| Medicine home | About | Future students | Current students | Research | Alumni | Contact us |
| Staff (Intranet) | Staff directory | A-Z index | Site map |
| Medicine home | About | Future students | Current students | Research | Alumni | Contact us |
| Staff (Intranet) | Staff directory | A-Z index | Site map |
|
|
Department of Medicine, Alfred HospitalInner and Eastern Clinical School Campus Bachelor of Science Honours Projects 2001
Transplantation Immunology / Renal Disease GroupTitle: Mechanisms of chronic injury in cardiac allograftsSupervisor: Dr Alicia Stein-Oakley (Tel: 9903-0539) Email: Alicia.Stein-Oakley@med.monash.edu.au Location: Dept of Medicine, Monash Medical School, Alfred Hospital Description: Cardiac transplantation is currently the therapy of choice for end-stage heart disease resistant to other medical or surgical therapy. The main cause of graft and patient loss after the first post transplant year is cardiac allograft vasculopathy. This project is part of an extensive prospective longitudinal study of endomyocardial biopsies from patients with cardiac allografts. The aim of the study is to analyse specific features of biopsies during the first year post transplantation, to determine their value as predictive factors for the subsequent development of cardiac allograft vasculopathy. The factors to be examined include fibroblast growth factor-2, fibroblast growth factor -9, plasminogen activator inhibitor-1, and genes of the renin-angiotensin and endothelin systems. Techniques: Immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, in situ hybridisation Title: Apoptosis in renal disease: Effect of treatment with angiotensin II (AT1) receptor antagonistSupervisors: Dr Alicia Stein-Oakley (Tel: 9903-0539), Prof Napier Thomson Email: Alicia.Stein-Oakley@med.monash.edu.au Location: Dept of Medicine, Monash Medical School, Alfred Hospital Description: Glomerulonephritis remains the most common cause of end stage renal failure in the adult and the second most common in children. We have developed a model of focal and segmental glomerulosclerosis (FSGS) induced by a dual dose of puromycin aminonucleoside (PAN) and a uninephrectomy. The model results in progressive glomerulosclerosis, tubular atrophy and interstitial fibrosis, a pattern of renal injury representative of several forms of human glomerulonephritis. Primary and secondary FSGS are major causes of end stage renal failure. Recent studies in our laboratory have demonstrated a strong correlation between apoptosis in the tubulointerstitial compartment and progression of renal disease in the PAN model. Apoptotic cell numbers and renal dysfunction were also found to be correlated with increased Bax, Fas and Fas L expression, and the latter was associated with the presence of infiltrating T cells. In contrast Bcl-2 expression was transiently increased in glomeruli, localised to areas of sclerosis. Treatment with the AT1 receptor antagonist, Losartan, ameliorates disease progression in the PAN model, delaying the development of glomerular and tubulointerstitial injury. Angiotensin II can induce apoptosis and both the AT1 and the AT2 receptors have been implicated. This project will examine the effect of Losartan treatment on apoptosis, and on the expression of apoptotic signaling, regulatory and effector molecules in the PAN model. Techniques: Immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, gel electrophoresis, in situ hybridisation, TUNEL technique Title: Apoptosis in cultured renal cellsSupervisors: Dr Alicia Stein-Oakley (Tel: 9903-0539), Prof Napier Thomson Email: Alicia.Stein-Oakley@med.monash.edu.au Location: Dept of Medicine, Monash Medical School, Alfred Hospital Description: This project will analyse the mechanisms involved in the delivery of apoptotic signals to renal cells, in mesangial and/or tubular cell culture systems in vitro. This will lead to a better understanding of the mechanisms involved in renal cell survival or loss, which contribute to the development of renal damage. Using glomerular mesangial and tubular epithelial cell cultures, specific aims will be:
Techniques: Cell culture, TUNEL, Annexin V, immunohistochemistry, confocal microscopy, quantitative reverse transcription-polymerase chain reaction, gel electrophoresis, in situ hybridisation, Western blot analysis Title: Apoptosis in tolerance inductionSupervisors: Dr Alicia Stein-Oakley (Tel: 9903-0539), Prof Napier Thomson Email: Alicia.Stein-Oakley@med.monash.edu.au Location: Dept of Medicine, Monash Medical School, Alfred Hospital Description: Peripheral transplantation tolerance can be induced by donor specific blood transfusions given under cover of cyclosporine A. Increased apoptosis has been implicated in the induction of tolerance through other mechanisms, such as increased antigen load. Spleen and thymus have been collected from AS rats, at different time points during the induction of tolerance to DA recipients, by donor specific blood transfusion. This project will assess changes in apoptotic cell numbers and in the regulation of apoptotic pathways in these tissues. Techniques: Immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, gel electrophoresis, in situ hybridisation, TUNEL technique Respiratory Medicine GroupMuch of Respiratory Medicine's research effort over the last six years has involved the collection and archiving of lung samples from asthmatic subjects and lung transplant recipients. The broad strategy that underpins the following projects is to make use of human samples that have already been collected. This represents a tremendous opportunity for students to take part in research that will potentially increase understanding of the pathogenesis of asthma and chronic rejection of lung allografts. These projects will utilise methodological approaches that have been proven in house, but which also represent work which is at the forefront of peer reviewed research. It is envisaged that these projects may lead to higher degree projects and in the past departmental scholarships have been made available in order to facilitate this. Transplantation ProjectsTitle:Airway RemodellingSupervisors:Mr C Ward (Tel: 9903 0622) Email: Chris.Ward@med.monash.edu.au Location:Dept. Medicine, Monash Medical School, Alfred Hospital Description: Having recruited and followed a cohort of lung transplant recipients we wish to investigate changes in airway structural components or‘airway remodelling’, over time. This will form part of the departmental program of transplant research that has been gaining international recognition over the last six years. Techniques: immunohistochemistry, interactive computerised image analysis, ELISA, potential for molecular biological work, especially qRT-PCR. Asthma ProjectsTitle: Long term airway changes in asthmaSupervisor:Prof E H Walters (Tel: 9276 2405) Email: Haydn.Walters@med.monash.edu.au Location:Dept. Medicine, Monash Medical School, Alfred Hospital Description: Asthma is a disease which affects much of the Australian population and which involves a significant load of morbidity and mortality. Prevalence in Australia and other developed societies is increasing. Asthmatic airway samples before and after treatments have been collected from patients by the Department. We wish to continue work looking at long term inflammatory and structural changes in asthmatic airways. The effect of treatment will be investigated. The project will form part of the departmental program of asthma research that is internationally recognised. Techniques: immunohistochemistry, interactive computerised image analysis, ELISA, potential for molecular biological work, especially qRT-PCR. Title: Immunostaining for inflammatory enzymes in airway biopsies in asthmaSupervisors:Prof E H Walters (Tel: 9276 2405) Email: Haydn.Walters@med.monash.edu.au Location:Dept. Medicine, Monash Medical School, Alfred Hospital Description: Our respiratory immunology group has collected a large number of biopsies from asthmatics. We have developed techniques for assessing a number of potentially relevant enzymes (e.g Nitric oxide synthases and steroid dehydrogenases) in these tissues. The project would involve quantitative assessment of staining in such material and its relation to clinical parameters, and responses to medication in our completed prospective studies. Techniques: immunohistochemistry, interactive computerised image analysis, assessment of clinical database information. Respiratory Physiology ProjectsTitle:Effect of Bronchodilation on the measurement of airway distensibilitySupervisors:Dr D Johns (Tel: 9276 3476) Email: d.johns@alfred.org.au Location:Lung Function Laboratory, Alfred Hospital Description: Respiratory medicine has developed a method for measuring the stiffness (distensibility) of the lung airways from changes in the volume of the anatomical dead space with lung inflation. Using this method we have found that the airways of mild asthmatics are stiffer than normal. We have evidence that the stiffer airways in asthma may be due to structural changes to the airway wall (e.g deposition of collagen, increased vascularity). The aim of the proposed study is to determine the effect of salbutamol on the measurement of airway distensibility in healthy subjects and asthmatics and to further develop the technique using Nitric Oxide as an airway product. Techniques: hands on "state of the art" airway physiology in patients, Nitric Oxide measurements in patients. Rheumatology GroupTitle:Inhibition of osteoclast-mediated joint destruction in collagen-induced arthritis modelSupervisors:A/Prof P Ryan, Dr E Romas Contact: Dr O Bakharevski 9903 0547 Email: Olga.Bakarevski@med.monash.edu.au Location:Dept. Medicine, Monash Medical School, Alfred Hospital Description: Rheumatoid arthritis (RA) is a chronic autoimmune disease, where joints are predominantly affected. RA is associated with persistent joint inflammation and progressive joint destruction. While the inflammatory component can be, in many cases, treated successfully, joint destruction often continues to progress. It has been shown earlier that osteoclasts play a major role in bone erosion in RA. We have established an animal model for RA, collagen-induced arthritis (CIA) in rats. The model shares a number of important features with RA, including osteoclast formation and subsequent joint erosion. Pamidronate is a drug which inhibits osteoclast-mediated bone resorption. PTHrP (parathyroid hormone-related peptide) is a cytokine that promotes osteoclast production. Neutralising PTHrP with a specific antibody may stop erosions from developing. The honours project offered will assess the effects of pamidronate and anti-PTHrP treatment on CIA. Techniques: induction of CIA (animal model), administration of pamidronate and anti-PTHrP antibodies, histology, immunohistochemistry and in situ hybridisation Dermatology GroupTitle:Female androgenetic alopeciaSupervisor: Dr R Sinclair Email: SINCLAIR@svhm.org.au Location:Dermatology Unit, Alfred Hospital Description: Hair loss is a common and often distressing problem affecting adult women. There are a number of possible causes of hair loss including childbirth, systemic illness, drug reactions, as