4742 Resaerch Profs 17 - University College Dublin research profiles 2004.pdfVaccine delivery...

Contents

RESEARCH PROFILES

001 Conway Synthesis & Chemical Biology

037 Conway Integrative Biology

097 Conway Molecular Medicine

CONWAY INTEGRATIVE BIOLOGY

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Our group is interested in abnormal

embryonic development and their

pathogenic mechanisms. Currently,

the following projects are in progress.

EFFECTS OF CADMIUM ON DEVELOPMENT

Environmental pollution with cadmium

is a serious problem in industrialised and

agricultural economies. The annual

production of cadmium in the EU, mainly

for the manufacture of batteries and

fertiliser, is 2,800 tonnes. We are studying

the teratogenic effects of cadmium using

chick embryos. We choose this species

because an important indicator of

cadmium accumulation in the environment

could be an effect on wild birds. So far, we

have found that exposure of chick embryos

to as little as 0.5μg cadmium causes

abnormalities of axial skeletal, body wall

and limb development. Most interestingly,

the body wall mesoderm grows in a dorsal

instead of a ventral direction. At a cellular

level, these changes are associated with

dis-adhesion of ectodermal cells and cell

death of somites and neural tissue. We are

examining the hypothesis that cadmium

interferes with cadherins in ectodermal

cells followed by an increase in intracellular

b catenin. This would be equivalent to

activation of the WNT pathway with

a consequent dorsalising effect.

ALCOHOL EFFECTS

ON CEREBELLAR DEVELOPMENT

Maternal alcohol abuse is the third

commonest preventable cause of mental

retardation. We are using the developing

cerebellum as a model to study the effects

of alcohol on a developing neuronal system.

In particular, we are using stereological

methods to document effects on sizes of

particular neurone populations and tracing

methods to examine patterns

of neuronal migration.

NEURAL CREST CELL MIGRATION

AND SURVIVAL IN THE CAUDAL GUT

Using transplants of quail neural crest into

chick embryos, we are tracing the relative

contributions of vagal and sacral neural

crest to the development of the intrinsic

neuron population of the caudal gut and

cloaca. We hope to gain insights that will

aid the understanding of Hirschsprung’s

disease and to see how the intrinsic

innervation of the bladder is formed.

AN EXPERIMENTAL MODEL

OF CONGENITAL GUT ATRESIA

Using the cancer chemotherepeutic

agent, adriamycin, we are examining

the pathogenesis of congenital atresias

of the oesophagus and small intestine.

In particular, we are examining the

ultrastructure of the tissues involved in

these malformations because our previous

findings indicated that an abnormal

adhesion between the notochord and

the developing gut was important for

the pathogenesis of these defects.

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PROFESSOR ALAN BAIRD


Mucosal biology deals with the interface

between individuals and their environment.

Our interest in the gastrointestinal tract

ranges from studying mechanisms of

nutrient absorption to mechanisms, which

prevent entry of potential pathogens. We

have developed methods with which to

study the form and function of epithelial

cells that line the intestine. These methods

lend themselves to a number of projects.

SENSORY PROCESSES

OF INTESTINAL EPITHELIA

Using avian and mammalian (including

human) epithelial sheets, we have addressed

specific questions concerning the barrier

nature of the gut. From interactions with

bacteria to translocation of macromolecules,

epithelial monolayers act as portals of entry

to the host organism. Outcomes of such

interactions range from altered epithelial

cell function to immunological responses.

Antigens include simple proteins as well

as actual pathogenic nematodes.

Immunological outcomes range from

tolerance to hypersensitivity states.

EFFECTOR PROCESSES

OF INTESTINAL EPITHELIA

The mammalian gut can be changed from

an organ of net water absorption to one of

net secretion as a consequence of altered

epithelial ion channel activity. Intracellular

signalling cascades can be triggered

by extracellular signals, which include

bacterial toxins as well as hormones,

neurotransmitters or inflammatory

mediators derived from populations

of cells in the lamina propria.

With colleagues, including Prof D

O’Donoghue, we have shown that signals

for recruitment of mast cells and

eosinophils are coupled to stimuli, which

increase nerve density (nerve growth

factor) and that these mechanisms are

common to parasite infection and to

chronic inflammatory bowel disease.

OTHER TISSUE TYPES

Smooth muscle function is studied directly

by measuring contraction in vitro. We have

used classical approaches to determine the

role of calcium in excitatory coupling.

Varying dependence of extracellular

calcium sources may account for different

side effects of calcium-blocking drugs in

smooth muscle from different tissue sites

(gallbladder and urinary bladder).

With Prof A McCormick, we have explored

the contribution of mast cells and nerves

to disordered function of liver. Specifically,

we have examined whether nerves/mast

cell interactions contribute to pruritis

(uncontrollable itching) associated with

liver disease.

RESEARCH PROJECTS

CURRENTLY ONGOING:

Regulation of epithelial function

by non-epithelial cells.

Peyer’s patch function in new

born lambs.

Function of intestinal mucus gels

(with Steve Carrington).

Bacterial interactions with human

epithelia (with University of Bristol).

Prion uptake by the mammalian gut

(with Institute of Animal Health).

Avian intestinal regulation by small

molecular weight messengers.

Vaccine delivery systems and

adjuvants (with David Brayden).

Tight junction contribution to liver

disease (with Ann Hopkins).LUMEN

TIGHT JUNCTIONS

EPITHELIAL MONOLAYER

BASEMENT MEMBRANE

G.A.L.T. E.N.S.

BLOOD LYMPH

LAMINA PROPRIA

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PROFESSOR JOHN BANNIGAN


Fig. 1. Lamina propria elements are dynamic.

Cells of the enteric nervous system and of the local

immune system are strategically located to interact

with epithelial cells.

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DR BRENDA BRANKIN


Our research group is interested in the

blood brain barrier (BBB) and blood retinal

barrier (BRB) in health and disease. Our

work involves characterising growth factor

-induced changes in the tight junction

protein ZO-1 and occluden phosphorylation

and junctional permeability. We are

carrying out research on an inflammatory

central nervous system disease (multiple

sclerosis) and are interested in determining

mechanisms of BBB breakdown.

PROJECT 1: THE ROLE OF PRO-

ANGIOGENIC GROWTH FACTORS ON

BLOOD RETINAL BARRIER FUNCTIONS

IN OCULAR DISORDERS.

During proliferative retinopathies,

vasogenic growth factors have been

implicated in the angiogenic process. We

hypothesise that members of the vascular

endothelial growth factor (VEGF) family

of peptides act in a synergistic manner to

mediate vessel permeability by inducing

phosphorylation of tight junction proteins.

We have found that the angiogenic

inhibitor endostatin increases expression

and phosphorylation levels of occludin

and antagonises VEGF-mediated retinal

vascular permeability. We are further

investigating mechanisms whereby these

growth factors transduce the effects

of increased blood retinal barrier

permeability.

PROJECT 2: MICROARRAY ANALYSIS OF

GENE EXPRESSION IN MULTIPLE

SCLEROSIS (MS).

We are carrying out a comprehensive

analysis of genes whose expression

levels are over or under expressed in MS

compared to controls. Microarray analyses

of samples have been performed using

the Affymetrix Gene Chip system, and

data analysed in conjunction with gene

ontology criteria. Identification of specific

genes, which are overexpressed in MS

compared to controls, will provide

information to explain mechanisms of

myelin destruction and this study will

contribute to our understanding of how

these genes influence susceptibility and

pathogenesis in MS. We have identified

increased transcripts of genes encoding

immune-related molecules, molecules

involved in maintaining blood-brain

integrity and molecules involved

in signal transduction.

PROJECT 3: ZONULAS OCCLUDEN 1

EXPRESSION IN RETINITIS

PIGMENTOSA

Retinitis pigmentosa (RP) represents a

major cause of progressive retinal disease

worldwide. We have found increased

expression of ZO-1 in Rho-/- mice compared

to wild type C129. ZO-1 interacting proteins

cdc42 and a-catenin are also elevated.

We are investigating the retinal changes

following photoreceptor death, which may

contribute to pathogenesis. This project

is being carried out in collaboration with

Professor Pete Humphries, Department

of Genetics, Trinity College Dublin.

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Control-No Injection

(n=12)

Control Injection

(n=12)

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60ng Endostatin

(n=10)

30ng VEGF

(n=10)

30ng VEGF + 60ng

Endostatin (n=8)

DR STUART BUND


My research interests are centered on

the structure and function of resistance

arteries and abnormalities in hypertension.

These particular blood vessels are pivotal

to the regulation of blood pressure and

local blood flow. Accordingly, functional

abnormalities of these blood vessels may

be critical in the initiation and progression

of a number of cardiovascular diseases

such as hypertension, stroke and heart

disease. Cardiovascular disease represents

a major cause of mortality and morbidity,

especially in developed countries.

In particular, my research interests

include the relationship between arterial

contractile responses and arterial

structure in models of systemic

hypertension. A collaborative venture

with Professor Paul McLoughlin, Conway

Institute has recently been established

to investigate the contractile properties

of pulmonary arteries and how these are

affected by hypoxia. The experiments

underway aim to establish mechanisms

responsible for the raised pulmonary blood

pressure associated with certain lung

diseases. Basic research interests include

an ongoing collaboration with Dr James

Jones, Conway Institute, which aims

to elucidate the means by which the

diameters of arteries that provide blood

flow to cardiac muscle are controlled by

the autonomic nervous system. We are

interested in the neural regulation of the

classical coronary arteries within the heart

and the accessory arteries that feed the

cardiac muscle in the great veins.

Experiments are also underway that aim

to determine the pressure-dependent

contractile functions of amphibian arteries.

My particular skills are in the use of

techniques for the in vitro assessment

of resistance arteries. These techniques

are the Halpern-Mulvany myograph and

the Living Systems arteriograph. These

assess arterial function under isometric

and isobaric conditions, respectively. The

myograph is better established but the

arteriograph is now widely accepted as

the most functionally relevant method for

assessment of resistance artery function

in vitro because the arteries are assessed

under pressurised conditions and they

can therefore attain their correct shape

and develop myogenic (pressure-

dependent) tone as they would in vivo.

Fig. 2. Endostatin antagonises VEGF-mediated Retinal Vascular Permeability.

Fig. 1. Fluorescent image of a retinal microvessel

from a mouse stained for the tight junction protein,

occludin, which allows for maintenance of the

integrity of the blood-retinal barrier.

Fig. 1. This Fig. illustrates a small artery (internal

diameter approximately 200 micrometres) mounted

in an arteriograph. It is cannulated at either end

by glass micropipettes and secured by fine nylon

strands teased from a nylon suture. The pressure

inside the artery is regulated and the diameter

is recorded by videometric means.

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INTERACTIONS OF AVERMECTINS

WITH P-GLYCOPROTEIN (P-GP)

EFFLUX PUMPS

This project is funded by Pfizer Animal

Health, UK. The anti-parasitic drug,

ivermectin, is neurotoxic to a cohort of

collies in high oral doses since they have

a mutation in their mdr1a gene coding

for P-gp on their blood-brain barrier

(BBB). In contrast to normal dogs and

humans, ivermectin penetrates the BBB

of these knock-out animals to cause toxicity.

We examined the approved topical agent

of the same class, selamectin, and proved

that it was also a potent substrate for P-gp.

The methods used were P-gp expressing

human Caco-2 epithelial monolayers and

freshly-derived canine lymphocytes.

The conclusion of the work was that all

avermectins have the potential to access

the BBB if given in high enough doses

by the oral or injected route of delivery.

Topically-applied selamectin never reaches

the systemic concentrations that might

cause problems to genotypically-sensitive

collies. Reference: Griffin J et al. (2005).

Selamectin is a potent substrate

and inhibitor of human and canine

P-glycoprotein. J. Vet. Pharm. Ther. 28, 1-9.

MYOSIN LIGHT CHAIN KINASE

INHIBITION – CLOSE SESAME!

Funded by the Health Research Board,

this project is examining the hypothesis

that epithelial tight junction dysregulation

is a contributory cause of inflammatory

bowel disease. The work has shown that

intestinal epithelial segments from IL-10

knock-out mice display lower transepithelial

resistance and higher permeability to

paracellular flux markers than tissue

derived from normal mice when mounted

in Ussing chambers. We are working with

a set of myosin light chain kinase (MLCK)

inhibitors and have shown that ML-9

can close the tight junctions when

pharmacologically opened by sodium

caprate and cytochalasins. The next part

of the project will examine whether MLCK

inhibitors can ‘repair’ the defect in

permeability of the IL-10 mouse and

to examine other potential therapies

including probiotics.

PRION INTERACTION WITH

PEYER’S PATCH M CELLS

Funded by the Department of Food

and Agriculture, we are examining the

hypothesis that food-derived prions enter

the body via M cells of the Peyer’s patch

across a range of species. Isolated Peyer’s

patch tissue is notoriously subject

to degradation. We have therefore

established two models of M cells:

a human co-culture and a horizontal

diffusion chamber for patches of different

sizes. That the patches display functionality

is suggested by their increased uptake of

microparticles over non-patch intestinal

regions. Currently, we are examining

the uptake of scrapie-infected ovine

homogenate by ovine patches in vitro.

Fig. 2 shows a section of rat Peyer’s patch.

Research in this drug delivery laboratory

is based on drug and pathogen interactions

with intestinal epithelia from the

standpoints of permeation, effects on

paracellular permeability and on efflux

transporters. We are also investigating

methods to promote prolonged mucous

adherence of drugs as topical agents, for

example, in the treatment of inflammatory

bowel disease. Finally, we are examining

intestinal Peyer’s patch M cells as routes

of entry for prion pathogens across several

veterinary species. Current research

projects include:

POLYMER INTERACTION WITH

MUCOUS-SECRETING INTESTINAL

EPITHELIA

This project is funded by Genzyme

Corporation, USA and involves

collaboration with the Chemistry

Department at the University of Warwick.

A filter-grown, mucous-secreting, human

intestinal epithelial cell line has been

established, which shows a correlation

with isolated rat intestinal sacs in the

mucoadhesion of novel polymers.

Chitosan- and methacrylate-based

polymers were shown to bind to both

the mucous and epithelial aspects of these

models and, moreover, cytotoxicity assays

showed that they were innocuous.

Fig. 1 shows evidence of polymer

attachment to overlying mucous.

Intriguingly, the combination of polymer

with mucous prevented the cell damage

normally induced by bacterial-derived

toxins and this raises the possibility

of developing anti-bacterial topical

mucoadhesives for eye, oral cavity and the

intestine. Reference: Keely S et al (2005).

In vitro and ex vivo intestinal tissue

models to measure mucoadhesion of

poly(methacrylate) and N-trimethylated

chitosan. Pharm. Res. 22 (1) 38-50. 043 <> 044

DR DAVID BRAYDEN


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Fig. 1. A. Light micrograph of alcian blue / neutral

red staining of mucous-secreting E12 intestinal

epithelia exposed to poly-diamino diethyl amino

methcrylate (pDMAEMA) -coumarin fluorescent

conjugate. X: Mucus gel layer; Y: Cell monolayer.

B. Fluorescent micrograph of pDMAEMA-coumarin

conjugate on same fixed section. Broken white

lines denote cell monolayer outline. Co-localisation

of pDMAEMA with mucus layer is evident by

superimposing micrograph B on A. Bars = 25μm.

50 µm

Fig. 2. H & E staining of a section of rat intestinal

Peyer’s patch. Inset shows follicle-associated

epithelium (FAE) overlying dome lymphocytes.

M cells are in the FAE and act to take up particles

and pathogens and to initiate the mucosal

immune response.

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DR SEAN CALLANAN


FELINE IMMUNODEFICIENCY VIRUS

INFECTION: THE DEVELOPMENT OF

AN IN VITRO FELINE BLOOD BRAIN

BARRIER SYSTEM

Feline immunodeficiency virus infection

(FIV) is a naturally acquired infection in

pet and feral cats worldwide and is also

an established model of HIV-1 infection

in man. Like HIV, this virus is neurotropic

with entry of the virus into the CNS within

the first month of infection. However,

there is a paucity of information on how

the virus enters the brain and how it

interacts with neurological tissue to induce

significant neurological impairment.

Research at UCD, in collaboration with the

University of Glasgow and University of

Vienna has centred on dissecting the

pathogenesis of the early stage of

infection and has focused on the

characterisation of the neuropathology,

viral distribution and load and on viral

entry mechanisms (Ryan et al, 2003). Our

recent research with FIV (Glasgow -8) has

shown that in the acute phase of infection,

the virus enters the brain in the majority

of animals and it is distributed initially in

a random fashion, with more diffuse brain

involvement as disease progresses. We

have also shown that virus in the CSF is

predictive of brain parenchymal infection.

During this period of time, prominent

lymphocyte trafficking through both the

blood-brain and blood-choroid plexus

barriers is noted and currently we have

developed in vitro systems to elucidate

pathogeneic mechanisms involved.

Currently, postgraduate researcher Nicola

Fletcher has developed and characterised

an in vitro model of the feline blood brain

barrier in collaboration with Dr Brenda

Brankin and Dr David Brayden. This in vitro

model of the feline blood-brain barrier

was developed using primary cultures

of brain-derived endothelial cells and

astrocytes. The barrier system has

developed tight junctions and occludin

and ZO-1 expression is present. Barrier

function has been determined by

transendothelial electral resistance and

permeabilities to dextran formulations.

