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SCHOOL OF CHEMISTRY AND FORENSIC SCIENCE

Bachelor of Science (Honours) in Applied Chemistry

HONOURS PROJECTS FOR 2014

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The value of an Honours degree An Honours degree provides an opportunity to get involved in a research program in an area that interests you, as well as providing training in research techniques and experience with modern research instrumentation. The Honours programme adds a new dimension to the skills that you have acquired during your undergraduate years and enhances your immediate employment prospects and, more significantly, your future career potential. An Honours degree can lead to an opportunity to proceed to a postgraduate research degree (MSc or PhD), with financial support from an Australian Postgraduate Award (APA) or some other postgraduate scholarship.

Eligibility

To be eligible for entry into the Honours degree, applicants should have a Bachelor of Science majoring in Chemistry or Forensic Chemistry and should have achieved a minimum of a credit average (65%) during their undergraduate programme.

Assessment In the Honours year, each student undertakes an original research project under the supervision of a member of academic staff, writes a thesis about the project and presents a talk on the outcomes. There is also a coursework component, with assessment tasks on advanced chemistry topics. Choosing a project

It is advisable to contact a potential supervisor and discuss a project during the semester prior to enrolment in the Honours project. A number of research projects are on offer in the school and are outlined in this booklet. Feel free to discuss any of these with the appropriate supervisor.

If you have an interest in carrying out a project in an area that is not listed, it may be possible to arrange suitable supervision. For instance, a number of previous students have carried out their work-based projects in conjunction with the CSIRO, ANSTO or an industrial partner.

How to apply

After discussing deciding on a project with a supervisor, fill out the forms available at http://www.uts.edu.au/future-students/science/go-further/honours-program/school-chemistry-and-forensic-science. Applications should be submitted by November 29, 2013 to be considered for a first round offer, but final round applications are accepted until January 31, 2014. Students generally begin work on their project in February. There are Autumn and Spring semester intakes for the programme.

Advice

If you have any questions about the programme, please feel free to discuss them with the Chemistry Honours Coordinator, A/Prof. Barbara Stuart (email Barbara.Stuart@uts.edu.au).

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Synthesis of stemofurans as FtsZ inhibitors: potential antibiotic agents

Supervisors: A/Prof. Alison Ung and Prof. Elizabeth Harry

Stemonafurans are natural products isolated from the roots of Stemona plants. These compounds were found to be antifungal and antibiotic against MRSA.1 Despite information on the antimicrobial activities of stemofurans, the actual mode of actions has not yet been established. Stemofurans are structurally similar to known FtsZ inhibitors, such as 8j, which has been shown to effectively killed Staphylococci aureus.2

Figure 1

FtsZ (Filamentous temperature sensitive mutant Z) is the cytoskeletal protein essential in bacteria cell division. It forms the cytokinetic Z-ring at the division site,3 and it has been viewed as an attractive target for the development of new antibiotics.4 Our main objective is the development of stemofurans as FtsZ inhibitors with the aims to use of these and for better understanding of bacterial cell division and as potential antibiotics. Specific aims of this project are:

1. To synthesise novel stemofuran derivatives from aromatic alkynes.

2. To study the FtsZ inhibitory activity of stemofuran derivatives.

3. To carry out biological screening of stemofuran derivatives for antibiotic activity.

References

1. T. Sastraruji, S. Chaiyong, A. Jatisatienr, S. G. Pyne, A. T. Ung, W. Lie, Phytochemical Studies on Stemona aphylla: Isolation of a New Stemofoline Alkaloid and Six New Stemofurans, Journal of Natural Products, 2011, 74, 60–64.

2. D. W. Adams, L. J. Wu, L. G. Czaplewski and J. Errington, Multiple effects of benzamide antibiotics on FtsZ function, Molecular Microbiology, 2011, 80, 68−84.

3. E. Bi and J. Lutkenhaus, FtsZ ring structure associated with division in Escherichia coli, Nature, 1991, 354, 161-164.

4. R. L. Lock and E. J. Harry, Cell-division inhibitors: new insights for future antibiotics. Nature Rev. Drug Discovery, 2008, 7, 324−338.

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Synthesis of berberine analogues as potential FtsZ inhibitors

Supervisors: A/Prof. Alison Ung and Prof. Elizabeth Harry

Berberine is a plant alkaloid obtained from various species of Berberis. Plants containing berberine have been traditionally used in Chinese and Native American medicine to fight a number of infectious organisms. Berberine is effective against multiple drug-resistant M. tuberculosis1 and methicillin-resistant S. aureus (MRSA).2 Berberine has been identified as a small molecule that selectively alters bacterial FtsZ Z-ring,3 without inhibiting the function of tubulin assembly, even at high concentration (100 μM).4,5 FtsZ (Filamentous temperature sensitive mutant Z) cytoskeletal protein, it plays a pivotal role in prokaryotic cell division and is present in most of the bacterial species.6 FtsZ inhibitors have been shown to effectively killed bateria.7

Scheme 1

In recent years, FtsZ has been identified as a promising target for the development of novel antimicrobials.8 Our main goal is the development of small molecule inhibitors of FtsZ as antibiotics and the use of small molecules for better understanding of bacterial cell division.

Specific aims of this project are: 1. To synthesise novel analogues 2 from isoquinolines 1 (scheme 1)

2. To explore the FtsZ inhibitory activity of the synthesised compounds.

3. To carry out biological screening of synthesised compounds for antibiotic activity.

References

1. Gentry, E. J., Jampani, H. B., Keshavarz-Shokri, A., Morton, M. D., Velde, D. V., Telikepalli, H, Mitscher, L. A., Shawar, R., Humble, D., and Baker, W. J. Nat. Prod. 1998, 61, 1187–1193.

