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School of Biomedical, Biomolecular and Chemical Sciences

Honours Projects 2010

BiochEmisTry, molEcUlar

Biology & gENETics


Welcome to Honours

School of Biomedical, Biomolecular and Chemical Sciences

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

Biochemistry and Molecular Biology

Professor Alice Vrielink

Phone: 6488 3162

alice.vrielink@uwa.edu.au

Chemistry & Nanotechnology

Professor Mark Spackman

Phone: 6488 3140

mark.spackman@uwa.edu.au

Genetics

Professor Lawrie Abraham

Phone: 6488 1148

labraham@cyllene.uwa.edu.au

Honours Co-ordinators

Forensic Science

Professor John Watling

Phone: 6488 4488

John.watling@uwa.edu.au

Microbiology and Immunology

Professor Barbara Chang

Phone: 9346 2288

Barbara.chang@uwa.edu.au

&

A / Professor Manfred Beilharz

Phone: 9346 2663

Manfred.beilharz@uwa.edu.au

Physiology

Dr Gavin Pinniger

Phone: 6488 3380

gavin.pinniger@uwa.edu.au


Table of Contents

Project Entries Page 1

How to Apply Page 37

Application Form Page 38

Preference Form Page 40


1B

PROFESSOR

LAWRIE ABRAHAM

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

1

Biochemistry

21BRoom

2.58, MCS building, Phone: 6488 3041,

22BEmail:

H

Human Molecular Biology Lab

Ulabraham@cyllene.uwa.edu.auU

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


etween 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 biotinstreptavidin

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).

Biochemistry

2


Biochemistry

9BASSOCIATE

PROFESSOR

PETER ARTHUR

10B

Room 2.41, MCS Building, Phone: 6488 1750

Email: HUparthur@cyllene.uwa.edu.auU

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 antiageing

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

3


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 endstage

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.

Biochemistry

4


Biochemistry

11BPROFESSOR

PAUL ATTWOOD

Room 3.69, MCS Building, Phone: 6488 3329

Email: HUpaul.attwood@uwa.edu.auU

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.

5


Biochemistry

6


Biochemistry

PROFESSOR CHARLIE BOND

Structural Biology

23BRoom

4.16, MCS Building, Phone: 6488 4406

BEmail: H

24

UCharles.Bond@uwa.edu.auUH

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)

7


2. Protein Structure Prediction: PPR Proteins

Collaboration with Ian Small, CoE for Plant Energy Biology

PPR proteins are modular proteins composed of tandem

repeats of 35 amino acid sequences. A number of these

proteins are known to bind and/or process RNA by

recognising the RNA sequence. We want to understand

how this sequence-specific recognition occurs. In similar

protein families (TPR and ankyrin proteins) for which the

structures are known, these repeats form alpha-helical

hairpins which assemble to make a long ‘solenoid’. We

have evidence from bioinformatics studies that PPR

proteins have a similar, but different structure. Based on a

specific type of bioinformatic (sequence covariation) data,

we can predict both the secondary and tertiary structure of

TPR and ankyrin proteins. We are interested in exploring

which other protein families can be investigated with these

Sequence covariation data can be used to

methods, but our main aim is to produce plausible models

of PPR protein structure which can be used to guide wet- predict the structure of helical repeat

lab experiments into the function of these proteins. proteins, with a relatively high accuracy.

This is a computation-based project which will involve

learning about protein structure, molecular dynamics and bioinformatic analysis of proteins.

3 Chaperones and Co-chaperones of the Malaria Pathogen, Plasmodium Falciparum

With Dr Will Stanley

Schematic of the interactions in the multichaperone complex.

Malaria is a widespread tropical disease killing about 2 million people annually, young children and pregnant

women being especially vulnerable. It is a disease associated with poverty and classed as a neglected disease –

no vaccine is available and prophylactic drugs are often too costly for those most at risk. The microbial

pathogen, Plasmodium falciparum, is the major cause of life-threatening malaria amongst humans.This project

explores a complex of P. falciparum chaperones – proteins essential for folding, stabilising and sorting of other

proteins – which is critical to survival and proliferation of the pathogen, and thus a target for new kinds of

antimalarial drugs. The complex consists of two housekeeping heat shock proteins, Hsp70 and Hsp90, and a

Hsp organiser protein, HOP, which are involved in a complex set of intermolecular interactions to facilitate

folding/sorting of a number of client proteins.

Components of the complex can be recombinantly expressed and purified with the aim of detailed biochemical

and References: biophysical studies of the assembly, structure and function of this multi-chaperone complex.

(1) A Shonhai, A Boshoff & GL Blatch (2007). . Protein Sci. 16(9):1803-18.;

For (2)SC other Onuoha, collaborative ET Coulstock, projects, JG please Grossmann see entries & SE for Jackson Dr Swaminatha (2008). J Mol Iyer Biol. and 379(4):732-44.

