Coláiste na hEolaíochta, Ollscoil na hÉireann, Gaillimh C C C o o o l l l l l l e e e g g g e e e o o o f f f S S S c c c i i i e e e n n n c c c e e e R R R e e e s s s e e e a a a r r r c c c h h h D D D a a a y y y Monday, 12 th April 2010 Venue: Bailey Allen Hall, (Áras na Mac Léinn) Poster Presentation Sessions: 9.30 am to 5.40 pm and Science Speak Presentations: 2.00 pm to 5.40 pm Science Speak is intended to brief the public on scientific research in a non-technical fashion. The winner will represent NUI, Galway at the National Competition to be held in the RDS, Dublin on April 27 th , 2010. Admission to the event is free of charge and open to everyone!
Transcript
Coláiste na hEolaíochta, Ollscoil na hÉireann, Gaillimh
Acute kidney injury (AKI) and chronic kidney disease (CKD) are associated with
activation of the immune system. T-helper 17 (Th17) cells are recruited which in turn recruit other cells causing further inflammation and damage.
In AKI or CKD, the kidney may be still responsive to repair. Mesenchymal stem
cells (MSCs) found in bone marrow can be used a therapeutic agents. MSCs preferentially migrate to and help repair damaged tissue, in part by dampening immune responses following inflammation.
MSCs potently inhibit the primary induction of Th17 cells. This was characterised
by preserved T-cell viability, reduced proliferation, suppression of surface activation markers and decreased production of pro-inflammatory factors.
While soluble factors secreted by MSCs contribute to the suppressive effect, cell-cell contact also plays an important role. Results indicate suppressive factors are not actively expressed by MSCs or are secreted at very low levels and require dynamic cross-talk between MSCs and Th17 cells for expression to be induced. MSCs administered to patients may act as anti-inflammatory agents. Early treatment has the potential to prevent progression of AKI and CKD to end stage renal disease. In turn, this will reduce the heavy burden on society due to expensive dialysis treatments and transplantation.
7
Michel M. Dugon Zoology, School of Natural Sciences
CENTIPEDES: Venom, fangs and predation
Present in all continents except Antarctica, centipedes are important generalist terrestrial predators thriving particularly in the tropical and temperate regions of the globe. The most conspicuous and unique feature of centipede anatomy is the pair of large stabbing fangs protruding from the first trunk segment. Each fang contains a fully independent venom apparatus, used as a predatory and defensive tool. While most specimens from temperate areas have moderate body size from one to ten centimetres, large tropical species may reach lengths close to thirty centimetres. Developmental and functional investigations of such a system will help to unveil the mechanisms involved in the specialisation of this segment. Molecular techniques, electronic microscopy and ecological experiments are among the investigation methods used here to address questions relating to the origin of venom glands in arthropods and, more generally, to the origin of evolutionary novelties in the animal kingdom. Furthermore, this study may help to gain a better understanding of the medical significance of centipede bites and the pharmaceutical potential of their venom.
8
Noel Fitzpatrick
School of Physics, NUI Galway
AIR – SEA CO2 EXCHANGE AND ITS MEASUREMENT
Emissions of greenhouse gases due to human activity, such as carbon dioxide (CO2), have increased significantly since the industrial revolution, primarily due to the combustion of fossil fuels and changes in land use. The world’s oceans play an important role as a sink of carbon, without which, the atmospheric levels of CO2 would be significantly higher than the ~380ppm observed today. This gas exchange or flux is controlled by a range of physical and chemical factors of both the ocean and atmosphere, including temperature, salinity, wind, sea state, and biological processes. In a changing system, an understanding of air – sea CO2 flux is vital for predicting the future behavior of the global climate. However, due to sparse coverage and difficulties associated with gas flux measurements, there are significant uncertainties and gaps in understanding relating to the magnitude and pattern of CO2 exchange. A measurement campaign is currently underway at the National University of Ireland, Galway, to help determine the relationship between air – sea CO2
flux, and more easily measured variables such as wind speed. This will help facilitate widespread measurements of CO2 flux, and lead to a better understanding of the Ocean’s role in the carbon cycle.
9
Liam Naughton School of Mathematics, Statistics and Applied Mathematics
CALCULATIONS IN SYMMETRIC GROUPS Group theory can be considered the study of symmetry. The collection of symmetries of an object which preserves some structure of that object forms what mathematicians call a group. In the picture below we can see that the tiger’s head is symmetrical about the central axis.
Of course, mathematicians don’t study the heads of wild animals but even this simple picture can illustrate the concept of symmetry. In fact a mathematician generally doesn’t care what the picture actually looks like. What is important to us is that we can label the structure in question in some way. Consider the graph shown below. If we label the vertices we can study the symmetry of the graph by examining which vertices can be “mapped” by symmetry to which other vertices. This gives us the group which describes the symmetry of our object.
For any group we can compute a matrix of numbers called a table of marks. This block of numbers completely describes all of the symmetries of the underlying object to which it is associated. The applications of this theory are many, but a particularly interesting example is in the study of isomerism in chemistry. The problem of connectivity isomers is to determine all essentially different ways in which a given set of atoms can be structurally combined into a molecule.
