Department of Pharmacology Honours Projects 2020 · Research Seminars (introductory & final) Thesis...

Department of Pharmacology, Honours 2020

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Department of Pharmacology

Honours Projects 2020

2019 Honours Students

www.monash.edu


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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 scientific 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. As course convenors, A/Prof Barb Kemp-Harper and I, 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 2020 and wish you all the best for a rewarding and exciting year of research. Good luck! Professor Robert Widdop Head, Department of Pharmacology


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Department of Pharmacology Honours Convenors

Prof Rob Widdop A/Prof Barb Kemp-Harper

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

Phone: 9905 4858 Phone : 9905 4674

Pharmacology Honours: Pre-requisites

BSc Biomedicine BMS

Pre-requisites A Distinction average (>70) in 24 points at 3rd year in relevant disciplines within the School of Biomedical Sciences*

A Distinction average (>70) in 24 points at 3rd year level, including 12 points in 3rd year core BMS units (BMS3021, BMS3042) and 12 points in other 3rd year units*

Application closing date

15th November 2019 15th November 2019

Application form https://www.monash.edu/science/current-students/science-honours

https://sites.google.com/monash.edu/biomedical-science-honours/home

Commencement date 24th February, 2020 24th February, 2020

* There is no pre-requisite in terms of 3rd 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]





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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)


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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 following steps:

These forms must be signed by the Honours Convenors of the Pharmacology Department,

A/Prof Barb Kemp-Harper or Professor Rob Widdop.

Apply on-line via E-admissions by Friday 15th November:

Science: Complete on-line application form via E-Admissions

http://www.monash.edu/science/current-students/science-honours.

Submit Departmental Project Preference form to Pharmacology Honours Convenor.

Biomedical Science: Complete on-line project application form and eAdmission form

https://sites.google.com/monash.edu/biomedical-science-honours/application-process

Submit Departmental Project Preference form to Pharmacology Honours Convenor.

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 to late December 2019.

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 2020.

http://www.monash.edu/science/current-students/science-honours

https://sites.google.com/monash.edu/biomedical-science-honours/application-process


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Honour Projects 2020 – On campus projects

LABS / SUPERVISOR(S)

PROJECT TITLE

Education-Based Projects Liz Davis, Klaudia Budzyn, Jennifer Irvine

Investigating how students make use of learning resources in Pharmacology

Fibrosis Group Chrishan Samuel, Tracey Gaspari, Anita Pinar, Padma Murthi

Investigating novel anti-fibrotic therapies

Integrative Cardiovascular Pharmacology Group Tracey Gaspari, Rob Widdop Rob Widdop, Mibel Aguilar, Mark Del Borgo Claudia Ireland, Brad Broughton, Rob Widdop

Exploring novel signalling mechanisms associated with insulin-regulated aminopeptidase (IRAP)

Drug discovery program for AT2 receptor ligands of the RAS

Inhibition of Acid Sensing Ion Channels (ASIC) as a novel treatment for stroke

Cardiovascular & Pulmonary Pharmacology Group Barb Kemp-Harper, Brad Broughton Barb Kemp-Harper, Rob Widdop, Brad Broughton Barb Kemp-Harper, Brad Broughton

Targeting the CCL18-CCR8 axis to treat pulmonary hypertension

Targeting the CCl18-CCR8 axis to treat hypertension-associated end organ damage

Targeting the NLRP3 inflammasome in the treatment of pulmonary hypertension

Respiratory Pharmacology Group Jane Bourke, Simon Royce Glen Westall, Jade Jaffar Jane Bourke, Rebecca Ritchie, Helena Qin Jane Bourke, Simon Royce

Precision cut lung slices as a platform for studying anti-fibrotic drugs

Exploring a novel treatment for pulmonary hypertension

Free fatty acid receptors as novel bronchodilator targets for asthma

Monash Venom Group Wayne Hodgson, Geoff Ibister

A pharmacological and biochemical examination of two Chinese snake venoms

Kidney Regeneration & Stem Cell Laboratory Sharon Ricardo, Andrea Wise

Kidney organoids for disease modeling of genetic kidney disease


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Honour Projects 2020 – Off campus projects

LABS / SUPERVISOR(S)

PROJECT TITLE

Baker Heart & Diabetes Institute Geoff Head, Kristy Jackson, Cindy Gueguen Judy de Haan, Rebecca Ritchie, Arpeeta Sharma Bing Wang, David Kaye, Chrishan Samuel Rebecca Ritchie & Miles De Blasio Rebecca Ritchie & Barb Kemp- Harper Rebecca Ritchie & Mitchel Tate Rebecca Ritchie & Mitchel Tate Waled Shihata, Bing Wang, Barb Kemp-Harper & David Kaye

Effects of administration of ganoxolone in spontaneous hypertensive rats

Targeting the Nrf2/NLRP3 axis in diabetic hypertension to improve cardiac function post myocardial infarction

Targeting receptor tyrosine kinase as novel anti-fibrotic therapy for cardiovascular and related diseases

Exploring the Role of Altered Cardiac Glucose Metabolism in the Cardiac Complications of Diabetes

Using the NO Redox Sibling Nitroxyl to Overcome

Diabetes‐induced Impairments in Cardiac and Vascular NO Signalling

To understand the role of bone morphogenetic protein 7 (BMP7) on cardiac fibrosis in the diabetic heart

Comparing a clinically-relevant experimental model of heart failure with preserved ejection (HFpEF) fraction to human disease

Investigating novel therapeutic targets for obesity-induced heart failure

Drug Discovery Biology Theme Monash Institute of Pharmaceutical Sciences Sebastian Furness Sebastian Furness Katie Leach, Andrew Keller, Tracy Josephs Karen Gregory, Shane Hellyer & Katie Leach Denise Wootten & Elva Zhao Denise Wootten & Elva Zhao Denise Wootten & Elva Zhao

Mechanisms for differential GPCR: Transducer Coupling

The effect of cholesterol on satiety

Targeting the calcium sensing receptor to treat asthma and endocrine disorders

Allosteric modulation of glutamate receptors for psychiatric and neurological disorders

Modulation of G protein-coupled receptor function by receptor activity modifying proteins

Alternative splicing of the pituitary adenylate cyclase-activating polypeptide (PAC1) receptor

Understanding the molecular mechanisms of glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide receptor dual agonists


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INVESTIGATING HOW STUDENTS MAKE USE OF LEARNING RESOURCES IN PHARMACOLOGY

Supervisors: A/Prof Liz Davis Dr Klaudia Budzyn Dr Jennifer Irvine Location: Pharmacology Education Research Initiative (PERI) Department of Pharmacology

Monash University, Clayton Background: The current trend in university teaching, is to move away from didactic teaching and instead engage students more in active learning via pre-class; in-class; and after-class activities. While there is evidence that active learning improves student performance in science, this is dependent on student engagement with their learning, including attendance, active participation in their classes and use of available learning resources. Student engagement, however, is influenced by motivation and experience of what has worked for them previously. As it is known that assessment drives learning, if students have been successful using a passive approach to learning, they may be reluctant to embrace active learning. In addition, the resources that are made available as learning tools, and the feedback they receive on their work can have an impact on how students engage with their studies. We therefore need to have a better understanding of how our students respond to different “modes” of teaching and how they use and value the resources we offer to support their learning. In particular, we want to know what they perceive as most useful and how they adapt their learning in response to different formats of teaching. Project aim: The aim of our studies is to investigate factors that influence the engagement of students with their learning of pharmacology, in particular their use of learning resources their evaluation of attitudes to, and use of assessment feedback Techniques: The projects will use a mixed methods approach, using both quantitative (e.g. surveys) and qualitative (e.g. focus groups, interviews and in-class observation) techniques. Contacts: Assoc Prof Elizabeth Davis Department of Pharmacology Monash University Phone: 9905 5755, Rm E123 [email protected]

