Honours Projects: SMMB/i3 The end of an era? The need for new antibiotics: Supervisors · ·...
49
Honours Projects: SMMB/i3 The end of an era? The need for new antibiotics: Supervisors Primary: Prof. Ian Charles Co-supervisor: Dr Dagmar Alber The use of antibiotics is under threat from the emergence of bacteria that have become resistant. Not only have some bacteria become resistant to a single antibiotic, but strains have emerged that are resistant to most or all clinically used drugs. These strains are known as ‘superbugs’ and they represent the most significant threat to human health that we currently face. Failure to find new methods to treat infection will result in a return to the ‘pre-antibiotic era’. The consequences for this are enormous and a failure to find new antibiotics will undo all the advances we have made in improving human health. The drug resistant bacteria have been collectively described as the ‘ESKAPE’ pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter species) as they effectively ‘escape’ the effects of antimicrobial drugs. The World Health Organisation (WHO), Gates Foundation and The Wellcome Trust all recognise antibacterial resistance as a major threat to human health and the global spread of antibacterial resistance was the fo cus of the ‘WHO World Health Day 2011’ to highlight the problem and the urgent need for consolidated efforts to avoid regressing to the ‘pre -antibiotic era’. We have extensive experience of successful antimicrobial drug discovery (1) and have already screened a compound library and identified novel antimicrobial drugs with potent antibacterial activity. The aim of this project will be to further characterise the most promising compound in various in vitro growth and biofilm assays to assess their potential in treating acute and chronic bacterial infections. Experiments will also be aimed on identifying the resistance profile of these compounds. Furthermore mechanistic studies will be carried out to elucidate the mode of action of these compounds as this will allow us to progress them into the development pipeline. Key references 1) Professor Charles was a founder and chief scientist of the antimicrobial drug discovery company Arrow Therapeutics, where Dr Alber was head of Virology http://www.astrazeneca.com/Media/Press- releases/Article/20070201--AstraZeneca-Acquires-Arrow-Therapeutics-To-Broaden-An
Transcript of Honours Projects: SMMB/i3 The end of an era? The need for new antibiotics: Supervisors · ·...
Honours Projects: SMMB/i3 The end of an era? The need for new antibiotics: Supervisors
Primary: Prof. Ian Charles Co-supervisor: Dr Dagmar Alber The use of antibiotics is under threat from the emergence of bacteria that have become resistant. Not only have some bacteria become resistant to a single antibiotic, but strains have emerged that are resistant to most or all clinically used drugs. These strains are known as ‘superbugs’ and they represent the most significant threat to human health that we currently face. Failure to find new methods to treat infection will result in a return to the ‘pre-antibiotic era’. The consequences for this are enormous and a failure to find new antibiotics will undo all the advances we have made in improving human health. The drug resistant bacteria have been collectively described as the ‘ESKAPE’ pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter species) as they effectively ‘escape’ the effects of antimicrobial drugs. The World Health Organisation (WHO), Gates Foundation and The Wellcome Trust all recognise antibacterial resistance as a major threat to human health and the global spread of antibacterial resistance was the focus of the ‘WHO World Health Day 2011’ to highlight the problem and the urgent need for consolidated efforts to avoid regressing to the ‘pre-antibiotic era’. We have extensive experience of successful antimicrobial drug discovery (1) and have already screened a compound library and identified novel antimicrobial drugs with potent antibacterial activity. The aim of this project will be to further characterise the most promising compound in various in vitro growth and biofilm
assays to assess their potential in treating acute and chronic bacterial infections. Experiments will also be aimed on identifying the resistance profile of these compounds. Furthermore mechanistic studies will be carried out to elucidate the mode of action of these compounds as this will allow us to progress them into the development pipeline. Key references
1) Professor Charles was a founder and chief scientist of the antimicrobial drug discovery company Arrow Therapeutics, where Dr Alber was head of Virology http://www.astrazeneca.com/Media/Press-releases/Article/20070201--AstraZeneca-Acquires-Arrow-Therapeutics-To-Broaden-An
Project Title: Development of a novel clinical genomic microbiological technique using next generation
sequencing. Supervisor/s name/s: Piklu Roy Chowdhury, Steven Djordjevic and Aaron Darling
Location: 6.34
Project description:
Whole-genome sequencing is becoming an integral part of clinical genomic microbiology. However, techniques which predate genome sequencing, like Multilocus sequence typing (MLST), are still considered to be gold standard techniques in molecular surveillance studies in the current literature. With our own next generation sequencing machine, our group at the i3 institute in UTS is perfectly placed to develop an alternative, cost effective, robust typing technique for clinical pathogens. Our model organism for this project will be a cohort of drug resistant clinical E. coli isolates, but once established the protocol has the potential to
be applied broadly to any clinically or industrially important micro-organism. The project/s, will generate data that would enable development and validation of a novel technique for molecular typing of clinically important bacteria. The expected project will also involve using a bioinformatic analysis pipelines to provide an alternative to MLST analysis for bacteria. The project will be an ideal opportunity for undergraduate student/s to pick up molecular biological techniques and couple them with next generation sequencing and bioinformatics analytical skills. This will include methods like DNA extraction and purification, polymerase chain reactions, gene sequencing and most importantly development of a bioinformatics protocol that will be of immense use in clinical genomics laboratories.
Project title: Population metagenomics of gut bacteria Supervisor name: Aaron Darling Location: CB04.06.34
Project Description: The human body is comprised of 10 times more bacterial cells than human cells. Newly developed sequencing and metagenomics technologies enable direct quantification and monitoring of the microbial component of the human body. When the human microbiome is sampled and sequenced over a time-series, the dynamics of how individual species grow and perish can be observed. When a human is born the body is essentially sterile and immediately becomes colonised by successive waves of bacteria. In breast-fed infants, members of the genus Bifidobacteria tend to dominate the gut ecosystem early in life, followed by a transition to a microbial community dominated by Bacteroides.
This project focuses on characterising population dynamics of the microbiome in a breast-fed infant. A dataset of 45 metagenomic samples taken over the first three months of life will be analysed. This will be an opportunity for a motivated student to develop skills ranging from bioinformatics to phylogenetic analysis, with
potential to eventually develop into Ph.D. research.
Project title: Phylogenetic analysis for metagenomics and epidemiology Supervisor name: Aaron Darling Location: CB04.06.34
Project Description: Microbes in the wild live in populations that are subject to continuous evolutionary pressure. Next generation sequencing and metagenomics methods provide a candid view of how microbes, including pathogenic bacteria and viruses, survive in the wild. Using sequence data generated with instruments such as the Illumina MiSeq at the ithree institute, phylogenetic inference methods can be applied to trace the epidemiological history of pathogen outbreaks, and understand how the pressures of natural selection relate to the genes in the organisms. This bioinformatics-focused project involves applying phylogenetic methods to understand the strength of natural selection on bacteria and the relationship between genotype and phenotype. Inference of epidemiological links via similarity of phylogenetic diversity across metagenomic samples will be explored. This will be an excellent opportunity for an advanced undergraduate to learn and further develop computational and bioinformatic skill sets.
Molecular characterisation of antibiotic resistant bacteria from hospitals and food production animals
Supervisor: Professor Steven Djordjevic Co-Supervisor: Dr Piklu Roy Chowdhury
The evolution and spread of multiple antibiotic resistance (MAR) arguably poses the most serious threat to human health. Despite extensive studies the problem is not well understood partially because there is a limited (1) understanding of the types of mobile genetic elements that spread MAR, (2) knowledge pertaining to where resistance genes are sourced and how resistance genes assemble on mobile elements. Antibiotics are used heavily in food animal production and in clinical medicine. Many antibiotics used in animal production are excreted unchanged and accumulate in sludge and waste ponds before being used as fertilizer on pasture. Consequently there is a growing concern that many antibiotic resistance genes are captured from the environment and from the gastrointestinal tracts of food animals. Thus many antibiotic resistance genes, and the mobile elements that move them around, found in clinical hospital isolates have their origins outside the hospital environment. Despite the groundswell of evidence to support this contention, the topic remains controversial (Nordstrom et al., (2013) Frontiers in Microbiology. March Vol 4:Article 29; Wellington et al., (2013) Lancet Infectious Diseases 13:155-165). This project will focus on characterising antibiotic resistance gene loci on mobile genetic elements from multiply antibiotic resistant bacteria derived from both the hospital environment and from food-producing animals. We will use next-generation sequencing and state of the art bioinformatics pipelines to characterise complex antibiotic resistance gene loci and the plasmids and genomic islands that carry them to determine how these mobile genetic elements move between food animals, the environment and human populations.
Analysis of non-classically secreted proteins and their functions in pathogenic Mycoplasma species Supervisor: Prof. Steven Djordjevic Project description:
Moonlighting proteins are subgroup of newly emerging multifunctional proteins that play important roles in bacterial pathogenesis. Often, moonlightling proteins perform different functions in different cellular locations. The functions of moonlighting proteins cannot be predicted and often the first evidence of a moonlighting function is the detection of a protein in a place where it is not previously been shown to have a function. We have been conducting extensive proteome analyses of the surfaces of a range of important bacteria disease causing agents (pathogens) and observing proteins on the cell surface that are not predicted to be there using standard bioinformatic tools. Detailed molecular biological and biochemical analyses have confirmed their presence of the cell surfaces of these bacteria and novel functions that are important in pathogenesis. We have identified a number of putative moonlighting proteins that reside on the surface of Mycoplasma hyopneumoniae, Mycoplasma pneumoniae, Staphylococcus aureus, Shewanella spp and Vibrio cholera. Our preliminary evidence suggests that these proteins play important roles as adhesins, proteases
and immune modulating functions and are important candidates for urgently needed vaccines. Aims:
1. To clone, express and purify recombinant moonlighting proteins in E. coli 2. To determine the functions of moonlighting proteins using biochemical assays, ligand blotting,
microtitre-plate binding assays and Thermophoretic analyses. 3. To determine if these moonlighting proteins play a role in adherence to porcine epithelial-like cell
monolayers using state of the art fluorescence microscopy. Key References
1. Copley, S.D. Moonlighting is mainstream: Paradigm adjustment required. Bioessays 34: 578 2. Henderson, B. & martin, A. Bacterial virulence in the moonlight: Multitasking bacterial moonlighting
proteins are virulence determinants in infectious disease. Infection & Immunity
The need for new vaccines Supervisors: Steve Djordjevic & Ian Charles Project description
The last few years has seen the emergence of ‘superbugs’ that are resistant to current antibiotics. Failure to find new methods to treat infection will result in a return to the ‘pre-antibiotic era’. The drug resistant bacteria have been collectively described as the ‘ESKAPE’ pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter species)
as they effectively ‘escape’ the effects of antimicrobial drugs. While the search for new antibiotics is part of the strategy to deal with ‘superbugs’, an important complementary approach is to develop vaccines. Despite the importance of Staphylococcal-mediated disease, there is no current vaccine to protect against a S. aureus infection. Over the last few years we have developed state-of-the-art approaches to identify new targets for vaccines and therapeutics. These approaches carry out genome-wide surveys of the Staphylococcal genome and characterise essential genes (1, 2). We have already identified the complete repertoire of essential genes (i.e. all the genes in the target genome that are required for survival and growth of the organism) in S. aureus and are using this information in parallel with next-generation proteomic approaches to identify novel vaccine candidates. Aims
4. To characterise the global repertoire of vaccine candidates in S.aureus. 5. To identify vaccine candidates and over-express the Escherichia coli to generate trial vaccines.
