Faculty of Medicine, Dentistry and Health Sciences School of Dental Science

HONOURS RESEARCH PROJECTS

ORAL BIOLOGY

The following honours research projects are offered for 2008. These projects are associated with the research conducted at the Cooperative Research Centre for Oral Health Science. Scholarship opportunities may be available for these projects.

Multiple projects are available in most of these fields:

A. Introduction to bacterial pathogenesis and vaccine development.

  1. Proteomics of oral bacteria.
  2. Gene expression studies of oral bacteria.
  3. Molecular biology of virulence-associated genes of oral bacteria.
  4. Immunology of disease and vaccine development.

B. Introduction to nanotechnology: structure-function studies of biomineralising proteins.

  1. Structural, biomimetic and modeling studies of phosphopeptides and proteins.

ORAL ANATOMY, MEDICINE AND SURGERY

There is one project available in this field for 2008.

ORAL BIOLOGY

A. INTRODUCTION TO BACTERIAL PATHOGENESIS AND VACCINE DEVELOPMENT

Periodontal diseases are chronic inflammatory diseases of the supporting tissues of the teeth caused by the anaerobic, Gram-negative bacteria Porphyromonas gingivalis and Treponema denticolagrowing as part of a polymicrobial biofilm on the surface of the tooth. Periodontal disease causes destruction of the supporting tissues of the tooth, is the main cause of tooth loss in adults and is a major health problem in Australia. We have an integrated program of research studying the virulence of these Gram-negative bacterial pathogens funded by the National Health and Medical Research Council (AUS) and the National Institutes of Health (USA). The aims of this program are to understand at a microbiological, immunological and molecular level how disease is caused and then use this information to develop novel diagnostics, therapeutics and vaccine technologies to prevent periodontal disease. We have already characterised many of the virulence determinants of P. gingivalis using a variety of molecular biology and biochemical analyses. We have recently developed techniques to study the proteomics and transcriptomics of these bacteria and have developed novel bacterial culture techniques to study how the interactions between these species contribute to the disease.

1. Proteomics of oral bacteria

We have a very well equipped proteomics lab, housing the first Australian MALDI TOF/TOF mass spectrometer as well as two LC-MS systems, and robotics for sample preparation. The honours student who takes on this project will receive in-depth training in proteomics while making an important contribution to our studies. A variety of specific projects can be tailored to suit your interests.

For more information e-mail Dr Paul Veith or Dr Yu-Yen Chen.

2. Gene expression studies of oral bacteria

2A. Effect of polymicrobial biofilm growth on P. gingivalis and T. denticola global gene expression.

To cause disease P. gingivalis and T. denticola must establish and proliferate as a bacterial biofilm on the tooth surface. We have developed novel methodologies for the growth and analyses of these bacteria as polymicrobial biofilms. DNA Microarray technology will be used to identify how the expression of genes encoding the major virulence associated genes, metabolic pathways and outer membrane proteins of P. gingivalis and T. denticola is affected when these bacteria are grown as polymicrobial biofilms. As part of this project you will learn and use techniques including; novel polymicrobial continuous culture techniques, confocal microscopy analysis of bacterial biofilms, bacterial mRNA isolation, reverse-transcription of mRNA, hybridization to oligoarray chips and scanning. Data analyses will be conducted using a range of computer programs.

For more information e-mail Dr Helen Mitchell, A/Prof Stuart Dashper, or Dr Nada Slakeski.

2B Identification and characterisation of iron and manganese-regulated genes of P. gingivalis.

Fur (Ferric uptake regulator) and MntR (Manganese transport regulator), transcriptionally regulate genes in response to the environmental levels of their metallic co-factors, iron and manganese, respectively. In many bacteria these proteins have been found to regulate the expression of a wide variety of genes, including those that encode bacterial toxins and other virulence determinants. The project will involve the creation of P. gingivalis mutants lacking these iron and manganese regulatory proteins using a broad range of techniques including anaerobic bacterial culture, DNA cloning and sequencing and site-directed mutagenesis. Characterisation of iron- or manganese-regulated genes of P. gingivalis will involve the use of state-of-the-art technology including DNA microarray, surface plasmon resonance, confocal microscopy, and mass spectrometry. This research will contribute to our understanding of the pathogenicity of this bacterium, and will also be one of the first studies of transcriptional regulation within P. gingivalis.

For more information e-mail Dr Samantha Byrne, Dr Christine Seers, Dr Nada Slakeski or A/Prof Stuart Dashper.

