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Department of Pharmacology, Honours 2015

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Department of Pharmacology

Honours Projects 2015

www.monash.edu



Welcome to the Pharmacology Department! The Honours year represents a new adventure, very different to your undergraduate experience, in which you will have the opportunity to undertake a research project, communicate your science to colleagues and peers and learn to critically evaluate scientif ic concepts and literature. Your supervisor(s) will be there to guide and advise you along this research journey. At the very least, you are expected to bring with you the following skills set, in no particular order: -enthusiasm -an enquiring mind -respect & humility -determination & persistence -a sense of humour -a collegial spirit -patience This booklet provides information about the research projects on offer in the Department of Pharmacology and we encourage you to identify the areas of research in which you are most interested, contact potential supervisors and discuss the projects with them. The course convenors, A/Prof Grant Drummond and Dr Barb Kemp-Harper, can advise on projects, guide you through the application process and help with any queries you may have. We look forward to welcoming you the Department of Pharmacology in 2015 and wish you all the best for a rewarding and exciting year of research. Good luck!

Professor Robert Widdop Head, Department of Pharmacology



Department of Pharmacology Honours Convenors

Dr Barb Kemp-Harper A/Prof Grant Drummond

Email: [email protected] Email: [email protected]

Phone: 9905 4674 Phone: 9905 4869

Pharmacology Honours: Pre-requisites

BSc Biomedicine BBiotech BMS

Pre-requisites A Distinction average (>70) in 24 points at 3

rd

year in relevant disciplines within the School of Biomedical Sciences*

A Distinction average (>70) in 24 points at 3

rd

year level, including BTH3012

A Distinction average (>70) in 24 points at 3

rd

year level, including 12 points in 3

rd year core

BMS units (BMS3021, BMS3042) and 12 points in other 3

rd year

units*

Application closing date

14th

November 2014 14th

November 2014 14th

November 2014

Application form http://monash.edu/science/current/honours/how-to-apply/

http://monash.edu/science/current/honours/how-to-apply/

www.med.monash.edu/biomed/honours/

Commencement date

23rd

February, 2015 23rd

February, 2015 23rd

February, 2015

* There is no pre-requisite in terms of 3

rd year PHA units, but the Pharmacology Honours Convenors

will need to be satisfied that you have the necessary background in pharmacology to undertake your

chosen research project.

mailto:[email protected]




Pharmacology Honours Course

The Pharmacology BSc Biomedicine Honours Course comprises 2 units:

BMH4100 (36 points) – Research Unit

BMH4200 (12 points) – Coursework Unit

BMH4100 This major focus of this unit is the research project you will conduct under the guidance of

your supervisor. The assessment tasks include:

Literature Review

Research Seminars (introductory & final)

Thesis & its defence

BMH4200 This unit provides you with the necessary skills to critically review and evaluate the scientific

literature and effectively communicate concepts related to the discipline of pharmacology

and your research area both in writing and orally. The assessment tasks include:

Journal club presentation & participation

Assessment of data exam

Written critique of scientific paper exam

Lay poster presentation

Bachelor of Biomedical Science (BMS) Honours students undertake

BMS4100 (identical to BMH4100)

BMS4200 (similar to BMH4200 but administered through the School of Biomedical

Sciences)



Choosing an Honours Project

The research projects on offer in the Department of Pharmacology and off-campus, with our

collaborators, are outlined in the following pages. Once you have identified a few projects

that you find interesting, then contact the potential supervisors by email or phone and

arrange to meet with them to find out more about the projects on offer. It’s a great idea to

visit the research labs and meet other members of the research group in order to get a ‘feel’

for the people you would be working with and type of research you would be undertaking.

Please note that the availability of a supervisor to sign you on for a project will depend on

that project still being available and the limit as to how many students a supervisor can take

on. At least one of your supervisors must be a member of staff or an adjunct member of

staff of the Department of Pharmacology.

How do I apply?

Once you, together with your potential supervisor, have identified a project that would be

suitable for your Honours research program, then you will need to complete the application

form from the specific faculty you are from (i.e. Faculty of Science Website for BSc students

and Faculty of Medicine Website for BMS students). These forms must be signed by one of

the Honours Convenors of the Pharmacology Department (Dr Barb Kemp-Harper or A/Prof

Grant Drummond).

Students enrolling through the Faculty of Science must go to the Faculty of Science office for

an eligibility check to ensure that they are/will be eligible to commence the relevant Honours

program in Semester 1, 2015. Students must obtain this eligibility check before they go to the

Honours coordinator to get their form signed.

Application forms must be submitted by Friday 14th November to:

Science Faculty Office (BSc, BBiotech students)

Submit application form on-line via E-Admissions (BMS students)

All applications will be reviewed and students who meet the eligibility criteria will be informed

of their success in obtaining an Honours place by letter, which will be sent out in mid-late

December 2014. Students must then notify the Faculty and supervisor of their intention to

accept or reject the place. Students will be able to enrol into the Honours course via WES in

January 2015.



Honour Projects 2015 – On campus projects

LABS / SUPERVISOR(S)

PROJECT TITLE

Education-based projects Liz Davis, Eva Patak

Evaluation & development of web-based pharmacology resources

Fibrosis Group Chrishan Samuel, Simon Royce

Investigating novel anti-fibrotic therapies Integrative Cardiovascular Pharmacology Group Tracey Gaspari, Rob Widdop Emma Jones, Mark Del Borgo, Rob Widdop

Exploring novel therapeutic strategies to prevent cardiac remodelling

Drug discovery program for AT2 receptor ligands

Cardiovascular Immunobiology Group Antony Vinh

Mechanisms of inflammation and immunity during the development of hypertension

Neuropharmacology Group Richard Loiacono, Siew Yeen Chai (Physiology)

Modulation of neuronal oxytocin – effects on social behaviour

Vascular Biology & Immunopharmacology Group Brad Broughton, Chris Sobey Grant Drummond Barb Kemp-Harper, Grant Drummond Chris Sobey, Helena Kim Sophocles Chrissobolis, Chris Sobey

Identifying the mechanisms underlying G protein-coupled estrogen receptor-mediated neuroprotection post-stroke

Targeting the immune system in the treatment of high blood pressure

Selective targeting of monocyte chemoattractant proteins to treat atherosclerosis

Inflammatory mechanisms in ischemic stroke

Investigating cardiovascular abnormalities during aldosterone-dependent hypertension

Oxidant and Inflammation Biology Group Stavros Selemidis

Roles of endosomal NADPH oxidases and TLRs in Influenza innate immunity

Respiratory Pharmacology Group Jane Bourke, Simon Royce, Chrishan Samuel Jane Bourke, Simon Royce, Rebecca Ritchie Jane Bourke, Tim Moss, Megan Wallace

Evaluating the antifibrotic and bronchodilator potential of relaxin

Overcoming steroid resistance – evaluation of the effects of annexin-A1 mimetics on airway inflammation

Early life influences on airway and vascular reactivity

Venoms & Toxins Group Sanjaya Kruppu, Wayne Hodgson Oded Kleifeld, Sanjaya Kuruppu, Wayne Hodgson

Muscle damage induced by African Puff Adder Venom

Development of proteomic system-wise approach for characterization of snake venoms



Honour Projects 2015 – Off campus projects

LABS / SUPERVISOR(S)

PROJECT TITLE

Australian Centre for Blood Diseases Justin Hamilton, Grant Drummond

Targeting the human platelet thrombin receptor, PAR4, as a novel anti-thrombotic therapy

Developing isoform-specific PI3K inhibitors as novel anti-platelet agents

Baker IDI Heart & Diabetes Institute Rebecca Ritchie, Barb Kemp-Harper Rebecca Ritchie, Grant Drummond Jennifer Rivera, Yung Chih Chen, Karlheinz Peter Bock Lim, Karlheinz Peter

Therapies to circumvent impaired nitric oxide function for managing the cardiovascular complications of diabetes

Therapeutic targeting of cardiac hexosamine biosynthesis – ROS interactions to protect the diabetic heart

Targeting cardiac inflammation as a novel therapeutic approach for the cardiac complications of diabetes

Exploring novel therapeutic strategies to prevent atherosclerotic plaque rupture

Targeting activated platelets as early marker for diagnosis and treatment of inflammatory diseases

Drug Discovery Biology Theme Monash Institute of Pharmaceutical Sciences Katie Leach, Arthur Christopoulos Karen Gregory, Arthur Christopoulos Sebastian Furness, Patrick Sexton Michelle Halls Dana Hutchinson, Bronwyn Evans Celine Valant, Arthur Christopoulos Erica Sloan, Scott Mueller David Thal, Arthur Christopoulos

Elva Zhao, Nigel Bunnett

Targeting the calcium sensing receptor in osteoporosis and hyerparathyroidism

Allosteric modulation of metabotropic glutamate receptors: novel treatment strategies for CNS disorders

The molecular basis of G protein-coupled receptor function: chemical biology approaches

Resolving the spatiotemporal signalling of G protein-coupled receptors

Pharmacological therapies to burn fat

Investigation of a ‘locked’ active conformation of a G protein-coupled receptor

Stress is making me sick! Neural regulation of viral immunity

Allostery and ligand bias at muscarinic receptors: structural insights towards understanding CNS disorders

Understanding pain from inside out



EVALUATION & DEVELOPMENT OF WEB-BASED PHARMACOLOGY RESOURCES

Supervisors: Dr Elizabeth Davis & Dr Eva Patak Location: Pharmacology Education Research Initative Department of Pharmacology

Monash University, Clayton

Background:

Our group aims to gain a better understanding of factors that influence the engagement of students with their learning of pharmacology and the development of resources and curricula to support students in their learning, particularly within the science and medical courses.