Her studies are now focusing on the

interaction of this barrier with FIV-

infected cells.

GENE REGULATION IN FELINE

HYPERTHYROIDISM

Feline hyperthyroidism is the most

common endocrine disease of cats

and toxic nodular goiter, a primary

hyperthyroid disease in humans, is

considered similar in many respects.

At University College Dublin, a collaborative

study has commenced with Trinity College

Dublin to evaluate the transcription and

expression of selected candidate genes

between normal thyroid tissue and

adenomatous thyroid lesions. Postgraduate

researcher, James O’Donovan, Petsavers

resident in veterinary pathology, is

responsible for this project.

NON-SUPPURATIVE

MENINGOENCEPHALITIS

IN YOUNG GREYHOUNDS IN IRELAND

We have detected a novel neurological

syndrome in juvenile greyhounds

(Callanan et al, 2002). As Ireland is one of

the world’s largest breeders and exporters

of greyhounds, an ideal situation exists to

detect emerging disorders, which may,

at first, appear to have only a breed-

association but may involve a broader host

range, involving other breeds within the

species or extending across species. The

disease is rapid in onset and usually fatal

presenting with a unique histopathological

profile, which is essentially a severe non-

suppurative meningoencephalitis.

To date research, in collaboration with

Akzo Nobel (Intervet UK), University

of Berne and University of Zurich, has

centred on the characterisation of this

lesion and clinical observations, serology,

histopathological, immunohistochemical

and molecular analysis have not implicated

the worldwide-known canine encephalitis-

inducing agents.

A non-suppurative meningoencephalitis

favours the involvement of a viral agent

and the object of this programme is to

centre on methodologies to detect this

agent and in parallel, dissect the

pathogenesis of CNS inflammation.

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Fig. 1. Immunofluorescence for GFAP in feline brain

astrocytes.

Fig. 2. Confluent culture of feline brain capillary

endothelial cells.

Fig. 3. TEM image of feline endothelial cells. Note

mitochondria (M) and tight junction (arrow).

Fig. 4. Section of cerebrum from a greyhound with

meningoencephalitis revealing perivascular cuffing,

gliosis and prominent gemistocytosis (GFAP-stained

astrocytes).

DR DEIRDRE CAMPION


The thin layer of epithelial cells lining

the gut and the inner organs protect them

from potentially harmful intestinal bacteria,

and from ‘foreign substances’ such as food

additives and drugs. Specialised cells

monitor this lining, monitoring suspicious

bacteria and recruiting cells of the immune

system. However, debilitating conditions

such as inflammatory bowel disease are on

the increase. One hypothesis suggests that

immunological challenges early in life

reduces the risk of developing allergies by

‘training’ the immune system. Conversely,

a relatively clean environment in early life

would sway the immune system towards

allergy- promoting responses. Is it possible

that modern practices of sanitation could

deprive people, and animals, of defences

needed to prevent allergic responses? We

set out to investigate this by infecting rats

with a parasite that caused a mild

inflammation of the gut wall.

Immunohistochemical analysis of colonic

tissues from both parasite-infected and

uninfected rats demonstrated nerves

serving the gut-associated lymphoid tissue

(GALT). These nerves extended to the

lymphoid follicle associated epithelium

(FAE). The polysialated form of the neural

adhesion molecule NCAM, an indicator

of neural plasticity, was demonstrated

in some of these enteric nerves. Nerve,

eosinophil and mast cell numbers

increased significantly with parasitic

infection. Eosinophils and mast cells were

frequently closely associated with nerve

fibres in both the regular and lymphoid

follicle-associated lamina propria. These

data indicate that the density of GALT

associated nerves are, as elsewhere

inthe gastrointestinal tract, raised as

a consequence of parasitism.

Colonic GALT epithelium was mounted in

Ussing chambers under voltage clamped

conditions. Electrical field stimulation of

GALT epithelium evoked a mean inward

SCC, which was neuronally mediated

and accounted for, at least in part,

by electrogenic chloride secretion.

In experiments to assess the effect of

chloride secretion on pathogen invasion,

in uninfected rat ileum, sustained

electrogenic chloride secretion was elicited

by forskolin or, in companion tissues from

the same animal, inhibited by bumetanide.

Salmonella invasion in actively secreting

tissues was significantly less than the level

observed in bumetanide-treated (quiescent)

tissues. Bacterial adherence levels were

similar in either state. Similar results,

although of a lesser magnitude, were found

in GALT epithelium.

Our results also show, for the first time,

that a functionally secreting phenotype

regulates bacterial internalisation by

intestinal epithelial cells. It now seems

that the effect of altered nerve density

is likely to result in an improved protective

response, in contributing to protecting

the bowel by an additive effect on the

secretory mechanism in both normal

and PP epithelium, thereby protecting

the bowel from pathogen invasion by

the enteric tear mechanism, hydration

of mucous gel, alteration of surface charge

or all of these in concert.

Having found a role for altered nerve

density, nerve activation, and chloride

secretion in reducing Salmonella invasion

in isolated rat tissues, we are now

investigating these effects in avian tissues.

The importance of this work lies in discovery

and implementation of measures to

counter poultry meat contamination with

pathogens such as Salmonella and may

form an important mechanism of control

of this serious zoonotic threat.

Conway investigators Professor Alan

Baird and Dr David Brayden collaborate

on this work.

DR MARK A CROWE


REPRODUCTIVE AND STRESS

PHYSIOLOGY IN CATTLE

The research theme in our area focuses

on two major areas: (i) reproductive

function in cattle and sheep, and (ii)

stress physiology associated with both

acute and chronic stressors.

Our current reproductive work focuses

on the mechanisms of ovarian follicle

selection in cattle. Specifically, we are

investigating the roles of the insulin

-like growth factor (IGF) system in the

mechanisms of follicle growth. Ovarian

follicles grow throughout the oestrous

cycle of cattle. During the luteal phase, there

are up to two sequential non-ovulatory

waves of follicle growth and during the

follicular phase, the ovulatory wave grows

and produces the ovulatory follicle. Growth

of a follicle wave involves emergence of

a cohort of 4 to 20 follicles, selection down

to the ovulatory quota (usually one follicle

in cattle); dominance during which the

selected follicle continues growing and

suppresses the growth of subsequent

follicles, and atresia (in the luteal phase)

or ovulation (in the follicular phase).

The end of dominance, signified by either

atresia or ovulation, allows a subsequent

wave of follicles to grow. These waves

of follicle growth are tightly regulated by

transient increases in follicle-stimulating

hormone (FSH). However, the mechanism

by which selection occurs during declining

FSH concentrations remains a subject

of intense research. Clearly, it has been

demonstrated by both our research and

that of others that increased bioavailability

of IGF-I, facilitated by decreased IGF-

binding protein (IGFBP) activity, is involved.

Our recent work has studied the roles of

both proteolysis on the IGFBPs and their

gene expression.

In the area of stress physiology in cattle,

using surgical and burdizzo castration as

acute stressors, we have studied the roles

of non-steroidal anti-inflammatory drugs

(NSAIDs) in the mediation of stress-

induced changes in the hypothalamus-

pituitary-adrenal axis and immune

function responses. Currently, our focus

is on the identification of key inflammatory

genes involved with the inflammatory

responses of bull calves following

castration. Ketoprofen (Ketofen) was

successful in suppressing cortisol secretion

and blocking immune suppression following

castration of cattle.

Current research group includes:

Sandeep Gupta, PhD student,

February 2002 to present.

Wanyong Pang, PhD Student,

June 2003 to present.

Kelly Buckham, PhD Student,

July 2004 to present.

Heather Haycock,

September 2004 to present.

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Figure. 1 Nerves in rat colonic mucosa were labelled

with antibodies raised against GAP-43. In this

section, nerves serving the follicular associated

epithelium (FAE) at the dome of a lymphoid follicle

are clearly identifiable. This is the region where

sampling of luminal antigens through differentiated

enterocytes (M cells) occurs.

IGFBP-2

IGFBP-2

IGFBP-2IGFBP-4

IGFBP-2

IGFBP-3

Insulin like growth factor binding protein (IGFBP) 2, 3 and 4 localisation in granulosa cells (GC), theca cells

(TC) and theca cells, respectively of bovine follicles using in situ hybridisation. A, D and G are dark field

images of the anti-sense hybridization B, E and H are dark field images of the sense hybridisation (negative

control) and C, F and I are light field images of the corresponding follicle tissue sections.

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PROFESSOR MICHAEL DUNN

PROTEOME RESEARCH CENTRE

My research group is working in the

Proteome Research Centre (PRC), directed

by Professor Stephen Pennington, which

was opened in newly refurbished

laboratories in July 2004. The PRC is

equipped with instrumentation for a range

of complementary proteomic strategies

for the analysis of proteins from different

biological samples with the aim of

identifying proteins that play important

roles in fundamental biological events in

health and disease. The PRC is equipped

with large-format two-dimensional gel

electrophoresis (2-DE), a fluorescent

imager that enables the application of

the 3-dye DIGE technology, and computer

software for differential protein expression

analysis. A suite of complementary mass

spectrometers (AB 4700 MALDI-Tof/Tof,

ThermoFinnigan LTQ ESI-MS/MS, LTQ-FT,

and Ciphergen SELDI-MS-MS/MS) has

been installed. In addition, a range of LC

equipment is available for off-line and

on-line to MS separation of proteins and

peptides. This instrumentation provides

the capacity for high-throughput

identification and characterisation

of proteins found to be differentially

expressed from the 2-DE platform. They

also enable the application of alternative

MS-based proteomic strategies, for

example stable-isotope labelling

techniques for differential expression

studies and chip-based approaches

for biomarker discovery, to projects

undertaken in the centre.

The aim of our research is to identify and

implement proteomic technologies and then

to apply them to a variety of biomedical

research programmes in collaboration

with other groups both within the Conway,

in other departments of UCD and other

laboratories in Ireland and worldwide.

My own research programme is focused

in three major areas of biomedicine:

(1) cardiovascular proteomics, (2)

transplantation proteomics, and (3)

neuroproteomics. The overall aim of these

studies is to further our understanding of

the molecular basis of biological processes

in health and disease and to identify

new biomarkers that can be exploited

as diagnostic/prognostic reagents

and/or as therapeutic targets.

My group established the first Internet

www database of heart proteins, which

is accessible at: http://www.doc.ic.ac.uk

/vip/hsc-2dpage/index.html (see

Fig. 1). The emphasis of the cardiovascular

research programme is on those heart

diseases that result in end-stage heart

failure as these diseases are among

the leading causes of morbidity and

mortality in the developed world. A

systematic study of protein expression

in the normal and diseased heart will

provide important new insights into

cellular mechanisms involved in cardiac

dysfunction and will also lead to the

identification of potential new diagnostic

markers and therapeutic targets.

Our transplantation proteomics research

programme, carried out in collaboration

with Professor Marlene Rose (Imperial

College London), is focused on processes

of acute and chronic rejection following

heart transplantation. In acute rejection,

we have used proteomics to identify novel

potential biomarkers that could form the

basis of a minimally invasive (ie blood)

test to replace the highly invasive and

expensive endomyocardial biopsy. Chronic

rejection, currently the most important

factor limiting the long-term survival

of heart transplant recipients, involves

an accelerated form of coronary artery

disease. We have been successful in

identifying a serum biomarker (circulating

antibodies reactive with the intermediate

filament protein, vimentin) that has the

potential to be used for effective routine

monitoring of chronic rejection as a

complement to angiography or intra-

vascular ultrasound.

We are also pursuing functional

proteomics studies, which have identified

a panel of proteins showing altered

patterns of expression associated with

long-term freedom from chronic rejection,

and are therefore candidate cytoprotective

proteins. The most significant of these

changes involves the 27 kDa heat shock

protein, Hsp27. We have found that a

specific diphosphorylated form of Hsp27 is

expressed strongly in the hearts of

patients free of chronic rejection, but is not

expressed in the hearts of patients with

this disease (Fig. 2).

We are now pursuing this finding

through analysis of in vivo and in vitro

model systems.

Our neuroproteomics research programme

is being carried out in collaboration with

Dr David Cotter (Royal College of Surgeons

in Ireland) in the area of the major

psychiatric disorders, schizophrenia,

bipolar disorder and major depressive

disorder. This group of diseases are

particularly complex and it is known

that multiple, largely unidentified genetic

and environmental risk factors interact to

lead to disease and disease progression.

Structural alterations occur within the

brain of affected patients, both during

early disease development and during

a later, possibly ‘degenerative’ stage.

However, the underlying functional

changes at the cellular level remain largely

unknown. Proteomic analysis of samples

of affected brain tissue has the potential

to provide important new insights into

the cellular mechanisms underlying these

psychiatric diseases. In addition, by

characterising disease-associated protein

profiles in body fluid samples (CSF, serum),

we may be able to identify proteins that

serve as diagnostic or prognostic markers

of disease. 049 <> 050C

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Late no TxCAD

Hsp 27 CD31 SMaA .ve control

100µmWeekly+ve

Strongly+ve

Late TxCAD

Fig. 2. Immunocytochemical staining of cardiac biopsies from transplant patients showing that Hsp27 is

strongly expressed by vessels within the heart of patients without chronic rejection, while it is not expressed

in the hearts of patients with this disease (De Sousa et al, unpublished).

Fig. 1. Computer screen shot of the human heart

ventricle 2-DE protein database from HSC-2DPAGE.

(http://www.doc.ic.ac.uk/vip/hsc2dpage/index.html)

051<> 052

DR ALEX EVANS


Reproduction is a fundamental process

of all living organisms and there has been

a significant decline in fertility in recent

years due to a number of factors in many

different species (including humans

and farm animals). Through the use

of established models and local and

international collaborations, we have

established a well defined programme

that is designed to make fundamental

discoveries directed at better understanding

the development of reproductive tissues.

The success of mammalian reproduction

relies on the ovulation of a healthy oocyte

from an ovarian follicle and, after

fertilisation, the development of a healthy

embryo. Many ovarian follicles develop

in parallel in the ovary, a species-specific

number of which is selected to ovulate.

Each follicle contains an immature oocyte

that undergoes final maturation just before

ovulation, preparing it for fertilisation.

The initial development of the embryo

is governed by gene transcripts and proteins

derived from earlier stages of oocyte

development and it is not until the embryo

has undergone several mitotic divisions

that it becomes reliant on its own embryonic

genome for survival. With this in mind, the

development of ovarian follicles and the

oocytes that they contain is central for

successful reproduction. To critically

examine the development of the cells

involved in ovarian follicle development,

established bovine and ovine models, both

in vivo and in vitro, are used.

While the biochemistry of intracellular

mechanisms regulating cell differentiation

has been well studied in simple organisms

and in well-defined culture systems,

reproductive tissues as they relate to

fertility in mammals have received

relatively little attention, particularly in

terms of the fundamental molecules and

genes that regulate function. At present,

the focus of the research is on understanding

the genomic regulation and follicle

development with particular emphasis

on the regulation of transcription and the

hormonal signalling pathways that control

the outcome of development. Knowledge

gained from this research will make it

possible to develop strategies that more

effectively control follicle development

and ultimately fertility enhancement

or contraception.

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Zygote

Ovulation

Follicular Wave

LH - surge

3mm

8mm

15mm

S.F.

D.F.

Oocyte

Prematuration

Oocyte Final

Maturation

Major ActivationMinor Activation

Embryonic mRNAMaternal mRNA

Fertilisation

Early Embryonic DevelopmentFollicle and Oocyte Development

2-cell 4-cell 8-cell Morulae Blastocyst

Schematic of the Pattern of fillicle development.

DR SHAY GILES


Our group is primarily involved in

investigating the underlying mechanisms

of teratogenesis. Teratogens are agents

that interfere with normal developmental

processes to produce birth defects following

exposure during pregnancy. They can

range from naturally occurring elements

such as mercury and cadmium to synthetic

drugs such as thalidomide. Several of our

group are investigating the mechanisms

of action of alcohol as a teratogen. Abuse

of alcohol during pregnancy can cause a

spectrum of birth defects collectively known

as the foetal alcohol syndrome (FAS)

consisting of growth retardation, craniofacial

anomalies, neurological disturbances and

a variety of major and minor organ system

malformations. Exposure to alcohol during

the first trimester can cause craniofacial

and ocular anomalies whereas exposure

during the third trimester causes

neurological deficits resulting in mental

retardation. Using the chick embryo as an

experimental model, we are interested in

the early cellular and tissue changes resulting

from alcohol exposure at gastrulation

through neurulation stages of development

with particular emphasis on ocular and

spinal cord development. Current projects

in this area include:

The effects of ethanol on sonic hedgehog

and its downstream signal response

genes during the critical period of eye

development in the chick embryo.

The role of L1 cell adhesion molecule

in alcohol-induced spinal cord anomalies.

Teratogens can also lead to the development

of invaluable model systems for research

into birth defects. We are currently

working on one such system, the adriamycin

rat model for VACTERL association.