Selected Recent Publications:

  1. Dashper SG and Reynolds EC. Effects of Organic Acid Anions on Growth, Glycolysis and Intracellular pH of Oral Streptococci. Journal of Dental Research 79:90-96 (2000).
  2. Dawson NF, Craik DJ, McManus AM, Dashper SG, Reynolds EC, Tregear W, Otvos L and Wade JD. Chemical Synthesis, Characterisation and Activity of RK-1, a Novel a-Defensin-related Peptide. Journal of Peptide Science 6:19-25 (2000).
  3. Slakeski N, Dashper SG, Cook P, Poon C Moore C and Reynolds, EC. A Porphyromonas gingivalis Genetic Locus Encoding a Heme Transport System. Oral Microbiology and Immunology 15:388-392 (2000).
  4. Dashper SG, Hendtlass A, Slakeski N, Jackson C, Cross KJ, Brownfield L, Hamilton R, Barr I and Reynolds, EC. Characterization of a Novel Outer Membrane Hemin-binding protein of Porphyromonas gingivalis. Journal of Bacteriology 182:6456-6462 (2000).
  5. Dashper SG, Brownfield L, Slakeski N, Zilm PS, Rogers AH and Reynolds, EC. Sodium Ion-driven Serine/Threonine Transport in Porphyromonas gingivalis. Journal of Bacteriology 183:4142-4148 (2001).
  6. O’Brien-Simpson NM, Paolini RA, Hoffmann B, Slakeski N, Dashper SG, Reynolds EC. Role of RgpA, RgpB and Kgp Proteinases in Virulence of Porphyromonas gingivalis in the Murine Lesion Model. Infection and Immunity 69:7527-7534 (2001).
  7. Veith PD, Talbo GH, Slakeski N, Dashper SG, Moore C, Paolini RA and Reynolds EC. Major Outer Membrane Proteins and Proteolytic Processing of RgpA and Kgp of Porphyromonas gingivalis W50. Biochemical Journal 363:105-115 (2002).
  8. O’Brien-Simpson NM, Veith PD, Dashper SG and Reynolds EC. Porphyromonas gingivalis Gingipains: the Molecular Teeth of a Microbial Vampire. Current Protein and Peptide Science 4:409-426 (2003).
  9. Dashper SG, Butler C, Lissel P, Paolini, RA, Hoffman B, Veith PD, O’Brien-Simpson NM, Snelgrove S, Tsiros JT and Reynolds EC. A novel Porphyromonas gingivalis FeoB Plays a Role in Manganese Accumulation. Journal of Biological Chemistry. 280:28095–28102 (2005).
  10. Seers C, Slakeski N, Veith P, Nikoloff T, Chen Y-Y Dashper SG and Reynolds EC. The RgpB C-terminal domain has a role in attachment of RgpB to the outer membrane and belongs to a novel C-terminal domain family found in Porphyromonas gingivalis.  Journal of Bacteriology 188:6376-6386 (2006).
  11. Diaz PI, Slakeski N, Reynolds EC, Morona R, Rogers AH, Kolenbrander PE. Role of oxyR in the oral anaerobe Porphyromonas gingivalis.  Journal of Bacteriology 188:2454-2462 (2006).
  12. Frazer LT, O'Brien-Simpson NM, Slakeski N, Walsh KA, Veith PD, Chen CG, Barr IG, Reynolds EC. Vaccination with recombinant adhesins from the RgpA-Kgp proteinase-adhesin complex protects against Porphyromonas gingivalis infection. Vaccine (2006).
  13. Dashper S, Byrne S, Orth R and Reynolds EC. Long in the Tooth: Oral Bacterial Communities and Chronic Periodontitis. Microbiology Australia (September 2007).
  14. Dashper SG, Ang CS, Veith PD, Peter Zilm and Reynolds EC. Applied genomics of oral bacteria. Chapter in “Oral Molecular Microbiology” 2007 Horizon Scientific Press. (Australia Publication date: February 2008).

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3. Molecular biology of virulence-associated genes of oral bacteria

3A. Factors involved in the export and attachment of P. gingivalis gingipains.

The cell surface proteinases and adhesins (gingipains) are major virulence factors of P. gingivalis.  The Arginine-specific (RgpA and RgpB) and a Lysine-specific (Kgp) gingipains have been shown to degrade many human proteins, including those involved in the innate immune response.  The gingipains are members of a novel C-Terminal Domain (CTD) family of proteins that are secreted to the cell surface, attached and processed by as yet poorly defined mechanisms. There are various projects on offer that aim to characterize the translocation, attachment and processing mechanisms of the P. gingivalis proteinases and adhesins.  These projects will utilize various techniques in molecular biology which may include: DNA cloning and sequencing, DNA soeing, site-directed mutagenesis, transposon insertion, gene deletion, RNA analysis, expression of recombinant proteins, protein purification and analysis, aerobic and anaerobic bacterial culture, cell fractionation, enzyme assays, 1D and 2D gel electrophoresis, western immunoassays, mass spectrometry and database information mining.

