, and the plasticity of plants in obtaining nutrients and water 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
The plants we use in our work are in two evolutionarily significant genera – Flaveria and Neurachne, the latter being native to Western Australia, and only found in Australia! These groups of plants are important model systems for examining molecular evolutionary questions because the individual species in the genera use the C3, C4 or an intermediate C3-C4 photosynthetic pathway and represent a living continuum from the ancestral C3 condition to the evolutionarily advanced C4 state. This allows us to discover the changes that occurred during the evolution of the C4 pathway at the level of the genes, transcripts and the proteins they encode. This involves:
1. Comparison of gene expression patterns of key enzymes in C3, C4 and intermediate C3-C4 species of Flaveria and/or Neurachne using qRT-PCR, transcriptome sequencing and/or in situ labeling techniques.
2. The biochemical characterisation of photosynthetic isoenzymes that function in the same intracellular compartment, and the identification of the proteins with which they interact.
26 3. Identification of regulatory regions that control the expression of genes encoding photosynthetic enzymes.
4. Exploring potential correlations between ploidy and survival under biotic and abiotic stress conditions.
27 ASSOCIATE PROFESSOR THOMAS MARTIN Room 3.47, Bayliss Building, Phone: 6488 3331
Email: [email protected] U H
The Signalling and Protein Interaction Group
We are interested in cellular signalling and how this impacts on plant development and function. Learning about this will help us to identify mechanisms by which plants can be improved to be for example drought, salt or stress resistant or to generate higher yields. These are desirable traits for plants growing under the harsh environmental conditions in Australia. To this end we investigate two gene families related to stress responses in plants:
a) One is a class of histone deacetylases (HD2) found only in plants. These are proteins involved in the regulation of gene expression by deacetylation of histones which causes changes in chromatin structure. Some of these plant specific histone deacetylases were reported to lead to increased drought and salt tolerance when overexpressed in Arabidopsis (1). b) The other is a family of nitrilases which are potentially involved in cyanide detoxification and plant hormone biosynthesis (2).
Using a state of the art protein interaction system named Bimolecular Fluorescent Complementation (Fig 1) we have shown that members of the plant specific histone deacetylases and the nitrilases interact with 14-3-3 proteins (Fig 2 a and b). These 14-3-3 proteins bind to other proteins and regulate their activity, cellular localisation or stability in response to intracellular or extracellular signals and thereby impact on protein activities and functions (3). Our interest is to understand what the impact of this regulatory interaction between 14-3-3 proteins and histone deacetylases and 14-3-3 proteins and nitrilases is and how this contributes to normal plant function, especially under stress conditions.
Figure 1: The principle of Biomolecular Fluorescence Complementation (BiFC). Two non-fluorescent parts of the Yellow Fluorescent Protein (YFP) are fused to two proteins assumed to interact, for example a 14-3-3 protein and a potentially 14-3-3 regulated protein (A and B). If these proteins do not interact (left) we will not observe fluorescence. Interaction of A with B (right) reconstitutes a functional YFP and fluorescence can be observed using fluorescence microscopy The great advantage of this system is that it can be used in living plants instead of looking at interactions in vitro. (from 4).
Figure 2: Interaction of 14-3-3 proteisn with histone deacetylase (a) and nitrilase 1 (b) demonstrated using
a b Biomolecular Fluorescence Complementation (BiFC). (a) 14-3-3 mu was tested for interaction with the plant specific histone deacetylase HD2C. Interaction was found to occur in the nucleus (N) and nucleolus (No). (b) Interaction of 14-3-3 proteins with nitrilase 1 after induction of cell death. The interaction occurs usually in the cytoplasm of plant cells but localises to round structures after cell death induction as shown in figure b.
28 PROJECTS
1. Investigating the regulation of plant specific histone deacetylases by 14-3-3 proteins Histone deacetylases regulate gene expression by deacetylating histones thereby leading to changes in chromatin structure. We are interested in a subfamily of histone deacetylases found only in plants some of which were reported to lead to increased drought and salt tolerance when overexpressed in Arabidopsis (1). The degree of salt and drought tolerance caused by overexpression can potentially be increased significantly by preventing the regulation of histone deacetylase activity caused by interaction with regulatory proteins such as 14-3-3 proteins which we identified (Figure 2a). Removing such regulation would potentially allow generating plants which are better able to cope with stresses such as salt and drought stress. We postulate that preventing 14-3-3 binding to histone deacetylases will increase the enzymes activity or prevent its inactivation. Overexpressing such mutated histone deacetylases, i.e. those which are not controlled on the protein level, may in turn increase tolerance of plants to stress conditions such as high salt and drought. The honours project will explore the regulation of histone deacetylases by 14-3-3 proteins. The aims of this project are: a) To identify and mutate 14-3-3 binding sites in histone deacetylase HD2a and HD2b b) To verify the loss of protein interaction in living plant cells using Bimolecular Fluorescence Complementation c) To test the mutated enzymes for changes in enzymatic properties and regulation.
2. Identifying novel protein interactions of plant specific histone deacetylases H2a/b Histone deacetylases interact with other proteins in order to achieve proper gene regulation control. Interacting proteins can be for example transcription factors, methyltransferases and protein kinases and phosphatases. Knowing these interacting proteins will point towards biological processes regulated by histone deacetylases and hence open up new approaches towards their biological role. The project will identify and test novel proteins interacting with histone deacetylases. The aims of this project are: a) To identify proteins interacting with histone deacetylases HD2a/b using a yeast two hybrid screen b) To localise in living plant cells on the cellular level the interaction of HD2a/b with the identified proteins using Bimolecular Fluorescence Complementation c) To identify domains in HD2a/b required for protein interactions by testing HD2a/b mutant forms for interaction with identified proteins.
3. Investigating the biological role of nitrilase interaction with 14-3-3 proteins during plant cell death Plant nitrilases are enzymes thought to be involved in cyanide detoxification and hormone biosynthesis (2). However, their true function is still under debate. My lab has shown that the nitrilases 1 to 4 interact with 14-3- 3 proteins. This indicates that the biological activities of these nitrilases are regulated by 14-3-3 proteins. This regulation will be explored during the proposed honours project. We have further shown that induction of cell death causes nitrilase 1: 14-3-3 complexes to localise to ER derived vesicles (figure 2b). The project will thus explore the reason for this relocalisation and whether any of the other three nitrilases also localises to ER derived vesicles during plant cell death. Finding these answers will help to understand similarities and differences between the functions of the four nitrilase isoforms and will help us to understand their biological roles. The aims of this project are: a) To investigate if 14-3-3 complexes with nitrilases 2, 3 and 4 localise to vesicular structures within the cell during plant cell death. b) To generate nitrilase proteins unable to bind to 14-3-3 proteins and to verify the loss of binding c) To investigate whether loss of 14-3-3 binding changes the ability of nitrilases to localise to ER bodies.
References (1.) Sridha and Wu, The Plant Journal 2006, 46, 124-133 (2.) Piotrowski 2008, Phytochemistry 69, 2655–2667 (3.) Comparot et al., 2003, Journal of Experimental Botany 54, 595-604 (4.) Bhat R.A. et al., Plant Methods 2006, 2:12 (5.) Cutler and Somerville, 2005, BMC Plant Biology, 5, 4
29 PROFESSOR ALLAN McKINLEY Room 2.11, Bayliss 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], HgCH3 [5] MgP, CdP and ZnP [6] are published in top international chemistry journals. We have also completed the experimental phase for, MgN, ZnN, 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 simples radicals such as OH, NH2 and O2 molecule and a water molecule. To date we have published four papers in this area [7-10]. We are interested in nitrogen containing radical adducts with water as these molecules could be important intermediates in the chemical reactions occurring in our atmosphere or those of solar system bodies such as Titan, one of the moons of the planet Saturn. The atmosphere of Titan is mainly nitrogen with traces of water and organic compounds. We are also interested in the chemistry leading to the formation of methanol. Methanol has been observed on comets and may be present on Titan. These experiments would involve matrix isolation IR and ESR experiments and could involve PES experiments in collaboration with Professor Duncan Wild.
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 [12] we have studied denitrification of ammonium nitrate contaminated groundwater[11]. There is a plume of ammonium nitrate flowing into Cockburn Sound and we have tested this remediation technique on this plume [13]. 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. Due to the scarcity of water there is also considerable interest in ways of recycling and reusing water. Of particular interest is purifying waste-water from sewage treatment plants with reverse osmosis equipment and using this water to recharge underground aquifers. Questions that need to be answered include: how long do contaminants persist if they get through the purification process and what chemical changes occur in the anoxic aquifer when oxygenated water is injected? 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.
30 4. Development of New Antimicrobials.
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 and resistant strains of TB are rapidly spreading throughout the world. Furthermore, many strains of enterococci have acquired resistance to vancomycin, one of the last lines of defence against such species. Last year many wards at Royal Perth Hospital were plagued by VRE (vancomycin resistant enterococci) and MRSA (methicillin resistant staphylococcus aureus) and a hospital in Melbourne reported the first cases of the hypervirulent Quebec strain of Clostridium difficile.
In a joint project with Professors Riley (Microbiology) and Stewart (Chemistry) we have synthesized a new compound which shows exceptional activity against gram-positive bacteria. The activity of this compound against MRSA is similar to the activity of vancomycin and other commercial antimicrobials. We hold a provisional patent on this compound and its analogues. Projects in this area could involve synthesis of analogues of the compound with Professor Stewart or, for an appropriately qualified student, experiments with Professor Riley to determine the mode-of-action of the compound and biological activity of its analogues.
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 12 12 13 12 111 12 113 and 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.Fuller, R. O; Chandler, G. S.; Davis, J. R.; McKinley, A. J. ―Matrix isolation ESR and theoretical studies of metal phosphides‖, J. Chem. Phys. 2010, accepted for publication. 7.Langford, V. S.; McKinley, A. J.; Quickenden, T. I. "Identification of OH∙H2O in argon matrices." J. Am. Chem. Soc. 2000, 122, 12859-12863. 8.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. 9.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. 10.Ennis, C. P.; Lane, J. R.; Kjaergaard, H. G.; McKinley, A. J. ―Identification of the water amidogen radical complex.‖ J. Am. Chem. Soc. 2009, 131, 1358-1359. 11.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. 12.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. 13.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.
31 WINTHROP PROFESSOR HARVEY MILLAR ARC Centre of Excellence in Plant Energy Biology (PEB) UWA Centre for Comparative Analysis of Biomolecular Networks (CABiN) (www.plantenergy.uwa.edu.au, www.cabin.uwa.edu.au) Bayliss Building, Room 4.74, Phone: 6488 7245
Email: [email protected]
Cellular processes are directed by genes, orchestrated by proteins and delivered through fluxes of metabolites. 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 and the networks of molecules that define cell energy metabolism and its impact on real-world problems. To see the latest publications from our group see: http://www.plantenergy.uwa.edu.au/publications/millar.shtml http://www.cabin.uwa.edu.au/publications
1. Senescence: remobilisation for plant productivity and yield. With Dr Julia Grassl The aging and dying of plant tissues (termed senescence) is an integrated and essential process in plant development and has a critical role in remobilisation of nutrients from leaves to both seeds and storage tissues. During this process nutrients are transported from the outer leaf areas to the central vascular systems that feed the growing plant. This can be seen as leaves turn colour in autumn. Re-localisation of proteins and other molecules in this process is a large and important research area in cereal crops. Finding molecular markers and genes that influence the senescence process could lead to plants that perform better even in challenging environments such as nutrient deficient soils or during drought. A project would use techniques like quantitative proteomic using isobaric labelling of proteins, molecular imaging using mass spectrometry, Western Blotting, 2D gel electrophoresis, and transcript analysis.
2. Characterization of plant specific complex II subunits With Dr Shoabai Huang The mitochondrion is the powerhouse of the eukaryote cell by synthesis of ATP via electron transport chain complexes coupled with the tricarboxylic acid (TCA) cycle. Complex II (succinate dehydrogenase; SDH) has a central role in mitochondrial metabolism as a component of both the electron transport chain and the TCA cycle. Complex II catalyses the oxidation of succinate to fumarate. We have recently shown that beyond its role in respiration, this protein complex is also involved in defense signalling in plants by helping plants to respond to invading organisms like pathogenic fungi. The objective of this project is to use T-DNA knockout lines of plant specific complex II subunits to characterise their functions at the physiological, proteomic and metabolomic levels and therefore to uncover the hidden role of these plant specific subunits .