This project is in collaboration with

the Childrens Research Centre, Crumlin

Hospital. We are primarily interested

in examining the molecular and cellular

changes that lead to adriamycin induced

tracheo-oesophageal atresias and fistulas.

In addition, we are developing an

adriamycin mouse model, which will allow

us to use a greater variety of experimental

tools to investigate the mechanism of

action of adriamycin.

1.

2.

DR CARMEL HENSEY


Our research uses embryos of the

amphibian Xenopus laevis to investigate

the cellular and molecular basis of

vertebrate development. Gene function

is studied in the context of the developing

organism by making use of overexpression

screens and antisense morpholino

oligonucleotides. Gene expression patterns

are followed by wholemount in situ

hybridisation and immunohistochemistry.

The control of cell proliferation and

cell survival is critical for the normal

development and tissue homeostasis of

multicellular organisms. Defects in these

processes underlie a number of human

diseases, in particular cancer, which

is associated with loss of controls over

cell division and evasion of cell death

by apoptosis.

The early Xenopus embryos have a very

simple cell cycle consisting only of S and M

phase. These embryos therefore provide a

system of choice for studying the increase

in complexity in cell cycle, particularly the

establishment of cell cycle checkpoints.

Our goal is to understand what determines

the balance between cell cycle progression,

checkpoint arrest and the induction of

apoptosis. Our work focuses on a number

of checkpoint proteins and apoptotic

regulators in Xenopus.

The Xenopus embryo is unique in that the

p53 tumour suppressor protein is naturally

expressed at high levels. p53’s role as a

transcription factor, mediating cellular

responses to diverse stresses such as DNA

damage, is well characterised. Additionally,

p53 is known to have a role in Xenopus

development, where the protein has a

function in patterning the body axes, an

effect mediated through its interaction

with the TGF‚ signalling pathway.

Dual roles or mutual interactions of

proliferation and developmental control

genes seem likely given the requirement

for strict coordination between embryonic

patterning and the cell cycle. Our analysis

of p53 expression patterns, in particular

the specific expression patterns of post-

translationally modified forms such

as acetylated p53, are providing insight

into the functions of the various post-

translationally modified forms of p53.

Differing expression patterns for different

forms of p53 fit with its dual role as

a cell cycle/apoptosis regulator and

developmental regulator.

We are also studying later stages of

development, particularly organogenesis

of the eye and kidney. Our emphasis is

on the molecular basis of organ formation

and in particular, we study genes that

re-emerge in adult disease. Much of our

work concentrates on the analysis of BMP

signalling in pronephric development (the

simple embryonic kidney) and specifically

the role of the BMP antagonist gremlin, an

important developmental regulator that

is also implicated in the pathogenesis of

diabetic nephropathy in adults. These

developmental signalling pathways

function to coordinate proliferation,

apoptosis and differentiation, and a

combination of gene overexpression and

gene knockdown approaches are being

used to unravel gene function during

organogenesis.

OTHER PROJECTS WHICH ARE

CURRENTLY BEING DEVELOPED

INCLUDE:

The role of the cyclo-oxygenase

/arachidonic acid pathway in

vasculogenesis and angiogenesis.

(with Dr K O Boyle).

The development of a chick embryo

rapid teratogen screening system

(with Dr J Hellwig, BASF, Germany).

1.

2.

Fig. 2. Wholemount immunohistochemistry stains

the simple tadpole kidney in blue.

Fig. 3. Green fluorescent protein is used as a tracer

and highlights half of the tadpole where gene

function is being manipulated.

Fig. 1. Wholemount immunohistochemistry for p53

protein in cleavage stage embryos. Cytoplasmic p53

is stained dark blue in panel A. Nuclear p53 shows

dark blue punctate staining in B.

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Recently, we have initiated a collaboration

with two groups at the Wellcome Trust

Sanger Institute to examine the evolution

of microRNA (miRNA) genes and their

regulatory targets in multiple nematode

genomes. These prevent translation by

binding to the 3’UTRs of mRNA moleculules

and it is estimated that there are hundreds

in the human genome, acting to control

expression of thousands of genes. Genes,

which are regulated by miRNAs have

conserved motifs in their 3’UTRs; the sites

at which the miRNAs bind by base pairing.

We are interested in finding these as well

as potential regulatory motifs in the

promoter regions of genes. Specifically,

we are searching for transcription factor

binding sites upstream of mouse

phenotypic plasticity genes and kidney

specific genes, in collaboration with

groups in the Conway Institute and the

Mater Misericordiae University Hospital.

CURRENT RESEARCH PROJECTS:

Multiple alignment methods

and software

- iterative multiple alignment

algorithms

- methods weighting

- alignment accuracy benchmarking.

Microarrays and Proteomics

- application of multivariate analysis

techniques

- analysis of 2D-gel data

(collaboration with Professor Mike

Dunn and Dr David Cotter, RCSI)

- analysis of cancer data sets

(collaborations with Dr Liam

Gallagher, Professor Joe Cassidy,

Dr Denise Sadlier, Professor Finian

Martin, Dr Ray Stallings,

Dr Linda McArdle)

- evaluation of gene/feature

selection methods.

Promoter motif analysis

- mouse phenotypic plasticity genes

(collaboration with Professor Ciaran

Regan and Dr Keith Murphy)

- kidney specific genes.

Molecular evolution and genome analysis

- evolution of miRNA genes and targets

in nematodes

- phylogeny of the main animal phyla

- analysis of the genome of

Bifidobacterium breve

(collaboration with Dr Douwe van

Sinderen, UCC)

- prediction of oncogenes in the

human genome.

055 <> 056

PROFESSOR DES HIGGINS


Bioinformatics is the application

of computational techniques to problems

in molecular biology. It has grown to be

of central importance partly due to the

sequencing of the complete genomes of

most model organisms but also due to the

huge quantities of data that are generated

by many modern experimental techniques

such as gene expression microarrays

or protein mass spectrometry. In the

bioinformatics laboratory, we are interested

in a range of bioinformatics problems from

how best to align DNA and protein sequences

to questions about how genes and

genomes have evolved.

Sequence alignment involves taking two

or more sequences and matching up the

homologous residues. This allows you to

predict some of the characteristics of one

sequence if you have information about

one or more of the others. In our group,

we have been working on multiple sequence

alignment for many years (see fig. 1).

I was the originator of the ClustalW

package, which is the most widely used

multiple alignment software. It is used on

a daily basis in thousands of laboratories

worldwide. In our group, we are interested

in developing the use of iterative algorithms

for making better alignments and doing it

faster. We are also working on weighting

methods that allow us to combine

information from many multiple alignment

programs using the T_Coffee package.

Some programs perform better on some

data sets than others. Combining them

allows you to combine the best

characteristics of each method in order

to make more generally useful alignments.

Gene expression microarrays are devices

that measure the expression levels of many

thousands of genes, simultaneously. We

are interested in applying computational

and statistical techniques to analyse array

data sets, especially from cancer studies.

We have been applying multivariate

analysis methods to two types of problem:

Using arrays as diagnostic tools to predict

the clinical characteristics of biopsy

samples or cell lines derived from tumours.

Techniques for integrating complex

and heterogeneous data sets. This has

resulted in a series of collaborations

with other groups within the Conway

Institute and abroad to develop the

techniques further or to analyse their

data sets. We are currently collaborating

with the proteomics groups within the

Conway to apply these techniques to

proteomics data sets.

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1.

2.

2MHB_A --------- VLSA ADKTNVKAAWSKV GGHAG EYGAEALERMFLGFPTTKTYFPHF ---- D 6HBW_A --------- VLSP ADK TNVKAAWGKV GAHAG EYGAEALERMFLSFPTTKTYFPHF ---- D 2MHB_B -------- VQLSG EEKAAVLALWDKV --NEE EVGGEALGRLLVVYPWTQRFFDSF GDLSN 4HHB_B --------- VLSP ADKTNVKAAWGKV GAHAG EYGAEALERMFLSFPTTKTYFPHF ---- D 5MBN --------- VLSE GEWQLVLHVWAKV EADVA GHGQDILIRLFKSHPETLEKFDRF ---- K 2LH7 -------- GALTE SQAALVKSSWEEF NANIP KHTHRFFILVLEIAPAAKDLFSFL KGTSE 3LHB_A PIVDTGSVAPLSA AEKTKIRSAWAPV YSTYE TSGVDILVKFFTSTPAAQEFFPKF ---- K *: : : * . : .: * : * : . 2MHB_A L ------ SHGSA QVKAHGKKVGDALTLAVG HLD ----- DLPGALSDLSNLH AHKLR VDPV 6HBW_A L ------ SHGSA QVKGHGKKVADALTNAVAHVD ----- DMPNALSALSDLH AHKLR VDPV 2MHB_B ---- PGAVMGNP KVKAHGKKVLHSFGEGVHHLD ----- NLKGTFAALSELH CDKLH VDPE 4HHB_B L ------ SHGSA QVKGHGKKVADALTNAVAHVD ----- DMPNALSALSDLH AHKLR VDPV 5MBN HL KTEAEMKASE DLKKHGVTVLTALGAILKKKG ----- HHEAELKPLAQSH ATKHK IPIK 2LH7 V ------ PQNNP ELQAHAGKVFKLVYEAAIQLE VTGVV VTDATLKNLGSVH -VSKG VADA 3LHB_A GLTTADELKKSA DVRWHAERIINAVDDAVASMD DT--EKMSMKLRNLSGKH AKSFQ VDPE . .:: *. : . : *. * . : 2MHB_A NFKLLSHCLLSTLA VHLPNDFTPAV HASLDKFLSSVSTVLTSK YR------ 6HBW_A NFKLLSHCLLVTLA AHLPAEFTPAV HASLDKFLASVSTVLTSK YR------ 2MHB_B NFRLLGNVLVVVLA RHFGKDFTPEL QASYQKVVAGVANALAHK YH------ 4HHB_B NFKLLSHCLLVTLA AHLPAEFTPAV HASLDKFLASVSTVLTSK YR------ 5MBN YLEFISEAIIHVLH SRHPGDFGADA QGAMNKALELFRKDIAAK YKELGYQG 2LH7 HFPVVKEAILKTIK EVVGAKWSEEL NSAWTIAYDELAIVIKKE MDDAA --- 3LHB_A YFKVLAAVIADTVA A--------- GDAGFEKLMSMICILLRSA Y------- : .: : .: ... . :

3.

4.

Fig. 2(B) ClustalW alignment of the same sequences. The coloured regions represent the structural core of 6

alpha helices. VAST and Cn3D are available from: http://www.ncbi.nlm.nih.gov/Structure/

1.

2.

Fig. 1(A) Structural alignment (using VAST) of

seven globins (human and horse alpha chains,

human and horse beta chains, whale myoglobin,

lamprey cyanohaemoglobin and lupin

leghaemoglobin) visualised using Cn3D.

057<> 058

MR ARNOLD HILL


INTRACELLULAR SIGNALLING AND

TRANSCRIPTIONAL REGULATION IN

HUMAN BREAST CANCER

The clinical use of an antiestrogen

for the treatment of breast cancer was

first reported by Cole et al in 1971, who

described the potential use of tamoxifen

(ICI 46,474). Since then, tamoxifen has

become the most widely prescribed anti-

cancer drug in the world. However, while

most estrogen receptor (ER) positive

patients will initially respond to tamoxifen,

approximately one third relapse within

a period of 18 months. While initial

response rates are high among ER-positive

tumours, most breast cancers that acquire

tamoxifen resistance do so while continuing

to express functional ER. Despite extensive

clinical use of tamoxifen, several aspects

of its mechanism of action and acquired

resistance remain unclear.

The ER is encoded for by two genes,

ER-a and ER-b. Both genes can function

as transcription factors to modulate target

gene expression. ER-a and ER-b‚ interact

with a number of nuclear proteins in vitro.

These ‘co-activator/co-repressor’ proteins

interact directly with steroid receptors at

their response element in the promoter

region of target genes to enhance or inhibit

transcription. Co-activators such as SRC-1

are thought to facilitate the activity of the

ER whereas co-repressors such as SMRT

maintain transcriptional silence. We

have described a significant association

between expression of co-activators SRC-1

and AIB1 in human breast cancer. At a

molecular level, we have observed distinct

transcription factor- co-regulatory protein

interactions and protein DNA interactions

in endocrine sensitive cells compared to

those that are resistant, in response not

only to the steroid environment but also

growth factors.

Co-regulatory proteins interact with

nuclear receptors, including ER, at a

conserved LXXLL motif within the receptor

interacting domain of the protein to drive

target gene expression. Although the

steroid co-regulatory proteins were

previously thought to exclusively associate

with nuclear receptors, we have identified

MAP kinase effector transcription factors

as novel co-activator protein targets.

These new interacting partners may

provide the basis for a new model

of endocrine resistance.

We feel that co-regulatory proteins are

pivotal to the development of endocrine

resistance and may ultimately be

responsible for the development

of steroid independent tumours.

RESEARCH TEAM 2004

I work with Dr Leonie Young, Conway

Institute and our current research

team is made up of the following people:

Postdoctoral fellows, Dr Marie McIlroy

and Dr Yvonne Buggy; PhD students,

Sinead Kelly and Niamh Cosgrave; MD

student, Eddie Myers; MCh students,

Dara Kavanagh, Ruth Pritchard and May

Cleary. Julie Watson is a research assistant

in the group.

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DR JAMES JONES


The areas of focus of my research are

autonomic neuroscience and comparative

physiology of respiratory and cardiovascular

systems. My current research interests

have expanded to include vagal paragan-

glionic chemoreceptors, accessory rat

coronary circulation, frog carotid labyrinths,

pulmonary venous system of muridae,

central regulation of crural diaphragm,

lower oesophageal sphincter and external

anal sphincter.

059 <> 060

PROFESSOR ALAN KEENAN


Our research group’s main interests are in

mechanisms/treatment of cardiovascular

disease. In recent years, this has included

investigation of roles for oxidant stress

and nitric oxide in endothelial dysfunction

and vascular injury. Our current focus is on

development of intelligent polymer-based

delivery systems for local drug delivery in

prevention of restenosis injury following

angioplasty.

While stenting at the time of percutaneous

transluminal coronary angioplasty (PTCA)

has significantly reduced the incidence of

restenosis in coronary vasculature, 20-30%

of patients undergoing coronary intervention

with stent implantation require repeat

procedures. This may be due to localised

neointimal proliferation at the site of

implantation, or to multiple effects

in distal/proximal arterial segments.

The luminal enlargement achieved with

stenting of the diseased vessel damages

the endothelial monolayer lining the blood

vessel wall, disrupts the internal elastic

lamina and causes dissection of the medial

layer. Exposure of the media to circulating

blood results in platelet activation and

aggregation, with the potential for formation

of a platelet-rich thrombus and acute

restenosis. Secretion of growth factors

and further inflammatory mediators

converts vascular smooth muscle to a

synthetic phenotype, leading to vascular

smooth muscle cell (VSMC) migration

and proliferation, neointimal hyperplasia,

critical lumen narrowing and late restenosis.

The biological processes described

above suggest a number of targets

for pharmacological intervention. These

therapies can be divided into categories

based on mechanisms of action; namely,

anti-thrombotic, anti-inflammatory, anti-

mitotic and pro-mitotic agents for targeting

of unwanted platelet deposition, VSMC

proliferation, or desirable endothelial cell

re-growth respectively. Optimal efficacy

of such agents can be achieved in principle

by local delivery. This achieves high

regional drug concentrations, with

prolonged retention at lower doses

and reduced systemic toxicity. It also

offers the advantage that drugs with low

bioavailability can be targeted directly

to the required site. The intravascular

stent is currently under intensive scrutiny

as a local drug delivery system for

targeting restenosis injury. The key factor

determining efficacy for drug-eluting

stents is the density of drug dose achieved

in the injured segment of the vessel wall,

rather than the overall concentration in

the coronary tree. One of the approaches,

which has been employed to date involves

incorporation of drugs into a polymer,

which is then used to coat the stent. The

coating procedure can also be tailored to

generate improved stent biocompatibility.

Thermoresponsive polymers are a class

of potential stent coatings, which our group

has been using as systems for elution of

drugs with a range of therapeutic activities.

To date, we have incorporated drugs into

a bulk gel medium from which they are

eluted, following a phase transition

undergone by the polymer with increased

temperature. A significantly more versatile

structure known as a ‘plum-pudding’ gel

has recently been characterised, which is

composed of responsive microgel particles

dispersed in the bulk gel medium. The

potential exists for incorporation of

individual drug types into different

populations of microgel particles and

differentially regulating their delivery.

We are currently exploiting this system

for multi-drug delivery in prophylaxis

of restenosis injury.

CURRENT RESEARCH INTERESTS

Hormonal regulation of signal

transduction in the cardiovascular

system.

Vascular endothelial biology in health

and disease.

Role of vascular endothelium

in inflammation.

Nitric oxide in vascular and

extra-vascular tissue.

Oxidant stress and endothelial

dysfunction in diabetes.

Vascular injury and repair in coronary

artery disease.

Therapeutic angiogenesis.

Local drug delivery in the

vascular system.

Development of intelligent

polymer systems for delivery

of anti-restenosis therapy.