well as androgenetic alopecia. In addition some cases are idiopathic. This project aims to study in detail the morbidity and natural history of hair loss in women. Having previously identified 330 female patients with diffuse hair loss, of whom 220 have histologically proven androgenetic alopecia, the focus of this research is to apply previously evaluated quality of life questionnaires to these women to assess morbidity, and the influence of treatment on their quality of life. In addition, as the natural history of androgenetic alopecia in females has not previously been studied, untreated patients will be recruited via media advertising, and the natural history of their hair loss monitored by standardized clinical photography and phototrichogram hair count data. Bone Marrow Transplantation / Clinical HaematologyTitle:Evaluation of apoptotic mechanisms in multiple myeloma cell lines and patient derived primary tumours.Supervisor: Dr A Spencer Email: aspencer@netspace.net.au Location:Bone Marrow Transplant Unit, Alfred Hospital Description: We have previously evaluated the efficacy of a novel potential anti-tumour agent TRAIL / Apo-2 (TNF-alpha receptor apoptosis inducing ligand) in primary tumours derived from patients with multiple myeloma. Our data suggests that approximately 30% of tumours are sensitive to TRAIL but that nearly all (>95%) express cell surface effector receptors for TRAIL. This suggests that functionally relevant abnormalities of the TRAIL receptors are present or alternatively that intra-cellular anti-apoptotic mechanisms prevent TRAIL induced tumour cell apoptosis. This study will investigate TRAIL apoptotic pathway related components in cell lines and primary tumours focusing on differences between TRAIL sensitive and insensitive cells and variations induced by exposure to effective concentrations of TRAIL. If a common mechanism for TRAIL resistance can be demonstrated combination therapeutic strategies including TRAIL could be explored. Title:Mutations in components of the TRAIL apoptosis pathway in multiple myelomaSupervisor: Dr A Spencer Email: aspencer@netspace.net.au Location:Bone Marrow Transplant Unit, Alfred Hospital Description: Multiple myeloma is a malignant expansion of plasma cells characterised by multiple genetic abnormalities and a high degree of resistance to conventional chemotherapeutic strategies. We have demonstrated that 30% of primary tumours show sensitivity to the potential novel anti-tumour agent TRAIL (TNF-alpha receptor apoptosis inducing ligand) despite apparent expression of cell surface effector receptors in most cases. Mutations of an alternative apoptosis inducing cell surface receptor FAS / Apo-1 have previously been demonstrated in multiple myeloma. This study will analyse the key components of the TRAIL apoptotic pathway in a search for functionally relevant mutations that may explain the TRAIL resistance seen in the majority of primary tumours. Clinical Pharmacology / Therapeutics GroupTitle: Autonomic Effects of Lung TransplantationSupervisor: A/Prof H Krum, Dr M Naughton, A/Prof T Williams Contact:A/Prof H Krum (Tel: 9903 0042 / 0417 325 834) Email: henry.krum@med.monash.edu.au Location:Clinical Pharmacology / Respiratory Medicine, Alfred Hospital Description: Background - Patients who have undergone lung transplantation may have disordered autonomic function, however this has been poorly characterised to date. Lung transplantation involves decentralisation of vagal afferent input whilst vagal input to the heart is preserved. Therefore lung transplantation provides an excellent model in which to assess the effect of vagal afferent denervation in man. Methods - We will study the autonomic effects of lung transplantation in 20 lung transplant recipients and compare this to 20 age-and sex-matched control subjects. Parameters to be assessed include heart rate variability as a measure of autonomic function, non-invasive measures of baroreflex sensitivity, cold pressure testing and effects of tilt. The effect of controlled breathing to simulate Cheynes-Stokes respiration on autonomic function will also be assessed in these groups. Implications - The information obtained from these studies will be of benefit in ascertaining the effect of afferent vagal denervation in man and by assessing autonomic derangements in lung transplant patients, may be of benefit in predicting potential for future cardiac events. Techniques: heart rate variability analysis (12 lead ECG data acquisition), baroreflex sensitivity gating 12-lead ECG with Finapres continuous BP monitoring forearm blood flow using venous occlusion plethysmograpahy cold pressor testing (BP, blood flow response to stress) tilt (80 degrees head-up tilting on tilt table) Clinical Neurosciences GroupTitle:Clinical correlates of recently developed otolith function testsSupervisor:Dr. John Waterston (Tel: 9276 2552) Email: John.Waterston@med.monash.edu.au Location:Oto-Neurology, Alfred Hospital Description: Traditional vestibular function tests assess only the functional integrity of the semicircular canals. Until recently, no practicable routine tests of otolith organ function existed. However, the Alfred Neuro-otology laboratory has now added two new tests of otolith function to its capabilities: static bias testing (judgement of the horizontal in the absence of visual environmental clues) and vestibular-evoked myogenic potentials (recorded in sternocleidomastoid after high intensity auditory stimuli, which cause saccular activation). This project would address the correlation of abnormalities on these new tests with clinical syndromes and with traditional tests of vestibular (semicircular canal) function. Techniques: clinical electrophysiology, statistics Title:Validation and clinical correlation of upper limb ballistic tracking movement recordings in cerebellar diseaseSupervisors:Prof E Storey, Ms Kate Tuck (Tel: 9276 2552) Email: Elsdon.Storey@med.monash.edu.au Location:Neurosciences / Dept of Medicine, Alfred Hospital Description: Quantitation of cerebellar dysfunction is difficult, although a number of subjective rating scales such as the International Cooperative Ataxia Rating Scale have been published. Movements which are "aimed and fired" - performed too rapidly for correction to occur during the movement - are termed "ballistic". Overshoot on ballistic tracking movements is a useful clinical test of cerebellar dysfunction, and should particularly reflect dysfunction of the lateral zone of the neocerebellum. In conjunction with a biomedical engineering student, we are developing a laboratory version of the ballistic tracking test, measuring peak velocity / peak acceleration / duration of acceleration /overshoot versus target displacement. This project would investigate the reliability of these measures in cerebellar disease, and explore the correlation with clinical and MRI findings and with the genotype in various dominant spinocerebellar degenerations Title: Novel proverbs test of frontal-executive functionSupervisor:Prof E Storey (Tel: 9276 2552) Email: Elsdon.Storey@med.monash.edu.au Location:Neurosciences, Alfred Hospital Description: One method whereby clinicians have traditionally tested abstraction is by asking patients to interpret proverbs. There are difficulties with this method, however, including differing exposure to the proverbs usually used due to differing sociocultural backgrounds, and the likelihood that interpretation of proverbs well-known to the patient may reflect semantic memory more closely than "abstraction". In an attempt to examine these points and increase test validity, a novel proverbs test has been developed together with an MCQ answer format, in which twelve novel proverbs are randomly intermixed with six known proverbs The purposes of this study are to refine the novel proverb battery by examining the contribution of each novel proverb to the overall score in elderly patients, to correlate the novel proverb score with other bedside measures of frontal-executive function and with estimated premorbid FSIQ, and to test the hypothesis that the score on novel proverbs will not correlate tightly with that on known proverbs, reflecting the contribution of semantic memory to the latter. Title: The role of APP and APLP2 in neurite outgrowthSupervisors:Dr L Kelly, Prof E Storey (Tel: 9276 2552) Email: Elsdon.Storey@med.monash.edu.au Location:Neurosciences / Dept of Medicine, Alfred Hospital Description: APP, the amyloid precursor protein of Alzheimer’s disease, and its homologue APLP2 are found in a segmental distribution on the surface of neurites in cortical neurons in primary culture. Earlier work on the function of APP suggests a role in neurite-substratum adhesion. We postulate that there will be functional redundancy between APP and APLP2 in this regard. This project aims to investigate the effect of removing APP, APLP2, or both on neurite outgrowth and branching using time-lapse videomicroscopy and computerised analysis of neurons cultured from gene knockout mice lacking APP, APLP2, or both. This is an entirely laboratory based project, which will also enable the acquisition of skills in tissue culture. Infectious Diseases GroupTwo or three vacancies. Title: Development of an oral plant-derived vaccine for measlesSupervisors:Prof S Wesselingh (Tel: 9276 3087) Email: S.Wesselingh@alfred.org.au Dr J Martin (Tel: 9903 0762) Email: Jenny.Martin@med.monash.edu.au Location:Depts Medicine & Microbiology, Monash Medical School Description: In the developing world over a million malnourished infants die annually from measles because of poor coverage of the existing measles vaccine making measles one of the main causes of infant mortality. To overcome these problems new measles vaccination strategies need to be developed. This project will investigate the development of an "edible" oral vaccine to the measles virus through the expression of measles antigens in plants. We plan to test the hypothesis that orally administered measles antigens derived from transgenic plants will evoke a protective immune response, and that oral immunogenicity can be enhanced by targeting measles H protein to the gastrointestinal mucosa, through genetic fusion of the protein to natural mucosal binding proteins. Techniques: Plant molecular biology, B+T cell immunology, viral culture Title: Neuropathogenesis of HIV DementiaSupervisor:Prof S Wesselingh (Tel: 9276 3087) Email: S.Wesselingh@alfred.org.au Dr J Martin (Tel: 9903 0762) Email: Jenny.Martin@med.monash.edu.au Location:Depts of Medicine & Microbiology, Monash Medical School Description: Dementia is an extremely common problem in the late stages of Human Immunodeficiency Virus (HIV) infection. We have previously demonstrated a correlation between the severity of dementia, macrophage activation, and the production of neurotoxins. However, recently we have obtained data that suggest a significant role for astrocytes in the development of dementia. We hypothesise that non-productive HIV infection of astrocytes leads to a loss of astrocyte function, increased astrocyte apoptosis and a consequence in the levels of neurotoxins. Techniques: Viral culture, in situ PCR, immunohistochemistry Title: