ORAL HEALTH RESEARCH PROJECTS
ORAL BIOLOGY
The following research projects are offered for 2012. These projects are associated with the research conducted at the Oral Health Cooperative Research Centre.
1.A. Introduction to bacterial pathogenesis, vaccine and antimicrobial peptide development
1.A.1.1 Proteomics of oral bacteria
1.A.1.2 Protein Modification and Bacterial Pathogenicity
1.A.2. Polymicrobial Biofilms and Disease
1.A.3. Bacterial protein secretion and surface attachment
1.A.3.1. Characterisation of a novel bacterial protein secretion system
1.A.4. Immunology of Disease and Vaccine Development
1.A.4.1. Mucosal and systemic immune response to bacteria
1.A.4.2. Vaccine development and delivery and peptide therapeutics
1.A.5. Antimicrobial Peptides and Proteins
1.B. Introduction To Nanotechnology: Structure-Function Studies Of Biomineralising Proteins
1.B.1. Bioinspired approach for the development of novel biomimetic nanoparticle materials with applications in bone/enamel tissue engineering
1.B.2. Bioinspired synthesis of hydroxyapatite nucleating peptides derived from milk peptides with applications in biomimetic materials used in bone/enamel tissue engineering
1.B.3. Molecular modelling of the binding of multi-phosphorylated peptides to hydroxyapatite
1.B.4. Diffusion model for mineralization
1.D. Inflammation and Host Defence
1.D.2 Using nanoparticles and siRNA to modulate the host immune response to bacterial pathogens
2. ORAL ANATOMY, MEDICINE AND SURGERY UNIT
ORAL BIOLOGY
1.A. Introduction to bacterial pathogenesis, vaccine and antimicrobial peptide development
Periodontal diseases are chronic inflammatory diseases of the supporting tissues of the teeth that have a polymicrobial aetiology. Three anaerobic, proteolytic bacteria Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia that grow as a consortium as part of a polymicrobial biofilm on the surface of the tooth are related to disease initiation and progression. Chronic periodontitis 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. Chronic periodontitis has also been linked to a variety of systemic diseases including cardiovascular disease and some cancers. We have an integrated program of research studying the virulence of these bacterial pathogens. 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 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.A.1.1 Proteomics of oral bacteria
We have a very well equipped proteomics lab, housing two MALDI TOF/TOF mass spectrometers 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 proteomic studies of dental pathogens and their virulence factors. 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
1.A.1.2 Protein Modification and Bacterial Pathogenicity
Lysine acetylation is an important regulatory post-translational protein modification found in eukaryotes and more recently, in some prokaryotes. In eukaryotes this modification regulates diverse protein properties including DNA-protein interactions, subcellular localization, transcriptional activity and protein stability. Dysregulation of lysine acetylation and its regulatory enzymes is associated with aging and several major diseases such as cancer, cardiovascular diseases and neurodegenerative disorders. Although lysine acetylation in Escherichia coli has recently been determined this area of research is still in its infancy in prokaryotes. We have confirmed that lysine acetylation does occur in the oral pathogen Porphyromonas gingivalis and we would like to identify the proteins which are affected by this important modification. This project involves P. gingivalis culture, protein extraction and digestion, enrichment of peptides containing acetylated lysines and mass spectrometric identification of proteins. This would not only be the first study to identify lysine acetylated proteins in P. gingivalis but the knowledge gained about this previously unknown form of regulation will further our understanding of P. gingivalis pathogenicity.
For more information e-mail Dr Catherine Butler, or Dr Paul Veith
1.A.2 Polymicrobial Biofilms and Disease
The objective of this project is to characterise the metabolic interactions that underpin symbioses of three pathogenic oral bacterial species, P. gingivalis, T. denticola and T. forsythia, which grow as a polymicrobial biofilm. The proposed investigation will characterise the alterations in bacterial metabolism that are associated with polymicrobial biofilm development in culture, and responsible for the elevated secretion of metabolites that contribute to bacterial growth synergies, as well as metabolites with potential to act as cytotoxic agents or promoters of pathogenic processes in host tissues and mediators of systemic diseases associated with periodontitis. We have developed novel methodologies for the growth and analyses of these bacteria as polymicrobial biofilms. Metabolomic and transcriptomic analyses will be used to identify how these bacteria grow as polymicrobial biofilms. This will identify metabolites and metabolic pathways essential for the synergistic growth of these bacteria and secreted metabolites with potential to contribute to metabolic co-operativity and signalling. As part of this project you will learn and use techniques including; novel polymicrobial continuous culture techniques, confocal laser scanning microscopy, transcriptomic and metabolomic analyses.
