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BiochEmisTry, molEcUlar Biology & gENETics
Honours Projects 2010
School of Biomedical, Biomolecular and Chemical Sciences
Welcome to Honours
School of Biomedical, Biomolecular and CChheemmiiccaall SScciieenncceess
2010 Honours
We hope that you will enjoy this event and that it will serve as a good introduction to the range of
Honours projects offered in the School for 2010.
If you are interested in doing an Honours year at UWA, you maybe are already asking about the
exciting prospects available within each of the Disciplines and sub-disciplines within the School.
These are Biochemistry and Molecular Biology, Biomedical Science, Chemistry, Forensic
Chemistry, Genetics, Medical Science, Microbiology and Immunology, Pharmacy, Physiology and
Structural Biology. This Honours Projects book will enable you to further explore the possibilities
and talk to staff that will be on hand. If you intend to enrol in Honours in 2010, this booklet will
provide you with a comprehensive overview of the interests of each of the research groups within
Biochemistry, Molecular Biology and Genetics as well as outlining suitable Honours projects. The
Honours Expo is designed to showcase the depth and diversity of research being undertaken in the
School. Here, you will be able to talk to staff who will be available to explain their research in
much detail.
Enjoy!
Professor GA Stewart
Head of School
Honours Co-ordinators
Biochemistry and Molecular Biology
Professor Alice Vrielink
Phone: 6488 3162
Chemistry & Nanotechnology
Professor Mark Spackman
Phone: 6488 3140
Genetics
Professor Lawrie Abraham
Phone: 6488 1148
Microbiology and Immunology
Professor Barbara Chang
Phone: 9346 2288
&
A / Professor Manfred Beilharz
Phone: 9346 2663
Physiology
Dr Gavin Pinniger
Phone: 6488 3380
Forensic Science
Professor John Watling
Phone: 6488 4488
Table of Contents
Project Entries Page 1
How to Apply Page 37
Application Form Page 38
Preference Form Page 40
1 Biochemistry
1B PROFESSOR LAWRIE ABRAHAM 21BRoom 2.58, MCS building, Phone: 6488 3041,
22BEmail: [email protected] U
Human Molecular Biology Lab
Our group is interested in the transcriptional regulation of gene expression.
We are also interested in the effects of genetic polymorphism (SNPs) on the expression of genes, particularly
promoter and other regulatory variants.The focus is on genes that are involved in regulating inflammatory
responses and understanding how genetically determined differences in expression contribute to diseases such as
autoimmune disease, cancer and cardiovascular disease. To this end we are involved in the identification of
transcription factors and upstream components of the signal transduction pathways that regulate these genes.
Our long-term aim is to develop therapeutic strategies to modulate the activity of these genes through
interference with such regulators in order to prevent disease. Prospective Honours students with a commitment
to excellence and a background in Molecular Biology, Biochemistry, Genetics or Immunology are particularly
encouraged to apply. Students will be exposed to a range of techniques including DNA sequencing, DNA
cloning, cell culture, transfection assays, RT-PCR, expression array analysis, siRNA knockdown, DNA binding
assays (EMSA), protein analysis, DNase I Footprinting, Chromatin immunoprecipitation (ChIP) and FACS
analysis.
12BPROJECTS
1. The Transcriptional control of the CD30 Gene in Anaplastic Large Cell Lymphoma
Anaplastic large cell lymphoma (ALCL) is a variant of immunoblastic lymphoma and tends to be clinically
aggressive, resulting in the destruction of the involved lymph node structure, the infiltration of the lymph node
sinuses by large transformed neoplastic cells with prominent
nucleoli. The major diagnostic marker of ALCL is strong
overexpression of the CD30 gene thought to result from a
transforming event that leads to neoplasia. Fundamental to our
understanding of the causes and treatment of ALCL is an
understanding of the mechanism of overexpression of CD30.
The CD30 gene promoter, including an ALCL-specific
hypersensitive site we have discovered in the 1st intron, will be
characterised with respect to transcriptional control elements
by EMSAs, CD30 reporter gene analysis and CHART
(chromatin accessibility by real-time PCR). The transcription
factors binding to the promoter and the 1st intron will be
identified by use of a 2-dimensional proteomics technique
developed in our group. Once cloned, the identified proteins
will be tested for the ability to repress endogenous expression
and reporter constructs by overexpression in cell lines and by
RNAi approaches. Chromatin immunoprecipitation (ChIP) assays will also be carried out to establish the in vivo
relationship between the various cis-elements and trans-acting factors, including sites of histone modification. The
long-term aim is to develop therapeutic strategies that interfere with the transcriptional regulation of CD30 and so
block the deleterious effects resulting from overexpression of CD30.
2. Cell Signalling in Anaplastic Large Cell Lymphoma following CD30 Receptor Stimulation
As outlined above, one of our objectives is to understand the neoplastic phenotype in ALCL and develop targeted
therapies. By understanding how CD30 gene regulation is aberrant in ALCL cells, one can develop appropriate
therapies to reverse the neoplastic phenotype that results from CD30 receptor signalling. We have recently shown
that when ALCL cells are stimulated via the CD30 receptor (using ligand CD153 - see Model above) some of the
cells undergo apoptosis. However, many cells escape cell death and instead differentiate by becoming adherent
and after extended culture begin to proliferate, thereby escaping the apoptotic signal. Due to the importance of
adhesion in neoplasia and metastasis, the aim of this project is to define, initially at the gene level, the
differentiation program by comparing the transcriptional profiles of apoptotic versus adherent cells following
ligand stimulation. Transcription profiles will be produced using Affymetrix U133A chips and comparisons made
2 Biochemistry
between control and CD153-treated cells induced for 0, 0.25, 4 and 24 hours; non-adherent and adherent cells will
be arrayed separately. Expression changes will be compared using the clustering programs available in the
Genesifter package. The objective is to identify uniquely expressed genes that correlate with the adhesion
response. Following identification and confirmation of
expression by quantitative PCR (qPCR) we will
establish whether these potential facilitators show
activation by coIP and immunoblotting. The results of
these studies will be used to identify downstream
effectors that are involved in the adhesion or apoptotic
responses resulting from ligand-receptor engagement.
3. Characterisation of functional polymorphisms of Vanin 1, a QTL controlling HDL-C Levels
This collaborative project with the Southwest Foundation for Biomedical Research, Texas, USA involves the
characterisation of the Vanin 1 gene, which has been shown to be genetically associated with low levels of High
Density Lipoprotein-cholesterol ("good" cholesterol) levels
in the blood. Low HDL levels are a strong risk factor for
cardiovascular diseases such as arthrosclerosis and heart
attack. Four promoter variants in the Vanin 1 gene (see Fig)
show significant correlations with HDL-C as well as Vanin 1
mRNA expression levels. The most likely functional
promoter variant at -137 exhibits a strong association with
HDL-C levels (p = 0.002). The project aims are to
characterise transcription factors that differentially bind to
the -137 SNP region using EMSA/peptide mass
fingerprinting, determine the effects of the -137 SNP on
transcriptional activity using reporter gene analysis, and to
identify modulators of VNN1 expression & determine their
effects on allele-specific transcription of VNN1 using
mRNA expression profiling. An understanding of how the
gene is controlled will inform the development of therapeutic strategies and/or drugs to modulate the activity of
the Vanin 1 gene with the objective of raising HDL-cholesterol levels in individuals at risk.
4. Mechanism of Action of Newly Synthesised Thalidomide Derivatives. (See also co-supervised project with
Dr Scott Stewart)
In a collaborative project with Dr Stewart, newly synthesised thalidomide-based drugs will be screened for novel
biological activities using TNF reporter gene assays. For those students interested in the functional aspects of
thalidomide and newly synthesised derivatives, transcriptional profiling will be carried out, using Affymetrix
microarrays to define novel cellular activities, with a focus on therapeutic application. The project also involves
the identification of the cellular targets of thalidomide. Photoactivatible biotin-derivatized thalidomide will be
used to treat cells, followed by UV-catalysed cross-linking. 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.
Also see co-supervised projects with Dr Daniela Ulgiati (Biochemistry & Molecular Biology).
3 Biochemistry
9BASSOCIATE PROFESSOR
PETER ARTHUR 10B
Room 2.41, MCS Building, Phone: 6488 1750
Email: [email protected]
Reactive Oxygen Species as modulators of signal transduction
pathways and biochemical systems
Where there is aerobic life, there are reactive oxygen species (ROS). ROS have the potential to disrupt cellular
function but are usually held in check by an array of antioxidant systems. In many chronic diseases this balance
is disturbed, and the resultant oxidative stress is thought to contribute to disease pathology. There is evidence of
oxidative stress in a diverse range of diseases including atherosclerosis, diabetes, Parkinson's disease and
Alzheimer's disease. There is also good evidence oxidative stress is an important contributor to dysfunction
associated with aging.
There has been an intensive research effort into understanding how ROS disrupts cellular function by damaging
cellular macromolecules such as DNA, protein and membrane lipids. An exciting development has be the
realization that ROS could be interfering with a variety of biochemical and genetic systems involved
maintaining cellular homeostasis. Of the various ROS, hydrogen peroxide (H2O2) has attracted interest because
it may be involved in modifying key intracellular proteins (e.g. signal transduction proteins) to influence many
aspects of cellular function (growth, metabolic rate, protein synthesis and catabolism, cell division to name a
few). If this concept is correct, then it should be possible to develop drugs which can prevent or reverse the
detrimental effects of this form of oxidative stress. One example serves to illustrate this point. Recently,
overexpression of catalase (an antioxidant enzyme) in the mitochondria was found to extend maximum lifespans
of mice by 20%. This exciting research also provides very strong support for the theory that oxidative stress is a
cause of aging. We are following up on this research by developing our own transgenic mouse and we are keen
to involve students in this research.
This research area is constantly developing, so I am happy to discuss the research area in general or work with
you to develop a project that suit your interests. I am an experienced supervisor with a preference for
collaborative projects so that you can gain the benefits of dual supervision. Please see below examples of
current research projects to give you an idea of the type of work we do.
PROJECTS
1. Does a potential therapeutic cause oxidative stress?
Collaborative with Dr Thea Shavlakadze & Professor Miranda Grounds, Anatomy
Insulin growth factor 1 (IGF-1) is widely investigated as a therapeutic agent for muscle ageing and muscular
dystrophy. In both conditions oxidative stress plays a significant damaging role, but it is not certain how
elevated IGF-1 affects ROS generation in skeletal muscle. Down-regulation of IGF-1/Insulin signalling has anti-
ageing properties (i.e. extends lifespan), which has been attributed to reduced oxidative stress. On the other
hand, IGF-1 has been shown to be protective against ROS induced apoptosis in cardiac muscle cells. The
interaction between IGF-1 and oxidative stress in skeletal muscle has not been investigated. The objective of
this project is to use proteomic technologies to establish the relationship between IGF-1 and oxidative stress in
dystrophic muscle (mdx/IGF-1 mice (Fig 1)) through
our oxidative profile measurements. This will be
significant because if IGF-1 does not affect oxidative
stress, then the protective effect of IGF-1 may be
enhanced by combination with appropriate
antioxidant therapies. Additional techniques may
include Immunohistochemistry, Western Blotting,
quantitative PCR and EMSA.
Figure 1. One year old male mdx/IGF-1 and mdx
littermate mice. Mdx/IGF-1 transgenic mice are much
bigger compared to their age matched litter mates and
they have a pronounced skeletal muscle hypertrophy.