10
Padraig O’Connor
School of Chemistry
CONTROLLED RADICAL POLYMERIZATIONS IN SUPERCRITICAL CARBON DIOXIDE
Modern day technological advancements are inextricably tied to the availability of new
materials. About 80% of all commercial synthetic polymers (plastics) are prepared by
radical polymerizations. In Ireland there are approximately 350 companies specialising in
the manufacture of plastics employing about 25,000 people. Volatile organic compounds
(VOCs) are required as reaction media and extraction solvents. VOCs are toxic,
hazardous, flammable (non-chlorinated), and contaminate aqueous effluents. This
research aims to prepare well-defined polymeric materials using heterogeneous
polymerizations in supercritical carbon dioxide (scCO2). ScCO2 is an attractive
replacement for VOCs, since it has tuneable solubility with pressure, low toxicity, and
provides a positive use for a green house gas. In the heterogeneous polymerization, initial
ingredients are soluble, but the polymer formed precipitates, and the polymerization is
said to continue in the particle phase. With an appropriate stabilizer, latex polymer
particles of narrow size distribution are obtained, while the controlled radical
polymerization allows narrow molecular weight distribution polymer to be formed. The
polymer can be isolated as a dry powder by simple venting (depressurisation) of the
reactor, thus circumventing the requirement for time-consuming and costly extraction
techniques. This presentation discloses practical and environmental advantages of using
benign scCO2 as a polymerization medium.
11
Paul ' O Reilly Biochemistry, School of Natural Sciences
CANCER; THE MAN THEY COULDN’T HANG
Cancer is an insidious disease of the cell, a cacophonous discord muffling cellular messages causing the cells to form tumours, to grow when and where they should not, invading and damaging other tissues. As an ailment it refers to over 100 different and distinct conditions which can arise in any tissue with varying degrees of malignancy. Owing to this variance in origin and design treatment of cancer has been focused on administrating broad spectrum chemotherapeutics. These “medicines” are poisons by definition; blighting healthy tissues, perverting and adulterating their development (nausea, hair loss ect.). Our research is geared towards the development and application of a newly discovered, naturally occurring chemotherapeutic, TRAIL, which has been shown to kill only cancerous cells leaving normal tissue unscathed. However, it is known that certain cancers are resistant to TRAIL treatment. The root cause is variable with no one molecule or collection of molecules, thus far, accounting for the tumour’s obstinate behaviour. We characterise resistant and sensitive cell lines, utilising novel methods, to better understand and elucidate which factors are involved in TRAIL resistance and thus pertinent for predicting whether a patient will respond to treatment. In doing so we hope to encourage and facilitate personalised patient care.
12
Alma Siggins, Microbiology, School of Natural Sciences
LOW-TEMPERATURE ANAEROBIC DIGESTION OF A TRICHLOROETHYLENE-CONTAMINATED WASTEWATER.
As interest in environmental protection and waste management becomes more commonplace in our everyday lives, the demand for processes to deal with an expanding amount of waste increases. Ideally these processes should yield a two-fold benefit: the removal of waste materials from our environments, coupled with the production of high levels of renewable energy. Research into the field of anaerobic digestion does just this, combining degradation of wastewaters ranging from industries such as food and chemical manufacturing, with the production of biogas, a valuable energy supply similar to natural gas. More specifically, this study investigated the treatment of trichloroethylene (TCE), a chemical used in the metal cleaning and degreasing industries. TCE is commonly found as a contaminant of groundwater, due to the limitation of treatment processes traditionally available. The EPA have imposed strict guidelines as to the level of TCE permitted in drinking water supplies, as it has been classified as ‘potentially carcinogenic to humans’. This research shows that the process of anaerobic digestion can be successfully employed for the treatment of TCE, at both traditional temperatures (37°C) and at more typical industrial discharge temperatures (15°).
13
Merry Zacharias Botany, School of Natural Sciences
MARINE ALGAE: THE MISSING LINK TO CLOUD FORMATION? INVESTIGATIONS ON THE EMISSION AND
EXUDATION OF ORGANIC COMPOUNDS
Cloud condensation nuclei (CCN) are small suspended particles (aerosols) in the atmosphere about which cloud droplets are formed. Many different types of atmospheric particles can act as CCN over the oceans like sea salt, sulphate and organic carbon. Recent field experiments have shown that marine algae emit significant quantities of organic matter (iodocarbons and volatile organic compounds) which could contribute to marine aerosol formation and thus have a direct effect on the earth’s climate. Algal metabolic stress compounds are released in potentially large quantities and contribute to the dissolved organic matter pool and also in the formation of CCN: organic matter in low size ranges can enhance cloud droplet formation and ultimately clouds. As part of a large-scale EPA climate change research programme, a series of algal life cycle and environmental response and emission experiments were conducted. Biological and emission data will be used in a coupled atmospheric-ocean model for climate prediction. Experiments were conducted to produce marine aerosols from artificially generated bubbles in a water tank using seawater with selected cultures of microalgae. A four-fold increase in organics was observed over 48h. Additionally, emissions of iodocarbons and different forms of iodine, including I2 from macroalgae and microalgae are being investigated. Further analysis of dissolved organic carbon (DOC) and particulate organic carbon (POC) in seawater and culture media of micro- and macroalgal species grown under different environmental conditions are also being carried out.