3. Plant Mitochondrial Responses to Thermal Variation. With Dr Nicolas Taylor Fluctuations in temperature affect the metabolic processes of photosynthesis and respiration and can have dramatic implications for biosynthesis, cellular maintenance and growth. This can be seen in the different ways plants grow at low and high temperatures. In this project you will be preparing cold and hot stressed Arabidopsis plants and the isolating mitochondrial proteins from these plants. You will then analyse these proteins by a quantitative proteomic technique using cutting edge Q-ToF mass spectrometry. You will also analysis the mass spectrometry data to determine changes in respiratory components in response to thermal variation.
4. Distinguishing Wheat Cultivars Using Mass Spectrometry. With Dr Nicolas Taylor Wheat flour is highly valued for its taste and dough-making properties. Because these traits differ between cultivars, there is a need to readily identify cereal grains according to cultivar especially with the development of cultivars that have endpoint royalties, genetic modification or specific “built in attributes” that provide agronomic or processing advantages. Currently it is almost impossible to distinguish
32 between cultivars from a seed sample prior to sowing or of a grain sample after harvest. This project aims to determine novel biomarkers for commercial West Australian wheat varieties and develop these markers to allow the distinction between these varieties. It will develop high throughput selective reaction monitoring (SRM) assays to distinguish between wheat varieties. Your role in this project will be the preparation of protein extracts from a range of commercial West Australian wheat varieties, analyse these samples using Q-ToF mass spectrometry and collect proteins identifications for each cultivar. The differences in the proteins found for each cultivar will be then used as biomarkers for each variety and SRM development
5. Proteomics of Rice phosphate stress-induced changes With Dr Ralitza Alexova Phosphate is an essential element in a wide range of cellular components such as macromolecular structures like nucleic acids, membrane lipids and proteins as well as simple molecules like ATP and sugar phosphates. As most of the phosphate is found in soils and rock deposits, organisms including plants have developed strategies to extract this element and efficiently incorporate it into organic molecules. This project will use high-throughput proteomic techniques to build a more complete picture of global protein expression changes that occur in phosphate-stressed and phosphate-replete rice seedlings. The rice phosphate stress response will be further investigated by developing novel mass spectrometry-based assays for the simultaneous detection and quantitation of multiple proteins without the need for antibodies or chemical labelling.
6. What determines nitrogen use efficiency in crop plants? With Dr Clark Nelson As nitrogen is the most expensive component in fertilizer production this nutrient is arguably the most important component of plant metabolism to study. We are exploring the biochemical machinery involved in nitrogen-use efficiency. In collaborations we are conducting greenhouse trials as well as field trials in wheat, barley, and rice to study the effects of various nitrogen regimes on metabolism. We are applying a discovery-based approach using stable-isotope labelling and LC-MS techniques to monitor the steady state proteome, and alteration in the metabolome of these plants. In this project you would be involved in metabolomic and proteomic analysis of these cereal grains in an attempt to dissect the molecular mechanisms of nitrogen metabolism.
7. Glutaredoxins as agents of redox homeostasis in plants With Dr Elke Stroher Posttranslational modifications (PTMs) of proteins, like formation of disulfide bonds or addition of glutathione (glutathionylation), are important for fast adjustment of protein activities – they can even serve as on/off switch for protein activity. Members of the thioredoxin superfamily, such as thioredoxins (Trxs) and glutaredoxins (Grxs) are the most likely candidates for re-reduction of oxidatively modified proteins and are considered „key players‟ in signalling networks. This project would consider Grxs in energy metabolism. Genetically modified Arabidopsis thaliana plants with either less or more Grx would be analysed using novel biochemical and genetic technologies to uncover the impact of this protein family in energy metabolism.
8. Biology of honeybee defence and reproduction With Assoc/Prof Boris Baer and Dr Reza Zareie Honeybees contribute to our economy and food industry by producing honey and more importantly by pollinating some of our major crops and fruit trees. Honeybee populations, however, have been declining in recent years, creating growing concerns amongst many farmers and the public. To safeguard bees, research into the bee immune system as well as their reproduction has been intensified on a global scale. We have recently found that proteins within the honeybee seminal fluid significantly increase sperm‘s life span. The next logical step is to isolate and identify which proteins are responsible. In this project you will fractionate the seminal fluid and test which fractions are responsible to keep sperm viable. We will then use mass spectrometry and other proteomics techniques to identify the proteins behind this activity.
33 DR MATTHEW PIGGOTT ASSOCIATE PROFESSOR Room 3.29, Bayliss Building, Phone: 6488 3170 Email: [email protected]
Synthetic Organic Chemistry, Medicinal Chemistry and Chemical Biology Our expertise in organic and medicinal chemistry is applied to the design and synthesis of therapeutic drug candidates and small molecule probes to help investigate complex biological systems. We have several active collaborations with more biologically orientated scientists and opportunities for cross-disciplinary projects exist. The synthesis of biologically active natural products and novel aromatic molecules with potential applications in organic electronics, supramolecular chemistry, and as components of molecular machines are other areas of interest. PROJECTS
1. Drug discovery for human African Trypanosomiasis Human African Trypanosomiasis (HAT), also known as Sleeping Sickness, is caused by subspecies of the protozoan parasite Trypanosoma brucei, transmitted by the Tsetse fly. Current treatments for HAT are toxic, have difficult administration regimes and limited effectiveness, so there is a considerable need to find better drugs. A recent high-throughput screen of the WEHI (Walter and Eliza Hall Medical Institute, Melbourne) chemical library unearthed several promising hits, including the thiazole WEHI-1203394. Preliminary medicinal chemistry in the Piggott group has identified the benzamide analogue MRK8 as having improved potency against the parasite in vitro. This project will involve an expansion of this medicinal chemistry project in the search for sub-nanomolar IC50 inhibitors of T. brucei.
S O S O
N N N N H H F WEHI-1203394 MRK8 0.48 M 0.25 M
2. Chemical biology of phosphohistidines with Professor Paul Attwood Histidine kinases are a family of enzymes that catalyse the phosphorylation of the N1- or N3-imidazole nitrogen of specific histidine residues in proteins. Their better-known cousins, the serine, threonine and tyrosine kinases, have been implicated in the regulation of almost all eukaryotic cellular functions. In prokaryotes and lower eukaryotes, histidine kinases play critical roles in the response to environmental stimuli. It is assumed that histidine kinases and their substrates are also important components of mammalian cell-signalling pathways; for example, Histone H4-kinase is upregulated in foetal, regenerating, and cancerous liver cells. However, none of the mammalian histidine kinases are well characterised and their exact roles remain to be elucidated.
O O O H H H N protein histidine N protein N protein protein N kinase protein N protein N H H OR H 3 ATP 1 N HN 3 N N 1 N O N 1 P O 3 O P O O O histidine residue N1-phosphohistidine N3-phosphohistidine residue residue
O O
H3N H2N O O 1 N N N N 3 N N 1 O P O 3 P O O O O stable triazole analogue stable triazole analogue
34 The N-P bond in phosphohistidines is hydrolytically labile, which makes their identification, purification and study challenging. For this reason, there are no antibodies to the phosphohistidine epitope, which impedes progress in the field. We have recently devised syntheses of stable phosphonotriazole analogues of both isomers of phosphohistidine. This project will involve efforts to exploit these compounds to learn more about phosphohistidine biochemistry. Goals include the generation and characterisation of generic phosphohistidine antibodies, affinity chromatography to purify histidine kinases and phosphohistidine-recognizing proteins, and investigating the biological activity of the phosphonotriazoles as inhibitors of histidine kinases. This project requires a combination of synthetic chemistry and biochemistry skills, but can be tailored to suit the strengths and interests of the student.
3. Novel aromatic molecular architecture The classes of compounds shown on the right are challenging and fundamentally interesting synthetic targets, but also have potential applications in organic electronics, supramolecular chemistry and crystal engineering, and as components of molecular machines. Opportunities to examine the metal coordination chemistry and electronic applications (OFETs, OPVs and OLEDs) of these compounds (once synthesised) are possible through collaboration with Professor George Koutsantonis and Dr Gavin Collis (CSIRO Materials Science and Engineering Division, Melbourne).
4. A dihydropyrene-based organometallic molecular switch with Professor George Koutsantonis Dimethyldihydropyrenes are fascinating molecules with a planar 14- -electron periphery, making them aromatic. They are easily converted to their valence tautomers, cyclophanedienes, by irradiation with certain wavelengths of light. This project will involve the synthesis of a novel diethynyl-substituted dihydropyrene that will be used to prepare organometallic complexes. Switching between the valence tautomers is expected to drastically change the conductivity of the organic ligand, which in turn will affect properties such as colour, crystallinity and redox potential.
5. Total synthesis of biologically active naphtho[2,3- OH O OH O c]furan natural products We recently achieved an efficient synthesis of the natural O O O product monosporascone. This project will use this MeO MeO starting material for the synthesis of a number of related O OH monosporascone dehydroxyarthrinone secondary metabolites, including the antifungal agent (MAO inhibitor) (antifungal) dehydroxyarthrinone.
35 WINTHROP PROFESSOR COLIN RASTON Founding Director, Centre for Strategic Nano-Fabrication (Incorporating Toxicology) and Fledgling Centre for Green Chemistry and Molecular Discovery Room 3.09, Bayliss Building, Phone: 6488 3045 Email: [email protected] http://www.strategicnano.uwa.edu.au/
Organic Synthesis, Tissue Engineering, Nano-chemistry, Graphene, Desalination, Solar and Fuel Cell Technology, Chemical Sensors, Drug Delivery, Microfluidics platforms Current research covers: (i) Process intensification using spinning disc/rotating tube, electrospinning and narrow channel processing, fabrication of nano-materials, nano-chemistry, supramolecular chemistry, and crystal engineering, with applications in tissue engineering. (ii) Benign process technology – process intensification in organic synthesis (controlling chemical reactivity and selectivity), and drug delivery. (iii) Device technology – sensors, desalination, solar and fuel cells. Integration of these areas has led to novel chemistry and applications. Projects for 2012 deal with these areas which are directed towards the major challenges facing humanity in the 21st Century – in being able to gain access to complex functional molecules and materials for tackling energy, health and environmental issues. The projects are excellent training in a wide range of techniques, including green chemistry, engineering, nano-technology, inorganic and organic synthesis, X-ray diffraction, NMR, electron and atomic force microscopy, analytical techniques, other characterisation techniques. Brief details of some projects are given below. Other projects are also available depending on the interests of the researcher.
PROJECTS 1. Controlling chemical reactivity and regio-selectivity in organic synthesis using microfluidic platforms (MP) with Dr Keith Stubbs We have established the remarkable utility of MP 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 MP: (i) Plug flow conditions which control chemo-selectivity without the need for protection and de-protection. (ii) The ability to control the 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 MP 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 used MP to prepare new classes of pyridine compounds which have application in medicine, including diabetes inhibitors, and anti-cancer and anti-inflammatory activity, having identified the binding prowess of molecules to G-Quadruplex (insert). 2. ‘Bottom up’ materials synthesis using dynamic thin films in microfluidic platforms (MP) with Dr Paul Eggers and Dr Selvi Dev. We have recently established that spinning disc processing (SDP) and rotating tube processing (RTP) 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), and more. Recently we patented a variable angle RTP which allows control over residence time, and is a powerful MP for controlling the formation different shapes and nano-arrays. New materials have potential in synthesis (eg. Heck reaction), medicine (e.g. multi- functional imaging and drug release), and fuel cell and solar cell technology. The MP facility at UWA is one of a few such facilities in the world, which is further enhanced with a larger SDP and with accessible temperatures ≥ 600oC. Au, Pd and PIRS Nano- Pt work is supported by The Perth Mint, with other projects in collaboration with Materials industry, eg drug delivery (with iCeutica), and tissue engineering (with Chimere PIRS Pearls). Combining SDP with RTP, and narrow channel processors has exciting possibilities in building complex functional nano-materials under continuous flow.