Host-foreign body responses to

implanted vascular biomaterials.

Peripheral vascular actions of novel

substituted amphetamines related

structurally to MDMA.

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061<> 062

DR BRENDAN KENNEDY


Our current research interests utilise

zebrafish to identify genes responsible for

survival and function of cone photoreceptors,

and involve generating zebrafish models

of cone photoreceptor disease that can

be incorporated into drug screens.

CONE PHOTORECEPTORS

Vision is mediated by rod and cone

photoreceptors of the retina. Rod

photoreceptors function during dim

nightlight conditions but are saturated

during daylight conditions. In contrast,

(red-, green-, blue- and ultraviolet-

sensitive) cone photoreceptors are

insensitive to low light levels but function

over a vast range of daylight conditions to

enable high visual acuity and colour vision.

Human blindness can result from genetic

mutations that cause the death of rod

and/or cone photoreceptors. The loss

of cone photoreceptors during numerous

forms of retinal degeneration constitutes

the principal reason for severe visual

handicap and human blindness such that

patients are no longer able to read or

discriminate detailed features such as

faces. In age-related macular degeneration

(AMD), cone photoreceptors concentrated

within the central human retina gradually

die AMD is the predominant reason for

blindness worldwide, currently affecting

~25 million individuals, and expected to

triple in incidence within 40 years. Thus,

research to understand cone photoreceptor

function and pathology is critically needed.

ZEBRAFISH

How can we identify genes and drugs

that enable normal cone photoreceptor

function or that can slow the onset of

degeneration? Mouse and rat models have

provided insights into the time course

and molecular basis of rod degenerations

as well as the efficacy of therapeutic

approaches However, the scarcity of cone

photoreceptors in mice and rats renders

these nocturnal models inappropriate

for cone photoreceptor studies.

The advent of zebrafish as a prolific model

and the abundance of cone photoreceptors

in zebrafish has provided us with novel

opportunities to investigate vertebrate

cone photoreceptors in vivo. Zebrafish are

small in size and regularly produce large

clutches of transparent offspring that

rapidly develop ex-utero. These properties

enable high-throughput genetic and

pharmacological screens to be performed

in vivo.

Zebrafish have abundant, layered cone

photoreceptors. This facilitates analysis of

the individual red-, green-, blue-, and UV

-sensitive cone types. Advantages of the

zebrafish system include the ability to

produce large numbers of experimental

samples. A single pair of zebrafish can

generate ~200 embryos per mating and

can be mated several times per week.

These embryos develop externally,

eliminating invasive procedures, and

develop rapidly (see movie of development

at (http://zfin.org/zf_info/movies/

Zebrafish.mov) such that organs, including

the eye, are visible at 1-day post fertilization

(dpf). In addition, adult fish only grow to

~2 inches in length and this compact size

facilitates the maintenance of large

numbers of fish.

Importantly for studies of vision, the

morphology of the zebrafish retina is

highly conserved with the human retina,

both comprising of neuronal (rod and

cone photoreceptors, amacrine, horizontal,

bipolar, and ganglion), epithelial (retinal

pigment epithelium) and glial (Müller)

cell types. Secondly, the genes involved

in eye development (pax6, rx, six3) and

phototransduction (opsins, transducins,

phosphodiesterase) appear conserved

between fish and humans. Finally, the

integrity of the zebrafish visual system can

be assayed by morphological, behavioural

and physiological approaches (see movie

at http://faculty.washington.edu/jbhhh/

Movies/fish.mov).

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Visualisation of retinal cell layers upon staining

sections with Methylene Blue-AzureII. rpe, retinal

pigment epithelium; pcl, photoreceptor cell layer;

opl, outer plexiform layer; inl, inner nuclear layer;

ipl, inner plexiform layer; gcl, ganglion cell layer;

cgz, circumferential germinal zone; on, optic nerve.

Photo of an adult zebrafish.

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Our main interest is in improving our

understanding of the basic mechanisms

controlling follicle growth, oocyte and

embryo development and the establishment

of pregnancy. Specifically, using in vivo

and in vitro, bovine and ovine models, we

can examine follicle-oocyte relationships,

oocyte maturation, fertilisation and early

embryo development.

It is well known that embryos produced

in vitro are of poorer quality than those

produced in vivo, in terms of morphology,

cryotolerance and their ability to establish

a pregnancy. The environment in which

an embryo develops can have a dramatic

effect on the relative abundance of gene

transcripts within it and on its quality,

manifested in terms of cryotolerance or

ability to establish a pregnancy. We have

shown that exposure to suboptimal

conditions for even a relatively short

period can lead to sustained changes

in mRNA expression. Such changes

undoubtedly have consequences for

the future developmental potential

of the embryo.

Currently, the focus of our work is to

compare differential gene expression in

oocytes and embryos derived in vivo or in

vitro in an effort to understand why those

produced in vitro are less developmentally

competent. We are doing this using

techniques such as suppressive subtractive

hybridisation, DNA microarray technology

and real-time RT-PCR.

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Fig. 1. Bovine 2 to 6-day-old embryos.

DR BARTHOLOMEW MASTERSON, PHD


MICROBIAL POLLUTION

Coherent integration of ecological,

physicochemical and microbiological

aspects in river basin management plans

is a substantial part of the challenge set

by the EU Water Framework Directive

(2000/60/EC). Compared to ecological

/chemical investigations though, relatively

few microbiological investigations have

been carried out at the river basin

(catchment) level in Ireland.

My research group completed recently

an INTERREG-II funded Irish-Welsh

study of the impact of storm-related

riverine microbial loads on bathing beach

hygiene centred on the Dargle catchment

which drains to the sea at Bray, Co. Wicklow.

Microbial ‘budgets’ were prepared to show

the numbers of organisms coming from

all the different catchment sources which

might impact on bathing water quality

— both in quiescent weather conditions

(low river flow) and in high rainfall

conditions when there was high run-off

and river flow. In general, the numbers

were greatly increased as a result of

rainfall events, and there was coincident

deterioration in bathing site microbial

water quality. Mathematical models were

used to represent the impacts of land use

and the influence of rainfall on river water

quality within the catchment, to make more

clear their likely effects on bathing sites.

Since then, the EU Commission has proposed

a revised Bathing Water Directive that

would classify bathing areas on the basis

of new microbial water quality standards,

having regard to other environmental hazards

that include relevant catchment impacts.

In association with colleagues in the

Department of Geography and the

Department of Civil Enginering, we are

now engaged in extended studies that

reflect the Water Framework Directive

2000/60/EC philosophy, centred on two

case-study estuaries (Liffey, Co. Dublin

and Bannow, Co. Wexford) and their

associated catchments. The study continues

the collaboration with colleagues in Wales.

We seek to develop predictive models

of the direct and indirect impacts of point

-source and diffuse microbial pollution

that will be generic, ie. suitable for wider

implementation. The overall objective

is to enhance the potential for sustainable

aquaculture (principally shellfisheries) and

tourism through the provision of predictive

modelling tools for amenity management.

In association with colleagues in the

Department of Industrial Microbiology

and the Department of Geography, and

with trans-national partners in France,

Portugal, Spain and the United Kingdom,

we are developing molecular biological

source-tracking techniques to phenotype

indicator microorganisms in the aquatic

environment. The objective is to be able

to distinguish between human and non-

human sources of diffuse microbial

pollution so as to inform appropriate

catchment management actions.

We advised the Irish Government in

the negotiations during the Irish EU

Presidency on the technical aspects of the

draft Bathing Water Directive; technical

consensus was achieved during that period.

INDUSTRIAL

Studies on the haemostatic action of

a constituent incorporated in a range of

healthcare products are continuing under

contract for an industrial client. Also the

cholesterol-lowering action of one of the

client’ products is underway.

2-cell 8-cell 16-cell

2-cell 8-cell 16-cell

DR PATRICK LONERGAN, B AGR SC, M AGR SC, PHD, D SC


DR JOHN LOWRY


A growing number of new methodologies

are being developed, including sampling

(eg. microdialysis), spectroscopic

and electrochemical, to study such

neurochemical phenomena in the living

brain. One such set of techniques focuses

on the detection of substances using

amperometric electrodes and voltammetric

techniques in vivo. By implanting a

microvoltammetric electrode (sensor

/biosensor, see below) in a specific brain

region, applying a suitable potential profile

and recording the resulting faradaic

current, changes in the concentration of

a variety of substances in the extracellular

fluid can be monitored with a sub-second

time resolution over extended periods.

This allows investigations of the functions

and roles of specific neurochemicals in

neuronal signalling, drug actions, and

well-defined behaviours, with a high time

resolution presently not available with

other methods.

In vivo voltammetry (IVV) using

electrochemical sensors (ie electrodes

which respond to particular electroactive

analytes in a selective way through a redox

reaction) can be used to target a wide

variety of electroactive compounds in the

extracellular fluid including: small, highly

permeable species such as dioxygen and

nitric oxide; ascorbic acid, which has a

range of general anti-oxidant and specific

neurochemical functions; neuromediator

catecholamines such as dopamine and

noradrenaline, as well as their metabolites

3,4-dihydroxyphenylacetic acid (DOPAC),

3-methoxytyramine and homovanillic acid;

the 5-hydroxyindole neuromediator 5-

hydroxytryptamine (serotonin) and its

metabolite 5-hydroxyindoleacetic acid;

and the purine metabolite uric acid.

Non-electroactive or poorly behaved

electroactive species, such as glucose,

glutamate and lactate, can now be

targeted in vivo using biosensors, in the

form of enzyme modified electrodes. Such

biosensors involve the immobilisation of

a sensitive and selective biological element

(eg. an enzyme) on, or within close

proximity to, an analytical detector

(the electrode). Their use has significantly

broadened the applications of IVV for

understanding the neurochemical basis

of behaviour. Neurochemical analysis in

intact tissue can also be performed using

in vivo microdialysis. This technique

involves the perfusion of a physiological

fluid (artificial cerebrospinal fluid)

through an implanted semi-permeable

dialysis probe. The low molecular weight

compounds that diffuse into the dialysis

fluid are collected and assayed ex-situ,

generally using HPLC methods. Although

the technique has limitations with respect

to time resolution and quantitative

determinations (the concentration

of analytes in the dialysate depends

on a variable in vivo recovery), it has the

advantage that specific substances (eg.

neurotransmitters and selective agonists

/antagonists) can be added to the

perfusion fluid thus enabling novel

pharmacological studies to be

performed in situ.

In the bioanalytics laboratory we combine

both in vivo techniques in order to capitalise

on the distinct advantages of the two

methodologies: high temporal resolution

(voltammetry), and the ability to administer

drugs directly into the local extracellular

environment (microdialysis). The laboratory

has two units: the sensors development

unit and the neurochemistry research

unit. The former focuses on the in vitro

development and characterisation of

microelectrochemical devices for real-time

monitoring of important biological species,

while in the neurochemistry unit, these

devices are used in targeted pre-clinical

and clinical neuroscience studies

(see Fig. 1).

Funding Agencies: Science Foundation

Ireland (SFI); Health Research Board

(HRB); The Wellcome Trust; Enterprise

Ireland; Royal Society of Chemistry

(RSC) & Engineering & Physical Sciences

Research Council (EPSRC), UK; European

Commission; Eli Lilly & Company Ltd., UK;

Solvay Pharmaceuticals, The Netherlands;

UCBPharma, Belgium.

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Fig. 1. A & B: Simultaneous measurement of

regional cerebral blood flow (rCBF, A) using the

H2 clearance technique (Pt electrode), and tissue

O2 (B) using a carbon paste electrode, in rat

striatum in response to neuronal stimulation

(5 min tail pinch, TP).

C: The effect of a 5 min tail pinch on brain

extracellular glucose levels recorded in real time

in the striatum of a freely moving rat using a

microelectrochemical glucose biosensor.

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Recent advances in high-throughput DNA

mapping and sequencing, gene expression

technologies and bioinformatics, originally

developed for human and model organism

research, have begun to profoundly impact

on farm animal genomics. Our research

group has obtained significant funding from

Science Foundation Ireland (SFI) and we

are using advanced genomic technologies

to investigate physiological, biochemical

and genetic regulatory networks that control

traits of economic or veterinary interest.

Current research themes are as follows.

BOVINE IMMUNOBIOLOGY

We are using massively parallel gene

expression technologies such as DNA

microarrays to study the host response

to infection with Mycobacterium bovis, the

causative organism of bovine tuberculosis.

Using an immunogenetic DNA microarray

capable of monitoring messenger RNA

(mRNA) abundance for thousands of genes

simultaneously, we have identified a gene

expression signature of infection in cattle

infected with M. bovis (Fig. 1). We have

also been able to reconstruct the Toll-like

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DR DAVID MACHUGH


receptor (TLR) signalling pathways that

detect microbial infection and induce

activation of inflammatory and antimicrobial

innate immune responses.

African bovine trypanosomiasis, caused

by the extracellular flagellate protozoan

trypanosome parasite (Trypanosoma spp.),

is endemic throughout the humid and semi-

humid zones of sub-Saharan Africa. The

disease is a major constraint on livestock

production in Africa. We are using DNA

microarrays and high-throughput proteomic

technologies to investigate the host response

to trypanosome infection and in particular,

to identify the genes responsible for the

phenomenon of trypanotolerance, a heritable

resistance to trypanosomiasis unique to

certain breeds of West African cattle.

POPULATION GENOMICS OF

ECONOMICALLY IMPORTANT TRAITS

IN DOMESTIC CATTLE

The objectives of this work are to study

the physiological pathways underpinning

milk production in dairy cattle, to define

the roles of key genes in the process

of lactogenesis and to investigate the

antagonistic relationship observed

between milk production and fertility.

We have analysed population haplotypes

composed of single nucleotide

polymorphisms (SNPs) and other

molecular markers spanning the bovine

growth hormone (GH1) and growth

hormone receptor (GHR) genes. This work

has provided novel information regarding

the long-term molecular evolution of these

genes. In addition, it has also shed light

on population variation and microevolution

due to intense artificial selection for milk

traits in dairy cattle.

LIVESTOCK GENOME MAPPING

Arthrogryposis multiplex congenita (AMC)

is a symptom complex characterised by

flexion rigidity or limitation in the range

of motion of joints at birth. Our research

group has characterised an inherited form

of ovine AMC (OAMC) and we have used

a whole genome scan of a large affected

pedigree to identify a candidate region

for ovine AMC on the short arm of sheep

chromosome 5 (OAR5). Comparative

genomics analysis indicates that the

mapped region is syntenic to a segment

of the long arm of human chromosome

5 (HSA5). Notably, a form of human AMC

neuropathic type has been fine-mapped

to a 442 Kb interval within this region.

These linkage results (in sheep and

humans) and the syntenic location of the

mapped regions provide strong evidence

that the ovine and human AMC loci

represent the same gene, or are part

of the same gene cluster.

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Fig. 1. BOTL-5 DNA microarray image and gene expression signature of Mycobacterium bovis infection

in cattle. A BOTL-5 DNA microarray image is shown from a tuberculosis-infected animal. Also shown is an

average linkage (UPGMA) cluster diagram showing a gene tree for 40 genes differentially expressed between

tuberculosis-infected cattle and uninfected control animals (P<0.0001). Gene expression fold change is

indicated by the colour scale.

Fig. 2. Field work on trypanosomiasis in Kenya.

Dr Emmeline Hill, Animal Genomics Laboratory,

Department of Animal Science, UCD, carrying out

fieldwork on bovine trypanosomiasis with Kenyan

colleagues at the International Livestock Research

Institute (ILRI) in Nairobi, Kenya.

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PROFESSOR FINIAN MARTIN

CONWAY INTEGRATIVEBIOLOGY

The work of my group focuses

on the following:

INTRACELLULAR SIGNALS THAT

DETERMINE THE ORGANISATION OF

MAMMARY EPITHELIAL CELLS INTO

3D-ASSEMBLIES:

Culture of epithelial cells as 3-D

assemblies is a powerful technique

with which to examine the molecular

requirements for tissue-like epithelial

organisation. Mammary epithelial cells

generate spherical acini when cultured

in a supporting laminin-rich extracellular

matrix (ECM). These assemblies lose their

internal cells to form spherical monolayers,

an arrangement reminiscent of in vivo

mammary epithelial acini of pregnancy

/lactation. ECM contributes to the spatial

organisation of the acini through

interactions with integrins. Essential cell-

cell interactions occur via tight junctions,

adherens junctions and desmosomes. Our

recently published work establishes that:

Dispersed mammary epithelial cells

harvested from mid-pregnant mice

and placed in primary culture on a

concentrated laminin-rich extracellular

matrix (Matrigel) form 3D-spherical

acini that mature to contain a single

layer of polarised epithelial cells

surrounding a lumen.

This process requires glucocorticoids

and that the glucocorticoids act

by triggering a novel intracellular

signalling cascade, a glucocorticoid

receptor (GR)-Brca1-GADD45bMEKK4-

JNK pathway, in supporting

acinus formation.

The GR-Brca1-GADD45b-MEKK4-JNK

pathway is essential for atl east some

critical phases of acinus formation:

inhibition of JNK activity with a

specific inhibitor disrupts functional

acinus formation.