Discrete polynuclear complexes and 1, 2 and 3-D extended networks (coordination polymers) comprising paramagnetic 1st row transition metals have attracted much interest for many years due to their potential applications in numerous fields including molecular magnetism (encompassing information storage,1 quantum computing,2 molecular spintronics3 and magnetic refrigeration4) and gas storage.5 Our own recent contributions to the field of molecular magnetism detail the investigation of the coordination chemistry of the polyaromatic 2,2-biphenol ligand. We describe here the synthesis and characterisation of two dimeric [Mn2] complexes and a family of 1 and 2-D MnIII and FeIII
based coordination polymers whose nodes connect via covalent and / or H-bonding interactions.
1. R. Sessoli, D. Gatteschi, A. Caneshi, M. A. Novak, Nature. 1993, 365, 141-143. 2. M. N. Leuenberger, D. Loss. Nature. 2001, 410, 789-793. 3. L. Bogani, W. Wernsdorfer. Nature Materials. 2008, 7, 179-186. 4. M. Manoli, A. Collins, S. Parsons, A. Candini, M. Evangelisti, E. K. Brechin. J. Am.
Chem. Soc., 2008, 130, 11129-11139. 5. X. Zhao, B. Xiao, A. J. Fletcher, K. M. Thomas, D. Bradshaw, M. J. Rosseinsky. Science,
2004 306: 1012.
18
Study into use of a decoupler for the analysis of saccharides by capillary
electrophoresis
Vincent Bonifay and Dόnal Leech*
Enviromental Change Institue & Centre for BioAnalytical Science (affiliated NCBES) &
School of Chemistry, National University or Ireland, Galway, Ireland
Proteomics is rapidly emerging as one of the dominant research areas in today’s pharmaceutical and biothechnology industries. An array of new peptides and proteins possessing unique activities and metabolic pathways has yet to be identified. Analytical glycobiology is a field which increasingly demands high sensitivity and accurate methods of structural determination. Capillary electrophoresis, (CE), a technique widely employed in analytical glycobiology, is a powerful tool for the separation of a variety of biological compounds. Complex carbohydrate analysis is particularly challenging in analytical glycobiology, since these compounds possess no unique chromophore or fluorophore necessary for direct detection. Two techniques were used:
Carbohydrates were separated by a CE coupled to a Laser Induced Fluorecence Detector (instrument commercially available). The carbohydrates were labelled with a fluorophore, 1-aminopyrene-3,6,8-trisulfonate (APTS) at the reducing termini by reductive amination.
The second technique was a homemade system using a CE coupled to a Pulsed Amperometric Detector (not commercially available), which does not require a prior derivatization of the carbohydrates to allow a detection.
The CE-Pulsed Amperometric Detection (CE-PAD) system utilizes a decoupling joint between the CE separation voltage and PAD allows for improvement of the signal-to-noise ratio, thus increasing detection sensitivity. The decoupler was constructed by etching holes in the capillary with a CO2 laser. Effects of several important factors, such as buffer concentration and separation voltage, were investigated to acquire the optimum condition for the separation of a few saccharides and a model glycoprotein (fetuin). The two techniques were compared in terms of time for the total assay and limit of detection. Schematic setup of capillary electrophoresis Laser etched capillary
High Voltage
buffer
buffer
Sample Detector
19
Novel Metal Complexes as Catalysts for Phosphate Diester Hydrolysis
Fergal Coleman, and Andrea Erxleben.
School of Chemistry, National University of Ireland , Galway, Ireland. E-mail: [email protected]
Currently there is much interest in the development of catalysts that hydrolyse the phosphodiester bonds of DNA and RNA. In biochemistry, gene and cancer therapy, there are many potential applications for DNA cleaving agents; footprinting, sequence analysis and the degradation of viral or tumor cell nucleic acids. In nature, many of the enzymes which catalyze DNA hydrolysis employ one or more metal ions in the activation process.1 Much effort has been dedicated to producing small molecule catalysts that mimic these highly efficient natural hydrolases.2 While DNases are usually activated by Zn or Mg ions, phosphatases that hydrolyse phosphate monoesters contain Fe(III). The similarity of Fe3+ and Ga3+ prompted us to study interactions between Ga3+ complexes and phosphate esters. Ga3+
can be considered a diamagnetic probe for Fe3+ that allows for the characterization of active species and investigation of substrate binding by NMR spectroscopy. Here we report the synthesis and structural characterization of three mononuclear Ga(III) complexes with tripodal ligands (Figure 1) which catalyse the hydrolysis of the activated phosphodiester BDNPP (Bis(2,4-dinitrophenyl) phosphate). In these complexes, there are two coordination sites at the catalytic centre occupied by labile water/hydroxo groups (depending on pH). Coordination of the electron-rich phosphate diester to the electrophilic Ga(III) centres is the first step in the catalytic process.
