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Department of Pharmacology, Honours 2014

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Department of Pharmacology

Honours Projects 2014

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 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. 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 2014 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 BBNS 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


rd year

level, including at least 18 points in 3

rd year PHA units


rd year level,

including 12 points in 3

rd year core BMS

units and 12 points in other 3

rd year units*

Application closing date

15th

November 2013 15th

November 2013 31st

October 2013 15th

November 2013

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

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

http://www.med.monash.edu.au/psych/course/4thyear/bbns-honours-apply.html

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

Commencement date

24th February, 2014 24

th February, 2014 24

th February, 2014 24

th February, 2014

* 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

Assessment of scientific writing

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, 2014. 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 15th November to:

Science Faculty Office (BSc, BBiotech students)

SOBS Office, Building 77 (BMS students)

For Bachelor of Behavioural Neuroscience Honours students, application forms must be

submitted by Thursday October 31st to:

Emily Cavanagh, School of Psychology & Psychiatry, Building 17

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

December 2013. 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 2014.



Honour Projects 2014 – On campus projects

LABS / SUPERVISOR(S)

PROJECT TITLE

Clinical & Experimental Toxicology Group Andis Graudins, Dimitri Gerostamoulos

The effects of the timing of intravenous lipid emulsion administration on absorption and bioavailability of orally administered lipophilic toxicants in a rodent model of oral amitriptyline toxicity

Education-based projects Eva Patak, Liz Davis

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 Claudia McCarthy, Rob Widdop Emma Jones, Mark Del Borgo, Rob Widdop Sanjaya Kuruppu (Biochemistry), Rob Widdop

Cardio-protective effects of incretin hormones: potential beyond glycaemic control?

Inhibition of acid sensing ion channels (ASIC) as a novel treatment for stroke

Drug discovery program for AT2 receptor ligands

Predicting complications associated with haemorrhagic stroke

Cardiovascular Immunology 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 Barb Kemp-Harper Barb Kemp-Harper, Grant Drummond Helena Kim, Chris Sobey Sophocles Chrissobolis, Chris Sobey

Are the adverse effects of hormone replacement therapy on stroke outcome mediated by G protein-coupled estrogen receptor signalling?

Exploring the vasoprotective actions of novel HNO donors

Exploring the role of NOX5 in vascular function

Inflammatory mechanisms in ischaemic stroke

Investigating the role of aldosterone in promoting inflammation during atherosclerosis

Oxidant and Inflammation Biology Group Stavros Selemidis

Investigations into the roles of NAPDH oxidases in Influenza A virus indcued lung injury

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 2014 – 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 Jennifer Irvine, Karen Andrews, Barb Kemp-Harper Geoffrey Head

Strategies to rescue diabetes-induced heart failure

Nitroxyl protects against the causes of heart failure

Annexin-A1 protects against cardiac inflammation

Effects of positive allosteric modulator of GABAA receptors on hypertension and stress

Central mechanisms in the control of blood pressure

Centre For Eye Research Australia Hitesh Peshavariya, Grant Drummond

Targeting NADPH oxidase using pharmacological inhibitors for treatment of ocular neovascularisation

Department of Forensic Medicine Dimitri Gerostamoulos, Jennifer Pilgrim

The use of dried blood spots (DBS) in forensic toxicology

Drug Discovery Biology Theme Monash Institute of Pharmaceutical Sciences Katie Leach Karen Gregory, Arthur Christopoulos Nigel Bunnett, Bill Graham, Nicholas Barlow

Nigel Bunnett, Chris Porter Nigel Bunnett, Dane Jensen Nigel Bunnett, TinaMarie Lieu Nigel Bunnett, Meri Canals, Daniel Poole Nigel Bunnett, Nicholas Barlow Nigel Bunnett, Paul Myles, Nicholas Veldhuis Nigel Bunnett, Elva Zhao Nigel Bunnett, Daniel Poole

Tissue-specific role of biased signalling at the CaSR

Probing an allosteric binding site in the human calcium sensing receptor

Allosteric pharmacoregulation of a “headless” calcium sensing receptor

Biased allosteric modulators of metabotropic glutamate receptor 5: a novel therapeutic strategy for CNS disorders

Illuminating inflammatory signalling

Intracellular drug delivery: a route to more selective and effective treatments for disease

Trafficking of G protein-coupled receptors: a new pathway to pain

Mechanosensation in the nervous system: implications for visceral pain

Compartmentalized signalling of opioid receptors

Protease-activated receptors: mediators of the inflammation and pain

A window into pain: protease signalling in pain states

Regulation of PAR2 signaling by RGS proteins

Bile acid signalling in the intestine: a new target for digestive and inflammatory diseases



THE EFFECTS OF THE TIMING OF INTRAVENOUS LIPID

EMULSION ADMINISTRATION ON ABSORPTION AND

BIOAVAILABILITY OF ORALLY ADMINISTERED LIPOPHILIC

TOXICANTS IN A RODENT MODEL OF ORAL AMITRIPTYLINE

TOXICITY

Supervisors: Professor Andis Graudins1

Dr Dimitri Gerostamoulos2

Location: Clinical and Experimental Toxicology Laboratory1


Monash University, Clayton

Victorian Institute for Forensic Medicine (VIFM)2

Kavanagh Street

Southbank, Melbourne

Background:

Intravenous lipid emulsion (ILE) is recommended as an adjunctive therapy in the treatment

of severe cardiovascular drug poisoning after overdose with lipophilic drugs such as the

tricyclic antidepressant amitriptyline. It is postulated to have a number of mechanisms of

action, however, the most accepted is the ‘lipid sink theory’. It is postulated that ILE

creates an intravascular ‘lipid sink’ of drug, preventing its delivery to target organs such as

the heart.

Little is known regarding the effect of timing of ILE administration with respect to timing

of oral toxicant ingestion. A pilot study in our lab suggested that early treatment with ILE

was associated with significantly higher blood amitriptyline concentration and poorer

survival than animals treated with standard antidotes for this poisoning. It is currently

unknown whether this effect is related to enhanced absorption or increased bioavailability

in the presence of ILE treatment.

Project aims: To assess the pharmacokinetics and toxicity of amitriptyline in moderate

overdose following early and late ILE administration in a rodent model. Does early ILE

administration increase drug bioavailability and worsen toxicity?

Techniques: Utilising anaesthetized whole animal model of amitriptyline toxicity with

invasive haemodynamic monitoring. Blood samples to be collected and defined time

intervals. ILE to be administered early at time of oral drug delivery and late after drug

absorption. Drug assays to be performed by the student after training using GC/MS, at

the VIFM, Southbank. Pharmacokinetic assessment of blood drug concentrations.

Assessment of physiological outcomes and survival.

Contact: Prof Andis Graudins: [email protected]




EVALUATION & DEVELOPMENT OF WEB-BASED

PHARMACOLOGY RESOURCES

Supervisors: Dr Elizabeth Davis & Dr Eva Patak

Location: Pharmacology Education Research Initative



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: Dr Chrishan Samuel & Dr Simon Royce

Location: Fibrosis Laboratory (Rms E101/E102)



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:

Dr Chrishan Samuel and Dr Simon Royce Department of Pharmacology

Monash University

Phone: 9902 0152 / 9905 0913

[email protected]

[email protected]



CARDIO-PROTECTIVE EFFECTS OF INCRETIN HORMONES:

POTENTIAL BEYOND GLYCAEMIC CONTROL?

Supervisors: Dr Tracey Gaspari & Prof Robert Widdop

Location: Cardiovascular Integrative Group



Collaborator: Dr Anthony Dear

Deputy Director, Eastern Clinical Research Unit, Monash University

Background:

Our research focuses on understanding the pathophysiological processes involved in the

development of cardiovascular disease, one of the major causes of illness and death in

Australia and worldwide.