Honours Project
We invite students interested in vaccine biologyto discuss the project with the supervisors Key references
1) Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH). Chaudhuri RR et.al., BMC Genomics. 2009 10:291.
2) Structure of Staphylococcus aureus guanylate monophosphate kinase. El Omari K,et.al.,2006, Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006;62:949-53.
What makes bacteria pathogenic? Supervisors: Steve Djordjevic & Ian Charles Project description
We can all make a ‘top ten’ list of bacteria that are serious pathogens. Infection with organisms like Mycobacterium tuberculosis (TB) Vibrio cholerae (Cholera), Yersinia pestis (Plague) and Salmonella enterica serovar Typhi (Typhoid) results in millions of deaths per year. Research into these organisms (and many other pathogens) has not only resulted in important information on targets for vaccines and antimicrobial drug discovery, but has also uncovered some general ‘virulence’ strategies and factors used by micro-organisms to become pathogenic. Living alongside these well -known pathogens are myriad other bacteria that appear harmless to man, although they may possess many of the ‘virulence determinants’ found in pathogens. This finding has raised an important question in microbiology: what makes a good bug (i.e. one that is happy to live alongside man – in the gut or on the skin) and what makes a bad bug (i.e. one that is pathogenic and cause disease)?
Understanding this process will give us enormous insights into the evolution of pathogenicity. Our research into these basic questions on the evolution of disease-causing micro-organisms is underpinned by examination of so called ‘emerging pathogens’. These are a class of organism that are normally considered ‘good’ but are increasingly associated with infectious disease. To study this important area of emerging infections we are using a Gram negative bacteria called Shewanella algae. This organism is halophilic and widely distributed in the environment, but over the last few years has
been associated with a range of infections (1) including a bacteraemia similar to that caused by Vibrio spp. (2). We are using NextGen sequencing and cutting edge proteomics to understand how micro-organisms become pathogenic. Aims
6. To characterise the global network of pathogenicity associated genes in S. algae
7. To identify candidate virulence genes for further study using a combination of over-expression of recombinant proteins and in vitro and in vivo assays.
Honours Project
We invite students interested in microbial pathogenicity to discuss the project with the supervisors Key references
1) Epidemiology and clinical features of Shewanella infection over an eight-year period. To KK et.al., Scand J Infect Dis. 2010 42: 757-62.
2) Primary Shewanella algae bacteremia mimicking Vibrio septicemia. Myung DS et.al., J Korean Med
Sci. 2009 Dec;24(6):1192-4. Epub 2009 Nov 9.
Microbial cell Dynamics and infection
Supervisor: Dr Iain Duggin
Our research group is interested in the function, dynamics and evolution of microbial cell morphology. We use a wide range of modern molecular biology methods to understand microbial cell shape changes that occur in important infectious disease and environmental processes. Methods currently being used by research students in the group include: Recombinant DNA technology, genetic engineering, molecular genetics and functional genomics, high-resolution fluorescence and time-lapse microscopy, cell culture, and protein functional analysis.
Much of our recent work has focused on pathogenic strains of E. coli that cause urinary tract infections
(UTIs). UTIs are one of the most common infections world-wide and are potentially very dangerous; an alarming rise in resistance to antibiotics seen recently is of major concern. E. coli changes its shape
dramatically during invasive infection of human bladder cells. We are discovering the function of genes and proteins that are essential for the infection process, with the hope of underpinning new therapies that will be needed in future. Please email [email protected] for further information about the latest projects on offer.
Prevalence of Angiostrongylus cantonensis in Sydney
UTS Supervsior: Prof John Ellis
Dr Damien Stark, Department of Microbiology, St. Vincent’s Hospital Sydney, Darlinghurst, NSW 2010
Angiostrongylus cantonensis (rat lung worm) is the most common cause of eosinophilic meningitis worldwide.
In Australia, the definitive hosts of the parasite are non-native rats in whose pulmonary arteries and cardiac cavities adult worms lay their eggs. These hatch in the lung capillaries and larvae pass from the alveoli up the trachea, and are then swallowed. Larvae passed in the rat's faeces are ingested by snails or other molluscs. Humans are infected by ingesting molluscs or possibly food contaminated with larvae, such as improperly cooked crustaceans or unwashed vegetables. Larvae penetrate the intestinal wall, entering the circulatory system before migrating to the CNS. Unable to complete their life cycle, the larvae die, leading to intense CNS inflammation resulting in self-limiting meningitis and in some cases death.
The first reported human infection in Australia was in 1971; although infection is well documented in animals. Recently two cases among young children from Sydney were reported. It is the aim of this study to document the prevalence of A. cantonensis in various mollusc species collected throughout the Sydney metropolitan area using molecular techniques.
Genotyping of Dientamoeba fragilis
Supervisors: Prof John Ellis, Dr Joel Barratt and Dr Damien Stark School of Medical and Molecular Biosciences, University of Technology Sydney and St Vincent’s Hospital Sydney. Over the last five years, the protozoan parasite Dientamoeba fragilis has emerged as a leading cause of
11iarrhea in the Australian population, and many other countries in the world where routine testing for this organism has been introduced. “Gastro” represents a major illness that affects many people and these studies will advance knowledge on this pathogen as well as the disease it causes. A central dogma in Parasitology is that morphologically identical parasite populations are often composed of different species. For example, Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii are all morphologically identical, and cannot be distinqushed from each other unless molecular techniques are used. This project will investigate gene sequences derived from RNA seq data. We have recently completed a draft transcriptome for D. fragilis, that has generated data on approximately 7000 independent genes. This data set will be mined by computational methods to identify genes showing significant nucleotide changes/insertions and deletions. PCR and DNA sequencing will be used to confirm these changes. The development of a genotyping system for D. fragilis will then be applied to clinical species of dientamoebiasis to study the divergence in parasite populations.
Molecular Phylogeny of Apicomplexa
Supervisors: Prof John Ellis, Prof Michael Reichel School of Medical and Molecular Biosciences, University of Technology Sydney and Adelaide University School of Veterinary Science Cases of epistaxis in sub adult Western Grey kangaroos (Macropus fuliginosus), Red kangaroos (Macropus rufus) and Common Wallaroo (Macropus robustus) have been reported recently by wildlife carers in South
Australia. Some clinical cases were reported to have even died from severe epistaxis but no necropsies were performed. Besnoitia-like parasites were visualised microscopically in Diff Quik stained smears made from nasal swabs
and flushes obtained from individuals examined in the autumn of 2011 and 2012. These contained small numbers of hypertrophied host epithelial cells, each with an enlarged displaced nucleus and a large (> 100 um) intracytoplasmic parasitophorous vacuole enclosing closely packed small (~4um long) protozoal zoites. The aim of this research study is to PCR and sequence the 18S ribosomal DNA of the Besnoitia-like parasites and to conduct a molecular phylogeny study to determine the evolutionary relationships of these organisms to the phylum Apicomplexa.
Differential gene expression in Neospora caninum
Prof John Ellis and Dr Joel Barratt School of Medical and Molecular Biosciences, University of Technology Sydney Neospora caninum is a parasite that is a major cause of abortion in cattle. Over the last 15 years we have evaluated the development of vaccines to prevent fetal loss due to neosporosis and demonstrated that vaccination with live N. caninum can protect against experimental infection of cattle and mice. The UTS naturally attenuated NC-Nowra strain of N. caninum has become a lead vaccine for cattle. Studies have commenced to investigate what makes NC-Nowra naturally attenuated. Next generation sequencing of the NC-Nowra transcriptome has been completed, and this project aims to use data mining techniques to identify the unique aspects of the NC-Nowra transcriptome that may be associated with avirulence. PCR and sequencing will be used to compare gene sequences from different N. caninum isolates, Real time PCR will be used to compare gene expression between N. caninum strains.
Variant gene detection in Neospora caninum
Prof John Ellis and Dr Joel Barratt School of Medical and Molecular Biosciences, University of Technology Sydney Neospora caninum is a parasite that is a major cause of abortion in cattle. Over the last 15 years we have evaluated the development of vaccines to prevent fetal loss due to neosporosis and demonstrated that vaccination with live N. caninum can protect against experimental infection of cattle and mice. The UTS naturally attenuated NC-Nowra strain of N. caninum has become a lead vaccine for cattle. Studies have commenced to investigate what makes NC-Nowra naturally attenuated. Next generation sequencing of the NC-Nowra genome is underway, and this project aims to use data mining techniques to identify mutations (insertions/deletions/SNPs) that may be associated with avirulence. We will focus on identifying genes that are polymorphic amongst strains as a result of mutational events leading to changes in gene sequences. PCR and sequencing will be used to confirm the observed gene sequences from different N. caninum isolates.
Honours Project (AMG) Project title: Bacterial biofilms and Cystic Fibrosis infection control
Supervisor: A/Prof Tony George Location: iThree and MMB, level 6, Building 4, UTS
Project description Objectives: To determine the efficacy of various drug combinations against cystic fibrosis bacterial isolates that are resistant to antibiotics. Background: Cystic fibrosis is the most common lethal inherited disorder in Caucasians, affecting 1 in 2,500 births. Whilst median survival has increased from 1-2 years (1960) to a current 36-38 years, chronic suppurative lung disease still causes the majority of the mortality associated with this disease. Infection with P. aeruginosa and B. cepacia are important causes of morbidity and mortality in CF, progressively damaging the lungs and
ultimately causing death from respiratory failure in the majority of patients. Most patients with CF are infected chronically with P. aeruginosa, the organism being sustained mainly in antibiotic-resistant biofilms. While non-mucoid strains are isolated initially, P. aeruginosa in the CF lung is soon seen to secrete a protective mucoid
layer that limits host defences. Resistance to most chemotherapeutic antibiotics, or combinations of antibiotics, is commonplace, with estimates that 25-45% of adult CF patients are infected with mutiresistant bacteria within their airways. We have demonstrated striking clinical efficacy for the combination of an aminoglycoside antibiotic, tobramycin, and a diuretic agent, amiloride, against B cepacia in a small clinical pilot study. We have also shown in vitro activity for amiloride and related compounds against both B. cepacia and P. aeruginosa. This project will determine the extent to which this is generally true for these bacterial
pathogens; and will determine feasibility for clinical trials. Methodology: Bacterial isolates from cystic fibrosis patients will be tested for resistance to common antibiotics in planktonic and ‘artificial’ biofilm contexts. Combinations of drugs will also be tested under these conditions to determine the most efficacious drug treatments for biofilm culture resistant to antibiotics, using a checkerboard analysis method. Resistance mechanisms will also be determined.