2. Yu, H., Kim, K., Cha, J., Kim, H., Lee, Y., Choi, N., and You, Y., J. Med. Food, 2005, 8, 454–461. 3. Domadia, P. N., Bhunia, A., Sivaraman, J., Swarup, S., and Dasgupta, D., Biochemistry, 2008, 47,

3225−3234. 4. Wolff, J., and Knipling, L. , Biochemistry, 1993, 32, 13334−13339. 5. Lopus, M., and Panda, D., FEBS J. 2006, 273, 2139−2150. 6. E. Bi and J. Lutkenhaus, Nature, 1991, 354, 161-164. 7. David E. Anderson, Michelle B. Kim, Jared T. Moore, Terrence E. O’Brien, Nohemy A. Sorto, Charles I.

Grove, Laura L. Lackner, James B. Ames, and Jared T. Shaw, ACS Chemical Biology, 2013, 8. R. L. Lock and E. J. Harry, Cell-division inhibitors: new insights for future antibiotics. Nature Rev. Drug

Discovery, 2008, 7, 324−338.

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Alkaloid-like molecules from terpenes as potential anti-cancer agents and AChE inhibitors

Supervisor: A/Prof. Alison Ung

Using natural terpenes in the Bridging Ritter reactions to prepare alkaloid-like compounds of drug-like properties is an innovative approach to Drug discovery. This approach addresses the limiting supply of natural alkaloids required for biological investigation. Our alkaloid-like compounds have shown significant biological activities.1

Our earlier work on the Bridging Ritter reactions of natural cyclic monoterpenes such (-)-β-pinene and (R)-(+)-limonene with a variety of nitriles under Bridging Ritter reaction conditions, provided optically pure cyclic imines 1 and 2, respectively (Scheme 1).2

Scheme 1

Methodology

The cyclic imines will be synthesis, using our standards condition from (R)-(+)-limonene and various nitriles. These compounds will be used as the central intermediates, to generate novel and relatively complex alkaloid-like molecules.

Biological assays Since the natural alkaloids often exhibit a rich spectrum of biological activity and it would be likely the case for the alkaloid-like molecules. Therefore, the potential biological activities of alkaloid-like compounds will be assessed, in particular for anti-cancer activity and AChE inhibitory activity.3,4

Aims

1. To synthesize optically active cyclic imines from (R)-(+)-limonene, via the Bridging Ritter reaction.

2. To synthesize alkaloid-like compounds from the cyclic imines. 3. To assess the biological activities of alkaloid-like compounds for anti-cancer activity and

acetylcholinesterase (AChE) inhibitors as potential treatment of Alzheimer’s disease (AD). References 1. C. Hemtasin, A. T. Ung, S. Kanokmedhakul, K. Kanokmedhakul. R. Bishop, T. Satraruji, D. Bishop,

Monatshefte fur Chemie, 2012, 143, 955-963. 2. A. T. Ung, S. G. Williams, A. Angeloski, J. Ashmore, U. Kuzhiumparambil, M. Bhadbhade, R. Bishop,

Tetrahedron, 2013, submitted. 3. P.J. Houghton, Y. Ren and M.-J. Howes, Nat. Prod. Rep. 2006, 23, 181–199. 4. M.C. Baird, S.G. Pyne, A.T. Ung, W. Lie, T. Sastraruji, A. Jatisatienr, C. Jatisatienr, S. Dheeranupattana, J.

Lowlam, and S. Boonchalermkit, J. Nat. Prod. 2009, 72 , 679-684.

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Alkaloid-like molecules as potential AChE inhibitors

Supervisor: A/Prof Alison Ung

Background

Our earlier work on the Bridging Ritter reactions of cyclic alkene 1 with a CH3CN then followed by reduction to give alkaloid-like compounds 2 and 3 (Fig.1). These alkaloid-like compounds were found to have strong AChE inhibitory activity.1

Figure 1

Methodology

The cyclic imines will be synthesis, using our Bridging Ritter reaction conditions from compound 1.2-4 Cyclic imines will be used to generate novel and relatively complex alkaloid-like molecules such as compound 4 as shown in Figure 1.

Biological assays

Since the natural alkaloids often exhibit a rich spectrum of biological activity and it would be likely the case for the alkaloid-like molecules. Therefore, the potential biological activity of compound 4 and its analogues will be assessed, in particular for anti-cancer activity and AChE inhibitory activity.5-7

References 1. C. Hemtasin, A. T. Ung, S. Kanokmedhakul, K. Kanokmedhakul, R. Bishop, T. Satraruji,

D. Bishop, Monatshefte fur Chemie, 2012, 143, 955-963. 2. I.C.C. Bong, A.T. Ung, D.C. Craig, M.L. Scudder, R. Bishop, Aust. J. Chem., 42, 1929-

1937 (1989). 3. Q. Lin, G.E. Ball, R. Bishop, Tetrahedron, 53, 10899-10910 (1997). 4. Q. Lin, D. Djaidi, R. Bishop, D.C. Craig, M.L. Scudder, Aust. J. Chem., 51,799-806

(1998). 5. P.J. Houghton, Y. Ren and M.-J. Howes, Nat. Prod. Rep. 23, 181–199 (2006). 6. A. Marston, J. Kissling and K. Hostettmann, Phytochem. Anal. 13, 51–54 (2002). 7. M.C. Baird, S.G. Pyne, A.T. Ung, W. Lie, T. Sastraruji, A. Jatisatienr, C. Jatisatienr, S.

Dheeranupattana, J. Lowlam, and S. Boonchalermkit, J. Nat. Prod. 72 , 679-684 (2009).