36 3. Applications of phosphonated calixarenes in tissue engineering and as anti-cancer targets with Dr Keith Stubbs, Prof Fiona Wood, Prof Sarah Dunlop, and Prof Lee Yong Lim. Relatively unexplored phosphonated calixarenes have been prepared, 1, Fig 1, allowing access to derivatives with alkyl chains attached to the phenolic O-centres, and various functionalised moieties in the same position, including unsaturated chains (for photolytic cross linking) and groups with specific binding prowess (towards metal centres and organic molecules). Long alkyl chain (R) derivatives assemble into intertwining nano-fibres with the overall material having nano-textured features suitable for application in tissue engineering - for skin regeneration and neurotrauma. In addition, the calixarenes can act as surfactants in stabilising nano-particles (for imaging / magnetic field manipulation), and binding drug and enzyme molecules, and as anti-cancer agents themselves. 4. New device technology for sensors, desalination and energy with Dr Swaminathan Iyer, Dr Paul Eggers and Dr Ela Eroglu We recently developed drop casting devise technology for detecting hydrogen gas and discriminating organic molecules in the gas phase. This has exciting possibilities in sensor technology for detecting chemical warfare agents, fuel cells (including hydrogen gas), forensics (explosives and their breakdown products) and solar cell technology. The core of the device is based on (i) carbon nano-tubes (CNT) which can be decorated with selected nano-materials to tailor specific applications, and (ii) bare Pd nano- particles, which are accessible using our recent advances in continuous flow technology microfluidics. Also included is the development of new device technology for desalination. 5. Application of diatoms in device technology with Dr Swaminathan Iyer and Dr Ela Eroglu Single cell diatoms have well ordered silica skeletons with regularly spaced pores all the same size with diameter di- mensions down to 40 nm. The skeletons have exciting potential in nano-technology, ranging from medical (drug delivery) through to solar and fuel cell technology, paint additives, water purification, and photonic devices. We recently established that the pores can be decorated with nano-
particles of gold (inset), with a very narrow size distribution. The proposed research focuses on extending it to decorating with superparamagnetic nano-particles, associated with advances in the above microfluidic platforms, as well as with several materials (different nano- particles) depending on the application. High temperature treatment of the skeletons is also possible using the new spinning disc reactor, >600oC. This has potential in replacing silicon atoms with other metals, titanium and magnesium, under scalable continuous flow conditions. 6. Materials chemistry of carbon with Prof Hui Tong Chua New forms of carbon nano-materials, including composites of fullerenes with carbon nano- tubes and graphene (as a recently established form of carbon), will be investigated using self-assembly strategies and innovative approaches such as high temperature continuous flow and scalable spinning disc processing. A detailed understanding of the structures of these is important in developing their potential applications. These include separating different diameter carbon nano-tubes with different properties (semi-conducting versus conducting), quantum dots, controlling chemical reactivity and selectivity inside the tubes, chip devices for gas sensing (including chemical warfare agents), devices for solar energy conversion, and desalination. Membranes based on specific diameter carbon nano-tubes, in combination with other material, will be developed to gain access to material for only water passing across the membrane (desalination). 7. „Top-down‟ materials synthesis using microfluidic platforms (MP) We have recently established variable angle rotating tube (VARTP) MP can be used to exfoliate graphite to graphene in water, as a benign process, and similarly for boron nitride (BN). Varying the conditions can result in the graphene and boron nitride sheets being „rolled up‟ into scrolls, which have applications in chemical doping, hydrogen storage, battery technology, and nano-mechanical devices. The intense shearing in the VARTP is responsible for the „scrolling‟, and it has potential for exfoliating and/or scrolling other laminar structures including mica and clays. The same shearing is also effective in removing DNA from virus molecules, as an entry to drug delivery and vaccination strategies using the resulting intact capsids, and also in controlling protein folding.
37 Dr. SAM SAUNDERS Room 3.10, Bayliss 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.
PROJECTS
Atmospheric Science and Air Quality Issues
1. Investigating reactive indoor air chemistry
This project will work towards extending the field of knowledge on indoor air chemistry. Particularly in reference to the types of photochemical degradation reactions of organic compounds that occur in the indoor environment and how these compare with those outdoor, for which there is currently very little research. The project will focus on tailoring the Perth ambient outdoor model to the indoor study region and work on further developing a new model for the simulation of the reactive indoor air chemistry based on the master chemical mechanism (MCM) framework.
2. Assessment of the Pearl River Delta emissions, monitoring and meteorological data to develop a regional chemical mechanism for simulating observed ozone formation and ambient VOC measurements
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 field campaigns in the Pearl River Delta region of China. A base case model has been developed in 2009, and this requires further refinements for this geographic location. 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.
3. 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, and there would be the possibility of a study visit to the facility at Lucas Heights in NSW. 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.
Other similar project developments may also be available in 2012, depending on collaborating partners. And note projects will only be available for semester 2 in 2012.
38
All projects require an interest in gas phase chemical kinetics, reaction mechanisms and computational chemistry, and to develop reaction schemes for volatile organic compounds. Background on the technology and methodologies involved can be found on the following web site.
http://mcm.leeds.ac.uk/MCM/
Recent related publications
H. R. Cheng, H. Guo, S. M. Saunders, S. H. M. Lam, F. Jiang, X. M. Wang, T.J.Wang Photochemical ozone formation in the Pearl River Delta assessed by a photochemical trajectory model Atmospheric Environment 44, 4199 (2010)
H. R. Cheng, H. Guo, X. M. Wang, S. M. Saunders, S. H. M Lam, F. Jiang, T. J. Wang, S. C. Lee, K. F. Ho – On the relationship between ozone and its precursors in the Pearl River Delta: Application of an Observation- Based Model (OBM). Environmental Science and Pollution Research 17:547–560 (2010)
S. Ho Man Lam , H. Cheng , H. Guo, S.M. Saunders X. Wang, I.J. Simpson, A. Ding, T. Wang, D. R. Blake. A tailored Master Chemical Mechnism (MCM) model for the Pearl River Delta (PRD) region of South China. 19th International clean air and environment conference, CASANZ, Perth, Sept. (2009) ISBN: 978-0-9806045
R.G. Hynes, D.E. Angove, S.M. Saunders, V. Haverd, M. Azzi – Evaluation of two MCM v3.1 alkene chamber mechanisms using indoor environmental chamber data. Atmospheric Environment 39, p7251-7262 (2005)
S. Maisey, S.M. Saunders, N. West, P.J. Franklin. Modelling Seasonal influences on Reactive Indoor Air Pollution Chemistry for Residential Environs in the Southern Hemisphere. 19th International Congress on Modelling and Simulation, Perth, December (2011) http://mssanz.org.au/modsim2011
S.Maisey, P.Franklin, N.West, S.M. Saunders. Assessment of the indoor/outdoor relationship of VOCs in residential properties in Perth, Western Australia. 19th International clean air and environment conference, CASANZ, Perth, Sept. (2009) ISBN: 978-0-9806045-1-1
39 PROFESSOR IAN SMALL ARC Centre of Excellence in Plant Energy Biology Room 4.03, Bayliss building, Phone: 6488 4499 Email: [email protected]
Organelle Gene Expression Group
Our group is studying the RNA world within the energy organelles of plants – the mitochondria and chloroplasts. These organelles contain the genes that code for the most important and abundant proteins on Earth, those that drive photosynthesis, the basis for most biological productivity. The regulation of the expression of these genes is crucial, yet still only poorly understood mechanistically. Our aim is to understand how the biogenesis and function of chloroplasts and mitochondria are controlled through alterations in gene expression, with the goal of making discoveries relevant to optimal use of plants in agricultural and environmental applications. Gene regulation in plant organelles primarily occurs through changes in RNA processing, which makes these expression systems unique. Much of our research builds upon the discovery of the PPR protein family, novel sequence-specific RNA-binding proteins found in all eukaryotes, but particularly prevalent in plants (Schmitz- Linneweber and Small, Trends Plant Sci, 13, 663-670). The experiments mostly involve the model plant Arabidopsis thaliana to make use of the full range of international collections and databases on the ‗lab rat‘ of the plant kingdom. Prospective Honours students with a background in Molecular Biology, Biochemistry, Genetics or Computer Science are particularly encouraged to apply. The projects will benefit from all the expertise and facilities available within the ARC Centre of Excellence and will be at the forefront of research in this field.
PROJECTS
1. Analysing RNA processing with single-base precision by deep sequencing
RNA-seq is revolutionising the study of the transcriptome, by providing unprecedented detail into the nature of every transcript in the cell. Although many RNA-seq studies limit themselves to simple quantitative measures of overall gene expression, through careful design of experiments it is now possible to quantitatively analyse every step of RNA processing (transcription, end-processing, splicing, polynucleotide tailing, editing…) in a single set of samples, which would have been impossible only a year or two ago. The Centre‘s brand new HiSeq1000 is ideal for this approach, and so there are a plethora of new possibilities. Some examples are listed below.
Mapping transcription start sites and processing sites. Primary transcripts differ from processed transcripts by their 5‘ triphosphate; this leads to contrasts in the sensitivity of the RNAs to exonucleases and their ability to be ligated. We can therefore distinguish these RNAs and use RNA-seq to map every transcription start and processing site across the genome.
Analysing RNA editing. Plant organelle RNAs have their sequence changed after transcription, a mysterious process referred to as RNA editing. These sequence alterations are highly specific and involve a particular set of RNA-binding factors. Much remains to be discovered about RNA editing, and RNA-seq offers great promise for analysing the process in more detail than ever before.
Identifying target sites for RNA binding proteins. Proteins bound to RNA can leave ‗footprints‘ by protecting the RNA from exonuclease attack. By sequencing these footprints, we can discover where on the RNA the protein was bound in the cell. RNA-seq allows us to do this across the entire transcriptome, mapping the binding sites of multiple proteins at once. This information is crucial for working out how proteins recognise their target sites (see ‗Deciphering the code‘, below).
Quantifying translation by ribosome footprinting. One set of particularly interesting ‗footprints‘ belongs to ribosomes. By using antibiotics to block the ribosome in the course of translation, we can collect footprints that show where the ribosomes were on each transcript. This gives unique information on the rate of translation of each transcript, and insights into translational control mechanisms.
These projects will give students the chance to work with some of the most modern technology available anywhere. It would suit students with a keen interest in biochemistry and/or genetics, especially those wanting to learn computational data analysis approaches. 40 2. Deciphering the RNA-binding code of PPR proteins in collaboration with Prof. Charlie Bond, Biochemistry
Studies of PPR proteins indicate that these proteins play key roles in plant development, crop breeding and human disease. In plants, the exact function of less than 10% of the 400-500 PPR proteins has been discovered. Several PPR proteins have been shown to be essential for the expression of genes required for the construction and function of the major protein complexes involved in photosynthesis and respiration. They are thus vital during germination and early seedling development and some are absolutely required for autotrophic growth. The current bottleneck in the study of these important proteins is discovering the RNA targets of each one.
Statistical analysis of PPR protein sequences has suggested hypotheses proposing which amino acid residues are required to recognise specific RNA targets. These hypotheses need to be tested experimentally by electrophoretic mobility shift assays in which purified recombinant protein is incubated with labelled RNA target and run on acrylamide gels - protein binding to the RNA oligonucleotide retards migration, giving a simple semi- quantitative measure of binding affinity. It is simple in such assays to modify the sequence of the RNA, the protein, or both, to investigate the importance of particular amino acids or particular bases in the target. These experiments will lead to an understanding of which features in the RNA are being recognized by the protein, and (we hope) to the ability to predict binding sites and even construct custom-designed proteins to bind desired targets. The biotechnological possibilities are endless if we could achieve this. The project would suit students interested in molecular biology, biochemistry or genetics.