Project aim:

Two classes of diabetic drugs targeting the glucagon-like peptide-1 (GLP-1) pathway are

used clinically to treat Type 2 Diabetes Mellitus. These are GLP-1 receptor agonists

(e.g., Liraglutide) and dipeptidyl peptidase-4 (DPP-4) inhibitors (prolong ½ life of

endogenous GLP-1). In collaboration with Dr Anthony Dear we aim to investigate the

vascular and cardio-protective effect of these classes of drugs in a number of

cardiovascular disease states, including atherosclerosis, vascular injury (neointimal

hyperplasia) and cardiac remodeling.

Techniques:

Projects will incorporate a range of methodologies including: animal dietary and

pharmacological treatments, surgical techniques for vascular injury models,

immunohistochemistry, biochemical measures (western blot, markers of cellular

proliferation and hypertrophy), histological and morphological analyses (such as fibrosis,

intimal to medial measurements).

Contacts:

Dr Tracey Gaspari and Prof Robert Widdop Department of Pharmacology

Monash University

Phone: 9905 4762, Rm E119

[email protected]

[email protected]

Liraglutide

I

M

Vehicle

Figure 1: Representative images of cross-

sectional lesion development in aortic arch of

ApoE KO mice. Liraglutide treatment shows

decreased intima (I) development. Intima (I) and

media (M) areas are identified by dotted line.



Untreated brain

Damage

Treated brain

INHIBITION OF ACID SENSING ION CHANNELS (ASIC) AS A

NOVEL TREATMENT FOR STROKE Supervisors: Dr Claudia McCarthy & Prof Robert Widdop.

Location: Integrated Cardiovascular Pharmacology Laboratory



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. Targeting these ASIC channels to prevent activation may be a new and

effective approach to reduce the severity of brain damage in patients with acute

stroke.

Project aim:

The aim of this project is to explore the therapeutic potential of ASIC blockers as a

novel treatment for stroke. The neuroprotective capacity of three ASIC inhibitors will

be evaluated in a clinically relevant model of stroke. This study will hopefully establish

ASIC as an effective target to prevent the spread of neuronal damage caused by

stroke and identify a new class of drug 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. In

addition, peripheral changes associated with stroke will be evaluated to; firstly

determine whether stroke severity can be accurately measured using these biomarkers;

and secondly to assess whether a drug effect is reflected by these endpoints.

Contacts: Dr Claudia McCarthy and Prof Robert Widdop


Monash University

Phone: 9905 0095, 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


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 (e.g. beta-substituted; D- substituted amino

acids). 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 assays

Contacts: Dr Emma Jones & and Prof Robert Widdop


Monash University

Emma.Jones @monash.edu

[email protected]




PREDICTING COMPLICATIONS ASSOCIATED WITH

HAEMORRHAGIC STROKE

Supervisors: Dr Sanjaya Kuruppu1 & Prof Rob Widdop2

Location: Departments of Biochemistry1 & Pharmacology2

Monash University, Clayton.

Background:

Haemorrhagic stroke (or Subarachnoid Haemorrhage) is characterized by the rupture

of a blood vessel on the surface of the brain. It is a medical emergency requiring

prompt neurosurgery, and nearly 50% of patients don’t survive. Nearly a third of the

patients who survive develop lifelong debilitating neurological deficits. At present there

are no methods to accurately identify patients who develop these complications. Early

identification will enable appropriate treatment strategies to be implemented, in order

to prevent or reduce the impact of these neurological deficits.

Project: Soluble Angiotensin Converting Enzyme-2 as a marker of Complications

Associated with Subrachnoid Haemorrhage (SAH).

Project aims: Angiotensin Converting Enzyme-1 (ACE-2) is one of the enzymes that play

a key role in the cardiovascular system. Its known functions include the breakdown of

Angiotensin I and II. The enzyme is expressed by endothelial cells lining blood vessels,

as well as in the heart and kidney. Although initially thought to be bound to the plasma

membrane, recent studies have shown the presence of a circulating form of ACE-2 in

human biological fluids. This project aims to examine if levels of ACE-2 in the biological

fluids of patients with SAH, can be used to predict the onset of complications

associated with SAH.

Techniques: The study will involve working with human biological fluids, enzyme assays

and various biochemistry techniques such as electrophoresis and mass spectrometry.

Contacts:

Dr Sanjaya Kuruppu1 and Prof Rob Widdop2

Departments of Biochemistry1 & Pharmacology2

Monash University

[email protected]

[email protected]





MECHANISMS OF INFLAMMATION AND IMMUNITY DURING

THE DEVELOPMENT OF HYPERTENSION

Supervisors: Dr Antony Vinh

Location: Cardiovascular Immunology Group



Background:

Inflammation and immunity continue to be a topical area of hypertension research. T cells

and the adaptive immune system are known to play a vital role in the development of

hypertension. We have reported that dendritic cells, which are also known as professional

antigen presenting cells (APCs), potentially present hypertension-specific antigens to

induce T c, dendritic cells can also induce tolerance to prevent T cells from attacking self-

antigens in the body. Therefore, harnessing this tolerogenic potential may represent a

novel antigen-specific approach to prevent hypertension.

Project aim:

The aim of this honours project is to investigate the ability of bone marrow-derived

dendritic cells that have been rendered tolerogenic (inhibits T cell activation), to induce

tolerance and inactivate hypertension specific-T cells.

Techniques:

Several techniques will be used in this project including harvesting bone marrow and its

differentiation into dendritic cells, primary cell culture techniques to induce tolerance

(with/without cell lysate antigens), flow cytometry to confirm tolerogenic DCs (tDCs)

have been created and mixed leukocyte reactions to detect whether tDCs will induce

tolerance in T cells isolated from a hypertensive mouse. Based on outcomes and if time

permits, we will perform adoptive transfer of tDCs into hypertensive mice to observe

whether tDCs can be used as a vaccine to prevent hypertension.

Contacts:

Dr Antony Vinh Department of Pharmacology

Monash University

Phone: 9902 4844, Rm E33

[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]




ARE THE ADVERSE EFFECTS OF HORMONE REPLACEMENT

THERAPY ON STROKE OUTCOME MEDIATED BY G PROTEIN-

COUPLED ESTROGEN RECEPTOR SIGNALLING?

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 treatments is vital to reduce the damage

caused by stroke. In recent years, growing evidence has revealed that estrogen is

neuroprotective against stroke, but confusingly hormone replacement therapy (HRT)

worsens stroke outcome in post-menopausal women. Interestingly, recent work from our

lab has found that activation of a novel estrogen receptor (G Protein-coupled Estrogen

Receptor, GPER), which is widely distributed throughout the brain, increases cerebral

infarct damage in males following stroke. Therefore, we hypothesise that GPER

activity affects outcome of HRT therapy in females after stroke.

Project aim:

The aim of this project is to investigate the importance of GPER in mediating the

adverse effects of HRT in stroke. The outcomes of the proposed project are expected to confirm that estrogen therapy has an adverse effect on stroke outcome in young ovariectomised mice due to activation of GPER signalling.

Techniques:

Techniques that will be used during this project include treating mice with estrogen and

various GPER ligands, histochemical approaches to measure cerebral infarct damage,

immunohistochemistry to examine the distribution of GPER in the brain and Western

blotting to determine GPER expression levels.

Contacts:

Dr Brad Broughton & A/Prof Chris Sobey Department of Pharmacology

Monash University

Phone: 9905 0915, Rm EG20

[email protected]

[email protected]

Vehicle Estradiol



EXPLORING THE VASOPROTECTIVE ACTIONS OF

NOVEL HNO DONORS

Supervisors: Dr Barbara Kemp-Harper




Background:

Nitric oxide (NO), is an important endogenous vasodilator and regulator of vascular tone.

In disease states such as hypertension and atherosclerosis, the NO signaling pathway is

impaired leading to reduced blood flow to vital organs such as the brain and heart. Whilst

nitrovasodilators can be used to overcome dysfunctional NO signaling, the clinical

application of these agents is limited due to their susceptibility to scavenging by

superoxide and tolerance development.