Dr Klaudia Budzyn Department of Pharmacology Monash University Phone: 9905 4857, Rm E116c [email protected]

Dr Jennifer Irvine Department of Pharmacology Monash University Phone: 9905 5745, Rm E116c [email protected]




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INVESTIGATING NOVEL ANTI-FIBROTIC THERAPIES

Supervisors: A/Prof Chrishan Samuel, Dr Tracey Gaspari & Dr Anita Pinar & Dr. Padma Murthi Location: Fibrosis Laboratory Rms E101/E102/E107 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. The eventual replacement of normal tissue with scar tissue leads to organ stiffness and failure. Despite a number of available treatments for patients with various heart and kidney 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.

Aims: The Fibrosis Lab aims to identify novel anti-fibrotic therapies (relaxin, stem cells, AT2 receptor agonists 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 and kidney disease) 2. Head-to-head and combination therapy efficacy studies 3. Targeting inflammasome activity to treat fibrosis Techniques: Depending on the project involved, animal/cell culture models of (heart/kidney disease), blood pressure and functional measurements, matrix biology, protein biochemistry, molecular biology and/or histological techniques will be utilized.

Contacts: A/Prof Chrishan Samuel, Dr Tracey Gaspari, Dr Anita Pinar & Dr Padma Murthi Department of Pharmacology, Monash Univ. Phone: 9902 0152 / 9905 4762 [email protected] / [email protected] [email protected] / [email protected]




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EXPLORING NOVEL SIGNALING MECHANISMS ASSOCIATED WITH INSULIN-REGULATED AMINOPEPTIDASE (IRAP)

Supervisors: Dr Tracey Gaspari & Professor Robert Widdop Location: Integrative Cardiovascular Pharmacology Group Department of Pharmacology Clayton Campus A/Prof Siew Yeen Chai, Dept Physiology Clayton Campus Background: Cardiovascular diseases (CVDs) remain the world’s leading cause of morbidity and mortality, claiming 17 million deaths annually. Risk factors such as ageing, excessive acute (e.g. myocardial infarct) or chronic (e.g. hypertension) cardiac injury, lead to increased cardiac fibrosis, chronic heart failure and/or end organ damage. However there are few effective treatments currently available and thus there is an urgent need to identify new targets/treatment given our aging population. We have compelling evidence that IRAP is upregulated in cardiac tissue in pathophysiological states, although we do not know if this is a cause or consequence of disease.

Project aim: The aim of this Honours project is to investigate signaling mechanisms in human cardiac fibroblasts, using this system as a surrogate in vitro fibrotic read-out to mimic in vivo conditions. This study will also aim to elucidate the underlying effects caused by IRAP inhibition using novel selective pharmacological inhibitors. Techniques: This project will involve a range of methodologies that will include predominantly cell culture work coupled with biochemical measurements and cell microscopy.

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


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DRUG DISCOVERY PROGRAM FOR AT2 RECEPTOR LIGANDS OF THE RAS

Supervisors: Prof Robert Widdop1, Prof Mibel Aguilar2 &

Dr Mark Del Borgo2

Location: Integrative Cardiovascular Pharmacology Group Group 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 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, based on in vitro binding. Project aim: The current projects will use novel ligands, together with clinically-used compounds, to assess the therapeutic potential of AT2R stimulation in two areas of huge unmet clinical need: - the treatment of hypertensive heart disease (HHD) - the treatment of diabetic nephropathy (DN)

Techniques: This project will involve

animal models of HHD or DN

Ex vivo analysis of collagen markers (production and breakdown)

Signaling assays (vasorelaxation, inflammatory, extracellular matrix, biomarkers) These studies will pave the way for future novel therapies that are likely to be beneficial against organ fibrosis in a number of common disease settings. Contact: Prof Robert Widdop Department of Pharmacology Monash University [email protected]


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INHIBITION OF ACID SENSING ION CHANNELS (ASIC) AS A NOVEL TREATMENT FOR STROKE

Supervisors: Dr Claudia McCarthy, Dr Brad Broughton & Prof Robert Widdop

Location: Integrated Cardiovascular Pharmacology Laboratory Department of Pharmacology

Monash University, Clayton Background:

Stroke is the third largest cause of death and the major cause of disability in industrialised countries. Despite this there is only one approved treatment for patients with stroke. Severe oxygen depletion during stroke forces the brain to switch from aerobic to anaerobic respiration, which subsequently results in the production of lactic acid and a drop in the extracellular pH. Studies have demonstrated a direct correlation between brain acidosis and the degree of damage following stroke. Recently it has been discovered that a group of acid sensing ion channels (ASIC), which are activated by acidosis, play a role in propagating the spread of neuronal damage caused by stroke induced acidosis. Recent work in our lab has shown that targeting these ASIC channels dramatically reduce the severity of brain damage in rats. We are currently exploring whether these exciting results may translate into an effective new therapy for the indication of stroke in humans.

Project aim: The aim of this project is to explore the therapeutic potential of ASIC blockers as an effective treatment for stroke. The neuroprotective capacity of a novel ASIC inhibitor will be evaluated in the photothrombotic model of stroke in mice. This study will hopefully establish ASIC as an effective target to prevent the spread of neuronal damage caused by stroke and identify a new drug candidate that may benefit stroke patients.

Techniques:

This study will utilize an integrated perspective and encompasses both in vivo and in vitro techniques. An animal model of stroke will be implemented in conjunction with behavioral tests which provide a symptomatic endpoint. Several physiological parameters will be monitored throughout the experimental protocol. Specific organs will be isolated and preserved to measure stroke-induced changes using histology. The mechanism of protection will be explored using cultured cells to provide further insight into any potential treatment effect.

Contacts: Dr Claudia McCarthy, Dr Brad Broughton Prof Robert Widdop Department of Pharmacology Monash University Phone: 9905 0095, Rm E119 [email protected] [email protected] [email protected]

Untreated brain

Damage

Treated brain



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TARGETING THE CCL18-CCR8 AXIS TO TREAT PULMONARY HYPERTENSION

Supervisors: A/Prof Barbara Kemp-Harper & Dr Brad Broughton Location: Cardiovascular & Pulmonary Pharmacology Group

Department of Pharmacology Monash University, Clayton

Background: Chronic hypoxia-induced pulmonary hypertension is a major cause of death and illness throughout the world. Whilst there has been advancement in the treatment of PH, current treatment is not optimal, and a range of different drug options is needed for better clinical management of PH. Our research group has recently demonstrated that the chemokine, CCL18, which activates the G-protein coupled receptor, CCR8, causes an elevation in systemic blood pressure and associated end-organ damage. Moreover, CCL18 has been found to increase in the lungs of patients with pulmonary disease. Therefore, we hypothesise that the CCL18-CCR8 axis contributes to the development of chronic hypoxia-induced pulmonary hypertension.