Project title: Ameliorating asbestosis
Supervisor: A/Prof Tony George Location: iThree and MMB, level 6, Building 4, UTS
Project description
Objectives: To ameliorate or eliminate the progressive decline in health associated with asbestos fibres. Background: When asbestos is mined or pulverized, small dust balls become suspended in the air as “parachutes”, and it is this propensity that leads to inhalation into the lungs where it can induce both fibrosis (asbestosis) and malignancy (mesothelioma). Once inside lung cells, asbestos-bound or released metal ions, especially iron, generate cytotoxic reactive oxygen radicals that cause most of the cellular and DNA damage that leads to asbestosis or mesothelioma. The presence of other heavy metals from exogenous sources, for example, cadmium from smoking, and mercury, aluminium, zinc, copper and tungsten from industry and mining, exacerbate the lung problems. We have made a serendipitous discovery for ameliorating the adverse effects of asbestos fibres that we now want to test in lung cell cultures, and then in animal trials. Our hypothesis is based on the premise that Compound ‘M’ will entrap the iron as it is released from asbestos particles. Iron sequestered in this way is effectively rendered chemically inert. “M” is harmless to humans, having been approved for human use for other conditions. After achieving proof-of-concept, we will forge a commercial partnership to conduct animal trials with a nebulizer formulation to reverse the effects of inhaled asbestos fibres. If these trials are successful, human clinical trials will be possible. Methodology: Cell culture for different cell lines (epithelial and mesothelial cells; macrophages). Flow cytometry assays for apoptosis and viability. Fluorescence assays for the generation of reactive oxygen species.
Project title: ABC transporters
Supervisor: A/Prof Tony George Location: iThree and MMB, level 6, Building 4, UTS
Project description
Objectives: Structure-function study of the multidrug resistance ABC transporter P-glycoprotein, the major cause of cancer chemotherapy failure. Background: (in collaboration with Dr Richard Callaghan, University of Oxford, UK; Dr Ian Kerr, University of Nottingham, UK; and Dr Megan O’Mara, University of QLD). ATP-Binding Cassette (ABC) transporters couple hydrolysis of ATP to vectorial translocation of substrates across cellular membranes. These integral membrane proteins are involved in diverse cellular processes including, maintenance of osmotic homeostasis, nutrient uptake, resistance to cytotoxic drugs, antigen processing, cell division, pathogenesis, cholesterol transport, and stem cell biology. ABC transporters are found in all phyla and form one of the largest protein families. The ability of some ABC transporters to efflux diverse cytotoxic compounds is a crucial problem in human medicine - cancer cells, pathogenic microbes and parasites employ this mechanism to evade chemotherapeutic drugs. Inhibition of these proteins will improve the efficacy of primary drug treatment and render these ABC proteins as targets for new drugs. We will address key mechanistic questions about ABC transporters, using recent atomic structures as the basis for molecular dynamics calculations, homology modelling, and cross-linking experiments designed to identify regions crucial to the functioning of ABC transporters, thereby enabling us to identify and test small molecules that interfere with the normal modes of movement of critical regions in these proteins. Methodology: Standard molecular biology, biochemistry, and cell culture techniques. Purification of membrane proteins, site-directed mutagenesis and transport activity assays.
Project Title: Neural progenitor cell reaction in the entire central nervous system following spinal cord injury.
Supervisor/s name/s: Dr Catherine Gorrie. Location: Medical and Molecular Biosciences. Faculty of Science, UTS. City Campus.
Objectives: To determine whether endogenous neural progenitor cells respond to spinal cord injury
throughout the central nervous system (lumbar, thoracic, cervical, brain regions) or just locally near the injury site. Background: The finding of endogenous neural progenitor cells (NPC) in the brain has caused neuroscientists to rethink repair strategies for degenerating neurological conditions and injury in the brain. A niche of neural progenitor cells is also located in the spinal cord but very little is known about this cell population. There are indications that the signals regulating NPC may be travelling through the CSF. We hypothesise that the endogenous neural progenitor cells will respond to spinal cord injury distal to the injury site, and possibly in the brain. The results of this study will impact on future treatment options, and may add to knowledge of the normal functioning of these cells. Methodology:
Assist with surgical procedures and behavioural assessments
Histology preparation of tissue samples (frozen sections)
Fluorescent immunohistochemistry
Microscopy and Image analysis
Quantification using image analysis software Additional considerations:
Students wishing to undertake this project must be prepared to conduct research using animals. All animal experiments will have ethics approval from UTS.
There may be occasional evening and weekend rosters for animal care.
Project Title: The response of endogenous neural progenitor cells to injury in the rat spinal cord.
Supervisor/s name/s: Dr Catherine Gorrie. Location: Medical and Molecular Biosciences. Faculty of Science, UTS.
City Campus. Objectives: To determine whether endogenous neural progenitor cells in young animals have an increased response to spinal cord injury compared to adult rats. Background: The finding of endogenous neural progenitor cells (NPC) in the brain has caused neuroscientists to rethink repair strategies for degenerating neurological conditions and injury in the brain. A niche of neural progenitor cells is also located in the spinal cord but very little is known about this cell population. We hypothesise that the endogenous neural progenitor cells in young animals will have an increased response to injury due to the higher levels of plasticity in developing animals. The results from this study may show that there is potential for novel treatments for spinal cord injury, aimed at manipulating these cell populations in vivo rather than introducing transplanted cells from other sources. Methodology:
Surgical procedures
Behavioural assessments
Histology preparation of tissue samples
Immunohistochemistry
Microscopy and Image analysis
Quantification using image analysis software Additional considerations:
Students wishing to undertake this project must be prepared to conduct research using animals. All animal experiments will have ethics approval from UTS.
There will be occasional evening and weekend work.
The identification of saliva-specific bacteria in samples of forensically relevant bacterial species
using advanced DNA techniques.
Supervisor: Associate Professor Peter Gunn
Project Description & Significance:
The forensic identification of saliva can be important in re-constructing the events surrounding a crime.
Current methods for the presumptive identification of saliva are often difficult to apply, especially in mixed
stains ( for example blood + saliva, sweat + saliva). One possible approach to identify saliva in mixed stains
is to identify saliva-specific bacteria in the stains. Previous work in this School (and in conjunction with NSW
Police) demonstrated the feasibility of identifying saliva-specific strains of Streptococcus in mock forensic
samples.
This project will further this work, by adapting this technique to the newly-acquired Applied Biosystems 3500
Genetic Analyser, so that the technology can be further automated and quantitated.
The project will aim to:
develop fluorescently-labelled DNA primers &/or probes that will identify saliva-specific bacterial
strains in a range of pure and mixed samples, by PCR followed by capillary electrophoresis.
optimise this technique, and the determine its sensitivity and specificity.
examine the range and distribution of the bacteria in body fluids other than saliva, and amongst
volunteers.
The work may also evolve into studies of buccal bacteria with epidemiological interest.
The recovery and identification of pollen from the sinus cavities of cadavers: can this help identify the place of death?
Supervisor: Associate Professor Peter Gunn / Professor Shari Forbes
Project Description & Significance:
At the time of death, a person’s sinuses may contain traces of pollen specific to the area in which they died.
These pollen grains may persist well after decay of the body has proceeded. If a body is moved after death,
or if the body is not discovered for some time, it is possible that the identification of this pollen will assist in
specifying the place &/or season of death. Pollen can be recovered from sinus cavities even long after death.
The pollen grains can then be identified morphologically (e.g. through Scanning Electron Microscopy).
In this project the student will investigate the recovery of pollen from the sinuses of pig cadavers that are
located in an off-site UTS facility managed by Prof Forbes. By comparing these to reference samples, we will
attempt to identify the time &/or location of the death of our pigs, in order to validate the technique.
Further identification of the pollen will be investigated by sequencing of the DNA contained in the pollen grain
(in particular, plastid DNA), utilising the Applied Biosystems 3500 Genetic Analyser recently acquired by the
School. The student will need to research & devise the optimum sequencing strategy for this approach.
Please note that the student will need to commence this project in mid- January 2014, and will be
required to have close contact with pig cadavers in various states of decay.
Low Copy Number DNA analysis – how reproducible is it?
Supervisor: Associate Professor Peter Gunn / Professor Claude Roux
Project Description & Significance:
The forensic analysis of tiny amounts of human DNA (less than ~ 100 picograms) is sometimes referred to as
“Low Copy Number” (LCN) or “Low Template” DNA analysis.
The use of this technique is controversial, and not universally accepted by the forensic community. Because
of the very low template levels, the interpretation of the resulting profiles can be complicated by, amongst
other factors:
the random appearance of extraneous alleles (“drop-in”)
the random disappearance of some alleles (“drop-out”)
extreme allele imbalance
Although interpretation rules exist, and are applied to LCN analyses by the practitioners, there remain doubts
as to the reproducibility of LCN results.
In this project, & using the newly acquired Applied Biosystems 3500 Genetic Analyser, the student will
construct a series of LCN profiles specifically designed to test the limits of discrimination, and the
reproducibility & accuracy of LCN interpretations. The student will build a “model system” that allows the
examination of several of the factors that may compromise the interpretations of LCN profiles , and assess
the reliability of this technique by these reference to these factors.
Cell Cycle Control in Bacteria and Drug Discovery
Supervisors Name: Professor Liz Harry Location: the ithree institute, Faculty of Science, UTS, City Campus. Cell Division
Cell division in bacteria is essential for survival and infection, and therefore represents an attractive antibacterial target for the development of new antibiotics. Cell division must occur at the right time and the right place within the cell to ensure equal partitioning of DNA into newborn cells. What are the cues that signal cells to divide at the right place and at the right time? How are the division proteins recruited to the division site and what is their function? Which division proteins make the best antibiotic targets? Research in my laboratory addresses these questions in bacteria to gain an understanding of the regulation of this vital process and to facilitate the design of novel antibiotics that target it. I have a lab group of 12 people, and there are several honours projects in cell division available:
1. We have identified a link between DNA replication and positioning of the cytokinetic ring in the Gram-
positive model bacterium, B. subtilis. The project will involve unraveling how this occurs using
molecular biology, bacteriology and fluorescence microscopy. 2. Using protein chemistry to characterize protein-protein interactions within the division machinery in
pathogenic bacteria. This includes the Gram-positive Staphylococcus aureus (Golden staph.) and the Gram-negative Acinetobacter baumannii. Both are resistant to multiple antibiotics and pose a
significant threat to human health. The aim of this project is to identify interactions that would make good drug targets for the development of new antibiotics.
3. Bacteria not only multiply quickly to cause infection but they are also adapted to stop dividing when signaled by the environment. This enables them to survive antibiotics, and to trick our immune system so that it can’t get rid of the infection. We have identified several genes of E. coli that inhibit division and one project would involve characterizing how they do this using molecular biology and fluorescence microscopy. This project would be co-supervised by Dr Cath Burke at UTS.
The Antibacterial Properties of Manuka Honey
Before antibiotics were discovered, honey was commonly used to treat skin infections. Given the alarming problem of antibiotic resistance, honey is now making a comeback as a treatment for skin and chronic wound infectons, and approved medical honey dressings are available. NZ manuka honey is a particularly potent antibacterial agent, has broad-spectrum activity, and unlike antibiotics, bacteria do not become resistant to honey. Honours projects would involve investigating the antibacterial activity of Australian manuka honeys to see if they are as good as those of NZ manuka. Methods would involve bacteriology, antibacterial assays and microscopy.