Importantly, NO can also exist in the reduced state as nitroxyl (HNO) and recent

evidence suggests that this nitrogen oxide is also generated endogenously and has distinct

pharmacological properties and therapeutic advantages over NO. Thus HNO is not

scavenged by superoxide, does not develop tolerance and can target distinct signaling

pathways to cause vasorelaxation. As such, the bioavailability of HNO is preserved in

disease. With a suite of vasoprotective actions, HNO donors have therapeutic potential in

the treatment of cardiovascular disease. Currently, however the clinical utility of these

donors is limited by their short-half lives and byproducts. Excitingly, new pure HNO

donors with longer half-lives are being developed and this study will elucidate the

therapeutic potential of these drugs in the setting of atherosclerosis.

Project aim:

The aim of this honours project is to investigate the ability of novel HNO donors to serve

as vasoprotective agents in the setting of atherosclerosis.

Techniques:

It is anticipated that the project will involve the use of techniques to assess the

vasodilator (small vessel myography), anti-aggregatory and superoxide suppressing

(chemiluminescence) actions of novel HNO donors in control and atherosclerotic mice.

Contacts:

Dr Barbara Kemp-Harper Department of Pharmacology

Monash University

Phone: 9905 4674, Rm E140

[email protected]

HNO VSMC Relaxation Proliferation

.

O2

-

.

O2

-

.

O2

-

Superoxide

Production

Platelet Aggregation



EXPLORING THE ROLE OF NOX5 IN VASCULAR FUNCTION

Supervisors: Dr Barbara Kemp-Harper & A/Prof Grant Drummond




Background:

Oxidative stress is a major cause of the vascular inflammation, remodeling and

dysfunction associated with hypertension and atherosclerosis. NOX5 is a recently

described reactive oxygen species (ROS)-generating enzyme, which in humans is

expressed in 3 of the major cell types involved in vascular disease (endothelial, vascular

smooth muscle and macrophages). Moreover, NOX5 expression is upregulated in the

vascular wall of coronary artery disease patients. However, the absence of NOX5 from

the rodent genome has hampered efforts to determine whether it directly contributes to

the pathogenesis of vascular disease. In this proposal, we will utilize 'humanized' mouse

models of NOX5 expression to assess NOX5 as a contributor to, and target for therapy

in, vascular injury during hypertension and atherosclerosis.

Project aim:

The aim of this honours project is to investigate the role of endothelial NOX5 in the

modulation of vascular function. This study may lead to the development of more effective

therapies for the treatment of vascular disease.

Techniques:

The project will utilize mice which transgenically express NOX5 in endothelial cells 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




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

triggers a cascade of complex events including excitotoxicity, oxidative stress, and up-

regulation of pro-inflammatory molecules and transcription factors that lead to apoptosis.

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. Nuclear factor kappa B (NF-kB) is a transcription

factor that modulates gene expression of a range of cytokines and other pro-inflammatory

molecules. Recent findings suggest that inhibition of NF-kB, especially those expressed in

neurons, provide neuroprotection following stroke.

Project 1 aim: Investigate the role of endothelial NF-kB on stroke outcome using mice

overexpressing NF-kB inhibitor, IkBk, in vascular endothelium. This study will elucidate

the potential therapeutic effect of blocking NF-kB and/or downstream pathways following

stroke and may lead to the development of more effective stroke therapies.

Project 2 aim: Investigate the importance of specific types of inflammatory responses –

e.g. Th1 versus Th2 - in contributing to stroke outcome. This study will identify the types

and numbers of activated immune cells infiltrating the brain following stroke, in order to

block effects of culprit cells and help develop new types of stroke therapies.

Techniques:

These projects will measure behavioural deficits, brain infarct size, 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 THE ROLE OF ALDOSTERONE IN PROMOTING

INFLAMMATION DURING ATHEROSCLEROSIS

Supervisors: Dr Sophocles Chrissobolis & A/Prof Chris Sobey




Background: Atherosclerosis, a chronic disease characterized by chronic inflammation of

the arterial wall, is the underlying pathological process for both coronary and cerebral

artery disease, which are the two most common forms of cardiovascular disease.

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. During atherosclerosis, the role of

aldosterone in promoting inflammation in the vasculature and other organs important in

cardiovascular disease (such as the kidney and brain) is not well characterized.

Project aim:

The aim of this honours project is to investigate the role of aldosterone in promoting

inflammation in the vasculature, brain and the kidneys during atherosclerosis. This may

reveal potential targets to minimize damage in these organs that occurs during

atherosclerosis.

Techniques:

It is anticipated that this project will involve surgeries on mice to administer aldosterone,

and the use of assays to measure blood pressure (tail cuff), atherosclerotic plaque

formation, inflammatory cell markers (RT-PCR), and immune cell numbers (flow cytometry)

in atherosclerotic mice.

Contacts:

Dr Sophocles Chrissobolis and A/Prof Chris Sobey Department of Pharmacology

Monash University

Phone: 9905 0914, Rm E139A

[email protected]

[email protected]



INVESTIGATIONS INTO THE ROLES OF NADPH OXIDASES IN

INFLUENZA A VIRUS INDUCED LUNG INJURY

Supervisor: Dr Stavros Selemidis

Location: Oxidant and Inflammation Biology Group,

Department of Pharmacology,

Monash University, Clayton.

Background:

Current drug therapies to treat

influenza pathology are focused

primarily on halting mechanisms of viral

infection and replication. Far less

attention has been directed to

identifying mechanisms of host

defense that lead to the acute lung

injury. Animal and human studies

provide compelling evidence that the acute lung injury following influenza A virus infection

is caused by reactive oxygen species (ROS) production such as superoxide anion.

Importantly, the primary sites of influenza A virus infection, airway epithelial cells, and

resident and infiltrating macrophages are key sites of ROS production via Nox1- and

Nox2-containing NADPH oxidases, respectively. Our preliminary studies have identified

the Nox1 and Nox2 enzymes as novel targets for modulating pathology irrespective of the

infecting influenza strain.

Project aims: The aim of this honours project is to investigate the roles of NADPH

oxidases in influenza A virus induced lung injury and inflammation. To achieve this a

multidisciplinary honours project has been devised. It is anticipated that these studies

will have a major impact on modern drug discovery focusing on oxidative stress caused by

influenza A viruses.

Techniques: This will involve the use of live influenza viruses, cell culture and in vivo

animal models; and assays to detect NADPH oxidase expression and localization (western

blotting, immunohistochemistry, QPCR), ROS generation (chemiluminescence,

immunocytochemistry) and inflammatory cell characterization (flow cytometry).

Contacts: Dr Stavros Selemidis, Rm E137

Department of Pharmacology, Monash University

Phone: 9905 5756,

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

Email: [email protected]

External supervision: A/Prof Ross Vlahos, Department of Pharmacology

The University of Melbourne. Tel: 8344 4221; Email: [email protected]

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




PHARMACOLOGICAL CHARACTERISATION OF VENOMS

Supervisors: Dr Sanjaya Kuruppu1, Dr Oden Kleifeld1,

Professor Wayne Hodgson2

Location: Monash Venom Group

Departments of Biochemistry1 & Pharmacology2


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

Supervisor: Dr Justin Hamilton, A/Prof Grant Drummond

Location: Platelet & Megakaryocyte Cell Biology Lab

Australian Centre for Blood Diseases

AMREP (Alfred Hospital Campus), Prahran

Background:

Arterial thrombosis is the most common cause of death and disability in Australia.

Platelets are the blood cells which form arterial thrombi. Therefore our research

examines platelet function in order to discover improved antithrombotic 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 (see Figure), suggesting PAR antagonists as novel antithrombotic agents. As a

result, PAR antagonists are currently in clinical trial 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 utilize 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:

Dr Justin Hamilton

Australian Centre for Blood

Diseases Monash University

Phone: 9903 0125

[email protected]

In vivo thrombosis: Shown are platelets (red) in arterioles of control or PAR4-deficient mice at various times after vascular injury. Note the limited thrombus growth in PAR4-deficient mice. (Vandendries, Hamilton, et al, PNAS, 2007).