Project aim:

This group is currently involved in several ongoing projects and prospective students could become involved in aspects of these. Projects involve the development and evaluation of web-based learning resources and activities to encourage active learning. In addition, we want to assess the use of communication tools (including social media) to encourage interaction and learning. Techniques:

Techniques used in education-based projects include development of teaching material, monitoring of use and student evaluation through surveys and focus groups.

Contacts: Dr Elizabeth Davis Department of Pharmacology Monash University Phone: 9905 5755, Rm E123 [email protected]

Dr Eva Patak Department of Pharmacology Monash University Phone: 9905 5783, Rm E117 [email protected]




INVESTIGATING NOVEL ANTI-FIBROTIC THERAPIES Supervisors: A/Prof Chrishan Samuel & Dr Simon Royce Location: Fibrosis Laboratory (Rms E101/E102) Department of Pharmacology


Background:

Fibrosis is defined as the hardening and/or scarring of various organs including the heart, kidney and lung; which usually arises from abnormal wound healing to tissue injury, resulting in an excessive deposition of extracellular matrix components, primarily collagen. The eventual replacement of normal tissue with scar tissue leads to organ stiffness and ultimately, organ failure. Despite a number of available treatments for patients with various heart/kidney/lung diseases, patients receiving these therapies still progress to end-stage organ failure due to the inability of these treatments to directly target the build-up of fibrosis. Hence, novel and more direct anti-fibrotic therapies are still required to be established.

Aims:

The Fibrosis Lab aims to identify novel anti-fibrotic therapies (relaxin, stem cells, trefoil factor 2; and combinations of these) that will more effectively prevent/reverse fibrosis progression. Additionally, by understanding the mechanisms of action of these potential therapies of future, we aim to delineate new targets that can be utilized to enhance their therapeutic potential and abrogation of organ scarring. Projects: 1. Signal transduction studies (in models of heart / kidney / lung disease) 2. Head-to-head and combination therapy efficacy studies 3. The influence of ageing and gender on fibrosis 4. Development of new approaches to target airway remodeling in asthma Techniques:

Depending on the project involved, animal/cell culture models of (heart/kidney/lung disease), blood pressure and functional measurements, matrix biology, protein biochemistry, molecular biology and/or histological techniques will be utilized.

Contacts: A/Prof Chrishan Samuel and Dr Simon Royce Department of Pharmacology Monash University Phone: 9902 0152 / 9905 0913 [email protected] [email protected]



EXPLORING NOVEL THERAPEUTIC STRATEGIES TO PREVENT CARDIAC REMODELLING

Supervisors: Dr Tracey Gaspari & Professor Robert Widdop Location: Cardiovascular Integrative Pharmacology Group Department of Pharmacology


Background:

Cardiovascular diseases (CVD) are the leading causes of death in more affluent societies, killing more than 7 million people worldwide each year. Development of cardioprotective agents to improve myocardial function, to decrease extracellular matrix (ECM) deposition and to limit the total extent of infarction during ischaemic heart disease is of great clinical importance especially with our ageing population. The renin angiotensin system (RAS) is well known to participate in the pathogenesis of cardiovascular diseases with overactivity of Ang II instrumental via its effects mediated by the AT1R. We now have evidence that indicates both shorter angiotensin peptides (Ang (1-7) and Ang IV) and activation of non-AT1Rs (AT2R, MasR

and AT4R) can oppose the AT1R-mediated pathophysiological effects seen in cardiovascular remodelling with these results collectively highlighting the potential of targeting the non-AT1R axis in treatment of CVD, particularly for difficult-to-treat end-organ damage.

Project aim:

The aim of this honours project is to investigate the cardio-protective effect of targeting the AT4R using novel ligands. This study will elucidate the underlying mechanisms involved and may lead to the development of more effective therapies for the treatment of cardiovascular disease. Techniques: It is anticipated that this project will involve a range of methodologies including: animal dietary and pharmacological treatments, surgical techniques for implantation of osmotic mini pumps, immunohistochemistry, biochemical measures (western blot, markers of cellular proliferation and hypertrophy), histological and morphological analyses (such as fibrosis, intimal to medial measurements) and cell culture experiments.

Contacts: Dr Tracey Gaspari and Prof Robert Widdop Department of Pharmacology Monash University Phone: 9905 4762, Rm E119 [email protected] [email protected]



DRUG DISCOVERY PROGRAM FOR AT2 RECEPTOR LIGANDS

Supervisors: Dr Emma Jones1, Dr Mark Del Borgo2 &

Prof Robert Widdop1 Location: Integrated Cardiovascular Pharmacology Laboratory

Departments of Pharmacology1 and Biochemistry2 Monash University, Clayton

Background:

The main effector hormone of the renin angiotensin system (RAS) is angiotensin II which can stimulate both angiotensin AT1 receptors (AT1R) and AT2 receptors (AT2R). There is evidence to suggest that there is cross talk between AT1R and AT2R at both functional and signaling levels. For example, AT1R activation is responsible for the classical effects of Ang II such as vasoconstriction, whereas AT2R activation opposes this effect by direct AT2R-mediated vasorelaxation or inhibition of AT1R-mediated signaling. There is currently intense interest focusing on the AT2R cardiovascular function, although there are few selective AT2R ligands available to delineate such effects.

We have a drug discovery program replacing natural amino acids in the Ang II molecule with synthetic amino acid derivatives that may act as lead compounds. Our preliminary data have identified a series of novel angiotensin peptide analogues that exhibit AT2R selectivity, evidenced from both in vitro and in vivo vasodilator activity.

Project aim: Therefore, the current project will continue this work and will involve biochemical and in vitro and in vivo cardiovascular experiments identifying lead compounds with AT2R agonist or antagonist activity. Techniques: This project will involve animal work including:

Surgical procedures

In vitro studies of vascular reactivity

In vivo measurement of blood pressure

Cell culture

Signaling and binding assays Contacts: Dr Emma Jones & and Prof Robert Widdop Department of Pharmacology Monash University Emma.Jones @monash.edu [email protected]




MECHANISMS OF INFLAMMATION AND IMMUNITY DURING THE DEVELOPMENT OF HYPERTENSION

Supervisors: Dr Antony Vinh & Prof Robert Widdop Location: Cardiovascular Immunobiology Group

Department of Pharmacology Monash University, Clayton

Background:

Inflammation and immunity continue to be a topical area of cardiovascular research. T cells and the adaptive immune system are known to play a vital role in the development of hypertension. It is well established that T cells and other innate immune cells such as macrophages are recruited by chemoattractants known as chemokines, to sites of inflammation including blood vessels during hypertension. Another vital system that contributes to the pathogenesis of both hypertension and immunity is the renin angiotensin system (RAS). We have previously shown that the vasodepressor arm of the RAS, which is largely mediated via the AT2-receptor, is known to exert anti-inflammatory effects on T cell function. However, due to its low expression, targeting the AT2-receptor remains challenging. During hypertension, endogenous chemoattractants recruit T cells that cause damage to blood vessels. If we could reprogram T cells to exert an anti-inflammatory phenotype (overexpress AT2R?), and then adoptively transfer them into hypertensive animals, we could potentially harness the endogenous chemokines to recruit our modified T cells to suppress the inflammation associated with hypertension.

Project aim:

This project aims to examine the effect of adenoviral overexpression of the AT2-receptor in primary mouse T cells, and validate reprogramming of these T cells as a potential cell therapy to suppress vascular inflammation. Techniques: Several techniques will be used in this project including harvesting T cells, primary cell culture techniques, adenoviral transduction to overexpress the AT2R, flow cytometry and cytometric bead arrays to measure T cell proliferation and cytokine production. Based on outcomes and if time permits, we will perform adoptive transfer of reprogrammed T cells into

hypertensive mice to observe whether AT2R-overexpressing T cells are recruited to sites of inflammation and validate their potential as a cell therapy to treat hypertension.