Project aim: The aim of this Honours project is to assess the therapeutic utility of pharmacological targeting the CCL18-CCR8 axis to protect against chronic hypoxia-induced pulmonary hypertension. The outcomes of the proposed research are expected to advance our understanding of the role of CCL18-CCR8 axis in pulmonary arteries following chronic hypoxia and thus, lead to the development of a novel therapy for the treatment of pulmonary hypertension.

Techniques: Techniques that will be used during this project include establishing a 4-week mouse model of chronic hypoxia-induced pulmonary hypertension, and involve the measurement of blood pressure and ex vivo assays to assess vascular function (myography), detect inflammation (RT-PCR, superoxide detection), collagen generation (zymography, immunohistochemistry) and pro-fibrotic factors (ELISA). Contacts: A/Prof Barbara Kemp-Harper Department of Pharmacology Monash University Phone: 9905 4674, Rm E117 [email protected] Dr Brad Broughton Department of Pharmacology Monash University Phone: 9905 0915, Rm E140 [email protected]

Stroke

Human amnion epithelial cells


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TARGETING THE CCL18-CCR8 AXIS TO TREAT HYPERTENSION-ASSOCIATED END ORGAN DAMAGE

Supervisors: A/Prof Barbara Kemp-Harper, Prof Rob Widdop,

Dr Brad Broughton

Location: Cardiovascular & Pulmonary Pharmacology Group Department of Pharmacology Monash University, Clayton

Background: Hypertension is a major cause of heart failure, heart attacks and strokes. Associated with the development of hypertension is the accumulation of macrophages in the arterial wall leading to fibrosis and vascular stiffening of central elastic arteries (i.e. aorta, carotid artery). This results in a reduced capacity of these vessels to buffer pulse pressure leading to peripheral vascular and end-organ damage. Whilst current antihypertensives are effective at lowering blood pressure, they don’t necessarily target vascular stiffening and as such new therapeutic approaches are sought. We have exciting new data to suggest that human macrophages release high concentrations of a chemokine, CCL18 which activates the G-protein coupled receptor, CCR8 leading to an elevation in blood pressure and associated end-organ damage. We hypothesise that pharmacological targeting of the CCL18-CCR8 axis will reverse hypertension and associated cardiovascular and renal fibrosis, and protect against end-organ damage. Project aim: The aim of this honours project is to assess the therapeutic utility of pharmacological targeting the CCL18-CCR8 axis to protect against hypertension and the associated end-organ damage. This study may lead to the development of more effective therapies for the treatment of hypertension and fibrosis in cardiovascular pathologies. Techniques: The project will utilize an angiotensin II-infusion model of hypertension in mice, and involve the measurement of blood pressure and ex vivo assays to assess vascular function (myography), detect inflammation (RT-PCR, superoxide detection), collagen generation (zymography, immunhistochemistry) and pro-fibrotic factors (ELISA).

Contacts: A/Prof Barbara Kemp-Harper Department of Pharmacology Monash University Phone: 9905 4674, Rm E117 [email protected]


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TARGETING THE NLRP3 INFLAMMASOME IN THE TREATMENT OF PULMONARY HYPERTENSION

Supervisors: A/Prof Barb Kemp-Harper & Dr Brad Broughton Location: Cardiovascular and Pulmonary Pharmacology Group


Research Interests: Pulmonary hypertension (PH) is an incurable disease and a major cause of death and illness throughout the world. Whilst there has been advancement in the treatment of PH, current treatment is not optimal, with 5 year survival of ~50%. New therapeutic strategies are urgently needed. Our research group has recently demonstrated that macrophages and, in particular the NLRP3 inflammasome, are associated with pathological pulmonary and cardiac remodelling in pulmonary hypertension. Hence, we are interested in studying the impact of NLRP3 inflammasome inhibition of the development of PH and the associated end-organ damage. Project: Assessing the therapeutic utility of pharmacological targeting the NLRP3 inflammasome to protect against chronic hypoxia-induced pulmonary hypertension. Techniques: Techniques that will be used during this project include establishing a 4-week mouse model of chronic hypoxia-induced pulmonary hypertension, and involve the measurement of blood pressure and ex vivo assays to assess vascular function (myography), detect inflammation (RT-PCR, superoxide detection), collagen generation (zymography, immunohistochemistry) and lung histology. Contacts: A/Prof Barb Kemp-Harper Department of Pharmacology Monash University Phone: 9905 4674, Rm E117 [email protected] Dr Brad Broughton Department of Pharmacology Monash University Phone: 9905 0915, Rm E140 [email protected]



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PRECISION CUT LUNG SLICES AS A PLATFORM FOR STUDYING ANTI-FIBROTIC DRUGS

Supervisor: Dr Jane Bourke, Dr Simon Royce1 Co-supervisor: Prof Glen Westall; Dr Jade Jaffar2 Location: 1Respiratory Pharmacology, Monash 2Dept Immunol, Alfred Hospital

Background:

The Respiratory Pharmacology Laboratory is focused on identifying new strategies to prevent and treat chronic diseases that impair lung function, such as idiopathic pulmonary fibrosis (IPF). One marker of disease pathology in IPF that is not targeted by current therapies is fibrosis, which causes scarring of the lung parenchyma, making it difficult to breathe.

Dr Jane Bourke has pioneered the application of precision cut lung slices (PCLS) as an in vitro platform for assessing novel bronchodilator treatments for asthma, while Dr Simon Royce has expertise in the assessment of airway and lung remodeling, including fibrosis. This project will extend the use of PCLS for studies of development and reversal of established fibrosis, comparing recently identified treatments such as pirfenidone (now in clinical use) and relaxin (under investigation). For these studies, PCLS will be prepared from animal models of IPF and from lung samples from patients undergoing transplantation for pulmonary fibrosis obtained through our collaboration with Prof Glen Westall (transplant surgeon) and Dr Jade Jaffar (Alfred Lung Fibrosis (ALF) Biobank).

Project aims:

This project will test the hypothesis that relaxin can inhibit and reverse lung fibrosis in IPF.

Specific aims to be tested may include (i) defining the antifibrotic efficacy of relaxin mimetics relative to and in combination with

pirfenidone in vitro; (ii) exploring the mechanisms underlying protective actions of these agents

Techniques:

This project will combine in vitro assessment of markers of fibrosis using a novel lung slice technique, combining biochemical and molecular assays such as Westerns, RT-PCR, ELISAs etc (to measure collagen expression and release) with established and novel imaging techniques (histology, Histoindex).

This project will be performed largely in Dr Bourke’s laboratory on campus, but will also include components in Prof Westall’s’s laboratory at the Alfred Hospital in Prahran.