DEVELOPING ISOFORM-SPECIFIC PI3K INHIBITORS

AS NOVEL ANTI-PLATELET AGENTS

Supervisors: Dr Justin Hamilton, A/Prof Grant Drummond

Location: Platelet & Megakaryocyte Cell Biology Lab

Australian Centre for Blood Diseases

AMREP (Alfred Hospital Campus), Prahran

Background:

Arterial thrombosis is the leading cause of death and disability in industrialized 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 prepare and screen a designed library of compounds for inhibitory

activity against purified PI3K proteins, and will thereby perform protein expression/

isolation techniques and 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:

Dr 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.



STRATEGIES TO RESCUE DIABETES-INDUCED HEART FAILURE

Supervisors: A/Prof Rebecca Ritchie & Dr Barbara Kemp-Harper

Location: Heart Failure Pharmacology, Baker IDI Heart &

Diabetes Institute, 75 Commercial Rd, Prahran

Background:

Our laboratory’s research focuses on identifying intra-cardiac regulators of growth and

function, to better prevent and treat the causes of heart failure. Diabetes impairs cardiac

muscle function and relaxation, increasing the risk of death from heart failure by more

than 2-fold. We have demonstrated the causal role of excess generation of reactive

oxygen species (ROS) in diabetes-induced heart failure, in both type 1 and type 2 diabetes

(Huynh et al Diabetologia 2012,55:1544-53; Huynh et al Free Rad Biol Med 2013,60:307-

17). Cardiac-selective activation of physiological growth signalling prevents both diabetes-

induced upregulation of cardiac reactive oxygen species, and diabetes-induced heart

failure (Ritchie et al Diabetologia 2012,55:3369-81). We are now exploring mechanisms

downstream of ROS and physiological growth signalling, in order to better understand, and

develop new treatments for, diabetes-induced heart failure.

Project aim:

The aim of this project is to investigate a novel potential therapeutic strategy for

rescuing cardiac function and structure in the diabetic heart. It will determine whether

post-translational protein modifications induced by high glucose play a causal role in

development of diabetes-induced heart failure. Whether treatments that limit these

protein modifications prevent diabetes-induced cardiac dysfunction (and the impact on

cardiac structure and morphology) will also be investigated. This work may lead to

development of more effective therapies for restoring cardiac function in diabetes.

Techniques:

This project uses rodent models of chronic diabetic cardiac disease. Changes in cardiac

function (either in the isolated heart ex vivo or in the intact heart in vivo), blood pressure

and cardiac structure/morphology (fibrosis, hypertrophy, inflammation, apoptosis, ROS

generation) will be measured. Assays include Westerns, chemiluminescence, ELISA, real-

time PCR, histology and/or immunohistochemistry.

Contact:

A/Prof Rebecca Ritchie, Baker IDI Phone: 8532 1392

[email protected]

cardiomyocyte

NADPH oxidase

Nox2

p47

p2

2

DIABETES

glucose

+

O2-

O2-

+

mitochondria

mitochondrial uncoup-ling & dysfunction

apoptosis

NO

ONOO- 3NT

Inflammation

hypertrophy

cardiac fibrosis

ACE

Ang IIAng I +

O2-

LV diastolic function




NITROXYL PROTECTS AGAINST THE CAUSES OF HEART

FAILURE




Background:

Our laboratory has identified the NO•/cGMP signalling system as a powerful cardiac

antihypertrophic mechanism (Ritchie et al Pharmacol Ther 2009;124:279-300). Nitroxyl

(HNO), a novel redox sibling of NO•, has therapeutic advantages for treatment of

cardiovascular diseases. We have shown that HNO prevents hypertrophy (abnormal

pathological growth) and generation of reactive oxygen species (ROS) in isolated

cardiomyocytes (Lin et al PLOS One 2012;7(4):e34892; Irvine et al Am J Physiol 2013;305:H365-77). HNO (but not NO•) also potentiates cardiac function, via direct

interactions with cardiac calcium handling proteins (e.g. SERCA2a, RyR2). Activity and

expression of these proteins is imapired in cardiac pathologies (cardiac hypertrophy,

heart failure, diabetes). Together with ROS upregulation, these changes contribute to

development of cardiac dysfunction. HNO is thus likely favourable for treating cardiac

pathologies; we are now exploring the potential mechanisms by which acute and chronic

HNO therapy protects cardiac function in hypertension, hypertrophy and/or diabetes.

Project aim:

The aims of this project are to investigate (i) whether the mechanisms by which HNO

acutely enhances cardiac function in the intact heart are different to those that prevent

hypertrophy and suppress ROS; and (ii) determine if acute or chronic HNO treatment is

cardioprotective in isolated cardiomyocytes and/or the intact myocardium in vivo in

settings of chronic cardiac impairment. The outcome of this project will be definitive

information regarding the mechanism(s) and effectiveness of HNO-mediated rescue of

myocardial dysfunction.

Techniques:

This project uses rodent models of cardiac disease, which may include cultured cells

(cardiomyocytes and/or cardiac fibroblasts), isolated hearts ex vivo or intact hearts in vivo. Changes in cardiac function, blood pressure and cardiac morphology (hypertrophy,

fibrosis, ROS) will be measured, using Westerns, chemiluminescence, ELISA, real-time

PCR, histology and/or immunohistochemistry analyses.

Contact:


[email protected]

heart failure

cGMP thiol reactivity

SERCA/RyRfunction

XX X

cardiac function

SERCA/RyRfunction

cardiac hypertrophy cardiac fibrosis

ROS

Heart disease (e.g. Hypertension, Diabetes)

Nitroxyl

X




ANNEXIN-A1 PROTECTS AGAINST CARDIAC INFLAMMATION




Background:

Myocardial ischaemic injury (such as heart attack) induces an inflammatory response,

resulting from both infiltration of circulating inflammatory cells, as well as direct actions

on myocardium and endothelium (via Ca2+ overload, reactive oxygen species (ROS)

upregulation or mitochondrial dysfunction). In addition to increased cardiac cell death,

recovery of cardiac function is impaired. We have shown that the diabetic heart also

exhibits an inflammatory phenotype (Huynh et al Free Rad Biol Med 2013,60:307-17). Both

disorders increase risk of heart failure. The endogenous anti-inflammatory protein

annexin-A1 (ANX-A1) protects against cardiac injury and loss of cardiac function (Ritchie

et al Eur J Pharmacol 2013;461:171-9; Ritchie et al Br J Pharmacol 2005;145:495-502; Qin

et al Br J Pharmacol 2013;168:238–52), at least in the short-term. We are now exploring

the potential for, and the mechanisms responsible, ANX-A1 to protect against cardiac

inflammation and resultant heart failure over the longer-term, in both myocardial

infarction (MI, heart attack) and diabetes.

Project aim:

The project will test the hypothesis that ANX-A1 represents a novel modulator of

inflammation, viability and function of the heart following MI and diabetes. It will also

investigate the receptors responsible (e.g. FPR1 or FPR2) for cardioprotection, and

examine potential therapeutic opportunities offered by synthetic ANX-A1 mimetics. This

work may lead to development of effective therapies for treating cardiac inflammation

resulting from diabetes and/or MI, to reduce progression to heart failure.

Techniques:

This project uses rodent models of chronic cardiac disease. Changes in cardiac function (in

isolated heart ex vivo or intact heart in vivo) & morphology (inflammation, apoptosis,

necrosis, fibrosis) will be measured, via Westerns, real-time PCR, histology,

immunohistochemistry and other assays. Cardiac myocytes

or fibroblasts may also be used.