Viral encephalitisSupervisor:Prof S Wesselingh (Tel: 9276 3087) Email: S.Wesselingh@alfred.org.au Dr J Martin (Tel: 9903 0762) Email: Jenny.Martin@med.monash.edu.au Location:Depts of Medicine & Microbiology, Monash Medical School Description: This project involves the use of Sindbis virus to investigate the immune response generated in the brain and the possibility of using an orally administered (transgenic plant) vaccine to prevent viral infections of the central nervous system. Techniques: Viral culture, in situ hybridisation, molecular biology, T+B cell immunology Title: Methicillin Resistant Staph aureusSupervisor:Prof S Wesselingh (Tel: 9276 3087) Email: S.Wesselingh@alfred.org.au Dr J Martin (Tel: 9903 0762) Email: Jenny.Martin@med.monash.edu.au Location:Depts of Medicine & Microbiology, Monash Medical School Description: The project involves the development of molecular typing methods which will then be used to aid the control of MRSA infections which continue to be a major problem in hospital settings. Techniques: Bacterial identification, bacterial resistance measurements, pulse field electrophoresis, PCR, RFLP The Australian Centre for Blood Diseases Department of Medicine Box Hill Hospital Title: Novel Therapies in the Treatment of CancerSupervisors:Dr A Dear, Dr R Medcalf Email: Anthony.Dear@med.monash.edu.au Location:Box Hill Hospital Description: Cancer represents one of the most common causes of death in western society. Much effort has been expended on developing new therapies for the treatment of cancer, a consequence of an increasing incidence of the disease together with the development of resistance of many cancers to conventional chemotherapy. Our research efforts have concentrated on the design and testing of new cancer treatments in an attempt to overcome the current limitations of conventional cancer treatments. We have been interested in identifying molecules which have the capacity to inhibit enzyme systems thought to be integral to the spread of cancer cells from the site of primary tumour growth to distant regions of the body, a process referred to as metastasis. The metastatic process is responsible for the vast majority of the morbidity and mortality associated with cancer. We have identified several molecules with the potential to inhibit the metastatic process and we are currently in the process of testing these molecules in vitro and generating animal models to assess the anti-metastatic activity of these agents in vivo. This project will involve both in vitro testing of new agents in assays developed in the lab, with the potential to identify new actions of these compounds, together with involvement in establishing and monitoring the effects of compounds in animal models. Students interested in understanding the process of new drug discovery together with the testing and application of drug discovery programs will find the project particularly attractive. This project will compliment much of the pre-clinical material students have been exposed to and in addition will augment much of the information provided in the clinical years ahead. Title: Regulation of Gene Expression in the Plasminogen Activating SystemSupervisors: Dr R Medcalf Email: robert.medcalf@med.monash.edu.au Location:Box Hill Hospital Description: Broad scope of laboratory: Our laboratory is interested in understanding the mechanisms that govern gene expression in human cells. The genes that we study encode the members of a proteolytic enzyme system known as the plasminogen activating system. The end product of this system is a powerful protease called plasmin. Plasmin can degrade a number of other proteins, including most proteins of the extracellular matrix. It is also very important in the removal of blood clots ("fibrinolysis") from the circulation and facilitates cell movement. Indeed, cancer cells harness this enzyme system to enable them to metastasize to other organs. The important role the plasminogen activating system plays in these (and many other) processes has fuelled much interest to understand how its individual components are regulated. Components of the plasminogen activating system: Plasmin is formed when its inactive precursor, plasminogen is cleaved by one of two proteases known as tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). These activating enzymes are themselves regulated by specific inhibitors known as plasminogen activator inhibitor type 1 (PAI-1) and PAI-2. Other components exist that allow plasmin generation to occur at specific sites and on the surface of some cells. This in part is achieved by cell-bound receptors for either t-PA or u-PA. To make matters even more intricate, these receptors can associate with other cell bound proteins to activate intracellular signalling processes which can in turn change the cells behaviour. An outline of this system is provided in the figure below. Our laboratory has a number of concurrent projects that address various aspects of the regulation of expression of essentially all components of the plasminogen activating system. The BSc. (Hons) project available addresses the biology of the inhibitor PAI-2. PAI-2 is expressed in many cell types, but is mainly found in monocytes, macrophages and in trophoblast cells of the placenta. It is an unusual protease inhibitor, mainly because it exists predominantly as an intracellular protein. Its target protease (u-PA) is present in the extracellular space or on the cell surface, bound to its receptor. There has been much speculation that PAI-2 plays an intracellular role in addition to its role as a protease inhibitor. The project available will investigate this possibility. Potential Projects :-(i)The intracellular role of PAI-2 in THP-1 monocytes[Supervisors: Dr Robert Medcalf and Dr Hong Yu] We have available a monocyte cell line (THP-1 cells) that contains a unique defect in the production of PAI-2. We have determined the molecular basis of this mutation (BSc. Hons project in 1999) and a paper describing this will be published in the September issue of Thrombosis and Haemostasis (Katsikis et al., 2000). Unlike all other monocytic cells available, THP-1 cells cannot make a functional PAI-2 protein. We have taken the opportunity offered by THP-1 cells to assess the intracellular role of PAI-2 by expressing an active PAI-2 in these cells. We observed that the THP-1 cells proliferation rates were significantly reduced and they did not respond to classical triggers of differentiation. Of particular interest was that an intracellular complex was formed in THP-1 cells expressing active PAI-2 indicating the presence of an intracellular target protein for PAI-2. The identity of this protein is unknown. The THP-1 cells expressing PAI-2 as well as other PAI-2 variants are established and available for further study. The project will address the mechanism by which PAI-2 influences the cells behavior and will also attempt to identify the intracellular protein target for PAI-2. Methods: Cell culture, Northern and Western blotting, PCR, DNA preparation and manipulation. It is also anticipated that microarray screening will be available in early 2001 to identify candidate genes altered as a consequence of PAI-2 expression. (ii) Studies on the regulation of the human tissue-type plasminogen activator gene in vivo using transgenic mice"[Supervisor — Dr Robert Medcalf] Tissue-type plasminogen activator (t-PA) is an important serine protease that is involved in the removal of blood clots from the circulation as well as the turnover of the extracellular matrix. More recently however, t-PA has been shown to be expressed at high levels in the brain where it plays a crucial role in the development of memory, visual processing, and neurodegeneration. Our laboratory has been studying the transcriptional regulation of the t-PA gene in vitro and we have identified many of the important control elements within the t-PA gene promoter. The project available is to assess the regulation of t-PA gene expression in vivo using transgenic mice as a model. Three lines of transgenic mice are available which express different lengths of the t-PA gene promoter (9 kb, 3 kb and 1.4 kb) fused to the LacZ reporter gene. To date, we have exciting data showing abundant t-PA promoter directed LacZ expression in the brains of transgenic mice harbouring the 9.0 t-PA gene promoter construct, while only limited expression is seen in animals harbouring the 1.4 promoter construct. Importantly, treatment of these animals with agents known to regulate expression of the t-PA gene in vitro also increases expression of t-PA promoter directed expression in vivo. The project will be to further map the promoter-dependent expression pattern of the t-PA gene in these animals and to study the change in the expression pattern of the t-PA gene in vivo after administration of tumor necrosis factor (a powerful modulator of t-PA expression) as well as neurostimulatory agents. This project will also incorporate in situ hybridisation and immunohistochemistry to detect endogenous t-PA mRNA and antigen, respectively, in these animals. In parallel to the in vivo aspects of the project, the transcriptional control of the t-PA gene will be assessed in neuroblastoma cells (transient transfection, DNase-1 hypersensitivity mapping etc). All methods required to implement this project are fully established in the laboratory. Title: Signal Transduction in PlateletsSupervisors:Dr Yuping Yuan, Dr S Jackson Email: yuping.yuan@med.monash.edu.au Location:Box Hill Hospital Description: Platelet adhesion to the site of vessel wall damage is essential for normal haemostasis and thrombosis. This process is mediated by the interaction between the subendothelial protein, von Willebrand Factor (vWf), and the platelet surface receptors, glycoprotein Ib/V/IX and integrin IIb3. Exaggerated adhesion can result in life threatening heart attacks and strokes, which represent the major cause of death in developed countries. On the other hand, defects in platelet adhesion can lead to severe bleeding disorders. Our current research aims to improve our understanding of the regulation of platelet adhesion, particularly by intracellular signalling proteins and pathways. Using cellular, molecular and biochemical approaches, we have discovered critical roles for multiple signalling proteins including protein tyrosine kinases, protein kinase C and the protease, calpain, in regulating platelet adhesion. Our recent studies have established for the first time the ability of GP Ib/V/IX to induce calcium (Ca2+) mobilisation from intracellular stores, as a necessary event for platelet cytoskeletal remodelling and integrin IIb3 activation. We are currently investigating, at a molecular and structural level, the important signalling events linking the vWf-GPIb/IX interaction to intracellular Ca2+ mobilisation. These studies will significantly enhance our current understanding of the mechanisms regulating platelet activation and may help identify key signalling molecules or pathways as novel antithrombotic targets. Title: The Dynamic Cytoskeleton — A Role in Haemostasis and ThrombosisSupervisor:Dr S Schoenwaelder Email: simone.schoenwaelder@med.monash.edu.au