Contacts:

Dr Jane Bourke, Dr Simon Royce Dr Jade Jaffar Pharmacology, Monash University Alfred Hospital Phone: 9905 5197 [email protected] [email protected]


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EXPLORING A NOVEL TREATMENT FOR PULMONARY HYPERTENSION Supervisor: Dr Jane Bourke1 Co-supervisors: Dr Helena Qin, Prof Rebecca Ritchie2 Location: 1Respiratory Pharmacology, Monash 2Heart Failure Pharmacology, Baker Heart & Diabetes Institute

Background:

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

Dr Helena Qin and Prof Rebecca Ritchie have shown that the glucocorticoid-regulated protein annexin-A1 (ANX-A1) and drugs that acts at the same receptor have cardioprotective effects (Qin et al., Front Pharmacol 2019; Nature Comm 2017). In an exciting new collaboration with Dr Jane Bourke, they have now shown that this drug class possesses novel vasodilator properties and relaxes pulmonary arteries. These results suggest that synthetic ANX-A1 protein and related non-protein mimetics may represent an alternative or adjunct therapy for the treatment of pulmonary hypertension (PH).

Project aims:

This project will test the hypothesis that ANX-A1 mimetics cause relaxation of pulmonary arteries in a mouse model of PH.

Specific aims to be tested may include (iii) defining the efficacy of ANX-A1 mimetics relative to and in combination with existing pulmonary

vasodilators in vitro; (iv) exploring the mechanisms underlying protective actions of ANX-A1 mimetics; and/or (v) assessing ANX-A1-mediated effects on vascular relaxation in a mouse model of PH.

Techniques:

This project will combine in vitro assessment of vascular reactivity using a novel lung slice technique (to visualise changes in the lumen area of small intrapulmonary arteries in situ), standard organ bath techniques and myography (to measure changes in contractile force in larger arteries) as well as biochemical and molecular assays such as Westerns, RT-PCR, ELISAs etc (to measure receptor expression and changes in cytokines that contribute to PH).

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

Dr Jane Bourke Pharmacology, Monash University Phone: 9905 5197 [email protected] Prof Rebecca Ritchie & Dr Helena Qin Baker Heart & Diabetes Institute 75 Commercial Rd, Melbourne Phone: 8532 1392 [email protected] [email protected]



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FREE FATTY ACID RECEPTORS AS NOVEL BRONCHODILATOR TARGETS FOR ASTHMA

Supervisor: Dr Jane Bourke Co-supervisor: Dr Simon Royce Location: Respiratory Pharmacology, Monash

Background:

The Respiratory Pharmacology Laboratory is focused on identifying new strategies to treat chronic diseases that impair lung function, such as asthma. Currently used bronchodilators such as salbutamol have limited efficacy in severe disease, so new treatments are required to provide improved relief of symptoms of breathlessness.

Dr Jane Bourke has pioneered the application of precision cut lung slices (PCLS) as an in vitro platform for assessing the effects of novel bronchodilator treatments on small intrapulmonary airways, while Dr Simon Royce has established expertise in the assessment of airway reactivity in mouse models of lung disease in vivo. This project will fully characterize the therapeutic potential of agonists of free fatty acid receptors 1 and 4 (FFA1, FFA4), recently identified by us as bronchodilators. Importantly, we have shown that these agonists have efficacy under conditions when the effectiveness of salbutamol is reduced. To support the clinical translation of these findings, their efficacy now needs to be confirmed in animal models of allergic asthma, both in vitro and in vivo.

Project aims:

This project will test the hypothesis that the bronchodilator efficacy of FFA1 and FFA4 agonists is greater than salbutamol in asthma.

Specific aims to be tested may include (vi) confirming airway inflammation, remodeling and hyperresponsiveness in a mouse model of

allergic asthma (vii) defining the dilator efficacy of FFA1 and FFA4 agonists relative to and in combination with

salbutamol in vitro; (viii) exploring the mechanisms underlying dilator actions in disease context.

Techniques:

This project will combine in vitro assessment of relaxation using a novel lung slice technique to visualize airway contraction and relaxation (phase-contrast microscopy) with in vivo studies measuring airway resistance (invasive plethysmography). Pharmacological approaches will be used to establish mechanisms of action (receptor antagonists, pathway inhibitors, calcium signaling). Westerns and RT-PCR will be used to assess FFA receptor expression, while inflammation and remodeling will be measured using ELISAs and morphometric analysis.

Contacts:

Dr Jane Bourke, Dr Simon Royce Pharmacology, Monash University Phone: 9905 5197 [email protected]; [email protected]


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PHARMACOLOGICAL AND BIOCHEMICAL EXAMINATION OF TWO CHINESE SNAKE VENOMS

Supervisors: Professor Wayne Hodgson

Professor Geoff Isbister (University of Newcastle) Location: Monash Venom Group


Background: The Chinese cobra (Naja atra) and sharp nosed pit viper (Deinagkistrodon acutus) are two medically important snakes found in China and surrounding countries. The Chinese cobra is from the family Elapidae (i.e. snakes with small, fixed front fangs) while the sharp nosed pit viper is from the family Viperidae (i.e. snakes with large mobile fangs). Both these species are highly venomous snakes responsible for considerable mortality and morbidity in the region. However, the venoms of these snakes has been poorly studied. Chinese cobra Sharp nosed pit viper

Project aim: The aim of this honours project is to undertake a details pharmacological and biochemical investigation of these venoms and, if possible, isolate and characterize key toxins. The efficacy of commercially available antivenoms against the activity of these venoms/toxins will also be investigated. The primary focus will be on (1) neurotoxicity, (2) cardiovascular activity and (3) pro-coagulant activity. Techniques: This will involve the use of in vitro and in vivo assays including the chick biventer cervicis nerve-muscle preparation (i.e. a skeletal muscle), isolated blood vessels as well as anaesthetized rats. Blood coagulation studies and enzymatic assays will be carried out at the University of Newcastle (NSW) so students will need to be willing to spend 1-2 weeks in NSW (NB. this trip will be funded by the laboratory).

Contact: Professor Wayne Hodgson Deputy Dean, Education, Faculty of Medicine, Nursing & Health Sciences Head, Monash Venom Group, Department of Pharmacology Monash University Phone: 99054861 [email protected]


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KIDNEY ORGANOIDS FOR DISEASE MODELING OF GENETIC KIDNEY DISEASE

Supervisors: Professor Sharon Ricardo and Dr Andrea Wise Location: Kidney Regeneration and Stem Cell Laboratory


Research Interests: The epidemic of chronic kidney disease (CKD) and end-stage renal failure represents a major epidemic world-wide. Inherited renal disease accounts for 50% of children and 20% of adults with the CKD. The landmark discovery of reprogramming adult cells to generate induced pluripotent stem cells (iPSC) has opened an unprecedented opportunity to study inherited kidney disease. Alport syndrome is one of these genetic kidney diseases; it affects 1 in 10,000 individuals and occurs as a result of collagen IVa5 (COL4A5) mutations affecting kidney podocytes. The absence of disease modeling presents a significant challenge in developing targeted therapies for patients with Alport syndrome.

We have generated iPSCs from patients with Alport syndrome that enable us to examine how mutations produce renal disease. Moreover, we are now establishing methods to generate kidney organoids from iPSCs obtained from Alport patients. Kidney organoids self-organize in culture and consist of “multiple cell types” arranged in a three-dimensional (3D) architecture.

This project will use kidney organoids to develop an in vitro 3-D model of X-linked Alport syndrome to facilitate the toxicology of drug screening for Alport diseased cells to better understand Alport podocyte biology, which overcomes inherent limitations in existing disease model systems. This ‘disease in a dish’ model will enable us to explore cell differentiation and how deregulation of gene expression due to Alport mutations causes disease.