Contact:


[email protected]

HONOURS PROJECT 2014

ANX-A1 mimetics

early rescue of contractile function

preservation of cardiac morphology

preservation of contractile function

inflammation in vivo

myocardial viability

FPR1

heart failuredeath

XX

FPR2

GGq/11 GP-Akt

Gi/o




THE ROLE OF HNO IN MACROPHAGE POLARIZATION

Supervisors: Dr Jennifer Irvine, Dr Karen Andrews &

Dr Barb Kemp-Harper

Location: Vascular Pharmacology Baker IDI Heart & Diabetes Institute

Prahran

Background:

Vascular inflammation is a critical early event in the development of atherosclerosis,

involving the recruitment and adhesion of monocytes to the endothelium. The monocytes

then transmigrate through the vessel wall into tissue, where they differentiate into

macrophages. Under physiological conditions, the monocyte-derived macrophage is of the

M2 type playing a normal protective role in the organism. When pathological factors

(including inflammation) are present, the differentiated macrophage possesses features

which damage tissue, and this type of macrophage is normally termed M1. M1 macrophages

play an important role in cardiovascular disease. For example, M1 macrophages turn into

foam cells through the uptake of ox-LDL in atherosclerosis, forming pathological lipid

lesions on the vessel wall. The vasoactive properties of nitric oxide (NO•) are well

recognized, yet it is becoming increasingly apparent that nitroxyl (HNO), a redox sibling

of NO•, exhibits distinct pharmacology from NO• and may itself play a significant

vasoprotective role.

Project aim:

This project aims to explore the potential effects of HNO on macrophage polarization.

We will isolate monocytes from human blood, differentiate them into macrophages, and

induce the macrophages into M1 or M2 states respectively. We will then study the

influence of treatment with HNO donors on the induction of M1 or M2 macrophages.

Techniques:

It is anticipated that this project will involve the use of cell culture techniques, flow

cytometry (surface marker expression), quantitative PCR (gene expression), cell

morphology and reactive oxygen species generation (chemiluminescence).

Contacts:

Dr Jennifer Irvine and Dr Karen Andrews

Vascular Pharmacology Baker IDI Heart & Diabetes Institute

Phone: 8532 1238

[email protected]

[email protected]



CENTRAL MECHANISMS IN THE CONTROL OF BLOOD PRESSURE

Supervisors: Professor Geoffrey A. Head

Location: Neuropharmacology Laboratory

BakerIDI Heart & Diabetes Institute

Prahran

Background:

Our laboratory explores the role of the sympathetic nervous system in the development

and maintenance of hypertension with a particular emphasis on the central pathways and

neurotransmitters which contribute to the long term adaptive changes which lead to

hypertension in obesity and during chronic stress.

Research Projects: For further information on research projects on offer please contact

Professor Geoff Head.

Contact:

Prof. Geoffrey Head Baker IDI Heart & Diabetes Institute

Phone: 03 8532 1332

[email protected]



TARGETING NADPH OXIDASE USING PHARMACOLOGICAL

INHIBITORS FOR TREATMENT OF OCULAR

NEOVASCULARISATION

Supervisors: Dr Hitesh Peshavariya1 & A/Prof Grant Drummond2

Location: Cytoprotection Pharmacology Group

Centre For Eye Research Australia1

Department of Pharmacology, Monash University2

Background:

Ocular neovascularisation (of the retina and choroid) underlies the pathogenesis of severe

vision loss and is a growing public health problem in Australia and worldwide, making major

contribution to diabetic retinopathy and macular degeneration, respectively.

Neovascularisation is involved in the repair of tissues such as the retina after ischemic

insult. This is an indispensable process for tissue repair which involves proliferation,

migration and capillary formation by endothelial cells. Redox signalling mediated by

NADPH oxidase has been implicated in neovascularisation in vivo of the retina and choroid.

We have discovered pharmacological candidates that specifically modulate the expression

of Nox4 type NADPH oxidase in endothelial cells and in vivo. Inhibition of Nox4 type

NADPH oxidase could create new opportunities for treatment of patients with diabetic

retinopathy and other proliferative retinopathies including wet age-related macular

degeneration

Project aim:

In this project first we will investigate the role of NADPH oxidase isoforms (Nox1, Nox2

and Nox4) in both oxygen-induced retinal neovascularisation or laser-induced choroidal

neovascularisation in mice. Second, we will investigate pharmacological intervention with

Nox4 inhibitors using in vitro and in vivo models of ocular neovascularisation, and

determine whether the same effects are seen in Nox4-deficient mice.

Techniques:

These studies will use pharmacological, molecular and cell culture tools as well as knockout

mice, and mouse models of ocular neovascularisation.

Contacts: Dr. Hitesh Peshavariya Ph.D

Centre for Eye Research Australia

University of Melbourne

Level 1, 32 Gisborne Street

EAST MELBOURNE VIC 3002

[email protected]



THE USE OF DRIED BLOOD SPOTS (DBS) IN FORENSIC

TOXICOLOGY

Supervisors: Dr Dimitri Gerostamoulos1,2 & Dr Jennifer Pilgrim1,

Location: 1 Department of Forensic Medicine, Monash University, 2 Victorian Institute of Forensic Medicine, Southbank, Victoria.

Background: The application of routine toxicology to coroners/medical examiner/police

cases is all but essential in medico-legal investigations. In order to determine whether

drugs and/or poisons may have contributed to an accident, assault or death, or whether

a person may have been under the influence of drugs at the time of an event, blood,

among other tissue samples, can provide valuable information.

Samples of blood collected from victims of crime, drug impaired drivers and deceased

persons undergo a routine toxicological analysis typically involving immunoassays and

other chromatographic/mass spectrometric techniques (GC/MS or LC/MS). The

specimen of blood can vary in volume from 0.1 – 1mL depending on the type of analysis.

Often multiple tests are conducted on one sample and so there is need for up to 5-10

mL to be collected for toxicological purposes. Dried blood spots (finger prick resulting

in a small volume of blood applied to a blotting card) may seek to provide an alternative

to traditional blood sampling.

The use of dried blood spots (DBS) is not new in toxicology but has recently been

examined more closely for its usefulness in forensic cases, especially in instances where

low volumes of blood are collected or only small amounts of blood have been collected as

evidence (Stove et al., 2012). Applications of DBS now included therapeutic drug

monitoring, environmental contaminants and trace elements. Recently DBS have been

used to detect novel psychoactive substances in forensic casework (Ambach et al.,

2013, Jantos et al., 2011).

Project aim: This honours project focuses on developing an extraction technique in DBS

for the isolation and identification of routine drugs for both living and deceased persons.

The developed method will be compared to traditional extraction techniques currently

used in the toxicology laboratory at VIFM. The investigations may lead to novel

applications for screening for drugs in victims of crime and deceased persons. The use

of DBS may be preferential in small children and neonates where the identification of

drugs is satisfactory for case investigations.

References

AMBACH, L., et al., 2013. Rapid and simple LC-MS/MS screening of 64 novel

psychoactive substances using dried blood spots. Drug Test Anal. JANTOS, R., et al., 2011. Analysis of 3,4-methylenedioxymetamphetamine: whole blood

versus dried blood spots. J Anal Toxicol, 35, 269-73.

STOVE, C. P., et al., 2012. Dried blood spots in toxicology: from the cradle to the

grave? Crit Rev Toxicol, 42, 230-43.

Contacts:

Dr Jennifer Pilgrim [email protected]

Department of Forensic Medicine, Monash Univ [email protected]

Phone: 9252 1582





TISSUE-SPECIFIC ROLE OF BIASED SIGNALLING AT THE CaSR

Supervisors: Dr Katie Leach

Location: Drug Discovery Biology

Monash Institute of pharmaceutical sciences, Parkville

Background:

The human calcium sensing receptor (CaSR) primarily acts to sense and regulate

extracellular calcium (Ca2+o) concentrations in the body, but it has a number of non-

canonical roles including control of neuronal and colon epithelial differentiation and

proliferation, nutrient sensing, hormone secretion from intestinal and thyroid cells and

regulation of cardiovascular events. Its diverse functions are related to its widespread

tissue distribution and its ability to bind multiple endogenous ligands. In addition, a

number of small molecule drugs bind the CaSR, one of which, cinacalcet, is used clinically

to treat certain instances of hyperparathyroidism. CaSR ligands exert diverse effects on

receptor signalling, in part due to their ability to stabilise discrete protein conformations,

with each conformation being able to activate its own signature of signalling and

regulatory pathways, termed biased signalling. Biased signalling at the CaSR may have

important implications for therapeutic strategies involving this receptor.