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From Australian environmental weeds to anti-cancer agents

Supervisors: A/Prof. Alison Ung and Dr Linda Xiao

Background Pampas Lily of-the-valley, Salpichroa origanifolia (Fig. 1a), a native of South America, is an environmental weed in NSW. Previous studies on the extracts of Salpichroa origanifolia (Lam.) Thell growing in Argentina presented a family of withanolides. These molecules are a group of C-28 steroidal lactones and lactols built on an intact or rearranged ergostane framework (Fig. 1b).1

(a) (b) Figure 1: (a) Pampas Lily of-the-valley (Salpichroa origanifolia); (b) withanolides The unique structural feature of these compounds, in comparison to other steroids, was an aromatic ring D. Preliminary studies have shown that these withanolides and derivatives possess significant pharmacological activities, including antitumor,2 cytotoxic,3,4 immunosuppressive,4 anti-inflammatory.5,6 Biological activities of withanolides show immensely valuable therapeutic targets. Hence Pampas Lily of-the-valley (Salpichroa origanifolia) deserves further investigation as a potential source of anticancer agents and bioactive molecules. The plant will be collected under the guidance of Randwick City Council. This project aims to make use of an environmental weed as a source of real important bioactive natural products for future pharmaceutical drug discovery. To achieve this objective we propose:

1. To investigate the biological role of Salpichroa origanifolia by the high-throughput screening of crude plant extracts and the pure isolated natural product compounds for their acetylcholinesterase (AChE) inhibitory and anticancer activities.

2. To isolate the chemical constituents that are shown to havebiological activities. 3. To identify the chemical constituents by GC/MS, LC/MS and NMR spectroscopy.

References

1. Ray, A. B.; Gupta, M. Prog. Chem. Org. Nat. Prod. 1994, 63, 1-106. 2. Jayaprakasam, B.; Zhang, Y.; Seeram, N. P.; Nair, M. G. Life Sci., 2003, 74, 125-132. 3. Minguzzi, S.; Barata, L. E. S.; Shin, Y. G.; Jonas, P. F.; Chai, H.-B.; Park, E. J.; Pezzuto, J. M.; Cordell,

G. A. Phytochemistry 2002, 59, 635-641. 4. Habtemariam, S. Planta Med. 1997, 63, 15-17. 5. Jayaprakasam, B.; Nair, M. G. Tetrahedron 2003, 59, 841-849. 6. Budhiraja, R. D.; Sudhir, S.; Garg, K. N. Planta Med. 1984, 50, 134-6.

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Volatile profiling of Pseudomonas aeruginosa as a screening tool for cystic fibrosis

using breath analysis

Supervisors: Prof. Shari Forbes and A/Prof. Peter Middleton (Westmead Millennium

Institute for Medical Research, University of Sydney)

Chronic Pseudomonas aeruginosa infection is a hallmark of cystic fibrosis lung disease. Current techniques used to screen patients for this infection are both cumbersome and highly invasive. New techniques involving the chemical analysis of volatile biomarkers in breath samples have demonstrated their potential as non-invasive screening tools. Importantly, Pseudomonas bacteria have a distinct metabolism which can produce bacteria-specific volatile organic compounds (VOCs) in exhaled air, allowing for rapid diagnosis and treatment.

This project will chemically profile the VOCs produced by P. aeruginosa which are characteristic of chronic lung infections in cystic fibrosis patients. The VOCs will be collected from breath samples using headspace solid phase micro-extraction (HS-SPME) and analysed using comprehensive two dimensional gas chromatography – time-of-flight mass spectrometry (GCxGC-TOFMS). The results of the project will assist in determining bacteria-specific biomarkers which can be used as a screening tool for detecting lung infection in cystic fibrosis patients. This information will assist with the long term goal of training medical dogs to rapidly detect this infection on the breath of cystic fibrosis patients.

The goals of this project are as follows:

To optimise a GCxGC-TOFMS method for the analysis of VOCs from P. aeruginosa isolates

To compare the volatile profiles of breath samples collected from a control group with a clinical group of cystic fibrosis patients

To identify P. aeruginosa specific volatile biomarkers which can be used to screen cystic fibrosis patients for bacterial lung infection

For further information please contact Prof. Forbes at shari.forbes@uts.edu.au

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Characterisation of wine aromas using comprehensive two dimensional gas

chromatography – time-of-flight mass spectrometry

Supervisors: Prof. Shari Forbes and Dr Tapan Rai (School of Mathematical Sciences)

Understanding the differences in the chemical composition and aroma of wines requires an enhanced analytical method to identify the large number of volatiles present in these complex mixtures. Volatile compounds are collected using headspace solid phase micro-extraction (HS-SPME) for subsequent chromatographic separation and identification. Comprehensive two dimensional gas chromatography – time-of-flight mass spectrometry (GCxGC-TOFMS) is a new separation method which has the capability to accurately profile wine volatiles. This project involves the optimisation of a GCxGC-TOFMS method for the collection and analysis of wine aromas. A range of Australian wines will be analysed to determine volatile differences between wine types. This information can assist in identifying a consumer’s preference for particular wines based on aroma and taste.

The goals of this project are as follows:

To optimise a GCxGC-TOFMS method for the analysis of wine volatiles

To compare the volatile profiles across different wine types

To identify key compounds which characterise a specific wine’s aroma

For further information please contact Prof. Forbes at shari.forbes@uts.edu.au

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Profiling the decomposition odour produced by buried remains

Supervisors: Prof. Shari Forbes and Leading Senior Constable David Cole (NSW Police Force Dog Unit)

Cadaver detection dogs are regularly used to locate human remains in various stages of

decomposition. When attempts have been made to conceal the remains in a clandestine

grave, the process of decomposition will typically slow down. This environment presents a

challenge to cadaver detection dogs since, depending on grave depth, the decomposition

odour is not always detectable on the surface. It is currently unknown to what extent buried

remains will produce a volatile profile aboveground and how long that odour persists. It is

typical for canine handlers to probe the soil around a suspected grave site in an attempt to

release the decomposition odour. However, this process is not ideal as probing is considered

an intrusive technique which can potentially destroy evidence.