3. Solving the mysteries of RNA editing in plant organelles
RNA editing is a site-specific modification of RNA molecules, occurring by nucleotide insertion/deletion, nucleotide substitution or nucleotide modification. RNA editing alters the sequence of many different types of RNAs in many organisms including plants and humans and 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. Thirty PPR proteins have been found to be essential for the editing of specific sites in organelle transcripts of Arabidopsis thaliana. These RNA-binding proteins probably 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. We are approaching an understanding of how plants edit their organellar RNAs, with plausible hypotheses to test, but experimental proof is lacking. There is an outstanding opportunity to for an Honours project to provide the final data to confirm (or disprove!) the currently preferred model. The project will involve constructing plasmids to express modified proteins in transgenic plants and then assaying for RNA editing. The project will give a thorough grounding in many essential molecular biology techniques, including purification of DNA, RNA and proteins; cloning, PCR amplification, bacterial and plant transformation, analyses of gene expression.
Relevant references from the group
Schmitz-Linneweber, C., and Small, I. (2008) Pentatricopeptide repeat proteins: a socket set for organelle gene expression, Trends Plant Sci 13, 663-670. Hammani, K., Okuda, K., Tanz, S. K., Chateigner-Boutin, A. L., Shikanai, T., and Small, I. (2009) A study of new Arabidopsis chloroplast RNA editing mutants reveals general features of editing factors and their target sites, Plant Cell 21, 3686-3699. Chateigner-Boutin, A. L., and Small, I. (2010) Plant RNA editing, RNA Biol 7, 213-219. Fujii S, Bond CS, Small ID (2011) Selection patterns on restorer-like genes reveal a conflict between nuclear and mitochondrial genomes throughout angiosperm evolution. Proceedings of the National Academy of Sciences USA 108(4):1723-8
41 PROFESSOR STEVE SMITH ARC Centre of Excellence in Plant Energy Biology and Centre of Excellence for Plant Metabolomics Room 4.05, Bayliss Building, Phone: 6488 4403 Email: [email protected]
Discovering genes for plant growth
Arabidopsis thaliana provides the most powerful platform for modern genomics-based research in eukaryotes. It provides us with the opportunity to discover genes and mechanisms by which plants grow, how they produce the food that we eat, how they cope with environmental stresses (eg caused by climate change), and how they resist diseases. Research using Arabidopsis can provide training in a range of disciplines including genomics, genetics, cell biology, biochemistry, and new multi-disciplinary areas such as bioinformatics, systems biology and metabolomics. The following projects are proposed but there is room for flexibility and originality, and the emphasis can be matched to your particular skills or interests.
PROJECTS
1. Discovery of a new mechanism of growth control
Mutants that cannot break down their oil supply in the seed, fail to grow from seedlings into adult plants. But wait! We have discovered two ways to persuade them to grow: 1) give them some sugar (ie. carbon, energy), or 2) take away their supply of nitrogen (nitrate or ammonium)! This is very strange because it means that a seedling deprived of carbon and nitrogen grows better than one that is deprived only of carbon! Our hypothesis is that the seedlings „sense‟ and „measure‟ the relative amounts of carbon and nitrogen, and only grow when the ratio is suitable. The original „oil mutant‟ is starved of carbon but has nitrogen. So either give it some carbon or take away the nitrogen and it is happy.
Next, we have subjected our original „oil mutant‟ to mutagenesis and screened the mutated progeny for seedlings that can now grow the same as wildtype (ie with some nitrogen but without added sugar). There is one shown in the picture among all its siblings that have not learnt the trick. This mutant should still be unable to breakdown its oil supply, but is altered in its ability to „sense‟ or „measure‟ the amounts of carbon and nitrogen. By identifying the new genes which are mutated in such mutants we expect to discover molecular components of the sensing or measuring pathway. In this way we should identify new mechanisms of growth control in plants.
You will be given one or more mutants to study, with the aim of discovering the mutated gene(s) and how it works. This will be an exciting journey of discovery, taking us in an unknown direction. The project will likely involve several techniques such as genetic analysis, molecular biology, metabolomics and cell biology, and offers the potential to make important discoveries.
References
Germain V, Rylott EL, Larson TR, Sherson SM, Bechtold N, Carde JP, Bryce JH, Graham IA, Smith SM. (2001) Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid beta-oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings. Plant J. 28:1-12.
Martin T, Oswald O, Graham IA. (2002) Arabidopsis seedling growth, storage lipid mobilization, and photosynthetic gene expression are regulated by carbon:nitrogen availability. Plant Physiol. 128:472-81.
42 2. Molecular mechanism by which karrikins from smoke promote seed germination
Karrikins are compounds discovered in smoke from bushfires, which promote seed germination. They were discovered at UWA in a collaborative effort between Kings Park botanists and UWA chemists. The original compound (structure 1, KAR1) is a butenolide (3-methyl-2H-furo[2,3-c]pyran-2-one) but a few other closely related compounds have also been found. We call them karrikins from „karrik‟, the first recorded Noongar word for „smoke‟. It has been established that KAR1 can also promote germination and seedling development 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. Karrikins have some structural similarity to a family of plant hormones called strigolactones, which can also promote seed germination in some species, so they might act through a similar molecular mechanism.
Figure. Mutants that respond differently to karrikins.
A. Karrikin insensitive (kai) mutant (top row) does not germinate. The „faker‟ (bottom row) is germinating on KAR1 just like wildtype.
B. A genetic screen using a transgenic line which is totally dependent on KAR1 for germination.
C. Wildtype is inhibited by growth on very high karrikin whereas a mutant grows normally (okr „overdose on karrikin resistant‟).
The goal of our research is to discover the molecular mode of action of karrikins in promoting seed germination and seedling development. We are using Arabidopsis for this, both by studying existing mutants (eg. in seed germination or hormone signaling) and by isolating new mutants. We have used transcript profiling with microarray technology to identify genes that respond to KAR1. This provides insights into karrikin action as well as a set of genes that can be targeted for mutation analysis. We have also carried out random mutagenesis of wildtype Arabidopsis and isolated novel mutants that do not respond correctly to karrikins (see Figure). The aim now is to discover the genes required for karrikin action and hence to discover its molecular mode of action. The research will involve a range of techniques in molecular biology and biochemistry, and also close collaboration with our chemistry friends.
References
Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD. (2004). A compound from smoke that promotes seed germination. Science. 305:977.
Nelson DC, Riseborough JA, Flematti GR, Stevens J, Ghisalberti EL, Dixon KW, Smith SM. (2009) Karrikins discovered in smoke trigger Arabidopsis seed germination by a mechanism requiring gibberellic acid synthesis and light. Plant Physiol. 149: 863-73.
Chiwocha SDS, Dixon KW, Flematti GR, Ghisalberti EL, Merritt DJ, Nelson DC, Riseborough JAM, Smith SM, Stevens JC. (2009) Karrikins: A new family of plant growth regulators in smoke. Plant Science 177: 252-256.
Nelson DC, Flematti GR, Riseborough JA, Ghisalberti EL, Dixon KW, Smith SM (2010) Karrikins enhance light responses during germination and seedling development in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA, 107, 7095-7100.
43 PROFESSOR MARK SPACKMAN Room 4.11, Bayliss 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 and Dr Mike Turner
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, including the program CrystalExplorer, developed in collaboration with Dylan Jayatilaka's group, can be found at the web site associated with this project: http://ra.bcs.uwa.edu.au/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 important interactions.
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2. Charge density analysis of fundamental host-guest supramolecular systems several projects, with A/Prof George Koutsantonis and Dr Alex Sobolev
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 A/Prof Dylan Jayatilaka and Dr Mike Turner
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, or at least a vehicle for mapping properties such as the magnitude of the LUMO orbitals, etc. This project will build on the results of Maram Susli, a 2009 Honours student, to further explore the correlation of void locations, volumes and orbital properties with experimental information on various kinds of reactivity involving molecules in crystals.
Another aspect of this project could focus on a more detailed investigation of void space (i.e. empty space) in reactive solids such as metal-organic frameworks (MOFs) and zeolites. An example of the void space in Linde type A zeolite is shown in the figure on the right. This project would exploit the recent implementation of tools in CrystalExplorer for visualising and mapping void surfaces and volumes.
45 DR SCOTT STEWART Room 3:30, Bayliss building, Phone: 6488 3180, Email: [email protected]
Research Overview
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. Several natural products Arboflorine (1),1 Ajamalicine, Pumiliotoxin B (2) ,2 Flinderole A,3 Epoxiquinol and BE-26554B (3) have been targeted within this group because of their interesting molecular architecture and biological activity. Related to this field, methodological studies involving the improvement various reactions including, Suzuki, Buchwald-Hartwig and intramolecular Heck reactions through the modification of nickel(0) and palladium(0) catalytic conditions are currently being explored. Research in the discovery of novel domino transformations (the execution of two or more bond-forming transformations under identical reaction conditions)4 mediated by palladium are routinely carried out within the group.1b,5 Medicinal chemistry interests include the synthesis of libraries of new thalidomide analogues for the inhibition of tumour necrosis factor (TNF) expression as well as determining the molecular mode of action.6,7
1. The Synthesis of Ngouniensine and Arbiflorine
The domino Tsuji-Trost/Heck reaction has been used devised within our group and used for the construction of the azepino[4,5-b]indole ring system 5 and 3-benzazepines. In this process the construction of the seven membered C-ring can be achieved in a single step. This project will involve using this domino reaction as a key step for the production of the natural product Ngouniensine (6). The natural product alkaloid 6 isolated from Strychnos ngouniensis has reported activity against several P. falciparum strains, a protozoan parasite responsible for the cause of malaria in humans. Although the IC50 value of 6 is moderate the epimer at C20 is more potent suggesting that analogues generated at C20 should be investigated.
Although the domino Tsuji-Trost/Heck reaction is sufficient for the construction of the azepino[4,5-b]indole ring system 5, the exocyclic olefin within this ring system is not amenable to use in the total synthesis of Arboflorine.1 The second part of this project is to investigate reactions, namely reaction between tryptamine and methyl chloropyruvate, in the production of unsaturated azepino[4,5-b]indoles and their use as precursors for Arboflorine.
2. The Synthesis of Amphibian Alkaloids through the Tsuji-Trost Reaction
Several classes of compounds can be found in the skin of Amphibians with a wide range of biological activity. One such class of compound includes the pumiliotoxins of the general indolizidine structre 8 where R is an alkyl side chain. Several pumiliotoxins have cardiotonic and myotonic activity through binding to unique binding sites
46 on the voltage dependent ion channel. This project will focus on the synthesis of the indolizidine ring system staring with proline 2 which includes the correct stereochemsistry at the -carbon. Previous group work has generated the indolizidine core through a key intramolecular Tsuji-Trost cross coupling reaction.8 Such palladium mediated cross coupling reactions have been used regularly in complex natural product synthesis.
3. Enantioselective and Diastereoselective Domino Reactions (with Dr F. Pfeffer)
In 2010, we reported a new domino reaction for the production of tetrahydro-β-carbolines 10 which involves an initial Heck reaction followed by an aza-Michael addition.5a This process, also amenable to many acrylate based reagents resulting in variation of the terminal functional group, is considered a process comparable to the Pictet- Spengler reaction. In this domino reaction, however the stereogenic centre at C* is not formed in a controlled manner. The aim of this project is to use a chiral pool variation of the toluenesulfonyl (Ts) protecting group at N10 to create a diastereoselective domino reaction firstly generating epimers at C*. A second part of this project will involve the investigation of an enantioselective domino process by altering the using enantiopure phosphines or quaternary ammonium salts. Once the generation of the stereocentre at C* is confirmed then an application in the synthesis of biologically active natural products such as Elaeocarpidine 11 is to be attempted.
4. The Preparation of New Reagents and Catalytic Systems in Organic synthesis Organocatalysis is a rapidly developing field in synthetic organic chemistry. In a simple organic reaction such as the Michael reaction new stereogenic centres can be generated asymmetrically in a single step in high yields and ee. The aim of this project is to create new organocatalytics reagents and reactions. In particular an asymmetric variant of Mander‘s reagent 13 and/or applied transformations will be investigated. This reagent has been effectively used for -keto ester formation in fine chemical and natural product syntheses.