GENE EXPRESSION AND

INTRACELLULAR SIGNALLING IN THE

MAMMARY EPITHELIUM AT PUBERTY

Expression microarray analysis identified

CITED1, an estrogen receptor coactivator,

among a group of genes specifically

upregulated in the pubertal mouse

mammary gland. At puberty, CITED1

localises to the luminal epithelial cell

population of the mammary ducts and

the body cells of the terminal end buds.

Generation of CITED1 gene knockout mice

demonstrated that female homozygous

null mutants exhibit retarded mammary

ductal growth in comparison to age matched

wild type females and heterozygous female

littermates at puberty. This mammary

gland phenotype is further characterised

by dilated ductal structures and a lack

of spatial restriction of the subtending

branches. Analysis of homozygous null,

heterozygous and wildtype pubertal

mammary gland gene expression using

microarrays suggests that the mammary

specific phenotype seen in the homozygous

null females is due to a disturbance in the

transcription of a number of key mediators

of pubertal ductal morphogenesis.

INTRACELLULAR SIGNALLING AND

ACTIN CYTOSKELETON TURN-OVER IN

THE RENAL MESANGIUM IN DIABETIC

NEPHROPATHY

Diabetic nephropathy (DN), a fibrotic

disorder of the kidney, is the leading cause

of end stage renal disease. High

extracellular glucose plays a pivotal role

in the development of glomerulosclerosis

in DN. Transforming growth factor beta

(TGF-b), a pleiotropic profibrotic cytokine

mediates many of the pathological

changes of diabetic kidney disease. We

have previously described several genes

whose expression are differentially

regulated in response to high glucose,

amongst these was IHG-1 (induced in high

glucose-1). IHG-1 is a novel evolutionary

conserved protein, whose expression

is significantly increased in patients

suffering from DN. IHG-1 is localised to

mitochondria in mammalian cells and its

expression is induced by reactive oxygen

species but not by TGFb1. Importantly,

IHG-1 amplifies TGFb1. induced

transcriptional activation associated

with enhanced Smad 3 availability

/activity as overexpression of IHG-1

results in increased levels and enhanced

phosphorylation of Smad 3. Deletion of

the mitochondrial localisation signal of

IHG-1 suppresses its amplification of

TGFb‚ activity. Thus, mitochondrial

location of this conserved intracellular

protein determines its ability to modulate

TGFb‚ signalling establishing a novel

mitochondrial-nuclear regulatory pathway.

Our group is engaged in a number of

translational research projects that examine

the pathophysiology of obstructive sleep

apnoea and its cardiovascular consequences.

The clinical and physiological components

of the research are based in the respiratory

sleep research laboratory at St. Vincent’s

University Hospital and the basic science

components in the Conway Institute under

the directions of Dr Cormac Taylor and the

Departments of Electronic and Electrical

Engineering, UCD in collaboration with

Dr Conor Heneghan and Professor

Mark O’Malley.

OBSTRUCTIVE SLEEP

APNOEA SYNDROME

(OSAS) affects 4% of adults and is

characterised by frequent transient

oxygen desaturations during sleep. It

is characterised by repetitive episodes

of apnoea or hypopnoea during sleep,

up to several hundred times per night in

severe cases, which lead to hypoxia, sleep

fragmentation, excessive sleepiness and

fatigue with associated risk of serious

sequelae such as road traffic accidents.

OSAS is an independent risk factor

for cardiovascular diseases, particularly

systemic hypertension, but the underlying

molecular mechanisms are poorly

understood. Hypoxia is likely to play

a significant role, particularly the

intermittent hypoxia and re-oxygenation

(IHR) that are characteristic of recurring

apnoeas. We are currently exploring the

potential role of IHR in OSAS to activate

molecular pathways that predispose to

vascular endothelial damage. We are

studying changes in a range of pro-

inflammatory cytokines and adhesion

molecules in addition to adaptive

HIF-1 dependent genes before and after

treatment with nasal continuous positive

airway pressure (nCPAP).

In collaboration with Dr Taylor in the

Conway Institute, we have developed

a unique model of intermittent hypoxia

/reoxygenation in short cycles for cell

cultures and have already utilised this

model to demonstrate that IHR activates

the adaptive HIF-1 pathway to a lesser

extent than sustained hypoxia whereas

both forms of hypoxia activate the

inflammatory NFKB-dependent pathway.

The transcriptional activity was measured

using a NFKB- and HIF-1-dependent

luciferase reporter gene. We have

confirmed the activation of NFKB by IH

using DNA binding assays and Western

blot. Furthermore, we have assessed the

impact of IH on the expression of typical

NFKB downstream genes encoding for

proinflammatory cytokines such as TNFK

and IL-8 in OSAS patients before and after

CPAP therapy and demonstrated elevated

TNFK a levels in OSAS patients compared

to matched controls that fall to control

levels after CPAP therapy.

This comprehensive translational project

should help clarify the molecular

mechanisms of IHR and relate these to

the cardiovascular consequences of OSAS.

In collaboration with Dr Heneghan of the

Department of Electronic and Electrical

Engineering, we have studied the ability

to identify sleep apnoea from digital signal

processing of the ECG signal, based on

characteristic changes in the ECG that

accompany apnoea. We are working

towards the development of a simplified

ambulatory diagnostic system for OSA

based on detailed analysis of the ECG,

oxygen saturation and related derived

signals such as pulse transit time. In

collaboration with Professor O’Malley in

the same department, we have developed

new surface electrode configurations to

record genioglossus EMG with a view to

studying the role of skeletal muscle fatigue

in the pathophysiology of OSA.

CURRENT ONGOING

RESEARCH PROJECTS:

Cell and molecular mechanisms

of cardiovascular complications of

sleep apnoea, in collaboration with

Dr Cormac Taylor.

Role of genioglossus fatigue in the

pathophysiology of sleep apnoea, in

collaboration with Professor Mark

O’Malley and Dr Philip Nolan.

New diagnostic techniques in sleep

apnoea based on bioimpedance

measurements and digital signal

processing of the ECG signal, in

collaboration with Dr Conor Heneghan.

Evaluation of cardiovascular outcomes

of continuous positive airway pressure

therapy (CPAP) in sleep apnoea.

Comparison of auto-adjusting and fixed

pressure therapy for sleep apnoea.

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PROFESSOR WALTER MCNICHOLAS MD, FRCPI, FRCPC


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DR GETHIN MCBEAN


Our area of interest is neurotransmitter

transport and metabolism, which currently

comprises three areas of investigation:

the regulation of glutamate transport,

the mechanism of action of gliotoxic amino

acids and understanding how drugs such

as 3,4-methylenedioxymethapmhetamine

(MDMA; ‘Ecstasy’) affect neurotrans-

mitter function.

Glutamate transporters are a family

of integral membrane proteins that are

essential for the removal of extracellular

glutamate following its release from pre-

synaptic terminals. The transport of

glutamate is therefore necessary to

prevent hyperactivation of glutamate

receptors leading to glutamate-induced

neurotoxicity. There are five subtypes

of mammalian glutamate transporters

(GLAST, GLT1, EAAC1, EAAT4 and EAAT5).

Gene knockout studies have indicated that

the bulk of glutamate transport is into

astrocytes, via GLT1 and, to a lesser extent,

GLAST. The EAAC1 transporter may have

a totally different role and we have recently

shown that cystine and cysteine are

preferred substrates for this transporter.

An emerging and highly interesting aspect

of glutamate transporters is that of

differential regulation by fatty acids.

We have shown, using transporters

overexpressed in HEK cells, that

docosahexaenoic acid (DHA) up-regulates

transporter activity by both GLT1 and

EAAC1, but down-regulates transport

by GLAST. The net effect, as well as the

timescale, differs according to the identity

of the transporter overexpressed in each

cell line. The effect of DHA on glutamate

transport also shows a differential

response following manipulation of

extracellular calcium and calcium-

calmodulin dependent kinase II (CaMKII).

Such differential modulation immediately

rules out non-specific effects of fatty acids

on membrane integrity, and a full analysis

of the mechanism of action of fatty acids

on transport is currently ongoing.

Gliotoxins are a group of a-amino di-

carboxylic acids that are structurally-

related to L-glutamate that are toxic

to glial cells. Several of the gliotoxins

(L-a aminoadipate, L-homocysteate,

L-cysteate) occur naturally as metabolic

intermediates in the brain, whereas

another, bN-oxalylamino-L-alanine,

which is a constituent of the chickling

pea (lathryus sativus), has been implicated

as a causative agent in the human disease

known as neurolathryism. We have

initiated a programme of research into

the mechanism of action of gliotoxins,

by determining the effects of these

compounds on intermediary metabolism

in C6 glioma cells using NMR spectroscopy.

We have already concluded that inhibition

of glutamate transport into astrocytes

per se is not central to toxicity, but that

pathways associated with metabolism

of glucose, alanine and glutathione may

be the primary targets. Our hypothesis

is that, whilst there are important

differences in the mechanism of action

amongst the toxins, they all compromise

the capacity of the astrocyte to carry out

vital functions such as detoxification of

ammonia through synthesis of glutamine,

or synthesis of the major cellular

antioxidant, glutathione.

MDMA is widely abused, particularly

amongst young people, and much research

has been done on its proposed mechanism

of action and potential toxicity. However,

the emergence of MDMA-like compounds

on the drug scene poses a new threat to

human health, because virtually nothing is

known of how they might affect neuronal

function. We have used an approach of

rational synthesis to construct approximately

30 novel structural analogues of MDMA,

which we are testing on aspects of

monoamine-mediated neurotransmission

that are known to be targets of MDMA

in the brain. These compounds include N-

alkyl derivates of MDMA, as well as a range

of compounds in which the methylenedioxy

functionality of MDMA has been altered.

In this way, information on the potential

toxicity of these compounds will be gained

in advance of their emergence as street

drugs. Our results to date show that there

are notable differences in the mechanism

of action of MDMA analogues.

RESEARCH PROJECTS

CURRENTLY ONGOING:

Analysis of the mechanism of

regulation of glutamate transport

by fatty acids.

Determination of mechanism of

transport of cystine and cysteine

by individual glutamate transporters

overexpressed in HEK cells (in

collaboration with Dr Thomas Rauen,

University of Münster).

Analysis of the metabolic disruptions

by gliotoxic amino acids on primary

cultures of astrocytes and glioma

cell lines by NMR spectroscopy (in

collaboration with Dr Lorraine Brennan).

Analysis of the effect of MDMA

analogues on noradrenaline-mediated

signalling in the vasculature (in

collaboration with Prof Alan Keenan

and Prof Pat Guiry).

Determination of the interaction of

MDMA analogues with monoamine

transporters in PC12 and HEKSERT-

TREX cells (in collaboration with Prof

Alan Keenan and Prof Pat Guiry).

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Our research involves the study of kidney

fibrosis and end-stage renal disease.

The incidence of end stage renal disease

(ESRD) is rising at a rate of 8-10% per

annum. While the initiating factors of

the disease may vary, tubulo-interstitial

fibrosis is a key event and indicates

a likelihood of progression to ESRD.

Therapeutic strategies to halt the

progression to ESRD are very limited

mainly because of a lack of understanding

of the mechanisms involved in tubulo-

interstitial fibrosis.

MOLECULAR AND CELLULAR BIOLOGY

OF RENAL FIBROSIS

We believe that a process known as

epithelial-mesenchymal transition (EMT),

whereby epithelial cells become more

fibroblast-like, may be involved in the

development of kidney fibrosis (Fig. 1).

EMT is a physiological process during

embryo development but in the adult can

play a fundamental role in the early stages

of transformation, invasion and metastasis

of cancer cells. This process involves the

loss of epithelial cell markers such as E-

cadherin and the increase in mesenchymal

markers such as a-smooth muscle actin

(Fig. 2). We are using DNA microarray

analysis to determine specific gene

changes in cell culture models of renal

fibrosis, including EMT models.

RENAL PHARMACOLOGY

AND TOXICOLOGY

We are also interested in the development

of models of renal cell injury, including

the investigation of the role of immuno

-suppressant agents in renal fibrosis

through the application of genomics

and proteomics techniques. Studies

on differentially regulated genes and

proteins provide insights not only into

immunosuppressant-induced renal

fibrosis, but also into the molecular

mechanisms underlying EMT in other

contexts, including tumour biology

and fibrosis of other organs. Further

examination of differentially regulated

genes may lead to the identification

of novel genes and potential

adjunct therapies.

The major focus of our research is

providing new models to develop our

understanding of the mechanisms of

nephrotoxicity and kidney damage, with

the aim of identifying new therapeutic

targets and strategies.

PROFESSOR PAUL MCLOUGHLIN


Our research group is focused on the

exploration and understanding of key

mechanisms in the development and

progression of lung disease, in order to

identify and validate novel therapeutic

strategies. Lung diseases including

asthma, chronic obstructive pulmonary

disease (COPD), emphysema, cystic

fibrosis, adult respiratory distress

syndrome and occupational lung diseases,

are amongst the most common causes of

death and disability worldwide. Reduced

oxygen (hypoxia) is a common complication,

which increases morbidity and mortality.

Unfortunately, our present treatment

options are limited, often providing

symptomatic relief and dealing with

recurrent infections but in most patients

they do not cure and often do not even slow

the progression of the underlying disease.

The responses of the pulmonary

circulation play a key role in the

development of many lung diseases.

These vascular responses, including the

development of pulmonary hypertension,

are mediated by hypoxic and inflammatory

pathways. We have a well-established

research programme investigating

structural and functional alterations in

the pulmonary vasculature in models of

chronic hypoxic and inflammatory lung

diseases. We have demonstrated the

important modifying effects of nitric

oxide and elevated carbon dioxide on

both hypoxic and inflammatory pulmonary

vascular disease and important interactions

of these two endogenously produced

gases. We have recently shown, for the

first time, angiogenesis in the pulmonary

circulation in response to hypoxia, a

vascular change that was previously

thought not to be possible in adulthood.

We are currently exploring the molecular

mechanisms underlying this response.

A key strategy in the approach to our

research is to use integrated in vivo disease

models that allow the assessment of

fundamental mechanisms in a context that

is directly related to disease in humans.

This positions us ideally to translate

discoveries at molecular level into

clinically relevant models and to test

their potential relevance to human lung

diseases. We have well-established

collaborations with respiratory clinicians

at UCD’s teaching hospitals, St Vincent’s

University Hospital and the Mater

Misericordiae University Hospital.

DR TARA MCMORROW


F-actinTEC

F-actinTEC

E-cadherin -SMA

E-cadherin -SMA

Fig. 2. Immune cell products induce genotypic

changes in kidney epithelial cells. TEC cells were

cultured in the absence or presence of immune cell

products for 48 hours. The effects on E-cadherin

expression and de novo a-SMA expression were

assessed by immuno-fluorescent microscopic

examination.

Fig. 1 Microscopic image (confocally acquired)

of lung tissue showing identification of endothelial

cells within the alveolar walls for the purpose of

detecting new vessel formation in response to

reduced oxygen. Nuclei of all cells were stained

with propidium iodide (red) while endothelial

cells were identified using anti-VEGFR-2 antibody

visualized with FITC (green) labelling. Scale bar

represents 10 μm.

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Fig. 1. Immune cell products induce phenotypic

changes in kidney epithelial cells. TEC cells were

cultured in the absence or presence of immune cell

products for 48 hours. Morphological changes were

examined by phase-contrast and immuno-

fluorescent microscopy.

PROFESSOR MARK ROGERS


Our research group is interested in the

neurodegenerative diseases caused by

a novel group of infectious agents called

prions. The prion is a misfiled form of a

normal host protein termed PrP. Diseases

caused by prions include Creutzfeldt-

Jacob disease (CJD) in humans, bovine

spongiform encephalopathy (BSE) in

cattle and scrapie in sheep. The human

diseases may be inherited or caused by

infection. The economic consequences

of this family of disease are of particular

significance in Ireland where a large

proportion of the cattle production

is exported. In addition, the health

consequences of the human prion diseases

extend beyond the affected individuals but

include risks to the blood supply and to

contamination of pharmaceuticals and

other products derived from animal sources.

Our work has currently been focused in

two areas, the normal function of the prion

protein in cells and in understanding the

processes at the cellular level, which are

involved in the formation of abnormal,

disease causing form of the prion protein.

Over the past few years, we have utilised

a cell culture model of infection in mouse

neuroblastoma cells that can be chronically

infected with mouse prions. Using these

cells we have been modelling the

consequences of different mutations

in the PrP gene on both normal function

of PrP and on the replication of the infectious

agent. In addition, we have been evaluating

the consequence of the infection of these

cells on their survival.

The long term objectives of the research

group is to understand the role of the prion

protein both in normal cellular metabolism

and the processes that occur during both

acute and chronic infection by prions.

RESEARCH CURRENTLY UNDERWAY

IN THE LABORATORY INCLUDES:

Determination of the temporal and

spatial distribution of the infectious

agent in cattle orally infected with BSE.

Development of scFv fragments that

recognise abnormal prion proteins

and that can interfere with the

replication events.

Defining the role of the prion protein

in oxidative stress.

Understanding the consequence of the

infection on cells chronically infected

with prions.