The goals of this project are as follows:

To compare the chemical odour produced above a grave site before and after probing the soil

To determine how long the odour is still detectable after burial

To chemically profile the decomposition odour using thermal desorption-two dimensional gas chromatography- time-of-flight mass spectrometry (TD-GCxGC-TOFMS)

To compare the chemical results to alerts by cadaver dogs during training

For further information please contact Prof. Forbes at shari.forbes@uts.edu.au

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Synthesis of RAFT mediated cationic glycopolymer for nucleic acid delivery

Supervisor: Dr Simon Ting (Centre for Health Technologies)

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) spectroscopy. This will be followed by performing polymerization in solution and resulting polymers will also be analysed using NMR spectroscopy and gel permeation chromatography (GPC).

Figure 1: Schematic of glucose-based glycopolymer with thiocarbonylthio end-functional group (bracketed) of RAFT agent.3

References 1Dorsett, Y.; Tuschl, T. Nat. Rev. Drug Disc. 2004, 3, 318-329. 2Ting, S. R. S.; Chen, G.; Stenzel, M. H. Polym. Chem. 2010, 1, 1392-1412. 3Ting, S. R. S.; Min, E. H.; Zetterlund, P. B.; Stenzel, M. H. Macromolecules 2010, 43, 5211-5221.

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New metal oxide core / gold shell nanoparticles

Supervisor: Dr Andrew McDonagh

Background

Nanoparticles with core−shell structures (Fig. 1) may be used as extremely sensitive bio-imaging probes if they possess an appropriate metal oxide core and gold shell. Inclusion of appropriate cores allows for multiple sites to be imaged using an inductively coupled plasma mass-spectrometer (ICP-MS). An appropriate metal is one that is (a) detectable at relatively low concentrations using an ICP-MS and (b) does not occur in biological systems. Thus, a large number of the transition and lanthanide metals are suitable for this purpose. Gold shells, on the other hand, will facilitate the use of mature bio-labelling technology to provide the particles with bio-specificity towards particular targets (e.g. proteins, DNA).

This project will explore new core/shell nanoparticles that incorporate transition and lanthanide metals in their core. The particles will be coated with a gold outer shell that can be functionalized with various thiol-containing compounds. This project will also investigate the detection of the new particles using laser ablation mass spectrometry techniques and extend this technique to include biological samples. It will also assess their suitability for use as markers in bio-imaging applications.

Project aims

This project will test the hypotheses that (a) transition and lanthanide metal oxide / gold shell nanoparticles can be synthesized such the core is coated in a continuous gold shell and that (b) the core/shell particles will behave in a similar fashion to all-gold nanoparticles in the context of the application of bio-labels.

Thus, the aims of this project are to:

Synthesise and characterise new core-shell nanoparticles Examine the physical and chemical properties of the new materials Examine the behaviour of the new particles in laser ablation inductively coupled

mass spectrometry experiments

Figure 1. Structure of a metal oxide core / gold shell nanoparticle.

Metaloxidecore

GoldShell

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Analysis of BMAA in neuroproteins by LC-MS/MS

Supervisors: Dr Ken Rodgers (School of Medical and Molecular Biosciences) and Dr David Bishop

Background β-methylamino-L-alanine (BMAA), an amino acid produced by cyanobacteria (blue-green algae), has been linked to neurodegenerative disease in indigenous communities in Australia and the South Pacific (Guam) and more recently to sporadic Amyotrophic Lateral Sclerosis (sALS) and Alzheimer’s disease (AD) in Canada and the USA. Cyanobacteria are ubiquitously distributed in terrestrial, fresh water and marine environments and due, in some cases, to mismanagement of these resources human exposure to BMAA is increasing. Of great concern is recent data showing BMAA is bioconcentrated by aquatic species raising the possibility that we could be subject to ‘silent’ exposure to BMAA in the absence of any readily identifiable cyanobacterial blooms.

The mechanism whereby BMAA triggers neurological disorders is not yet known. BMAA is most commonly found in a protein-associated form. The nature of this association was not known but it has generally been considered to contribute to the chronic toxicity attributed to BMAA. We have recently shown for the first time that BMAA (a non-protein amino acid) competes with the protein amino acid L-serine for incorporation into proteins by protein synthesis and this promotes intracellular protein misfolding and aggregation (PLOS ONE in press). BMAA could therefore specifically ‘target’ aggregate-prone proteins and could be an environmental trigger that could impact on individuals with a genetic susceptibility to protein misfolding disorders. We believe that this provides, for the first time, a creditable mechanism of BMAA toxicity and its association with a range of disorders associated with protein misfolding such as amyotrophic lateral sclerosis (ALS/MND), Parkinson’s disease (PD) and dementia. Aims of the project In this project human motor neurons and astrocytes will be cultured in the presence of BMAA. The rate of uptake of BMAA by cells and the level of incorporation of BMAA into proteins will then be assessed under different culture conditions by LC-MS/MS. A cell-free protein expression system will be used to examine BMAA incorporation into specific neuroproteins and the position of the L-serine substitution for BMAA in the protein sequence determined. Suggested reading: 1. Rodgers, K.J. and N. Shiozawa, Misincorporation of amino acid analogues into

proteins by biosynthesis. Int J Biochem Cell Biol, 2008. 40(8): p. 1452-66. 2. Bradley, W.G. and D.C. Mash, Beyond Guam: the cyanobacteria/BMAA hypothesis of

the cause of ALS and other neurodegenerative diseases. Amyotroph Lateral Scler, 2009. 10 Suppl 2: p. 7-20.

3. Cox, P.A., Conclusion to the Symposium: the seven pillars of the cyanobacteria/BMAA hypothesis. Amyotroph Lateral Scler, 2009. 10 Suppl 2: p. 124-6.

Please contact Ken (Kenneth.rodgers@uts.edu.au) for pdf files of these articles.

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Atomistic simulations on graphene-based materials for hydrogen storage and in Li-ion batteries

Supervisors: Dr Zhimin Ao and Prof. Guoxiu Wang

Graphene was experimentally fabricated for the first time in 2004 and was found with excellent electrical, mechanical and thermal properties. Graphene has shown promising applications as ultra-sensitive gas sensors, transparent electrodes in liquid crystal display devices, large capacity electrodes in Li-ion batteries and hydrogen storage materials. In this project, to further explore graphene applications in electronic devices, especially in Li-ion batteries, and hydrogen storage materials, atomistic simulation method (using Materials Studio software and other first principle calculation softwares) is used to predict the electronic and magnetic properties of graphene in the presence of substrates and different kinds of defects, the interaction between hydrogen or Li-ion and graphene related materials, then to determine the hydrogen storage behaviors or the performance of Li-ion batteries.