Mdx/IG
F-1-1
Md
x
4 Biochemistry
2 Does oxidative stress cause muscle wasting?
As skeletal muscle ages it loses strength and power leading to reduced mobility and deleterious changes in
lifestyle. The relentless loss of muscle mass and function in elderly individuals impairs daily functions such as
walking, using stairs and rising from chairs and results in an increased incidence of falls. Muscle wasting is also
associated with immobility and diverse pathologies such as cancer, bacterial sepsis, AIDS, diabetes, and end-
stage heart, kidney, and chronic obstructive pulmonary disease. We are using transgenic mouse models of
muscular dystrophy (which we already have) and ageing (which we are developing) to investigate the role of
oxidative stress in muscle wasting. Transgenic mouse models are particularly significant in biomedical research
because they reflect the complexity of human disease processes. This work is supported by a grant from the
National Health and Medical Research Council.
One signaling protein associated with muscle wasting is NFkB and there is evidence indicating that oxidative
stress can activate NFkB. Muscle wasting has also been linked to increased rates of protein breakdown and
increased oxidative stress. The objective of this project is to establish whether oxidative stress causes an
increase in the rate of protein breakdown via a signaling pathway involving NFkB. For this work a muscle cell
line (C2C12) will be used, as cell culture systems are particularly useful experimental systems to pin point the
precise molecular mechanisms involved in disease processes. Techniques likely to be required (you will be
trained) for this project include proteomic techniques, tissue culture, quantitative PCR for atrophy related genes
and measurement of oxidative stress. This project is also related to our larger effort to understand the effects of
mild oxidative stress (particularly ageing) by developing a transgenic mouse over-expressing catalase.
3. Oxidative stress in the heart Collaborative with Dr L. Hool, Physiology
Mild oxidative stress is a feature of a variety cardiovascular disease states including ischemic heart disease,
hypertension, and congestive heart failure. The L-type calcium channel plays a key role in contraction and is a
likely target of mild oxidative stress. This year, an honours student established a method (immunoprecipitation)
to isolate the L-type calcium channel from tissue. The objective of this project is to use this technique to
evaluate whether the L-type calcium channel is oxidized in vivo. Establishing that the L-type calcium channel is
oxidized would be an important step towards understanding the role of oxidative stress in the development of
pathology. This work is supported by a grant from the National Health and Medical Research Council.
4. Developing proteomic technologies to identify proteins responding to mild oxidative stress in chronic
diseases (collaborative with Proteomics International)
Proteomics is an exciting and rapidly expanding field which seeks to unravel biochemical and physiological
processes by focusing on proteins. Encoded proteins carry out most biological functions, and to understand how
cells work, we need to study what proteins are present, how they interact with each other and what they do.
The West Australian-based biotechnology company Proteomics International and the University of Western
Australia are associated with the “Diabesity Research Program” (DRP), which is flagship node of the
comprehensive “Centre for Food and Genomic Medicine (CFGM)” based in Perth.
Diabetes mellitus often referred to simply as diabetes, is a HUsyndromeUH characterized by disordered HUmetabolismUH
leading to abnormally high HUblood sugarUH (HUhyperglycaemiaUH). Diabetes is a common condition, on the rise
worldwide. It has been estimated that 940,000 Australians have diabetes and that about half of these are not
aware of it. If uncontrolled, diabetes can lead to a wide range of serious, long-term health complications which
contribute to illness, disability and early death. Its onset is often associated with overweight. Diabesity is a
relatively new term for diabetes caused by excessive weight, or the condition of having both diabetes and
excessive weight.
As part of the DRP, we are undertaking identification of novel protein biomarkers for diabesity in human
plasma. Such biomarkers are measurable proteins whose detection can be used clinically to enhance prediction
of disease, diagnosis or prognosis.
The challenge is to identify valuable biomarkers from amongst the many thousands of proteins in human
plasma. Oxidative stress has been implicated as a mechanism underlying hyperglycaemia-associated cellular
damage and could play a role in the development of diabetes-related complications. This suggests that focussing
on oxidised forms of particular plasma proteins may be the key to identifying valuable biomarkers for diabesity.
This project will involve using proteomic technology including protein separation techniques (HPLC, 2D gel
electrophoresis, antibody technology) and protein identification techniques (mass spectrometry).The effect of
oxidative stress on proteins we will be evaluated using a patented technique developed by Proteomics
International and Dr. Arthur.
5 Biochemistry
11BPROFESSOR PAUL ATTWOOD Room 3.69, MCS Building, Phone: 6488 3329
Email: [email protected]
The research focus of A/Prof. Attwood's laboratory is the structure and function of enzymes in general.
However, there is a particular focus on two enzymes:
(i) pyruvate carboxylase, a 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 are investigating the structure-function relationships in this enzyme, with a
combination of site-directed mutagenesis, kinetic and physical methodologies;
(ii) mammalian histidine kinases which catalyse the phosphorylation of histidine residues in substrate
proteins. This is a little understood form of phosphorylation in mammalian cells and its biological roles
are not yet clear, although we have established a link between enhanced histone H4 histidine kinase
activity and hepatocellular carcinoma in human liver.
Honours projects for 2010 will be in these two areas of research.
6 Biochemistry
7 Biochemistry
PROFESSOR CHARLIE BOND 23BRoom 4.16, MCS Building, Phone: 6488 4406
24BEmail: [email protected] UH
Structural Biology
Structural Biology research involves building a three-dimensional picture of biological molecules to shed light
on the molecular interactions and events which drive many of the fundamental processes of life. Investigations
in my lab address proteins of relevance to human health, including DNA repair enzymes and other nucleic acid
processing proteins, and enzymes essential to the survival of life-threatening parasites, which may be drug
targets.
Different aspects of this research can be tailored to students with strengths in Biochemistry, Chemistry, and
Biophysics. Structural Biology research typically involves the opportunity to learn from a diverse set of useful
techniques including molecular biology, protein purification and crystallisation, spectroscopy, X-ray
crystallography, molecular modelling, bioinformatics, unix computing. The Structural Biology lab is equipped
with state-of-the-art equipment including a crystallization robot and X-ray data collection facilities.
For further information, reprints of papers, a colour version of this page, or to find out about other research in
the lab come and see me (MCS Lab 4.16) and look at HUhttp://xtal.uwa.edu.au/px/charlieUH .
13BPROJECTS
14BNOTE: IN ADDITION TO PROJECTS LISTED HERE, IT MAY BE POSSIBLE TO TAILOR A
STRUCTURAL BIOLOGY PROJECT TO YOUR SPECIFIC INTERESTS.
15B1. How do proteins recognise RNA molecules? Rop mutants
Collaboration with Dr Daniel Christ, Garvan Institute, Sydney
Rop is a small alpha-helical protein which plays a critical role in bacteria where it regulates the number of
copies of a DNA plasmid that the bacteria can accommodate. Rop does this by binding to a complex of two
RNA hairpins – called the 'kissing' complex. By binding and stabilising this interaction, it stops the RNA being
used to prime replication of the plasmids.
As it is a small protein and is known to crystallise, Rop makes an excellent target for research to understand the
basis of protein:RNA interactions. We have a panel of mutant proteins, which were selected using in vitro
evolution methods, that have higher affinity for the RNA than the wild-type protein. This project will involved
expressing some of these mutant proteins, generating heterodimers of the mutants, measuring their affinity for
RNA, attempting to crystallise them and solving their structures. Skills learned will include molecular biology
(mutagenesis), protein expression and purification, protein:RNA interaction assays, crystallisation and protein
crystallography.
A predicted model of the Rop:RNA
complex.(from Christ, D and Winter, G, Proc
Natl Acad Sci U S A. 2003 November 11;
100(23): 13202–13206)
8 Biochemistry
2. Protein Structure Prediction: PPR Proteins
Collaboration with Ian Small, CoE for Plant Energy Biology
PPR proteins are modular proteins composed of tandem
repeats of 35 amino acid sequences. A number of these
proteins are known to bind and/or process RNA by
recognising the RNA sequence. We want to understand
how this sequence-specific recognition occurs. In similar
protein families (TPR and ankyrin proteins) for which the
structures are known, these repeats form alpha-helical
hairpins which assemble to make a long ‘solenoid’. We
have evidence from bioinformatics studies that PPR
proteins have a similar, but different structure. Based on a
specific type of bioinformatic (sequence covariation) data,
we can predict both the secondary and tertiary structure of
TPR and ankyrin proteins. We are interested in exploring
which other protein families can be investigated with these
methods, but our main aim is to produce plausible models
of PPR protein structure which can be used to guide wet-
lab experiments into the function of these proteins.
This is a computation-based project which will involve
learning about protein structure, molecular dynamics and bioinformatic analysis of proteins.
3 Chaperones and Co-chaperones of the Malaria Pathogen, Plasmodium Falciparum
With Dr Will Stanley
Malaria is a widespread tropical disease killing about 2 million people annually, young children and pregnant
women being especially vulnerable. It is a disease associated with poverty and classed as a neglected disease –
no vaccine is available and prophylactic drugs are often too costly for those most at risk. The microbial
pathogen, Plasmodium falciparum, is the major cause of life-threatening malaria amongst humans.This project
explores a complex of P. falciparum chaperones – proteins essential for folding, stabilising and sorting of other
proteins – which is critical to survival and proliferation of the pathogen, and thus a target for new kinds of
antimalarial drugs. The complex consists of two housekeeping heat shock proteins, Hsp70 and Hsp90, and a
Hsp organiser protein, HOP, which are involved in a complex set of intermolecular interactions to facilitate
folding/sorting of a number of client proteins.
Components of the complex can be recombinantly expressed and purified with the aim of detailed biochemical
and biophysical studies of the assembly, structure and function of this multi-chaperone complex.
For other collaborative projects, please see entries for Dr Swaminatha Iyer and Prof Ian Small
Sequence covariation data can be used to
predict the structure of helical repeat
proteins, with a relatively high accuracy.
Schematic of the interactions in the multichaperone complex.
References:
(1) A Shonhai, A Boshoff & GL Blatch (2007). . Protein Sci. 16(9):1803-18.;
(2)SC Onuoha, ET Coulstock, JG Grossmann & SE Jackson (2008). J Mol Biol. 379(4):732-44.
9 Biochemistry
3BDR BERNARD CALLUS 25BRoom 3.49, MCS building, Phone: 6488 1107
26BEmail: [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 express wild-type and mutant genes,
e.g. oncogenes and tumour suppressors genes, in cells to examine how they impact on apoptotic mechanisms
and the signalling pathways that regulate them. The aim of our research is to identify novel regulators involved
in apoptosis and cancer as candidates for drug design leading to the development of new therapies to be used
either alone or in combination with existing chemotherapeutics to kill cancer cells.
Our research group is new to the School and offers new areas of research to prospective students. Feel free to
come along and discuss your research interests and to learn more about our ongoing projects in the lab.
PROJECTS
1. The role of the miRNAs in the regulation of the YAP oncogene.
Yes-associated protein p65 or YAP is a transcriptional co-activator that has recently been identified as a
oncogene. Its gene is upregulated and/or amplified in tumours of breast, liver, prostate and colo-rectal cancers,
as well as in hepatocellular (HCC) and squamous cell carcinomas (SCC). YAP is regulated by cell-cell contact
through the Hippo/Sav tumour suppressor pathway 1. Over-expression of YAP in cells causes increased cell
proliferation, loss of cell-cell contact inhibition and resistance to apoptotic stimuli, which are key traits of cancer
cells. Proper regulation of YAP is therefore essential in healthy cells. YAP protein is regulated by
phosphorylation and degradation, however, the transcriptional and post-transcriptional regulation of YAP has
not been well characterized.