Project aim:

To evaluate biased signalling at the CaSR by a range of endogenous orthosteric agonists

(Ca2+o, Mg2+o, Sr2+o, Fe2+o, Gd3+o), endogenous allosteric ligands (L-phenylalanine,

spermine and γ-glutathione) and small molecule allosteric modulators (cinacalcet, calindol,

NPS2143, calhex) in distinct cellular backgrounds that represent the diverse tissue

distribution of the receptor, including human embryonic kidney, colon epithelial, neuronal-

like and thyroid derived cell lines. This information will be used to elucidate the distinct

signalling pathways that are important for the (patho)physiological processes mediated by

the CaSR and may lead to the development of more effective therapies for the treatment

of disorders such as neurodegeneration, colon and thyroid cancers and disorders of

calcium homeostasis.

Techniques:

Tissue culture and high throughput signalling assays including ERK1/2 phosphorylation and

intracellular calcium mobilisation to measure receptor signalling events, and fluorescence

activated cytometry to detect cell surface receptor expression levels. The project will fit

data obtained from signalling assays to pharmacological models to quantify parameters

such as ligand binding affinity, allosteric cooperativity and biased agonism.

Contacts:

Dr Katie Leach Drug Discovery Biology

Monash Institute of Pharmaceutical

Sciences Parkville

Phone: 9903 9089

[email protected]

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PROBING AN ALLOSTERIC BINDING SITE IN THE HUMAN

CALCIUM SENSING RECEPTOR



Monash Institute of pharmaceutical sciences

Monash University, Parkville

Background:

The human calcium sensing receptor (CaSR) is a family C G protein coupled receptor

(GPCR) that plays a pivotal role in extracellular calcium (Ca2+o) homeostasis and parathyroid

hormone (PTH) secretion. However, over 200 clinically relevant polymorphisms have been

identified in the CaSR and unfortunately they can result in a number of disorders, such as

autosomal dominant hypocalcaemia (ADH), Bartter syndrome type V, familial hypocalciuric

hypercalcaemia (FHH), familial benign hypercalcaemia (FBH) and neonatal severe

hyperparathyroidism (NSHPT). Cinacalcet, a positive allosteric CaSR modulator prescribed

for the treatment of primary and secondary hyperparathyroidism, was recently shown to

successfully normalise serum Ca2+o in individuals with loss-of-function CaSR mutations,

suggesting that cinacalcet and other allosteric CaSR modulators could be invaluable in the

treatment of disorders linked to CaSR mutations. However, recent molecular modelling and

mutagenesis studies have predicted that the binding site(s) for allosteric CaSR

modulators comprises residues located in the transmembrane (TM) domains and

extracellular loops of the receptor, where a growing number of naturally occurring

mutations are being identified. Thus, it is imperative that we understand how drugs bind

to the CaSR.

Project aim:

To probe the location of an allosteric binding site on the CaSR using mutagenesis of amino

acid residues. This information will lead to a better understanding of how drugs interact

with the CaSR.

Techniques:

Alanine substitution of amino acids will be used to determine the involvement of amino

acids in the function of allosteric modulators. Culture of human embryonic kidney

(HEK293) cell lines expressing the wild type and mutant CaSRs wil be required to evaluate

receptor function using high throughput assays that measure intracellular Ca2+o

mobilization to determine receptor signalling events, and fluorescence activated

cytometry to detect cell surface receptor expression levels. The project will fit data

obtained from signalling assays to pharmacological models to quantify parameters such as

ligand binding affinity and allosteric cooperativity.

Contacts:


Monash Institute of Pharmaceutical Sciences, Parkville

Phone: 9903 9089

[email protected]




ALLOSTERIC PHARMACOREGULATION OF A “HEADLESS”

CALCIUM SENSING RECEPTOR



Monash Institute of pharmaceutical sciences, Parkville

Background:

The human calcium sensing receptor (CaSR) is a family C G protein coupled receptor

(GPCR) that plays a pivotal role in extracellular calcium (Ca2+o) homeostasis and

parathyroid hormone (PTH) secretion. It is expressed on the surface of a variety of cell

types throughout the body, where it responds to small increases in free Ca2+o

concentrations. Although the primary Ca2+ (orthosteric) binding site has been localised to

the large extracellular N-terminal region of the receptor, otherwise known as the venus

flytrap (VFT) domain, evidence has pointed towards an additional binding site within the

transmembrane spanning regions of the receptor. This second site is topographically

distinct from the region that binds allosteric calcilytics and calcimimetics, which modulate

the activity of Ca2+o at the CaSR, although it is unclear which amino acids form this

second binding site for Ca2+o.

Project aim:

To investigate the activity of orthosteric and allosteric ligands at a “headless” CaSR that

lacks the first 599 amino acids that comprise the VFT domain and is thus composed of the

TM domains and a functional but truncated C-terminal tail, which lacks the last 176 amino

acids of the receptor. The extent to which allosteric modulators retain their ability to

regulate CaSR activity in the absence of the primary Ca2+o binding site will be

investigated to gain an understanding of the mechanism of allosteric modulation at the

human CaSR. This information will lead to a better understanding of agonist and allosteric

modulator activity at the CaSR.

Techniques:

Growth and culturing of human kidney embryonic (HEK293) cell lines expressing the wild

type and headless CaSR to evaluate receptor function using high throughput assays that

measure extracellular signal regulated kinases (ERK1/2), intracellular Ca2+o mobilization

to measure receptor signalling events, and fluorescence activated cytometry to detect cell

surface receptor expression levels. The project will fit data obtained from signalling

assays to pharmacological models to quantify parameters such as ligand binding affinity

and allosteric cooperativity.

Contacts:


Monash Institute of Pharmaceutical Sciences,

Parkville

Phone: 9903 9089

[email protected]

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BIASED ALLOSTERIC MODULATORS OF METABOTROPIC

GLUTAMATE RECEPTOR 5: A NOVEL THERAPEUTIC STRATEGY

FOR CNS DISORDERS

Supervisors: Dr Karen Gregory & Prof Arthur Christopoulos


Monash Institute of Pharmaceutical Sciences

Monash University, Parkville

Background:

The metabotropic glutamate receptor subtype 5 (mGlu5) is a G protein-coupled receptor

(GPCR) that has emerged as an exciting new target for treating schizophrenia and

depression. Enriched in brain regions implicated in both disorders, numerous studies have

suggested that modulation of mGlu5 is a viable approach for therapeutic intervention. An

exciting development in the GPCR field is the phenomenon of stimulus-bias, whereby

different ligands induce differential pharmacological profiles for receptor activation.

Therapeutically, this creates a scenario where distinct signalling pathways could be

targeted using two different compounds that bind to the same receptor.

Project aim:

The aim of this honours project is to test the hypothesis that mGlu5 allosteric modulators

engender stimulus-bias in measures of acute and chronic receptor activation. This study

will probe the potential for known modulators to display bias, setting the stage for

rational development of novel biased allosteric modulators.

Techniques:

This project will utilize multiple assays for receptor activation, including intracellular

calcium mobilization, kinase phosphorylation, inositol phosphate accumulation and label

free technologies.