Hydrogen storage in 3D graphene structure

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Detection of toxic food contaminants by surface-enhanced Raman spectroscopy

Supervisors: Dr Shanlin Fu, Dr Linda Xiao and Dr Annette Dowd (School of Physics and Advanced Materials)

In recent years, food safety issues caused by contamination of toxic chemical substances or microbial species have raised a great deal of concern in the world. Conventional chromatography-based methods for detection of chemical contaminants and microbial plating methods for detection of food-borne pathogens are time-consuming and labour intensive. In this project, we aim to explore the use surface-enhanced Raman spectroscopy (SERS) (e.g. commercial SERS substrates, fractal-like metal nanosponges, metal nanodendrites) as alternative and speedy analytical tools to detect and characterise various toxic food contaminants, including patulin, melamine and its analogues, some restricted antibiotics, prohibited dyes and fertilisers found in food matrices. SERS is a technique that enhances the Raman signal from Raman-active analye molecules and reduces the fluorescence from food matrices. The limit of detection of SERS could reach parts per billion level for chemical samples. These results indicate a great potential of using SERS techniques for rapid detection, classification, and quantification of chemical and biochemical contaminants in food products.

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Development of novel methods to characterize and quantify oxidative stress in cells/tissues

Supervisors: Dr Shanlin Fu, Professor Roland Stocker (Victor Chang Institute) and Dr Ghassan Maghzal (Victor Chang Institute)

It is now well recognized that many age-related diseases including cardiovascular diseases, diabetes and cancer are associated with increased oxidative stress, and that different endogenously generated reactive oxygen and nitrogen species (RONS) such as superoxide

anion radical (O2-) and hydrogen peroxide (H2O2) can have physiological and pathological

roles, depending on the circumstances involved. Oxidative stress may cause oxidative damage and result in cell injury, dysfunction and/or death leading possibly to disease. However, large-scale controlled interventions with antioxidant supplements have been largely disappointing with little if any benefit in limiting age-related diseases.

A potential explanation for why antioxidant supplements have proven ineffective in preventing such diseases is that our current understanding of the oxidative stress and the RONS involved is insufficient and, thus appropriate antioxidant treatments have not been tested. Secondly, we now know that many important redox processes are confined to specific cellular compartments such as the mitochondria. Also, the detection and quantification of RONS in biological systems has proven challenging due to the oxidizing species being short-lived and present at very low concentrations. A serious, yet general, limitation of presently available methods for the measurement of cellular oxidative stress is the lack of specificity, quantification and spatial information.

The aim of this project is to establish mass spectrometry (LC-MS) based methods employing

novel fluorescent probes such as hydroethidine and aryl boronates that are specific for O2- and

H2O2, respectively and that can also be targeted to the mitochondria. The methods to be developed will allow quantification of both parent and reacted probes in cells and tissues. This includes the chemical characterization of the different products formed to facilitate the establishment of footprints for the different oxidants/probes involved. This project will provide the basis for a better understanding of redox-modulated pathways in health and disease.

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Characterisation of singlet oxygen derived products of selenium-containing amino acids and proteins

Supervisors: Dr Shanlin Fu, Dr David Pattison (The Heart Research Institute, Sydney)

and Prof. Michael Davies (The Heart Research Institute, Sydney) Singlet oxygen (the first excited singlet state of oxygen; 1O2) is generated by multiple

chemical reactions in vivo including photosensitization reactions with exogenous or endogenous sensitizers. 1O2 reacts with most biological molecules with proteins believed to be a major target in vivo [1]. The damage induced by these reactions has been implicated in the development of a number of human pathologies (e.g. skin aging, cataract, sunburn [2]).

Previous studies have shown that 1O2 reacts rapidly with the nucleophilic sulphur-containing amino acids, cysteine (Cys) and methionine (Met), and many of the products of these reactions have been characterized. Selenium compounds are typically better nucleophiles than sulphur centres, thus we hypothesized that selenocysteine (Sec) and selenomethionine (SeMet) residues might be important targets for 1O2. Our recent studies have shown that key antioxidant enzymes with Sec residues at the active sites (e.g. glutathione peroxidase (GPx)) are readily inactivated by 1O2 [3], but the mechanisms of inactivation have not been fully resolved.

In this project the products of reaction of 1O2 with low-molecular-mass seleno compounds such as SeMet, diselenides (RSe-SeR), Sec and other selenols will be investigated by HPLC and mass spectroscopic techniques. The initial studies with free amino acids will be extended to isolated selenoproteins such as GPx, where the damage to the protein will be analysed by peptide mass mapping studies following enzymatic digestion of the protein.

The data obtained will give insight into the mechanisms of photo-oxidative damage to selenoproteins, thereby providing a basis for the development of putative protective agents for the pathologies described above. References 1) Pattison, D. I., Suryo Rahmanto, A., Davies, M. J. (2012) “Photo-oxidation of proteins” Photochem. Photobiol. Sci. 11, 38-53 2) Davies, M. J. (2005) “The oxidative environment and protein damage” Biochim. Biophys. Acta 1703, 93-109 3) Suryo Rahmanto, A., Pattison, D. I., Davies, M. J. (2012) “Photo-oxidation-induced inactivation of the selenium-containing protective enzymes thioredoxin reductase and glutathione peroxidase” Free Radic. Biol. Med. 53, 1308–1316

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Development of microfluidic paper-based analytical devices to detect amino acids in urine

Supervisors: Dr Lucas Blanes and Prof. Claude Roux

Microfluidic paper-based analytical devices (µPADs) offer rapid and multiplexed analysis of small volumes of fluid in an inexpensive platform [1-2]. µPADs combine the capabilities of traditional microfluidic devices with the simplicity of diagnostic strip tests. The presence of certain amino acids present in urine offers important clinical data on metabolic, nutritional, and neurological disorders and provides a detailed explanation of the possible causes and consequences of detected abnormalities, along with nutritional information. In this project the student will design innovative µPads to detect amino acids using different chemical derivatization techniques to produce colour and fluorescent patterns that will be used to identify and quantify these substances.