Project Title: Molecular characterisation of membrane bound free immunoglobulin light chains Supervisor: Dr Andrew Hutchinson Location: Ultimo, Building 4, Level 6 (School of Medical & Molecular Biosciences) Project Description:
Objectives: To characterise the interaction between free immunoglobulin light chains and cell membranes Background Information Free immunoglobulin light chains (FLC) belong to a class of proteins that readily form pathological protein aggregates termed amyloids. Amyloid formation by FLC is dependent on a number of factors including primary amino acid sequence, post translational modifications, concentration and pH. Furthermore, it is also well known that seeding of amyloids can occur on cell membranes, however this mechanism is yet to be fully elucidated. This project will characterise membrane bound FLC via molecular techniques such as atomic force microscopy in an attempt to better understand the mechanisms behind amyloid formation. Methodology - Size exclusion chromatography - Protein – lipid ELISA - Preparation of liposomes - Sample preparation for atomic force microscopy - Atomic force microscopy Supervisor Dr Andrew Hutchinson (MMB)
Project Title: Characterisation of a cell death pathway in multiple myeloma cells Supervisor: Dr Andrew Hutchinson Location: Ultimo, Building 4, Level 6 (School of Medical & Molecular Biosciences) Project Description:
Objectives: To determine the cell death pathway induced by a small molecule inhibitor in multiple myeloma cells. Background Information Multiple myeloma (MM) is hematological cancer characterized by the infiltration of malignant plasma cells into the bone marrow. It is the second most common type of blood cancer and accounts for 2% of all cancer deaths. Despite a number of novel compounds entering the clinic in the past decade, MM remains incurable with median life expectancy of approximately five years. The primary issues with current treatment regimes are toxicity and drug resistance. Therefore there is an unmet need for the development of novel compounds that are both safe and effective for the treatment of MM. We have identified a key survival enzyme expressed by MM cells. Blockage of the enzyme activity with a small molecule inhibitor shows selective cell death for MM cells and this inhibitor can delay MM tumor growth in a relevant animal model of the disease. This project will attempt to elucidate the MM cell death pathway that is induced by the small molecule inhibitor. Methodology - Cell culture - Western blots - Transfection (siRNA, cDNA) - Flow cytometry - Microscopy Supervisor Dr Andrew Hutchinson (MMB)
The Role of miRNAs in Microparticle Formation and Microparticle Mediated Multidrug Resistance
Co-Supervised by: A/Prof Mary Bebawy (School of Pharmacy, Graduate School of Health) and A/Prof Gyorgy Hutvagner (School of Engineering, Centre for Health Technologies), UTS. Microparticles (MPs) are small membrane vesicles (0.1-1 mm in diameter) derived from the ubiquitous cellular phenomenon of membrane budding MPs are released, under normal physiological conditions, from the plasma membranes of various cell types including; platelets, macrophages, monocytes, T-cells, endothelial cells and erythocytes MPs are now important clinical mediators of inflammation, coagulation and vascular homeostasis We have shown that MPs shed by malignant cells confer chemoresistance by virtue of their role in the intercellular exchange of resistance proteins and various nucleic acid species. miRNAs are small (21nt long) non coding RNAs that are key regulators of all biological processes, including the regulation of the cell cycle and the immune response. An increasing amount of evidence shows that miRNAs and the machinery that processes miRNAs are involved in extracellular signaling because they are secreted from the cells with exosomes and microparticles. miRNAs packed in exosomes are great biomarkers of a wide range of diseases and miRNA containing microparticles were shown to be involved in the retemplating of the protein and transcriptional landscape of cancer cells. The packaging of miRNAs into extracellular vehicles is not random. A process of selective packaging allows for only a few miRNAs to be incorporated into MPs from the donor cells out of the several dozens that are actually expressed. In this project we would like to study two interrelated phenomena. First, we would like to reveal the mechanism of how miRNAs are selectively packaged in the MPs and how they are transported to MPs from within the intercellular space. We have preliminary data showing that miRNAs are packaged in MPs with the protein Argonaute, which binds miRNAs and are absolutely required for their biological functions; and therefore, the secreted miRNA complexes are ready to reprogram the targeted cells without further processing. We would like to investigate which step of miRNA processing and maturation decides that an miRNA is packaged into a MP or engaged in intracellular regulation. Secondly, we would like to describe the molecular mechanism of how miRNAs contribute to the reprogramming of the recipient cells that bind shed MPs. In this honour’s project, the student will be exposed to state of the art RNA biology, biochemistry and cell biology techniques and be a part of two dynamic collaborative research consortium
Project title: Evolution of pathogenic Vibrio cholerae Pathogenesis in Vibrio species and the role of mobile DNA
UTS Supervisor: Dr Maurizio Labbate
Location: Department of Medical and Molecular Biosciences & The ithree Institute BACKGROUND: Bacteria of the Vibrio genus show a wide range of niche specialization, from free-living
forms to those attached to biotic and abiotic surfaces, from symbiotic to pathogenic interactions and from estuarine to deep-sea habitats. They encompass the human pathogens Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus and a range of marine pathogens infecting fish, coral, shellfish and
prawns. The adaptability of vibrios relies on their capacity to generate genetic diversity at high rates, largely because of Lateral Gene Transfer (LGT). LGT is the method by which DNA moves between bacterial cells. It allows sharing of DNA between bacteria and is a major evolutionary driving force. In Vibrio cholerae, the major
genes responsible for causing the disease cholera are on DNA elements that are mobile. RESEARCH QUESTIONS: How does mobile DNA drive the evolution of vibrios? What role does mobile DNA
have in the emergence of new vibrio pathogens? What role does environmental selection play in creating pathogenic vibrios that can infection humans? METHODS: Honours candidates will be exposed to a range of scientific techniques including molecular biology techniques (PCR, cloning etc), proteomics based techniques (polyacrylamide gel electrophoresis, protein extraction and identification), bioassays (eg. bacterial stress assays) and bioinformatics. MORE INFORMATION: Interested students can visit the following websites for more information:
1. https://sites.google.com/site/thevibrioresearchgrouputs/home/welcome 2. Search for “The Vibrio Research Group at UTS” on FACEBOOK
Students can also contact Maurizio at [email protected] to organise a meeting.
Supervisor names: Dr Maurizio Labbate and Professor Ian Charles Location: The ithree Institute Project description: Cholera is a disease endemic in many developing countries resulting in thousands of
deaths per year and responsible for orders of magnitude more over human history. Cholera is primarily confined to endemic regions of the globe, mostly in South Asia, punctuated by outbreaks elsewhere as a consequence of natural or human induced disruption as evidenced most recently in Haiti and Zimbabwe. The causative agent for cholera is the bacterium Vibrio cholerae, an organism that is indigenous to the aquatic
environment. Of the 200+ diverse serogroups present in the aquatic environment, only serogroups O1 and O139 are reported to cause epidemic and pandemic cholera, with the former being the most common. However, some non-O1/O139 V. cholerae are capable of causing outbreaks or sporadic cases of non-cholera
gastroenteritis. From Sydney waters, we have isolated diverse V. cholerae strains, including non-O1/O139 serogroup strains
that are able to cause disease symptoms in animal models despite lacking the common known virulence factors. Most strains lack the type three secretion system considered a mediator of diarrhoea in non-O1/O139 V. cholerae indicating the presence of novel virulence factors. Furthermore, the Sydney strains are genetically distinct from O1/O139 toxigenic strains and are themselves diverse grouping with various environmental strains from other geographic regions. Out laboratory is focused on identifying the novel virulence factors in these Sydney V. cholerae strains and comparing how seemingly diverse V. cholerae strains travel down different evolutionary routes to become
pathogenic. Honours candidates will be exposed to a range of scientific techniques including molecular biology techniques (PCR, cloning etc), proteomics based techniques (polyacrylamide gel electrophoresis, protein extraction and identification), bioassays (eg. bacterial stress assays) and bioinformatics.
Broad research topics: Understanding physiological associations with clinical and other human factors
(Discuss a specific topic of interest with the supervisor) Supervisor: A/Prof Sara Lal Other potential co-supervisors and advisors: Jenny Wyndham Location: School of Medical and Molecular Biosciences, Neurosearch Research Unit Studies into physiological associations for identifying biomarkers (e.g. for fatigue, depression etc.) and/or clinical associations (diabetes, hypertension, cognitive activity, cardiac risk factors etc.) are continuously underway. There is lack of information in the literature in many such areas of physiological and neurophysiological associations. Such studies can help identify neurophysiological and lifestyle factors in samples that make them more predisposed to particular states such as fatigue, cardiac risk factors, cognitive decline, disease states, anxiety, depression etc. The impact of these states on a variety of physiological changes can be investigated. These types of studies can lead to development of human state indicators or countermeasures, identification of biomarkers and early clinical intervention in some cases. If interested a specific or focussed project should be discussed with the supervisor. Laboratory studies will include attaching non-invasive electrodes such as electroencephalogram (brain) for measuring EEG , electrocardiogram (ECG), electro-oculogram (eye), respiration, etc. Cognitive tests may be performed. The study will enable you to interpret physiological data as well as administer and interpret questionnaires. Student’s may propose a topic of interest and discuss with supervisors.
Neuroscience and medical physiology investigations: Honours projects in areas of cognitive function, fatigue, cardiovascular diseases, anxiety, affective states etc. Supervisor: A/Prof Sara Lal Location: School of Medical and Molecular Biosciences, Neurosearch Research Unit
Neuroscience and physiology projects are available in areas of investigating cognitive function, fatigue, cardiovascular diseases, anxiety, affective states etc. Projects include neurophysiology and medical physiology investigations using electroencephalography, electrocardiogram, blood pressure, etc. data obtained from human samples. Samples can range from the normal population to specific sample types such as nurses, drivers, police, and other sample types. Lab or filed based data collection utilises non-invasive physiological measures and also subjective tests and tools. Some specific honours projects in broad terms: ‘Heart rate variability as a biomarker of cognitive function in shift worker nurses’ ‘Cognition function assessment using electroencephalography and cognitive tests: gender assessment’ ‘Cardiac autonomic evaluation of diabetes using heart rate variability: a potential non-invasive marker’. ‘Chronic and acute fatigue in nurses (or drivers): medical physiology and questionnaire assessment’ ‘Cardiac autonomic and other medical physiology associations with anxiety and affective state disorder (depression) in different sample types’ You can also discuss a different neuroscience based project of interest with the supervisor. ‘Blood pressure associations to sleep, work and lifestyle factors in shift workers’
Associations between heart rate variability and blood pressure and cognitive function in different
racial groups: proposing cardiac predictors for cognitive impairment
Supervisor: A/Prof Sara Lal Location: School of Medical and Molecular Biosciences, Neurosearch Research Unit Cardiovascular factors such as heart rate variability (HRV) may have links to cognitive function and investigating cardiac variables may provide insight into preventing or delaying onset of cognitive impairment. Vascular pathology (such as hypertension) may also impair cognition. High blood pressure has been linked to poor performance at neuro-cognitive tasks Studies with non-demented samples suggest that higher levels of blood pressure may be attributable to cognitive dysfunction and that this may lead to higher risks for dementia in the future. However studies in the area of HRV association to cognitive function is scarce and there are further calls for research in the area. HRV can provide a measure of cardiac autonomic (sympathetic and parasympathetic) activity, using low and high frequency spectral components of HRV, respectively. Sympathovagal balance may also be derived from the ratio of the low and high frequency HRV activity. Past honours research identified some cardiovascular links with cognitive function. The present study aims to advance previous research by investigating HRV and BP links to cognitive function in different racial groups. Assessing non-invasive cardiac autonomic nervous system associations to glucose levels: implications for a heart rate variability based marker of diabetes Supervisor: A/Prof Sara Lal Location: School of Medical and Molecular Biosciences, Neurosearch Research Unit
Heart rate variability (HRV) provides a beat to beat non-invasive marker of the cardiac autonomic nervous system. Via spectral analysis (frequency domain analysis) of R-R intervals low frequency and high frequency components of HRV can be extracted. This provides measures of cardiac sympathetic and parasympathetic activities. Research suggests that heart rate variability (HRV) using beat-to-beat R-R variability could be a potential indicator of various medical conditions such diabetes, hypertension and other cardiac disease. Identifying a tool to assess glucose level fluctuations (as apparent in diabetes) based on HRV parameters could provide a continuous monitoring of changes in the glucose levels. In patients with diabetes mellitus who tend to develop autonomic neuropathy, there have been links with reduced heart rate variability. However, a thorough study assessing the different HRV parameters extracted from ECG such as low and high frequency components, total HRV power and sympathovagal balance (symaptehtic: parasympathetic ratio) would provide information of the variables most strongly linked to changes in glucose levels. This study has the potential to address and provide further information in the area of early intervention for preventing diabetes, continuous monitoring of glucose levels, ongoing diabetes management as well as HRV changes linked to the different disease states linked to diabetes such as cardiac and renal problems.