Recent studies have shown that many oncogenes
have shortened 3'UTRs in cancer 2 and that miRs can
regulate oncogene expression and function in
cancer3. Bioinformatic analysis has revealed several
potential miRNA (miR) binding sites within the 3'
untranslated region (3'UTR) of YAPmRNA (see
figure). MiRs bind to mammalian 3'UTRs usually
resulting in inhibition of translation or deadenylation
of message causing decreased mRNA stability. This
project will investigate whether the YAP 3'UTR
regulates protein translation or message stability thus
providing an additional level of YAP regulation.
The human YAP 3'UTR will be cloned and inserted 3' to luciferase in reporter constructs to see whether
luciferase expression is affected by the presence of the 3'UTR. Serial truncations from either end of the 3'UTR
will also be generated and cloned 3' to luciferase to map functional regions of the 3'UTR and potential miR
binding sites. Individual miRs, identified bioinformatically, will be co-expressed with the luciferase reporter
constructs to ascertain whether they repress luciferase expression. The use of mutant 3'UTRs that abolish
specific miR binding will be used to demonstrate specificity for particular miRs. Once a role for the 3'UTR in
regulating luciferase expression has been established the mechanism by which the 3'UTR imparts its effect by
either inhibiting translation or destabilising mRNA will be investigated. This work could have important
implications for the regulation of YAP in cancer.
10 Biochemistry
References
1. Harvey, K. and Tapon, N. Nat. Rev. Cancer (2007) 7: 182-91
2. Janaiah Kota et al Cell (2009) 137: 1005–1017
3. Christine Mayr and David P. Bartel Cell 138: 673–684
2. The role of p53 in the transformation of liver progenitor cells.
With Professor George Yeoh, Biochemistry and Molecular Biology (School of BBCS)
The tumour suppressor, p53, dubbed the "Guardian of the Genome" is commonly mutated in cancer with
mutations or deletions of p53 found in more than 80% of all solid tumours. Thus loss of p53 function is one of
the most common events in the transformation of cells leading to cancer. p53 is a transcription factor and
following certain genotoxic stresses, e.g. DNA damage, p53 is activated resulting in increased transcription of
the cell-cycle inhibitor, p21, leading to cell-cycle arrest, and the pro-apoptotic proteins, Puma and Noxa leading
to cell death. Thus loss of p53 function results in abnormal cell-cycle regulation and apoptotic resistance, two
key traits of cancer cells.
Liver progenitor cell (LPC) lines are an excellent system in which to study cellular transformation leading to
cancer. Their continuous culture results in their transformation into tumourigenic cells. Associated with this are
chromosomal defects, loss of basal p53 expression, and elevated expression of oncogenes cIAP1 and YAP 1. We
hypothesize that loss of p53 function is a key transforming event in these cells. The aim of this project is to
formally determine the p53 status in transformed versus non-transformed LPCs. p53 levels will be examined in
cells before and after DNA-damage induced by UV-irradiation or cisplatin. The expression and function of p53
will be determined by using Western blot analysis and p53-responsive GFP reporter constructs, respectively.
Changes in p53 status will be compared with changes in karyotype (chromosomal abnormalities), and
expression of transformed markers cIAP1, YAP and PKM2 to determine whether loss of p53 function precedes
or correlates with these markers of transformation.
We hypothesize that loss of p53 function is also required for the generation of LPC lines by the "plate and wait"
method 2. To test this, primary LP cells will be purified and treated with pifithrin-α, a p53 inhibitor, or infected
with a dominant-negative p53 expressing lentivirus to determine whether p53 inhibition enhances the generation
of LPC lines by the "plate and wait" method.
References
1. Zender, L et al Cell (2006) 125: 1253-67
2. Dumble, M.L et al Carcinogenesis. (2002) 23: 435-45
11 Biochemistry
PROFESSOR PETER HARTMANN Room 2.03, MCS Building, Phone 6488 3327
Email: [email protected] U
The long-term objective of the Human Lactation Research Group at The University of Western Australia is to
facilitate successful breastfeeding, as defined by the WHO, by providing an evidence base for the clinical
management of human lactation. Currently in Western Australia ~ 95% of mothers express a desire to
breastfeed and ~ 93% leave hospital fully breastfeeding their babies. However, by 6 months postpartum only ~
60% of these mothers are still breastfeeding. Thus, almost all mothers know that breastfeeding is best for their
babies but many of these mothers do not achieve a successful lactation. Therefore, to achieve our objective a
fundamental understanding of the physiology and biochemistry of milk synthesis, milk secretion, milk ejection,
the mechanics of breastfeeding and infant appetite are required so that appropriate clinical assistance can be
given to mothers who are not achieving the WHO recommendation to exclusively breastfeed babies for the first
6 months of life. These studies are particularly relevant to mothers who have delivered prematurely because the
outcomes for premature babies who receive breastmilk are very much better than those who receive only infant
formula.
Prof Hartmann’s research covers a broad spectrum of topics in the area of human lactation and infant
nutrition. Honours candidates may participate in a variety of projects that fall within the following
categories.
PROJECTS
1) The effect of ultraviolet irradation on human milk
Ultraviolet (UV) is widely used in the purification of water, air and food (fruit juice and beer) pasteurization. It
effectively reduces the levels of microbes by damaging the bonding of their DNA structure. However, the effect
of UV damage could also extend to bioactive components, such as protein, vitamins and cells, in human milk.
Therefore the use of UV as the pasteurizing method on human milk could cause damage to these bioactive
components and reduce their biochemical functions when received by the babies. Hence this project has been
set out to investigate the effect of UV irradiation on the bioactive components of human milk using varies
proteomic, and molecular cell biology techniques.
2) Is the initiation of lactation affected by birth mode?
While exclusive breastfeeding is recommended for the first six months of an infant‟s life, few women are able to
achieve this. Perceived insufficient milk supply is often cited as a reason for premature weaning. Furthermore,
weaning or the introduction of complimentary milks (i.e. infant formula) often occurs in the first 6 weeks after
birth. Interestingly, the number of caesarian section deliveries has risen dramatically in the past 20 years and
preliminary results from our laboratory are the first to indicate that milk production may be affected in these
women. Observational studies indicate the „coming in of milk” may be delayed in mothers that have caesarian
section births. This project involves determining the timing of the initiation of lactation using biochemical
markers in a group of women that have give birth via caesarian section compared to a group that have had a
normal vaginal birth.
3) Persistent pain during breastfeeding – is the breast inflamed?
Next to insufficient milk supply, pain during breastfeeding is the second most common reason for giving up
breastfeeding. Currently there are few tests available to diagnose the cause of the pain, thus there exists a
distinct lack of evidence-based treatments for this condition. Milk samples have been collected from of cohort of
mothers experiencing persistent nipple pain of which the cause is unknown. It is anticipated that assays to
determine the presence of blood as well as markers of inflammation such as lactate will shed light on the health
of the breast. This study may contribute valuable information that may assist in the medical treatment of
lactating women.
12 Biochemistry
4) Determining macronutrients of human milk using near infrared spectroscopy (NIR)
The current nutritive care of preterm infants involves feeding with fortified human milk. Fortification involves
boosting the protein and energy content of human milk by the addition of commercially available fortifiers. Pre-
term infants are born with very immature digestive system and thus have a limited tolerance in accepting the
amount of nutrients given by clinicians. Current method of human milk fortification for these infants does not
taking any consideration of the large variations in the macronutrients of human milk between women and over
the duration of lactation. Therefore it is likely that current method of human milk fortification is resulting in an
under- or overestimation of the actual required level of macronutrients for these infants, which posts a potential
risk on these infants. Hence in order to eliminate such a risk, it is important for clinicians to have access to a
rapid, accurate method for the measurement of baseline macronutrients in human milk. Thus this project has
been set out to investigate the use of near NIR as a rapid method of determining the macronutrients of human
milk.
All of the above projects will provide information so that evidence based procedures can be developed
that will directly impact on the care and health outcomes of both pre-term and term infants.
13 Biochemistry
ASSOCIATE PROFESSOR
MARTHA LUDWIG Room 3.05, MCS Building, Phone: 6488 3744
Email: [email protected] U
The Evolution of Photosynthetic Pathways
Terrestrial plants are typically grouped according to the biochemical pathway they use to fix atmospheric
CO2 into carbohydrates – the so-called C3 plants, which include crop species such as rice and wheat as well
as nearly all trees and herbs; the C4 plants, which include crop plants like corn and sugarcane, and some of
the world‟s worst weeds; and the Crassulacean Acid Metabolism (CAM) plants, which include cactuses,
orchids and pineapple. C4 and CAM plants evolved from C3 plants, and some groups of plants have left
“evolutionary footprints” that give us insights into how this process has occurred at the molecular level.
Other plants are able to “switch” between pathways, depending on the environmental conditions and/or their
developmental stage. Still other plants produce photosynthetic roots when flooded, allowing them to maintain
oxygen levels in tissues that would otherwise become anoxic and die, and use dissolved CO2 in the flood
waters to produce carbohydrates as an energy source.
We are using tools of cell and molecular biology such as differential cDNA library construction and
screening, quantitative reverse transcription PCR (qRT-PCR) and in situ hybridisation to identify key
proteins involved in the above processes and examine the expression patterns of their genes. These studies
will give insight into the evolution of photosynthesis, the process on which all life depends, and the plasticity
of plants in obtaining nutrients from their environment. This information will open avenues for manipulating
these pathways in economically valuable plants and will increase our knowledge of how plants may respond
and cope with predicted future climate scenarios.
Projects include:
1. Examining gene expression patterns of key enzymes in evolutionarily closely related plants that use the
C3, C4 and intermediate C3-C4 photosynthetic pathways.
2. Identifying genes showing differential expression during photosynthetic pathway switching.
3. The biochemical characterisation of photosynthetic isoenzymes that function in the same intracellular
compartment, and the identification of the proteins with which they interact.
4. Identifying genes showing differential expression during the development of photosynthetic aquatic roots.
This project is in collaboration with Assoc Prof Tim Colmer, School of Plant Biology.
Pollen Cell and Molecular Biology – Effects of Global Climate Change
Pollen-related allergy is a significant public health problem, which has increased in recent decades.
Accumulating data indicate that this increase may be a consequence of climate change. Grasses are major
contributors to airborne pollen levels and pollinosis worldwide; however, very little information exists on the
effects of climate change on grass pollen biology, especially with regard to allergen content and dispersal.
Ryegrass, a clinically important allergenic species, is being used as a model grass species to examine the
impact of increased CO2 and temperature on the number, structure and allergen content of grass pollen.
Outcomes of this work will include a greater understanding of the prevalence of grass pollen allergens and
the mechanism of their dispersal under future climate change scenarios, which will contribute to the
development of rational strategies for pollen-related allergy prevention, management, and therapies.
We are producing recombinant ryegrass pollen allergens to use in cell culture assays and quantitative assays
that will ultimately compare the levels of allergens in pollen from plants grown under climate scenarios
differing in CO2 and/or temperature.
14 Biochemistry
PROJECTS
1. Cloning of recombinant ryegrass allergens and their expression in Escherichia coli, and the development
of quantitative and functional cell culture assays. This project is in collaboration with Prof Geoff Stewart,
School of Biomedical, Biomolecular and Chemical Sciences.
2. Examination of ryegrass pollen characteristics under increased temperature. Do plants produce more
pollen or is the pollen structurally and/or biochemically modified under increased growth temperature
relative to control plants?