Figure 1- Example of analysis of glucose and bovine serum albumin using a µPAD.

References

1- R. V. Taudte, A. Beavis, L. Wilson-Wilde, C. Roux, P. Doble, L. Blanes, A portable explosive detector based on fluorescence quenching of pyrene deposited on coloured wax-printed µPADs. Labon a Chip, 2013,13, 4164-4172 (paper selected as front cover of the journal)

2- A. W. Martinez, S. T. Phillips, Z. Nie, C.-M. Cheng, E. Carrilho, B. J. Wiley, G. M. Whitesides, Programmable diagnostic devices made from paper and tape. Lab on a Chip, 2010, 10, 2499-2504.

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Development of self-powered electrochemical microfluidic devices

Supervisors: Dr Lucas Blanes and Prof. Philip Doble

Microfluidic paper-based analytical devices (µPADs) present a new generation/class in microfluidics. Filter paper is used as a fabrication substrate on which hydrophobic microfluidic patterns are imparted to control fluid movement and compartmentalize chemical reactions [1]. In comparison to other micro devices, µPADs have particular benefits such as, no requirement for pumps, adsorption of aqueous fluids, availability in a range of thickness, easy disposal, flexibility, and suitability for colorimetric tests. Recently new µPAD devices using online electrochemical detectors have been reported in the literature [2]. In this project the student will develop a µPAD with an internal battery embedded on the paper that will power the microchip device. This device will be initially used to detect hydrogen peroxide and noradrenaline [3]. It’s not necessary but students that have a bit of knowledge in electronics or computer programming will be able do more sophisticated projects.

Figure 1- Example of an µPAD with internal electrochemical detection.

References

1- R. V. Taudte, A. Beavis, L. Wilson-Wilde, C. Roux, P. Doble, L. Blanes, A portable explosive detector based on fluorescence quenching of pyrene deposited on coloured wax-printed µPADs. Labon a Chip, 2013,13, 4164-4172 (paper selected as front cover of the journal)

2- Hong Liu, Yu Xiang, Yi Lu, and Richard M. Crooks. Aptamer-Based Origami Paper Analytical Device for

Electrochemical Detection of Adenosine. Angew. Chem, 2012, 124, 1 – 5

3- Lucas Blanes, Maria F. Mora, Claudimir L. do Lago, Arturo Ayon and Carlos D. Garcia Lab-on-a-Chip

Biosensor for Glucose Based on a Packed Immobilized Enzyme Reactor. Electroanalysis 19, 2007, No. 23, 2451 – 2456

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The analysis of superoxide dismutase (SOD) by chipLC-ICP-MS and chipLC-QTOF-MS in motor neuron disease mouse models

Supervisor: Prof. Philip Doble and Dr David Bishop

The burden of motor neuron disease (MND) is very significant with a disease incidence of approximately 2 in 100,000; it is the most common neuromuscular disorder. The aetiology of MND is still unclear, but several suggested mechanisms involve glutamate excitotoxicity[1], excessive generation of reactive oxygen and nitrogen species[2], mitochondrial dysfunction[1], induction of endoplasmic reticulum (ER) stress and in non-SOD-1 MND, ubiquitinated neuronal inclusions where TDP-43 is the major protein component[1, 3]. Alterations in the essential micronutrients Cu and Zn and their metalloenzymes have been reported.

About 10% of MND cases are familial, with mutations in the protein SOD1, which plays a key role in destroying superoxide radicals in the body. SOD1 requires copper and zinc ions for activity. Significantly, multiple data describe defective Cu binding by various SOD1 mutants[4-6] and the ability of Cu chelators diethyldithiocarbamate and penicillamine to reverse mutant SOD reactivity[7]. Despite a general belief in a ‘toxic gain of function’, attributed to redox phenomena, it remains unclear how SOD1 mutations result in neuronal degeneration. An intriguing suggestion is that undermetallation of SOD1 could be a cause of SOD1 aggregation in vivo; SOD1 fractions isolated from spinal cords that were insoluble (i.e., aggregated) were Cu and Zn depleted, whereas soluble SOD1 fractions were highly metallated[8]. A re-distribution of Cu from gray to white matters correlated to areas of high SOD1 and increased Zn was also found in the white matter in all mutant SOD1 mice[8].

Recently, chipLC was first hyphenated to ICP-MS for the analysis of phosphorylated peptides. This technique has potential to identify and quantify metalloprotein bases on the elemental tag. When combined with the complementary information of chipLC-QTOF-MS for positive protein identification, chipLC-ICP-MS is a powerful tool for metallomics analyses.

The aims of the project are to use chipLC-ICP-MS and chipLC-QTOF-MS to identify and quantify SOD1 in the spinal cord and brain of SOD1 transgenic mouse models of MND and to determine the metallation status of SOD1.

For more information please contact David at david.bishop@uts.edu.au, Philip.Doble@uts.edu.au

1. Rothstein, J.D., Current hypotheses for the underlying biology of amyotrophic lateral sclerosis. Ann Neurol, 2009. 65 Suppl 1: p. S3-9.

2. Barber, S.C. and P.J. Shaw, Oxidative stress in ALS: key role in motor neuron injury and therapeutic target. Free Radic Biol Med, 2010. 48(5): p. 629-41.

3. Arai, T., et al., TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun, 2006. 351(3): p. 602-11.

4. Eum, W.S. and J.H. Kang, Release of copper ions from the familial amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutants. Mol Cells, 1999. 9(1): p. 110-4.