References
1.a) Lim, K-H.; Kam, T-S. Org. Lett., 2006, 8, 1733; b) S. G. Stewart, C. H. Heath, E. L. Ghisalberti, Eur. J. Org. Chem, 2009, 1934. 2. J. Daly, T. Spande and H. Garraffo, J. Nat. Prod., 2005, 68, 1556-1575. 3. Fernandez, L. S., Buchanan, M. S.,et al., Org Lett 2009, 11 (2), 329-332 4. L. F. Tietze.; G. Brasche.; K. M. Gericke, Domino Reactions in Organic Synthesis, Wiley-VCH 2006. 5a) D. L. Priebbenow, L. C. Henderson, F. M. Pfeffer, S. G. Stewart, J. Org. Chem. 2010, 75, 1787; c) S. G. Stewart, E. L. Ghisalberti, B. W. Skelton, C. H. Heath, Org. Biomol. Chem, 2010, 8, 3563. 6.a) S. G. Stewart, L. A. Ho, M. E. Polomska, A. T. Percival, G. C. T. Yeoh, ChemMedChem, 2009, 4, 1657; b) S. G. Stewart, M. E. Polomska, R. W. Lim, Tetrahedron Lett. 2007, 48, 2241. 7.a) S. G. Stewart.; D. Spagnolo, M. E. Polomska, M. Sin.; M. Karimi.; L. J. Abraham, Bioorg. Med. Chem Letts. 2007, 17, 5819; b) S. G. Stewart.; C. Braun.; S-L. Ng.; M. E. Polomska.; M. Karimi.; L. J. Abraham, Bioorg. Med. Chem. 2010, 18, 650; c) S. G. Stewart, C. J. Braun, M. E. Polomska, M. Karimi, L. J. Abraham, K. A. Stubbs, Org. Biomol. Chem., 2010, 8, 4059; 8. R. E. Martin, M. E. Polomska, L. T. Byrne, S. G. Stewart Tetrahedron Lett. 2011, 4878.
47 DR KEITH STUBBS Room 4.18/Lab 4.22, Bayliss Building, Phone: 6488 2725 Email: [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.
The laboratory is a highly collaborative environment where researchers work to solve problems in chemical glycobiology. Depending on the project, you will have the opportunity to gain exposure to methods ranging from carbohydrate synthesis, protein expression, molecular biology and enzymology. The laboratory enjoys extensive collaborations and researchers are provided with mentoring so as to aid their scientific development and enable them to realize their professional goals. All the summaries of projects outlined below will initially involve the synthesis of compounds and once prepared, investigation(s) using biochemical and microbiological assays will be conducted.
If you are excited about interdisciplinary science, enjoy experimental research in chemistry or biochemistry and are interested in joining the laboratory feel free to contact Dr. Stubbs by email or come and chat with me about these and any other projects and research interests you are interested in.
PROJECTS
1. Development of new scaffolds to inhibit carbohydrate-processing enzymes.
The enzymes that regulate the structures of glycans are extremely important and have been implicated in a wide variety of diseases and thus are targets for therapeutics. For example, carbohydrate- processing enzymes are important for bacterial growth and invasion of our cells. Project(s) described here will be to design and synthesize new inhibitor scaffolds that can be used to investigate the role these carbohydrate-processing enzymes play in human disease. The prepared compounds will be tested for their potency against the human enzymes in question and they will also be tested in vitro to determine their effectiveness at the cellular level. As well, through strong international collaborations, these compounds will also be co- crystallized with proteins of interest (example on left). This research is funded by the Australian Research Council (ARC).
Students with interests in synthetic chemistry or both synthetic chemistry and biochemistry are very well suited for this project.
2. Investigations into the glycobiology of Helicobacter pylori.
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. Increased insight into these interactions would be of use in the design of new therapeutics to treat H. pylori infections.
48 In collaboration with Professor Alice Vrielink, Associate Professor Mohammed Benghezal and Professor Barry Marshall, projects under this heading will investigate, through chemical synthesis and molecular biology, what roles carbohydrates and larger glycan structures play in the pathogenesis of H. pylori and to use this information in the design of new therapeutics. This research is funded by the National Health and Medical Research Council (NHMRC).
Students with interests in synthetic chemistry or both synthetic chemistry and microbiology are very well suited for this project.
3. Investigations into the glycobiology of Neisseria sp.
Neisseria sp. are Gram-negative bacteria that colonize the mucosal surfaces of many animals. Of interest are the two pathogens Neisseria meningitidis, which causes bacterial meningitis, and Neisseria gonorrhoeae, which causes gonorrhoea. These two pathogens have developed unique mechanisms of invading host cells many of which involve carbohydrates and their associated enzymes. Insight into these interactions would be of use in the design of new therapeutics to treat Neisseria sp. infections. In collaboration with Professor Charlene Kahler and Winthrop Professor Alice Vrielink projects under this heading will investigate, through chemical synthesis and molecular biology, what roles carbohydrates and larger glycan structures play in the pathogenesis of Neisseria sp. This research is funded by the National Health and Medical Research Council (NHMRC).
Students with interests in synthetic chemistry are very well suited for this project.
49 Dr. K. Swaminathan Iyer ARC Australian Research Fellow Deputy Director, Centre for Strategic Nano-Fabrication, School of Biomedical Biomolecular and Chemical Sciences Phone: 6488 4470, Bayliss, Room: 4.41. Email: [email protected]
BioNanoChemistry: Interdisciplinary research encompassing Chemistry, Physics and Biology. PROJECTS
1. Magnetically responsive polymeric scaffolds for wound healing with Prof. Fiona Wood, Prof. Tim St. Pierre, Dr. Rob Woodward and Dr. Mark Fear. Despite recent therapeutic advances, the mortality and morbidity from major burns remains high. Consequently, there is a pressing need to develop economical, efficient and widely-available therapeutic approaches to enhance the rate of wound re-epithelialization and restoration of the protective epithelial barrier. Skin, the largest organ of the human body, provides an essential protective barrier and serves several homeostatic/sensory functions vital to health and its functional recovery post burn injury remains the ultimate goal of wound healing research. Polymer nanoscaffolds have been extensively utilized in the design of tissue engineered constructs in delivering several growth factors for the correction of a wide range of medical conditions. A variety of polymeric scaffolds have been used to deliver growth factors, including natural or synthetic polymers that generally form either hydrogels or solid polymer scaffolds. However extended release of proteins is not easily achieved due to the release kinetics of growth factor through hydrogels being mainly diffusion controlled via the numerous aqueous channels within the hydrogels. Immobilization of the growth factor within the biodegradable hydrogel seems to improve the release kinetics, with release being controlled by the degradation of the hydrogel. Here the release kinetics are slow and progressive necrosis sets in post injury. A novel modulated delivery system would indeed be ideal, allowing the release profiles of payloads to be manipulated to match the physiological requirements of the patient. The project will explore the utility of magneto-responsive scaffolds for on-demand delivery of payloads. 2. Exploring nanoparticles as biomarkers in evolutionary biology with Dr. Boris Baer. This project is collaboration with Collaborative Initiative for Bee Research (CIBER: http://www.ciber.science.uwa.edu.au/) and the BioNano Research Initiative in Chemistry. Researchers in CIBER have been investigating honeybee reproduction, which is quite spectacular, as queens only mate at the beginning of their life, during one or very few mating flights. Following this they are able to store millions of sperm for years, and use them in very economic ways to fertilize millions of eggs. Currently there is very little information how social insect queens are able to keep sperm alive for years, how active sperm remain during storage, and how queens are able to economize their use of sperm during egg fertilization. This research project explores the possibility of developing nanoparticles as markers in an attempt to unravel this phenomenon by tagging sperms. The project will involve synthesis of magnetic nanoparticles and semiconductor quantum dots as markers, exposure to the bee research team in CIBER and training at the nanotechnology and biology interface. 3. Antibody conjugation of nanoparticles for cell specific drug delivery in the central nervous systems with Dr. Lindy Fitzgerald and Prof. Sarah Dunlop. In the nervous system, all sensations and behaviors are encoded in dynamic patterns of activity in cellular networks. Through a sequence of neural networks, sensory information is transmitted to higher, associative brain areas. Following integration in these areas, specific activity patterns are eventually formed in the relevant motoneuron pools to produce adequate behavior. In this chain of events, key processing steps are thought to
50 occur on the level of local microcircuits that contain on the order of 1,000–10,000 cells. These local circuits form highly connected three-dimensional networks. Calcium ions (Ca2+) have been a favorite target in molecular imaging studies because of the important role of calcium as a second messenger in cellular signaling pathways. Neurotrauma, such as traumatic brain or spinal cord injury, encompasses both acute damage induced by the primary injury and chronic progressive secondary degeneration of intact, but highly vulnerable, tissue, results in a drastic change in the cellular signalling pathways. Reactive oxygen and nitrogen species (ROS and RNS) are implicated to play a vital role in this, as their production is reported to exceed a cell‘s antioxidant capacity following injury. After neurotrauma, calcium fluxes are uncontrolled and spread to intact but vulnerable tissue. Triggers for uncontrolled calcium fluxes are varied but include ROS/RNS which activate Ca2+ channels and repress Ca2+ pumps. Astrocytes in particular are chiefly responsible for the brain‘s antioxidant defense whereby they play a pivotal role in protecting neurons and oligodendrocytes from oxidative stress. The project explores developing cell specific targeting of astrocytes to develop drug delivery vehicles as a mode to combat secondary degeneration following neurotrauma. This site specific targeting is projected to have high efficacy regulation in the calcium flux. 4. Developing a nanoscale therapy to alleviate oxidative stress in placental-related diseases of pregnancy with Prof. Jeff Keelan and Prof. Brendan Waddell. Pregnancy is a state of oxidative stress arising from increased placental mitochondrial activity and production of reactive oxygen species (ROS), mainly superoxide anion. The placenta also produces other ROS including nitric oxide, carbon monoxide, and peroxynitrite which have pronounced effects on placental function including trophoblast Diagram of a gestational sac proliferation and differentiation and vascular reactivity. Excessive at the end of the 2nd month production of ROS may occur at certain windows in placental showing the myometrium (M), development and in pathologic pregnancies, overpowering antioxidant the decidua (D), the placenta defences with deleterious outcome. For example: miscarriage and pre- (P), the exo-coelomic cavity eclampsia are the most common disorders of human pregnancy. There is (ECC), the amniotic cavity mounting evidence that oxidative stress or an imbalance in the (AC) and the secondary yolk oxidant/antioxidant activity in utero–placental tissues plays a pivotal role sac (SYS). Ref: Human in the development of placental-related diseases. This project explores Reproduction Update, the application of magnetic nanoparticles as antixodant delivery agents in Volume12, Issue6, Pp. 747- placenta via a systematic approach. 755. 3+ 5. Colloidal Upconverting NaYF4 Nanocrystals Doped with Er , Yb3+ and Tm3+ for biomedical imaging and diagnostics with Prof D. D. Sampson. Upconversion nanocrystals are luminescent nanomaterials that convert a near-infrared excitation into a visible emission through lanthanide doping. Compared to organic fluorophores and semiconducting nanocrystals, upconversion nanocrystals offer high photochemical stability, sharp emission bandwidths, and large anti-Stokes shifts (up to 500 nm) that separate discrete emission peaks from the infrared excitation. Along with the remarkable light penetration depth and the absence of autofluorescence in biological specimens under infrared excitation, these upconversion nanocrystals are ideal for use as luminescent probes in biological labelling and imaging technology. Organic dyes and semiconductor quantum dots that emit at higher energies via two-photon absorption processes require expensive high energy pulse lasers. Due to the relative high efficiency of the upconversion process in lanthanide-doped materials, inexpensive 980 nm NIR diode lasers may be employed as the excitation source. The realization of efficient NIR to visible upconverting nanocrystals can be exploited to develop novel dual modality drug carriers. The project will explore the synthesis and properties of doped NaYF4 nanosystems and their utility as biomarkers in vitro. See for reference: Analyst, 2010, 135, 1839-1854.
51 ASSOCIATE6B PROFESSOR ROBERT TUCKEY
Room29B 3.71, Bayliss Building, Phone: 6488 3040,
30B Email: [email protected] U
Molecular Steroidogenesis Group
Current research involves the metabolism of vitamins D2 and D3 by cytochrome P450scc, and the activation and inactivation of vitamin D by other mitochondrial-type cytochromes P450 including CYP27A1, CYP27B1 and CYP24.