Characterising the acute stage and

strain specific factors involved in the

infection process using mouse cell

culture as a model.

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DR PAUL MOYNAGH


My research group currently consists of

three postdoctoral fellows and five PhD

students with extensive national and

international collaborations. Work in

my laboratory has focused increasingly

on proinflammatory stimuli and the signal

transduction pathways that they employ

in effecting an inflammatory phenotype.

Studies especially concentrate on human

Toll-like receptors (TLRs). TLRs recognise

pathogen associated molecules. As examples,

TLR2 recognises peptidoglycan and

bacterial lipoprotein from Gram-positive

bacteria, TLR3 mediates responses to

double-stranded RNA, TLR4 is involved

in recognition of Gram-negative

lipopolysaccharide (LPS), TLR-5

recognises bacterial flagellin and TLR9

functions as a receptor for bacterial DNA

containing CpG motifs. The engagement

of TLRs by pathogenic components results

in induction of co-stimulatory molecules

that facilitate T-cell activation and pro-

inflammatory proteins that effect elimination

of the pathogen from the body. TLRs

employ many of the same signalling

components as the type I IL-1 receptor

(IL-1RI). This is hardly surprising since

the intracellular regions of TLRs and IL-

1RI share a conserved Toll / IL-1R (TIR)

domain that is important in initiating

various signalling pathways especially

that regulating the transcription factor

NFkB. My research group is currently

exploring the signal transduction pathways

employed by the TLR members and their

roles in the pathogenesis of rheumatoid

arthritis and inflammatory bowel disease.

We have identified a number of novel

regulators of the TLR pathways and

hope to explore their potential value

as therapeutic targets in these diseases.

We also have a continuing interest

in characterising the effects of pro-

inflammatory cytokines in brain. This

area of research probes the effects of the

cytokines IL-1 and TNF in brain cells. We

have shown that both cytokines induce

the cell adhesion molecules VCAM-1 and

ICAM-1 and chemokines such as IL-8 in

glial and neuronal cells. The induction of

these genes appears to play important

roles in cerebral recruitment of leukocytes,

which may lead to neuropathology. Indeed

IL-1 and TNF promotes sustained

expression of these genes and this

may underlie the chronic cerebral

inflammatory responses seen in

neurological disorders such as multiple

sclerosis. We are thus very interested in

exploring the mechanism by which IL-1

and TNF can cause sustained expression

of the genes and we have published findings

that show that the sustained expression

is likely to be due to prolonged activation

of the transcription factor NFaB by IL-1

and TNF. We have also resolved the

mechanism underlying this prolonged

activation of NFaB. This work is currently

being extended in my laboratory with a

view to identifying novel agents to block

the expression of adhesion molecules and

chemokines in brain cells. Such agents would

have much therapeutic value in the treatment

of various neuropathological conditions.

Fig. 1. - IL-1 IL-1 causes nuclear translocation

of NF·B in human glial cells

Fig. 2. +IL-1

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PROFESSOR GRACE MULCAHY


VACCINES TO PROTECT AGAINST

PARASITIC INFECTIONS

Helminth parasites are a cause of very

significant morbidity and mortality on a

global scale. In man, important helminth

diseases include geohelminthosis such

as ascariosis and hookworm infections,

schistosomiosis, and filarial diseases

such as river blindness. The World Health

Organisation estimates that over a third

of the world’s populatation are affected

by such diseases. In veterinary medicine,

helminth parasites are of constant concern

to livestock producers and, indeed to

owners of companion animals, and this

is reflected in the fact that 27 % of sales

of veterinary medicines are anti-parasitic

drugs and this market was worth 3.1

billion dollars in 2002. However, resistance

among helminth populations to these

drugs has now reached epidemic

proportions and, in sheep and horses,

render drug-based control difficult.

Most worryingly, reports of multi-drug

resistance in helminths of sheep raise

the spectre depopulation of affected farms

being the only viable control strategy.

To date, immunoprophylaxis for these

diseases has not been developed. The

prevalence of helminth infections in

animals has consequences for consumers

as well as producers, in terms of the quality

of food produced, risks of chemical residues

in products, and environmental residues.

Our research group is working to develop

vaccines as an alternative to drug use in

controlling parasitic infections. Working

with liver fluke infection as our first target,

we have demonstrated that cattle and

sheep can be vaccinated with recombinant

forms of liver fluke enzymes, to protect

them against infection. Ongoing work is

aimed at optimising the dose and timing

of vaccination, and the prospects of this

development being translated into a

commercial product are good.

Recently, in collaboration with the Marine

Institute in Galway, the group has begun

work, which they hope will lead to the

eventual production of a vaccine to protect

farmed fish against sea lice. Although in its

early stages, this project has the potential

in the long term to reduce the need for

drug use in the aquaculture industry. The

vaccine development projects are funded

by Enterprise Ireland.

In addition to the vaccine work, the group

are also studying the mechanisms whereby

parasite infection can damage the ability

of animals and man to deal with bacterial

and viral infections and the role of

immunoregulation in host-parasite

interaction, the immunogenetics of

resistance to helminth infection, and the

biological relevance of strain variation in

the protozoan parasite Cryptosporidium.

CURRENT RESEARCH

PROJECTS INCLUDE:

Recombinant vaccines for the

protection of cattle and sheep against

liver fluke infection.

Immunoregulation by helminth

parasites.

Immunogenetics of resistance to

helminth infection in sheep (with Dr

Torres Sweeney, Faculty of Veterinary

Medicine and Conway Institute).

Novel diagnostics for parasitic diseases

of livestock.

Vaccines to protect farmed salmon

and trout against sea louse infestation

(with Dr Dave Jackson, Marine Institute).

Molecular correlates of biological

variability in Cryptosporidium

(with Drs Billy Bourke and Marguerite

Clyne, Children’s Research Centre and

Conway Institute).

Molecular epidemiology and

pathogenesis of Angiostrongylus

vasorum in dogs (with Prof Boyd Jones,

Dr Sheila Brennan and Dr Carmel

Mooney, University Veterinary Hospital).

Innate immunity in chickens (with Prof

Cliona O’Farrelly, Education & Research

Centre, St.Vincent’s University Hospital

and Conway Institute).

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Faecal Fluke Egg Counts

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week 5 week 6 week 7 week 8 week 9 week10 week 11 week 12 week 13 week 14 week 15

Week post infection

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There is an urgent need for the

development of alternative control

strategies for helminth diseases in

livestock, as drug-resistance is now

widespread. Liver fluke populations,

in particular, have developed resistance

to the most efficacious drugs which up to

now have been widely used. Our strategy

for vaccination uses recombinant versions

of enzymes secreted by the parasite as

they migrate within the host. As we have

now achieved ‘proof of concept’ that

the recombinant molecules can deliver

substantial protection, we are now focussing

on the mechanisms of protection, and on

optimising adjuvant and delivery strategies.

Fig. 1. Evidence of protection of sheep by vaccination with recombinant cathepsin-L1 from the liver fluke,

Fasciola hepatica. (a) Reduction in fluke faecal egg counts in vaccinated animals (yellow) as compared with

controls. (b) Reduction in liver damage as assessed by serum glutamate dehydrogenase (GLDH) levels.

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W0 +inf W2 pi W4 pi W6 pi W8 pi W10 pi W12 pi W15 pi

I.U

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DR EVELYN MURPHY


SIGNAL TRANSDUCTION PATHWAYS

INVOLVED IN THE PATHOGENESIS OF

INFLAMMATORY DISEASES

Long-term physiologic stress responses

are achieved by integration of the autonomic

nervous, HPA, cardiovascular, metabolic

and immune protective responses.

Achieving this dynamic stability requires

considerable ‘tissue wear and tear’

resulting from sustained overactivity

(or underactivity) of these systems.

Disturbances in endocrine-immune

interactions upset the normal regulatory

homeostatic balance and may alter

susceptibility to a variety of disease states

associated with immune dysregulation.

Inflammatory stimuli reliably elicit HPA

activation, and it is now established that

the immune and HPA systems are

mutually regulatory and that their

interactions partly determine stress

effects on immune function (Fig. 1). What

has not been extensively studied, however,

is the interactive nature of the endocrine

and immune systems and whether this

might alter predisposition to inflammatory

disease or stress-related pathologies. The

long-term aim of this research programme

is to further explore the physiological and

molecular interactions occurring at the

neuroendocrine-immune interface.

Recent advances have highlighted the

efficacy of IL1 and TNF blocking strategies

in the treatment of inflammatory diseases,

including rheumatoid arthritis (RA),

psoriasis and colitis. However, it is now

clear that redundancy of cytokine actions

will necessitate combination therapy to

interrupt positive feedback loops. The

identification of signal transduction

pathways involved in the pathogenesis

of these diseases has sparked a search

for novel therapeutic strategies. These

intracellular signalling systems transduce

extracellular signals from the cell surface

to the nucleus, where they are integrated

at the level of transcription factor activity.

The aberrant functions of downstream

transcription factors have been implicated

as critical regulators of gene expression

involved in chronic inflammation.

Therefore, specific signal transduction

targets or transcription factor targets may

interrupt the perpetuation mechanisms in

RA and help re-establish homeostasis. We

have evidence to support the conclusion

that modulation of the nuclear

transcription factor NURR1 by pro-

inflammatory mediators is an important

component of the inflammatory process

in human arthritis. Our data demonstrates

that NURR1 induction occurs downstream

to TNFa and IL-1b‚ signalling and may

therefore be an effective target for

anti-cytokine therapy in inflammatory

conditions. The ongoing aim of our research

is to gain further insight into the cytokine

signalling pathways regulating NURR1

expression and to understand the regulatory

targets of NURR1 transcriptional activity.

These studies will provide important

information on disease mechanisms

and pathways involved in the

inflammatory process.

RESEARCH GROUP:

My research group currently consists of

two postdoctoral fellows, Dr Kimberlee Mix

and Dr Darren Ennis as well as two PhD

students; Jennifer Ralph and Carol Aherne.

SPECIFIC RESEARCH PROJECTS:

Modulation of matrix

metalloproteinase expression

by the nuclear receptor NURR1.

Understanding nuclear orphan

receptor function.

Transcriptional events in human

inflammatory joint disease.

Proinflammatory actions of

corticotropin-releasing hormone.

Neuroendocrine regulation

of inflammatory joint disease.

Model systems to dissect the bioactivity

of novel anti-inflammatory agents.

Stress

hypothalamus

Infection, disease

Inflamed tissueadrenal CS

pituitary

Inflammatory mediators/

cytokines

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Fig. 1. The hypothalamic-pituitary-adrenal (HPA) axis plays an important regulatory and modulatory role

in normal immune function, permitting adequate responses to invading organisms and other inflammatory

stimuli. The HPA axis potently modulates the inflammatory response through the actions of corticosteroids

(CS). Pro-inflammatory cytokines (including IL1, IL6 and TNF) can activate the HPA axis. Dysfunction of this

bi-directional communication has been implicated in the development and pathogenesis of chronic

inflammatory disease.

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DR KEITH MURPHY


Our laboratory is part of the Applied

Neurotherapeutics Research Group

(ANRG), a research cluster that was

assembled in January 2004 funded by

Science Foundation Ireland and Wyeth

Research. The major goal of the ANRG

is the identification of novel drug targets

for the treatment of neurological and

neurodegenerative diseases (www.ucd.ie

/neurotherapeutics). Our research strives

to understand the molecular underpinnings

of plasticity occurring at synaptic

connections between nerve cells during

information storage. In particular, we focus

on the hippocampus and prefrontal cortex.

In parallel, we characterise behavioural

models of schizophrenia, depression

and neurodegeneration and probe these

for deficits in proteins that regulate

synaptic plasticity.

We bring several different approaches

together to address these challenging

issues including electron microscopy, site-

directed interruption of function with

siRNA, radio telemetry, electrophysiology,

DNA microarray, proteomic and

bioinformatic analyses. My research group

currently consists of 1 postdoctoral fellow,

5 PhD students and 1 MD student. To date

our work has lead to 24 papers in peer-

reviewed publications, a book chapter

and numerous published abstracts.

RESEARCH PROJECTS

CURRENTLY ONGOING:

Large scale transcriptional profiling

of memory consolidation-associated

synaptic plasticity following passive

avoidance and water maze paradigms

using Affymetrix microarray

technologies. (In collaboration with

Drs Gene Brown, David von Schack

and Rob Ring, Wyeth, Cambridge, USA).

Transcriptional profiling of the

hippocampus and prefrontal cortex in

animal models of schizophrenia. These

studies hope to relate the emergence

of behavioural deficits characteristic

of the disease with molecular deficits

in pertinent brain regions.

Large-scale validation of protein

expression change associated with

transcriptional regulation across the

first 24h of memory consolidation.

Here, a conventional isotope coded

affinity tagging (ICAT) strategy

is being combined with on-line

liquid chromatography and mass

spectrometry (MS/MS) and off-line LC

MALDI-Tof/Tof MS/MS to characterise

protein level change following passive

avoidance learning. (In collaboration

with Prof Stephen Pennington

and Prof Des Higgins).

Comparative analyses looking at

the nature of memory-associated

plasticity in the hippocampus and

prefrontal cortex. These studies focus

on molecular aspects of plasticity

deployed by these distinct yet connected

brain structures during different types

of learning (see Fig. 1).

Identification of transcription factors

regulating gene clusters during

memory-associated synaptic plasticity.

Here, a bioinformatics approach

is being used to study promoter

sequences common to temporal and

functional clusters of regulated genes

identified through our microarray

studies of memory consolidation.

(In collaboration with Prof Ciaran

Regan and Prof Des Higgins).

Exploration of pro-inflammatory

intracellular signalling pathways

that modulate synaptic transmission:

Relevance to physiological regulation

of learning and memory. (In

collaboration with Dr Paul Moynagh

and Dr John O’Connor).

Radio telemetric studies evaluating

the Eeg. patterns of activity along the

ventral tegmental-prefrontal cortex

and hippocampal-entorhinal cortex

pathways in rodent models of

schizophrenia.

We are currently developing

procedures to allow direct viral vector

delivery of short interfering RNA

(siRNA) to the hippocampus to

selectively silence a single gene

transcript. We hope to use this

approach to offer direct evidence

of a given protein’s involvement in

memory-associated synaptic plasticity.

Identification of cortical sites involved

in the commitment of information

to long-term memory. These studies

involve metabolic labelling techniques

to identify specific cortical areas active

during memory retrieval. Several such

areas of cortex are now being

interrogated by electron microscopic

techniques for ultrastructural correlates

of long-term information storage.

Studies on drug addiction and the

changes that occur within the memory

systems to mediate such long-term

perturbations to normal cognitive

function.

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NCAM PSA NeuN NCAM PSA & NeuN

Fig. 1. Neuroplastic cells in the prefrontal cortex

of the Wistar rat. The Fig. shows cells that express

the polysialylated version of the neural cell adhesion

molecule (NCAM PSA: green) co-express the

neuronal marker NeuN (red). The co-expression

is evident from the overlapping image: yellow).

The sections are counter stained withthe nuclear

marker DAPI (blue) to aid identification of cells.

We have identified these cells in various

subdivisions of the rodent prefrontal cortex and

shown them to co-regulate with similar neuroplastic

cells in the hippocampus during consolidation of the

water maze spatial task.

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DR TORRES SWEENEY


The overall objective of my research group

is to characterise the genetic factors that

influence host-pathogen interactions.

Based in the Faculty of Veterinary Medicine,

the animal models that we use are farm

animals, predominantly sheep. The

specific research areas that are currently

funded include:

Genetic susceptibility to scrapie and

molecular mechanisms to discriminate

scrapie and bovine spongiform

encephalopathy (BSE) in sheep.

The genetic and immunological basis

influencing susceptibility to

gastrointestinal nematodes in sheep.

The genetic factors influencing host-

pathogen interactions in

verocytotoxigenic E. coli.

Genetic susceptibility of cattle

to brucellosis.

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DR KATHY O’BOYLE


Our research group has a broad interest

in the mechanisms by which small,

biologically active molecules modify

activity of their cellular targets. We have

two main research priorities: the first

is the investigation of G-protein coupled

receptors (GPCRs) and their neurotrans-

mitters in the central nervous system

(CNS); the second focuses on the

development of cell biological techniques

for the investigation of novel glycoconjugate

modulators of angiogenesis.

GPCRS AND THEIR

NEUROTRANSMITTERS

IN THE CNS

Communication between neurons in

the CNS usually occurs through chemical

synapses wherein neurotransmitter

substances, released from the pre-synaptic

terminal, activate specific post-synaptic

receptors. The largest group of receptors

are those that couple to G-proteins;

G-proteins, in turn, activate or inhibit a

variety of intracellular second messengers

and thereby elicit many different types

of cellular effects. The first GPCR to be

cloned was the ß-adrenoceptor, which

was cloned in 1986. Since then, most of

the GPCRs that have been identified by

their pharmacological properties have

been cloned. G-proteins themselves exist

in several forms (eg. Gs, Gi, Gq) that

interact with different receptors (eg.

dopaminergic, serotonergic, cholinergic)

and control different effector molecules

(eg. adenylyl cyclase, phospholipase C,

ion channel). The conventional view that

one GPCR binds to one G-protein, which

activates/inhibits one effector pathway

is now being superseded by evidence that

under, certain conditions one GPCR can

activate more than one G-protein and G-

proteins can modify activity of more than

one effector pathway. The complexity

of GPCR signalling pathways is further

evidenced by recent data that demonstrate

that GPCRs often dimerise in order to

create the functionally active receptor.