Location:Box Hill Hospital Description: Platelets are small specialised blood cells, which play a key role in clot formation and the arrest of bleeding (Haemostasis). Under normal conditions, platelets circulate throughout the blood stream without interacting with the vessel wall endothelium. However, upon vessel wall damage, platelets are recruited to exposed subendothelial proteins, where they adhere, spread and form a thrombus that effectively seals off the site of injury and prevents further blood loss. Many of the changes platelets undergo in response to vessel injury are driven by reorganisation of their cytoskeleton. Remodelling of the platelet cytoskeleton leads to changes in cell shape and behaviour, which in turn affect the ability of these cells to attach to an injured vessel wall and stop bleeding. We are working towards a better understanding of the mechanisms that regulate cytoskeletal reorganisation, so that we may more effectively regulate platelet function in vivo. Potential Projects :-(i) Regulation of the Cytoskeleton by a Protein-Tyrosine Phosphatase, Shp-2[Supervisor — Dr Simone Schoenwaelder] The regulation of cytoskeletal remodelling involves a complex array of signalling pathways, most of which are not fully characterised. One well described signalling pathway involves RhoA, which is responsible for promoting the formation of actin bundles and cell adhesions (focal adhesions). The role of RhoA is thought to lie primarily in maintaining a stable contact between the cell and the underlying matrix. Consistent with their specialised adhesive role in haemostasis, platelets contain abundant levels of RhoA, however its regulation and function in these blood cells remains unclear. Our previous studies have described a novel pathway whereby a protein-tyrosine phosphatase regulates RhoA-mediated cytoskeletal remodelling in fibroblasts. This project will investigate how Shp-2 regulates RhoA, both in cultured cells and in platelets, and examine the role of the Shp-2/RhoA signalling pathway in platelet function. (ii) Investigate the Role of Shear Stress on Platelet Cytoskeletal Changes[Supervisors — Drs Simone Schoenwaelder and Shaun Jackson] Platelets participate in "damage control" when vessels are injured. Therefore, it is envisaged that cytoskeletal remodelling leading to platelet spreading should be executed rapidly in vivo following injury. However, in vitro analysis of platelet spreading has revealed that this process can take up to 60 minutes on a vWf matrix. This prolonged time frame would be highly undesirable in vivo, as it would allow for the continued loss of blood. Therefore, additional regulatory mechanisms must exist to ensure that rapid spreading of platelets ensues. The majority of studies examining platelet spreading thus far have been performed under static, non-physiological conditions. Therefore, one hypothesis would be that the shear conditions experienced by platelets in vivo may exert regulatory effects on these blood cells to regulate their function. In this research project, the effect of increasing shear rate, such as those found in veins (150s-1), arteries (600s-1) and arterioles (1800s-1), on cytoskeletal remodelling, platelet spreading, and signalling events will be investigated. Title: Haemostasis and ThrombosisSupervisors:Prof H Salem, Dr S Jackson Email: shaun.jackson@med.monash.edu.au Location:Box Hill Hospital Description: In these studies the role of platelet glycoproteins in mediating platelet adhesion to fibrinogen and von Willebrand factor is examined. The role of novel drugs in the modulation of platelet adhesion to these ligands is also assessed. This work will help elucidate the basic mechanism for mediating adhesion and allow the identification of new drugs that may help reduce platelet adhesion and hence thrombosis. A new system that measures platelet adhesion and thrombus formation is currently being developed. This system will be tested in patients with bleeding and thrombosis and healthy controls. These investigations will define a role for this new system in the assessment of patients with abnormal haemostasis and thrombosis. Title: Structure and Functional Interactions of Platelet Surface ReceptorsSupervisor:Dr D Williamson Email: david.williamson@med.monash.edu.au Location:Box Hill Hospital Description: Platelets are specialised blood cells that play a critical role in blood clotting. For these cells to initiate the formation of a blood clot, they must first stick to the blood vessel wall at the site of injury. They do this through the action of specific platelet receptors. The interaction between the platelet glycoprotein (GP) Ib/V/IX receptor complex and von Willebrand factor (vWf) exposed on the injured vessel wall is of particular significance in this process. Once GP Ib/V/IX adheres to vWf, changes are initiated inside the platelet that allow it to stick firmly, change its shape and go on to form a firm clot that can "plug" the area of injury and stop bleeding. The importance of GP Ib/V/IX in the clotting process is highlighted in certain rare bleeding disorders in which they are abnormal or absent. GP Ib/V/IX has "external domains" that are involved in sticking to the blood vessel and other platelets, as well as "internal domains" that bind to other proteins inside the platelet. We are currently investigating the role of specific structural domains in the interaction of this receptor with intracellular structural and signalling proteins. Current studies in transfected cell lines will be complemented by in vitro protein binding studies using purified normal and mutant receptor proteins. These studies will help to further define receptor interactions that regulate platelet adhesion and activation and contribute to our overall understanding of the processes that lead to normal and pathological blood clot formation. The same basic processes that are involved in normal blood clotting (haemostasis), also apply in thrombosis, the formation of harmful blood clots that can be the cause of a heart attack or stroke. Therefore, understanding in detail the processes that control clot formation can help to identify therapeutic strategies for the prevention of thrombotic diseases Title: Haematopoiesis — The Role of Haemopoietic Serpins in Cell Growth and DifferentiationSupervisor:Dr P Coughlin Email: paul.coughlin@med.monash.edu.au Location:Box Hill Hospital Description: The blood of a normal person contains three main components — red cells, white cells and platelets which are made within the bone marrow. All mature cells are generated from a small group of primitive "stem cells". It is now thought that leukaemia, and related bone marrow diseases, are caused by malfunction of the stem cells, often as a result of faulty genes. We are seeking new insights into the origins of leukaemia by investigating the genes which are normally active in these blood precursors. One such gene, serpin2A, produces a protein which we have found in the nucleus of "stem cells". The nucleus is a special compartment where genes are kept and controlled. In order to understand the biology of this unusual serpin we are analysing its molecular interactions with target proteins using the yeast-2-hybrid system. We are also examining its effects on growth and differentiation in model cell lines. Our hypothesis is that serpin2A is a component of the mechanism which regulates copying of genes and may also control the way cells grow and divide. Our studies link serpin2A to several other proteins in the nucleus which take part in these control mechanisms. We are investigating the way that these proteins interact with each other to co-ordinate the development of primitive blood cells. Our results so far suggest new and exciting roles for this serpin which require further detailed study. We believe this will lead to new insights into the causes of blood diseases and ultimately give rise to safer and more effective treatments. Baker Medical Research InstituteMolecular SignallingTitle: Function and regulation of human telomerase in cellular ageing and immortalisationSupervisor: Dr Jun-Ping Liu (Tel: 9522 4385) Email: jun-ping.liu@baker.edu.au Location:Baker Medical Research Institute Description: Telomerase, a specialised RNA-directed DNA polymerase at telomeres, plays a key role in controlling cell survival and immortalization. Human telomerase contains the catalytic subunit telomerase reverse transcriptase (hTERT). The aim of the project is determine how telomerase activity is regulated by interacting with other proteins in cell ageing, using molecular biology and immunological techniques. Recombinant TERT fusion proteins will be produced and used to demonstrate putative interacting proteins in a pull-down assay. Up- and down-regulation of hTERT or its regulatory proteins will be performed in cultured cells using established methods. Title: A novel muscle-specific mitotic checkpoint proteinSupervisor: Dr Jun-Ping Liu (Tel: 95224385) Email: jun-ping.liu@baker.edu.au Location:Baker Medical Research Institute Description: A cDNA coding for a novel 75-kDa protein in heart and skeletal muscle has been cloned. Subcellular localisation study suggests the protein at the kinetochore of mitotic cells. The aim of the project is to determine cellular function of the new gene product in heart. Cardiac muscle cells from neonatal rats will be cultured and transfected to force dominant expression of mutated genes or under-expression of antisense cDNAs. Immunofluorescent staining, cell growth and apoptosis will be analysed. Cardiomyocyte cultures and all methods have been available in our laboratory. Title: A novel neuronal ATPaseSupervisor: Dr Jun-Ping Liu (Tel: 9522 4385) Email: jun-ping.liu@baker.edu.au Location:Baker Medical Research Institute Description: A cell membrane ATPase in human brain has been isolated. Sequence analysis suggests that the ATPase be regulated by protein phosphorylation at a large intracellular loop in the vicinity of plasma membrane. Subcellular localisation and ATPase activity will be analysed as a function of protein phosphorylation by certain protein kinases. All techniques and methodologies have been established. Title: Profiling genetic changes in human hypertensionSupervisor:Dr K M Curnow (Tel: 9522 4367) Email: Kathleen.Curnow@baker.edu.au Location:Molecular Endocrinology Laboratory, Baker Institute Description: The Human Genome Project has identified new mutations in genes encoding proteins that regulate blood pressure. An Honours/BMedSci project will involve introducing these missense mutations into wild type cDNA clones by site directed mutagenesis and testing their effects on protein function as a means of determining their relative roles in modulating human blood pressure. The initial genes under study will be those encoding the angiotensin AT1 receptors, aldosterone synthase and the mineralocorticoid receptor, these being key functional elements of the renin-angiotensin-aldosterone axis. A PhD student would additionally commence a longer term study investigating the genetic basis of hypertension characterised by high aldosterone/renin levels. Patient collection for this study commenced in 1998 and sufficient