Techniques: During the project you will gain hands on experience in the culture of iPSCs and develop new methods for generating 3D kidney organoids in culture. Characterisation of the organoids will involve immunofluorescence and confocal microcopy, and qPCR for the detection of kidney markers. The project will involve the testing of known drug therapies on organoids to compare control and patient-derived kidney organoids.

Contact: Professor Sharon Ricardo Department of Pharmacology Monash University Phone: 9905 0671 [email protected]

Human kidney podocyte from iPSCs



21

EFFECTS OF ADMINISTRATION OF GANAXOLONE IN SPONTANEOUS HYPERTENSIVE RATS

Supervisors: Prof. Geoff Head, Dr Kristy Jackson, Dr Cindy Gueguen Location: Neuropharmacology Laboratory Baker Heart and Diabetes Institute

75 Commercial Rd, Melbourne

Background: There is much evidence that reduced GABA function may be a common finding in neurogenic hypertension such as that observed with Spontaneous Hypertensive Rats (SHR). SHR have reduced GABA turnover in hypothalamus (brain) and altered functioning of GABA receptors compared with their normotensive counterparts. Our lab has shown that allopregnanolone (AlloP), an endogenous neurosteroid and allosteric modulator of GABA receptors, reduced blood pressure in genetically hypertensive Schlager mice, another model of neurogenic hypertension. The mechanism may be related to GABA receptor function improvement leading to reduction in sympathetic nerve overactivity. Project aim: Our aim is to investigate the effectiveness and mechanism of action of treatment with an AlloP analogue ganaxolone, for the treatment of neurogenic hypertension. In this study ganaxolone or vehicle will be administered via minipump for 2 weeks to SHR and normotensive Wistar-Kyoto (WKY) rats. Blood pressure and heart rate will be continuously recorded via radiotelemetry probes. At the end of the experimental period, sympathetic nerve activity will be assessed and GABA receptors contribution to hypertension will be investigated using intracerebral administration of GABA agonists. Techniques: It is anticipated that this will involve the use of radiotelemetric measurement of blood pressure, drug administration using subcutaneous osmotic minipump and stereotaxic injections, recording of sympathetic nerve activity.

Contact: Dr Cindy GUEGUEN Neuropharmacology laboratory Baker Heart and Diabetes Institute Phone: 03 85321390, Level 4, Lab1 [email protected]


22

TARGETING THE Nrf2/NLRP3 AXIS IN DIABETIC HYPERTENSION TO IMPROVE CARDIAC FUNCTION POST MYOCARDIAL INFARCTION

Supervisors: A/Prof Judy de Haan, Prof Rebecca Ritchie, Dr Arpeeta Sharma Location: Oxidative Stress Laboratory,

Baker Heart and Diabetes Institute 75 Commercial Rd, Melbourne.

Background: Cardiac remodeling post myocardial infarction (MI) worsens cardiac function, an often fatal condition

for patients who survive the initial heart attack. Patients who experience a heart attack often present

with several risk factors. Indeed, a worse outcome is predicted for patients with co-morbid diabetes

and hypertension. In this project, the diabetic and hypertensive Schlager mouse will be used to

investigate the interplay of diabetes and hypertension on oxidative stress and inflammation in the

diabetic heart post MI. Mice will be treated with a drug that activates the master regulator of oxidative

stress, namely the transcription factor Nrf2. This project will investigate the effect of drug treatment

on oxidative stress, inflammation and in particular, will focus on the interplay of the Nrf2 transcription

factor and the NLRP3 inflammasome in the diabetic/hypertensive heart post MI.

Project aim: This project aims to investigate whether targeting the Nrf2/NLRP3 inflammasome pathway in a diabetic hypertensive model post MI, will lessen inflammation and oxidative stress to improve endpoints of cardiac remodeling post MI.

Techniques: The student will receive training in in

vivo mouse models of diabetes and

hypertension, Ischemia/reperfusion

injury, cell culture, immune-

histochemistry, histology, real time

PCR and Western Blotting.

Contacts: A/Prof Judy de Haan Oxidative Stress Laboratory Baker Heart and Diabetes Institute Phone: 85321520 [email protected]


23

TARGETING RECEPTOR TYROSINE KINASE AS NOVEL ANTI-FIBROTIC THERAPY FOR CARDIOVASCULAR AND RELATED DISEASES

Supervisors: A/Prof Bing Wang, Prof David Kaye and A/Prof Chrishan Samuel Location: Biomarker Discovery Laboratory and Heart Failure Research

Group, Baker Heart and Diabetes Institute

Background: The pathophysiologic progression to heart failure is a complex process. Initial insults such a myocardial infarction or chronic hypertension, trigger the activation of neurohormonal systems in the body in an attempt to restore cardiac function. However, long-term activation of this system becomes maladaptive by causing cellular and molecular changes in the heart which leads to cardiac remodeling and eventually leading to heart failure. Cardiac fibrosis is one of the key cardiac remodeling processes. Thus, anti-fibrotic therapy could be of particular important in the management of heart failure. We have evidence to suggest a novel receptor tyrosine kinase (RTK) plays an important in fibrosis particularly important for cardiac fibrosis. We have also developed new compound/s that are targeted inhibitors of this pathway that may be of therapeutic potential.

Project aim: The aim of this honours project is to investigate the ability of the RTK inhibitor developed in house and siRNA strategy to blockade the signaling pathway. This study will elucidate the potential anti-fibrotic effects of the RTK inhibitors or siRNAs and may lead to the development of more effective therapies for the treatment of cardiac fibrosis. Techniques: It is anticipated that this will involve the use of assays to detect fibrosis (Western blotting, immunohistochemistry, collagen synthesis analysis), gene expression (RNA extractions and qPCR), primary cell culture of both human and animal cells and immunohistochemistry.

Contacts: A/Prof Bing Wang

Biomarker Discovery Laboratory Baker Heart and Diabetes Institute 75 Commercial Road, Melbourne, 3004 Phone: 9903 0648, or 8532 1178 [email protected] or [email protected]




24

EXPLORING THE ROLE OF ALTERED CARDIAC GLUCOSE METABOLISM IN THE CARDIAC COMPLICATIONS OF DIABETES

Supervisors: Prof Rebecca Ritchie and Dr Miles De Blasio Location: Heart Failure Pharmacology Laboratory Baker Heart and Diabetes Institute

75 Commercial Road Melbourne 3004

Background: Diabetes affects almost 2 million Australians, increasing heart failure risk and accelerating its onset. Our laboratory has an established track record for identifying mechanisms of diabetes- induced cardiomyopathy, many of which target reactive oxygen species (ROS, also known as free radicals). Building on this experience, we have obtained recent evidence that maladaptive cardiac glucose metabolism, via hexosamine biosynthesis (an alternative fate of glucose), has now emerged as a contributing factor to the cardiac complications of diabetes. GENERAL HYPOTHESIS: that the combined impairments in both systemic glucose handling and cardiac levels of ROS together provide an additional drive towards maladaptive cardiac glucose metabolism, negatively impacting cardiac function and mitochondrial integrity. Project aim: To demonstrate that cardiac-directed therapeutic targeting of this axis delays or even overcomes diabetes-induced cardiac dysfunction in the intact heart in vivo. Techniques: In vivo models of diabetic cardiac disease, assessment of cardiac and mitochondrial function, mitochondria isolation. Biochemical techniques: Westerns, ELISA, ROS detection, Seahorse Bioanalyzer, real-time PCR, histology, immunofluorescence.