Contacts:

Dr Karen J. Gregory & Prof. Arthur Christopoulos Monash Institute of Pharmaceutical Sciences

Monash University, Parkville Campus

Rm 4.346

[email protected]

[email protected]


ILLUMINATING INFLAMMATORY SIGNALING

Supervisors: Professor Nigel Bunnett, Dr Bim Graham and Dr. Nicholas Barlow Location: Monash Institute of Pharmaceutical Science, Royal Parade Background: Chronic inflammation underlies diseases of global relevance that affect all organ systems. Inflammatory diseases are poorly treated due to an incomplete understanding of the mechanisms that transmit inflammatory signals and an inability to detect the early signs of disease prior to irreversible organ damage. Our team seeks to discover new approaches to study inflammatory signalling and to thereby identify therapeutic targets and diagnostic indicators. This project will define the role of proteases in chronic inflammation. Project aim: Working with a team of medicinal chemists and biologists, you will develop approaches to localise and identify proteases that are activated during inflammatory diseases, and to determine their causative role in disease. You will design and synthesize activity-based probes that “light up” when they react with inflammatory proteases. In collaboration with biologists, you will administer probes to mice with chronic inflammatory diseases and thereby localize activated proteases by non-invasive whole animal imaging and cellular confocal imaging. You will use mass spectrometry to identify probe-bound proteases in tissue extracts from animals and patients with chronic inflammation. By studying mice lacking key proteases or treated with protease inhibitors, you will define the causative role of proteases in inflammatory diseases. Techniques: Design and synthesis of activity based probes, proteomics and mass spectrometry, collaboration with biologists studying chronic inflammation in mice. References: Cattaruzza F, et al. Gastroenterology 2011;141:1864-74. Contact: Nigel Bunnett. [email protected]. 0407 392 619

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INTRACELLULAR DRUG DELIVERY: A ROUTE TO MORE SELECTIVE AND EFFECTIVE

TREATMENTS FOR DISEASE

Supervisors: Professor Nigel Bunnett, Professor Chris Porter Location: Monash Institute of Pharmaceutical Science, Royal Parade Background: Receptors convey distinct signals from specific subcellular domains that give rise to unique patho-physiological outcomes. This “compartmentalized signalling” underlies the selective actions of receptors that, at first sight, activate the same signalling cascades. Efforts in drug delivery have focussed on targeting drugs to particular tissues and cells. However, the rational and precise targeting of drugs to defined subcellular domains offers the exciting prospect of manipulating the compartmentalized signaling that underlies key patho-physiological processes, resulting in more selective and effective treatments for diseases that are a major cause of human suffering. This project will develop methodologies of targeting compartmentalized signaling by G protein-coupled receptors that transmit inflammation and pain. Project aim: You will work with a team of medicinal chemists and cell biologists to design and evaluate strategies for targeted subcellular delivery of agonists and antagonists of receptors for peptides and proteases that mediate inflammation and pain. By localizing drugs in specific subcellular domains and examining their effects on compartmentalized signaling, you will develop highly selective treatments, which you will then evaluate in experimental models of disease. The project will allow you to develop a new paradigm of drug delivery, with widespread implications. Techniques: Confocal and super-resolution microscopy, compartmentalized signaling assays using fluorescence-based sensors in cell lines, murine disease models. Key reference: Murphy, JE et al. Endosomes: a legitimate platform for the signaling train. Proc Natl Acad Sci USA, 106: 17615-17622, 2009. Contact: Nigel Bunnett. [email protected]. 0407 392 619

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TRAFFICKING OF G PROTEIN-COUPLED RECEPTORS:

A NEW PATHWAY TO PAIN Supervisors: Professor Nigel Bunnett, Dr Dane Jensen Location: Monash Institute of Pharmaceutical Science, Royal Parade Background: G protein coupled receptors (GPCRs) are the largest family of cell-surface receptors, contribute to most patho-physiological processes, and are a major therapeutic target. Upon activation with agonists, most GPCRs traffic from the plasma membrane to the endosomes, a dynamic tubulo-vesicular network that ramifies throughout the cytoplasm. Once regarded as a mere conduit for receptor trafficking to degradatory or recycling pathways, endosomes are now considered a major site of signal transduction. However, the contribution of endosomal signaling to complex patho-physiological processes, such as pain, are poorly understood. This project investigates the contribution of endosomal trafficking and signaling of the substance P or neurokinin 1 receptor (NK1R) to pain transmission. Project aim: The project will investigate the role of endosomal trafficking and signaling in pain transmission by spinal neurons. By using novel pharmacological and genetic approaches to disrupt key mediators of endosomal trafficking and signaling (clathrin, dynamin/Rab GTPases, arrestins, endosomal peptidases), you will examine the mechanisms and functional relevance of NK1R trafficking in spinal neurons. You will study compartmentalized NK1R signal transduction in primary neurons, and will define the importance of these signals for pain transmission in behavioural studies of mice. Techniques: Confocal and super-resolution microscopy, compartmentalized signaling assays in primary neurons, behavioural assessment of pain in mice. Reference: Murphy, JE et al. Endosomes: a legitimate platform for the signaling train. Proc Natl Acad Sci USA, 106: 17615-17622, 2009. Contact: Nigel Bunnett. [email protected]. 0407 392 619

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MECHANOSENSATION IN THE NERVOUS SYSTEM: IMPLICATIONS FOR VISCERAL PAIN

Supervisors: Professor Nigel Bunnett, Dr TinaMarie Lieu Location: Monash Institute of Pharmaceutical Science, Royal Parade Background: Sensory neurons innervating the intestine detect mechanical stimuli and convey information to the central nervous system. This mechanosensation is necessary for the normal reflex control of digestion. However, inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) amplify mechanosensation and lead to debilitating visceral pain by unknown mechanisms. This project examines mechanisms of normal mechanotransduction and investigates how it becomes dysregulated in disease. Project aim: The project will examine the expression and function of novel receptors and channels that transduce mechanical force into biological signals in sensory neurons innervating the intestine. You will identify mechanoreceptors (e.g., Piezo, ENac/DEG, TREK1, TRP channels) in sensory neurons innervating the mouse colon, and will assess the impact of IBD/IBS on expression levels. By studying intestinal sensory neurons in culture, you will determine the functional importance of mechanoreceptors and determine how they are regulated during inflammation. Through experiments with genetically-modified mice with IBD/IBS, you will investigate the contribution of these channels to neuronal sensitization and visceral pain. Techniques: In situ hybridization, immunofluorescence, single cell PCR, signaling assays in primary neurons, behavioural experiments. Key references: Basbaum et al. Cell 139:267-284, 2009; Brookes et al. Nat Rev Gastroenterol Hepatol 10:286-296, 2013. Contact: Nigel Bunnett. [email protected]. 0407 392 619

CLR$

NK1R$SP$

CGRP$

Pain%

Dorsal%horn,%Spinal%cord%Spinal$neuron$

Periphery%

PAR1,2,4%

SP$ CGRP$Pep8dergic%Nociceptor%

SP$CGRP$

CLR$ NK1R$

Arteriolar$dila7on$

Plasma$extravasa7on$Granulocyte$infiltra7on$

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Proteases%

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TRPV1,%V4,%A1%

COMPARTMENTALIZED SIGNALLING OF OPIOID RECEPTORS

Supervisors: Professor Nigel Bunnett, Dr. Meri Canals, Dr. Daniel Poole Location: Monash Institute of Pharmaceutical Science, Royal Parade Background: Opiates, such as morphine, are the mainstay treatment for pain. However, their use is restricted by side-effects, including constipation and respiratory depression, and by development of tolerance and dependence. Opioids and opiates activate receptors on primary spinal afferent neurons to induce analgesia, and on enteric neurons to cause constipation. This project seeks to define the mechanisms by which opioid receptors signal in spinal afferent and enteric neurons, with the intent of identifying opiates that relieve pain without causing constipation. Project aim: The project will examine trafficking and signaling of µ- and ∂-opioid receptors in spinal afferent and enteric neurons, and will identify the key pathways and mechanisms responsible for analgesia and constipation. By examining the actions of endogenous opioids and opiate drugs that differentially control receptor trafficking and signaling, you will identify pathways that transmit analgesia and constipation. You will examine pain-related behaviour and intestinal functions in mice to investigate whether these pathways mediate analgesia and constipation. By examining mice lacking key signaling mediators, such as arrestins, you will determine the functional importance of these pathways. The project will provide critical insight into the mechanisms of opiate-induced analgesia and constipation, leading to the development of more effective therapies both for pain and for motility disorders. Techniques: Immunofluorescence, confocal and super-resolution microscopy, signaling assays in primary neurons, behavioural experiments in mice, study of intestinal function in mice. Key references. Wood JD et al. Neurogastroenterol Motil 16 (Suppl 2):17-28, 2004. Poole DP et al. Gastroenterology, 141: 982-991, 2011. Contact: Nigel Bunnett. [email protected]. 0407 392 619