total numbers for study will be available in early 2000. Techniques: Site directed mutagenesis, plasmid DNA preparation, DNA sequencing, use of software to analyse DNA sequences, cell culture, transpection, protein assays Title: Preeclampsia (hypertension of pregnancy) and inappropriate activation of the angiotensin AT1 receptorSupervisor:Dr K M Curnow (Tel: 9522 4367) Email: Kathleen.Curnow@med.monash.edu.au Location:Molecular Endocrinology Laboratory, Baker Institute Description: Preeclampsia, hypertension presenting in late pregnancy, is a serious complication of gestation that can result in maternal and/or foetal death. The disorder is inherited, occurs in 5% of pregnancies, and is characterised by an increased sensitivity to the vasoconstrictor hormone, angiotensin II. The levels of this circulating hormone are not elevated. Our aim, to determine the genetic basis of this disease, has focussed on whether angiotensin AT1 receptors of affected women differ in their structure, function or regulation by investigating polymorphism in the AT1 receptor gene. We have identified several genetic changes, both in the coding sequence and the untranslated regions, and the effects of these on receptor function and regulation are currently being tested. A recent study has determined that women with preeclampsia produce antibodies to the AT1 receptor, this being consistent with the idea that the receptors of affected individuals are altered. The studies proposed for an Honours/BMedSci project are to determine the functional capacities of these antibodies by studying to what degree they replicate the actions of the natural hormone, angiotensin II. Functions investigated will include the determination of the ability of the antibodies to activate the classical AT1 receptor intracellular signalling pathways, as well as their ability to stimulate smooth muscle contraction, cardiomyocyte contraction and aldosterone production. The efficacy of newly developed AT1 receptor antagonists to block these actions will also be tested. Should these studies form the first year of a PhD project, the ongoing area of interest will be to continue characterising the genetic abnormalities believed to trigger this disease, ultimately allowing prediction of disease susceptibility. Techniques: Preparation of antibodies from blood samples, IP-3 assays, cell culture, physiological assays of AT1 receptor activation Title: Amino acids involved in activation of the human angiotensin AT1 receptorSupervisor:Dr K M Curnow (Tel: 9522 4367) Email: Kathleen.Curnow@med.monash.edu.au Location:Molecular Endocrinology Laboratory, Baker Institute Description: Angiotensin AT1 receptors play a pivotal role in mediating the effects of the renin-angiotensin system, signalling the extracellular presence of angiotensin II to the intracellular signalling pathways activated by this peptide hormone in target tissues. Research activity on these receptors has intensified because drugs that block binding of angiotensin II to the AT1 receptors are under development and are currently being introduced for treatment of essential hypertension (high blood pressure). The projects proposed aim to expand our understanding of the structural and functional biology of these receptors. The Honours/BMedSci project will focus on residues involved in activation of the receptors, specifically determining the requirement of four transmembrane domain (TMD) cysteine residues (in TMDs 2, 3, 4, and 7). These are likely to form disulphide crosslinks important in determining the alignment of the TMDs for ligand binding and/or mediating the conformational change permitting signalling. The techniques employed will be site directed mutagenesis of the cysteine residues in cloned cDNAs, transfection of these into cells lacking AT1 receptors, and characterisation of the effects of the mutations on ligand binding and signalling. Follow on mutagenesis studies are proposed for an ongoing PhD. Such mutational analyses have routinely been used to identify residues required for function of the AT1 receptors, as well as other members of this large family of 7 TMD receptors, but no information on the physical changes occuring during receptor activation have been obtained for this receptor family by other means. A PhD student would therefore commence the biochemical mapping of these receptors by a novel method. Receptors will be synthesised using standard in vitro translation techniques in microsomal membranes. In this way, the cytoplasmic face of the receptor will be exposed to degradation by proteases. It will thus be possible to determine the regions of the receptor exposed, before and after ligand interaction, by mass spectrometry analysis of the regions resistant to protease degradation. Effects of disease causing mutations on the receptor will be examined with both the traditional and new assay systems. Techniques: Site directed mutagenesis, plasmid DNA preparation, DNA sequencing, use of software to analyse DNA sequences, cell culture, transpection, protein assays, IP-3 assays Title: Sequences regulating expression of the human angiotensin AT1 receptorSupervisor:Dr K M Curnow (Tel: 9522 4367) Email: Kathleen.Curnow@med.monash.edu.au Location:Molecular Endocrinology Laboratory, Baker Institute Description: Preliminary studies have shown the 5’UTR and 3’UTR of the angiotensin AT1 receptors to determine the level of synthesis of these receptors by several different mechanisms. An Honours/BmedSci project would utilise deletion and replacement mutagenesis studies to identify the specific exon 1 sequence motifs required to maintain basal transcription, identify sequences in the 5’UTR that interact with RNA binding proteins, investigate the putative presence of "zip code" sequences in the 3’UTR and further characterise sequences in the 3’UTR affecting stability of the AT1 receptor mRNA. Mutations in the 5’UTR and 3’UTR of the receptor have been identified in human hypertension and it will be important to determine the role of these mutations in determining receptor expression. An ongoing PhD would further identify and characterise the proteins interacting with the regulatory regions, improving our understanding of the mechanisms regulating AT1 receptor synthesis at the levels of transcription, translation, location of protein synthesis and RNA degradation. Techniques: Deletion mutagenesis, plasmid preparation, DNA sequencing, transpection, angiotensin binding assays, luciferase assays, Northern Blotting Title: Proteoglycan biosynthesis by human vascular smooth muscle cellsSupervisor:Dr P J Little (Tel: 9522 4361) Email: peterjlittle@hotmail.com Location:Cell Biology of Diabetes Laboratory, Baker Institute Description: Atherosclerosis of coronary arteries leads ultimately to a need for coronary artery bypass grafting (CABG). In CABG, blocked vessels are by-passed using alternative non-diseased vessels. The utility of the procedure is limited by the redevelopment of atherosclerosis in the grafted vessels. One of the determinants will be the production of matrix molecules, and in particular protoeglycans, which retain lipoproteins as an early phase in the development of atherosclerosis. The plan is to grow vascular smooth muscle cells from the three different vessels used for CABG, characterise the cells by immunohistochemical techniques and study the production of proteoglycans. The atherosclerotic potential of the cells will be assessed by studying, in vitro, the binding of the proteoglycans to lipoproteins. Because of the interest of the laboratory in diabetes we will investigate several mechanisms relevant to diabetes (e.g. insulin, hyperglycemia and anti-diabetic drugs). Techniques: Cell and tissue culture, multiple electrophoresis, ligand binding analyses, proteoglycan biochemistry Title: Molecular CardiologySupervisor:A/Prof Elizabeth A. Woodcock Location:Baker Institute Description: We currently have a transgenic mouse line that is resistant to the development of pressure overload hypertrophy. These mice remain healthy after thoracic aortic constriction and retain contractile function without hypertrophy. The studies will investigate how this peptide expressed in the hearts of these animals prevents hypertrophic growth. These mice can also be investigated for functional changes after banding, using echo cardiography and ultrasound as well as isolated tissue responses. Techniques:ECG, ultrasound, tissue culture Title: Mitochondrial regulation of cell death in human myocardiumSupervisor:Dr S Pepe (Tel: 9522 4353) Email: salvatore.pepe@baker.edu.au Location:Cardiac Surgical Research Unit, CJOB Cardiothoracic Surgery & Transplantation Dept, Alfred Hospital & Baker Medical Research Institute Description: How is mitochondrial function in the human heart specifically altered in disease states such as cardiac ischaemia, heart failure or hypertrophy and with increased age? This project involves the use of physiological, histological, biochemical and electrochemical techniques to study living, freshly isolated cardiac trabeculae, myocytes and mitochondria from clinical cardiac tissue. Specific projects are available in mitochondrial enzyme function, cell death, redox systems, antioxidant and free radical metabolism. Mitochondrial genetics projects are available in collaboration with Prof. Phillip Nagley, Dept Biochemistry & Molecular Biology, Monash University. Techniques: physiological, histological, biochemical and electrochemical techniques Title: Opioid peptide-mediated protection of human heart against ischaemic injurySupervisor:Dr S Pepe (Tel: 9522 4353) Email: salvatore.pepe@baker.edu.au Location:Cardiac Surgical Research Unit, CJOB Cardiothoracic Surgery & Transplantation Dept, Alfred Hospital & Baker Medical Research Institute Description: Opioid proteins, produced by nerve afferents to the heart and by heart muscle itself, specifically alter cardiac excitation-contraction coupling mechanisms and intracellular signaling pathways. Opioids have been shown to afford protection against ischemia and reperfusion injury via intracellular pathways identical to those that mediate ischemic preconditioning. Exactly how remains undefined, particularly in human heart. We are presently examining the functional role of Proenkephalin opioid metabolites and synthetic agonists/antagonists in trabeculae and individual myocytes isolated from human hearts. Biochemical studies will characterise in human heart the specific intracellular signalling pathways that are activated after opioid receptor stimulation. Techniques: molecular pharmacology, opioid metabolic and cardiovascular physiological techniques Title: Biology of human aging and pathology with relevance to new strategies for improved outcomes in cardiac surgery and heart transplantationSupervisor:A/Prof F Rosenfeldt (Tel: 9522 4353) Email: frankr@baker.edu.au Location:Cardiac Surgical Research Unit, CJOB Cardiothoracic Surgery & Transplantation Dept, Alfred Hospital & Baker Medical Research Institute Description: Bachelor of Medical Science (Hons), MD, MS, and PhD clinical projects are available for those with medical background. Title: Beta carbolines : are they endogenous ligands for CNS imidazoline receptors?Supervisor:Dr