Our interest in this area resides around

the role GPCRs and their neurotransmitters

play in the regulation of normal behaviour,

and in disease states.

NOVEL GLYCOCONJUGATE MODULATORS

OF ANGIOGENESIS

Angiogenesis is a process that provides

developing tissue with new blood vessels,

and, therefore, with the nutrients essential

for further tissue growth. It relies on the

up-regulation of cell proliferation/survival

pathways. The process of angiogenesis

is tightly regulated in healthy adults and

occurs for the most part during events

such as pregnancy or wound healing.

However, it is an important event in many

disease states such as cancer, diabetes

and arthritis; hence, drugs that can block

angiogenesis have great therapeutic

potential. One of several strategies being

pursued for the development of anti-

angiogenic agents is inhibition of growth

factors such as bFGF, VEGF and PDGF,

all of which stimulate the growth and

proliferation of various cell types. The

cellular receptors for bFGF are tyrosine

kinases and these are activated by the

oligosaccharide, heparin, or heparin

sulphate proteoglycans. The hypothesis

underlying our studies is that small,

carbohydrate compounds that resemble

some of the structural features of heparin-

like oligosaccharides will modify cell

proliferation and migration. This project

is an interdisciplinary collaboration

between our group and Dr Paul Murphy

in the Chemistry department, UCD. Dr

Murphy has carried out the synthesis of

a collection of novel, structurally diverse

glycoconjugates and the aim of our

studies is to systematically screen these

for biological activity. The strategy is

to evaluate the ability of novel glycocon-

jugates to compete with heparin for

binding to physiologically important

proteins. Compounds shown to have activity

in this basic screen are then further

investigated in a variety of increasingly

complex models, all of which are designed

to identify potential modulators

of angiogenesis.

RESEARCH PROJECTS

CURRENTLY ONGOING:

Systematic study of structure-activity

relationships for thyrotropin-releasing

hormone (TRH).

Long-term effects of caffeine on

synaptic plasticity and implicit memory.

Methylenedioxymethamphetamine:

toxicity versus lethality

The biological evaluation of novel

glycoconjugates

Towards novel glycoconjugate based

therapeutics for angiogenesis

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Examining the regulation of MAP

kinase phosphatase 1 (MKP1)

expression by CCE; thus, our recent

studies have revealed the growth

factor induced expression of MKP1

is attenuated completely by inhibition

of CCE. We have cloned 820bp of the

murine MKP-1 promoter sequence by

PCR of genomic DNA. Analysis of the

promoter region has revealed that

there are several potential transcription

factor binding sites, including two

putative Ca2+ response elements

(CRE). We intend to identify the

regions of the promoter that confer

CCE-regulated expression and identify

the protein(s) that bind to these regions

and explore the extent to which these

elements are present in other growth

factor induced genes.


PROJECTS

As well as our own research programmes,

the PRC also supports a range of

collaborative projects. Hence, we are

actively engaged in a wide range of on-

going collaborative projects in a diverse

range of areas – the brief descriptions

below give an illustration of just some

of these projects. Many of our projects

are in the area of cancer biology. One of

our most advanced cancer biology projects

is on the identification of potential diagnostic

biomarkers and therapeutic targets for

pancreatic ductal adenocarcinoma (in

collaboration with Professor J Neoptolomos

and Dr E Costello, University of Liverpool).

Pancreatic cancer is the 4th most common

cause of cancer death in the Western world

and potential curative surgery has a very

low success rate. So, new biomarkers and

targets need to be identified. We have

therefore used laser capture microdissection

to isolate normal and tumour pancreatic

ductal cells (from the same individual)

and subjected these to 2-DE based

proteomic analysis. We have identified

several proteins whose expression changes

and the potential role of these proteins in

the tumour phenotype of pancreatic ductal

cells is currently being investigated.

More recently, we have begun a project

on the identification of serum biomarkers

of organ confined prostate cancer (in

collaboration with Dr William Watson,

Conway Institute and as part of an Irish

Cancer Society funded Prostate Cancer

Consortium). In this project, plasma and

serum from patients with prostate cancer

or benign hyperplasia of the prostate are

being subjected to relatively sophisticated

sample preparation prior to 2-DE analysis.

This study is running alongside serum

and plasma proteomic analysis of patients

in a phase II clinical trial for evaluation of

combined hormonal and radiation therapy

with or without drug treatment (with

Professor J Armstrong).

085 <> 086

PROFESSOR STEPHEN PENNINGTON


My research group and that of Professor

Mike Dunn are located in the Proteome

Research Centre (PRC) within the Conway

Institute. Mike and I work together to lead

a group of researchers who are applying

proteomics to a range of different

biomedical research projects.


The PRC is equipped with state-of-the-art

instrumentation and software to support

a number of complementary proteomics

workflows. The resources in the mass

spectrometry laboratory in the PRC include

a MALDI-Tof/Tof mass spectrometer with

autoloader for high throughput MALDI-

MS/MS and off-line liquid chromatography

(LC) MS/MS workflows. We also have

a linear ion trap electrospray mass

sprectrometer and a linear ion trap

electrospray mass spectrometer coupled

to a Fourier Transform Ion Cyclotron

Resonance detector.

The PRC also has a large separations

laboratory that contains instrumentation

for 1-D gel electrophoresis, 2-D gel

electrophoresis (2-DE), image scanners

including a three colour laser fluorescence

scanner (Typhoon), which supports the use

of the latest three colour difference in gel

electrophoresis (DIGE) 2-DE. LC based

workflows are supported by the availability

of a multi-dimensional LC system and a

nano-LC system. The latter is coupled to

a MALDI target loading robot to support

off-line LC MALDI-MS.

These facilities are used to support the

research projects of our groups and a

range of collaborative biomedical research

projects. In general, our aims are to apply

proteomics to basic biomedical sciences

and clinical proteomics projects for the

purpose of gaining mechanistic insights

into biological processes, identifying

diagnostic and prognostic biomarkers

of diseases and identifying potential targets

for therapeutic intervention in human

diseases. To achieve this, we are particularly

interested in applying newly developed

leading edge technologies for proteomics

including methods for quantitative

measurement of protein expression.

The latter we are achieving in close

collaboration with Applied Biosystems

who have funded a Newman scholarship.

PENNINGTON GROUP

RESEARCH PROJECTS

My research interests are focused on

the investigation of signal transduction

pathways that regulate entry into and

progression through the G1 phase of

the mammalian cell cycle. Using a novel

experimental approach to control the

spreading (and shape) of individual

cells, we have shown that restricting cell

spreading prevents progression through

G1 to S phase and profoundly influences

the temporal pattern of Ca2+ signalling

by inhibiting capacitative calcium entry

(CCE) across the plasma. Importantly,

others have shown that changes in either

the temporal or spatial patterns of Ca2+

signalling can differentially regulate gene

expression. Our findings therefore suggest

that (i) CCE plays an important role in

G1 progression and S phase entry and,

(ii) the regulation of the temporal profile

of mitogen-induced intracellular Ca2+

signalling by cell shape may serve to

control gene expression during G1. We

are now therefore interested in the role

of CCE in the regulation of growth factor

induced gene.

Our research plans are therefore to

use complementary proteomic and

transcriptomic approaches to investigate

CCE-dependent gene expression by:

Using two-dimensional gel

electrophoresis (2-DE) and isotope-

coded affinity tagging stratgies to

examine the effect of SKF-96365,

an inhibitor of CCE, on growth factor

induced changes in protein expression

and post-translational modifications

early in G1. Initial experiments have

revealed changes in protein expression

induced by inhibition of CCE by 2-DE

and using antibody arrays (see Fig1).

We now intend to undertake a

systematic investigation of these

changes, generate a 2-DE database

and identify the proteins by MALDI-

Tof/Tof mass spectrometry and nano

liquid chromatography electrospray

mass spectrometry (ESI-MS/MS).

Undertaking detailed analysis of the

effect of CCE on the transcriptional

profile in response to growth factors

using Affymetrix arrays.

1.

2.

FCS

pH3 NL IPG pH10 pH3 NL IPG pH10

FCS+SKF

Fig. 1. Effect of inihibition of CCE (by SKF) on growth factor (FCS) induced protein expression by 2-DE and

(b) antibody arrays. In (a) arrows mark protein’s whose expression is influenced by inihibition of CCE. In (b)

proteins cell lysates from growth factor stimulated cells were labelled with Cye3 and those from growth factor

stimulated cells in which CCE was inhibited were labelled with Cye5.

3.

The lablled lysates were combined and applied

to an antibody array containing 242 antibodies

to different proteins to allow differential analysis

of protein expression.

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Neuroproteomics projects include the

analysis of human and mouse brain

samples as part of a pilot project for the

Human Proteome Organisation (HUPO)

Brain Proteome Project and the analysis

of white matter of the prefrontal cortex

in schizophrenia and bipolar disorder

(with Dr D Cotter, RCSI) and the large-

scale validation of protein expression

changs that are associated with the

known transcriptional regulation that

occurs across the first 24h of memory

consolidation (with Dr K Murphy).

OTHER PROJECTS INCLUDE:

Analysis of protein expression changes

during epithelial to mesenchymal

transition (with Professor F Martin,

Conway Institute).

Kidney proteomics (with Professor

Michael Ryan, Conway Institute).

’Lipobox’ independent lipid

modification of lipoproteins in mycolic

acid containing bacteria (with Dr W

Meijer, Conway Institute).

Protein expression changes during

Xenopus development (with Dr C

Hensey, Conway Institute).

Analysis of protein expression changes

in chronic lymphocytic leukaemia (with

Dr E Vanderberghe and A Hayat)

Identification of new diagnostic

biomarkers and therapeutic targets

in diabetic nephropathy (with Dr D

Brazil, Conway Institute)

Proteomic and functional analysis of

p21-mediated paracrine effects (with

Dr W. Gallagher, Conway Institute)

1.

2.

3.

4.

5.

6.

7.

Before

After laser pulse

Cap view

(a)

800 1140 1480 1820 21600

10

20

30

40

50

60

70

80

90

100 915.497907.426

845.6961000.6171149.574 1358.811 1821.9151643.929

1477.761

1098.0 1272.6 1447.2 1621.8 1796.4 1971.00

10

20

30

40

50

60

70

80

90

100 1149.5741107.9 92

1358.811

1142.6361213.769

1821.9151205.713 1643.9291477.761

**

*

Mass (m/z)

2500

70 343 616 889 1162 1435

186.3

0

10

20

30

40

50

60

70

80

90

1001 1 6 1 .9 0

7 4 1 .3 7

8 6 .0 6 7 4 7 .3 8

2 4 3 .1 46 1 8 .3 4

8 4 .0 4 8 5 4 .4 5

8 7 5 .4 8 9 5 5 .5 01 0 1 .0 3

2 4 0 .1 35 0 5 .2 8

1 2 9 .0 8 1 0 6 8 .6 02 5 7 .1 7

3 5 6 .2 0 1 0 5 0 .5 81 4 7 .7 4 5 8 3 .3 01 1 5 9 .8 97 1 3 .4 07 1 .2 9

9 3 7 . 5 62 2 2 .3 2 5 9 7 .9 23 4 3 .1 1 4 9 9 .2 41 3 8 .6 1 1 1 7 3 .8 08 0 4 .7 87 2 3 .3 4

ELIQKELTIGSK

5 .2E+4

428 .2 607.8 787 .4 967.0

1 .3E+4

40

50

60

70

80

90

1001 7 5 .0 9 5 5 9 .2 3

8 6 .0 7

9 1 5 .4 9

7 2 9 .8 2

3 5 9 .1 8

5 5 7 .1 78 4 3 .4 1

4 8 4 .2 53 1 6 .0 74 2 8 .1 4

6 7 4 .2 8 8 7 4 .9 54 4 4 .1 03 1 5 .1 2 7 2 7 .8 3 8 0 0 .3 7

4 8 8 .2 2 5 9 7 .8 5 6 5 7 .2 42 9 8 .0 9 4 0 3 .1 8 8 1 3 .3 95 4 3 .2 4 8 7 2 .9 57 3 2 .8 25 9 5 .8 7

LQDAEIAR

Normal

(LCM)

Tumour

(LCM)

Normal

Undissected

Mass (m/z)

MS/MS 1358.881

MS/MS 915.497

Kidney

Kidney

Normal

Colon

Colon

Lung

PDAC

Fig. 2. Pancreatic ductal cells were isolated by laser capture microdissection (a) and subjected to

2-DE and protein spots that altered in expression identified by MALDI-Tof/Tof mass spectrometry

(b). One of the protein spots identified (in b) was S100A and its upregulation in pancreatic ductal

adenocarcinoma was confirmed by use of tissue microarrays (c).

(b)

(c)

DR MARGARET WORRALL


Our research is primarily focused onthe serpin (serine protease inhibitor)superfamily of proteins and the role ofparticular serpins in cancer. Serpins areinvolved in a wide range of physiologicalprocesses including coagulation,inflammation and cell migration. Thereare 35 human serpins and most regulateproteases through a suicide inhibitionmechanism, which has been elucidatedusing the archetypal plasma serpina1-antitrypsin. However some serpin geneproducts lack the ability to inhibit proteasesand have other functions such as hormonetransport, tumour suppression or growthfactor activity. These have been coined non-inhibitory serpins and include PEDF (pigmentepithelial derived factor), maspin (mammaryserpin) and thyroxine binding globulin.

We are interested in PEDF andmaspin since both of these serpinshave therapeutic potential as inhibitorsof angiogenesis. PEDF is of particularimportance in the eye and may be usefulfor treating diabetic retinopathy and age-related macular degeneration. Maspinis known to have tumour suppressorproperties in breast and prostate. Themechanism of these serpins is not wellelucidated, and although they can exerttheir function as exogenous proteins addedto cellular assays, there is also evidencethat they are found in significant amountsintracellularly and in the nucleus.

We have carried out yeast-2-hybridstudies to identify target proteins forboth maspin and PEDF in order to gaina greater insight into their mechanismof action. In the case of maspin, we haveshown binding with collagen types 1 and3, which may facilitate cell adhesion inthe extracellular matrix. For PEDF, wehave identified an interaction with thenuclear transport protein transportin-SR2and we have found that PEDF is activelytransported to the nucleus. We have also

identified a novel nuclear localisationsignal sequence common to PEDF andother transportin substrates and, followingmutagenesis of this motif, PEDF isexcluded from the nucleus.

The nuclear uptake of PEDF may be criticalfor its function as an antiangiogenic factorand this is currently being assessed usinga mutagenesis approach. Little is knownabout cellular trafficking of maspin or themechanism of maspin tumour suppression,and we are also investigating maspinlocalisation, structure and function.

A further focus of the research group isto characterise two orphan human A-cladeserpins, which we have identified onchromosome 14. Expression studies showthat both are made in the liver and arelikely to be present in human plasma,but their function has yet to be elucidated.Using purified recombinant proteins, weare investigating conformational stabilityand inhibitory profiles of these proteins.

RESEARCH PROJECTS CURRENTLY

ONGOING:

Cellular uptake and nuclear localisationof PEDF: importance of phosphorylationand relevance to anti-angiogenic andneurotrophic function.

Investigation into a novel nuclearlocalisation signal sequencefor transportin cargo proteins.

Cellular localisation of the related non-inhibitory serpin, maspin and isolationof a putative maspin receptor.

Structure-function studies on maspin(in collaboration with Dr J Whisstock,Monash University).

Characterisation of the orphan humanA-clade serpins.

Fig. 1. (a) Tranfection of HEK-293 cells with GFP

tagged PEDF, showing a nuclear localisation and

(b) following mutagenesis of a YRVRS motif, GFP

tagged PEDFmut is excluded from the nucleus.

(a)

(b)

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PROFESSOR CIARAN REGAN


Our research focuses on the role of

structural plasticity in normal and disease-

affected brains. Structural plasticity refers

to the ability of neurons to remodel their

connections and contribute to ongoing

adaptations in neuronal circuits. Such

adaptations underlie our ability to learn

new facts and recall existing memories.

Many illnesses affecting the brain, such

as Alzheimer’s and schizophrenia, are

characterised by problems in learning

and recall and these problems are related

to underlying deficits in the brain’s ability

to remodel connections. Therefore,

a better understanding of the process

underlying structural plasticity will yield

insights into mental illness with the

promise of more effective treatments.

My approach to the complex problem of

better understanding structural plasticity

and developing new drug therapies

directed against mental illnesses and other

neurological conditions has been to draw

together the diverse research strengths

that exist in the Conway Institute at

University College Dublin, the Trinity

College Institute of Neuroscience

and Wyeth Discovery in Neuroscience

under the umbrella of the Applied

Neurotherapeutic Research Group

(ANRG). ANRG represents a unique

interface between academia and industry

that is committed to identifying novel drug

targets relevant to structural plasticity

(www.ucd.ie/neurotherapeutics).