Figure 1. Gallium (III) complexes synthesised in this study.
Subsequent attack by Ga-bound hydroxide or solvent hydroxide on the phosphorus centre occurs, leading to cleavage of a phosphate diester bond and generation of the dinitrophenolate. Ligand effects on the hydrolytic reactivity toward BDNPP have been investigated. We have observed that stronger ligand electron donating groups lead to greater catalytic activity, which suggests that the efficiency of the nucleophile prevails in determining the intrinsic reactivity of the Ga complexes. References
1 Strater, N.; Lipscomb, W. N.; Klabunde, T.; Krebs, Angew. Chem. Int. Ed. 1996, 35, 2024-2055. 2 Mancin, F.; Scrimin, P.; Tecilla, P.; Tonellato, U. Chemical Communications (Cambridge) 2005, 2540-2548.
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PROPERTIES AND PERMEABILITY OF HYPERICIN IN LIPID MEMBRANES WITH AND WITHOUT CHOLESTEROL
Emma S.E. Eriksson and Leif A. Eriksson* School of Chemistry, National University of Ireland – Galway, Galway, Ireland
hydroxyl and alkyl groups that is found naturally in the
Hypericum species. The compound displays promising
photosensitizing properties that have led to the
proposal that it can be utilized in photodynamic
therapy. Neither the detailed mechanism behind the
powerful action of hypericin, arising as a result of light
excitation, nor the intracellular localization and
transportation of the molecule is still fully understood. The behavior of hypericin and
brominated hypericin derivatives in a pure dipalmitoylphosphatidylcholine (DPPC) lipid
membrane model has recently been studied theoretically by means of molecular dynamics
simulations.1 Natural membranes however contain many important constituents -
cholesterol being one of the most essential - that influence the function and structure of
the membrane, and thereby also the behavior of drug molecules therein. Our previous
study was therefore extended in order to investigate hypericin and its brominated
derivatives in a membrane containing up to 25 mol% cholesterol. The results from the
two studies are compared and discussed. All simulations were performed using the
Gromacs program (versions 3 and 4).2-4
References
(1) Eriksson, E. S. E.; dos Santos, D. J. V. A.; Guedes, R. C.; Eriksson, L. A. J. Chem. Theor. Comput. 2009, 5, 3139. (2) Lindahl, E.; Hess, B.; van der Spoel, D. J. Mol. Model. 2001, 7, 306. (3) Van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C. J. Comput. Chem. 2005, 26, 1701. (4) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. J. Chem. Theor. Comput. 2008, 4, 435.
21
ONE-POT DOUBLE RADICAL CYCLIZATION ROUTES TO DIALICYCLIC RING-FUSED IMIDAZOBENZIMIDAZOLEQUINONES
AND PRELIMINARY ANTI-CANCER ACTIVITY
Vincent Fagan,a Sarah Bonham,a Michael P. Cartyb and Fawaz Aldabbagh*,a aSchool of Chemistry and bSchool of Natural Sciences,
Bu3SnH / azoinitiator mediated intramolecular homolytic aromatic substitution is now a valuable protocol in organic synthesis.1-3 We reported the use of the protocol in the synthesis of highly potent alicyclic [1,2-a] ring fused benzimidazolequinones via five to seven-membered radical cyclizations of nucleophilic N-alkyl radicals onto the benzimidazole-2-position activated by quaternizing the pyridine-like 3-N of imidazole with camphorsulfonic acid (CSA).3 The pyrido[1,2-a]benzimidazolequinone 1 was the most potent compound prepared being more than 300 times more cytotoxic than the clinically used drug, mitomycin C (MMC) towards hypoxic (low pO2) human skin fibroblast cells (GM00637). Moreover, compound 1 was more cytotoxic than other benzimidazolequinones containing “DNA-damaging functionality”, such as aziridine 24,5 or cyclopropane 33.
N NH
NH2
O
O
OMe
Me
OCONH2
N
O
O
N N
O
O
N N Tr
N
O
O
N
N
N
O
O
N
N
N
N
X
O
N
N
MMC 1 2
3 4, X = NH, O 5 The following presentation details the first one-pot double intramolecular homolytic aromatic substitutions onto heteroarenes: five, six and seven-membered double alkyl radical cyclizations onto the imidazobenzimidazole 2- and 6-positions. The cytotoxicity of iminoquinone and quinone derivatives 4 and 5 is evaluated against human normal (GM00637), cervical (HeLa) and prostate (DU145) cancer cell lines.
1. W. R. Bowman and J. M. D. Storey, Chem. Soc. Rev., 2007, 36, 1803. 2. D. C. Harrowven, I. L. Guy and L. Nanson, Angew. Chem. Int. Ed., 2006, 45,
2242. 3. M. Lynch, S. Hehir, P. Kavanagh, D. Leech, J. O’Shaughnessy, M. P. Carty and
F. Aldabbagh, Chem. Eur. J., 2007, 13, 3218. 4. L. O’Donovan, M. P. Carty and F. Aldabbagh, Chem. Commun., 2008, 5592. 5. K. Fahey, L. O’Donovan, M. Carr, M. P. Carty and F. Aldabbagh, Eur. J. Med.