G A Head (Tel: 9522 4322) Email: geoff.head@baker.edu.au Location:Neuropharmacology Laboratory, Baker Institute Description: Recent evidence suggests that the hypotensive effect of centrally-acting antihypertensive drugs is not due to stimulation of a 2-adrenoceptors but to an action on "imidazoline receptors". The existence of the latter sites has led to the development and recent clinical use of second generation agents such as rilmenidine and moxonidine which possess a much greater selectivity toward these non-adrenergic receptors. We have been examining this suggestion using a conscious rabbit preparation and have developed an antagonist protocol which can separate the a 2-adrenoceptor action from the "imidazoline receptors" action. We have mainly studied second generation drugs such as rilmenidine and moxonidine but this year we have been examining a new agent AGN19403 which is an isofuran with high affinity for imidazoline receptors and not a 2-adrenoceptors. One of the exciting very recent developments was the finding that b -carbolines are potentially endogenous ligands for imidazoline receptors and possess nanomolar affinity for IR and micro-molar affinity for a 2-adrenoceptors. While most b -carbolines are found in certain plants and can be absorbed from the diet, some have been shown to exist in the mammalian brain as endogenous substances. One of these, Harmane (1-methyl-beta-carboline) appears to have the highest affinity for IR according to the binding studies. Harmane has been previously reported as exerting locomotor and psychoactive effects and binds reversibly to the active site of monoamine oxidase type A in vivo. At present little is known about their central cardiovascular effects. The aim of the project therefore will be to examine central cardiovascular effects selected Beta-carbolines and also of other agents with selectivity for imidazoline-preferring receptors rather than a 2-adrenoceptors. The project involves assisting and learning the surgical preparation of rabbits with indwelling intracisternal catheters and acute cannulations of blood vessels for measurement of blood pressure and heart rate. Experiments will be performed to test the effect of specific antagonists which will determine the relative contribution of imidazoline or a 2-adrenoceptors. This project will require the handling of conscious animals and a degree of technical and practical skill as well as a good knowledge of computers. The environment at the Baker Institute provides an excellent level of equipment and space, a friendly environment with technical advice and training from not only myself but also from other members of the laboratory. Techniques: Blood pressure measurement, implantation of catheters, conscious animal pharmacology, computer data acquisition and analysis Title: Regulation of ribosomal gene transcription during hypertrophic and hyperplastic growthSupervisor:Dr R Hannan (Tel: 9522 4388) Email ross.hannan@baker.edu.au Location:Molecular Physiology Laboratory, Baker Institute Description: Cells need to tightly regulate their capacity to make proteins during growth and differentiation. In the long term this is controlled by altering the cellular content of ribosomes. Abnormal growth such as that associated with tumors or pathological hypertrophy of the heart is characterised by accelerated ribosomal synthesis regulated at the level of transcription of the ribosomal genes (rDNA). One factor that is thought to play a pivotal role in the regulation of ribosomal transcription is a nucleolar factor termed Upstream Binding Factor (UBF). UBF can activated rDNA transcription in vitro and interacts in vivo with a number of growth-related proteins including the tumor suppressor "pocket proteins", Rb and p130. We have recently shown that an increased expression and/or phosphorylation of UBF accompanies the mitogenic growth of 3T3 fibroblasts and the hypertrophic growth of cardiac myocytes. In addition we have demonstrated that transient overexpression of recombinant UBF stimulates growth in these systems. A number of projects are available which will further investigate the regulation of UBF activity and ribosomal gene transcription during models of growth and quiescence including: 1) determination of the DNA sequences and corresponding transcription factors that regulate UBF gene transcription during the differentiation of L6 myoblasts and during the hypertrophic growth of terminally differentiated neonatal cardiomyocytes; 2) regulation of UBF activity by cyclin dependent kinases in cultured cardiomyocytes; 3) determination of the sequence(s) sufficient and necessary to target UBF to the nucleolus using deletions constructs of UBF fused to the green fluorescent protein (GFP); and 4) identification of the signaling pathways that link G-protein coupled receptors to accelerated ribosomal gene transcription rates during the hypertrophic growth of cardiomyocytes and the mitogenic growth of 3T3 cells in culture. Techniques: recombinant DNA approaches, in vitro site directed mutagenesis, antisense expression, inducible bacterial, insect, and mammalian expression systems, introduction of recombinant proteins into cells and tissues using adenoviruses, tagging of proteins using green fluorescence protein, isolation and maintenance of primary cultures of cardiomyocytes in culture, and various immunoprecipitation and immunohistochemical techniques Title: Pressure dependence of arterial complianceSupervisor:Dr A Dart (Tel: 9276 3321) Email a.dart@alfred.org.au Location:Alfred Baker Medical Unit, Alfred Hospital Description: Arterial compliance is the capacity of vessels to expand with increase in pressure and is dependent, in part, on the intrinsic stiffness of the arterial wall. It is an important physiological parameter determining cardiac afterload and coronary perfusion. The stiffness of the arterial wall is not constant but is distension (ie pressure) dependent. Therefore, compliance itself varies with the arterial pressure. The exact relationship between these variables in man is however not known. Assessment of the pressure/compliance relationship usually requires pharmacological agents to alter pressure and is therefore difficult to interpret because of possible direct drug effects on the arterial circulation. In the current project, we plan to investigate subjects with autonomic neuropathy in whom arterial pressure can be altered without drugs by tilting. Measurements will be made of blood pressure compliance at different pressure levels. Techniques: Protocol writing/ethics submission, patient recruitment, study management, measurement of blood pressure and arterial compliance, (requiring use of external pressure sensors and determination of blood flow by Doppler ultrasound). Title: LDL cholesterol particle and coronary diseaseSupervisor:Dr A Dart (Tel: 9276 3321) Email a.dart@alfred.org.au Location:Alfred Baker Medical Unit, Alfred Hospital Description: We have previously found that patients with coronary heart disease, as a group, have a greater spread in the size of their low-density lipoprotein particles than in normal subjects. This may mean that coronary subjects have more variable LDL particles than normal. In the current study, we will measure LDL diameters at an interval of one and two weeks in subjects with and without coronary disease and compare the variability between these groups. LDL will be separated by oxygen ultracentrifugation and LDL diameters will then be determined following electrophoresis. Patients will be recruited from the Heart Centre following investigation of their coronary arteries or recruited by advertisement (normal volunteers). Techniques: Protocol writing/ethics submission, patient recruitment, study management, blood sampling, lipoprotein separation, LDL particles size measurement, data analysis Hormones and the Vasculature LaboratoryTitle: Variation of cutaneous vascular reactivity among patients with risk factors for heart diseaseSupervisors: A/Profs P. Komesaroff and K. Sudhir email: : paul.komesaroff@baker.edu.au Location: Hormones and the Vasculature Laboratory, Baker Medical Research Institute Description: Cutaneous vascular reactivity (CVR) is a new non-invasive method for measuring the health of small blood vessels in the skin. It uses a low power laser beam to detect changes in blood flow in response to tiny doses of hormones administered directly through the skin with the help of small electric currents. It has promise as a technique for predicting cardiovascular risk but its variation in relation to other known risk factors, such as high cholesterol levels, high blood pressure and family history, is unknown. This project will investigate the differences in CVR among these groups, compare them with changes in other physiological measures of blood vessel function, and explore the mechanisms underlying this variation. Techniques: Development and execution of a clinical research protocol; measurement of cutaneous vascular reactivity using laser Doppler velocimetry with low voltage iontophoresis of vasoactive substances, systemic arterial compliance and flow mediated dilatation; processing and analysis of data; opportunities for acquisition of laboratory-based techniques. Eleanor Shaw Centre for the Study of Medicine, Society and Law (Baker Institute and Department of General Practice)Title: Ethical issues in the use of complementary medicinesSupervisor: A/Prof P. Komesaroff and Prof L. Piterman email: : paul.komesaroff@baker.edu.au Location:Baker Institute and Department of Community Medicine and General Practice Description: Although nearly two thirds of Australians have used various techniques associated with complementary medicines and about half use them on a regular basis, for the most part the efficacy of these techniques and their possible side effects have not been examined. This study will explore the ethical and social issues underlying the use of complementary medicines and consider the role of government regulation to control this use. Techniques: Examination of evidence regarding the use and effects of complementary medicines; analysis of ethical arguments; qualitative and/or quantitative study of the use of complementary medicines of one or more forms. Macfarlane Burnet Centre for Medical ResearchTitle: Localisation of Vpr protein during HIV-1 infectionSupervisors: A/Prof D McPhee, Dr M Bateson Tel: 9282 2111 Location:AIDS Cellular Biology Unit, Macfarlane Burnet Centre for Medical Research, Fairfield Description: The Vpr protein is a 96 amino acid "accessory protein" of HIV-1 which has been found to be essential to productive infection. Although the function of Vpr within the life cycle of the virus remains unclear, a number of functions such as G2/M cell cycle arrest, LTR transactivation and nuclear localisation of the viral genome have been reported. The Vpr protein is expressed late in the viral cycle and packaged within the nucleocapsid of maturing virus particles via an interaction with the p6 domain of Gag. Previously in our laboratory we have constructed a Vpr protein fused to the green fluorescence protein (GFP) of the jellyfish Aequorea victoria. This enabled the visualisation of the