A New Therapy for Autoimmune Disease Using Novel Molecules from Parasitic Worms.
Supervisory Team: Assoc. Prof. Bronwyn O’Brien, Dr. Sheila Donnelly, Dr. Andrew Hutchinson The interaction between the vertebrate immune system and the major parasitic worm (helminth) groups began hundreds of millions of years ago, coincident with the evolution of adaptive immunity and before the emergence of humans as a species. During this co-evolution, helminth parasites have adapted to survive in their hosts for periods of up to several decades. They have achieved this by developing a range of mechanisms, which regulate both innate and adaptive immune responses in the host to prevent the development of host protective Thelper 1 (Th1, pro-inflammatory) responses, which would lead to their expulsion. The broad spectrum of regulatory mechanisms elicited by helminths also acts to limit immune-mediated pathology during infection. Over recent decades there have been marked improvements in social and economic conditions in the developed world. As a consequence, humans have reduced exposure to a range of infectious agents, in particular helminths. It has been proposed that reduced exposure to certain helminths might be responsible for the increased incidence of autoimmune diseases. Indeed, the incidence of several autoimmune conditions is inversely correlated with the occurrence of endemic helminth infections. Therefore, helminths and their secreted molecules likely play an important role in education of the immune system. On the basis of these observations we are investigating the use of parasitic worms to re-address the imbalance of the immune response and thus prevent the development of autoimmune disease. We have isolated, and produced in recombinant form, novel molecules secreted by the helminth, Fasciola hepatica. Using the clinically relevant model of type 1 diabetes, the nonobese diabetic (NOD) mouse, we have obtained proof-of-concept that delivery of a single protein (named HDM) from Fasciola hepatica, over a short time
course (only 6 doses administered over 12 days), prevents the autoimmune destruction of beta cells and therefore autoimmune diabetes. We are currently characterizing the immunological mechanisms underlying this protective effect. Our long-term goal is to develop and commercialise immune therapeutics for autoimmune disease. Our research team (Helmedix Therapeutics) includes academics, postdoctoral fellows, research assistants and PhD students, located at UTS and overseas. There are several projects available incorporating techniques in molecular biology, immunology, microscopy, cell culture, and animal experimentation. Specific research projects can be designed to include techniques of interest. For more information or to organize a meeting with the Helmedix team please email Assoc. Prof. Bronwyn O’Brien ([email protected]).
Project Title: Mechanisms of damage to the extracellular matrix proteins of the artery wall by myeloperoxidase-derived oxidants during chronic inflammation Supervisors: Dr. Matthew Padula Proteomics Core Facility, School of Medical and Molecular Sciences, University of
Technology, Sydney, PO Box 123, Broadway, NSW 2007 Tel: 02 9514 8374; Fax: 02 9514 8206; Email: [email protected]
Dr. David van Reyk School of Medical and Molecular Sciences, University of Technology, Sydney, PO Box
123, Broadway, NSW 2007 Tel: 02 9514 2221; Fax: 02 8206; Email: [email protected] Prof. Michael Davies The Heart Research Institute, 7 Eliza Street, Newtown, Sydney, NSW 2042. Tel: 02 8208 8900; Fax: 02 9565 5584; Email: [email protected]
Dr. Christine Chuang The Heart Research Institute, 7 Eliza Street, Newtown, Sydney, NSW 2042. Tel: 02 8208 8900; Fax: 02 9565 5584; Email: [email protected]
Project Background: Chronic inflammatory diseases such as atherosclerosis is responsible for ~40% of all deaths in
developed countries including Australia. Atherosclerosis is characterized by endothelial dysfunction (an early and defining marker for atherosclerosis), the accumulation of lipids in macrophage cells within the artery wall, the migration and proliferation of underlying smooth muscle cells into the intima, and, in many cases, lesion rupture and thrombosis. It is well established that the extracellular matrix (ECM) of the artery wall consists primarily of fibronectin, laminin, type IV collagen, and heparan sulfate (HS) proteoglycans including perlecan, which interact with growth factors and enzymes to regulate endothelial cell adhesion, proliferation and migration. These interactions are perturbed in atherosclerotic lesions, where activated monocytes and macrophages generate reactive oxidants, including the myeloperoxidase (MPO)-derived oxidants including hypochlorous acid (HOCl, the major component of household bleach) and hypothiocyanous acid (HOSCN), which subsequently alters the ECM proteins in the artery wall.
Enzymatically active MPO protein and elevated levels 3-chlorotyrosine have been shown to be
present in human atherosclerotic lesions. Our studies have shown that oxidized proteins are present in the ECM of human atherosclerotic plaques, and these materials are consistent with HOCl-mediated oxidation. Furthermore, we have demonstrated that MPO-derived HOCl disrupts the protein core of a key component of the subendothelial ECM, perlecan, which inhibits its biological interaction with fibroblast-growth factor (FGF) 2 and cellular proliferation. Most recently, we have also demonstrated that HOCl-mediated oxidation modulates the structure of other important ECM proteins, including fibronectin and laminin. Furthermore, cell adhesion binding sites of fibronecin was damaged by HOCl-mediated oxidation.
Collectively, these data provide compelling evidence that MPO-derived oxidants, and particularly
HOCl, play a key role in altering the vascular basement membrane by targeting the ECM proteins that underlies arterial endothelial cells during the development of atherosclerosis. Thus, we hypothesise that oxidative modification of ECM proteins contributes to endothelial cell dysfunction and weakening of the mechanical properties of the artery wall, which is associated with plaque rupture, the major cause of most heart attacks and strokes. The information gained through these studies will provide vital information on the key processes and factors that contribute to oxidant-mediated damage in vivo and aid the
targeted development of strategies to limit cellular dysfunction and chronic inflammatory diseases such as atherosclerosis.
Aim: To investigate and characterise the effects of damage of MPO-derived oxidants on the structure of purified and native ECM proteins that are functionally important in the basement membrane of the artery wall. This project will employ a range of techniques to study and characterise the effects of these oxidants on purified fibronectin, laminin and native endothelial cell-derived ECM using experimental techniques including separation of protein fragments using electrophoresis (1D and/or 2D), Western blotting to look for loss of antibody reactivity against biologically active sites on the purified proteins as well identification of oxidant-mediated damaged sites via mass spectrometry at the UTS Proteomics Core Facility with Dr. Matt Padula.
Optimisation of terminal labelling of protein samples for high throughput and comprehensive proteogenomics.
Supervisors: Dr Matt Padula, Proteomics Core Facility. Prof Steve Djordjevic, The iThree Institute. Project summary. Despite the fact that next generation genome sequencing technology has rapidly decreased the time and cost of sequencing genomes, Open Reading Frames (ORFs), the predicted protein products of the genome are still merely predictions. It is well known that when genes are translated to messenger RNA, splicing events can occur that are not predicted by the gene sequence. In addition, when the protein is produced from the mRNA, post-translational processing and cleavage events occur that are also not predicted by the gene sequence. These modifications can direct cellular localisation, protein activity and the half life of the protein. The extent of proteolysis ranges from degradation-to-completion to finely regulated and specific proteolysis such as initiator methionine removal, peptide chain generation from single translation products during protein maturation, export sequence cleavage, elimination of the pro-peptide for protein activation, and protein processing of mature proteins to switch activity. Proteolysis generates proteins with new N- and/or C-termini not originally present in the initially translated polypeptide. This characteristic can be exploited to identify protease-generated cleavage products and so provides an important layer of functional annotation of the proteome. Thus, there is a real need to definitively determine the true beginning and ending amino acids in the mature functional protein. This project will be carried out within the Proteomics Core Facility and involve the use of:
Multi-dimensional protein separations using electrophoretic and chromatographic methodologies.
Extensive use of MALDI and nanoelectrospray mass spectrometry.
Bioinformatic analysis of resulting datasets and protein interaction network construction.
Characterisation of the functional proteome of a model bacteria and re-annotation of the protein sequences.
Development and application of Imaging Mass Spectrometry techniques for the study of host-pathogen interactions.
Supervisor: Dr Matt Padula. Proteomics Core Facility. Prof Steven Djordjevic. The i3 Institute. Project Description. In cell biology, nearly all analytical techniques require the disruption of the architecture of a cell or tissue in order to analyse the components present in the system. The main exception to this is immunofluorescence microscopy where labelled antibodies specific for a target molecule, usually a protein, are used to ascertain the location of the molecule in a section of tissue or monolayer of cells. While immunofluorescence microscopy has produced a great wealth of information on the spatial location of molecules in cells and tissues it has some drawbacks, mainly:
1) Prior knowledge of the target molecule and availability of an antibody specific to the target. 2) A limitation in the number of targets that can be probed in a single experiment.
An emerging technique for determining the spatial location of molecules in cells or tissues is imaging mass spectrometry (IMS). IMS is a non-targeted techniques where the molecules at a specific location can be ionised by Matrix Assisted Laser Desorption/Ionisation (MALDI) mass spectrometry. By incrementally rastering across a piece of tissue and sequentially acquiring a mass spectrum at each point, a dataset is generated that can be interrogated to generate “heat maps” indicating the location of specific ions or molecules in the tissue section. Unlike immunofluorescence microscopy, no prior knowledge of the sample is required allowing the generation of data without a hypothesis. The ultimate application of IMS would be the spatial profiling, at sub-micrometre resolution, of the interactions between cells of tissues in a variety of disease states and the interactions between host cells and invading pathogens. As IMS is an emerging technique, many experimental issues need to be addressed to ensure the comprehensive and sensitive profiling of the sample. At present, the main issues are:
1) Sample preparation and the removal of interfering substances. 2) Matrix application on the sample to ensure homogeneous coverage with no “hot spots”. 3) Spatial resolution of the images and data generated, which is determined by the homogeneity of
matrix application and spot size of the ionising MALDI laser. This project will be conducted within the Proteomics Core Facility and involve the application of the following techniques:
MALDI mass spectrometry.