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5. Carri, M.T., et al., Impaired copper binding by the H46R mutant of human Cu,Zn superoxide dismutase, involved in amyotrophic lateral sclerosis. FEBS Lett, 1994. 356(2-3): p. 314-6.

6. Hayward, L.J., et al., Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral sclerosis. J Biol Chem, 2002. 277(18): p. 15923-31.

7. Wiedau-Pazos, M., et al., Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis. Science, 1996. 271(5248): p. 515-8.

8. Lelie, H.L., et al., Copper and zinc metallation status of copper-zinc superoxide dismutase from amyotrophic lateral sclerosis transgenic mice. J Biol Chem, 2011. 286(4): p. 2795-806.

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Development of ChipLC-ICP-MS

Supervisors: Prof. Philip Doble, Dr David Bishop and Dr Lucas Blanes

In this project, UTS and Agilent Technologies, the world’s leading vendor of analytical instrumentation, will develop the world’s first hyphenated microfluidic chip liquid chromatography–inductively coupled plasma–mass spectrometer (chipLC-ICP-MS) for the field of metallomics.

Trace elements (<0.01% of human body weight) are critical in biological processes. For example, protein phosphorylation cascades play a central role in cell signaling and development, particularly in cancer cells. Cyanocobalamin (vitamin B12), which forms two coenzymes responsible for biological transformations, contains cobalt. Iron in haemoglobin binds oxygen and carbon dioxide, reactants and products of cellular respiration. Copper and zinc are key components of superoxide dismutase, an important enzyme in oxidation–reduction reactions. It is currently thought that around one-third of all proteins in the human body contain at least one metal ion. These ions can act as structural features or active sites for catalysis [1]. Trace metals are so important to cell function that cell chemistry must be characterised by the distribution of the metals and metalloids among different biomolecules – defined as the ‘metallome’.

Hyphenated technologies such as liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS) are the most effective way to detect trace elements in biological samples. LC separates the sample fractions prior to detection by ICP-MS. ICP-MS has isotope specificity, versatility (virtually any element can be detected), high sensitivity, and enormous linear dynamic range (105–106) needed for efficient element detection. Figure 1 is one example of the analysis of metallothionein isoforms by LC-ICP-MS. These isoforms are metal-binding proteins associated with numerous disease states [2-4]. Recent improvements in ICP-MS instrumentation also detect non-metals such as phosphorous, expanding LC-ICP-MS analysis to phosphorylated proteins [5-6].

Traditional standard (3 to 5 mm internal diameter) and narrow bore (1 to 2 mm internal diameter) analytical LC columns are relatively easy to couple to ICP-MS due to compatible flow rates.

Figure 1: Separation metallothionein isoforms by LC-ICP-MS

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There are significant advantages in reducing the internal diameter of the separation column including improved separation efficiencies, higher sensitivity and less solvent consumption. However, reducing the internal diameter of the LC columns to micrometre-widths, also known as nano-LC, presents new challenges. Hyphenation of nano-LC requires transport of eluents with flow rates as low as 10 nL min-1 into the ICP-MS. This requires dedicated interfaces with sheath flows to boost flow rates to levels capable of nebulisation [7]; or employment of sheathless total-consumption direct-injection nebulisers [8]. Further challengers include frequent blocking of the capillary columns, requirements of sample pre-concentration prior to analysis with concentrator columns and the minimisation of dead volumes to avoid band broadening leading to poor separation efficiency. All of these issues may be overcome by incorporation of the LC components into a microfluidic lab-on-a-chip device.

The aim of this project is to design and fabricate an interface between a microfluidic liquid chromatograph (chipLC) and ICP-MS.

For further information please contact David at david.bishop@uts.edu.au, Lucas Blanes mimlucas@gmail.com, or Philip Doble Philip.Doble@uts.edu.au

1. Dudev, T. and C. Lim, Principles governing Mg, Ca, and Zn binding and selectivity in proteins. Chemical Reviews, 2003. 103: p. 773-787.

2. Aschner, M., et al., Metallothioneins in neurodegeneration. Metal Ions and Neurodegenerative Disorders, 2003: p. 307-322.

3. Cherian, M.G., et al., Contemporary Issues in Toxicology : Role of Metallothionein in Carcinogenesis. Toxicology and Applied Pharmacology, 1994. 126(1): p. 1-5.

4. Chung, R.S. and A.K. West, A role for extracellular metallothioneins in CNS injury and repair. Neuroscience, 2004. 123(3): p. 595.

5. Bandura, D.R., O.I. Ornatsky, and L. Liao, Characterization of phosphorus content of biological samples by ICP-DRC-MS: potential tool for cancer research. Journal of Analytical Atomic Spectrometry, 2004. 19(1): p. 96-100.

6. Bandura, D.R., V.I. Baranov, and S.D. Tanner, Detection of ultratrace phosphorus and sulfur by quadrupole ICPMS with dynamic reaction cell. Analytical Chemistry, 2002. 74(7): p. 1497-1502.

7. Agilent 1200 Series HPLC-Chip/MS System Data Sheet. 2006, Publication no. 5989-5492EN.

8. Profrock, D., et al., Development and characterisation of a new interface for coupling capillary LC with collision-cell ICP-MS and its application for phosphorylation profiling of tryptic protein digests. Analytical and Bioanalytical Chemistry, 2005. 381(1): p. 194.

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Three dimensional multiplexed protein atlas of the mouse brain

Supervisors: Prof. Philip Doble and Dr David Bishop

The brain is the most complex organ in the human body. Animal models like the laboratory mouse are commonly used to study diseases affecting the human brain. Structural brain atlases are universal features of neuroscience laboratories. Atlases are used to correlate features of disease with specific brain regions. Improving technology has seen a move to functional brain atlases, where regional gene and protein expression can be observed in conjunction with basic neuroanatomy via interactive software. This project will develop the first interactive model of various proteins in the mouse brain using advanced mass spectrometry techniques.