Contacts: Prof Rebecca Ritchie Heart Failure Pharmacology Baker Heart and Diabetes Institute Phone: 8532 1392 [email protected]


25

USING THE NO REDOX SIBLING NITROXYL TO OVERCOME DIABETES‐INDUCED IMPAIRMENTS IN CARDIAC AND VASCULAR NO SIGNALLING

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


Background: In patients with cardiovascular disease, impaired NO signalling predicts poor outcomes, including mortality. This loss of NO-responsiveness (termed ‘NO-resistance’) is particularly debilitating in type 2 diabetes, where cardiovascular emergencies occur more frequently, but NO-based pharmacotherapies are unable to effectively counteract platelet aggregation and vasoconstriction. We have now obtained the first evidence that the myocardium, like platelets and vessels, is also susceptible to NO-resistance such that NO can no longer enhance cardiac relaxation. However, the novel NO redox sibling, nitroxyl (HNO), may overcome this. This project explores the extent of NO resistance in type 2 diabetes, and whether HNO can overcome this, in the short-term. Whether HNO over the longer-term limits diabetes-induced myocardial dysfunction and changes in cardiac structure (and whether HNO is superior to NO in this context). Putative independent mediators of HNO cardioprotection include cGMP- mediated ROS suppression, and thiol-mediated preservation of cardiac calcium handling proteins, whose activity is abnormally affected in cardiac pathologies such as diabetes. Project aim: To demonstrate that HNO-based strategies may offer new treatment options for cardiac disease. Techniques: In vivo models of diabetic cardiac disease, isolated rodent hearts, assessment of cardiac and vascular function, biochemical techniques: Westerns, ROS detection, ELISA, real-time PCR, histology.

Contacts: Prof Rebecca Ritchie Heart Failure Pharmacology Laboratory Baker Heart and Diabetes Institute Phone: 8532 1392 [email protected]


26

TO UNDERSTAND THE ROLE OF BONE MORPHOGENETIC PROTEIN 7

(BMP7) ON CARDIAC FIBROSIS IN THE DIABETIC HEART

Supervisors: Prof Rebecca Ritchie and Dr Mitchel Tate Location: Heart Failure Pharmacology Laboratory Baker Heart and Diabetes Institute


Background: Diabetes affects almost 2 million Australians, increasing heart failure risk and accelerating its onset. One of the key structural changes in the diabetic heart is cardiac fibrosis, which contributes to the impaired cardiac function evident in diabetes patients. The main signaling pathway driving fibrosis in the diabetic heart is the transforming growth factor-β (TGF-β) pathway. We have recently discovered that cardiac-selective approaches to inhibit this pathway using bone morphogenetic protein 7 (BMP7) have beneficial effects in mouse models of diabetes-induced heart failure. In order to understand these interesting findings further, H9C2 cardiomyocytes will be used to interrogate the BMP7/TGF-β signaling pathway using adeno-associated viruses and BMP7 “superagonists”. Experiments will mimic the diabetes setting by incorporating high glucose conditions into the experiments. These interventions may ultimately limit progression to heart failure and death in diabetes-affected patients. Project aim: To understand the role of bone morphogenetic protein 7 (BMP7) on cardiac fibrosis in the diabetic heart. Techniques: In vitro cell culture, agonists and gene therapy, western blotting, ELISA, real-time PCR, histology, immunofluorescence. Contacts: Prof Rebecca Ritchie Heart Failure Pharmacology Laboratory Baker Heart and Diabetes Institute Phone: 8532 1392 [email protected]


27

COMPARING A CLINICALLY-RELEVANT EXPERIMENTAL MODEL OF

HEART FAILURE WITH PRESERVED EJECTION (HFPEF) FRACTION TO

HUMAN DISEASE

Supervisors: Prof Rebecca Ritchie and Dr Mitchel Tate Location: Heart Failure Pharmacology Laboratory Baker Heart and Diabetes Institute


Background: Heart failure with preserved ejection fraction (HFpEF) is becoming more common globally, accounting for approximately half of all heart failure hospital admissions. Clinically, HFpEF is a very complex disease due to the contribution of several underlying comorbidities, including obesity, hypertension and diabetes. It is generally accepted that the lack of treatment options in HFpEF, is partly due to the lack of a preclinical model that suitably mimic the pathophysiological mechanisms that underlie the disease. The model to be tested utilises high-fat diet and L-NAME to drive metabolic and hypertensive stress, respectively, recapitulating several systemic and cardiovascular features of HFpEF in humans. Identification of a suitable model of HFpEF will help understand disease pathogenesis and drive development of effective therapeutics. Project aim: To compare this animal model of HFpEF to human disease. Techniques: in vivo models of HFpEF, assessment of cardiac function (echocardiography and cardiac catheterisation), metabolic caging, western blotting, ROS detection, real-time PCR, histology, immunofluorescence Contacts: Prof Rebecca Ritchie Heart Failure Pharmacology Laboratory Baker Heart and Diabetes Institute Phone: 8532 1392 [email protected]


28

INVESTIGATING NOVEL THERAPEUTIC TARGETS FOR OBESITY-INDUCED HEART FAILURE

Supervisors: Dr Waled Shihata, Dr Bing Wang, A/Prof Barbara Kemp-Harper & Prof David Kaye

Location: Heart Failure Research Group, Baker Heart and Diabetes Institute, Melbourne

Background: Heart failure, the most common chronic cardiovascular condition, can be divided into two main subtypes: heart failure with reduced ejection fraction (HFrEF), where systolic or the pumping function of the heart is impaired, and heart failure with preserved ejection fraction (HFpEF), which is characterised by impaired filling capabilities (known as diastolic dysfunction). Whilst the incidence of HFrEF is declining as a result of effective treatments, HFpEF continues to be a growing problem due to the increasing ageing population as well as a lack of targeted and effective treatments (Figure 1). Moreover, both patients and animal models of HFpEF present with cardiac fibrosis which may promote heart stiffening and diastolic dysfunction. However, the mechanisms by which these cellular events lead to the pathogenesis of HFpEF remain unclear. Importantly, clinically HFpEF patients present with multiple cardiovascular risk factors, including obesity, which exacerbates their condition. There have been reports of increased cardiac and renal fibrosis, increased blood pressure and inflammation in the setting of obesity, which are all evident in HFpEF. Targeting secondary risk factors such as obesity and subsequent inflammatory and fibrotic events may represent a novel strategy to treating patients with HFpEF. We have evidence that a novel anti-fibrotic, anti-inflammatory agent known as VCP979 may prevent cardiac stiffening and improve cardiac function in the setting of heart failure.

Project aim: The aim of this honours project is to investigate the mechanisms by which obesity promotes heart failure and the effects of VCP979 on the condition. This study will have significant implications for the treatment of HFpEF associated with cardiovascular risk factors by identifying a novel therapeutic treatment strategy for this patient population.

Techniques: It is anticipated that this project will involve developing and monitoring an animal model of diet-induced CVD, histological and biochemical analysis of fibrosis and inflammation, behavioural and functional assessments (echocardiography, metabolic cages, echoMRI), and assessing immune populations via flow cytometry.