NK1R%Endocytosis%in%Neurons%

PROTEASE-ACTIVATED RECEPTORS: MEDIATORS OF THE INFLAMMATION & PAIN Supervisors: Professor Nigel Bunnett, Dr. Nicholas Barlow Location: Monash Institute of Pharmaceutical Science, Royal Parade Background: Inflammation and injury result in the activation of multiple proteases that can regulate cells by cleaving protease-activated receptors (PARs), a subfamily of G protein-coupled receptors. Activation of PAR2 promotes inflammation and pain, and PAR2 antagonists are under development for treatment of inflammatory and painful diseases. However, antagonist development is complicated because proteases can activate PARs by diverse mechanisms. Thus, proteases that cleave PAR2 at distinct sites stabilize unique receptor conformations and act as “biased agonists” of unique signaling pathways. This project seeks to define the molecular mechanisms and biological consequences of protease-biased PAR2 signaling. Project aim: The project will focus on cathepsin S, a macrophage and microglial protease that has been implicated in inflammation, pain and cancer. By studying model cell lines expressing wild-type and PAR2 mutants, you will identify the site at which cathepsin S cleaves PAR2 and will characterize the pathways of cathepsin S- and PAR2-dependent signaling. You will identify the pathways that underlie the proinflammatory and algesic actions of cathepsin S by studying primary cells from PAR2 wild-type and knockout mice. Through studies of mice lacking cathepsin S and PAR2, you will determine the importance of these pathways in inflammatory and painful diseases. These studies will identify new paradigms of protease and PAR signaling and facilitate the informed design of antagonists of specific aspects of protease signaling, with widespread utility for the treatment of inflammatory and painful diseases. Techniques: Receptor expression and mutagenesis, signal transduction assays, studies of genetically modified mice, experimental models of inflammation and pain. Key references: Ossovskaya VS, Bunnett NW. Physiol Rev, 84: 579-621, 2004; Cattaruzza F et al. Gastroenterology, 141: 1864-1874, 2011. Contact: Nigel Bunnett. [email protected]. 0407 392 619

CLR$

NK1R$SP$

CGRP$

Pain%


Periphery%

PAR1,2,4%


SP$CGRP$

CLR$ NK1R$

Arteriolar$dila7on$




Proteases%

Noxious%S8muli%

TRPV1,%V4,%A1%

A WINDOW INTO PAIN: PROTEASE SIGNALING IN PAIN STATES

Supervisors: Professor Nigel Bunnett, Professor Paul Myles, Dr. Nicholas Veldhuis Location: Monash Institute of Pharmaceutical Science, Royal Parade Background: Pain is a protective mechanism essential for survival. However, chronic pain is a major cause of human suffering that is poorly understood and inadequately treated. Proteases that are generated during injury/inflammation can signal to sensory neurons by activating protease-activated receptors (PARs) and transient receptor potential (TRP) ion channels, key mediators of pain. This project examines the contribution of proteases, PARs and TRPs to chronic pain. Project aims: The project will identify proteases that are activated in chronic pain, and will determine the causative role of proteases, PARs and TRPs in chronic pain. You will use small molecule activity-based probes, coupled with proteomics, non-invasive whole animal imaging and confocal cellular imaging, to identify and localize proteases that are activated in disease tissues and in cerebrospinal fluid of patients with chronic pain. By examining the effects of proteases in cell lines and nociceptive neurons expressing PARs and TRPs, you will determine the capacity of proteases to activate PARs and TRPs and sensitize neurons. Studies of mice lacking proteases, PARs and TRPs will allow you to determine the importance of these mediators in chronic pain. You will thereby discover new paradigms of chronic pain that will inform development of novel therapies for diseases that are a major cause of suffering. Key techniques: Cellular and whole animal imaging, proteomics, signaling assays, disease models in experimental animals. Key references: Steinhoff M et al. Nat Med, 6: 151-158, 2000; Cattaruzza F et al. Gastroenterology, 141: 1864-1874, 2011; Poole DP et al. J Biol Chem, 288: 5790-5802, 2013. Contact: Nigel Bunnett. [email protected]. 0407 392 619

CLR$

NK1R$SP$

CGRP$

Pain%


Periphery%

PAR1,2,4%


SP$CGRP$

CLR$ NK1R$

Arteriolar$dila7on$




Proteases%

Noxious%S8muli%

TRPV1,%V4,%A1%

REGULATION OF PAR2 SIGNALING BY RGS PROTEINS

Supervisors: Professor Nigel Bunnett, Dr. Elva Zhao Location: Monash Institute of Pharmaceutical Science, Royal Parade Background: G protein coupled receptors (GPCR) are cell surface receptors that play critical role in signal transduction. Protease-activated receptors (PARs) are a family of GPCRs that promote inflammation and pain. Different proteases that cleave PAR2 at distinct sites may regulate PAR2 signaling via biased agonism. Meanwhile, GPCR signaling can be mediated by intracellular accessory proteins. For example, regulator of G protein signalling (RGS) proteins play critical role in controlling the magnification and duration of GPCR signalling. The project explores the potential involvement of RGS proteins in PAR2 signaling. Project aim: The project will focus on RGS2 and RGS4, both of which have been linked to inflammation and pain via unknown mechanisms. The student will investigate whether the effects of RGS2 and RGS4 on pain response are PAR2 dependent, and will further identify specific pathways that underlie RGS protein activity by studying primary cells from wild-type and RGS knockout mice. The involvement of RGS proteins in PAR2 signaling, or pain transmission has not been systematically studied. A deeper understanding of the mechanisms of initiation, regulation and termination of protease-mediated pain will have implications for the treatment of inflammatory pain. Key techniques: baculovirus generation; real time PCR; insect and mammalian cell culture; enzyme kinetic assays, GTP hydrolysis assays; cAMP accumulation assays, Ca2+ imaging, ERK1/2 activation assays; confocal microscopy. Key references: Zhao P, Cladman W, Van Tol HH, Chidiac P. 2013. Prog Mol Biol Transl Sci 115: 421-53 Contact: Nigel Bunnett. [email protected]. 0407 392 619

PAR2%

Gα#Gβγ#

Gα#

Effectors#

Cell%response%

PAR2%

Gα#Gβγ#

Gα#

RGS% GEF%GPSM%

GDP% GDP%

GTP% GTP%

Effectors#

Gβγ# Gβγ#

BILE ACID SIGNALING IN THE INTESTINE: A NEW TARGETS FOR DIGESTIVE AND

INFLAMMATORY DISEASES

Supervisors: Professor Nigel Bunnett, Dr. Daniel Poole Location: Monash Institute of Pharmaceutical Science, Royal Parade Background: In addition to their role in digestion and absorption of dietary lipids, bile acids (BAs) are signaling molecules that regulate cells via plasma membrane and nuclear receptors. The presence of BAs in the intestinal lumen is required for normal digestion and metabolism, and abnormalities in the luminal concentrations of BAs, secondary to defects in bile secretion or BA metabolism by the colonic microbiome, cause digestive, inflammatory and metabolic diseases by unknown mechanisms. This project examines the contribution of TGR5, a novel G protein-coupled receptor for BAs, to diseases associated with defects in BA secretion and metabolism. Project aim: By studying mice with a loss or gain of TGR5 function, you will explore whether TGR5 contributes to diseases associated with defects in bile secretion of BA metabolism by colonic bacteria. You will determine whether TGR5 mediates BA-evoked fluid and electrolyte secretion from enterocytes, and will explore TGR5-mediated inflammatory signaling in the epithelium. Through studies of TGR5 expression in diseased tissues from experimental animals and patients with inflammatory bowel disease, you will determine whether alterations in TGR5 expression contribute to mechanisms of disease. These studies will identify new paradigms of BA signaling and disease mechanisms, and will determine whether TGR5 is a new therapeutic target. Key techniques: Generation of tissue-selective knockout mice, signaling assays of primary cells, disease models in experimental animals. Key references: Poole et al. Neurogastroenterology & Motility 22:814-25, 2010; Alemi F et al. Gastroenterology, 2012; Alemi F et al. J Clin Invest, 123: 1513-1530, 2013. Contact: Nigel Bunnett. [email protected]. 0407 392 619

department of pharmacology honours projects 2014 · department of pharmacology, honours 2014...

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