For more information e-mail Dr Nada Slakeski, Dr Christine Seers or Dr Benjamin Peng.

Relevant publications:

Other publications:

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4. Immunology of Disease and Vaccine Development

4A. Mucosal and systemic immune response to bacteria.

We are offering a number of projects investigating the mucosal and systemic immune responses to single and multi-bacterial species colonization and infection. We have already found that there is pathogenic synergy between pathogenic and non-pathogenic bacteria and their ability to cause disease and immunopathology. These projects will use a number of techniques and in-house equipment such as Flow cytometry, ELISPOT, T-cell proliferation, real-time PCR and cytokine DNA microarray. Areas in which projects are available are;

For more information e-mail Dr Neil O’Brien-Simpson or Dr Katrina Walsh.

4B. Vaccine development and delivery and peptide therapeutics

We currently are offering a number of projects investigating the synthesis of peptides and proteins for the development of a oral vaccine for periodontitis as well as designing peptide inhibitors to bacterial enzymes. These projects will involve the chemical synthesis of peptide vaccines or the production of recombinant protein vaccines and evaluating their efficacy using a number of immunological techniques. These projects will use a number of techniques and in-house equipment such as peptide synthesis, HPLC, FPLC, Mass spectrometry, polymer chemistry, Flow cytometry, ELISPOT, T-cell proliferation, real-time PCR and cytokine DNA microarray. Areas in which projects are available are;

For more information e-mail Dr Neil O’Brien-Simpson or Dr Troy Attard.

Relevant Publications

  1. O’Brien-Simpson NM, Paolini RA, Reynolds EC. “RgpA-Kgp peptide-based immunogens provide protection against Porphyromonas gingivalis challenge in a murine lesion model.” Infection and Immunity 68 4055-4063 (2000).
  2. Malkoski M, Dashper SG, O’Brien-Simpson NM, Talbo G, Macris M, Cross KJ, Reynolds EC “A novel antibacterial peptide from bovine milk, Kapacin, Ser(P)149 k-Casein (106-169)” Antimicrobial Agents and Chemotherapy 45 (8) 2309-2315 (2001).
  3. O’Brien-Simpson NM, Paolini RA, Hoffmann B, Slakeski N, Dashper SG, Reynolds EC. “Role of RgpA, RgpB and Kgp proteinases in virulence of Porphyromonas gingivalis in the murine lesion model.” Infection and Immunity 69 (12) 7527-7534 (2001).
  4. Rajapakse PS, O’Brien-Simpson NM, Slakeski N, Hoffmann B, Reynolds EC.  “Immunization with the RgpA-Kgp proteinase-adhesin complexes of P. gingivalis protects against periodontal bone loss in the rat periodontitis model. Infection and Immunity  70 (5) 2480-2486 (2002).
  5. O’Brien-Simpson NM, Veith PD, Dashper SG, Reynolds EC. “Antigens of bacteria associated with periodontitis.” Periodontology 2000. Invited Review 35:101-134 (2002).
  6. O’Brien-Simpson NM, Veith PD, Dashper SG, Reynolds EC. “Porphyromonas gingivalis gingipains: The molecular teeth of a microbial vampire”  Current Protein and Peptide Science 4 (6) 409-426 (2003). Invited Review
  7. Dashper SG, O’Brien-Simpson NM, Cross KJ, Paolini RA, Hoffmann B, Catmull DV, Malkoski M, Reynolds EC.  Divalent metal cations increase the activity of the antimicrobial peptide Kappacin.  Antimicrobial Agents and Chemotherapy. 49:2322-2328 (2005).
  8. Dashper SG, Butler CA, Lissel JP, Paolini RA, Hoffmann B, Veith PD, O'Brien-Simpson NM, Snelgrove SL, Tsiros JT, Reynolds EC.  A novel Porphyromonas gingivalis FeoB plays a role in manganese accumulation. Journal of Biological Chemistry Jul 29;280(30):28095-102 (2005).
  9. O'Brien-Simpson NM, Pathirana RD, Paolini RA, Chen YY, Veith PD, Tam V, Ally N, Pike RN, Reynolds EC.  An immune response directed to proteinase and adhesin functional epitopes protects against Porphyromonas gingivalis-induced periodontal bone loss. Journal of Immunology. 2005 Sep 15;175(6):3980-9.
  10. Pathirana RD, O’Brien-Simpson NM, Veith PD, Riley PF, Reynolds EC. Characterization of the proteinase-adhesin complexes of Porphyromonas gingivalis. Microbiology (SGM). 2006 Aug;152(Pt 8):2381-94.
  11. Frazer LT, O'Brien-Simpson NM, Slakeski N, Walsh KA, Veith PD, Chen CG, Barr IG, Reynolds EC.  Vaccination with recombinant adhesins from the RgpA-Kgp proteinase-adhesin complex protects against Porphyromonas gingivalis infection. Vaccine. 2006 Jun 23;
  12. Pathirana RD, O’Brien-Simpson NM, BramMAR GC, Slakeski N, Reynolds EC.  Kgp and RgpB, but not RgpA, are important for Porphyromonas gingivalis virulence in the murine periodontitis model. Infection and Immunity. 200775(3) 1436-42.
  13. Pathirana RD, O’Brien-Simpson NM, Visvanathan K, Hamilton JA, Reynolds EC. Flow cytometric analysis of the adherence of Porphyromonas gingivalis to oral epithelial cells. Infection and Immunity. 2007 Accepted.
  14. O’Brien-Simpson NM, Attard TJ, Loganathan A, Huq NL, Cross KJ, Riley PF, Reynolds EC. Synthesis and Characterisation of a Multi-phosphorylated Phosphophoryn Repeat Motif; h-DpSpSDpSpSDpSpSD-cooh. International Journal of Peptide Research and Therapeutics 2007 Provisionally accepted.
  15. Attard TJ, O’Brien-Simpson NM, Reynolds EC. Synthesis of phosphopeptides in the Fmoc mode. International Journal of Peptide Research and Therapeutics 2007 Provisionally accepted.
  16. O’Brien-Simpson NM, Walsh KA, Lam R, Reynolds EC. The RgpA-Kgp complex is crucial for Porphyromonas gingivalis binding to macrophages. Frontiers In Bioscience 2007 Invited manuscript Provisionally accepted.
  17. Pathirana RD, O’Brien-Simpson NM, Visvanathan K, Hamilton JA, Reynolds EC. Innate immune responses to Porphyromonas gingivalis. Frontiers In Bioscience 2007 Invited manuscript Provisionally accepted.
  18. Cross KJ, Huq NL, O'Brien-Simpson, NM, Perich JW, Attard TJ, Reynolds EC. The role of multiphosphorylated peptides in mineralized tissue regeneration. International Journal of Peptide Research and Therapeutics 2007 Provisionally accepted.