Nanoscale trypsin digestion using Chemical Inkjet Printer (ChIP) technology.
Cell culture of cell monolayers and their infection by pathogenic organisms such as Mycoplasma.
Confocal and fluorescence microscopy of infected monolayers.
Dissecting the interactions between bacterial pathogens and their hosts using Imaging Mass Spectrometry.
Supervisor: Dr Matt Padula. Proteomics Core Facility. Prof Steven Djordjevic. The i3 Institute. Project Description. The interaction between an infectious pathogen and its host is, not surprisingly, extremely complex and involves numerous different molecules such as proteins, peptides, DNA, RNA and small molecules. Traditional methods of co-culturing the pathogen and host cells in-vitro often lead to the complete disruption of the sample to recover the molecules in liquid for characterisation by Liquid Chromatography coupled to Mass Spectrometry (LC/MS). This invariably delocalises and separates important interacting molecules and knowledge of these interactions may be crucial to understanding and developing treatments. An emerging technique for determining the spatial location of molecules in cells or tissues is imaging mass spectrometry (IMS). IMS is a non-targeted technique where the molecules at a specific location can be ionised by Matrix Assisted Laser Desorption/Ionisation (MALDI) mass spectrometry. By firing a laser across a sample, such as co-cultured host and pathogen cells, a mass spectrum can be acquired at each point. Using this data, “heat maps” can be generated indicating the location of specific ions or molecules in the tissue section. No prior knowledge of the sample is required allowing the generation of data with or without a hypothesis.
An image of a Staphyloccus colony (USA300) on the left followed by heat maps created using IMS. These three images show the localization of the various molecules in the colony. This clearly shows dPSMa1 and dPSMa4 are on the perimeter of the colony[1]. This project aims to use established Imaging Mass Spectrometry techniques to characterise the molecules involved during infection of a host cell by a pathogen such as Staphylococcus. The project will be conducted within the Proteomics Core Facility and involve the application of the following techniques:
MALDI mass spectrometry.
Cell culture of cell monolayers (single layers of host cells) and their infection by pathogenic organisms such as Staphylococcus.
Confocal and fluorescence microscopy of infected monolayers. 1. Novel phenol-soluble modulin derivatives in community-associated methicillin-resistant
Staphylococcus aureus identified through imaging mass spectrometry. Gonzalez DJ, Okumura CY, Hollands A, Kersten R, Akong-Moore K, Pence MA, Malone CL, Derieux J, Moore BS, Horswill AR, Dixon JE, Dorrestein PC, Nizet V. J Biol Chem. 2012 Apr 20;287(17):13889-98.
2. Microbial competition between Bacillus subtilis and Staphylococcus aureus monitored by imaging mass spectrometry. Gonzalez DJ, Haste NM, Hollands A, Fleming TC, Hamby M, Pogliano K, Nizet V, Dorrestein PC. Microbiology. 2011 Sep;157(Pt 9):2485-92
3. Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis. Liu WT, Yang YL, Xu Y, Lamsa A, Haste NM, Yang JY, Ng J, Gonzalez D, Ellermeier CD, Straight PD, Pevzner PA, Pogliano J, Nizet V, Pogliano K, Dorrestein PC. Proc Natl Acad Sci U S A. 2010 Sep 14;107(37):16286-90
Project Title: Understanding the mechanisms of the platelet storage lesion following platelet
cryopreservation Supervisors:
Dr Lacey Johnson, Australian Red Cross Blood Service; Dr Matthew Padula, UTS; Dr Denese Marks, Australian Red Cross Blood Service. Location: Primarily at the Applied and Developmental Research Laboratory, Australian Red Cross Blood Service, Alexandria. Background: Platelets for transfusion are typically stored at 22-24 °C for up to 5 days. However, the shelf-life can be extended to 2 years when frozen at -80 °C in 5-6% dimethylsulfoxide (DMSO). Frozen platelets have been used in military applications for more than 30 years [1] and in vivo data suggests that frozen
platelets are more effective than liquid-stored platelets in restoring haemostasis and reducing nonsurgical blood loss [2]. Despite being effective, the freeze/thawing process is known to induce alterations to platelet in vitro quality, including morphological changes, reduced aggregation, and increased activation [3]. In addition,
the expression profile of numerous platelet receptors is affected. These receptors are essential for platelet function, via the activation of multiple signal transduction pathways, including the Mitogen Activated Protein Kinase (MAPK) signalling pathway [4]. Little is currently known about the mechanisms regulating these changes and understanding them may allow for improvement of the platelet cryopreservation techniques. Objectives: To understand the changes occurring in platelet receptor proteins and down-stream signal
transduction proteins in platelets following freezing and thawing. Methodology:
Freezing and thawing platelet concentrates Platelet stimulation with collagen and/or ADP to activate signalling pathways Phospho-kinase protein arrays (R&D systems) 2D gel electrophoresis and mass spectrometry Western blotting ELISA Flow cytometry
Ethics: Ethics approval has already been granted for this project by the Blood Services Human Research
Ethics Committee. Available Resources: The student will have full access to the facilities within the research and testing
laboratories of the Blood Service. This includes use of platelets and other blood products, bench and desk space, consumables and reagents, western blotting apparatus, ELISA reagents and plate reader and a FACSCanto II flow cytometer with FACSDiva analysis software. Further, they will have access to equipment and expertise at the Protein and Proteomics Core Facility at UTS. Key References: 1 Lelkens et al. Transfusion and Apheresis Science 2006;34: 289-98. 2 Khuri S et al. Journal of Thoracic and Cardiovascular Surgery 1999;117: 172-83. 3 Johnson LN et al. Cryobiology 2011;62: 100-6. 4 Adam F et al. Journal of Thrombosis and Haemostasis 2008;6: 2007-16
Fighting emerging bacterial diseases Supervisor: Dr Nico Petty Research in the Microbial Genomics Group is focused on the evolution and spread of clinically important bacterial pathogens and their viruses (bacteriophages). We use high throughput DNA sequencing, genomics, bioinformatics and molecular microbiology to track disease outbreaks and investigate the contribution of phages and other mobile genetic elements to the emergence of new pathogens and antibiotic resistance, with the aim of developing new strategies for the diagnosis, treatment and prevention of infectious diseases. We mainly study pathogenic E. coli, but also other bacteria that are important causes of public health concern, including Vibrio cholera (in collaboration with Dr Maurizio Labbate) and Legionella.
We have several projects currently available including:
What makes a pathogen: genome evolution in emerging pathogens.
Interactions and horizontal gene transfer between phages and their bacterial hosts.
Development of new bioinformatics tools for analysis of microbial genome data.
Please contact Nico ([email protected]) to discuss research opportunities.
Project title: The role of microbes in controlling leakage of carbon from seagrass ecosystems
Supervisors: Peter Ralph (C3), Peter Macreadie (C3/CENS), and Justin Seymour (C3). The student will be jointly supervised by MMB and C3 staff. Summary: Microbial priming is a fundamental process controlling underground ecosystem carbon storage. It
has become the new paradigm for understanding the dynamics of carbon release in terrestrial systems; however, it has been largely ignored in the aquatic fields. This project will provide a first assessment of microbial priming in Australian seagrasses ecosystems, which are among the planet’s most effective natural systems for removing greenhouse gases. This project will test how microbial abundance and community composition changes with habitat disturbance, and the relationship between these changes and carbon leakage from degraded seagrass meadows, thereby testing the hypothesis that microbes control carbon leakage in seagrass ecosystems. Ultimately, the goal of this project is to generate new knowledge on the role of microbes in the seagrass carbon cycle, and to provide new information for managing these powerful yet threatened carbon sinks. Approach: This project will involve fieldwork in shallow subtidal seagrass meadows to sample microbes from degraded and healthy meadows, and laboratory work to enumerate (flow cytometry) and characterise (ARISA, pyrosequencing) microbial communities. The student will be jointly supervised by MMB and C3 staff.
Chemical warfare: caught in the crossfire
Supervisor: Dr Ken Rodgers, MMB, UTS. Co-supervisor: Dr Rachael Dunlop, MMB, UTS. Location: UTS, Building 4.
Animals, in common with plants and bacteria, synthesise proteins from a pool of 20 protein amino acids. 100s of amino acids exist in nature that are close structural analogues of protein amino acids and can be mistakenly used for protein synthesis.1,2 Proteins synthesised using nonprotein amino acids misfold and are often toxic or can result in intracellular accumulation of protein aggregates3,4. Plants cannot run away so often use nonprotein amino acids in chemical warfare. Canavanine made by a number of leguminous plants, including alfalfa, is mistakenly incorporated into proteins in place of L-arginine and kills larvae of predatory insects. The roots of certain grasses release meta-tyrosine which is mistaken for L-phenylalanine and kills surrounding plants competing for territory.
Nonprotein amino acids are present in legumes, fruits, seeds and nuts and are ubiquitous in the diets of human populations around the world. Their consumption has been implicated in a broad spectrum of human diseases such as neurodegeneration and autoimmune disorders. The drug levodopa is a close structural analogue to the protein amino acid L-tyrosine and we have detected proteins containing incorporated levodopa in the brain of levodopa-treated individuals.5 The toxicity of nonprotein amino acids, that are mistaken for protein amino acids and used in protein synthesis, is poorly understood but clearly important.
In this project we will examine at a cellular level the earliest changes that occur in response to the synthesis of proteins containing nonprotein amino acids. We will examine the potential of non-protein amino acids to cause disease and also their potential as theraputic agents in diseases such as cancer. We will also examine the ability of the parent protein amino acid to remove nonprotein amino acids from proteins. Techniques that could be used: Molecular biology (quantitative PCR), human cell culture, high performance liquid chromatography (HLPC), electrophoresis and western blotting, fluorescent staining and microscopy, functional and toxicity studies using transgenic zebrafish. Suggested reading (please email ken for pdfs of any references: [email protected]) 1 Rubenstein, E., Biologic effects of and clinical disorders caused by nonprotein amino acids. Medicine
(Baltimore) 79 (2), 80-89 (2000). 2 Rodgers, K.J. & Shiozawa, N., Misincorporation of amino acid analogues into proteins by
biosynthesis. Int J Biochem Cell Biol 40 (8), 1452-1466 (2008). 3 Ozawa, K. et al., Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins. Febs J 272
(12), 3162-3171 (2005). 4 Dunlop, R.A., Brunk, U.T., & Rodgers, K.J., Proteins containing oxidized amino acids induce
apoptosis in human monocytes. Biochem J 435 (1), 207-216 (2010). 5 Rodgers, K.J., Hume, P.M., Morris, J.G., & Dean, R.T., Evidence for L-dopa incorporation into cell
proteins in patients treated with levodopa. J Neurochem (2006).
Could motor neurone disease be triggered by an unusual amino acid made by blue-green algae?