15 Biochemistry
ASSOCIATE PROFESSOR
THOMAS MARTIN Room 3.47, MCS Building, Phone: 6488 3331
Email: [email protected] UH
The Signalling and Protein Interaction Group
We are interested in eukaryotic cellular signalling. As a model, we are investigating a gene family known as 14-
3-3 genes. In Arabidopsis thaliana, at least 12 genes encoded for 14-3-3 proteins. The proteins form homo and
heterodimers. These dimers bind to other proteins and regulate their activity, cellular localisation or stability in
response to intracellular or extracellular signals.
To date, a great number of potentially 14-3-3 regulated proteins have been identified using in vitro and in silico
approaches. This suggests that 14-3-3s are important global regulators of cellular processes in all eukaryotes
including the regulation of mammalian cell cycle and apoptosis as well as hormone responses and metabolism in
plants. For example, in humans, epigenetic down-regulation of 14-3-3 gene expression was found in a great
number of cancers indicating the importance of 14-3-3 proteins for correct cell function (Hermeking, 2003,
Nature Reviews 3, 931-943). In plants, 14-3-3 proteins regulate development, responses to hormones and to the
environment and metabolic pathways (for a review see: Comparot S, Lingiah G and Martin T, 2003, Journal of
Experimental Botany 54, 595-604).
Despite intensive research into potential 14-3-3 target proteins, many crucial questions remain unanswered. For
example, most of the predicted interactions with 14-3-3 proteins lack experimental in vivo evidence. The
projects proposed here, will address this problem. To approach this, we are using a protein interaction system
named Bimolecular Fluorescent Complementation (BiFC, Bhat R.A. et al., Plant Methods 2006, 2:12) that can
be used in living plants. This system is based on the detection of protein interaction by the generation of a
fluorescent signal which can be observed using fluorescent and confocal microscopy (Figure 1).
Figure 1: The principle of Biomolecular Fluorescence Complementation (BiFC). Two non-fluorescent parts
of the Yellow Fluorescent Protein (YFP) are fused to two proteins of interest 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 (from Bhat R.A. et al., Plant Methods 2006, 2:12).
We have used this system extensively to investigate the formation of 14-3-3 dimers and discovered novel
intracellular localisation patterns for 14-3-3 proteins (Figure 2).
Figure 2: 14-3-3 dimerisation visualised using Bimolecular Fluorescence Complementation. A. 14-3-3 dimer
formation was observed in the Nucleus and cytoplasm of transient transformed Nicotiana benthamiana cells. B.
14-3-3 dimer formation in roots of stable transformed Arabidopsis plants. C. Association of 14-3-3 dimers with
globular ER structures formed during plant cell death.
PROJECTS
16 Biochemistry
1. Identification and localisation of 14-3-3 target protein interactions using BiFC
The aims of this project are:
1) To test a set of predicted 14-3-3 regulated proteins for interaction with 14-3-3 proteins using BiFC
analysis.
2) To identify the subcellular localisation of 14-3-3 interactions with target proteins.
In order to achieve these aims, you will clone a set of genes encoding for predicted 14-3-3 regulated proteins
from cDNAs and genomic DNAs of Arabidopsis thaliana using PCR based techniques. You will select a set of
genes were the proteins are known or predicted to be localised in mitochondria, chloroplasts, the nucleus and the
endoplasmic reticulum. The cloned DNAs will be used to construct translational fusion genes with N and C
terminal YFP fragment (Figure 1). You will use these constructs to test for interaction of the putative target
proteins with 14-3-3 proteins using fluorescent and confocal microscopy. You will further investigate if these
interactions are restricted to a specific subcellular localisation using co-expression with fluorescently labelled
marker proteins.
2. Establishing a map of 14-3-3 dimer formation in a developmental and environmental context
The aims of this project are:
1) To clone 14-3-3 BiFC constructs under the control of native 14-3-3 promoters
2) To generate a 14-3-3 protein interaction map for Arabidopsis thaliana
We have cloned a set of 14-3-3 gene promoters and generated a library of 14-3-3 BiFC constructs suitable for
the analysis of protein interaction. You will use and combine these resources and generate a set of 14-3-3 gene
BiFC constructs which are under the control of the native 14-3-3 promoters. The advantage of such constructs is
that they will only be expressed under the same circumstances as the endogenous 14-3-3 genes. Therefore, once
you have transformed these constructs into Arabidopsis, you will be able to visualise and investigate the
interaction of two 14-3-3 proteins. Using fluorescence and confocal microscopy, you will be able to identify the
plant tissues and cells in which these dimers form, how formation of these dimers is regulated and to establish a
map of 14-3-3 protein interactions for Arabidopsis.
3. Investigating the regulation of 14-3-3 dimer formation using BiFC analysis
14-3-3 protein dimers are likely to form, destabilise or change binding partners in response to signals or
treatments applied. In theory, such changes can be observed using Bimolecular Fluorescence Complementation
(BiFC). However, one of the problems associated with BiFC analysis is the stability of protein dimers once they
are formed. This stability makes it difficult to investigate the impact of treatments on existing protein
interactions, e.g. on preformed 14-3-3 protein dimers, and to discover the formation of new interactions. One
potential way to overcome this problem is the application of photobleaching. It obliterates existing fluorescence
and allows for newly appearing fluorescence to be observed. Thus this approach is suitable for the analysis of
dimer formation in response to a treatment. A second possible way to investigate the impact of treatments on 14-
3-3 dimer formation is the use of inducible promoters. This will allow expressing the interacting proteins only
after the treatment was applied. Thus, all interaction observed would be under the influence of the treatment and
can be compared to non-treated (control) conditions.
Investigating the suitability of the two outlined approaches is one of the aims of this project. The second aim is
the application of this approach to test the impact of treatments on 14-3-3 dimerisation and subcellular
localisation. You will start your work using transgenic Arabidopsis plants expressing two 14-3-3 protein BiFC
constructs. We used such plants to demonstrate dimerisation of 14-3-3 proteins (Figure 2b). You will test the
impact of photobleaching and investigate the appearance of new dimers using fluorescence microscopy. You
will further select inducible promoter, clone these promoters so that they regulate the expression of 14-3-3 BiFC
construct and transform plants. You will then test these plants for 14-3-3 dimer formation before and after
induction of the promoter and in response to treatments likely to impact on 14-3-3 dimer formation.
17 Biochemistry
WINTHROP PROFESSOR
HARVEY MILLAR ARC Centre of Excellence in Plant Biology
(http://www.plantenergy.uwa.edu.au/aboutus/scholarships_uwa.html)
Room 4.74, Phone: 6488 7245
Email: [email protected] U
Using a combination of protein separation techniques, mass spectrometry and informatics my research group is
seeking to understand the compartmentation of cellular functions in cellular organelles. The major organelles in
green plant cells are the chloroplast (for photosynthesis), the mitochondrion (for respiration), and the
peroxisome (for carbon and nitrogen metabolism). These three organelles divide and organize their energy
conversion operations in a cooperative fashion. This cooperation is pivotal to directing energy capture and
storage in the form of sugars, starch, oils, protein and fibre. The metabolism of plant organelles also underlies
the growth and performance of plants including their ability to withstand environmental stresses. Further, the
synthesis of key antioxidants, vitamins and cofactors is central to their development of products that are vital for
human nutrition (e.g. vitamins A, C, and E, biotin, folic and lipoic acid, carotenoids). We are also studying
honeybee biology and reproductive success through proteomics in order to protect the critical role of these
social insects in plant pollination.
PROJECTS
1. Modification of Mitochondrial Proteins and Function by Oxidative Stress
Aerobic organisms exploit the redox chemistry of oxygen to efficiently derive energy from oxidation of
substrates. However, due to the tendency of molecular oxygen to gain single electrons and form reactive oxygen
species (ROS) this energy production comes at a price. ROS, and the hydroxyl radical in particular, are highly
reactive and can cause rapid and deleterious oxidation of biomolecules such as proteins, lipid and DNA. The
mitochondrial electron transport chain produces significant quantities of ROS. However, despite this knowledge
that plant mitochondria can produce ROS and contain mechanisms that may limit ROS accumulation, we are
only beginning to understand the antioxidant system of plant mitochondria and the consequences for metabolism
in this organelle if oxidative stress occurs. We have been studying specific proteins that are damaged by ROS,
lipid peroxidation products and antioxidant defence enzymes. This project will follow up on our past work using
mitochondrial function assays, proteomics and transcript analysis to further unravel the damage done to
mitochondria by oxidative stress and the response of mitochondria to protect themselves and repair damage.
Publications
1. Taylor NL, Heazlewood JL, Day DA, Millar AH (2005) Differential Impact of Environmental Stresses on the
Pea Mitochondrial Proteome. Molecular & Cellular Proteomics 4:1122-1133.
2. Winger, AL, Taylor NL, Heazlewood JL, Day DA, Millar AH (2007) The cytotoxic lipid peroxidation
product 4-hydroxy-2-nonenal covalently modifies a selective range of proteins linked to respiratory function in
plant mitochondria. The Journal of Biological Chemistry 282:37436-47.
2. Plant Mitochondrial Proteomics, Databases and Bioinformatics
The mitochondrion is the organelle within the eukaryotic cell that is primarily concerned with the synthesis of
ATP in the fundamental process known as respiration. It is predicted that mitochondria synthesise 3-5% of the
proteins required for their function, with the remaining 95-98% of proteins required encoded by the nuclear
genome and targeted back to the mitochondria as protein precursors using encrypted targeting information in the
protein sequence. The detection of these encryptions and thus identification of the full set of these genes within
the nuclear genome is a major challenge for biologists. We have established a database of these genes and
proteins and incorporated bioinformatic targeting prediction tools and experimental data (see
HUwww.suba.bcs.uwa.edu.au UH ). This honours project will utilise the bioinformatic database of predicted
mitochondrial proteins and experimental proteomics to further uncover the mitochondrial proteome. This will
involve specifically targeting a particular group of proteins, such as RNA binding proteins, phosphoproteins,
specific proteins in the intermembrane space, metal binding proteins or NADH binding proteins. UAlternativelyU,
strongly bioinformatic projects utilizing current data and exploring novel biological insights can be designed, for
this some interest and background in computer science, web interface design and script writing will likely be
required.
Publications
1. Heazlewood JL, Verboom RE, Tonti-Filippini J, Small I, Millar AH. (2007) SUBA: the Arabidopsis
Subcellular Database. Nucleic Acids Res. 35:D213-8.
18 Biochemistry
2. Huang S, Taylor NL, Whelan J, Millar AH. (2009) Refining the definition of plant mitochondrial
presequences through analysis of sorting signals, N-terminal modifications, and cleavage motifs. Plant Physiol.
150:1272-85.
3. Ito J, Taylor NL, Castleden I, Weckwerth W, Millar AH, Heazlewood JL (2009) A survey of the Arabidopsis
thaliana mitochondrial phosphoproteome Proteomics (in press)
3. Rice Proteomics and Oxygen as a Trigger for Energy Metabolism
The structure and functional status of mitochondria in the absence of O2 has intrigued researchers for decades.
Mitochondrial structures appear to proliferate in rice seedlings even when they are grown under anoxic
conditions from dry seed. We are using rice as a model system for studying the mitochondrial proteome in rice,
the oxygen trigger in mitochondrial biogenesis, the establishment of aerobic metabolism, and the energy
generating processes in rice during long-term anaerobic metabolism. An honours project in this area would
involve proteomic analysis of rice tissues or isolated mitochondria under anoxia or during return to air and
attempts to unravel specific aspects of the events and signalling process that regulate this process in rice cells.