Prof Ian Small

Biochemistry

8


Biochemistry

3BDR

BERNARD CALLUS

25BRoom

3.49, MCS building, Phone: 6488 1107

26BEmail:

HUBernard.Callus@uwa.edu.au

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

cancer 3 . 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.

9


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

Biochemistry

10


Biochemistry

PROFESSOR PETER HARTMANN

Room 2.03, MCS Building, Phone 6488 3327

Email: HUhartmannp@cyllene.uwa.edu.auU

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.

11


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. Preterm

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.

Biochemistry

12


Biochemistry

ASSOCIATE PROFESSOR

MARTHA LUDWIG

Room 3.05, MCS Building, Phone: 6488 3744

Email: HUmludwig@cyllene.uwa.edu.auU

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.

13


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?

14


Biochemistry

ASSOCIATE PROFESSOR

THOMAS MARTIN

Room 3.47, MCS Building, Phone: 6488 3331

Email: HUthomas.martin@uwa.edu.auUH

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

15


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.

Biochemistry

16


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: HUhmillar@cyllene.uwa.edu.auU

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

This honours project will utilise the bioinformatic database of predicted

HUwww.suba.bcs.uwa.edu.auUH

).

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.

17


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)

Biochemistry

18


Biochemistry

5B

PROFESSOR

IAN SMALL

ARC Centre of Excellence in Plant Energy Biology

27BRoom

4.03, MCS building, Phone: 6488 4499

28BEmail:HUiansmall@cyllene.uwa.edu.auU

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

19


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 multimolecular

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 highfidelity

macromolecular characterisation is available. The project would suit a chemistry, biochemistry or

biophysics student.

Biochemistry

20


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: HUssmith@cyllene.uwa.edu.auU

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.

1. Discovery of a new mechanism of growth control

PROJECTS

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.

important discoveries.

References

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

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.

21


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.

Biochemistry

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.

22


Biochemistry

ASSOCIATE PROFESSOR

NAOMI TRENGOVE

Room 2.04, MCS Building, Phone: 6488 4421

Wound Healing Research Group

Email: HUnaomi.trengove@uwa.edu.auUH

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 factoralpha)

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 TNFalpha

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 TNFalpha

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

23


Biochemistry

24


Biochemistry

30B

Email:

6BASSOCIATE

PROFESSOR

ROBERT TUCKEY

Molecular Steroidogenesis Group

BRoom 3.71, MCS Building, Phone: 64883040,

29

HUrobert.tuckey@uwa.edu.auU

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 innermitochondrial

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.

Figure 1. Model of the interaction

of cytochrome P450scc with the

inner-mitochondrial membrane

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, 1αhydroxylation

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 17,20,23trihydroxyvitamin

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-

25


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 7dehydrocholesterol

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.

Biochemistry

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 7dehydropregnenolone.

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 27position

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.

26


Biochemistry

DR DANIELA ULGIATI

Room 3.03, MCS Building, Phone: 6488 4423

Email: HUdaniela.ulgiati@uwa.edu.auUH

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.

27


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

FIGURE 1

A. Induced C4 Expression

Upstream

Repressor

element

B. Repressed C4 Expression

Biochemistry

Upstream

Repressor

element

BKLF

Sp1

BKLF

Sp1

Extra-cellular

signal

Initiation

complex

- Activation

Dissociated

complex

- no activation

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 BKLFbinding

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.

28


Biochemistry

WINTHROP PROFESSOR

ALICE VRIELINK

31BRoom

4.31, MCS Building, Phone: 6488 3162

32BEmail:

H

Ualice-vrielink@cyllene.uwa.edu.auU

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.

1. Endotoxin Biosynthesis in Neisseria.

18BPROJECTS

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

An image of the bacterium

Helicobacter pylori, the causative

agent of gastric ulcers and gastric

carcinoma

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

29


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.

Biochemistry

Structure of the dimeric L-amino acid

oxidase from the snake venom of

Malayan pit viper. The glycosylations

are also indicated.

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:

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.

(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.

30


Biochemistry

PROFESSOR JIM WHELAN

ARC Centre of Excellence in Plant Energy Biology

33BRoom

4.73, MCS Building, Phone: 6488 1749

34BEmail:

H

Useamus@cyllene.uwa.edu.auU

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.

31


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.

Biochemistry

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

32


Biochemistry

35B

Room

36B

Email:

PROFESSOR

MICHAEL J WISE

2.09, MCS Building, Phone: 6488 4410

HUmichael.wise@uwa.edu.auUH

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

1. Viral Codons

20BPROJECTS

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=cH 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.

33


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.

Biochemistry

34


Biochemistry

PROFESSOR GEORGE YEOH

37BRoom

2.59, MCS building, Phone: 6488 2986

38BEmail:

H

Uyeoh@cyllene.uwa.edu.auU

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 downregulated

(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 betagalactosidase.

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)

35


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.