Contacts: Dr Antony Vinh & Prof Robert Widdop Department of Pharmacology Monash University Phone: 9902 4844, Rm E33 [email protected] [email protected]





MODULATION OF NEURONAL OXYTOCIN – EFFECTS ON SOCIAL BEHAVIOUR

Supervisors: Dr Richard Loiacono1 & Dr Siew Yeen Chai2 Location: Neuropharmacology Lab & Neurophysiology Team Departments of Pharmacology1 & Physiology2


Background:

Metallo-peptidases cleave amino acids from either the N- and C-termini of peptide hormones to either generate or degrade biologically active peptides. These enzymes play important roles in the body and alterations in their activities can impact on a diverse range of physiological processes in both healthy and diseased states. This project is focussed on one such enzyme known as insulin-regulated aminopeptidase (IRAP) or oxytocinase. Oxytocin is known as the social hormone, regulating complex social behaviours including promoting trust, pair-bonding and has been explored as potential therapy for social behaviour impairments observed with autism spectrum disorders. Aim:

This project will investigate the behavioural phenotypes in the oxytocinase deficient mice to determine if regulation of endogenous oxytocin levels will affect social behaviour. Techniques:

Behavioural testing, cell culture, immunohistochemistry

Contacts: Dr Richard Loiacono1 & Dr Siew Yeen Chai2 Departments of Pharmacology1 & Physiology2 Monash University Phone: Richard 9905 4859 Siew Yeen 9905 2515 [email protected] [email protected]




IDENTIFYING THE MECHANISMS UNDERLYING G PROTEIN-COUPLED ESTROGEN RECEPTOR-MEDIATED

NEUROPROTECTION POST-STROKE

Supervisors: Dr Brad Broughton & A/Prof Chris Sobey Location: Vascular Biology & Immunopharmacology Group


Background:

Stroke is a debilitating disease that can cause permanent neurological damage, complications, and death. At present, there are very few treatment options available for patients, thus the development of new therapies is vital to reduce the damage caused by stroke. Interestingly, recent work from our lab has shown that activation of a novel estrogen receptor (G Protein-coupled Estrogen Receptor, GPER), powerfully limits the degree of brain

injury and functional impairment when administered to females at the time of an ischemic stroke. However, further research is required to identify the mechanisms and signalling pathways underlying GPER-mediated neuroprotection following stroke Project aim:

The aim of this project is to determine if neurons may be the cellular source of GPER activity affecting stroke outcome. The outcomes of the proposed project are expected to

confirm that GPER agonist treatment has a direct protective effect on neuronal survival following stroke. Techniques: This project will use an in vitro oxygen-glucose-deprived neuronal model of stroke to

examine the direct effects of GPER agonists on neuronal survival. Histochemical approaches will be used to assess cell viability, and Western blotting will determine GPER expression and ERK phosphorylation levels. Furthermore, an in vivo mouse model of stroke

will be treated with GPER ligands and histochemical approaches will be used to measure cerebral infarct damage. Contacts: Dr Brad Broughton & A/Prof Chris Sobey Department of Pharmacology Monash University Phone: 9905 0915, Rm E140 [email protected] [email protected]

Vehicle GPER

Agonist

Vehicle GPER

Agonist



TARGETING THE IMMUNE SYSTEM FOR THE TREATMENT OF HIGH BLOOD PRESSURE

Supervisors: A/Prof Grant Drummond Location: Vascular Biology & Immunopharmacology Group


Background: Hypertension (high blood pressure) is a major cause of heart failure, heart attacks and strokes. Over 2 million people in Australia suffer from hypertension and alarmingly, in up to 30% of these individuals, the condition is not controlled by current blood pressure medications. A recent development in the field of hypertension research is the recognition that the disease is caused by an influx of immune cells (i.e. T cells, B cells and macrophages) into the artery wall. Activation of these immune cells leads to the release of free radicals and cytokines that promote an inflammatory response, causing the arteries to become ‘stiff’ and constricted and exacerbating elevations in blood pressure. Hence, strategies that reduce inflammation in the vascular wall hold promise as future treatments for hypertension. Project aims: The aim of this project is to examine whether antibodies directed against B-lymphocytes prevent hypertension in mice by exerting anti-inflammatory effects in the artery wall and in other organs that regulate blood pressure, such as the kidney and brain. Techniques:

This project will involve surgeries on mice to induce hypertension, in vivo blood pressure monitoring, and the use of assays to measure immune cell numbers (flow cytometry, immunohistochemistry), and expression of inflammatory mediators (real-time PCR, ELISA, Western blotting) and free radicals (luminescence) in blood and tissues.

Contact: A/Prof Grant Drummond Department of Pharmacology Monash University Phone: 9905 4869, Rm E112 [email protected]



SELECTIVE TARGETING OF MONOCYTE CHEMOATTRACTANT PROTEINS TO TREAT ATHEROSCLEROSIS

Supervisors: Dr Barbara Kemp-Harper & A/Prof Grant Drummond Location: Vascular Biology & Immunopharmacology Group


Background: Atherosclerosis is the leading cause of death in the western world. Atherosclerotic plaque rupture can lead to thrombus formation, vessel occlusion and myocardial infarction or stroke. Current therapies include statins and anti-hypertensive agents and, whilst such treatments are efficacious, they only reduce the incidence of cardiovascular events by ~30%. As such, novel therapeutic strategies are sought. An important pathological feature of atherosclerosis is macrophage-mediated inflammation in the arterial wall. Macrophages are recruited to the vascular wall by chemokines; small chemoattractant cytokines that are released from sites of injury and infection to direct leukocyte trafficking. Once in the vessel wall, macrophages can become polarised towards either a pro-inflammatory (M1) phenotype or an anti-inflammatory and reparative (M2) phenotype. The balance between M1 and M2 macrophages is thought to govern plaque development and stability with a high M1 to M2 ratio favouring the formation of large, unstable plaques.

We have exciting new data to suggest that the CCR2 family chemokines, Monocyte Chemoattractant Protein-1 (MCP-1) and MCP-3 while similarly promoting macrophage trafficking, differentially affect macrophage polarisation. Whilst MCP-1 upregulates the expression of several M1 markers on human macrophages, MCP-3 by contrast has minimal effect on macrophage phenotype. As such selective inhibition of MCP-1 should alter the balance of M1 to M2 macrophages in favour of the latter thereby reducing vascular inflammation and improving plaque stability.

Project aim:

The aim of this honours project is to compare the effect of selective MCP chemokine inhibition on macrophage polarisation and vascular inflammation. This study may lead to the development of more effective therapies for the treatment of atherosclerosis. Techniques: The project will utilize atherosclerotic ApoE-/- mice and human primary macrophages and will involve the use of assays to detect inflammation (RT-PCR, western blotting, immunohistochemistry), reactive oxygen species generation (chemiluminescence) and vascular dysfunction (small vessel myography).

Contacts: Dr Barbara Kemp-Harper and A/Prof Grant Drummond Department of Pharmacology Monash University Phone: 9905 4674, Rm E140 [email protected] [email protected]



INFLAMMATORY MECHANISMS IN ISCHEMIC STROKE

Supervisors: A/Prof Chris Sobey & Dr Helena Kim Location: Vascular Biology & Immunopharmacology Group


Background: Stroke is the second leading cause of death and is due an interruption of brain blood flow by a blockage (ischemia) or a rupture (haemorrhage) in an artery. Cerebral ischemia leads to up-regulation of pro-inflammatory molecules. Inflammatory mechanisms, associated with the infiltration of numerous types of immune cells, are now a major focus of stroke research because they are suspected to contribute substantially to secondary brain damage. We have found that mice that naturally have a less inflammatory immunity show milder functional impairment and brain inflammation when compared to mice that naturally have a strong inflammatory immune system. This may help explain why pre-stroke infection can lead to a worse stroke outcome in some individuals.

Project 1 aim: Investigate whether acute administration of anti-inflammatory cytokines can

induce a shift towards an anti-inflammatory environment and their role in macrophage polarisation. This study will elucidate the underlying mechanism involved in the acute treatment with key interleukins following stroke. Project 2 aim: Investigate whether treatment with interleukins contributes to post-stroke

infection and thus affect stroke outcome. This study will identify the potential complication involved with shifting towards an anti-inflammatory environment and develop the concept of combination therapy in stroke therapies. Techniques: These projects will measure behavioural deficits, brain infarct size, real-time PCR, immune cell infiltration and inflammatory markers in mice following stroke.