Mitochondrial Cytochrome P450 Enzymes There are 7 mitochondrial cytochrome P450 enzymes encoded by the human genome. They catalyse hydroxylation reactions involved in steroid hormone synthesis and the metabolism of vitamin D. The mitochondrial P450s receive electrons to support their hydroxylation reactions from NADPH via adrenodoxin reductase and adrenodoxin. These P450s appear to be anchored to the mitochondrial membrane primarily by a region involving the F-G loop and bind substrate from the hydrophobic domain of the inner-mitochondrial membrane (Figure 1). Cytochrome P450scc (CYP11A1) catalyses the conversion of cholesterol to pregnenolone, termed the cholesterol side-chain cleavage reaction. This reaction involves three hydroxylations, all of which occur at a single active site on cytochrome P450scc. Pregnenolone serves as the precursor of all the steroid hormones such as corticosteroids, androgens and estrogens.
Figure 1. Model of the interaction of cytochrome P450scc with the inner- mitochondrial membrane
In collaboration with Professor Andrzej Slominski at the University of Tennessee, Memphis, we tested the ability of P450scc to metabolize vitamins D2 and D3. 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 did not cleave the side chain of vitamin D3 but hydroxylated the side chain producing 20-hydroxyvitamin D3, 20,23-dihydroxyvitamin D3 and 17,20,23-trihydroxyvitamin D3. P450scc converted vitamin D2 to 20-hydroxyvitamin D2 and 17,20-dihydroxvitamin D2, again with no cleavage of the side chain occurring.
We have carried out biological testing of several of the novel P450scc-derived hydroxyvitamin D3 products in collaboration with Professor Slominski in Memphis. 20-Hydroxyvitamin D3 has been found to be as effective as the hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3, in inhibiting cell proliferation and promoting differentiation of a variety of cells including skin, leukaemia, breast and prostate carcinomas. Importantly, it does not raise serum calcium levels in rats and consequently lacks the toxic side effect of hypercalcaemia caused by high doses of 1,25-dihydroxyvitamin D3. 20-Hydroxyvitamin D3 thus shows promise as a therapeutic agent for the treatment of hyper-proliferative disorders including cancer.
Other mitochondrial P450s we are studying are CYP27A1, CYP27B1 and CYP24, all of which can act on vitamin D. CYP27A1 catalyses the first step in the activation of vitamin D which is hydroxylation in the 25- position. CYP27B1 catalyses the second step in the activation of vitamin D, 1α-hydroxylation of 25- hydroxyvitamin D3 to produce 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D. 1,25- Dihydroxyvitamin D3 not only 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 causes its inactivation. We are
52 expressing CYP27A1, CYP27B1 and CYP24 in E. coli, and studying their catalytic properties in a reconstituted system that utilizes phospholipid vesicles to mimic the inner-mitochondrial membrane. We are also using these enzymes to hydroxylate the P450scc-derived vitamin D analogues such as 20-hydroxyvitamin D3 to see if 1-, 24- or 25-hydroxylation of these compounds enhances their potency without returning their calcaemic activity, with the aim of further improving their therapeutic potential. Of the 7 mitochondrial P450s in humans, only one remains whose function is unknown, CYP27C1. Since this is in the same family as CYP27A1 and CYP27B1, both of which can act on vitamin D derivatives, is likely that CYP27C1 acts on a vitamin D analogue or a steroid with similar structure. Mitochondrial P450s are also found in invertebrates but to date are poorly characterized. There are three mitochondrial P450s in insects that catalyse steroid hydroxylations, similar to what occurs in mammals for the synthesis of active steroids from cholesterol. In the case of insects the final active hormone is 20-hydroxyecdysone, also known as insect moulting hormone. The final activating step in the synthesis of ecdysteroids is the addition of the 20-hydroxyl group by mitochondrial CYP314A1.
PROJECTS
1. Can human CYP27C1 metabolize vitamin D or steroids?
CYP27C1 is the only mitochondrial P450 in humans whose function is yet to be determined. It has been expressed in E. coli and purified but no substrate for this enzyme has yet been identified. The CYP27C1 is expressed in a number of tissues including liver and kidney, known sites of vitamin D activation. The other two members of the CYP27 family, CYP27A1 and CYP27B1, are known to metabolise vitamin D hydroxylating it in the 25- and 1- positions, respectively. It would seem most likely that CYP27C1 will also act on a vitamin D derivative, but the only ones tested to date are vitamin D, 25-hydroxyvitamin D and 1-hydroxyvitamin D, where no metabolism was found. The aim of this project is to express human CYP27C1 in E. coli, purify the expressed enzyme and examine its ability to hydroxylate a large range of hydroxyvitamin D derivatives available in my laboratory. Some steroids which are structurally similar to vitamin D3 will also be tested. HPLC will be used to detect product formation and if products are detected reactions will be scaled up to permit sufficient product to be made to enable their identification by mass spectrometry and NMR (to be carried out by collaborators). Subsequent studies will involving testing the biological activity of products.
2. Expression and characterization of the 20-hydroxylase, CYP314A1
CYP314A1 is a mitochondrial P450 that catalyses the 20-hydroxylation of ecdysone producing 20- hydroxyecdysone, also known as insect moulting hormone. It is encoded by the gene known as shade, a member of the Halloween family. This steroid hormone controls moulting of immature insects and differentiation into pupae and adult. Thus CYP314A1 is a potential target enzyme for specific inhibitors to control insect pests. CYP314A1 has some properties very similar to P450scc including catalysing 20- hydroxylation of sterols, but the enzyme has not been purified for full characterization. The aim of this project is to express CYP314A1 in E. coli, purify the expressed enzyme and study its ability to 20-hydroxylate ecdysone. A range of other potential substrates including cholesterol and vitamin D will also be tested since their 20-hydroxy products have anti-cancer properties. HPLC will be used to measure product formation.
53 DR DANIELA ULGIATI Room 3.03, Bayliss Building, Phone: 6488 4423 Email: [email protected] UH
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 identification 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.
54 3. Understanding the Role of Notch Signalling and associated Transcription Factors in Lineage Commitment.
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.
4. 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 have analysed the promoter region of C4 in reporter gene assays, using deletion and mutant reporter constructs and in 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 two-part regulatory element within the promoter which appears critical for C4 expression in hepatic cells. The 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 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 unknown, but our hypothesis is that interaction with the –140 region prevents interaction of the upstream element 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 searching for such an agent we found an activity in serum 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.
55 WINTHROP PROFESSOR ALICE VRIELINK
31BRoom 4.31, Bayliss Building, Phone: 6488 3162
32BEmail: [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 bacterial immune evasion mechanisms hence present interesting opportunities for the development of vaccines against the organism. A large number of enzymes are involved in LOS biosynthesis including the additions of carbohydrate moieties to the endotoxin molecule and enzymes involved in modification of LOS to provide it with the molecular features that facilitate recognition by the host organism. A greater 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 Professor Charlene Kahler of the Department of Microbiology and Dr. Keith Stubbs of the Department of Chemistry at UWA we have begun a study to establish the structural and functional relationships of these enzymes. Towards this aim, overexpression systems for these enzymes must be developed in order to produce sufficient amounts of protein for structural and kinetic studies.
This project will involve cloning, protein expression, purification, crystallization and structure determination using crystallographic techniques. Kinetic assays for the enzyme will be established in collaboration with Dr. Stubbs and biophysical methods will be undertaken to characterize the protein. This project will be correlated with functional studies carried out by Dr. Kahler and coworkers.
2. Studies of Snake Venom L-amino acid oxidase L-amino acid oxidase is a flavoenzyme catalyzing the stereospecific oxidative deamination of L-amino acids to give the corresponding -keto acids. It is found in high concentrations in a number of different snake venoms, constituting up to 30% of the total venom proteins and is thought to contribute to the toxicity of the venom. The enzyme has also been shown to possess antibacterial, anti-HIV and antineoplastic or apoptosis-inducing activity. The general mechanism of cytotoxicity by the enzyme is thought to be due to the generation of H2O2. Indeed, studies have shown that the addition of catalase, a scavenger of H2O2, protects the cell from the toxic effects of the enzyme. However other factors may also contribute to the apoptotic activity including the glycosylation moiety of the enzyme and an increase in the presence of Structure of the dimeric L-amino acid substrate. The structure of the enzyme in the presence of a oxidase from the snake venom of Malayan substrate and an inhibitor have been determined in our pit viper. The glycosylations are also laboratory and reveal a channel that may act as the peroxide exit indicated. route from the active site. The channel exits near to the location of one of the two glycosylation sites on the protein surface. Further characterization of this enzyme and its mechanism of apoptosis will require production of wild type enzyme as well as specific mutants, which affect catalytic activity. The protein is not able to be expressed in a functional form in a bacterial expression system due to the presence of extensive glycosylation. Thus it must be
56 expressed in a eukaryotic system. Towards this aim a yeast expression system for the enzyme has been established and provides a basis for production of both wild type and mutant forms of the protein for further biological studies.
In this project you will use the yeast expression system to produce functional protein. Site directed mutagenesis, kinetic analysis, crystallographic studies and apoptosis studies will be undertaken to establish the roles of discrete residues in oxidation chemistry and its relationship to apoptosis.
3. Structural Studies of an Engineered Cephalosporin Acylase. Cephalosporin C was originally isolated from the microorganism Cephalosporium sp in 1945 as the first -lactam fused to a six-membered ring. Thereafter a number of semi-synthetic analogues were developed from the initial lead compound with fourth generation cephalosporins being used currently. Many of the semi-synthetic analogues of cephalosporin C (CephC) are synthesized starting with the conversion of CephC to 7-aminocephalosporanic acid (7-ACA). This conversion however involves a series of expensive chemical steps that require highly reactive chemicals resulting in chemical wastes, which must be safely disposed of. Hence altering the production method of semi-synthetic cephalosporins to overcome these disadvantages is of great interest to the pharmaceutical industry. An enzymatic method to produce semi-synthetic cephalosporins from 7-ACA using D-amino acid oxidase and glutaryl-7- Crystal structure of the H296S- amino cephalosporanic acid acylase is also possible and, although it H309S double mutant of gl-7- eliminates the problems associated with toxic waste products, it is ACA acylase. expensive and inefficient for industrial production. Therefore a one-step conversion of CephC to 7-ACA is highly desirable. For this conversion, utilization of glutaryl-7-amino cephalosporanic acid acylase (gl-7-ACA acylase) and altering its substrate specificity and activity for the substrate CephC rather than glutaryl-7-amino cephalosporanic acid (gl-7-ACA) offers an ideal solution. Towards these aims we are working with Prof Pollegioni (Universita degli Studi dell‟Insubria, Italy) to design and characterize mutants of gl-7-ACA acylase with switched substrate specificity. A double mutant of gl-7-ACA acylase (H296S-H309S) which exhibits 22 fold enhanced specificity and reactivity of CephC over the natural substrate gl-7-ACA has already been designed. Our laboratory has determined the crystal structure of this mutant and the wild type enzyme in order to establish the structural consequences of the mutation that facilitate altered specificity.
The project involves further engineering of the active site through a mutagenesis approach to identify other mutations that could enhance substrate specificity. The designed mutants will be prepared by site directed mutagenesis, protein expressed, purified and crystallized. Substrate complexes of crystals will be prepared and structures determined by X-ray crystallography methods.
57 WINTHROP PROFESSOR R JOHN WATLING Forensic Chemistry Forensic Science Building, Phone: 6488 4488 Email: [email protected]
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 (<100 m 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 <10 m 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.
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The science of “Spectral Fingerprinting” is on 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.
PROJECTS
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.
59 W/PROFESSOR JIM WHELAN ARC Centre of Excellence in Plant Energy Biology Room 4.73, Bayliss Building, Phone: 6488 1749 Email: [email protected]
Molecular Genetics and Genomics
We use a variety of post-genomic approaches to carry out discovery based investigations concerning the development and stress tolerance of plant model organisms, primarily rice and Arabidopsis. The main projects running in the laboratory focus on the biogenesis and function of plant mitochondria, and on the role of signaling events involved in plant phosphate uptake. Both mitochondria and phosphate metabolism are key players in energy production in plants, making our investigations highly relevant for fundamental and applied research. The students will be trained in a wide variety of molecular and cellular biology techniques, ranging from gene expression analysis, quantitative proteomics and metabolomics, physiology to bioinformatic analyses. This research is carried out in the ARC Centre of Excellence in Plant Energy Biology providing students with training and hands-on use of state-of-the-art equipment. Previous students have received international fellowships (EMBO, Human Frontiers, Australian Research Council) to carry out their own research projects in Europe, USA and Australia. National and international research agreements with the Australian National University, the University of Sydney, Zhejiang University (Hangzhou, China), The Max- Planck Institute of Molecular Plant Physiology (Potsdam, Germany), Ludwig-Maximilian University (Munich, Germany), and Umeå and Stockholm Universities (Sweden) provide students with the opportunity to study overseas, supported by a variety of grants or scholarships provided by the Centre.