NOVEL XYLIBOX LIGANDS: SYNYHESIS AND ENANTIOSELECTIVE APPLICATION
Fiona Kirbya, David Frain a, Patrick O’Leary*, a aSmaċt , School of Chemistry, National University of Ireland, Galway, Ireland [email protected], [email protected] We have prepared novel type of Bis(oxazoline) ligand.1 We will report on the synthesis and structure of these XyliBOX Ligands and on their catalytic activity in transition metal catalysed reactions.2 Metal complexes of 2,2-bis(oxazoline) ligands are well established asymmetric catalysts applicable to a wide variety of synthetically important reactions such as cyclopropanation, Diels-Alder cycloaddition, ene reactions, aldol reactions etc. Catalytically active complexes of these ligands have been reported using metals such as Fe, Cu, Co, Mg, Zn and Pd. A wide variety of ligands based on this structural motif have been reported. Such variations have largely concentrated on the functionalisation of the oxazoline rings and some variation at the bridgehead linkage.
Figure 1: X-Ray crystal structure of Cu(II) phenyl XyliBOX complex. Our aim was to create a new family of bisoxazoline ligands where the chiral centers were moved close to the bridgehead. This would have the consequence of introducing a twist into the ligand backbone which could have benefits in the catalysts’ activity in certain reactions. To that end we have successfully synthesised 4,4-bis(oxazoline) ligands which combine similar coordination chemistry to that of the 2,2-bis(oxazoline) ligands, with the intriguing feature of having the chiral centers of the ligand within the metallocycle in the catalytic complex. We will present the synthesis of the ligand shown via a novel one-pot double deprotection/activation/ring-closure protocol, the ligand-metal complex crystal structure and its catalytic performance in standard test reactions, comparing its performance to that of established 2,2-BOX complexes. 1) ‘The Synthesis of AraBOX, a New 4,4’-bis(oxazoline), from Novel Pentitol-Derived Bis β-Aminoalcohols.’ Frain, D.; Kirby, F.; McArdle, P.; O’Leary, P. Synlett 2009. 2)‘Preparation, Structure and Catalytic activity of Copper(II) complexes of novel BOX ligands’. Frain, D.; Kirby, F.; McArdle, P.; O’Leary, P. Advanced Synthesis and Catalysis, in review.
23
GENERATION OF CARBON RADICALS USING
SUPPORTED PHOTOMEDIATORS
Mary Treasa Lohan and Niall W.A. Geraghty* School of Chemistry, National University of Ireland, Galway
[email protected] The generation of carbon radicals for the formation of carbon-carbon bonds is a key step in synthetic organic chemistry. Standard procedures employ radical initiators, tin compounds or peroxides. All of these methods are environmentally unfriendly because of the toxic nature of the reagents or they present safety issues. An alternative approach involves the use of a photomediator such as benzophenone or a polyoxometalate (POM) to produce a nucleophilic carbon radical through hydrogen abstraction which can then attack an unsaturated system such as acetylene; particularly those with an electron withdrawing group. This functionalises the substrate and forms a new carbon-carbon bond (Scheme 1).
OO O
Scheme 1
It is synthetically important as the process allows the generation of carbon radicals directly from unactivated systems such as cycloalkanes, ethers and dioxolanes. Reactions have also been successful with cyclic and acyclic secondary alcohols. Although a useful method, the use of benzophenone as a photomediator has its drawbacks. Column chromatography is required to remove the photomediator which is unattractive from a synthetic viewpoint. POMs while offering increased reactivity result in reduced selectivity due to the formation of by-products. Therefore to overcome these deficiencies and enhance the synthetic importance of these reactions, a series of supported photomediators will be developed. By attaching benzophenone to a solid support such as silica or an organic polymer, the need for column chromatography can be eliminated as simple filtration allows the product to be isolated. This would also allow the photomediator to be recycled. POM supported photomediators will also be developed with a view to producing a supported photomediator of increased selectivity through an appropriate choice of support.
OMeMeO
hO
O
O
OMe
H
HO
H
OMe
OMe
O
24
A family of host-guest double-bowl pseudo metallocalix[6]arene discs
Seán T. Meallya and Leigh F. Jones*a
a, School of Chemistry, University Road, National University of Ireland, Galway, Ireland [email protected]
Much research in recent years has focused on the development and extension of supramolecular chemistry towards important applications such as catalysis,1 gas storage,2 and medical imaging (MRI contrast agents).3 A rapidly growing research field incorporating supramolecular methods, and the focus of our studies, is that of the development of host-guest systems via the targeted structural manipulation of polymetallic complexes and their associated molecular assemblies. Our research has recently focused on the synthesis and characterization of a family of heptanuclear planar discs of general formula [MII
7(OH)6(L1)6](NO3)2 (M = Ni, Zn, Co) (Fig. 1), for use as magnetically interesting host units; a field of host-guest chemistry still in its infancy. The complexes described here consist of a central MII ion surrounded by a further six MII ions to give the disc-like topology. These structures are linked by the Schiff base ligands IMMP (2-iminomethyl-6-methoxyphenol) and Br-IMMP (2-iminomethyl-4-bromo-6-methoxyphenol) (Fig. 2), which ligate via their imine, phenolic and methoxide functional groups and exhibit a μ2-η
1:η2:η1 coordination motif.