Contacts: A/Prof Chris Sobey and Dr Helena Kim Department of Pharmacology Monash University Phone: 9905 4189, Rm E148 [email protected] [email protected]




INVESTIGATING CARDIOVASCULAR ABNORMALITIES DURING ALDOSTERONE-DEPENDENT HYPERTENSION

Supervisors: Dr Sophocles Chrissobolis & A/Prof Chris Sobey Location: Vascular Biology & Immunopharmacology Group Department of Pharmacology


Background: Aldosterone, synthesized from cholesterol in the adrenal cortex, acts on the

kidney to promote sodium reabsorption, water retention and potassium excretion, thus modulating electrolyte and fluid homeostasis and blood pressure. However, elevated aldosterone levels are an independent cardiovascular risk factor, and up to 20% of hypertension cases are the result of elevated aldosterone levels. The role(s) of aldosterone in promoting vascular dysfunction and inflammation in organs important in hypertension (such as the kidney and brain) is not well characterized.

Project aim: The aim of this honours project is to investigate the pathophysiological actions of aldosterone in the vasculature, kidneys and brain during hypertension. This may reveal potential targets to minimize damage in these organs that occurs during aldosterone-dependent hypertension. Techniques:

It is anticipated that this project will involve surgeries on mice to administer aldosterone, measurement of blood pressure in conscious mice (tail cuff), and the use of assays to measure blood vessel function (myography), inflammatory markers (RT-PCR), and immune cell numbers (flow cytometry).

Contacts: Dr Sophocles Chrissobolis and A/Prof Chris Sobey Department of Pharmacology Monash University Phone: 9905 0914, Rm E139A [email protected] [email protected]



ROLES OF ENDOSOMAL NADPH OXIDASES AND TLRs IN INFLUENZA INNATE IMMUNITY

Supervisor: Dr Stavros Selemidis Location: Oxidant and Inflammation Biology Group, Department of

Pharmacology, Monash University, Clayton. Background:

Influenza A viruses are environmental pathogens which have evolved to effectively gain entry into mammalian cells and, if left unchecked, to replicate by using the replicating machinery of the host cell. To combat this, host cells internalize the virus into specialized compartments of the cell called endosomes from which a cellular response involving activation of proteins

that sense virus i.e. Toll-like receptors (TLRs) is mounted in an attempt to eliminate the virus. Although this host response is paramount for killing the invading pathogen and conferring immunity for future protection, this attack on the pathogen can result in significant inflammation and tissue injury to the host. However, how this occurs is poorly understood. Our laboratory now provides pivotal evidence that highly reactive and often toxic, oxygen containing molecules i.e. reactive oxygen species (ROS) produced by cells of the immune system, such as macrophages, are culprit chemical mediators of this response to influenza A virus. Despite this, several knowledge gaps still remain. Project aims: To identify: 1) the enzymes that generate subcellular ROS, 2) the primary subcellular compartment of the cell where this ROS is generated and 3) the protein targets and consequence of this subcellular ROS. Overall this project will further our understanding of virus host interactions that could be exploited for treatment of disease caused by influenza viruses. Techniques: This will involve the use of live influenza viruses, cell culture and assays to detect NADPH oxidase expression and localization (western blotting, confocal microscopy, immunohistochemistry, QPCR), ROS generation (chemiluminescence, immunocytochemistry).

Contacts: Dr Stavros Selemidis, Rm E137 Department of Pharmacology, Monash University Phone: 9905 5756. Website: http://www.med.monash.edu.au/pharmacology/staff/stavros-selemidis.html http://www.med.monash.edu.au/pharmacology/research/oxidant.html Email: [email protected]

http://www.med.monash.edu.au/pharmacology/research/oxidant.html




EVALUATING THE ANTIFIBROTIC AND BRONCHODILATOR POTENTIAL OF RELAXIN

Supervisor: Dr Jane Bourke Co-supervisors: Dr Simon Royce, Dr Chrishan Samuel Location: Respiratory Pharmacology & Fibrosis Groups


Background:

The Respiratory Pharmacology and Fibrosis Laboratories focus on identifying improved therapeutic strategies in chronic diseases such as asthma, pulmonary hypertension and fibrosis.

Fibrosis is the major component of the changes in lung structure that occur in both asthma and idiopathic pulmonary fibrosis. Relaxin is a peptide with strong antifibrotic activity currently in clinical trials for cardiac fibrosis but information about its effects in the lung is limited. Drs Simon Royce and Chrishan Samuel have shown that relaxin can prevent and reverse airway remodelling in mouse models of allergic airways disease that mimic key features of asthma. Recent work with Dr Jane Bourke provides preliminary evidence that relaxin may also have direct effects on airway contractility.

Project aims:

This project will test the hypothesis that relaxin inhibits lung fibrosis and causes relaxation of small airways. Specific aims to be tested may include defining the in vitro efficacy of relaxin relative to current

treatments for fibrosis and asthma on (i) fibrotic changes in lung fibroblasts and (ii) inflammation-induced changes in airway reactivity; (iii) exploring the mechanisms underlying the actions of relaxin; and/or (iv) assessing relaxin-mediated effects on chronic fibrosis and impaired bronchodilator sensitivity in vivo.

Techniques:

This project will combine in vitro and in vivo assays of airway reactivity using a novel lung slice technique (to visualise changes in small airway lumen area in situ), standard organ bath

techniques and myography (to measure changes in contractile force) and lung mechanics (to measure airway resistance) as well as biochemical and molecular assays such as Westerns, RT-PCR, ELISAs etc (to measure markers of fibrosis such as collagen synthesis).

Contacts Dr Jane Bourke, Dr Chrishan Samuel, Dr Simon Royce Department of Pharmacology Monash University Phone: 9905 5197, Rm E147 [email protected]



OVERCOMING STEROID RESISTANCE - EVALUATION OF THE EFFECTS OF ANNEXIN-A1 MIMETICS ON AIRWAY INFLAMMATION

Supervisor: Dr Jane Bourke1 Co-supervisor: Dr Simon Royce1, A/Prof Rebecca Ritchie2 Location: 1Respiratory Pharmacology Group


2Baker IDI Heart and Diabetes Institute

Background:

The Respiratory Pharmacology and Heart Failure Laboratories focus on identifying improved therapeutic strategies to prevent and treat chronic diseases that impair lung and myocardial function.

Although steroids are usually effective in controlling airway inflammation, a significant proportion of people with asthma are steroid-resistant. A/Prof Rebecca Ritchie has shown that the glucocorticoid-regulated protein annexin-A1 (ANX-A1) protects against inflammation-induced injury and impaired contractile function in cardiac muscle (Qin et al., 2013). These

results suggest that synthetic ANX-A1 protein and non-protein mimetics may represent a novel alternative to bypass steroid-resistant pathways in the treatment of asthma.

Project aims:

This project will test the hypothesis that ANX-A1 mimetics inhibit inflammation, reduce airway hyperresponsiveness (AHR) and preserve dilator sensitivity in steroid-resistant asthma.

Specific aims to be tested may include (i) defining the efficacy of ANX-A1 mimetics relative to steroids on inflammation-induced changes in airway reactivity in vitro; (ii) exploring the

mechanisms underlying protective actions of ANX-A1 mimetics; and/or (iii) assessing ANX-A1-mediated effects on chronic inflammation, AHR and impaired dilator sensitivity in vivo.

Techniques:

This project will combine in vitro and in vivo assays of airway reactivity using a novel lung slice technique (to visualise changes in small airway lumen area in situ), standard organ bath techniques and myography (to measure changes in contractile force) and lung mechanics (to measure airway resistance) as well as biochemical and molecular assays such as Westerns, RT-PCR, ELISAs etc (to measure markers of inflammation such as cytokines).

This project will be performed largely in Dr Bourke’s laboratory on campus, but will also include components in A/Prof Ritchie’s laboratory at the Baker Institute in Prahran.

Contacts Dr Jane Bourke Department of Pharmacology Monash University Phone: 9905 5197, Rm E147 [email protected]

A/Prof Rebecca Ritchie Baker IDI Heart and Diabetes Institute Phone: 8532 1392 [email protected]




EARLY LIFE INFLUENCES ON AIRWAY AND VASCULAR REACTIVITY

Supervisor: Dr Jane Bourke1 Co-supervisors: A/Professor Tim Moss2, Dr Megan Wallace2 Location: 1Respiratory Pharmacology Group Department of Pharmacology

Monash University, Clayton 2The Ritchie Centre Monash Institute of Medical Research

Background:

In utero and early life influences on the structure and function of small intrapulmonary airways and arteries contribute to serious lung diseases including: respiratory distress syndrome (RDS, characterized by inadequate surfactant), bronchopulmonary dysplasia (BPD, whereby respiratory support at birth injures the lung altering lung development leading to pulmonary hypertension) and childhood asthma (characterized by inflamed, remodelled airways with increased sensitivity to bronchoconstrictor stimuli).