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B. INTRODUCTION TO NANOTECHNOLOGY: STRUCTURE-FUNCTION STUDIES OF BIOMINERALISING PROTEINS

Recaldent® is the marketed product developed in this laboratory, consisting of tryptic peptides of milk caseins (known as casein phosphopeptides) complexed with calcium phosphate. This mixture replaces the calcium and phosphate in demineralized enamel. Since it is an anticariogenic agent, it is put into toothpaste, mouth washes, and chewing gum. We are studying the structure-function relationships and mechanisms of these peptide-mineral complexes and their interaction with salivary proteins on the tooth surface. These studies are funded through the NH&MRC, the CRC, and local industries, and are also part of international collaborations.

1. Structural, biomimetic and modelling studies of phosphopeptides and proteins

1A. Molecular modelling of the binding of multi-phosphorylated peptides to hydroxyapatite.

Many organisms form mineralised structures by the process of biomineralisation. Several multiple phosphoseryl-containing proteins have been identified in mineralising tissue or associated in vivo with calcium phosphate phases. We are investigating the binding of multi-phosphorylated peptides to hydroxyapatite using computer-based simulations. This project involves the use of molecular modelling software run on SGI Octane and Tezro workstations.

For more information e-mail Dr Laila Huq or Dr Keith Cross.

1B. Diffusion model for mineralization.

Casein phosphopeptides amorphous calcium phosphate (CPP-ACP™), marketed as Recaldent®, have been demonstrated to have anticariogenic activity in laboratory, animal and human in situ experiments. The aim of this project is to mathematically model the remineralization of enamel by CPP-ACP™ in the oral cavity.
The project involves the development of a finite element diffusion model for the processes involved in demineralization of enamel by acid challenge and remineralization by the casein phosphopeptides.

For more information e-mail Dr Keith Cross or Dr Laila Huq.

Relevant publications:

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ORAL ANATOMY, MEDICINE AND SURGERY UNIT

Determination of ethnicity from 3D facial mapping

For more information e-mail supervisors: Mr David Thomas and Prof John Clement.

We have already produced average faces for young adult Japanese and Caucasian subjects, both male and female. We now feel that the construction of an average face for ethnic Chinese participants would add considerably to our knowledge. The hons student would be required to take existing data and create a facial archetype representing both male and female Chinese subjects. They would then use existing methods to compare quantitatively the Chinese archetype with the Japanese and Caucasian archetypes.
Next, the student would have to recruit subjects who self-declare their ethnic origins by questionnaire. (We have ethical approval for this already).

Then, for each individual the affinity (or otherwise) relative to the 3 ethnic archetypes can be determined using comparative measurements and compared with the self-declared perception of ethnic group by the individual concerned.
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