The individual [MII
7] units pack in 1-D columns in their crystal lattices to form double-bowl conformations in which the [MII
7] core is the basal plane (Fig. 3), akin to the metallocalix[6]arene concave unit. Hydrogen bonding between pairs of [MII
7] units result in molecular cavities which allow for the incorporation of a variety of different guest molecules; demonstrating their effectiveness as inorganic host species and thus allowing for direct comparison to supramolecular calix[n]arene behaviour.4
(1) Magrans, J. O., Ortiz, A. R., Molins, M. A., Lebouille, P. H. P., Sanchez- Quesada, J.,
Prados, P., Pons, M., Gago, F., de Mendoza, J., Angew. Chem. Int. Ed. 1996, 35. (2) Dalgarno, S. J., Thallapally, P. K., Barbour, L. J., Atwood, J. L., Chem. Soc. Rev., 2007,
36. (3) Pierre, V. C., Botta, M., Aime, S., Raymond, K. N., J. Am. Chem. Soc., 2006, 128. (4) (a) Meally, S. T., Karotsis, G., Brechin, E. K., Papaefstathiou, G. S., Dunne, P. W.,
McArdle, P., Jones, L. F., CrystEngComm 2010, 12. (b) Meally, S. T., McDonald, C., Karotsis, G., Papaefstathiou, G. S., Brechin, E. K., Dunne, P. W., McArdle, P. Power, N. P. Jones, L. F., Dalton Trans, 2010, DOI:10.1039/b926704b.
25
ELECTRICITY GENERATION FROM WASTEWATER IN MICROBIAL FUEL CELL –BACTERIAL-ELECTRODE INTERACTIONS IN A BIO-ELECTROCHEMICAL CELL
Rengaraj Saravanan, Krishna Katuri, Paul Kavanagh, and Dónal Leech
Bimolecular Electronics Research Laboratory (BERL) School of Chemistry & ECI
National University of Ireland, Galway Ireland
E-mail: your e-mail http://www.nuigalway.ie/chemistry/staff/donal_leech/personnel.html
Microbial electrogenesis is one of the most promising research fields with the potential to
deliver sustainable energy through microbial fuel cell (MFC) and microbial electrolysis
cell (MEC) technology, based on oxidation of fuel (waste) substrates by biofilms of
electroactive bacteria (EAB) on anodes. A barrier to application of the technology is the
low power densities observed, predominately caused by poor electron transfer from
bacterial communities to the anode. A range of mechanisms of electron transfer from
bacteria to anode is proposed such as indirect transfer using low molecular weight
electron shuttles (e.g. mediators) or electron shuttling proteins (e.g. cytochromes), and
direct electron transfer from cell surface redox active proteins or via electron conductive
“nano-wires” (pili) produced by the bacteria. For example, recent analysis suggests that
Geobacter sulfurreducens, a metal-reducing bacterium that is the focus of intense MFC
research, is wired to electrodes via outer cell surface expressed c-type cytochromes
during electricity generation. Here we report on Geobacter sulfurreducens biofilm
characteristics grown on carbon electrodes, probed using cyclic voltammetry (figures),
chemical analysis and microscopy.
Bio-electrochemical cell setup Cyclic voltammograms of Geobacter sulfurreducens
STRUCTURE AND DYNAMICS OF LISTERIA MONOCYTOGENES SORTASE A ENZYME: INSIGHT INTO CATALYTIC MECHANISM
AND INHIBITOR DESIGN
Boxue Tian and Leif A. Eriksson* School of Chemistry, National University of Ireland – Galway, Galway, Ireland
[email protected] Listeria monocytogenes is one of the most virulent foodborne pathogens. Listeria
Monocytogenes Sortase A (SrtA) enzyme, which catalyzes the cell wall anchoring reaction of the LPXTG-based internalin proteins, is known to be a target for developing anti-listeriosis drugs. In this study, the structure of the Listeria Monocytogenes SrtA enzyme-substrate complex was obtained using homology modeling, molecular docking and molecular dynamic simulations. Explicit enzyme-substrate interactions in both the inactive and the active state of the enzyme are discussed. The catalytic role of the active site arginine (Arg 197) is explained, which provides insight into the catalytic mechanism and enables rational design of inhibitors against SrtA.