A/Prof Tim Moss and Dr Megan Wallace are internationally recognized for their validated animal models investigating mechanisms contributing to RDS and BPD. However, their highly significant work has yet to address functional airway and artery responses within the lung. This will be addressed in this project in collaboration with Dr Jane Bourke, who has unique expertise with an innovative technique to assess intrapulmonary contraction and relaxation responses within lung slices, which she has previously applied in the context of asthma.

Project aims:

This project will test the hypothesis that intrapulmonary airway and vascular reactivity is altered in established animal models of RDS, BPD and asthma.

Specific aims to be tested may include (i) characterizing in vitro reactivity to constrictor and

dilator agents in lung slices from neonatal animals (ii) investigating the effects of premature birth, in utero inflammation, ventilation-induced lung injury and/or hyperoxia on reactivity (iii) assessing the in vitro efficacy of novel broncho/vasodilators under conditions of altered

reactivity.

Techniques:

This project will combine in vitro assays of reactivity using a novel lung slice technique (to visualise changes in small airway and artery lumen area in situ), as well as biochemical and

molecular assays such as Westerns, RT-PCR, ELISAs etc (to measure mechanisms underlying altered reactivity).

Contacts Dr Jane Bourke Department of Pharmacology Monash University Phone: 9905 5197, Rm E147 [email protected]



PHARMACOLOGICAL CHARACTERISATION OF VENOMS

Supervisors: Dr Sanjaya Kuruppu1, Dr Oden Kleifeld1,

Professor Wayne Hodgson2 Location: Monash Venom Group

Departments of Biochemistry1 & Pharmacology2 Monash University, Clayton

Background:

Snakebite is a global public health problem. Venoms are composed of many proteins and detailed knowledge of these components is of importance, for both antivenom manufacture and identification of new leads for future pharmaceuticals. Our lab uses cutting edge techniques to purify these proteins and characterize them pharmacologically using both in vitro and in vivo techniques. Project 1 : Muscle Damage Induced by African Puff Adder Venom Supervisors : Dr Sanjaya Kuruppu and Prof Wayne C Hodgson

The African Puff Adder (Bitis arietans) is described as one of the most dangerous snakes in

the continent. Life threatening effects of envenoming include haemorrhage, and damage to muscle tissue which in some cases leads to limb amputation. This project will use various types of chromatography to purify the toxin(s) responsible for causing damage to muscle tissue. The purified toxins will be tested in a range of in vitro and in vivo pharmacological assays. The project will also examine the ability of currently available antivenoms to neutralise the effects of these toxins.

Project 2 : Development of Proteomic System-Wide Approach for Characterization of Snake Venoms Supervisors: Drs, Oded Kleifeld, Sanjaya Kuruppu and

Prof Wayne C Hodgson.

The routine methods to determine the identity of venom proteins can be labor intensive, and in many cases provide only partial sequence information due to technical limitations and/or lack genomics information. The aim of this honours project is to develop and test new proteomic workflow for that will allow full protein sequencing and characterization of snake venom proteome. Experiments will involve protein extraction, proteolytic digestion, peptide separation, liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) and bioinformatics (MS/MS data analysis, de novo protein sequencing and more).

Contact:

1) Dr Sanjaya Kuruppu; Email : [email protected] 2) Dr Oded Kleifeld; Email : [email protected] 3) Prof Wayne C Hodgson; Email : [email protected]

Suggested workflow






TARGETING THE HUMAN PLATELET THROMBIN RECEPTOR, PAR4, AS A NOVEL ANTI-THROMBOTIC THERAPY

Supervisors: Drs Justin Hamilton & Grant Drummond Location: Platelet & Megakaryocyte Cell Biology Lab Australian Centre for Blood Diseases

AMREP, Prahran (Alfred Hospital Campus)

Background:

Arterial thrombosis is the most common cause of death and disability in the world. Platelets are the blood cells which form arterial thrombi. Therefore our research examines platelet function in order to discover improved anti-thrombotic therapies. Thrombin is the most potent platelet activator and functions via protease-activated receptors (PARs). We have shown that PAR knockout mice are protected against thrombosis, suggesting PAR antagonists as novel anti-thrombotic agents. As a result, the first PAR antagonist has just been approved by the US FDA for the prevention of arterial thrombosis. However, human platelets have two thrombin receptors, PAR1 and PAR4. Current antagonists target only PAR1, and PAR4 function is poorly understood. To address this, we have recently developed an antagonist of human PAR4.

Project aim:

This project will utilise our newly developed PAR4 antagonist to determine the contribution of PAR4 to thrombus formation and stability in human blood. The major outcome of these studies will be a determination of the importance of PAR4 on human platelets and of the utility of PAR4 antagonists in preventing arterial thrombosis. Techniques: The project will use functional experiments on isolated platelets, ex vivo whole blood thrombosis experiments, and confocal microscopy, and will appeal to students interested in researching and developing future therapies for heart attack and stroke.

Contact: Justin Hamilton Australian Centre for Blood Diseases Monash University Phone: 9903 0125 [email protected]

mailto:Justin.Hamilton@monash



DEVELOPING ISOFORM-SPECIFIC PI3K INHIBITORS AS NOVEL ANTI-PLATELET AGENTS

Supervisors: Drs Justin Hamilton & Grant Drummond Location: Platelet & Megakaryocyte Cell Biology Lab Australian Centre for Blood Diseases

AMREP, Prahran (Alfred Hospital Campus)

Background:

Arterial thrombosis is the leading cause of death and disability in industrialised nations. Anti-platelet agents are the primary preventative therapy for this, but remain limited in their effectiveness. Identifying improved anti-platelet approaches is the major focus of our group. We have shown that a family of intracellular signalling enzymes, the phosphoinositide 3-kinases (PI3Ks), is important for platelet function during thrombosis. We developed a series of PI3K-deficient mice and showed that these mice have impaired platelet function, providing proof-of-principle evidence that inhibiting these enzymes may lead to new anti-platelet drugs. However, there are currently no pharmacological inhibitors of these PI3K isoforms. This project will develop and test the first isoform-specific PI3K inhibitors.

Project aim: To develop the first isoform-specific inhibitors of Class II PI3Ks, and to test lead compounds in order to determine the suitability of targeting Class II PI3Ks as an anti-thrombotic approach in humans. Techniques:

The student will screen a designed library of compounds for inhibitory activity against purified PI3K proteins using an established in vitro kinase activity screen. Lead compounds from this initial work will then be tested for anti-platelet activity in functional assays routinely used in our lab (see Figure). This project will appeal to students interested in drug design & development and in testing potential heart attack and stroke therapies.

Contact: Justin Hamilton Australian Centre for Blood Diseases Monash University Phone: 9903 0125 [email protected]

Examining thrombosis: A 3D reconstruction of a platelet thrombus

made using confocal microscopy

A platelet at work: F-actin of the cell’s cytoskeleton stained with

TRITC-phalloidin.



THERAPIES TO CIRCUMVENT IMPAIRED NITRIC OXIDE FUNCTION FOR MANAGING THE CARDIOVASCULAR COMPLICATIONS OF

DIABETES

Supervisors: A/Prof Rebecca Ritchie & Dr Barbara Kemp-Harper Location: Heart Failure Pharmacology Laboratory

Baker IDI Heart & Diabetes Institute 75 Commercial Rd, Melbourne

Background:

The global epidemic of diabetes mellitus is imposing an exponential burden on society – not only because of the substantial associated healthcare costs, but also because of the poor health outcomes for those with the condition, particularly as a result of diabetic cardiovascular complications-induced morbidity and mortality. Impaired nitric oxide (NO) signalling is an independent marker of poor prognosis. Defined as a loss of the protective cardiovascular actions downstream of NO, the phenomenon is particularly seen in diseased human vasculature, likely as a consequence of elevated oxidative stress (see diagram). Loss of NO responsive-ness is particularly debilitating in diabetes, where cardiovascular emergencies (acute myocardial infarction, transient ischaemia, cardiogenic shock) occur more frequently, yet the ability of NO-based pharmaco-therapies is often deficient. We have identified nitroxyl (HNO) as a putative strategy for enhancing NO signalling in the heart over the short- and longer-term, which we believe can potentially address this important clinical problem.

Project aim:

GENERAL HYPOTHESIS: Diabetes-induced NO resistance in the heart is exacerbated by poor glycaemic control, and can be circumvented by HNO. AIMS: To determine whether diabetes induces NO resistance in the myocardium (not previously known), to investigate the relationship between hyperglycaemia and NO responsiveness in the diabetic heart and to demonstrate that HNO circumvents this impaired NO signalling induced by diabetes. Techniques: It is anticipated that this will involve in vivo rodent models of diabetic cardiac disease, isolated heart studies, assessment of cardiac function and several biochemical techniques: Westerns, reactive oxygen species (ROS) detection, ELISA, real-time PCR, and cardiac histology.