Contacts: Dr Waled Shihata Heart Failure Research Group Level 3, Lab 4 Baker Heart and Diabetes Institute Phone: 8532 1486 [email protected]

Figure 1



29

MECHANISMS FOR DIFFERENTIAL GPCR:TRANSDUCER COUPLING

Supervisors: Dr. Sebastian Furness Location: Receptor transducer coupling lab Monash Institute of Pharmaceutical Sciences

Monash University, Parkville

Background: G protein coupled receptors can be activated to a varied extent by different ligands. This is the basis for partial efficacy, which has been exploited for a subset of medicines. We have previously shown that the calcitonin receptor, when activated by different peptides, stimulates distinct rates of nucleotide exchange at its G protein transducer. This occurs due to a ligand-promoted difference in the nucleotide affinity of the G protein. Unanswered is the question of whether this is due to absolute conformational differences or differences in protein dynamics.

Project aim: The project will aim to establish the relative contribution of ligand promoted differences in transducer conformation compared with ligand promoted differences in transducer conformational dynamics in determining signalling efficacy. Techniques: The project will involve the use of bi-orthogonal fluorescent labelling strategies on G proteins. Interaction of these fluorescent labels will then be investigated using single molecule dynamics with an emphasis on the utility of (Fluorescence Lifetime Imaging Microscopy – Fluorescence Resonance Energy Transfer) FLIM-FRET in addition to a suite of other techniques.

Contacts: Dr Sebastian Furness Monash Institute of Pharmaceutical Sciences Monash University, Parkville (399 Royal Parade) Phone: 9903 9055, Building 4 Rm 8.09 [email protected]


30

THE EFFECT OF CHOLESTEROL ON SATIETY

Supervisors: Dr. Sebastian Furness Location: Receptor transducer coupling lab Monash Institute of Pharmaceutical Sciences


Background: There has been a poor record of success in the pharmacological treatment of obesity. The cholecystokinin 1 receptor (CCK1R) regulates some gastrointestinal functions and also performs an anorexigenic role as part of the gut-brain axis. This appetite supressing role makes CCK1R a good target as an anti-obesity therapeutic. In spite of this, an orally available small molecule CCK1R agonist failed to perform better than diet and exercise in a clinical trial on overweight and obese patients. We know that CCK1R signalling is modulated by membrane cholesterol. Paradoxically high cholesterol increases CCK1R agonist affinity whilst simultaneously decreasing response potency (efficacy). Thus, high membrane cholesterol is interfering with coupling of the receptor (CCK1R)

to its transducer (Gq).

Project aim:

The project will involve the investigation of the biophysical basis of CCK1R:Gq in the presence of different levels of membrane cholesterol along with investigation of a cholesterol insensitive mutant of the CCK1R. This project will form part of an ongoing collaboration with Larry Miller M.D. at the Mayo clinic in Arizona. Techniques: It is anticipated that this will involve the use of calcium flux assays, a variety of biophysical assays such as conformational measurement using bioluminescence resonance energy transfer, fluorescence resonance energy transfer, and native PAGE along with fluorescence lifetime imaging microscopy.

Contacts: Dr Sebastian Furness Monash Institute of Pharmaceutical Sciences Monash University, Parkville (399 Royal Parade) Phone: 9903 9055, Building 4 Rm 8.09 [email protected]


31

TARGETING THE CALCIUM SENSING RECEPTOR TO TREAT ASTHMA AND ENDOCRINE DISORDERS

Supervisors: Drs Katie Leach, Andrew Keller & Tracy Josephs Location: Endocrine and Neuropharmacology lab Drug Discovery Biology Theme

Monash Institute of Pharmaceutical Science, Parkville

Background: The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that responds to multiple stimuli, including cations (e.g. Ca2+) and polyamines (e.g. spermine). It is a current drug target in hyperparathyroidism, where positive allosteric modulators (PAMs; e.g. cinacalcet) potentiate CaSR signalling. The CaSR is also a putative target for PAMs that rescue signalling at ~200 loss-of-function/expression naturally occurring CaSR mutations that cause endocrine disorders. Negative allosteric modulators (NAMs) are also sought to inhibit signalling at ~200 gain-of-function mutations, and polyamine-mediated CaSR signalling in the lungs of asthmatic patients. In conjunction with medicinal chemists, we have developed novel CaSR PAMs and NAMs, however, CaSR drug discovery remains impeded by limited understanding of CaSR structure, function and drug actions.

Project aims: Multiple project areas are available that aim to facilitate our development of CaSR-targeting drugs for asthma and endocrine disorders, by seeking to:

1. characterise PAMs and NAMs that correct naturally occurring mutation signalling and expression to treat endocrine disorders;

2. characterise NAMs that inhibit polyamine-mediated CaSR signalling to treat asthma; 3. elucidate CaSR structures, drug binding sites and structural changes upon agonist and drug

binding;

Techniques: 1. HEK293 cell high throughput signalling assays, bioluminescent trafficking assays, in-house

fluorescent PAMs, and analytical pharmacology will be used to evaluate the effect of PAMs and NAMs on naturally occurring mutant signalling and expression;

2. HEK293 cell high throughput signalling assays and analytical pharmacology will be used to characterise signalling pathways important for polyamine-mediated airway contraction, and the ability of NAMs to inhibit these pathways;

3. CaSR structures and binding sites will be studied using mutagenesis, cryoEM, x-ray crystallography and mass spectrometry techniques;

Contacts: Dr Katie Leach Phone: 03 99039089 [email protected]

Figure 1 The CaSR. The CaSR is a dimer consisting of two protomers, each possessing multiple agonist and drug binding sites. Different agonists and drugs stimulate or inhibit different downstream signalling pathways mediated by G proteins (e.g. Gq, G11 etc). We seek to elucidate CaSR structure, function and drug actions.



32

ALLOSTERIC MODULATION OF GLUTAMATE RECEPTORS FOR PSYCHIATRIC AND NEUROLOGICAL DISORDERS

Supervisors: Dr Karen Gregory, Dr Shane Hellyer &

Dr Katie Leach Location: Endocrine & Neuropharmacology lab Drug Discovery Biology


Background: Our lab is interested in the biology and therapeutic potential of targeting metabotropic glutamate receptors using small molecule allosteric modulators. We are predominantly focused on metabotropic glutamate receptor subtype 5 (mGlu5), as it is an exciting new target for schizophrenia, Alzheimer's disease, and depression. We are pursuing a novel class of therapeutics, called allosteric modulators, to selectively target these receptors. To facilitate rational drug design and discovery efforts, a better understanding of the functional consequences and structural basis of allosteric modulation is needed. Project aim: Multiple projects are available within our team to investigate:

the structural basis of mGlu5 activation and allosteric modulation

the influence of dimerisation on allosteric modulator pharmacology

the impact of chronic vs acute exposure to allosteric modulators on mGlu5 activity

how different compounds influence receptor trafficking and signalling from different subcellular compartments as well as in different brain cell types .

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 labelling, immunoblotting, computational modelling, medicinal chemistry and single cell imaging.