localisation pattern of this protein when it is expressed within human cells. The project will extend this work by co-transfecting cells with both the Vpr-GFP protein and a HIV-1 virus lacking native Vpr function. It is expected that by visualising the migration of the fusion protein during infection that more can be learnt about events and interactions that are important to the function of Vpr. This approach will lead to learning more novel ways to stop HIV-1 replication. Title: Interactions between the M and M-1 proteins of the human respiratory syncytial virusSupervisors: Dr R Ghidhyal, Prof J Mills, Dr J Meanger Tel: 9282 2111 Location:Children’s Virology Research Unit, Macfarlane Burnet Centre for Medical Research Description: Respiratory syncytial virus (RSV) is the major cause of viral pneumonia in infants. It is also the cause of lower respiratory disease in the elderly and immunocompromised adults. Annual epidemics of RSV are well documented, occuring in winter in temperate climates and in the rainy season in tropical regions. These epidemics can cause substantial morbidity and mortality. There is no vaccine available for RSV and no efficacious therapy. We, at the Children’s Virology Research Unit are studying the assembly of RSV within the host cell with a view to identifying novel targets for therapy and possible vaccine candidates. RSV is an enveloped, negative strand RNS virus belonging to the family Paramyxoviridae and the genus Pneumovirus. Assembly of Paramyxovirus virions during the final stages of infection is initiated by the interaction of nucleocapsids with specific components of the viral envelope, leading to bud formation and subsequent viral release. The M (matrix) protein of paramyxoviruses facilitates assembly by associating with nucleocapsids and virion envelope glycoproteins (F and HN). Previous work in the laboratory has demonstrated a specific interaction of the RSV M protein with the envelope glycoproteins and with the nucleocapsid complex. The interaction with nucleocapsids is facilitated by a specific interaction of the M protein with the M2-1 protein of RSV. In this project, we will attempt to define the domains of the interaction between these two proteins using in vitro binding assays. This will involve extensive cloning of truncated mutants of the two proteins followed by expression and purification from bacterial cells. The purified proteins will be used in ELISA and protein overlay protein binding assays. Title: Studies in international healthSupervisor: Dr M Toole Tel: 9282 2111 Location:International Health Unit, Macfarlane Burnet Centre for Medical Research Description:
Title: Cloning, expression and interaction of the respiratory syncytial virus subgroup B G glycoprotein with M proteinsSupervisors: Dr J Meanger Dr R Ghidhyal, Prof J Mills Tel: 9282 2111 Location:Children’s Virology Research Unit, Macfarlane Burnet Centre for Medical Research Description: Respiratory syncytial virus (RSV) is the major cause of viral pneumonia in infants. It is also the cause of lower respiratory disease in the elderly and immunocompromised adults. RSV is an enveloped, negative stranded RNA virus belonging to the family Paramyxoviridae and the genus Pneumovirus. Viral assembly of paramyxoviruses during the final stages of infection is initiated by the interaction of the nucleocapsids with specific components of the viral envelope, leading to bud formation and subsequent viral release. The single M protein of paramyxoviruses facilitates assembly of virus particles by associating with virion glycoproteins (F and HN/G) on intracellular membranes followed by localisation to the plasma membrane or by forming patches of M protein-glycoprotein complexes at the plasma membrane. A similar role in viral assembly has been hypothesised for the two RSV matrix proteins, M and M2. Two antigenic subgroups (designated A and B) have been described for RSV. Recent work in our laboratory has shown that the subgroup A, strain A2, G glycoprotein interacts with the M protein, and that the interaction is localised within the N-terminus of the G glycoprotein. The sequence of the subgroup B RSV G gene is slightly different than that of the A group, but interestingly there are only two substitutions in N-terminal amino acids. It is interesting that this maintains the charge balance pretty well and thus may not be critical to the interaction. In this project we would like to confirm if these changes have any effect on M-G interaction. Studies in the epidemiology of infectious diseases:
Centre for Harm Reduction
Title: HIV-1 Viral factors associated with delayed progression to AIDSSupervisors:A/Prof N Deacon, Dr M Churchill Tel: 9282 2111 Location:AIDS Molecular Biology Unit, Macfarlane Burnet Centre for Medical Research, Fairfield A small percentage (<5%) of persons infected with HIV-1 have a significantly delayed progression to disease (AIDS). This may be due to host or viral factors, or a combination of both. Host factors include HLA haplotype, a strong immune response to HIV antigens or altered genotype of co-receptor molecule (e.g. CCR5D 32 allele of chemokine receptor). Viral factors include mutations of a number of HIV-1 genes. We have demonstrated that deletions of the HIV-1 nef gene and the LTR region (viral gene promoter) are associated with slow/non-progression in 4 independent Australian strains of HIV-1. In one of these strains further deletion of LTR sequence has led to a progression to disease. In some other cases of slow progression without antiviral therapy nef/LTR deletions have not been found, suggesting that other factors are involved. Current research is aimed at (1) investigating LTR sequence changes that are associated with the switch from non-progression to progression and the mechanism by which these changes may lead to a change in viral gene expression and (2) determining the genomic sequence of HIV-1 strains from therapy naïve slow/non-progressors to examine possible associations of sequence changes with non-progression. Title: Inhibition of phagocytosis by HIV-1 infection of human macrophagesSupervisors:Dr A Jaworowski & Prof Suzanne Crowe Email: anthonyj@burnet.edu.au / crowe@burnet.edu.au Location:AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria. Description: Our laboratory has shown that HIV infection inhibits the ability of professional phagocytes such as macrophages to ingest and destroy certain micro-organisms (particularly those responsible for HIV-related opportunistic infections in patients with AIDS). This is a major mechanism contributing to the development of opportunistic infections in AIDS patients. We have recently shown that HIV infection of human monocyte-derived macrophages drastically inhibits their ability to phagocytose both complement- and IgG-opsonised particles. A project is offered to investigate the mechanism by which HIV infection of human monocyte-derived macrophages inhibits phagocytosis. Specifically, we are interested in (a) whether HIV infection inhibits phagocytosis by elevating intracellular [cAMP] and (b) whether inhibition is at the level of target binding, or phagosome formation and maturation. The project involves a wide variety of techniques including cell culture, growing and maintenance of HIV-1 virus stocks, measurement of intracellular cAMP levels and confocal microscopy. Title: Importance of tat in HIV infection in macrophagesSupervisor:Dr Con Sonza Email: sonza@burnet.edu.au Location:AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research Description: When compared to infection in CD4+ T lymphocytes, HIV infection in macrophages does not kill the cells and they remain infected for their lifetime. In vivo, tissue macrophages are thought to be important reservoirs of virus and their infection contributes to the pathogenesis of HIV infection. We have shown that even long-term treatment with highly active antiretroviral drugs, which reduce circulating virus to undetectable levels, does not prevent blood monocytes, which home to various tissues in the body, from remaining or becoming infected. When blood monocyte-derived macrophages are infected with HIV in the laboratory, the virus replicates to reasonable levels over a 2 to 3 week period but then the levels of virus produced decline and remain low for the life of the culture. We have shown that this decline in virus production from the macrophages is coincident with a specific down-regulation of the production of the messenger RNAs which code for an important viral regulatory protein, Tat. Tats main role in replication is as a transactivator of viral transcription. Supplying the infected macrophages with an exogenous source of Tat produces a burst of virus production from them. In this project we are interested in further characterising the role of Tat in the regulation of HIV infection in macrophages. Areas to be explored include: the characterisation of Tat protein production in macrophages during long-term infection determining whether the tat gene becomes defective over the course of infection and determining the levels of a variety of cellular proteins known to interact with Tat protein or RNA and to affect its function to see which may explain the observed decline in tat mRNA. Title: The impact of HIV-1 protease mutations on the arrangement of HIV-1 RNASupervisors:Dr J Mak & Prof S Crowe Email: mak@burnet.edu.au / crowe@burnet.edu.au Location:AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria. Description: The HIV-1 encoded protease (PR) protein is an essential participant in the assembly process of HIV-1. The recent successful use of PR inhibitors (PIs) as part of highly active antiretroviral therapy (HAART) in patients with HIV infection highlights both the importance of viral assembly in the HIV-1 replication cycle and its potential as an anti-retroviral target for drug development. The emerging clinical evidence of drug failure in HAART suggests that development of safe, novel, and effective antiretroviral agents, such as those which target HIV-1 assembly, is highly desirable. The focus of this proposal is to closely examine the molecular biology of how the HIV protease enzyme is involved in HIV-1 assembly, in particular the formation of virion RNA dimers, and to identify important events in HIV-1 assembly for the development of new drugs. The clinical significance of HIV-1 virion RNA dimerization will be examined using patient isolates of HIV-1 that are resistant to PIs. We predict that mutations conferring HIV-1 PI resistance will inhibit the formation of stable virion RNA dimers, and that multiple or increasing numbers of PI resistance mutations will further destabilise the virion RNA dimers. Title: Functional and structural requirement of sphingomyelin in the assembly of HIV-1Supervisors:Dr J Mak & Prof S Crowe Email: mak@burnet.edu.au / crowe@burnet.edu.au Location:AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria. Description: The envelopes of retroviruses are derived from the plasma membrane of the virion-producing cell. Analysis of HIV-1 and other retroviruses has shown that an unusually high cholesterol:phospholipid ratio is found in the retroviral envelope in comparison of the host cell