Contact: A/Prof Rebecca Ritchie Heart Failure Pharmacology Laboratory Baker IDI Heart & Diabetes Institute Phone: 8532 1392 Email: [email protected]




THERAPEUTIC TARGETING OF CARDIAC HEXOSAMINE BIOSYNTHESIS – ROS INTERACTIONS TO PROTECT THE DIABETIC

HEART

Supervisors: A/Prof Rebecca Ritchie & Dr Barbara Kemp-Harper Location: Heart Failure Pharmacology Laboratory Baker IDI Heart & Diabetes Institute

75 Commercial Rd, Melbourne

Background: Diabetes affects almost 2 million Australians; diabetes increases heart failure risk 2.5-fold and accelerates its onset. There is however a lack of existing management of heart failure in the context of diabetes. Our laboratory has identified several new therapies for diabetic cardiomyopathy, many of which target reactive oxygen species (ROS). The hexosamine biosynthesis pathway (HBP) is an alternative fate of glucose (see schema) that under normal conditions protects cells. In diabetes however, sustained upregulation of HBP has emerged as a contributing factor to the cardiac complications of diabetes. We propose that high glucose combined with high ROS explains why sustained activation of cardiac HBP signalling is detrimental. Using cardiac-selective gene therapy, we can regulate HBP signalling in the diabetic heart. Project aim:

GENERAL HYPOTHESIS: that the double triggers of elevated glucose and ROS provide an additional drive towards unchecked HBP dysregulation, switching to a maladaptive impact on cardiac function and mitochondrial integrity. AIMS: To demonstrate that cardiac-directed therapeutic targeting of the ROS-HBP axis, in either the early or later stages of diabetes, will delay or even reverse diabetes-induced cardiac dysfunction. Techniques: It is anticipated that this will involve in vivo rodent models of diabetic cardiac disease,

assessment of cardiac and mitochondrial function, gene therapy, isolation of cardiac myocytes, Westerns, ROS detection, real-time PCR, histology and immunofluorescence.

Contact:

A/Prof Rebecca Ritchie Heart Failure Pharmacology Laboratory Baker IDI Heart & Diabetes Institute Phone: 8532 1392 Email: [email protected]




TARGETING CARDIAC INFLAMMATION AS A NOVEL THERAPEUTIC APPROACH FOR THE CARDIAC COMPLICATIONS OF DIABETES

Supervisors: A/Prof Rebecca Ritchie & A/Prof Grant Drummond Location: Heart Failure Pharmacology Laboratory

Baker IDI Heart & Diabetes Institute 75 Commercial Rd, Melbourne

Background:

Diabetes is Australia’s fastest growing chronic disease. The disease affects almost 2 million Australians; diabetes increases heart failure risk 2.5-fold and accelerates its onset, giving rise to an ever-increasing heart failure burden. Our laboratory has an established track record for identifying new pharmacotherapies for diabetic cardiomyopathy. We have obtained recent evidence that cardiac inflammation is a key contributor to myocardial damage in the diabetic heart. We have previously shown that the glucocorticoid-regulated anti-inflammatory mediator annexin-A1 is a key regulator of cardiac viability and function. Annexin-A1 offers an attractive approach to minimise the detrimental consequences of diabetes on the heart, potentially addressing the unmet clinical need of the extra burden posed by concomitant diabetes, inflammation and cardio-myopathy. Thus, cardioprotective annexin-A1 mechanisms may ultimately limit progression to heart failure and death in diabetes-affected patients. Project aim: GENERAL HYPOTHESIS: Annexin-A1-based therapies limit diabetic cardiomyopathy by reducing cardiac inflammation and protecting cardiac contractile function. AIMS: To compare the time-course of cardiac inflammation and impaired cardiac function in diabetes, and to investigate annexin-A1 cardioprotection for the cardiac complications of the disease in vivo. Techniques: It is anticipated that this will involve in vivo models of diabetic cardiac disease, assessment of cardiac function and several biochemical techniques: Westerns, ELISA, real-time PCR, histology and immunofluorescence in diabetic rats or mice.

Contact: A/Prof Rebecca Ritchie Heart Failure Pharmacology Laboratory Baker IDI Heart & Diabetes Institute Phone: 8532 1392 Email: [email protected]




EXPLORING NOVEL THERAPEUTIC STRATEGIES TO PREVENT ATHEROSCLEROTIC PLAQUE RUPTURE

Supervisors: Dr Jennifer Rivera, Dr Yung Chih Chen & Professor Karlheinz Peter Location: Atherothrombosis and Vascular Biology Laboratory Baker IDI Heart and Diabetes Institute

Background:

Atherosclerosis is a chronic inflammatory disease characterised by the build up of lipid-filled plaques within arteries. While atherosclerotic plaques can remain silent for many years, some plaques can become unstable and rupture, resulting in thrombus formation and vessel occlusion, which can lead to life-threatening complications such as myocardial infarction or stroke. It is now well established that inflammatory pathways can influence plaque rupture and thrombus formation, making them ideal therapeutic targets. Our laboratory has established a unique mouse model of atherosclerotic plaque rupture that closely resembles human plaque pathology. We have demonstrated that this mouse model is a useful tool for testing of drugs with potential to treat plaque instability and prevent plaque rupture. Project aim:

The aim of this honours project is to use our mouse model of plaque rupture to characterise the ability of novel anti-inflammatory and anti-thrombotic inhibitors to stabilise plaques and prevent plaque rupture. This drug discovery project may lead to the identification of novel therapies that may be further translated to clinical use in humans. Techniques:

By the end of their Honours year, students will be well-trained in the following techniques: immunohistochemistry and histology (to assess the stability of atherosclerotic plaques), flow cytometry (characterisation of immune cells), cell culture and quantitative real-time PCR (microRNA and mRNA pro-inflammatory marker expression).

Contacts: Dr Jennifer Rivera, Dr Yung Chih Chen & Professor Karlheinz Peter Atherothrombosis and Vascular Biology Laboratory Baker IDI Heart and Diabetes Institute Phone: 9852 1235, Level 4, Lab 2 [email protected] [email protected] [email protected]

Mouse model of plaque rupture

Source: Chen et al., Circ Res 2013, Libby et al., NEJM 2013





TARGETING ACTIVATED PLATELETS AS EARLY MARKER FOR DIAGNOSIS AND TREATMENT OF INFLAMMATORY DISEASES

Supervisors: Dr Bock Lim & Prof Karlheinz Peter Location: Atherothrombosis & Vascular Biology Program Baker IDI Heart & Diabetes Institute Prahran, Melbourne

Background:

Besides their critical role in haemostasis, platelets are recently associated with immune defence and inflammation. Activated platelets are shown to play a critical role in various inflammatory diseases such as atherosclerosis, pulmonary embolism, rheumatoid arthritis and multiple sclerosis. Hence early detection of these cells during inflammatory progression (diagnosis) and effective inhibition of their function (therapy) are both critical in the clinical combat against these conditions. We have generated a single chain protein which binds activated platelets. When linked with a suitable detector, such as a near-infra red fluorescent probe, this single-chain can be used as a diagnostic marker. The linked protein can be used to detect the presence of activated platelets in areas of the body where disease associated inflammation is occurring by using a highly specialized, novel and world’s first FLuorescence Emission Computed Tomography scanner (FLECT/CT). The Figure below shows the detection of fluorescence signal by the FLECT/CT scanner in the liver of a mouse. In addition, when linked to a specific drug, this single-chain antibody can direct treatment to the site of inflammation where activated platelets are present, and thereby reduce drug dosage and undesirable side-effects.

Project aim:

The aim of this honours project is to examine the diagnostic and therapeutic capabilities of this single-chain antibody directed at activated platelets in the above diseases by using different mouse models. The established imaging tools and drug candidates have the potential to be directly applied in patients. Techniques: The following techniques will be taught and applied in this project: basic laboratory molecular biology techniques (DNA work, cloning, PCR), protein expression/purification, molecular-conjugation, flow-cytometry, mouse inflammatory disease models, FLECT/CT scanning and data analysis, Immuno-histochemistry, and fluorescence microscopy, antibody drug development.