Publications:
1. Millar AH, Trend AE, Heazlewood JL (2004) Changes in the rice mitochondrial proteome during the anoxia
to air transition focus around cytochrome containing respiratory complexes. The Journal of Biological
Chemistry 279:39471-39478
2. Howell KA, Cheng K, Murcha MW, Jenkin LE, Millar AH, Whelan J (2007) Oxygen initiation of respiration
and mitochondrial biogenesis in rice. Journal of Biological Chemistry 282:15619-31
3. Huang S, Colmer TD, Millar AH (2008) Does anoxia-tolerance involve altering energy currency towards PPi?
Trends in Plant Science 13:221-227.
4. Narsai R, Howell KA, Carroll A, Ivanova A, Millar AH, Whelan J (2009) Defining core metabolic and
transcriptomic responses to oxygen availability in rice embryos and young seedlings Plant Physiol. (in press)
4. Subcellular Proteomics in Arabidopsis
Central metabolism, biosynthesis of high quality and high quantity products and cellular signalling pathways in
defence from the physical environment and invading pathogens, are all essentially compartmented processes in
plant cells, but only a small number of the proteins in plants have known location. The overall aim of my
research in this area is to identify the intracellular location of proteins in the model plant Arabidopsis using
proteomics in eight subcellular compartments isolated by cellular fractionation. This will allow a view of
subcellular proteomes for plastids, peroxisomes, nuclei, plasma membrane, endoplasmic reticulum, golgi,
tonoplast and cytosol. An honours project in this area would aim to biochemically purify one of these
compartments based on modification of existing methods and to begin the mass spectrometry analysis of its
constituent proteins using shot-gun proteomics (using LC-MS/MS analysis).
Publications:
1. Heazlewood JL, Tonti-Filippini J, Verboom RE, Millar AH (2005) Combining experimental and predicted
datasets for determination of the subcellular location of proteins in Arabidopsis. Plant Physiology 139:598-
2. Millar AH, Carrie C, Pogson B, Whelan J (2009) Exploring the Function-Location Nexus: Using Multiple
Lines of Evidence in Defining the Subcellular Location of Plant Proteins Plant Cell (in press)
5. Honeybee Proteomics
With Dr Boris Baer, ARC COE in Plant Energy Biology & School of Animal Biology (FNAS)
Apart from being used for honey production, honeybees are the worldwide most important species for crop
pollination. However, we currently face a dramatic and global decline in honeybee populations with severe
expected consequences for agricultural yields. To counter the dramatic losses of honeybee colonies, detailed
studies about honeybee reproduction will ultimately allow optimizing breeding and allowing to compensate for
the current losses. Honeybee reproduction is quite spectacular, as queens only mate at the beginning of their
lives, during one or very few mating flights. Afterwards they are able to store millions of sperm for years and
use them in very economic ways to fertilise millions of eggs. We have very little information how queens are
able to keep sperm alive of years, how active sperm remains during storage and how sperm potentially interacts
with the female. This honours project will use proteomic tools to isolate and identify proteins that are relevant
during sperm storage and reveal their possible effects on sperm survival and paternity success. The project is
supported by Australian beekeepers and will use gel electrophoresis, mass spectrometry and the recently
published bee genome sequence. 1. Baer B, Heazlewood JL, Taylor NL, Eubel H, Millar AH (2009) The seminal fluid proteome of the honeybee
Apis mellifera. Proteomics 9:2085-97.
2. Baer B, Eubel H, Taylor NL, O'Toole N, Millar AH (2009) Insights into female sperm storage from the
spermathecal fluid proteome of the honeybee Apis mellifera. Genome Biology (in press)
19 Biochemistry
5B PROFESSOR IAN SMALL ARC Centre of Excellence in Plant Energy Biology
27BRoom 4.03, MCS building, Phone: 6488 4499
28BEmail:[email protected] U
Organelle Gene Expression Group
Our group is studying the regulation of gene expression inside mitochondria and chloroplasts and how this is
coordinated with nuclear gene expression. Our focus is on genes that are involved in photosynthesis and
respiration and include those that code for some of the most important and abundant proteins on the planet.
These genes are mostly regulated post-transcriptionally but the control loops involved need to be linked to
transcriptional control of nuclear genes via signalling pathways that are still to be discovered. Our aim is to
understand how energy metabolism in plants is regulated, with the goal of generating discoveries relevant to
optimal use of plants in agricultural and environmental applications. Much of the research will build upon the
discovery of the PPR protein family, a novel family of 450 sequence-specific RNA-binding proteins implicated
in these processes (Schmitz-Linneweber and Small, Trends Plant Sci, 13, 663-670). The experiments will be
carried out on the model plant Arabidopsis thaliana to make full use of the existing data and resources.
Prospective Honours students with a background in Molecular Biology, Biochemistry, Genetics or Computer
Science are particularly encouraged to apply. The projects will benefit from the full-range of expertise and
equipment on plant energy biology within the ARC Centre of Excellence and will be at the forefront of research
in this field.
16BPROJECTS
1. Linking chloroplast and nuclear gene expression
Several PPR proteins have been found to be needed for the correct activity of the chloroplast-encoded RNA
polymerase (RPO) that transcribes almost all of the photosynthesis genes. These include pTAC2 and GUN1
(which contain DNA-binding domains in addition to their RNA-binding PPR motifs) as well as CLB19 and
FLV, which are required for editing of the RPO transcripts. The phenotypes of mutants in these genes strongly
suggest that some or all of them are involved in a retrograde signalling pathway from the chloroplast to the
nucleus that controls the activation of nuclear photosynthesis genes.
This project will try to elucidate of the role of these PPR proteins and the chloroplast RNA polymerase during
chloroplast biogenesis in early seedling development. It will involve the detailed characterization of chloroplast
and nuclear gene expression in wild-type and mutant plants using Affymetrix microarrays and quantitative RT-
PCR. The assembly and activity of
the RNA polymerase will be
followed by immunodetection and
transcription assays. The DNA and
RNA-binding activities of the PPR
proteins will be analysed using
RIP-Chip or EMSA assays. Protein
synthesis and accumulation inside
chloroplasts will be followed by
western blot with a suite of
antibodies against the most
important photosynthesis proteins.
1. Koussevitzky et al. Signals from chloroplasts converge to regulate nuclear gene expression (2007) Science
316: 715-719
2. Pogson et al. Plastid signalling to the nucleus and beyond (2008) Trends Plant Sci 13: 602-609
20 Biochemistry
2. Discovering the function 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. Different types of editing have been described, generally
involving a specificity factor (RNA or protein) that recognizes the editing site and an enzyme catalyzing the
reaction. RNA editing alters the sequence of many different types of RNAs in many organisms including
plants and animals and thus constitutes a form of epigenetic gene regulation. In many cases, RNA editing is
essential for correct production of the protein encoded by the RNA, whilst in other cases, RNA editing
changes the functional properties of the encoded protein. In higher plants, RNA editing consists of C to U
changes and has been reported only in organelle transcripts, where over 500 different editing sites are now
known. Thirteen PPR proteins have been found to be essential for the editing of specific sites in chloroplast
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.
Despite big advances in understanding the mechanism involved, we still only have a very hazy idea about the
functions of RNA editing. In principle, it could be an extremely unusual way of regulating gene expression
or even protein function. To explore these questions, this project will test several Arabidopsis mutants
defective for specific editing events under a range of environmental stresses (high or low temperature; high
or low light, etc.). We expect physiological differences to be revealed under extreme conditions that will
explain how editing events are selected for in the natural environment. The lab techniques employed will
include detailed analysis of gene expression via real-time PCR and physiological characterisation of various
parameters of photosynthesis and respiration.
1. Kotera et al. (2005) A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature
433: 326-30.
2. Okuda et al. (2009) Pentatricopeptide Repeat Proteins with the DYW Motif Have Distinct Molecular
Functions in RNA Editing and RNA Cleavage in Arabidopsis Chloroplasts. Plant Cell 21: 146-156 3. In vitro structural and functional analysis of Arabidopsis PPR proteins
With Dr. Will Stanley and in collaboration with Prof. Charlie Bond, Biochemistry
Little is known about the molecular mechanisms behind the functions of PPR proteins in processing of
organelle encoded RNA transcripts. Some PPR proteins may specifically recognise RNA molecules and
carry out the processing by themselves (route 1 in the figure) while others may recruit additional protein
factors to catalyse processing (route 2 in the figure). Questions arise as to exactly how a given PPR interacts
with a given RNA molecule – is there an RNA sequence-specific interaction, or does the RNA adopt a
secondary structural arrangement amenable to PPR binding? Do some PPR proteins interact directly with
both RNA and other protein factors? Could there be cooperativity in assembly of this type of multi-
molecular complex?
Work is underway to study the atomic
resolution structure and chemistry of RNA
recognition for a selection of PPR proteins,
using X-ray crystallography. Additional
biophysical techniques, such as circular
dichroism (CD) and fluorescence spectroscopy
and isothermal titration calorimetry (ITC) are
in use to dissect structural and thermodynamic
parameters of multi-molecular complex
assembly. Recombinant protein production
and purification are prerequisites for
biophysical analysis and specialised
crystallisation techniques are required to make
PPR crystals. Thus, an honours project covering basic molecular biology, protein biochemistry and high-
fidelity macromolecular characterisation is available. The project would suit a chemistry, biochemistry or
biophysics student.
21 Biochemistry
PROFESSOR STEVE SMITH ARC Centre of Excellence in Plant Energy Biology
and Centre of Excellence for Plant Metabolomics
Room 4.05, MCS Building, Phone: 6488 4403
Email: [email protected] U
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.
22 Biochemistry
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.
23 Biochemistry
ASSOCIATE PROFESSOR
NAOMI TRENGOVE Room 2.04, MCS Building, Phone: 6488 4421
Email: [email protected] UH
Wound Healing Research Group
The investigation of the causes of impaired wound healing in chronic wounds in humans represents a
challenging area of medical research. Fortunately, with the emergence of new techniques in both cellular and
molecular biology it is now possible to use the small tissue samples obtained from humans to understand the
process more accurately.
Venous leg ulceration is a debilitating chronic wound that occurs most often in the elderly and is the result of
venous hypertension in the lower limb (venous disease). The pathogenesis of ulceration is not well
understood, but there is evidence that elevated levels of inflammatory mediators (e.g. tumor necrosis factor-
alpha) are involved (1,2). Susceptibility to ulceration in patients with venous disease varies. Professor
Stacey’s team has identified a polymorphism in the tumor necrosis factor-alpha gene (TNFA-308A) that is
associated with increased risk of ulceration (3). Further studies are required to determine whether carriage of
this allele is part of the cause of venous leg ulceration or just a marker of another causal allele in close
proximity on the chromosome.
PROJECT
With Professor Mike Stacey and Dr Hillary Wallace, School of Surgery and Pathology
This project will be undertaken in collaboration with the wound healing research group of the School of
Surgery and Pathology (Faculty of Medicine and Dentistry), which undertakes clinical and laboratory studies
into chronic venous leg ulceration.
The proposed Honours project will investigate whether the tumor necrosis factor-alpha (TNFA) genotype in
humans with chronic venous ulceration is associated with differences in the TNF phenotype. That is, do
individuals with different TNFA genotypes produce different amounts of TNF-alpha? The levels of TNF-
alpha protein and mRNA will be assessed in the wound and in stimulated peripheral blood leukocytes of
patients with leg ulcers who have been genotyped in a previous study. The working hypothesis is that TNF-
alpha protein and mRNA levels will be increased in patients carrying the TNF-308A allele compared to the
wild-type TNF-308G allele. Patients homozygous for the A and G alleles will be compared to minimise
individual variation. This project will provide opportunities to interact with patients in a clinical setting, as
well as giving a sound grounding in laboratory research techniques applicable to medical research.