REACTIVITY OF MOLYBDENUM COMPLEXES TOWARDS PEPTIDE CHAINS
Robert Whiriskey and Andrea Erxleben*
School of Chemistry, NUI Galway, University Road, Galway, Ireland. E-mail: [email protected]
The biological chemistry of metallocene dichlorides has attracted significant interest since the discovery of the anti-tumour properties of titanocene dichloride. This drug has been widely studied and is believed to inhibit nucleic acid synthesis. This has paved the way for it to become the first metallocene dichloride to enter clinical trials.1 Comparatively little work has been performed on other cytotoxic metallocene dichlorides. Molybdenum analogues of these metallocene dichlorides (molybdocenes) exhibit some antitumour activity. Their excellent chemical stability over a broad pH range combined with the different coordination chemistry and hydrolytic stability of the cyclopentadienyl ligands (Cp) makes them ideal candidates for study.2 Unlike titanocene dichlorides where DNA is the major target, molybdocene dichloride has a much stronger affinity for thiol groups of cysteine residues in glutathione and other oligopeptides. Much focus has been diverted to the interactions between molybdocenes and peptide chains.3 Studies on the reaction behavior towards short cysteine containing peptides pursues two objectives: gaining insight into the biological behavior of the molybdocene dichloride and using it as a cleaving agent for peptide bonds. These bonds are extremely stable, often the half life for the hydrolysis of simple peptides in biological conditions is several hundred years. The objective of this project was to synthesize molybdocenes which will hydrolyse peptide bonds under biological conditions. Due to its thiophilic behavior, the Cp2Mo+ moiety deprotonates the thiol group of the cysteine residue. This brings the nucleophilic group (Mo-OH) into close proximity with the scissile peptide bond. Nucleophilic attack is induced by the electron donating Cp groups, thus hydrolyzing the peptide chain selectively at the cysteine residue. Binding of these complexes to simple dipeptides and tripeptides has been studied using 1H NMR.4 The potential of molybdocene dichloride and other molybdocene derivatives as cysteine specific peptidases along with biochemical applications will be discussed. References 1Kopf-Maier, P European Journal of Clinical Pharmacology. 1994, 47, 1-16. 2Waern, J.B; Harding M.M.; Journal of Organometallic Chemistry 2004, 689, 2540-2548. 3Waern, J.B.; Harding M.M.; Inorganic Chemistry, 2004, 43, 206-213. 4Erxleben A. Inorg. Chem. 2005, 44, 1082-1094.
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RIBOFLAVIN ABSORPTION SPECTRA – A DIFFICULT CASE FOR COMPUTATIONAL MODELLING
Min Wu and Leif A. Eriksson* School of Chemistry, National University of Ireland, Galway, Ireland
Riboflavin (RBF; Figure 1), also known as vitamin B2, is one of the most easily absorbed micronutrients, present in a wide range of organisms. RBF is an important part of a healthy diet in human beings and animals, to maintain integrity of mucous membranes, skin, eyes and nervous systems. It also relieves diseases such as anaemia, migraine, and cataracts. Nowadays, several medicinal products containing riboflavin are generally available, in order to prevent or treat nutrient deficiencies. Flavin-related molecules are key cofactors in DNA photolyase to repair the photocycle of UV-damaged DNA, and RBF plays a key role in many enzymatic reactions and processes like phototropism and photo taxis. When dry, RBF is quite stable but in alkaline solution it deteriorates rapidly, and the deterioration to produce the reactive oxygen species 1O2 is accelerated by light. The absorption maxima of riboflavin in the body are at 445-450 (447) nm. In consequence, blue visible light will cause photodegradation of riboflavin. This results in diminished erythrocyte glutathione reductase, which indicates generalized tissue riboflavin deficiency and red cell lysis. Therefore, phototherapy will cause a deficiency of riboflavin.
B
Figure 1. A) RBF molecule B) RBF model in 10Å water environment
In the current study, time-dependent density functional theory using hybrid functions have been employed to investigate how well we can model the ultraviolet and visible spectra of RBF by computational means. The photochemical properties were obtained using three different functionals: MPWB1K, B3LYP and PBE0 both in vacuum and in implicit and explicit solvent models (cf Figure 1). The aim is to develop an approach that will enable prediction of spectral properties for modified RBF, as a tool in drug design.
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PULSATILE SMALL MOLECULES ELUTION FROM UV CROSS-LINKED THERMORESPONSIVE COPOLYMER THIN FILM
R. Yang1&2
, A. Gorelov3, M. Nash
1&2, W. Carroll
1 , F. Aldabbagh
1 and Y. Rochev
2*,
1Department of Chemistry, National University of Ireland, Galway, Co. Galway, Ireland
2National Centre of Biomedical Engineering Science, National University of Ireland,
Galway, Co. Galway, Ireland 3Department of Chemistry, University College Dublin, Co. Dublin, Ireland
In the development of pharmaceutics, some drugs require a periodic fluctuating concentration in the blood to reach an optimal therapeutic. Thus, a switchable drug delivery system whereby one could switch on and off the release could be used to obtain a pulsatile release. The polymer used in this body of work is poly (N-isopropylacrylamide-co-acrylamideobenzophenone) (poly (NIPAm-co-ABzPh)) which possesses a phase transition in aqueous solution at a lower critical solution temperature (LCST) of 28°C. UV cross-linked copolymer films1 were prepared [2] and loaded with the model drugs, fluorescein isothiocyanate (FITC) and Rhodamine B respectively.