membrane. Cholesterol and sphingomyelin are two lipids that are enriched in the HIV-1 envelope, and they represent approximately 15% and 4% of the total mass to HIV-1, respectively. We have evidence that removal of cholesterol from HIV-1 envelope alters the virion structure and inhibits virion infectivity, but the contribution of sphingomyelin in HIV-1 replication is less well understood. The focus of this proposal is to examine the structural and functional importance of sphingomyelin in HIV-1, and to identify important events in HIV-1 assembly for the development of new antiretroviral drugs. Various forms of fractionation procedures will be used in combination with different lipases to delineate the roles of sphingomyelin in the HIV-1 replication cycle and to identify new targets for anti-HIV-1 therapy. Title: A new role of primer binding site (PBS) in HIV-1 replicationSupervisors:Dr J Mak & Prof S Crowe Email: mak@burnet.edu.au / crowe@burnet.edu.au Location:AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria. Description: The primer binding site (PBS) of HIV-1 is found near the 5’end of the HIV-1 RNA genome, and it is essential for the annealing of tRNALys3 onto the RNA genome to initiate HIV-1 replication via reverse transcription. The HIV-1 PBS is part of the 5’ untranslated region (5’UTR) which has been shown to be important in multiple parts of HIV-1 replication, eg stability of RNA genome, mRNA transcription, mRNA splicing, protein translation, genomic RNA dimerization and cDNA synthesis of genomic RNA via reverse transcription. The 5’UTR has extensive RNA secondary structures, and it is probable that PBS is involved in other aspects of the HIV-1 replication cycle. The focus of this project is to explore the potential importance of PBS mRNA stability, transcription, and protein expression. We have constructed a series of 12 HIV-1 PBS deletion mutants, which consist of various deletions within PBS. Preliminary data have shown that a PBS 25 deletion expresses wild type level of HIV-1 protein whereas a PBS 13 deletion does not express HIV-1 protein with similar efficiency. These preliminary data suggest for the first time that the HIV-1 PBS sequence has a role in the regulation of viral protein production, and the precise contribution of the PBS to HIV-1 viral protein production will be initially mapped using the existing mutants. The precise RNA secondary structure and RNA sequences that are required for viral protein production will also be delineated via site-directed mutagenesis. Title: Role of disulphide interactions in hepadnaviral envelope stability and entry into the host cellSupervisors: Dr E Grgacic (Tel. 9282 2109) and Dr D Anderson Email: grgacic@burnet.edu.au Location:Hepatitis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield Description: Hepatitis B virus (HBV) is responsible for most hepatocellular carcinomas worldwide. Despite increasing use of an effective vaccine, there remain 350 million carriers who will continue to progress to severe disease for the next 30-40 years. There is thus a need to develop effective antiviral drugs, for which a detailed understanding of viral replication is essential. A major obstacle to HBV research is the lack of a cell culture infection system for human HBV, precluding studies of the early events of the virus life cycle. The best system to study these events are animal models of the related members of the family, hepadnaviridae, of which the duck hepatitis B virus (DHBV) model has been a major source of our understanding of hepadnaviral replication. We have been using this model to study the two viral envelope proteins, their topology, interactions and modifications and their role in the entry process. The small (S) and the large(L) envelope proteins are multi-spanning transmembrane proteins, synthesised from the same open reading frame. Both proteins contain three highly conserved cysteine residues, at least two of which are involved in intramolecular disulphide bonding which may serve to stabilise the entire envelope. We also have evidence that reduction of these disulphide bonds may play a role in conformational changes in envelope proteins associated with viral fusion. In this project, we will determine the role of these disulphide bonds in stabilising the virus envelope by examining the temperature sensitivity of native and reduced viral particles, and we will examine the role of the cysteine residues in promoting correct protein folding and S:L interactions in the membrane during virus assembly and disassembly/fusion. Title: Interactions of hepatitis B virus with polarized hepatocytesSupervisors: Dr D Anderson (Tel: 9282 2239) and Dr E Grgacic Email: anderson@burnet.edu.au Location:Hepatitis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield Description: In order to interact with both external (lumenal) and internal compartments of the body, epithelial cells are highly polarized, with discrete protein and lipid compositions in the surfaces facing the lumen (apical domain, AP) and the serosa and neighbouring cells (basolateral domain, BL). This polarity is established through the formation of tight junctions (TJs) at the points of contact between cells, and the network of TJs between neighbouring cells provides an effective barrier to simple diffusion of small molecules and viruses. Polarized cells can provide a barrier to viral entry by diffusion, yet the respiratory, genital and intestinal mucosa are also the primary sites of infection for many viruses. In such cases cell polarity can modulate the pathogenesis of viruses, many viruses being released in a vectorial fashion, predominantly to either the AP or BL domain. This has been extensively studied for many viruses such as influenza A and poliovirus. In addition, it is now clear that some viruses, such as the human immunodeficiency virus (HIV), can penetrate the epithelial barrier by the process of "transcytosis" (trans-cellular transport), gaining access to internal sites of the body without infecting the epithelial cells. Nothing is known of the interactions between the viruses which infect the liver (hepatitis A, B, C, and E viruses) and the polarized hepatocytes of the liver. Such studies have not been possible in the past because polarized hepatocytes have a complex cellular architecture which prevents experimental access to the separate AP and BL domains of the cell. We have recently developed a subline of liver-derived HepG2 cells which exhibit cell polarity but with the simple cellular architecture more typical of kidney, intestinal and respiratory epithelia which have been used to study virus/polarized cell interactions in the past. In this project, interactions between the human and duck hepatitis B viruses (HBV and DHBV) and polarized HepG2 cells will be studied, focussing initially on three main questions. (1) Is the expression of viral receptors and virus binding restricted to either AP or BL domains? (2) Is the export of virus from infected cells similarly (or differently) restricted? (3) Is such export affected by the presence or absence of virus receptors and the morphological state of the viral particle? This study will involve cell culture, virus growth, various molecular biology techniques and immunofluorescence and confocal laser scanning microscopy. These studies will have direct implications for understanding the pathogenesis of hepatotropic viruses. Title: Drug resistance in hepatitis BSupervisor: Dr D Anderson (Tel: 9282 2239) Email: anderson@burnet.edu.au Location:Hepatitis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield Description: Hepatitis B virus (HBV) is responsible for most hepatocellular carcinomas worldwide. Despite increasing use of an effective vaccine, there remain 350 million carriers who will continue to progress to severe disease for the next 30-40 years. Chronic hepatitis B virus (HBV) infection is increasingly being treated with the use of antiviral drugs (nucleoside analogues), but drug-resistant viruses are rapidly emerging with subsequent treatment failures. Improved therapies, including new targets with less cross-resistance, are required. The mutations in HBV associated with resistance to different drugs and the pattern of cross-resistance between drugs has been largely evaluated, but it is not known how these drug-resistant viruses spread through the liver. Because almost every cell in the target organ is infected with HBV, and HBV infection of cells normally prevents "superinfection" by another (eg drug-resistant) HBV, the question arises of how the drug-resistant virus becomes "fixed" in the patient, with important implications for treatment. The closely related virus of domestic ducks, DHBV, has a replication cycle, structure, genome, and profile of drug sensitivity and resistance almost identical to that of human HBV. Using infectious cDNA clones of DHBV, drug-resistant viruses will be constructed in which an antigenic epitope "tag" will allow us to detect the spread of drug-resistant virus through the liver in the presence or absence of ongoing wild-type virus infection, and the presence or absence of antiviral therapy (applying selective pressure in favour of the tagged virus). These studies will directly contribute to an improved understanding of drug resistance and ultimately to improved treatment strategies for chronic hepatitis B. Title: Antigenic structure of hepatitis E virusSupervisor: Dr D Anderson (Tel: 9282 2239) Email: anderson@burnet.edu.au Location:Hepatitis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield Description: The enterically transmitted hepatitis E virus (HEV) causes acute and often severe hepatitis, with a mortality of around 30% during pregnancy. HEV is the most common cause of acute hepatitis in many developing countries, accounting for as much as 70% of hepatitis in Nepal, India and similar countries. Infection is rare in developed countries, although there is evidence that zoonotic (eg pig) strains cause some disease. We have developed improved serological assays for diagnosis of HEV infection and an experimental subunit vaccine which is currently being trialled in animals. These advances have been based on the unique structure of one antigen derived from the virus, known as ORF2.1. Monoclonal antibodies (MAbs) to ORF2.1 have allowed us to dissect its antigenic structure in some detail, showing that it is highly conformational and immunodominant. In this project, we will use molecular biological methods to gain a more detailed understanding of the ORF2.1 antigenic epitope. Sequence differences between animal and human strains of HEV will be reconstructed in the ORF2.1 clones by site-directed mutagenesis, and the reactivity of these mutant proteins with MAbs, patient and animal sera will be determined. Domains of the protein which have been implicated in protein dimerisation will also be targetted to determine the role of dimerisation in formation of the epitope. Finally, panning of phage display peptide libraries with MAbs will be used to isolate "mimotopes" which mimic the antigenic structure of this immunodominant epitope. These studies will contribute to further improvements in the diagnosis and control of HEV infection. |