Contact: Dr Karen Gregory Department of Pharmacology & Drug Discovery Biology Monash Institute of Pharmaceutical Sciences Monash University Phone: 9903 9243, Rm 4.346 Building 4, Parkville [email protected]


33

MODULATION OF G PROTEIN-COUPLED RECEPTOR FUNCTION BY RECEPTOR ACTIVITY MODIFYING PROTEINS

Supervisors: A/Prof Denise Wootten and Dr Elva Zhao Location: Metabolic Receptor Biology group Drug Discovery Biology

Monash Institute of Pharmaceutical Sciences, Parkville.

Background: Receptor activity modifying proteins (RAMPs) are a class of single pass transmembrane accessory proteins consisting of three members (in mammals) that are ubiquitously expressed in the body where they interact with G protein coupled receptors (GPCRs); the largest group of signal-transmitting cell surface membrane protein’s in the human genome. RAMPs have substantial capacity for introducing functional diversity through their interactions with GPCRs where they can act as chaperones, alter ligand specificity, alter signalling and/or regulate trafficking in a receptor-dependent manner. This has the potential to result in differing selectivity’s for both ligand interactions and signalling outputs from a given GPCR depending on the tissue localisation and the RAMP expression profiles in different tissue beds. The majority of identified RAMP interacting receptors belong to the class B GPCRs with nine (of fifteen) receptors from this family published to interact with at least one of the RAMPs. Class B receptors are well established targets for multiple diseases including metabolic, cardiovascular and neurogenerative diseases, however for the majority of these receptors, the influence of RAMPs on their pharmacology is still poorly understood. Given the broad expression profiles of RAMPs within the body and the clinical importance of Class B GPCRs, it is important to consider their influence on class B GPCRs for drug discovery. Project aim: The aim of this project is to determine the influence of RAMPs (RAMP1-3) on the pharmacological profiles of a range of class B GPCRs where physical interactions with RAMPs have been identified (calcitonin receptors, glucagon receptor, secretin receptor, corticotropin releasing factor receptor 1, vasoactive intestinal receptors (1 and 2)). Techniques: This project will use a broad range of techniques including cell culture, transient transfections to express different receptor/RAMP combinations in recombinant cells, ligand-receptor interactions using radioligand binding techniques, interactions with transducer and regulatory proteins using a range of luminescence and resonance energy transfer assays, signalling efficacy using a range of state-of-the-art signalling assays (ie, cAMP, ERK1/2 phosphorylation, Ca2+ mobilisation) and receptor trafficking using cell surface enzyme-linked immunosorbent assays. Contacts: A/Prof Denise Wootten Monash Institute of Pharmaceutical Sciences Monash University Parkville Campus [email protected]


34

ALTERNATIVE SPLICING OF THE PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PAC1) RECEPTOR



Background: PAC1 receptor belongs to the class B G protein-coupled receptor superfamily and is pleiotropically coupled to a diverse range of downstream signalling pathways. This receptor is activated by the peptide ligand PACAP with high affinity, including PACAP38 and PACAP27, and by the ligand VIP with lower affinity. The distribution of PAC1 and its ligands PACAP and VIP in a variety of cell types is reminiscent of the pleiotropic functions of their receptors in development and physiology where PAC1 receptor signalling controls a variety of cellular and physiological responses, such as differentiation, proliferation, cell cycle regulation, neurotransmitter, and hormone release and adaptation to stressful challenges. One hypothesis is that this abundance of PAC1 receptor-mediated physiological activities may arise through the fine-tuning of signalling by the generation of a set of alternatively spliced PAC1 receptor variants. The PAC1 gene contains multiple exons that undergo extensive alternative splicing with the most common splice variants including (i) variations in the receptor N-terminal domain that can alter ligand binding specificity and affinity and (ii) variations in the exon encoding intracellular loop 3 (ICL3) that have the potential to alter interactions of the receptor with intracellular signalling partners; this altering its overall signalling profile. While alternative splice variants have been identified in different cell types and tissues, the effects of this splicing on the downstream signalling profiles mediated by the PAC1 receptor are still poorly understood. Project aim: The aim of this project is to determine the downstream signalling and regulatory profiles mediated by a range of ligands at different PAC1 receptor variants. This will provide greater understanding of the importance of alternative splicing on PAC1 receptor function. Techniques: This project will use a broad range of techniques including cell culture and transient transfections to express different receptor splice variants in recombinant cells, competition radioligand binding assays to assess ligand-receptor interactions, a range of luminescence and resonance energy transfer assays to assess interactions with transducer and regulatory proteins, alphascreen, HTRF and fluorescence based assays to assess second messenger (ie, cAMP , Ca2+ mobilisation) and kinase signalling (ie, ERK1/2 and Akt phosphorylation) and cell surface enzyme-linked immunosorbent assays to assessed receptor internalisation and recycling. Contacts: A/Prof Denise Wootten Monash Institute of Pharmaceutical Sciences Monash University Parkville Campus [email protected]


35

UNDERSTANDING THE MOLECULAR MECHANISMS OF GLUCAGON-LIKE PEPTIDE-1 AND GLUCOSE-DEPENDENT INSULINOTROPIC

POLYPEPTIDE RECEPTOR DUAL AGONISTS



Background: The glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) are clinical targets for treatment of type 2 diabetes and obesity. Combining the beneficial effects of targeting each receptor, poly agonists capable of activating both the GLP-1R and GIPR have recently gained attention with three GLP-1R/GIPR dual-agonist peptides in different phases of clinical trials. While these are reported to have superior body weight- and glucose- reducing efficacy in comparison to approved GLP-1R therapies, these three agonists differ in their vivo efficacies and the severity of their gastrointestinal side-effects. These peptides were specifically developed with respect to their potencies in cAMP production with the different peptides having distinct efficacies at each receptor. This suggests that an optimised relative potency ratio toward each receptor may be required to reach the maximal beneficial therapeutic outcome. Both the GLP-1R and GIPR are capable of coupling to a diverse array of signalling cascades and the extent to which the dual agonists in clinical trials alter the full signalling profile of these receptors has not been explored. Additionally, these receptors are co-expressed and whether the improved metabolic outcomes from these agonists is due to a synergistic combination of GLP-1R and GIPR signalling crosstalk or receptor oligomerisation is not known. These are major knowledge gaps in the development and optimal therapeutic exploitation of dual GLP-1R/GIPR agonists.

Project aim: The aim of this project is to establish detailed pharmacological profiles for different GLP-1R/GIPR dual agonists through comprehensive characterisation of the signalling and regulatory profiles at either the GLP-1R or GIPR alone when both are co-expressed. This will provide a mechanistic framework for understanding distinct activities of different dual agonists and will facilitate development of GLP-1R/GIPR dual with optimised pharmacology. Techniques: This project will use a broad range of techniques including cell culture, fluorescent ligand binding assays, cellular signalling assays (ie, cAMP, ERK1/2 phosphorylation, Ca2+ mobilisation) and resonance energy transfer assays to detect receptor-receptor interactions, receptor interactions with transducers and regulatory proteins such as G proteins and β arrestins, and to track receptor trafficking through different cellular compartments. Contacts: A/Prof Denise Wootten Monash Institute of Pharmaceutical Sciences Monash University Parkville Campus [email protected]

Department of Pharmacology Honours Projects 2020 · Research Seminars (introductory & final) Thesis...

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Transcript of Department of Pharmacology Honours Projects 2020 · Research Seminars (introductory & final) Thesis...