Laboratory techniques for this study will include: Cell culture, RNA extraction from cells and tissue, ELISAs
and real-time PCR and Immunohistochemistry.
References
(1)Trengove NJ, Bielefeldt-Ohmann H, Stacey MC. Mitogenic activity and cytokine levels in non-healing
and healing chronic leg ulcers. Wound Repair Regen. 2000 Jan-Feb;8(1):13-25.
(2)Wallace HJ, Stacey MC. Levels of tumor necrosis factor-alpha (TNF-alpha) and soluble TNF receptors in
chronic venous leg ulcers--correlations to healing status. J Invest Dermatol. 1998 Mar;110(3):292-6.
(3)Wallace HJ, Vandongen YK, Stacey MC. Tumor necrosis factor-alpha gene polymorphism associated
with increased susceptibility to venous leg ulceration. J Invest Dermatol. 2006 Apr;126(4):921
24 Biochemistry
25 Biochemistry
6BASSOCIATE PROFESSOR
ROBERT TUCKEY 29BRoom 3.71, MCS Building, Phone: 64883040,
30B Email: [email protected] U
Molecular Steroidogenesis Group
Current research involves structure-function studies on cytochrome P450scc, metabolism of vitamins D2 and
D3 by cytochrome P450scc, and the activation and inactivation of vitamin D by other mitochondrial-type
cytochromes P450.
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) is a mitochondrial P450 that
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.
Other mitochondrial P450s we are studying are 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1) and
vitamin D 24-hydroxylase (CYP24). CYP27B1 catalyses the final step in the activation of vitamin D, 1α-
hydroxylation of 25-hydroxyvitamin D to produce 1,25-dihydroxyvitamin D3, which occurs primarily in the
kidney. 1,25-Dihydroxyvitamin D3 is the hormonally active form of vitamin D that regulates calcium
metabolism, but also has many other important effects including inhibiting proliferation and promoting
differentiation of a range of cells, plus regulating the immune system. We have expressed mouse CYP27B1
in E. coli and shown that the purified enzyme can rapidly hydroxylate 25-hydroxyvitamin D3 incorporated
into the phospholipid bilayer of synthetic vesicles. We are currently working on human CYP27B1. CYP24
acts on 1,25-dihydroxyvitamin D3, hydroxylating it at C24 which causes its inactivation. We are expressing
the human CYP24 in E. coli, and studying its catalytic properties.
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. P450scc did cleave the side chain of 7-
Figure 1. Model of the interaction
of cytochrome P450scc with the
inner-mitochondrial membrane
26 Biochemistry
dehydroccholesterol, the vitamin D3 precursor, producing 7-dehydropregnenolone. The cleavage of the side
chain of 7-dehydrocholesterol by P450scc to produce 7-dehydropregnenolone provides an explanation for the
accumulation of 7-dehydrosteroids in Smith-Lemli-Opitz syndrome where there is an excess of 7-
dehydrocholesterol due to a 7-dehydrocholesterol reductase deficiency. We plan to directly test this
hypothesis by examining the subsequent metabolism of 7-dehydropregnenolone by other steroidogenic
enzymes such as P45017 (see Project 1).
We have carried out biological testing of several of the novel P450scc-derived hydroxyvitamin D3 products
in collaboration with Professor Slominski. 20-Hydroxyvitamin D3 has been found to be as effective as the
hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3, in inhibiting cell proliferation and
promoting differentiation of a variety of cells including skin, breast and prostate carcinomas. Importantly, it
does not raise serum calcium levels in rats and consequently lacks the toxic side effect of hypercalcaemis
caused by high doses of 1,25-dihydroxyvitamin D3. 20-Hydroxyvitamin D3 thus shows promise as a
therapeutic agent for the treatment of hyperproliferative disorders including cancer.
17BPROJECTS
1. Can 7-dehydropregnenolone be metabolized by steroidogenic enzymes and if so what are the
products?
We have shown that P450scc can convert the vitamin D3 precursor, 7-dehydrocholesterol, to 7-
dehydropregnenolone. The observation that various 7-dehydrosteroids are produced in the Smith-Lemli-
Opitz syndrome, where there is an excess of 7-dehydrocholesterol due to a 7-dehydrocholesterol reductase
deficiency, suggests that other steroidogenic enzymes can act on 7-dehydropregneolone to produce a number
of novel steroids. Enzymes to be tested will include P45017, P45011 and 3 -hydroxysteroid
dehydrogenase. Initial studies will involve incubating bovine adrenal microsomes which contain P45017 and
3 -hydroxysteroid dehydrogenase, with 7-dehydropregnenolone, isolating the products and analysing them
by HPLC. Subsequent studies will involve purifying the enzymes (from adrenals or expressed in bacteria)
and examining their ability to act on 7-dehydropregnenolone in a reconstituted system. Products of
enzymatic metabolism of 7-dehydrocholesterol will be identified by mass spectrometry and/or NMR.
2. Can CYP27A1 metabolize novel P450scc-derived hydroxyvitamin D derivatives?
CYP27A1 is a mitochondrial P450scc that can hydroxylate vitamin D3 in the 25-position, a necessary step in
the activation of vitamin D to 1,25-dihydroxyvitamin D3. It can also hydroxylate cholesterol in the 27-
position as a step in the synthesis of bile salts. Hydroxyvitamin D3 compounds produced by P450scc such as
20-hydroxyvitamin D3 are of interest for the treatment of cancer but may undergo 25-hydroxylation in vivo
producing products with modified activities. The aim of this project therefore is to test the ability of
CYP27A1 to metabolize P450scc-derived hydroxyvitamin D compounds. The project will involve setting up
the expression of human CYP27A1 in E. coli, purifying the expressed enzyme and testing its ability to
hydroxylate hydroxyvitamin D derivatives incorporated into the membrane of phospholipid vesicles. It will
also involve scaling up of successful reactions to produce adequate amounts of product for biological testing
to determine whether 25-hydroxylation modifies the antiproliferative activity of the parent compounds.
Note; other projects related to my field of study are possible and new projects arise as research progresses. I
am happy to discuss other projects with students expressing an interest.
27 Biochemistry
DR DANIELA ULGIATI Room 3.03, MCS 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 undentification of the unidentified binding factors
will be achieved using 2D gel/proteomics based approaches.
2. The role of CR2 promoter polymorphisms in Systemic Lupus Erythematosus (SLE) and
Rheumatoid Arthritis (RA).
Complement receptor 2 (CR2) is an important receptor that is required for a normal B cell immune response.
It is expressed at a critical stage in B cell development and has been implicated in a number of autoimmune
diseases. The significance of mechanisms that regulate CR2 expression is apparent by studies of human B
cell CR2 expression in patients with SLE and RA. Both patient groups have abnormalities in the expression
of CR2 on B cells (~50% of normal) and this decrease correlates with disease activity. With the recent
advent of transgenic and knockout mice, several groups have examined the importance of CR2 in a lupus
prone mouse model. Studies of these mice have also found an early decrease in CR2 expression that is
initially detected prior to any major clinical manifestations. We have recently sequenced the CR2 promoter
in a number of SLE patients and have found several single nucleotide polymorphisms (SNPs) within
functional regions of the promoter. We are currently assessing the functional implications of these
polymorphisms on the transcriptional regulation of CR2. This project involves determining the expression
status of CR2 on patient B cells by correlating cell surface expression with mRNA levels and transcriptional
activity. Furthermore, collating the expression and transcriptional data with the promoter phenotypes will
ultimately determine whether these promoter polymorphisms are indeed having an effect on CR2 expression
in patients with autoimmune diseases.
28 Biochemistry
3. Characterisation of the Upstream Repressor Element in the Complement C4 Gene and its control by
Lupus-associated Factors. (Co-supervised with Prof Lawrie Abraham)
The fourth component of human complement (C4) is a serum protein involved in initiation of immune and
inflammatory reponses. Previously, we have analysed the transcriptional regulation of the C4 gene. To
determine the requirements for basal and regulated
expression, we 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.
4. Understanding the Role of Notch Signalling and associated Transcription Factors in Lineage
Commitment. (Co-supervised with Prof Lawrie Abraham)
Notch signaling is an evolutionarily ancient mechanism which plays a critical role in dictating cellular fates.
Signals transmitted via Notch receptors control how cells respond to developmental cues and in turn control
lineage commitment. Notch signalling is intimately involved in lineage specification and differentiation of
lymphocytes.
Commitment to the B-lineage requires inhibition of Notch signals in lymphoid progenitors. Notch signals in
this context repress Pax5 expression thereby blocking B-cell differentiation. On the other hand, negative
regulation of Notch signals by the inhibitory Notch modulator deltex1, skews commitment of lymphoid
progenitors to the B-lineage. While, Notch1 signaling must be down-regulated to permit B-cell commitment,
the involvement of Notch signaling at subsequent stages of B-cell development in bone marrow have not
been clearly defined. Notch signaling also has important consequences for T lymphocytes. Dysregulated
Notch1 signaling leads to T cell leukemia in humans and mice. The ability of Notch to cause T cell neoplasia
results from aberent expression during thymocyte development, where Notch receptor expression and
signaling occur at distinct developmental stages. There is evidence that Notch expression at very early stages
of lymphoid development commit progenitors to the T cell lineage. Recent evidence indicates that Notch
may also influence mature T cell development.
We have recently developed an ex vivo model in which to study Notch signaling. Cells are co-cultured with
stromal cell lines ectopically expressing the Notch ligand, delta-like-1 (OP9-DL). Cells attached to the
stroma or in suspension following co-culture were harvested and can be analysed for differentiation and
neoplastic markers and associated transcription factors. Since Notch signaling is known to upregulate the
bHLH factor HES-1, we can also measure transcript abundance of this marker of Notch activation to ensure
proper induction of Notch by dela-like-1 ligand in the co-cultures.
Extra-cellular signal
Initiation complex
- Activation
Dissociated complex
- no activation
A. Induced C4 Expression
B. Repressed C4 Expression
Sp1
BKLF
BKLF
Upstream Repressor element
Upstream Repressor element
Sp1
FIGURE 1
29 Biochemistry
WINTHROP PROFESSOR
ALICE VRIELINK 31BRoom 4.31, MCS Building, Phone: 6488 3162
32BEmail: [email protected]
0BProtein 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.
18BPROJECTS
1. Endotoxin Biosynthesis in Neisseria.
The gram negative bacteria, Neisseria meningitidis, is the causative agent of meningitis and is responsible for
significant mortality throughout the world. A characteristic feature of these bacteria is the presence of
lipooligosaccharide (LOS) molecules on their outer membranes. These complex molecules, also called
endotoxins, are structural components that play a role in the pathogenicity of the organism. Twelve different
immunotypes are found depending on the structure of the LOS moiety. One aspect of complexity of the LOS
group that plays a role in defining the specific immunotype is the presence and location of
phosphoethanolamine (PEA) residues. The enzyme responsible for adding the PEA residue to the LOS
group is phosphoethanolamine transferase. Different forms of PEA transferase are present depending on the
precise location of the PEA moiety on the LOS molecule. Knowledge of the biosynthesis and regulation of
meningococcal lipoooligosaccharides will provide a more detailed understanding of the role of this molecule
in pathogenesis and disease. In collaboration with Dr. Charlene Kahler of the Department of Microbiology at
UWA we have begun a study to determine the three dimensional structure of the enzyme LPTA, the
phosphothanolamine transferase specific for phosphorylation of the lipid A core of LOS.
This project will involve protein expression, purification, crystallization and structure determination using
crystallographic techniques. The structural results will be correlated with functional studies carried out by
Dr. Kahler and coworkers.