7 Days 14 Days

a

c

e

Biochemistry

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)

b

d

f

This project will exploit differences between a nontumorigenic

PIL4 cell line which does not grow in semisolid

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 subculture

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.

36


UWA Applicants

Biochemistry

HOW TO APPLY

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.

37


Admissions Centre, M353, 35 Stirling Highway, Crawley WA 6059 P: (08) 6488 2077 F: (08) 6488 1226

1 Personal Details

Mr/Ms/Miss/Mrs etc Family Name

CRICOS Provider Code: 00126G

HONOURS APPLICATION FORM

(External Applicants

Student ID: (Office Use Only)

Given Names Date of Birth (dd/mm/yy) Sex M F

Former Family Name (if applicable) Please attach evidence of change of name to application

Notification Address

Home Address

Country/Postcode

Country/Postcode

Business Phone Home Phone

Mobile Facsimile

Email

2 Enrolment

Are you currently enrolled or have you previously enrolled as a student of The University of Western Australia?

YES NO If YES, state student number: Year last attended:

3 Course Information

1. Application for commencement: (Please tick)

Note: Applications are only permitted for the next available intake.

Start of Year (February commencement)

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]

Preference Course Title

(eg. Bachelor of Science Honours)

1st

2nd

Course Code

(eg. 5011H)

Major/Programme

if appropriate

(eg. Neuroscience)

Major/Programme Code

(eg.MJ-GRMAN)

3. Are you applying for Joint or Cognate Honours within your degree course? YES NO

If YES, list combination (eg Anthropology and Mathematics)

Note that you must have discussed this combination with both Schools responsible for the programme

4 Secondary School Qualifications

Please attach correctly certified copies of your results (not necessary for WA TEE results from 1976 onwards)

Qualification

Example:

Year

FACULTY

USE

ONLY

State/Country School Candidate No.

(if known)

TEE 2004 Western Australia Applecross S.H.S. 98122456

Office Use Only

ENTERED ON CALLISTA _________________

SENT TO FACULTY _________________________

OFFER NO OFFER NOTIFICATION SENT ___________________________

CONDITIONAL OFFER CONDITION SATISFIED; LIFTED ON CALLISTA


5 Post-secondary & Tertiary Qualifications

Please provide details of ALL study you have undertaken at a tertiary institution, and attach correctly certified copies of your results.

Official Academic Transcripts are required, NOT statements of examination results (UWA results are not required)

Years

Undertaken

Name of Course/Award

Institution, Country Course

Type

Example:

1996 - 1999 Bachelor of Arts University of Sydney, Australia Bachelor �

Course

Completed

YES NO

6 Personal Statistical Details

You must attach proof of citizenship/permanent residence status to your application. Acceptable documents include: original or certified

copy of an Australian or New Zealand Birth Certificate; ID page of your passport (and relevant visa pages, if you are an Australian PR)

1. Are you of Aboriginal or Torres Strait Islander origin? YES NO

2. What is your Citizenship or Residency Status? (tick applicable box below)

Australian Citizen

Possess a

humanitarian visa

New Zealand Citizen

(or diplomat or consular representative)

Possess a temporary entry visa (or

diplomat or a dependent of a diplomat) Other

3. In what country were you born? (if not born in Australia)

4. Year of arrival in Australia (if applicable)

Possess a permanent residency visa

(permitted to stay in Australia indefinitely)

5. Do you speak a language other than English at your permanent home residence? YES NO

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

Honours or GradDipSci in 2010

PROJECT PREFERENCE FORM

The purpose of this form is to ascertain your interest in our Honours/GradDipSci courses. It is appreciated that students may be

exploring Honours/GradDipSci in more than one discipline. Phone the BBCS School Office (64884402) to be referred to the

appropriate Coordinator to discuss any questions you may have.

Please return form to BBCS School Office by Wed 8th Dec 2009

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

Name…………………………………………………………………………………………………………………………

Address(es) (during period November/December 2009 – January 2010):

……………………………………………………………………………………………………………………………...…

………………………………………………………………………………………………………………………………...

Phone No (during same period) ……………………………..…………………………………….………...

Mobile No (during same period) ……………………………………………………….……………………..

Email address …………………………….………………………………………………..

2. PROJECT PREFERENCES

In order of preference:

1 Project No [ ] Supervisors ……………………………………………………………

2 Project No [ ] Supervisors ……………………………………………………………

3 Project No [ ] Supervisors ……………………………………………………………

4 Project No [ ] Supervisors ……………………………………………………………

5 Project No [ ] Supervisors …………………………………………………………...

6 Project No [ ] Supervisors …………………………………………………………...

If there are any points you would like us to take into consideration please note them below:

………………………………………………………………………………………………………………………………

………...………………………………………………………………………………………………………………………

Signature………………………………………………………Date………………………………………………………

The Faculty’s End-on Honours on-line application form must be completed by December 8 th 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 admin@bcs.uwa.edu.au

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