For more information e-mail A/Prof Stuart Dashper1.A.3 Bacterial protein secretion and surface attachment
1.A.3.1 Characterisation of a novel bacterial protein secretion system
The P. gingivalis proteinases and adhesins (gingipains) are major virulence factors of P. gingivalis that are secreted to the cell surface, attached and processed by poorly defined mechanisms. The gingipains are members of a novel family of proteins that are characterised by a highly conserved C-Terminal Domain (CTD). There are currently seven identified protein secretion systems used by bacteria to export proteins to the outer cell surface, into the extracellular milieu or into the cytosol of host cells. However, P. gingivalis secretes its cell-surface-associated proteins via a novel, as yet poorly-defined mechanism. This novel type of secretion system is also likely to be used by other bacteria, particularly of the Bacteroidetes phyla. We have identified several protein candidates that form part of this secretion system and are currently investigating their roles. Our results also suggest that the CTD is essential for export and attachment of proteins to the cell surface. There are various projects on offer that aim to investigate the function of candidate proteins in outer membrane protein secretion in P. gingivalis and identify further possible components of the secretion apparatus and to characterize the role of the CTD in translocation, attachment and processing of the proteinases and adhesins. Students undertaking this project will learn an array of techniques including anaerobic and aerobic microbial cell culture, DNA cloning, gene targeting, recombinant protein expression/antibody production, co-immunoprecipitation, protein purification and analysis, protein analysis techniques including 2D-PAGE, mass spectrometry and western immunoassay and enzyme kinetics.
For further information contact Dr Christine Seers, Dr Paul Veith, Dr Nada Slakeski, Dr Benjamin Peng, Dr Michelle Glew or Dr Yu-Yen Chen
1.A.3.2 Processing and surface presentation of the Porphyromonas gingivalis proteinase/adhesin complexes
The P. gingivalis gingipains, RgpA and Kgp, are encoded as large proteins that are cleaved post-translation to produce separate proteinase catalytic domains and several sequence related adhesins. The major haemagglutinin HagA is also cleaved post-translation to produce separate domains that have sequence similarity to the RgpA and Kgp adhesins. The various RgpA, Kgp and HagA domains remain associated at the cell surface in large non-covalent complexes. It is not yet elucidated how all these domains remain in association. We have noted that there are a series of repeated sequences scattered throughout the domains that we have hypothesized may be interdomain binding sites, otherwise called adhesin binding motifs (ABMs). The aim of this project is to elucidate the function of the various ABM motifs in the associations of the RgpA-Kgp cell-surface protein complexes. Students undertaking this project will learn an array of techniques including anaerobic and aerobic microbial cell culture, DNA cloning, purification and analysis, protein analysis techniques including mass spectrometry and western immunoassay and enzyme kinetics.
For further information contact Dr Nada Slakeski or Dr Christine Seers1.A.4 Immunology of Disease and Vaccine Development
1.A.4.1 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:
- What and how bacterial factors activate or induce apoptosis of immune cells such as CD4+, CD8+, αβ or γδ T-cells, B-cells, Macrophages, NKT cells and dendritic cells.
- How do bacteria effect immune cell trafficking and infiltration into the mucosa of bacterial infected tissue and its effect of multiple bacteria infection.
- What is the effect of multi-bacterial inocula on adherence and invasion of host cells such as oral epithelial cells.
- Are cytokines such as IL-10, IL-4, IL-6, IL-13, iNOS, IFNγ important for mucosal protection or disease induced by one or multiple bacterial species.
- What role do particular immune cells such as CD4+, CD8+, αβ or γδ T-cells, B-cells, Macrophages, NKT cells and dendritic cells have on bacterial infection and ability to cause disease and the underlying immune mechanisms involved.
1.A.4.2 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:
- Polymeric delivery of synthetic peptides to the immune system.
- Conjugation chemistries for assembly of multi-peptide constructs.
- Synthesis of peptide inhibitors for bacterial proteases.
- The use of specific bacterial antibody for the delivery of antibiotics.
- Recombinant proteins and chimeric protein vaccines.
1.A.5 Antimicrobial Peptides and Proteins
We currently are offering a number of projects investigating the synthesis of peptides and proteins for the development of antimicrobial peptides towards oral bacterial pathogens. These projects will involve the chemical synthesis of peptides and purification of proteins. These projects will use a number of techniques and in-house equipment such as peptide synthesis, HPLC, FPLC, Mass spectrometry, polymer chemistry, flow cytometry, bacterial culture, biofilm culture and proteolysis assays. Areas in which projects are available are:
1.A.5.1 Peptide Synthesis
- Synthesis of antimicrobial peptide and peptide mimetics
- The use of specific antimicrobial peptides for the delivery of chemical antibiotics.
1.A.5.2 Bioactive Peptides and Natural Inhibitors
- Novel delivery mechanisms of antimicrobial peptides
- Bioactive proteins and peptides derived from milk that inhibit biofilm formation and protease activity.
- Natural inhibitors of bacterial pathogen proteases
For more information e-mail A/Prof Neil O’Brien-Simpson, Dr Troy Attard, A/Prof Stuart Dashper or Dr Laila Huq
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1.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 wash 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.B.1 Bioinspired approach for the development of novel biomimetic nanoparticle materials with applications in bone/enamel tissue engineering
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 have the expertise and facilities to investigate novel, non-toxic, self assembling, injectable, nanoparticle biomaterials for potential applications in bone/enamel tissue engineering.
Recaldent™ is a non-toxic self-assembling delivery vehicle consisting of tryptic peptides of milk caseins encapsulating the mineral calcium phosphate for targeted release at the enamel surface. This project involves exploring the interactions between casein peptides and calcium and phosphate ions in different conditions.