Contacts: Prof Karlheinz Peter or Dr Bock Lim Thrombosis & Atherosclerosis Lab Baker IDI Heart and Diabetes Institute Phone: 8532 1490 or 8532 1447 [email protected] [email protected]



TARGETING THE CALCIUM SENSING RECEPTOR IN OSTEOPOROSIS AND HYPERPARATHYROIDISM

Supervisors: Dr Katie Leach & Professor Arthur Christopoulos Location: Drug Discovery Biology Laboratory Monash Institute of Pharmaceutical Sciences

Monash University, Parkville

Background:

The extracellular calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) that is expressed widely in tissues of the body. Its principal role is to detect subtle changes in the concentration of extracellular calcium ions (Ca2+

o) to maintain Ca2+o

homeostasis, primarily via regulation of parathyroid hormone (PTH) synthesis and secretion, calcium reabsorption in the kidney, and calcitonin secretion from thyroid C cells. CaSRs expressed on osteoblasts and osteoclasts also regulate bone resorption and formation. Thus, CaSR activity in the bone in conjunction with its control over the secretion of hormones vital for bone health and Ca2+

o homeostasis (PTH and calcitonin), make the CaSR is an attractive drug target in the treatment of osteoporosis and hyperparathyroidism. Indeed, calcilytics (CaSR negative allosteric modulators) have been tested in human clinical trials for osteoporosis due to their ability to stimulate PTH release. Furthermore, cinacalcet is a calcimimetic (CaSR positive allosteric modulator) that is used clinically to treat hyperparathyroidism. Please visit http://www.monash.edu.au/pharm/research/areas/drug-discovery/laboratories/leach.html for further information. Project aim:

Projects available in this area aim to investigate: 1. The molecular pharmacology of calcilytics and calcimimetics, using receptor mutagenesis studies to probe the interaction of these drugs with the receptor and high throughput pharmacological assays to determine drug effects on receptor signalling. 2. The impact of allosteric modulators on CaSR expression and desensitisation, using assays that measure receptor phosphorylation and trafficking. 3. The potential impact of calcilytics and calcimimetics on bone health using an osteoblast cell culture model to study cell differentiation and osteoblast-like activity. Techniques: This project will involve cell culture of recombinant HEK293 cells and osteoblast cells derived from human long bone. It will employ numerous cell-based assays that measure receptor function, such as intracellular calcium mobilisation, ERK1/2, AKT and beta-catenin phosphorylation, receptor phosphorylation and receptor trafficking.

Contacts: Dr Katie Leach Drug Discovery Biology Monash Institute of Pharmaceutical Science Phone: 9903 9089, Rm 4.344 [email protected]

PTH

Ca2+

Ca2+

Ca2+

Ca2+

Fig. Cinacalcet (sensipar) activates the CaSR in the parathyroid gland leading to PTH release



ALLOSTERIC MODULATION OF METABOTROPIC GLUTAMATE RECEPTORS: NOVEL TREATMENT STRATEGIES FOR CNS

DISORDERS

Supervisors: Dr Karen Gregory & Prof Arthur Christopoulos Location: Drug Discovery Biology Monash Institute of Pharmaceutical Sciences


Background:

The metabotropic glutamate receptor subtype 5 (mGlu5) is a family C G protein-coupled receptor that has emerged as an exciting new target to treat a number of CNS disorders including schizophrenia, Alzheimer's disease, autism spectrum disorders and depression. We are pursuing a novel class of therapeutics, called allosteric modulators, to selectively target mGlu5. To facilitate rational drug design and discovery efforts, a better understanding of the functional consequences and structural basis of mGlu5 allosteric modulation is needed. Project aim:

Multiple projects are available studying the molecular pharmacology of diverse allosteric modulators. Specifically we are interested in understanding: the structural basis for mGlu5 activation and allosteric modulation, the influence of receptor dimerization on mGlu5 pharmacology, and the impact of chronic vs acute exposure to allosteric modulators on mGlu5 activity. Techniques:

To test our hypotheses, we have access to a diverse allosteric modulator collection. Techniques applied may include molecular biology, high-throughput second messenger assays, primary neuronal culture, recombinant cell culture, protein chemistry, photoaffinity labeling, immunoblotting, computational modelling and single cell imaging.

Contacts: Dr Karen Gregory & Prof Arthur Christopoulos Drug Discovery Biology, MIPS Monash University, Parkville Phone: 9903 9243, Rm 4.346 [email protected] [email protected]



THE MOLECULAR BASIS OF G PROTEIN−COUPLED RECEPTOR FUNCTION: CHEMICAL BIOLOGY APPROACHES

Supervisors: Dr Sebastian Furness & Prof Patrick Sexton Location: Drug Discovery Biology Laboratory Monash Institute of Pharmaceutical Sciences


Background:

The membrane at the surface of cells is their defining feature, maintaining internal organisation in the face of the disorder of the environment. All cellular requirements and communication must cross this membrane. G protein-coupled receptors (GPCRs) represent the largest class of molecular machines that provide the means of communication across this barrier. This family of receptors allow cells to sense local and distant signals sent from other cells, nutrients in the gastro-intestinal tract, odours & tastes and even allow us to see. Chemical biology is a holistic approach to understanding the underlying molecular basis of biological events. My research seeks to develop a deeper understanding of the molecular basis for communication across the cell membrane by GPCRs using a variety of biochemical, molecular, cell biological and pharmacological methods – in essence a chemical biology approach. This molecular description of how GPCRs work is used to inform our understanding of whole organism physiology and pathology which we pursue via active collaborations.

Project aim:

Efficacy is a very well defined pharmacological concept. In analytical pharmacology, differences in the efficacy of different ligands can be quantified. This allows useful predictions to be made about how different biological systems will respond to particular ligands. In this analytical pharmacological approach the ligand:receptor:effector are treated as a black box to which values from the operational model are assigned, however there is very little data that allows us to understand efficacy from a molecular basis. This project aims to address the underlying molecular basis of efficacy. It will inform biased signalling and will provide insights that can be applied to developing better drugs.

Techniques:

Approaches will include cell culture, transfection techniques, real-time BRET assays and the use of high-end microscopy techniques.

Contacts: Dr Sebastian Furness and Prof Patrick Sexton Monash Institute of Pharmaceutical Sciences Monash University, Parkville Phone: 9903 9055, Rm 4.509 [email protected] [email protected]

A cartoon depicting the changes in shape that a GPCR undergoes in order to transmit signals across the cell membrane.



RESOLVING THE SPATIOTEMPORAL SIGNALLING OF G PROTEIN-COUPLED RECEPTORS

Supervisors: Dr Michelle L Halls Location: GPCR Signalosome and Compartmentalisation Group Drug Discovery Biology Theme

Monash Institute of Pharmaceutical Sciences

Background:

Cells endogenously express a variety of different receptors that can activate the same second messenger but with remarkably diverse physiological outcomes. This suggests a high degree of organisation and regulation of intracellular signalling, which is achieved by the spatiotemporal compartmentalisation of signals –the restriction of second messengers in space and time. In this way, G protein-coupled receptors (GPCRs) can direct the assembly of focused “platforms” for specific signalling. These signalling platforms facilitate second messenger production, the organisation and scaffolding of effectors, and co-ordination of regulatory elements.

Project aim: There are a number of projects available that examine the role of compartmentalised signalling:

Opioid receptors and pain transmission (ML Halls, M Canals, DP Poole)

Relaxin receptor signalosomes and compartmentalisation (ML Halls, RJ Summers)

GPCR compartmentalisation in breast cancer metastasis (ML Halls, M Canals, EK Sloan)

Techniques: By using targeted Förster Resonance Energy Transfer (FRET) biosensors, we can resolve sub-cellular spatial and temporal dynamics of GPCR signalling at a single cell level. This can reveal intricate detail about the role of receptor trafficking and regulation in the transmission of signalling, and how this is disrupted in various disease states.

Contact: Dr Michelle L Halls Drug Discovery Biology Theme Monash Institute of Pharmaceutical Sciences Phone: 9903 9094, Building 4 Room 324 [email protected]

Targeted biosensors measure compartmentalised signalling in

single live cells.