PROJECTS
1. Mitochondrial biogenesis and regulation
Mitochondria are key organelles in eukaryotic cells that play essential roles in energy production, various biosynthetic pathways and in cell death. Thus mitochondria play key roles in the life and death of cells. As most mitochondrial proteins are encoded in the nuclear genome and need to be imported into the mitochondria, the biogenesis of mitochondria is a complex and well regulated process. The aim of our research is to characterise the protein complexes that allow import of proteins from the cytosol to the mitochondria, the function of the proteins involved in energy metabolism itself, and how these processes are regulated during development and stress conditions. Using transcriptomic and proteomic approaches we have identified several novel mitochondrial proteins that are important for mitochondrial function, but their mode of action is currently poorly understood.
Mitochondrial dysfunction caused by a variety of environmental changes and stresses results in altered nuclear gene expression in plants, called mitochondrial retrograde regulation. Overall this altered nuclear gene expression results in mitochondria-mediated ability of the plant to cope with those stresses. However the identity of the genes involved in mitochondrial retrograde regulation is largely unknown. We use genetic approaches combined with state-of-the-art sequencing technologies to discover new genes that regulate the ability of the plant to respond to stress. The overall aim is to analyse the functions of these different proteins and pathways, thereby contributing to an integrated understanding of the biogenesis and regulation of mitochondria. A variety of projects are available in this area. Each project has a post-doctoral team leader and 1 to 2 Ph.D students. Honours students working on these projects will join these small teams with their own individual projects:
- Exploring the role of WRKY transcription factors in the molecular regulation of plant stress responses. Dr Olivier Van Aken – [email protected] - Studying the gene network that regulates mitochondrial stress response in Arabidopsis thaliana. Dr. Aneta Ivanova - [email protected]
- Determining the functions of novel protein transporters. Dr Monika Murcha ([email protected]) and Yan Wang -([email protected]) - Investigating the role of temperature and climate zones in grape berry development using next generation transcriptomic profiling technologies. Dr Estelle Giraud - [email protected] - From seed to plant: understanding rice development on a transcript level. Dr Reena Narsai – [email protected]
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- The mitochondrial bacterium 'The elephant in the room' - Exploring the adaptation of bacterial signalling pathways for the successful development of higher plants. Owen Duncan - [email protected]
2. Using –omics and biotechnology to develop crops with improved phosphate use efficiency
Phosphate is an essential element for life, required for all energy-dependent processes in a cell, and as a component of organic molecules, such as ATP, nucleic acids, membrane lipids and proteins. Plants have developed various strategies to cope with the limited bioavailability of this element and the molecular mechanisms that drive these processes have only recently started to become unravelled. Phosphate supply is of particular importance for crops such as rice that are grown in nutrient-poor weathered soils. Current farming practice requires application of large amounts of fertiliser derived from non-renewable phosphate rock. These fossilised phosphate deposits are predicted to become exhausted in the next century and drive plant production costs up, an issue of international economic importance as global demand for food increases and the environmental damage of fertiliser application becomes apparent.
We use high-throughput molecular approaches to search for key regulators of the response to phosphate stress in rice and the model plant Arabidopsis. Understanding the way plants adapt to a limiting nutrient environment will allow us to develop novel biotechnology based solutions in cereal crops that can use the phosphate pool in soil more efficiently and reduce the need for fertiliser application.
These projects are part of an international collaboration funded by the Australian Research Council SuperScience program and the Chinese National Science Foundation between UWA and Zhejiang University. Projects will be supervised by SuperScience fellows with expertise in –omic techniques, including transcriptomics, proteomics and metabolomics.
- Using laser capture microdissection and next generation sequencing to analyse cell-specific responses to phosphate starvation in rice. Dr David Secco – [email protected] - Tracking phosphate stress-induced proteomic changes in rice with mass spectrometry. Dr Ralitza Alexova – [email protected] - Getting the message across: coordinating retrograde and anterograde signalling of mitochondrial protein import upon phosphate deficiency in rice and Arabidopsis. Dr Marna van der Merwe – [email protected]
References Refer to http://www.plantenergy.uwa.edu.au/ for all publications and more details about scholarships.
Giraud E, Ng S, Carrie C, Duncan O, Low J, Lee CP, Van Aken O, Millar AH, Murcha M and Whelan J (2011) TCP transcription factors link the regulation of genes encoding mitochondrial proteins with the circadian clock in Arabidopsis thaliana. The Plant Cell 22:3921-3934. Giraud E, Van Aken O, Ho L and Whelan J (2009) The transcription factor ABI4 is a regulator of mitochondrial retrograde expression of Alternative oxidase 1a Plant Physiol 50: 1286-1296 Zheng L, Huang F, Narsai R, Wu J, Giraud E, He F, Cheng L, Wang F, Wu P, Whelan J, Shou H (2009) Physiological and transcriptome analysis of iron and phosphorus interaction in rice seedlings Plant Physiol 151:262-274
61 ASSISTANT PROFESSOR DUNCAN A. WILD Room 3.31, Bayliss building, Phone: 6488 3178, Email: [email protected]
Laser Spectroscopy & Computational Chemistry
Research interests include: Spectroscopic investigations of gas phase ionic clusters, ab initio calculations to predict infrared and photoelectron spectra, apparatus design and development.
5 projects are offered for prospective students. Projects 1-3 are concerned with spectroscopy of fundamental, yet important, gas phase species using the TOF-PES apparatus. Project 4, in collaboration with Assoc. Prof. Scott Stewart, deals with the synthesis and spectroscopy of novel carotenoids. Project 5 is theoretical in nature, and involves modelling photoelectron and infrared spectra via ab initio methodologies.
PROJECTS
1. Photoelectron spectroscopy of atmospherically and astronomically important species The spectroscopy projects are based on a time of flight (TOF) mass spectrometer coupled to a PhotoElectron Spectrometer (PES) which is now operational and churning out results in the Wild Lab. The idea behind the experiment is: 1) Create exotic gas phase anion-molecule clusters. 2) Mass select a specific cluster using TOF mass spectrometry 3) Record a photoelectron spectrum using the fourth harmonic of a pulsed Nd:YAG LASER ( = 266nm).
The rate and direction of chemical reactions is determined by the potential energy surface governing the interactions between the species. Using photoelectron spectroscopy of anion-molecule 1:1 complexes allows us to probe the neutral potential energy surface. Spectra are shown to the left for the chloride and bromide-carbon monoxide complexes.[1] In this project, you will extend our studies to look at fundamental species with Nitrogen and Sulphur containing molecules attached to an anion. These species have relevance for the chemistry occurring in our atmosphere, and that of distant celestial bodies. This project is flexible in that you can choose the systems to investigate! We are currently developing an oven source which will allow for more flexibility in our ion production techniques.
2. The inception of solvation Ever wondered what is occurring on the microscopic scale when solutes dissolve in a solvent? What are the dominant forces at play? How many solvent molecules are in close contact with the solute, or in subsequent solvation shells? Using the tof-pes we are in a position to answer these questions! With mass spectrometry we can probe one cluster size at a
62 time and build a picture of size dependent properties, eventually seeing the closing of a solvation shell. In this - project you will record the photoelectron spectra of clusters of the form X …(M)n with n=1,2,3,… and
supplement the spectra with ab initio calculations. We will target ligands such as C2H2 and CO as prototypical - solvent molecules. Shown in the figure are photoelectron spectra recorded recently of the I …(CO)n with n=0-4. The shift in the peak positions is determined by the intermolecular interactions between the solvent molecules (CO) and solute (I-).
3. Electronic Spectroscopy of cation-molecule complexes Space is not empty!! In fact there are many regions which are dense with interesting molecules that so far have not been unambiguously identified. Some potential candidates are polycyclic aromatic hydrocarbons, i.e. naphthalene, anthracene, and so on. In this project you will create clusters between these molecules and Argon cations, and then obliterate them with UV radiation. The project will be run on the TOF-PES, however by operating it in cation mode rather than anion mode. The photoelectron spectrometer will not be used, instead we will infer absorption of a photon by the neutral dissociation products that result. This project will utilise our newly acquired laser system, which is a dye laser pumped by a Nd:YAG laser, with a tunable range of 210-710nm (cool toys to play with!).
4. Synthesis and ultra-fast spectroscopy of novel carotenoids (co-supervised by Assoc. Prof. Scott Stewart) If you can‟t decide between a synthetic or physical chemistry project, then why not have the best of both worlds? In this project you will be involved with the synthesis of novel apo-carotenoids, with nitrogen containing functional groups. As part of the project you will collaborate with researchers at the University of Sydney and utilise their femto-second laser system to record transient absorption spectra to determine the energy relaxation pathways of these important molecules. Carotenoids are prevalent in nature, and notably are found in the photosynthetic system. Their role is to both protect the system from oxidative attack by singlet O2 and also to funnel energy into the PS system to aid photosynthesis. Carotenoids feature alternating C-C and C=C bonds along a carbon back bone, with various functional groups and ring systems attached at the ends.
5. Modeling 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 reference [2] and citations within). In this project you will model photoelectron of small dimer clusters. We will start with basic approximations, and then extend to producing multi- dimensional potential energy surfaces! (sounds impressive, heh?) We have a healthy allocation of computing time with IVEC [3] and the NCI [2] facilities. This project is ideal for those who are interested in theoretical chemistry, spectroscopy, computing, code production, fooling Potential describing the H- around with Unix(Linux), and working with multiple CPUs! bonded S-H stretching mode - of Cl …H2S
References: 1. K.M. Lapere, R.J. LaMacchia, L.H.Quak, A.J. McKinley, D.A. Wild, Chem. Phys. Lett., 504, 13-19 (2011) 2. D.A. Wild and T. Lenzer, Phys. Chem. Chem. Phys., 2005, 7, 3793-3804 3. http://www.ivec.org/ & http://nci.org.au/
Come by and see Duncan for more information, or drop by the lab to see “The Beast” (aka the TOF-PES) and have a chat with students in the group: Kim Lapere (PhD), Marcus Kettner (PhD), and Stephen Dale (Hons) about what life is like as a laser spectroscopist.
63 PROFESSOR MICHAEL J WISE
35B Room 2.09, Bayliss Building, Phone: 6488 4410
36B 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
PROJECTS
1. Systems Approaches to Oxidative Stress (Jointly supervised with Assoc. Prof. Peter Arthur)
Oxidative stress is caused by reactive oxygen species (ROS) and is thought to exacerbate pathology associated with many chronic diseases and conditions. Examples include Alzheimer‟s disease, atherosclerosis, dementia, diabetes, emphysema, heart disease, HIV/AIDS, kidney disease, liver disease, muscular dystrophy, Parkinson's disease, Rheumatoid arthritis, some cancers and aging. However, preventing the harmful effects of oxidative stress is not a simple matter, as antioxidant treatments have generally been ineffective in the treatment of these conditions.
One challenge has been the lack of understanding of the various molecular mechanisms by which oxidative stress causes pathology. We have established that cysteine residues on proteins are particularly sensitive to oxidative stress and our laboratory is now playing a leading role in identifying proteins sensitive to oxidative stress. Our work, and the work of others, has established that multiple proteins are sensitive to oxidative stress, which means oxidative stress could have a widespread impact on many cellular processes (metabolic pathways, ion transport, protein synthesis, protein degradation, gene expression, signal transduction pathways).
The objective of this project is to develop and use bioinformatic methods to identify the cellular processes and organelles that are particularly sensitive to oxidative stress. This will involve categorizing the involvement of proteins (those identified as sensitive to oxidative stress) in different cellular processes. You will be using pathway analysis software such as IPA (www.ingenuity.com), keyword clustering software (Protein Annotators Assistant) and databases such as BioCyc, Reactome and Kegg to look for common themes/processes. Protein- protein interaction data and data about predicted location may also be useful.