Students will learn various biochemical and analytical techniques and have access to the facilities at Bio21.
1.B.2 Bioinspired synthesis of hydroxyapatite nucleating peptides derived from milk peptides with applications in biomimetic materials used in bone/enamel tissue engineering
The bioinspired approach will generate biomaterials that can integrate with the hard tissues promoting regrowth of hard tissue with optimal architecture and biomechanical properties.
To develop biomimetic materials that can nucleate hydroxyapatite, the potential of casein derived peptides to promote mineralization of hydroxyapatite will be investigated. This project also involves the synthesis of designer peptides to delineate the peptide region important for nucleation.
Students will learn various biochemical methods including peptide synthesis and analytical techniques including TEM and X-ray crystallography.
1.B.3 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 Cross1.B.4 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.
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1.C. Cariology
1.C.1. Novel Preventive Products/Functional Foods
Projects are available to test novel products and functional foods designed to assist in the prevention of dental caries and/or dental erosion. The products may include toothpastes, dental cremes, mouthrinse solutions, gels, and varnishes for topical application. The preventive products will contain anti-caries/erosion agents. Alternatively, commonly consumed foods and beverages will be modified to minimize their potential to cause loss of mineral from teeth such as during dental caries and dental erosion or modified to provide a positive health effect. Projects may include laboratory and/or in situ studies.
For more information e-mail Dr Nathan Cochrane or Dr Glenn Walker1.C.2. Enhancement of remineralization
Projects are available to study the process of enamel remineralization of early tooth decay. Enamel remineralization is the process of net mineral uptake into partially demineralized tooth structure. Projects will study ways of enhancing remineralization and quantify the effects of these treatments using a number of state of the art quantification methods. These projects may include laboratory and/or in situ studies.
For more information e-mail Dr Nathan Cochrane or Dr Peiyan Shen
1.D. Inflammation and Host Defence
1.D.1 Regulation of the inflammatory response of the oral mucosa to bacterial pathogens in periodontal disease
The epithelial cells of the oral mucosa express innate immune receptors (e.g. Toll-like receptors) that allow them to directly participate in the host immune response to infection by periodontal pathogens. Given that oral epithelial cells are the first host cells to encounter periodontal pathogens, the inflammatory factors they secrete (e.g. cytokines and chemokines) is likely to be important for periodontal immunity. However, dysregulation of the inflammatory response of oral epithelial cells to periodontal pathogens is thought to be critical in the development and severity of periodontal disease. The objective of this project is to identify the inflammatory factors produced by oral epithelial cells in response to different periodontal pathogens and to then characterise the cell signalling pathways that regulate their production.
Techniques: Expertise in a variety of cell biology (cell culture, ELISA assays), microbiological (culturing of pathogens), molecular biology (Real-Time PCR, siRNA-mediated gene silencing) and cell signalling (Western blotting) techniques will be acquired.
For more information e-mail A/Prof Glen Scholz (glenms@unimelb.edu.au)
1.D.2 Using nanoparticles and siRNA to modulate the host immune response to bacterial pathogens
The immune system plays a fundamental role in protecting us from infection by pathogens (e.g. bacteria and viruses). At the molecular level, this largely occurs through the integration of the cell signalling pathways that control the production of inflammatory factors (e.g. cytokines and chemokines) by cells within the innate and adaptive immune systems. However, the dysregulated production of inflammatory factors plays a pivotal role in the development and severity of a wide range of diseases (e.g. chronic periodontal disease, septic shock). The objective of this project is to validate the therapeutic potential of using nanoparticle-delivered short-interfering RNAs (siRNAs) that target specific cell signalling molecules or inflammatory factors to modulate the inflammatory responses of oral epithelial cells to periodontal pathogens using cell culture (in vitro) and animal-model (in vivo) based systems.
Techniques: Expertise in a variety of cell biology (cell culture, ELISA assays), microbiological (culturing of pathogens), molecular biology (Real-Time PCR, siRNA-mediated gene silencing) and cell signalling (Western blotting) and animal model techniques will be acquired.
For more information e-mail A/Prof Glen Scholz (glenms@unimelb.edu.au)
2. ORAL ANATOMY, MEDICINE AND SURGERY UNIT
2A. Evaluation of Age-Progression Software for Human Faces
This project seeks to begin evaluation of a recently developed in-house, computer-based age-progression system for human faces that has obvious important potential applications for Missing Persons Investigations. The honours student would be required to collect 3D datasets of participants using optical scanners and the extract 2D images from the dataset of people at various ages that correspond to historical 2D photographs of the same people. These images will be compared to see how closely calculated age changes correspond with the real photographs taken at earlier stages of life. In essence this involves driving the age-progression software backwards.Data acquisition will involve the use of 3D scanners and the collection of archival photographs from participants and therefore an important component of the project will be for the student to obtain ethics approval for the study. They will be given considerable help with this and will have the benefit of close supervision throughout the study.
For further information contact John Clement (forensic dentist and anatomist), Peter Claes or David Thomas (engineers)