The aim of this project is to construct a standard three-dimensional atlas of metal -transporter proteins in the C57BL/6 mouse brain using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and tagged antibodies.

For further information please contact David Bishop at david.bishop@uts.edu.au, or Philip Doble Philip.Doble@uts.edu.au

Frogs as bio-indicators of environmental quality

Supervisors: Prof. Philip Doble and Prof. Graham Pyke (Australian Museum)

Frogs are often touted as being excellent bio-indicators of environmental quality, and yet the supporting evidence is meagre and they are not much used as indicators of ‘environmental health’. This project would address this issue by evaluating the chemical composition of various water bodies, especially in terms of various kinds of pollution, and comparing such evaluations with various aspects of frog biology, such as which species are present, their abundances, the nature and extent of physical abnormalities, and which body regions carry chemical signs of pollution.

For further information please contact Professor Graham Pyke Graham.Pyke@austmus.gov.au, or Philip Doble Philip.Doble@uts.edu.au

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Chemistry and function of frog secretions

Supervisors: Prof. Philip Doble and Prof. Graham Pyke (Australian Museum)

Little is known about the nature and function of frog secretions. Anecdotal evidence suggests that some frog secretions may sometimes occur when animals are handled by humans or held captive for periods of time, especially if held in groups. That some frog species are susceptible to fungal attack, while others are not, suggests the possibility that frog secretions may provide some protection from pathogens. This project would explore such aspects of frog secretion by evaluating the level and chemical composition of frog secretions across a range of frog species and under different conditions. It could also involve testing the effects of frog secretions, or various chemical constituents of such secretions, on frog pathogens.

For further information please contact Professor Graham Pyke Graham.Pyke@austmus.gov.au, or Philip Doble Philip.Doble@uts.edu.au

Other projects available with Prof. Doble – please contact him at philip.doble@uts.edu.au for further information

Analysis of Seal Whiskers by Laser Ablation – Inductively Coupled Plasma Mass Spectrometry

ChipLC-QqQ / QTOF Analysis of Explosives

ChipLC-QqQ / QTOF / ICP-MS Analysis of Tri-butyl tins

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Hydrothermal synthesis of amorphous silica from industrial wastes

Supervisor: Dr Paul Thomas

Amorphous silica is a growing commodity product used in a range of applications such as high surface area fillers for rubber in car tires resulting in significantly reduced fuel consumption to use as a thickening agent in toothpastes and cosmetics. Due to the high surface area and porosity, amorphous silica also has potential application as catalysts supports and may be used in a range of nutrient and drug delivery applications. Currently, silica is sources through the calcining of quartz at high temperature in the presence of soda to form a water soluble silica glass which is then neutralised to precipitate the high surface area amorphous silica. The high temperature route results in high energy consumption which can be avoided if hydrothermal (autoclaving) techniques are employed.

Hydrothermal methods for the production of waterglass (soluble silica glass) from aluminosilicates are known, but are not currently commercially available. This study proposes to investigate the conditions of hydrothermal synthesis of silica waterglasses by autoclaving aluminosilicate industrial wastes such as fly ash, bottom ash and pitchstone fines in the presence of sodium hydroxide. Subsequent precipitation of the silica with nitric acid yields high surface area high purity amorphous silicas. Methods of characterisation will be Raman spectroscopy which is sensitive to silica structure, gas adsorption to determine surface area and porosity characterisation and differential scanning calorimetry which can also be applied to porosity characterisation. Scanning electron microscopy will also be pursued.

In addition to the high surface area silica, by-products of the process are aluminosilicates which depending on the hydrothermal conditions can yield zeolites. Zeolites are important minerals in that they have many industrial applications in particular catalysis. These aluminosilicate by-products will be characterised by Raman spectroscopy, crystallography and thermogravimetric analysis.

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Calorimetric investigation of the hydration of high sulphate cement at elevated temperature

Supervisors: Dr Paul Thomas and Dr Kirk Vessalas (School of Civil and Environmental Engineering)

Precast concrete elements are commonly manufactured for the construction of bridges, tunnels, culverts, pipes and drains as high strength elements and are cured at elevated temperature within a 24 hour period (as oppose to 28 days at ambient temperature). The chemistry of the cement curing, however, is altered by the use of elevated temperature. In particular, the hydration of tricalcium aluminate (C3A) present in cement as a by-product of clinker process is affected. C3A hydrates rapidly on the addition of water and is extremely exothermic causing acceleration of Portland cement hydration. The hydration of the C3A may be inhibited by reaction of the hydrated C3A with sulphate to form the mineral ettringite. Sulphate is added typically in the form of gypsum which is interground with the cement clinker, a glassy calcium silicate formed in the kiln during cement manufacture. The sulphate ions react with the calcium aluminate hydrate to form ettringite which inhibits further hydration of the calcium aluminate through the formation of an impermeable layer.

In the manufacture of precast concrete elements at elevated temperature, however, the sulphate becomes more soluble and the ettringite decomposes as the sulphate is dissolved into solution. Further hydration of the C3A results in increased temperature and flash setting of the cement. At elevated temperature the formation of ettringite is inhibited by the solubility of the sulphate, however, after curing, and once the temperature has returned to ambient, the solubility of the sulphate decreases and sulphate becomes available once again for ettringite formation. This delayed ettringite formation, which is accompanied by a large volume increase, occurs in the hardened state and can result in the cracking and failure of precast concrete elements. In order to prevent these deleterious processes from occurring, an understanding of the formation conditions is necessary. This project, therefore, investigates the reaction chemistry of high sulphate cements at elevated temperature using adiabatic calorimetry and differential scanning calorimetry in conjunction phase analysis using crystallography. A greater understanding of the reactions involved, in particular in the formation of delayed ettringite, will help to minimise hazardous degradation of precast concrete elements in construction.

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A number of projects based at the National Measurement Institute (NMI) are also available.

Details are provided in the attached NMI document