2. 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.
64 3. Is Genome Plasticity a Cofactor of Microbial Virulence?
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. In other words, durability genes – like vector based transmission – give the pathogen “other options” beyond the survival of the host. An extension of this thesis is to include long-term dudrable energy storage 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. In this project you are to examine another possible cofactor: genome plasticity. Bacterial with plastic genomes leave themselves open to being parasitized. On the other hand, having a plastic genome gives the organism other options, in this case import of useful genes from other organisms, e.g. coresident in a biofilm. The overall aim of the project is to find any protein coding genes that may enable greater plasticity, linking these firstly to the difference organisms and then also to published mortality data as a proxy measure for virulence.
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. 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 the models generated by an existing genome analysis application as it traverses whole bacterial chromosomes. Then, after normalising the elements of the data vectors, you will 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.
5. Low Complexity Protein Domains in Bacteria
Globular proteins, e.g. enzymes, have sequences whose sequences appear to be random. That is, at any point in the sequence it is hard to predict what the next amino acids will be based on those you have seen to this point. These are called high complexity sequences. Low complexity proteins and protein domains, on the other hand, are peptide sequences whose compositions appear to be far from being random. A well-known example is the tandem GPP repeats found in collagen sequences. Amino acid stutters (tandem repeats of the same amino acid) are another type of low complexity sequence. In eukaryotes, low complexity proteins are often found in structural proteins, such as collagen and mucin in vertebrates and glutenins in plants. Low complexity proteins are also associated with a number of diseases, e.g. Huntingdon‟s disease is due to a pathological expansion of a poly-glutamine stutter. Low complexity proteins are also often natively unfolded – they have little or no organised structure at ambient temperature and pH, but may nonetheless still be functional. A survey in Wise (2002) found that low complexity sequences are rare in bacterial and phages, but more common in eukaryotes and their viral parasites. However, low complexity bacterial proteins do exist in bacteria, so the task in this project will be apply predictors of low complexity and natively unfolded proteins to a range of bacterial proteomes to determine where such domains are found and are there any functions that are associated with bacterial proteins which have low complexity or natively unfolded domains. Further more, to what extent do the archael proteomes follow any trends you observe in bacterial proteomes.
65 PROFESSOR GEORGE YEOH Room 2.59, Bayliss 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. We envisage an enormous potential for this cell as the vehicle for cell and gene therapy to treat liver disease. We contend it is superior to other cell types such as the hepatocyte and the embryonic (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. Most importantly, the LPC is developmentally close to the hepatocyte and the cholangiocyte in contrast to the ESC or ASC, which will require many more steps and much coaxing to produce useful cells for liver therapy. Our long-term vision is to hasten the day when human LPCs are utilised to treat liver disease, especially end-stage liver disease for which currently organ transplant is the only solution. A realistic expectation in the short term is to use LPCs to ―bridge‖ patients thereby extending their survival and enhances their probability of finding a suitable organ donor. A more ambitious and longer-term aim is to use these cells to circumvent the requirement for organ transplant. This may be possible with some liver diseases. To utilise LPCs we must identify and understand the action of growth factors and cytokines, which influence them. To accomplish this, we have characterised the pattern of cytokine expression in two mouse model of liver disease that induces the appearance of LPCs. These studies indicate that a subset of inflammatory cells, the macrophages and cytokines they produce namely TNF alpha and TNF like weak inducer of apoptosis (TWEAK) are LPC regulators. To understand both the cellular and molecular mechanism of action mediated by inflammatory cells we are using cultures of LPCs and LPC lines. This knowledge can be used to increase their contribution to liver regeneration in vivo which can lead to positive outcomes for liver disease patients. Both in vivo and in vitro, extended growth of LPCs results in transformation to cancer; in this context hepatocellular carcinoma. Therefore it is important to document changes in gene expression that are responsible for transformation. Recent developments in our laboratory which underpin the projects on offer are: 1 Isolation and characterisation of LPCs from adult human liver 2 Establishment of LPCs from a transgenic mouse which expresses beta-galactosidase when it becomes a hepatocyte and LPCs which express EGFP which facilitates cell tracing. 3 Acquisition of the Cellavista instrument which allows for progressive, accurate, high throughput and comparative growth characteristic of multiple cell cultures 4 Identification of chromosomal alterations (See Fig 1) and gene expression pattern differences between normal and transformed LPCs as a result of expression profiling.
Accordingly research projects will exploit these new developments for they are designed to increase our understanding of LPCs and establish their utility for treating liver disease.
Fig 1: (A) Chromosomal alterations during culture of an LPC line (BMEL) at passage 5 (A), 10 (B) and 15 (C). Chromosome loss (red arrows), gain (blue arrows) and consistent mars (small arrowhead) seen between passage 5 and 10. The chromosome in passage 5 and 10 remain telocentric, consistent with normal mouse structure. The cells are hypotetraploid, however, there are less than 4 copies of chromosomes 4 (red dotted circle) and more of chromosome 9 (blue solid circle). Massive transformation of chromosome structure has occurred between Passages 10 and 15 and typical mouse chromosomes can no longer be identified. The chromosomes have been assembled into a karyotype using traditional cytogenetic methods. They are grouped according to size and similar banding patterns then arranged from largest to smallest. Structural changes include duplication/translocation and increase in mars.
66 GENERATING FUNCTIONAL LIVER CELLS FROM LPCs
Assessing the ability of LPCs to synthesise urea
Ornithine transcarbamylase (OTC) is a urea cycle enzyme that is mutated in individuals with a metabolic disorder - OTC deficiency. The consequence of accumulating ammonia affects many tissues and the liver particularly is damaged. The condition affects young children with neurologic consequences, hence liver organ transplant is necessary to treat those severely affected. Cell therapy using normal hepatocytes may also be possible, but hepatocytes are difficult to maintain and store; and once transplanted may not survive for very long. In contrast LPCs are robust and have the added advantage of long-term survival and the ability to proliferate and continue to generate hepatocytes in situ.
This project evaluates the effectiveness of utilising LPCs to treat OTC deficiency. First, the ability of LPCs to express OTC following differentiation into hepatocytes will be determined. Then their ability to synthesise urea will be compared with hepatocytes.
Availability of the Spf-ash mouse model of human OTC deficiency through our collaboration with Professor Ian Alexander of the Childrens Medical Research Institute in Sydney allows us to directly test our LPC lines in these mice. This will be undertaken if the cells induce OTC and acquire the ability to synthesise urea following differentiation.
We are also attempting to generate LPC lines from the Spf-ash mouse. They will serve as negative controls for the differentiation studies. The CMRI group will use these cells to establish methods to correct the gene deficiency in OTC-/- LPCs as proof of concept studies in advance of applying this method to children with OTC deficiency.
WHAT MAKES LPC’s BECOME CANCEROUS?
Comparing tumorigenic and non-tumorigenic LPCs
LPC lines have been established from p53 -/- as well as +/+ mice. Some grow in soft agar and produce tumours when injected subcutaneously into nude mice; some do not. We are defining the differences beween these cell lines at the molecular and cellular level to identify features which are causative and those which are consequential in terms of cancer. Specifically we are documenting chromosomal changes and focusing on oncogene candidates raised by gene profiling. Two anti-apoptotic genes IAP and Yap are prime suspects and their expression at the mRNA level (through qPCR) and protein level (by Western Blot) are being be defined for a range of cell lines and during tumorigenesis during culture. Current studies follow changes in LPCs as they are passaged and progressively become tumorigenic. We are also documenting changes in expression of p53 and the level of its activity by measuring the expression of downstream genes such as p21. We are also testing the effects of culture conditions on tumorigenesis. In particular, we will determine whether the level of oxygen and the composition of the culture medium with respect to growth factors contribute to transformation.
Does the level of ROS contribute to transformation of LPCs as they are maintained in culture?
LPC lines which are initially non-tumorigenic will become tumorigenic following repeated passaging in culture. This project tests the hypothesis that ROS is an important contributor to the mutagenic events by passaging cells in 20% O2 and 2% O2. It predicts that cells maintained in hypoxic conditions will less readily transform. Another approach is to maintain cells in the presence of antioxidants (vitamin C or desferrioxamine) which should produce the same outcome. Alternatively, the hypothesis would be also be supported if it can be shown that transformation will occur sooner if cultures are maintained under conditions which produce higher levels of ROS such as in the presence of ethanol or H2O2.
The tumorigenic state of the LPCs will be assessed by their capacity to grow in soft agar. This can be confirmed by their ability to form tumours in nude mice. TBARS assay will be used to ascertain the ROS levels under different experimental conditions. Tumorigenic LPCs also display gross chromosomal abnormalities and this can be documented by karyotyping the resulting cell lines.
67 HOW TO APPLY
UWA Applicants
If you completed your undergraduate studies at UWA you should lodge an on-line application via StudentConnect by clicking on the Apply for Honours link in the left hand menu bar of StudentConnect.
Applications will open online on Monday 10th October and close on Tuesday 20th December.
Non-UWA Applicants
If you have not previously been enrolled at UWA, you apply through one of the following centres, depending on your circumstances.
Domestic Students
Australian citizens, permanent residents and/or holders of a humanitarian visa or New Zealand citizens apply through the UWA Admissions Centre via the UWA‟s Online Application System (OASys).
International Students
International Students apply through the UWA International Centre.
Honours Project Preference Form
All applicants must complete the BBCS Honours Project Preference Form and return it to the MCS Building reception by Friday November 11th 2011.
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Biochemistry & Chemistry
Honours or GradDipSci in 2012 PROJECT PREFERENCE FORM The purpose of this form is to ascertain your interest in our Honours/GradDipSci courses. It is appreciated that students may be exploring Honours/GradDipSci in more than one discipline. Phone the BBCS School Office (6488 4402) to be referred to the appropriate Coordinator to discuss any questions you may have. Please return form to BBCS School Office by Fri 11th Nov 2011
I am interested in Honours/GradDipSci in 2011 within the Discipline of: Biochemistry & Molecular Biology Biomedical Science Chemistry Genetics Nanotechnology
Note: You need to fill out a separate form for each Discipline if you are considering projects in more than one. Include projects for any Programme (e.g. Genetics, Chemistry, Biomedical Science etc) that will be located within one of the above Disciplines
I am considering mid-year entry to Honours in 2012
I am considering deferring Honours until 2013
I will will not be available for interview during the week 5 December - 9 December 2011
1. CONTACT DETAILS Name………………………………………………………………………………………………………………………… Address(es) (during period November/December 2011 – January 2012): ……………………………………………………………………………………………………………………………...… ………………………………………………………………………………………………………………………………... Phone No (during same period) ……………………………..…………………………………….………... Mobile No (during same period) ……………………………………………………….…………………….. Email address …………………………….………………………………………………..
2. PROJECT PREFERENCES In order of preference: 1 Project No [ ] Supervisors …………………………………………………………… 2 Project No [ ] Supervisors …………………………………………………………… 3 Project No [ ] Supervisors …………………………………………………………… 4 Project No [ ] Supervisors …………………………………………………………… 5 Project No [ ] Supervisors …………………………………………………………... 6 Project No [ ] Supervisors …………………………………………………………... If there are any points you would like us to take into consideration please note them below: ……………………………………………………………………………………………………………………………… ………...……………………………………………………………………………………………………………………… Signature………………………………………………………Date………………………………………………………
The Faculty’s End-on Honours on-line application form must be completed by December 20th 2011. Prospective candidates will be interviewed 5 December - 9 December 2011, although other arrangements can be made if candidates are unavailable. Those students who have submitted this project preference form and who are eligible to enrol in the course will be emailed a confirmation of eligibility as soon as exam results are known [approximately 20 December], and allocation of projects will be advised as soon as possible after this. Student Administration will send you an Authority to Enrol letter in January 2012. 69
Faculty of Life and Physical Sciences The University of Western Australia M310, 35 Stirling Highway Crawley WA 6009 Tel: +61 8 6488 4402 Fax: +61 8 6488 7330 Email: [email protected] www.biomedchem.uwa.edu.au
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