2. The Design of Therapeutic Agents to Treat Gastric Ulcers and Gastric Cancer
The bacterium Helicobacter pylori, is the leading cause of gastric
ulcers, infecting over half of the world population. Furthermore,
patients infected with the bacteria exhibit an increased risk of
developing gastric cancer, with 900,000 new cases diagnosed yearly.
The current treatment for H. pylori infection consists of a one week
triple therapy of antibiotics. Due to the widespread use of antibiotics
however, the bacteria are able to develop resistance resulting in
increasing rates of failed antibiotic treatment. Consequently there is a
pressing need for the development of new treatment options that will
allow for a continued high quality of life for those people suffering
from peptic ulcers. One mechanism by which H. pylori establishes
infection is by extracting cholesterol molecules from the host’s epithelial cells, and modifying them in such a
manner that the bacterium is able to evade phagocytosis, an essential effector response of the host’s immune
system. This alteration is carried out by a specific cholesterol -glucosyltransferase (CGT), which transfers a
single glucose molecule to cholesterol. Crystallographic studies of CGT will provide a detailed view of the
enzyme active site, which will facilitate the development of an anti-H. pylori drug therapy based on the
inhibition of this enzyme.
The enzyme has been cloned into a bacterial expression vector in our laboratory. In this project expression
of the enzyme from E. coli will be optimized and the protein purified by chromatographic methods.
Crystallization trials will be carried out on the enzyme with the aim of obtaining X-ray diffraction quality
An image of the bacterium
Helicobacter pylori, the causative
agent of gastric ulcers and gastric
carcinoma
30 Biochemistry
Electron density view of the isoalloxazine
ring of cholesterol oxidase. The density
clearly shows single electron differences
for individual atoms.
Difference electron density showing the
positions of hydrogen atoms in a region of
the structure.
Structure of the dimeric L-amino acid
oxidase from the snake venom of
Malayan pit viper. The glycosylations
are also indicated.
crystals. In addition, circular dichroism and other biophysical studies will be carried out on the enzyme in
order to establish the protein stability and elucidate whether structural changes occur to the enzyme upon
ligand binding.
3. 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
substrate. The structure of the enzyme in the presence of a
substrate and an inhibitor have been determined in our
laboratory and reveal a channel that may act as the peroxide exit
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 expressed in a eukaryotic system. In this project you will develop a Bacculovirus expression system
for the enzyme to produce functional protein. Site directed mutagenesis, kinetic analysis and
crystallographic studies will be undertaken to establish the roles of discrete residues in oxidation chemistry
and its relationship to apoptosis.
4. Probing the Structure of Cholesterol Oxidase – A Novel Antibiotic Target.
The flavoenzyme cholesterol oxidase constitutes an
important virulent factor in immunocompromised patients
prone to Rhodococcus equi lung infections. The
longstanding problem with antibacterial drug resistance
calls for a continued need to probe new targets for the
design and development of novel antibiotic treatments. The
design of novel antibacterial drugs is facilitated by a
detailed knowledge of the architecture of active site,
including the positions of hydrogen atoms, the ionization
state of titratable groups and the precise conformational
state of side chains and cofactors through the substrate
binding and catalysis events.
Our laboratory uses a combination of crystallographic,
mutagenesis and kinetic methods to understand these
events. We have crystals of cholesterol oxidase that diffract
to sub-Ångstrom resolution providing an unprecedented
view of the enzyme structure. Different redox states of the
enzyme can be followed spectrophotometrically in the
crystal and ligands bound to induce oxidation chemistry
while maintaining sub-Ångstrom diffraction. This provides
us with a unique opportunity to visualize transient states and
establish structural changes as a function of the redox state.
Honours projects, focused on testing hypotheses on redox
activity and oxygen reactivity for the enzyme include:
(i) Investigating the structural and electronic differences between the oxidized and reduced enzyme and
(ii) Examining whether a tunnel is involved in oxygen access to the active site during the oxidative half
reaction.
31 Biochemistry
PROFESSOR JIM WHELAN ARC Centre of Excellence in Plant Energy Biology
33BRoom 4.73, MCS Building, Phone: 6488 1749
34BEmail: [email protected] U
Molecular Genetics and Genomics
We use a variety of ‘omic’ approaches to carry out discovery based investigations concerning the control of
gene expression in cells, primarily using the plant models, rice and Arabidopsis, and to a lesser extent yeast.
In addition to the characterisation of motifs and transcription factors that control gene expression, we use
quantitative proteomic and metabolomic approaches, along with bio-informatic analyses coupled with
transgenic approaches to obtain a global view of the factors that control gene expression. Overall these
approaches will lead to a greater fundamental understanding of Molecular Cell Biology.
This research is carried out in the ARC Centre of Excellence in Plant Energy Biology provides 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 (Shanghai, China), The Max-Plank Institute in
Golm (Berlin, Germany), Ludwig-Maxmillians University (Munich, Germany) and Umea and Stockholm
Universities (Sweden) provide students with the opportunity to study overseas, supported by a variety of
grants or scholarships provided by the centre. The Centre provides a variety of scholarships to support
students in their studies.
19BPROJECTS
1. Identifications of transcription factors controlling stress responses
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. Due
to these roles, mitochondria are both a target of stress and play a central role in the stress response in cells. A
variety of proteins are induced in mitochondria in response to various stresses. The role of many of these
proteins is unknown. However, the absence of some of these proteins leads to a greater sensitivity to stresses
and an altered response to stress. Using whole genome transcriptome approaches we have defined the core
response of mitochondria to stress, i.e. the groups of genes that are induced under all stresses. The overall
aim now is to identify the transcription factors that regulate these core stress responsive genes.
A variety of projects are available in this area. Each projects has a team leader of a post-doctoral researcher
and 1 to 2 Ph. D. students. Honours students working on these projects will join these small teams with their
own individual projects. The approaches used will be to clone the promoters and determine the role of
specific sequence elements in regulating expression in response to stress using reporter genes, site directed
mutagenesis and transformation of plants. The binding properties of these sequences will be investigated
using Yeast 1-hybrid analysis and electromobility shift assays.
32 Biochemistry
2. Molecular dissection of the response of rice to anoxia
Rice is one of a handful of higher eukaryotic organisms that can withstand a prolonged period of anoxia.
This is achieved by a variety of molecular responses that result in biochemical and morphological
adaptations leading to survival under anoxia. We have used microarrays to investigate the changes in
transcript abundance following alterations in oxygen availability and can identify a number of primary
(within 1 hour) and secondary effects (after 1 hour). This has allowed the identification of core responses to
anoxia. Thus, we now have a set of genes that responsed to anoxia under several different experimental
conditions and transcriptome analysis has identified a core set of approximately 100 transcription factors that
regulate the expression of these genes. Thus, the overall aims of this project are to identify promoter regions
and cognate transcription factors that regulate expression of anaerobic genes.
This will be achieved as follows:
1) Cloning promoters in front of reporter genes and testing expression in the presence and absence of
oxygen after transformation into rice.
2) Deletion analysis of predicted elements to determine the role they play in regulating gene
expression
3) Testing the ability of selected transcription factors to bind promoter regions using Yeast 2-Hybrid
screening.
Different experimental conditions allow the genes involved in the core responses to aerobic and anaerobic
conditions to be defined – Providing a platform to characterize the regulatory events that defined these
changes.
References
Refer to HUhttp://www.plantenergy.uwa.edu.au/ UH for all publications and more details about scholarships.
Van Aken O, Giraud E, Clifton R and Whelan J (2009) Alternative oxidase: a target and regulator
of stress responses Physiologia Plantarum (in press) PMID: 19470093
Olivier Van Aken, Botao Zhang, Chris Carrie, Vindya Uggalla, Ellen Paynter, Estelle Giraud, and
James Whelan (2009) Defining the Mitochondrial Stress Response in Arabidopsis thaliana.
Molecular Plant Advance Access published on July 24, 2009.
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 Physiology 50: 1286-1296
Giraud E, Ho LH, Clifton R, Carroll A, Estavillo G, Tan YF, Howell KA, Ivanova A, Pogson BJ,
Millar AH and Whelan J 2008 The absence of Alternative Oxidase 1a in Arabidopsis thaliana
results in acute sensitivity to combined light and drought stress Plant Physiology 147: 595-6
Narsai R, Howell KA, Carroll A, Ivanova A, Millar AH, Whelan J (2009) Defining core metabolic
and transcriptomic responses to oxygen availability in rice embryos and young seedlings Plant
Physiology (in press) PMID: 19605549
Howell KA., Narsai R.,Carroll A., Ivanova A., Lohse M., Usadel B., Millar AH., Whelan J. (Feb.
2009) Mapping metabolic and transcript temporal switches during germination in Oryza sativa
highlights specific transcription factors and the role of RNA instability in the germination process
Plant Physiology 149(2):961-80
33 Biochemistry
PROFESSOR
MICHAEL J WISE 35B Room 2.09, MCS 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
20BPROJECTS
1. Viral Codons
You are no doubt aware that the "Universal" codon translation table in fact only applies to eukaryote
genomes, and even then not to all of them; slime mold has a different table. The set of different tables can be
found at: Hhttp://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c H If you look at that site you
will notice that there is no mention of viruses. One may assume, however, that because viruses are dependent
on the replication machinery of their hosts that their genes will be encoded like their hosts, i.e. use the same
codon translation tables. So, for example, MUMPS will use the Universal table, while lambda phage will use
a bacterial table.
The Codon Adaptation Index was developed some years ago and reflects the observation that some codons
are far more used than other codons for a given amino acid, arguably reflecting greater numbers of the
corresponding anti-codons. The authors also observed that highly expressed genes tend to use the most
abundant codons. The Codon Adaptation Index was developed to reflect these observations.
The project is to examine viral genes in terms of their Codon Adaptation Index to gauge the extent to which
the codon usage biases of a virus mirror that of its host. Is it possible to see significant differences between
codon usage in the different isolates of the same virus which target different species, e.g. influenza virus
affecting humans and birds.
2. Durability and Energy-Storage Genes – Under-Recognised Cofactors of Microbial Pathogenicity
The Sit-and-Wait hypothesis of microbial pathogenicity for non-vector-borne pathogens (Walther and Ewald
2004) suggests a correlation between the durability of a non-vector borne microorganism and its
pathogenicity. (See also the review: Brown et al. (2006).) Under the hypothesis, durability – the ability to
survive the stresses associated with existing for a period outside a host – is, in effect, a cofactor for
pathogenicity, in concert with the necessary presence of conventionally understood virulence factors. That is,
without an assortment of virulence factors, a microorganism is unable to colonise a host, but if the
microorganism is labile, virulence will be tempered over time because an immobilised infective host is
unable to move and thus unable to spread the infection. An extension of this proposal is to include long-term
energy storage, particularly those used in stress situations, as a cofactor for pathogenicity because unless an
energy store has been maintained the organism may have survived, but it will not have the energy to produce
the range of invasion mechanisms it requires, such as pili. The overall aim of the project is to assemble a set
of Hidden Markov Models (HMMs) that represent proteins involved long-term energy storage. The HMMs
are then used to search the protein coding genes across a range of bacterial chromosomes to see which
species use which mechanisms, which is then linked to published mortality data as a proxy measure for
pathogenicity.
34 Biochemistry
3. 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.
4. 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.
35 Biochemistry
PROFESSOR GEORGE YEOH 37BRoom 2.59, MCS building, Phone: 6488 2986
38BEmail: [email protected]
Liver Research Group Our research group focuses on the biology of the liver progenitor cell (LPC) called an “oval cell” which
describes its shape. This has enormous potential as the vehicle for cell and gene therapy to treat liver disease.