Supervisor: Dr Ken J. Rodgers, MMB, UTS. Other co-supervisors: Dr Rachael A. Dunlop, MMB, UTS. Location: UTS, Building 4. Amyotrophic lateral sclerosis (ALS) also known as motor neurone disease (MND) is a rare neuromuscular condition. It is characterised by progressive paralysis of the muscles controlling the limbs, speech and respiration due to the degeneration of nerves controlling voluntary muscles. Whilst multiple genes have been implicated in ALS (familial or fALS), in more than ninety percent of cases the causes are unknown (sporadic or sALS). There is no cure for either sALS or fALS and death commonly occurs three to five years after diagnosis. β-methylamino-L-alanine (BMAA), an amino acid produced by cyanobacteria (blue-green algae), has been linked to sALS in indigenous communities in Australia, Japan, and the south pacific island of Guam and was recently found to be deposited in the brains of ALS patients in the USA. Cyanobacteria are ubiquitiously distributed in terrestrial, fresh water and marine environments and there are multiple routes of human exposure including biomagnification through the food chain. Indeed, high concentrations of BMAA have been found in several types of seafood, including crabs, mussels and shark fin. Critically, the vast majority of BMAA present in extracts of cyanobacteria and brain tissue of ALS patients is in a protein-associated form. Our lab has been exploring the mechanisms of toxicity mediated by proteins containing BMAA, including their impact on gene expression and cell survival. This project has already produced a worldwide patent and clinical trials for a potential therapy will commence in November 2012. Techniques that could be used: Molecular biology (quantitative PCR), human cell culture, high performance liquid chromatography (HLPC), electrophoresis and western blotting, fluorescent staining and microscopy, functional and toxicity studies using transgenic zebrafish. Further reading: The Emerging Science of BMAA: Do Cyanobacteria Contribute to Neurodegenerative Disease? Was Lou Gehrig’s ALS Caused by Tap Water
Title: Liver-directed gene therapy of diabetes: Investigation of pancreatic trandifferentiation in a rodent liver cell line following expression of insulin and the beta cell transcription factor Neurod1: H4IIEins/ND Supervisor: Prof. Ann Simpson
Type I diabetes mellitus is caused by the autoimmune destruction of the beta cells of the pancreas that secrete insulin. Gene therapy is a promising strategy being explored to correct blood glucose concentrations in patients with Type I diabetes. In the current study, we used a bicistronic retroviral vector to deliver either the human insulin gene alone or the human insulin gene plus the rat NeuroD1 gene to the rat liver cell line H4IIE to determine if storage of insulin and pancreatic transdifferentiation occurred. Stable clones were selected and expanded into cell lines: H4IIEins cells (insulin gene alone), H4IIEins/ND (NeuroD1 gene alone)
and H4IIEins/ND (insulin and NeuroD1 genes). The H4IIEins/ND cells (107) were subsequently transplanted subscutaneously into diabetic NOD/ Scid mice to examine if the cells could reverse diabetes.
H4IIEins cells did not store insulin, however, H4IIE/ND and H4IIEins/ND cells stored 65.5+5.6 and
1,475.4+171.8/ pmol insulin per 5x106 cells (n=5), respectively. Unlike the parent cell line or H4IIEins cells, several beta cell transcription factors (Pdx1, NeuroD1, Nkx2.2, Nkx6.1) and pancreatic hormones (insulin 1
and 2, glucagon, somatostatin, pancreatic polypeptide) were expressed in both H4IIE/ND and H4IIEins/ND cell lines. Transmission electron microscopy revealed large numbers of insulin storage vesicles in the H4IIEins/ND cell line. Regulated secretion of insulin in response to increasing concentrations of glucose (0-20 mM) was seen only in the H4IIEins/ND cell line. The glucose response curve of the H4IIEins/ND cell line was near physiological; insulin secretion commenced in the presence of 2.5mM glucose. The blood glucose levels of all 9 diabetic mice that were transplanted became normal, 5.6 + 0.1 mM, in 5-10 days after the cells were implanted. Removal of the grafts at 21 days resulted in a prompt increase in the blood glucose levels to hyperglycaemic values, 17.9 + 4.2 mM. Our results suggest that expression of NeuroD1 together with insulin
may be a useful strategy for inducing islet neogenesis and reversing diabetes without exocrine differentiation. The aim of the honours project is to characterise the expression profiles of pancreatic genes that have
been genes that have been expressed in the insulin-secreting liver cells by quatitative RT-PCR and analyse the process that has led to the pancreatic transdifferentiation of the liver cells more thoroughly. This will be done by growing the liver cells in culture, isolating RNA and performing the quatitative RT-PCR assays. Quantitative real time PCR validation of differentially expressed genes will be performed. Relative quantification will be calculated using the comparative threshold cycle (CT) method. Protein analysis of selected genes will be followed up by western analysis.
FORENSIC FACE AND BODY MAPPING RESEARCH
The Forensic Face and Body Mapping Unit of the School of Medical and Molecular Biosciences and Centre for Forensic Science, is focussed on research into reviewing, refining current protocols and developing novel and innovative techniques of identification from images such as photos on identification documents (e.g. driver’s licence and passport) and CCTV surveillance footage. The ultimate aim is to equipped experts and law enforcement officials (e.g. Police, Prosecutors, Department of Immigration and Citizenship and Interpol) with the forensic intelligence and tools (e.g. manual and training) to combat crime such as identity theft and fraud and offensive acts recorded by CCTV surveillance cameras. Currently, face and body mapping evidence are only admissible in the Australian Court of Law for outlining the similarities and differences and not to confirm identity. The Unit is pro-active in standardising Face and Body Mapping protocols that would provide evidence which ultimately be admissible as identification evidence. Our research predominantly involves the morphometric study of the human anatomy (i.e. head, face and body), gait, personal behavioural and habitual characteristics to establishing population and frequency of occurrence database from which correlations with respect to age, race and sex are obtained. Another aspect of our research is image distortion assessments for qualifying the use in the Face and Body Mapping analyses. Honours Projects 2014
1. Mapping the human eyes and eyebrows: A forensic morphometric study. 2. Mapping the human mouth and lips: A forensic morphometric study. 3. Quantifying image distortion of ID photos to qualify the use in Face Mapping. 4. Quantifying image distortion of CCTV surveillance footage to qualify the use in Body Mapping. Supervisor: Dr Meiya Sutisno Advisors: Ms Jennifer Wright, Ms Joanna Spasojevic, E/Prof Richard Wright Collaborators: Prof Shari Forbes (UTS), A/Prof Masa Takatsuka (USYD), Dr Hayley Green (UWS)
Synthesis of RAFT Mediated Cationic Glycopolymer for Nucleic Acid delivery Supervisor: Dr S. R. Simon Ting (Centre for Health Technologies, UTS)
Locations: Research work will be conducted primarily at the Faculty of Science UTS level 4 and 6 laboratories with minor use of instrument at UNSW.
Background:
Diseases cause by genetic disorder has affected the health and lifestyle of many individuals around the world. The current method of treatment involves very high doses of drugs to suppress the symptoms. Small interfering RNA (siRNA) is the most recent nucleic acid molecule employed to silence gene expression, thereby, arresting the disease-causing genes.1 However, the effective delivery of siRNA is an arduous task. Glycopolymers are polymers bearing carbohydrate moieties and they have very strong binding abilities with cell receptors due to their multivalent interactions between the sugar repeating unit from the glycopolymers and the cell surface receptors.2 There have already been delivery systems (e.g. lipid based system) out in the market concerning gene knockdown technology, but none of these are specific toward targeting the affected areas. Receptor mediated delivery of siRNA using targeting glycopolymers paves the way to efficient therapeutic window for delivering siRNA.
Objectives: In this project, students will have hands on experience on performing reversible addition-
fragmentation transfer (RAFT) polymerization to generate cationic glycopolymers. Due to the controlled/living nature of RAFT polymerization, complex glycopolymer architectures such as diblock copolymers could be synthesized. Glycomonomer synthesis will initially be carried out and analysed using the nuclear magnetic resonance (NMR). This will be followed by performing polymerization in solution and resulting polymers will also be analysed using NMR and gel permeation chromatography (GPC).
Figure 1: Glycopolymers multivalent interactions with cell receptors (Left) and polymers without sugar
moieties with no cell surface receptor interaction (Right).2 References: 1 Dorsett, Y.; Tuschl, T. Nat. Rev. Drug Disc. 2004, 3, 318-329. 2 Ting, S. R. S.; Chen, G.; Stenzel, M. H. Polym. Chem. 2010, 1, 1392-1412.
The impact of E6 and E7 HPV-16 oncogenes on microRNA expression in oral cancers
Supervisor: Nham Tran ([email protected]) Background: Human papillomavirus (HPV), notably type 16, is a risk factor for up to 50% of oral squamous
cell carcinomas (SCC)s. For many decades, oral cancers were considered to be a disease associated with the older generation. Older men were the major demographic group with smoking and alcohol consumption being the main risk factors for oral SCC. Today this paradigm has shifted. HPV16 is now a major cause of oral cancers in the US and this rate is growing around the world. The fastest growing segment for oral cancers, are young women in the 20-24-age range, who are never smokers but are predominantly HPV16 positive (+). Similar trends for HPV16 incidence are seen in the UK and it is also on the rise. There is now significant public heath concern for the spread of HPV16 in oral cancers. These HPV16 positive tumours are localized to the posterior of the mouth; in the oropharynx, tonsils, and at the base of the tongue. Small non-coding RNAs (microRNAs-miRNAs) are known to play a vital role in the transformation process and recent evidence suggests that HPV16 may impact miRNA pathways in oral cancers. Objectives: To explore the associations between HPV16 and the expression of miRNAs in oral cancers.
Methodology: These projects cover a range of skills from cell biology to molecular techniques. The candidates will be fully trained in all aspects of the work. The projects are offered as honours by research or a full PhD program.
Tissue culture: Growing cancer cells.
Working with a major hospital to collect patient samples and tissue banking
Isolation of small RNAs from in vitro cells and patient samples.
Molecular biology skills such as Real Time PCR, cloning and bacterial work.
Regulation of genes using siRNAs and LNA antisense.
Detection of proteins using western blotting.
Presentation of results at regular lab meetings.
Selection criteria: The laboratory is seeking an enthusiastic and highly motivated individual to undertake this
research. This person must have a passion to learn and be very open to innovative methods of research. Please contact the supervisors above for further information.
The expression and function of exosomal long non-coding RNAs in prostate cancer
Supervisor: Nham Tran ([email protected]) Background and aims: Each year in Australia, close to 3,300 men die of prostate cancer, and each day
about 32 men learn news that they have prostate cancer. An estimate 20,000 new cases will be diagnosed each year and this rate is increasing. One of the major challenges for the treatment of prostate cancer is early detection and currently there are no robust clinical biomarkers. Our laboratory has identified a set of novel gene(s), which have the potential to become future biomarkers. These genes are unlike the traditional genes but are non-coding RNAs longer then 1 KB. These long ncRNAs are enriched in secreted vesicles, but their current function and application, as biomarkers are unknown. This project will have two major Aims A) to confirm the expression of these ncRNA in micro-particles and exosomes and B) to determine their function in the progression of cancer. Methodology: These projects cover a range of skills from cell biology to molecular techniques. The
candidates will be fully trained in all aspects of the work. The projects are offered as master by research or a full PhD program.
Tissue culture: Growing cancer cells.
Working with a major hospital to collect patient samples and tissue banking
Isolation of long RNA from in vitro cells and patient samples.
Molecular biology skills such as Real Time PCR, cloning and bacterial work.