PHARMACOLOGICAL THERAPIES TO BURN FAT

Supervisors: Dr Dana Hutchinson & Dr Bronwyn Evans Location: Drug Discovery Biology Monash Institute of Pharmaceutical Sciences


Background:

The high prevalence of obesity and type 2 diabetes worldwide has provoked substantial interest in adipose tissue research. As outlined by The World Health Organisation, government and health professionals would ideally manage obesity by encouraging increased physical activity and reduced intake of energy rich foods, but these measures have a low success rate amongst many individuals, highlighting the needs for additional pharmacological approaches to weight loss. There are two types of adipose tissue: white adipose tissue (WAT) that stores energy in the form of triglycerides, and brown adipose tissue (BAT) that releases chemical energy in the form of heat (thermogenesis), thereby counteracting obesity and related disorders. Over the past few years, a third type of adipose tissue, termed brite/beige adipose tissue, has been described. Under certain stimuli this type of adipose tissue can possess BAT functions including thermogenesis, and can be pharmacologically manipulated. Our aim is to investigate a range of pharmacological agents for their ability to turn on these brite/beige adipocytes. BAT depots are highly innervated and are activated by centres in the brain responsive to cold exposure or food intake, leading to release of noradrenaline (NA) from sympathetic nerves. NA

binds to -adrenoceptors that lead to increased expression of UCP1 (uncoupling protein-1 (UCP1), and breakdown of triglycerides to free fatty acids that activate UCP1, resulting in thermogenesis. Beige/brite adipocytes have negligible levels of UCP1 (especially in obese/ overweight/diabetic subjects), but diabetic therapeutic agents such as rosiglitazone can substantially increase UCP1 expression in these adipocytes, enabling NA to activate UCP1. Thus agents that can increase UCP1 levels in adipocytes may be of therapeutic benefit. Project aim:

Our aim is to investigate a range of pharmacological agents that are used therapeutically for their ability to turn on these brite/beige adipocytes. Techniques:

We will culture adipocytes from mice in the presence of a range of drugs used therapeutically for obesity/diabetes, and assess their ability to (1) increase UCP1 mRNA levels (2) their ability to increase cellular oxygen consumption rates ie. Cellular respiration that is required for thermogenesis (3) their impact on adrenoceptor function using high throughput assays

Contacts: Dr Dana Hutchinson and Dr Bronwyn Evans Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences Monash University, Parkville Phone: 9903 9077 & 9903 9086, Building 4, Rm 343 [email protected] [email protected]



INVESTIGATION OF A ‘LOCKED’ ACTIVE CONFORMATION OF A G PROTEIN-COUPLED RECEPTOR

Supervisors: Dr. Celine Valant & Prof. Arthur Christopoulos Location: Drug Discovery Biology theme

Monash Institute of Pharmaceutical Sciences Monash University, Parkville

Background & Project:

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors encoded by the human genome, and participate in all aspects of pathophysiological control. Unfortunately, the development of new therapies targeting GPCRs has been hampered by a high attrition rate, predominantly due to an inadequate understanding of fundamental aspects of GPCR activation, signalling, and importantly, dynamics. GPCRs are highly flexible proteins, constantly exploring a continuum of conformational states, from inactives to actives, that can be transiently stabilised by inverse agonists or agonists, respectively (Fig1A). Regrettably, the ‘isolation’ of active conformational states requires the purification of a complex made of the receptor, the ligand, and the G protein, and remains a highly difficult task (Fig1B). Understanding the pharmacological properties of active conformational states of these receptors could have a major impact on GPCR−based drug discovery. Here, we propose to genetically modify a GPCR to artificially generate a ‘locked’ active receptor state, amongst the panoply of conformational states available to it. Using the muscarinic M2 acetylcholine receptor (M2 mAChR), we have fused this GPCR to a mimic of its own G protein, in order to engender a stable active conformation of the receptor (Fig1C). We will

pharmacologically characterise this ‘locked’ active conformation, by investigating the binding and functional properties of a variety of structurally distinct muscarinic ligands. Techniques:

In this project you will learn the basics of cell culture, such as splitting and seeding cells, as well as cell transfection. Additionally, multiple cell-based assays will be utilised during this project, including radioligand binding and various signalling assays. Finally, as part of the project you will also learn basic molecular biological procedures including isolation and preparation of DNA. Figure 1. By-passing the inherent dynamic nature of G protein−coupled receptors.

A. Specific ligands can stabilise

distinct conformational states of receptors. B. A stable active state of a GPCR

requires the simultaneous binding of an agonist and a G protein to the receptor. C. The ‘locked’ active conformation of

the M2 mAChR can be pharmacologically studied by evaluation of both binding and

signalling properties of structurally diverse muscarinic ligands.

Contact: Dr. Celine Valant Phone: 9903 9091 Building: 404 Room: 344 Email: [email protected]

A!

B!

C!

GPCR%G&protein&mimic&fused&chimera&

Structurally&dis9nct&ligands&Inverse&Agonist& Agonist&

G&protein&mimic&

Unstable& Stable&




STRESS IS MAKING ME SICK! NEURAL REGULATION OF VIRAL IMMUNITY

Supervisors: Dr Erica Sloan & Dr Scott Mueller Location: Cancer & Immunology Groups Drug Discovery Biology


Background

Pre-clinical and clinical studies have shown that viral immunity is compromised during times of chronic stress. However, surprisingly little is known about the impact of stress neurotransmitters on T cell function or their roles in adaptive immune responses to pathogens. Using intravital 2-photon microscopy (2PM) we have made the unexpected observation that SNS neurotransmitters induce rapid and profound changes in T cell migration in intact lymph nodes in live mice. Physiological concentrations of SNS neurotransmitters immediately and reversibly halted T cell migration in vivo. Together, these

findings suggest that SNS signalling impairs immunity by impacting T cell activation, migration or effector functions. Moreover, this raises the intriguing possibility that α-blockers or β-blockers – safe and inexpensive drugs currently used to treat multiple conditions including hypertension – might be novel therapeutic approaches to boost immune responses to viral infections.

Project aim:

This honours project will begin define how stress impairs our ability to fight infection by investigating the effect of adrenergic signaling on T cell motility and migration in vivo. The project will use pharmacological and genetic techniques to explore the effect of stress signaling on anti-viral T cell responses.

Techniques:

This project uses state-of-the-art two-photon microscopy, pharmacological agents and adrenergic-deficient mice to track the effect of neural stress signaling on T cell function.

Contacts: Dr Erica Sloan and Dr Scott Mueller Department of Drug Discovery Biology Monash University, Parkville Phone: 03 9903 9707 [email protected] [email protected]




ALLOSTERY AND LIGAND BIAS AT MUSCARINIC RECEPTORS: STRUCTURAL INSIGHTS TOWARDS UNDERSTANDING CNS

DISORDERS

Supervisors: Dr David Thal & Prof Arthur Christopoulos Location: Drug Discovery Biology Laboratory Monash Institute of Pharmaceutical Sciences


Background:

The M1 – M5 muscarinic acetylcholine receptors constitute an important family of class A G protein-coupled receptors (GPCRs) activated by the neurotransmitter, acetylcholine. Muscarinic receptors are exciting targets for the treatment of various central nervous system (CNS) disorders, including Alzheimer’s disease, schizophrenia and drug addiction. Unfortunately, the clinical translation of compounds targeting these receptor subtypes has remained unsuccessful due to insufficient selectivity of candidate drugs targeting the highly conserved acetylcholine-binding orthosteric site. Encouragingly, muscarinic receptors possess spatially distinct allosteric binding sites that offer greater potential for selective receptor targeting, and compounds specifically targeting the M4 receptor subtype represent one of the first examples for which highly selective positive allosteric modulators (PAMs) with central nervous system activity and preclinical efficacy have been identified. However, the structural basis of orthosteric and allosteric drug interaction with this therapeutically relevant group of receptors remains largely unknown.

Project aim:

Despite recent success with the crystallization and structure determination of several family A GPCRs, including the muscarinic receptors, our understanding of the molecular mechanisms by which these receptors function is still quite limited. In this project we will take a reductionist approach and isolate and examine receptor function in an environment free from the cell. The aim will address the structural and molecular aspects by which allostery and signaling specificity is achieved at muscarinic receptors. The insight gained from these studies will provide a foundation for future drug discovery.

Techniques:

Approaches used may include the following: mammalian and insect cell culture, molecular biology, protein expression and purification, radioligand binding assays, high-throughput second messenger signaling assays, x-ray crystallography, molecular visualization and computational analysis.

Contacts: Dr David Thal and Prof Arthur Christopoulos Monash Institute of Pharmaceutical Sciences Monash University, Parkville Phone: 03 9903 9097, Rm 4.509 [email protected] [email protected]




Understanding pain from inside out

Supervisors: Dr Elva Zhao and Prof. Nigel Bunnett Location: Drug discovery biology

Monash Institute of Pharmaceutical Sciences Monash University, Parkville

Background:

Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage. In general, Pain triggers protective action from damaging situations. However, uncontrolled and persist pain can significantly interfere with a person’s quality of life and general functioning. You will be investigating various mechanisms that involved in pain management; in particular, proteases mediated inflammation and pain. Project aim:

1) Investigating the dynamics of protease activated receptors (PARs) signal transduction and regulation; 2) Evaluating fluorescence activity based protease probes both in vitro and in vivo; 3) Explore the mechanisms of PARs-dependent inflammation and pain; Techniques: Familiar with various cell biology, biochemistry, and imaging tools such as second messenger assays, BRET assays, proteolytic digestion assays, and confocal microscopy.

Contacts: Dr. Elva Zhao and Prof. Nigel Bunnett Monash Institute of Pharmaceutical Sciences Monash University [email protected] Nigel.Bunnett @monash.edu

Documents

Department of Pharmacology Honours Projects 2015 › pharmacology › docs › pharmacology... · the Honours Convenors of the Pharmacology Department (Dr Barb Kemp-Harper or A/Prof