Liver disease has become a significant health issue because its causes which are mainly lifestyle related –
alcohol, viral infection (HBV and HCV) and obesity are increasing at an alarming rate. All lead to chronic
liver disease and liver cancer (HCC) is a common final outcome. Liver transplantation is the only option
currently available for treating end-stage liver disease. This option is severely limited by the availability of
livers for organ transplant, hence we are exploring the potential of the LPC for use in cell therapy.
We contend it is superior to other cell types such as the differentiated hepatocyte or stem cells such as the
embryonic stem cell (ESC) or adult stem cell (ASC) for many reasons. In particular, it is robust and simple
to freeze and store, then thaw and grow by in vitro culture when required. It can be differentiated into either
hepatocytes or cholangiocytes (bile duct cells) quite easily and rapidly when maintained under appropriate
conditions, therefore it is more versatile than the hepatocyte. Accordingly, one of the objectives of our
research is to identify cytokines that regulate LPCs and to understand their mechanism of action. This will
underlie strategies to increase the contribution LPCs make to liver repair in vivo as well as to grow large
numbers of functional liver cells; hepatocytes or cholangiocytes in culture for cell therapy applications. We
are defining cytokine expression patterns in mice which are subject to chronic liver damage in which LPCs
are induced and participate in liver repair. The aim of these studies is to identify factors which induce
growth and the sebsequent differentiation of LPCs. We then test candidate cytokines in cell culture models.
Our laboratory has developed cell culture models based on cells isolated from mice placed on a diet which
induces fatty liver (primary cultures) as well as cell lines which we established which are now used
internationally.
There is also the potential to use our LPC lines in liver bioreactors and artificial organs. For this application
they must be differentiated into hepatocytes which are functionally equivalent to mature adult hepatocytes so
they are able to detoxify drugs, produce serum proteins and urea as well as perform the metabolic
interconversion between lipids, carbohydrates and proteins. The LPC is also of interest to cancer researchers
for it may be a target cell for transformation leading to the development of hepatocellular carcinoma (HCC).
Our laboratory has established LPC lines from mice which have the p53 gene deleted. These are called UpU53
UiUmmortalised UlUiver (PIL) cells. Interestingly we have PIL cell lines which are transformed and tumorigenic
while others are normal (Ref 1). We are comparing these cell lines to identify important genes which are
responsible for transformation of liver cells to hepatocellular carcinoma (HCC). Gene expression analyses
have highlighted several dozen genes which are upregulated (oncogenes?) and as many that are down-
regulated (tumour suppressor?). The overall aim of projects related to cancer is to identify genes which are
causal in terms of HCC and also to reconcile the cancer phenotype with the altered pattern of gene
expression.
1. Documenting the effect of cytokines on proliferation and differentiation of LPCs and determining
their mechanism of action
This project takes advantage of three resources available in our laboratory. First we have established a LPC
line (BMOL TAT) which expresses beta-galactosidase when it differentiates into a hepatocyte (Fig 1).
Second we have an instrument which measures cell growth continuously in cultured LPCs maintained in a
96-well format which allows for testing of many replicates under a variety of culture conditions such as the
exposure to a variety of cytokines. We have published the application of this instrument called the
Cellscreen to monitor growth of LPCs (Ref 1). Third we have identified several cytokines which are
associated with liver inflammation induced by a choline deficient
ethionine supplemented diet which are associated with the
induction of LPCs in mice following liver damage (Ref 2 & 3).
This project will focus on the role of TNF alpha, IL6 and IFN
alpha in cultured BMOL TAT cells in terms of their respective
effects on cell proliferation (using the Cellscreen instrument) and
differentiation by quantifying the expression of beta-
galactosidase. To confirm the effects of each cytokine on
proliferation and/or differentiation, their ability to induce cyclin D
(proliferation) and/or HNF4 alpha (differentiation transcription
factor) will be determined by qPCR and Western blotting. Fig 1. BMOL TAT cell line in growth conditions (top left) in which do not express
beta-galactosidase (top right) Following differentiation induced by high density
culture (bottom left) clusters of cells express beta-galactosidase (bottom right)
36 Biochemistry
References
1. Viebahn, C.S., J.E. Tirnitz-Parker, J.K. Olynyk, and G.C. Yeoh, Eur J Cell Biol, (2006).
2. Lim, R., B. Knight, K. Patel, J.G. McHutchison, G.C. Yeoh, and J.K. Olynyk, Hepatology, 43:
1074-83 (2006).
3. Knight, B., V.B. Matthews, B. Akhurst, E.J. Croager, E. Klinken, L.J. Abraham, J.K. Olynyk, and
G.Yeoh, Immunol Cell Biol, 83: 364-74 (2005).
2. Assessing the drug metabolising capacity of LPCs during growth and after differentiation into
hepaotcytes
This project will evaluate the drug metabolising ability of LPCs before and after differentiation. It will be
addressed at two different levels. First the overall efficiency of drug metabolism will be measured by the
conversion of a model substrate 7-Benzyloxyquinoline to its fluorescent product. This reflects the activity of
CYP3A family – the major form of cytochrome p450 involved in drug metabolism. The CYP3A family
comprises many isoforms and in the context of this project two are of special interest. CYP3A16 is the
dominant form in fetal liver whereas CYP3A11 is the adult form (Ref 1). Besides documenting the overall
change in CYP3A activity we wish to know if there is a swtitch from the fetal to adult form when LPCs
differentiate. To accomplish this the relative expression levels of mRNA coding for the respective isoforms
of CYP3A will be measured by real time PCR.
References
1. Sakuma, T., M. Takai, Y. Endo, M. Kuroiwa, A. Ohara, K. Jarukamjorn, R. Honma, and N. Nemoto,
Arch Biochem Biophys, 377: 153-62 (2000).
3. Documenting changes in expression of genes during transformation of liver progenitor cells into
hepatocellular carcinoma.
This project will exploit differences between a non-
tumorigenic PIL4 cell line which does not grow in semi-
solid medium in contrast to the tumorigenic PIL2 line
which does (see Fig 2). To identify genes that may be
causal in the transformation of LPCs we have profiled the
pattern of gene expression of PIL2 (transformed) and
PIL4 (normal) cells. The list of genes which have been
up-regulated (potential oncogenes) and down-regulated
(potential tumour suppressor genes) is extensive. The PIL
cells are derived from p53 null mice and suggest p53
plays an important role in LPC transformation. We also
have a cell line derived from wild type mice. Interestingly
this cell line becomes transformed during repeated sub-
culture This project will adopt two approaches to identify
which genes are important in transformation. One is to
correlate the loss of p53 with other oncogenes and tumour
suppressor genes during transformation. In this regard, we
will focus on two anti-apoptotic genes, namely the
inhibitor of apoptosis (IAP), the yes associated protein
(Yap) and the M2 isoform of pyruvate kinase (M2-PK).
Three approaches will be used to assess gene expression;
qPCR to measure mRNA abundance, Western blot for
protein and immunohistochemistry to detect and localise
the proteins in cells. This follows up on our recent findings which indicate that transfomred LPCs express
higher levls of IAP and Yap (Ref 2). Confirmation that these gene products are causative would be obtained
by their ability to transform normal LPCs by focing their over-expression by gene transfection.
References
1. Dumble, M.L., Croager, E.J., Yeoh, G.C.T., and Quail, E.A., Carcinogenesis. 23: 435-45 (2002).
2. Jellicoe, M.M., S.J. Nichols, B.A. Callus, M.V. Baker, P.J. Barnard, S.J. Berners-Price, J. Whelan, G.C.
Yeoh, and Filipovska, A., Carcinogenesis, 2008. 29:1124-33.
a
c
e
b
d
f
FRL
19
PIL2
PIL4
Fig. 2 PIL2 cells (c&d) are positive in the colony forming
assay whereas PIL4 cells (e&f) are negative.
FRL19 = positive control (a&b)
7 Days 14 Days
37 Biochemistry
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 Wednesday 7 October and close on Tuesday 8
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.
Applications close on Friday 18th December 2009
Domestic Students
Australian citizens, permanent residents and/or holders of a humanitarian visa or New
Zealand citizens apply through the UWA Admissions Centre.
International Students
International Students apply through the UWA International Centre.
All applicants need to complete the BBCS Honours Preference Form.
CRICOS Provider Code: 00126G
HONOURS APPLICATION FORM(External Applicants
Admissions Centre, M353, 35 Stirling Highway, Crawley WA 6059 P: (08) 6488 2077 F: (08) 6488 1226
Student ID: (Office Use Only)
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3 Course Information
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Mid-Year (July commencement)
2. Please list all the UWA Honours degree courses that you intend to apply for in order of preference: [Please submit separate School / Faculty Approval Form for each course]
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1996 - 1999 Bachelor of Arts University of Sydney, Australia Bachelor
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If yes, what is the other language?
7 Admission Statistical Details Please attach to the application original or certified copies of documentation to support Admission Statistical Details
1. Entry Qualifications - What is your highest educational attainment? (Please mark one box only)
Completed Higher Education postgraduate level course Completed Higher Education bachelor level course Completed Higher Education sub-degree level course (eg diploma) Incomplete Higher Education course Completed TAFE award course Completed final year of secondary education course at school or TAFE Completed other qualification or certificate of attainment or competence No prior educational attainment
2. In what year did you achieve your highest educational attainment?
8 Declaration All applicants must read, sign, and date the declaration below
I declare that I have read and understood the Information Sheet for Domestic (External) Honours Applicants. I declare that the information provided by me in connection with this application is true and complete. I understand that UWA reserves the right to vary or reverse any decision regarding admission or enrolment made on the basis of incorrect or incomplete information provided by me, and that any such act on my part will be placed on record and will form part of confidential information forwarded to selectors in assessing any subsequent applications. I authorise UWA to obtain results and records from any examining body or educational institution, and to disclose information to the Australian Vic-Chancellors’ Committee and its member institutions.
I understand that the University’s authority to collect the information on this form is given by the Higher Education Support Act 2003; that the information is collected to allow the University to properly administer its course programmes; that the information may be shared for these purposes between the Australian Taxation Office and the Department of Education, Science and Training; and that the information may not otherwise be disclosed without my consent, unless authorised or required by law.
APPLICANT’S SIGNATURE:________________________________________________________ DATE:___________________________
School of Biomedical, Biomolecular
and Chemical Sciences
HHoonnoouurrss oorr GGrraaddDDiippSSccii iinn 22001100
PPRROOJJEECCTT PPRREEFFEERREENNCCEE FFOORRMM
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 (64884402) to be referred to the appropriate Coordinator to discuss any questions you may have.
PPlleeaassee rreettuurrnn ffoorrmm ttoo BBBBCCSS SScchhooooll OOffffiiccee bbyy WWeedd 88tthh DDeecc 22000099
I am interested in Honours/GradDipSci in 2010 within the Discipline of:
Biochemistry & Molecular Biology Chemistry Microbiology & Immunology Physiology
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, Green Chemistry, Biomedical Science etc) that will be located within one of the above Disciplines
I am considering mid-year entry to Honours in 2010
I am considering deferring Honours until 2011
I will will not be available for interview during the week 14 December - 18 December 2009
1. CONTACT DETAILS
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Address(es) (during period November/December 2009 – January 2010): ……………………………………………………………………………………………………………………………...…
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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 …………………………………………………………...
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The Faculty’s End-on Honours on-line application form must be completed by December 8th 2009. Prospective candidates will be interviewed 14 December - 18 December 2009, 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 21 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 2010.
School of Biomedical, Biomolecular and Chemical 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] Web www.biomedchem.uwa.edu.au
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