In the first year of work, ANRG research

has focused on improving our understanding

of schizophrenia. Schizophrenia is commonly

understood as a mental illness, affecting

1% of the population regardless of culture

or class, that induces personality changes,

hallucinations, cognitive deficits and

suicidality. Current treatments do not

effectively manage all the symptoms

and are ineffective in a significant

minority of sufferers. It has been proposed

that impaired synaptic plasticity in specific

neural circuits during childhood and

adolescence ultimately results in altered

synapse formation or pruning, or both,

which mediates the onset of psychosis.

Currently, we are striving towards a better

understanding of the plasticity deficits

observed in schizophrenia and evaluating

the therapeutic effects of the best

currently available treatment, clozapine.

It is hoped that this endeavour will yield

a comprehensive profile of successful drug

intervention, which will provide a template

for the design of new, more effective ligands.

Currently, the group is also engaged

in a number of other studies including

the following:

SCREED – SCREENING

FOR ENDOCRINE DISRUPTORS

A substantial need exists for validated

in vitro methods for the screening of

environmental and industrial chemicals

for estrogenic and androgenic activity.

There is growing concern regarding the

long-term health and environmental

effects associated with continuing exposure

to potential endocrine disruptors. As

hormonally mediated events play a key

role in central nervous system development

and function, concerns exist that

inappropriate exposure to endocrine

disruptors during brain development

might affect neuronal cell differentiation

and function. For example, prenatal

exposure to endocrine disruptors, such

as organohalogens, has been shown to

lead to cognitive deficits. To measure these

potential endocrine disruptor effects in

vitro, we are currently developing a high-

throughput automated microscope system

using two cell models of neuronal

differentiation:

Firstly, we have investigated a number of

differentiation markers in a stem cell-like

model that can be induced to undergo

differentiation along glial and neuronal

lineages. Using automated image analysis

on immunolabeled cells, this model allows

us to look for lineage-specific effects

of endocrine disruptors.

Secondly, we are using the murine

Neuro2A cell line to look at possible

effects by endocrine disruptors on neurite

outgrowth. We have developed an image

analysis procedure (RAMON – Rapid

Automated Measurement Of Neurites)

that allows for automatic detection of

neurites and quantitation of various

morphometric parameters.

PSYCHOSCREEN

Since the introduction of

psychopharmacology in 1952, thousands

of compounds have been synthesised and

shown to be safe and of varying benefit

in the treatment of major mental illness,

such as depression and schizophrenia.

In the years 1968-74, the DESI program

resulted in thousands of pharmaceuticals

being withdrawn as a result of FDA

regulatory action. Over 50% of all

psychopharmaceutical agents were

believed to be less than effective on the

specious acceptance of open-label trials

and underpowered controlled trials, often

with heterogenous populations. Moreover,

as clinical efficacy trials on these drugs

were initiated around 1955, few provided

a convincing basis for their full clinical

potential. The drugs withdrawn were

never systematically listed but announced

in various issues of the Federal Register.

In view of the current slowdown in the

development of innovative psychophar-

maceuticals, revisiting some of these drugs

to establish their behavioural and receptor

profile is likely to provide new chemical

leads that, in turn, may provide patentable

and effective antipsychotic agents.

This project intends to: 1) data mine

this drug repository for ‘drug signatures’

of effective antidepressants and

antipsychotics using behavioural, receptor

and synaptic plasticity markers; 2) render

the lead compounds patentable by chemical

database searching of related compounds

and their custom synthesis; and 3) provide

proof-of-principle in models of target

diseases. Through intensive data

mining, we have uncovered 26 promising

compounds that were withdrawn under

the DESI programme.

0

1

2

3

4

5

6

7

# N

EU

RIT

ES

/P

ER

IME

TE

R

NEURO 2A

TREATMENT

SOVENT 5uM 500nM 50nM 5nM 0.5nM

Fig. 1. Estradiol treated Neuro2A cells evaluated

using Ramon. The highest concentrations of

estradiol (5 uM and 500 nM) reduce the number

of neurites per cell, whereas lower concentrations

of under 50 nM prove neuritogenic.

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Solvent

5 µM E2

5 nM E2

Phorbol EsterCrystal

of C1B

Brookhaven Protein Library

Fig 3. Images from in

silico modelling using

Cerius2. This set of

images shows the

crystal structure of

the ligand binding

domain of PKC (left)

and a close up view

of a phorbol ester

bound in the binding

pocket (right).

091 <> 092

MODEM

The development of drug therapies

for dementias including Alzheimer’s

disease (AD) has, to date, focused on

the enhancement of neurotransmitter

function. As these approaches have failed,

there is an urgent need to identify

alternative therapeutic strategies that

will provide drugs with disease-modifying

capacity. The major objective of this grant

is to incorporate in vitro endpoints of

drug action to facilitate the strategic

development of novel compounds with

cognition-enhancing and disease-

modifying efficacy for the treatment

of dementia. We have previously

demonstrated that compounds with the

capacity to increase the polysialylated

form of neural cell adhesion molecule

(NCAM PSA), through inhibition of its

regulatory enzyme protein kinase C delta

(PKCa), can induce cognition enhancement.

To expand our pool of potential PKCa

compounds, we have put in place an

SGI/Accerlys computer system that allows

substructure screening of chemical data

bases based on comparison of the chemical

structure of a known PKCa inhibitor -

rottlerin. The crystal structure of the PKCa

ligand binding site is known and, using the

Cersius2 package from Accerlys, potential

inhibitors can be docked and ranked based

on interaction with the PKC ligand

binding site (Fig. 2). Favourable

candidates are then tested in vitro to

assess activity. This involves the capacity

of the compounds to increase NCAM-PSA

expression, to down-regulate PKCa

expression and to induce neuritogensis in

Neuro 2A cells.

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MA

OI

5%

Antipsychotic

26%

Barbituate 7%

Stim

ulan

t 15

%

An

tid

ep

ress

an

t 3

%

Mis

cell

an

eo

us

2%

Antineurotic 42%

Miscellaneous 2%

MAOI 5%

An

tip

sych

otic

19

%

Stimulant 15%

Antineuro

tic 69%

Fig. 2. A list of the compounds evaluated during the DESI program and their fates.

DR LEONIE YOUNG


INTRACELLULAR SIGNALLING

AND TRANSCRIPTIONAL REGULATION

IN HUMAN BREAST CANCER

The clinical use of an antiestrogen

for the treatment of breast cancer was

first reported by Cole et al in 1971, who

described the potential use of tamoxifen

(ICI 46,474). Since then, tamoxifen has

become the most widely prescribed anti-

cancer drug in the world. However, while

most estrogen receptor (ER) positive

patients will initially respond to tamoxifen,

approximately one third relapse within a

period of 18 months. While initial response

rates are high among ER-positive tumours,

most breast cancers that acquire tamoxifen

resistance do so while continuing to express

functional ER. Despite extensive clinical

use of tamoxifen several aspects of its

mechanism of action and acquired

resistance remain unclear.

The ER is encoded for by two genes,

ER-a and ER-b. Both genes can function

as transcription factors to modulate target

gene expression. ER-a and ER-b‚ interact

with a number of nuclear proteins in vitro.

These ‘co-activator/co-repressor’ proteins

interact directly with steroid receptors at

their response element in the promoter

region of target genes to enhance or inhibit

transcription. Co-activators such as SRC-1

are thought to facilitate the activity of the

ER whereas co-repressors such as SMRT

maintain transcriptional silence. We have

described a significant association between

expression of co-activators SRC-1 and

AIB1 in human breast cancer. At a

molecular level, we have observed distinct

transcription factor- co-regulatory protein

interactions and protein DNA interactions

in endocrine sensitive cells compared to

those that are resistant, in response not

only to the steroid environment but also

growth factors.

Co-regulatory proteins interact with

nuclear receptors, including ER, at a

conserved LXXLL motif within the receptor

interacting domain of the protein to drive

target gene expression. Although the

steroid co-regulatory proteins were

previously thought to exclusively associate

with nuclear receptors, we have identified

MAP kinase effector transcription factors

as novel co-activator protein targets.

These new interacting partners may

provide the basis for a new model

of endocrine resistance.

We feel that co-regulatory proteins are

pivotal to the development of endocrine

resistance and may ultimately be

responsible for the development of steroid

independent tumours.

RESEARCH TEAM 2004

I work with Mr Arnie Hill, Consultant

General Surgeon / Lecturer in Surgery

in St Vincent’s University Hospital and

UCD. Our current research team is made

up of the following people:

Postdoctoral fellows, Dr Marie McIlroy

and Dr Yvonne Buggy; PhD students,

Sinead Kelly and Niamh Cosgrave; MD

student, Eddie Myers; MCh students, Dara

Kavanagh, Ruth Pritchard and May Cleary.

Julie Watson is a research assistant

in the group.

RESEARCH FUNDING

Current research is being funded by the

following agencies: Health Research Board,

Cancer Research Ireland and St Luke’s

Institute of Cancer Research.

093 <> 094

PROFESSOR MICHAEL P RYAN


IMMUNE-MEDIATED AND DRUG

INDUCED RENAL DISEASE:

TOXICOGENOMICS AND PROTEOMICS

The focus of our research group is on

immune-mediated and drug induced

renal disease including toxicogenomics

and proteomics. The incidence of end stage

renal disease (ESRD) is rising at a rate

of 8-10% per annum. While the initiating

factors of the disease may vary, tubulo-

interstitial fibrosis is a key event and

indicates a likelihood of progression to

ESRD. Therapeutic strategies to halt the

progression to ESRD are very limited

mainly because of a lack of understanding

of the mechanisms involved in tubuloin-

terstitial fibrosis. In our laboratory, we

have developed a model of epithelial-

mesenchymal transdifferen-tiation (EMT),

whereby epithelial cells become more

fibroblast-like. EMT is believed to play a

major role in renal fibrosis. We are using

genomic and proteomic analysis to

determine specific gene and protein

changes in cell culture models of renal

fibrosis. We have identified a number

of genes that are up-regulated during

this process and also a number of genes

that are down-regulated. Some of these

genes/proteins are possible new

therapeutic targets in renal fibrosis.

RENAL GENOMICS AND PROTEOMICS

In addition to cell culture models of

renal fibrosis, we are also investigating

proteomics in renal transplant patients

(in cooperation with the National Renal

Transplantation Unit, Beaumont Hospital)

and in liver transplant patients (in

cooperation with the National Liver

Transplant Unit, St Vincent’s University

Hospital). These studies should lead to

identification of novel biomarkers of renal

injury in transplant patients on immuno-

suppressive drug treatment.

As part of an EU–funded FP6 project

involving partners from a number

of universities and pharmaceutical

companies in Europe, we are establishing

genomic and proteomic profiles of kidney

cell responses to drugs. This EU project

entitled Predictomics will facilitate

development of novel systems for predicting

toxicity of drugs and chemicals with

renal effects.

MARINE BIOTOXINS

We are also involved in projects in

cooperation with the Marine Institute

to develop new approaches to establishing

mechanisms of toxicity of marine toxins.

This approach is now also funded by

the EU –FP6 programme in a project

entitled Biotoxins.

We have established a model of the

gastrointestinal tract in which human cells

are cultured. This allows us to establish

mechanisms of the toxic effects of shellfish

contaminants on the gastro-intestinal tract

using genomic profiling techniques and

trans-epithelial electrical resistance.

Additional studies into tight junction

regulation are also being pursued. The

models being evaluated are also intended

as alternatives to existing in vivo testing

methods used in assessing shellfish

contaminants.

IN VITRO MODELS OF ACUTE TOXICITY

We are also involved in a major FP6

Integrated Project entitled A-CUTE-TOX

involving 35 partners from across Europe

which aims to develop in vitro model

systems to replace in vivo toxicity studies

and therefore reduce the use of animals in

toxicity testing. We are the lead laboratory

for renal toxicity.

control treated

Fig. 1. Induction of EMT in kidney epithelial cells. Human renal tubular epithelial cells undergo EMT in the

presence of toxic agents for 48 hours. Morphological changes were examined by phase-contrast microscopy

and immuno-fluorescent microscopy. The pictures show changes in (i) morphology (top panel), (ii)

rearrangement of F-actin (middle panel) (iii) de novo expression of alpha smooth muscle actin, a marker

for myofibroblasts (bottom panel).

The WPI-REMS Autosampler automates

measurements of trans-epithelial electrical resistance.

We have developed this system to allow rapid assays

of the novel marine toxins called azaspiracids. The

mechanism of the toxicity of these compounds

seems to be related to altering the proteins in the

junctions between the models of gastrointestinal cells.

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DR PETER SMYTH


The research activities of the endocrine

laboratory are directed towards the

investigation of thyroid and breast

diseases with particular reference to the

role of iodine both as an essential nutrient

for development, a constituent of thyroid

hormones and a therapeutic agent

in thyroid and perhaps other cancers.

THYROID DISEASE IN PREGNANCY

AND THE NEONATE

Iodine deficiency disorders (IDD) can

give rise to thyroid hypofunction and have

particularly serious consequences in the

first trimester of pregnancy at which

time the fetus has not as yet developed

a thyroid gland and is totally dependent

on maternal thyroid hormones to achieve

normal neuropsychological development.

Maternal iodine deficiency in early

pregnancy can result in a deficit in thyroid

hormones leading in more severe cases

to irreversible mental retardation or,

even when the iodine intake is marginally

low, can lead to deficits in cognitive

development in the infant. Studies

on iodine and thyroid status in early

pregnancy are being pursued in

collaboration with Professor Colm

O’Herlihy, Department of Obstetrics

and Gynecology, UCD and the National

Maternity Hospital, Holles Street. The

endocrine laboratory is an iodine reference

laboratory and is a partner in the Cardiff

Antenatal Thyroid Survey, UK.

COINCIDENCE OF BREAST

AND THYROID DISEASE

Despite a widespread perception of the

association between diseases of the

thyroid and breast cancer, no definitive

evidence of a causal linkage currently

exists. The endocrine laboratory has been

involved for the past decade in studying

this linkage in collaboration with Professor

NJ O’Higgins, Dept of Surgery, St Vincent’s

University Hospital.

THYROID CANCER

The release of the thyroid hormone

storage protein (Thyroglobulin; Tg) into

the circulation acts as a marker for disease

outcome in thyroid cancer. Studies on Tg

protein and RNA expression in thyroid cancer

are carried out in the endocrine laboratory

in collaboration with Dr Michael Moriarty,

St Luke’s Hospital and St Vincent’s

University Hospital, Dublin.

RADIOIODIDE THERAPY FOR CANCER

The ability of the human thyroid gland to

concentrate iodine from the bloodstream

forms the basis for the use of radioactive

iodine as an adjuvant ablative agent in the

treatment of thyroid cancer. However, the

malignant thyroid gland often loses the

ability to accumulate radioiodine. In vivo

iodide transport is dependent on the

functional expression of transmembrane

solute transporters, the sodium iodide

symporter (NIS), Pendrin and human

apical thyroid transporter hAIT genes.

Understanding the role of these transporters

and investigating means of restoring

or enhancing their ability to promote

radioiodide uptake and retention in both

thyroid cancer and extrathyroidal tissues

such as the breast forms the basis of cancer

studies in the endocrine laboratory. It

is hoped that in the near future these

investigations will be enlarged to include

gene transfection studies in collaboration

with the Mayo Clinic, Minnesota, USA.

IODINE IN SEAWEEDS

The role of seaweed in human medicine

is well established. Many of the beneficial

health effects have been attributed to its

iodine content. Although the thyroid has

by far the greatest affinity, being able to

concentrate iodide by a factor 20-40 times

that of the bloodstream, seaweed is much

more efficient as it is capable of

accumulating iodide against a concentration

gradient by a factor up to 106 times that

of seawater. Despite the strong evidence

for active uptake of iodide from seawater

into seaweed, the mechanism through

which this is achieved remains to be

elucidated. The features of dietary intake

and iodine handling described in human

tissues have close parallels in seaweed and

it is apparent that studying the seaweed

model could significantly assist an

understanding of control of I- uptake/efflux

with the possibility of future prophylactic

or therapeutic applications. Seaweed studies

in our laboratory are in collaboration

with Professor Mike Guiry, Martin Ryan

Institute, NUIG.

IODINE UPTAKE AND LOSS

FOODSTUFFS:

The role of foodstuffs or leisure pursuits

in providing bioavailable iodine is being

studied by looking at the effects of iodine

rich (seaweed containing) diets on iodine

or thyroid status in human subjects.

SEAWEED BATHS:

The bioavailability of iodine and its

absorption through the skin in the course

of a seaweed bath is being studied in

collaboration with Celtic Seaweed Baths,

Strandhill, Co. Sligo.

SWEAT IODINE:

The major source of iodine loss in the body

is through urinary excretion. However

another pathway is by excess sweating.

In the absence of iodine replacement,

this has the potential of causing iodine

deficiency in athletes undergoing regular

strenuous exercise particularly in hot

climates. We have been investigating

this phenomenon in collaboration with

the Sports Centre, UCD and sports

organisations in Canada and Greece.

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4742 Resaerch Profs 17 - University College Dublin research profiles 2004.pdfVaccine delivery...

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