Regulation of genes using siRNAs and LNA antisense.
Isolation of micro-particles and exosomes from bodily fluids.
Detection of proteins using western blotting.
Presentation of results at regular lab meetings.
Selection criteria: The laboratory is seeking an enthusiastic and highly motivated individual to undertake this
research. This person must have a passion to learn and be very open to innovative methods of research. Please contact the supervisors above for further information.
Project Title: Effects of myeloperoxidase-derived oxidants on the biological function of the extracellular matrix
in the artery wall during chronic inflammation
Supervisors: Dr. David van Reyk
School of Medical and Molecular Sciences, University of Technology, Sydney, PO Box 123,
Broadway, NSW 2007
Tel: 02 9514 2221; Fax: 02 8206; Email: [email protected]
Prof. Michael Davies
The Heart Research Institute, 7 Eliza Street, Newtown, Sydney, NSW 2042. Tel: 02 8208 8900; Fax: 02 9565 5584; Email: [email protected]
Dr. Christine Chuang The Heart Research Institute, 7 Eliza Street, Newtown, Sydney, NSW 2042.
Tel: 02 8208 8900; Fax: 02 9565 5584; Email: [email protected]
Project Background:
Chronic inflammatory diseases such as atherosclerosis is responsible for ~40% of all deaths in developed
countries including Australia. Atherosclerosis is characterized by endothelial dysfunction (an early and defining marker for atherosclerosis), the accumulation of lipids in macrophage cells within the artery wall, the migration and
proliferation of underlying smooth muscle cells into the intima, and, in many cases, lesion rupture and thrombosis. It is
well established that the extracellular matrix (ECM) of the artery wall consists primarily of fibronectin, laminin, type IV collagen, and heparan sulfate (HS) proteoglycans including perlecan, which interact with growth factors and enzymes to
regulate endothelial cell adhesion, proliferation and migration (Figure 1). These interactions are perturbed in
atherosclerotic lesions, where activated monocytes and macrophages generate reactive oxidants, including the
myeloperoxidase (MPO)-derived oxidants including hypochlorous acid (HOCl, the major component of household bleach) and hypothiocyanous acid (HOSCN), which subsequently alters the ECM proteins in the artery wall.
Enzymatically active MPO protein and elevated levels 3-chlorotyrosine have been shown to be present in human atherosclerotic
lesions. Our studies have shown that oxidized proteins are present in the
ECM of human atherosclerotic plaques, and these materials are
consistent with HOCl-mediated oxidation. Furthermore, we have demonstrated that MPO-derived HOCl disrupts the protein core of a key
component of the subendothelial ECM, perlecan, which inhibits its
biological interaction with fibroblast-growth factor (FGF) 2 and cellular proliferation. Most recently, we have also demonstrated that HOCl-mediated oxidation modulates the structure of other
important ECM proteins, including fibronectin and laminin. Furthermore, cell adhesion binding sites of fibronecin was
damaged by HOCl-mediated oxidation.
Collectively, these data provide compelling evidence that MPO-derived oxidants, and particularly HOCl, play a
key role in altering the vascular basement membrane by targeting the ECM proteins that underlies arterial endothelial
cells during the development of atherosclerosis. Thus, we hypothesise that oxidative modification of ECM proteins
contributes to endothelial cell dysfunction and weakening of the mechanical properties of the artery wall, which
is associated with plaque rupture, the major cause of most heart attacks and strokes. The information gained
through these studies will provide vital information on the key processes and factors that contribute to oxidant-mediated damage in vivo and aid the targeted development of strategies to limit cellular dysfunction and chronic inflammatory
diseases such as atherosclerosis.
Aim: To investigate and characterise endothelial and smooth muscle cell adhesion on oxidised ECM proteins, which
are functionally important in maintaining the mechanical properties of the artery wall.
This project will employ a range of techniques to study and characterise the effects of endothelial and smooth muscle
cell activity (adhesion/spreading, proliferation and migration) on untreated and HOCl-treated ECM proteins. The experimental techniques involved in this project will include enzyme-linked immunosorbent-assay (ELISA), cell
culture, immunocytochemistry and microscopy.
Role of LDL modification in the endothelial dysfunction in atherosclerosis Supervisors: Dr. David van Reyk School of Medical and Molecular Sciences, University of Technology, Sydney, PO Box
123, Broadway, NSW 2007 Tel: 02 9514 2221; Fax: 02 8206; Email: [email protected] A/Prof. Clare Hawkins The Heart Research Institute, 7 Eliza Street, Newtown, Sydney, NSW 2042. Tel: 02 8208 8900; Fax: 02 9565 5584; Email: [email protected] Introduction Atherosclerosis is characterised by the accumulation of cholesterol and cholesteryl esters in monocyte / macrophage cells within the artery wall, with the resulting lipid-laden (“foam”) cells a hallmark and defining feature of this disease. Low-density lipoproteins (LDL) are the source of most of this lipid, as modification of LDL results in recognition and subsequent uncontrolled uptake of the modified particles by “scavenger” receptors. Moreover, LDL modified by oxidation (oxLDL) can also cause endothelial activation and dysfunction. Human lesions contain significant amounts of LDL modified by myeloperoxidase (MPO), an enzyme released under inflammatory conditions that is responsible for the formation of the potent chemical oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) [1]. MPO is a major risk factor for the development of coronary artery disease and a powerful prognostic agent for predicting the outcome of patients with cardiac symptoms [2, 3]. Increasing evidence shows that LDL modified by MPO-derived oxidants is highly pro-atherogenic [4]. Although MPO-modified LDL is highly relevant to human disease, there are a lack of data relating to the mechanisms involved in the cellular processing and accumulation of MPO-modified LDL in human cells and the resulting detrimental consequences, which will be examined in this project. We hypothesise that LDL modified by MPO-derived oxidants endothelial dysfunction, which exacerbates inflammation and accelerates atherosclerosis. Project Overview
In this project, we will examine the role LDL modified by MPO-derived oxidants on endothelial cell function using cultured primary human coronary artery endothelial cells (HCAEC). Our preliminary studies show that exposure of HCAEC to LDL modified by HOCl induces the increased expression of cellular adhesion molecules and pro-inflammatory cytokines, which may help to propagate inflammation in vivo. The role of the
inflammatory transcription factor NF- B in the HCAEC expression of adhesion molecules and cytokines will be examined by measuring the nuclear translocation of p65 by Western blotting. A blotting approach will also be used to examine the role of mitogen-activated protein kinase (MAPK) stress response pathways, by measuring changes in the extent of phosphorylation of p38, ERK and JNK proteins. Endothelial dysfunction will also be examined in HCAEC treated with each modified LDL. Changes in the activity of endothelial nitric oxide synthase (eNOS) will be assessed by examining changes in the ratio of eNOS dimer (active) to monomer (inactive) forms by Western blotting. eNOS activity will be determined by quantifying the conversion of Arg, the substrate for eNOS, to citrulline. Changes in eNOS activity will affect nitric oxide production, which will be assessed indirectly by measuring the accumulation of NO2
-/NO3- and directly using EPR spectroscopy
with spin trapping. eNOS uncoupling can result in superoxide production (O2-), which will be examined using
HPLC with dihydroethidium. The project will provide training and experience with cultured primary cells and will utilise a range of laboratory techniques including Western blotting, ELISA, real-time RT-PCR, flow cytometry, HPLC and EPR spectroscopy. References (1)Davies, M. J., Hawkins, et al. (2008) Mammalian heme peroxidases: from molecular mechanisms to health implications. Antioxid. Redox Signal. 10, 1199-1234; (2) Zhang, R., Brennan, et al. (2001) Association between myeloperoxidase levels and risk of coronary artery disease. J. Am. Med. Assoc. 286, 2136-2142 (3) Brennan, M. L., Penn, M. S., et al. (2003) Prognostic value of myeloperoxidase in patients with chest pain. N. Engl. J. Med. 349, 1595-1604 (4) Malle, E., Marsche, G., et al. (2006) Modification of low-density lipoprotein by myeloperoxidase-derived oxidants and reagent hypochlorous acid. Biochim. Biophys. Acta. 1761, 392-415
Do selenium-containing amino acids prevent damage by reactive oxidants produced during chronic inflammation ?
Supervisors: Dr David van Reyk (University of Technology Sydney), email: [email protected] Dr David Pattison (Heart Research Institute, Sydney); email: [email protected] Prof Michael Davies (Heart Research Institute, Sydney), email: [email protected]
Activated white cells play a key role in the immune response to invading pathogens. These cells
generate large fluxes of reactive oxidants via an oxidative burst that leads to pathogen killing. Inappropriate or misdirected stimulation of this system however, has the capacity to damage host tissues, and this has been linked to a large number of human pathologies [1,2,3]. These include cardiovascular disease, asthma, arthritis and some cancers.
Peroxidase enzymes (e.g. myeloperoxidase, MPO) are major sources of these reactive oxidants. MPO uses hydrogen peroxide (H2O2) to form powerful oxidants, including hypochlorous acid (HOCl, the major component of bleach), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN). Other oxidants including peroxynitrous acid (ONOOH) and singlet oxygen (an excited state of O2) are also formed at sites of inflammation.
We have shown that these oxidants react rapidly with sulphur-containing amino acids (cysteine (Cys) and methionine (Met)) and some of the products of these reactions have been characterized (reviewed [1,3]). Recent kinetic studies have shown that selenium compounds are considerably more reactive than their sulphur analogues [4,5], and we have therefore hypothesized that seleno compounds will be key targets for oxidants in vivo. Key antioxidant enzymes such as glutathione peroxidase (GPx) and thioredoxin reductase rely on a
selenocysteine (Sec) residue for their activity, and are readily inactivated by inflammatory oxidants [4,6], but the mechanisms and products of oxidative damage are unclear.
In this project the products of reaction of inflammatory oxidants with physiologically relevant low-molecular-mass seleno compounds (diselenides, selenides and selenols, including Sec) will be investigated by HPLC and mass spectroscopic techniques. These studies will be extended to investigate the fate of the Sec residues in isolated selenoproteins such as GPx, where the damage to the protein will be analysed by peptide mass mapping following enzymatic digestion of the protein.
The data obtained will give insight into the mechanisms of damage to selenoproteins by inflammatory oxidants, thereby providing a rational basis for the development of protective agents against inflammation-induced damage.
Key references 1) Davies et al (2008) Mammalian heme peroxidases: from molecular mechanisms to health implications. Antioxid. Redox Signal. 10, 1199.
2) Pattison et al (2012) Reactions and reactivity of myeloperoxidase-derived oxidants: Differential biological effects of hypochlorous and hypothiocyanous acids, Free Radic. Res., 46, 975. 3) Davies MJ (2005) The oxidative environment and protein damage Biochim. Biophys. Acta 1703, 93.
4) Skaff et al (2012) Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage, Biochem. J. 441, 305.
5) Storkey et al (2012) Preventing protein oxidation with sugars: Scavenging of hypohalous acids by 5-selenopyranose and 4-selenofuranose derivatives, Chem. Res. Toxicol., 25, 2589. 6) Suryo Rahmanto et al (2012) “Photo-oxidation-induced inactivation of the selenium-containing protective enzymes thioredoxin reductase and glutathione peroxidase” Free Radic. Biol. Med. 53, 1308.