The pulsatile elution (fig 1) was obtained between two states by using ambient temperature as the trigger: the collapsing state (above the LCST) and the swelling stage (below the LCST). The inconsistent elution rates between these two states can be explained by the different surface roughness of the films which was observed by atomic force microscope (AFM). In addition to the temperature, film reservoir thickness and the amount of drug loaded into the film are also critical parameters for controlling the elution profile. In the collapsing state, the releasing experiment presented the same regulation as the mathematical model. Conversely, the regulation was lost in the swollen state. In conclusion, thickness and amount of drug loaded are essential variables in regulating the drug elution.
Fig 1. Pulsatile elution profile: graph presented the pulsatile release controlled by temperature (4°C and 40°C) from a model drug loaded UV cross-linked film.
1 Films can be prepared in both nano-scale and micron-scale.
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MANIPULATION OF THE POINT-SPREAD FUNCTION OF A HIGH NUMERICAL APERTURE LENS BY TAILORING THE INPUT
POLARISATION STATE
Authors – Fiona Kenny1, Oscar Rodríguez1, David Lara2 and Chris Dainty1
1Applied Optics Group, School of Physics, NUI Galway 2The Blackett Laboratory, Imperial College London.
[email protected] When using a high numerical aperture lens to focus light, it is necessary to employ vectorial diffraction theory, in place of scalar, to describe the field at the focal region. This effectively means that the polarisation state of the input beam must be considered in the calculation, and was first described by Richards and Wolf [1] using diffraction integrals. A number of alternative methods have been developed since then, using both the Fast Fourier Transform algorithm [2] and the chirp-z transform [3]. A comparison of these numerical methods to calculate the field at the focal region was carried out. The results were compared to the rigorous Richards and Wolf calculation, and are presented here. Two variable retarders in sequence can be arranged to produce any pure state of polarisation [4] (i.e. no depolarisation). By a similar principle it is envisaged that by using a 2D array of these variable retarders any inhomogeneous distribution of state of polarisation can be obtained across the waist of a light beam. A spatial light modulator (SLM), consisting of a pixelated liquid crystal display, is such an array where the birefringence, and thus the retardance, of each pixel, is proportional to the voltage applied. An optical design that will be used to implement this principle in a vectorial polarimeter [5], along with the current status of the experimental work, is also presented. [1] B. Richards and E. Wolf. Electromagnetic diffraction in optical systems II. Structure of the
image field in an aplanatic system. Proceedings of the Royal Society of London Series A-Mathematical and Physical Sciences, 253(1274):358–379, 1959.
[2] C. W. McCutchen. Generalized aperture and the three-dimensional diffraction image (vol 54,
pg 240, 1964). Journal of the Optical Society of America A, 19(8):1721–1721, August 2002. [3] Marcel Leutenegger, Ramachandra Rao, Rainer A. Leitgeb, and Theo Lasser. Fast focus field
calculations. Optics Express, 14(23):11277{11291, 2006. [4] D. Lara and C. Dainty. Axially resolved complete Mueller matrix confocal microscopy.
Applied Optics, 45(9):1917–1930, March 2006. [5] O. G. Rodríguez-Herrera, D. Lara, and C. Dainty. Far-field polarization-based sensitivity to
sub-resolution displacements of a sub-resolution scatterer in tightly focused fields. Opt. Express, 18(6):5609–5628, 2010.
Léger et al. (2004) and Küüchner (2003) hypothesised that Ocean planets, Super-Earth planets with liquid-water oceans covering their whole surfaces may exist. Some may have already been discovered, such as GJ 1214b. However at this time we need predictions of observables to uniquely identify ocean planets. On such planets, the climate will be dominated by the nature of the ocean, which is currently poorly understood. However, Super-Earths which orbit close-in to dwarf M-stars will have very large tidal forcings, several orders of magnitude larger than Earth. The current work explores the effects of such a tidal forcing on the ocean circulation of such a planet. In particular, we aim to answer two questions: (1) Is there a deep circulation connecting the surface to the ocean floor, and (2) what is the scale of heat redistribution in the ocean. The nature of the circulation is crucial for determining the composition of the ocean. In the lack of a connecting circulation, most solids would be expected to precipitate to the ocean floor, hence inhibiting life. We examine the case of GJ-1214b, and evaluate the prospects for a liquid-water ocean. For a air-liquid phase transition to exist, a high albedo and hence cloud cover is required. We investigate the heat transfer required to generate cloud cover with a water ocean and H/He dominated atmosphere, and make preliminary predictions on the observables in such a climate, in particular the albedo from cloud layers and thermal emission profile.
EXPERIMENTAL DETECTION OF PHASE SINGULARITIES USING THE SHACK-HARTMANN WAVEFRONT SENSOR
Kevin Murphy and Chris Dainty
Applied Optics, School of Physics, NUI, Galway, Galway, Ireland [email protected]
Phase singularities (or optical vortices) occur at the points in an optical wavefront when the intensity of the beam drops to zero and the phase of the wavefront is undefined. These points can be seen in the cross-section of the laser beam at the receiver of an optical system. Phase singularities occur in pairs of opposite sign (or rotation) and are joined by a wave dislocation, called a branch cut, with a corresponding 2mπ radian jump in the phase. The m denotes the topological charge of the vortex. When a laser beam is propagated on a horizontal or near-horizontal path through the atmosphere, optical vortices will occur naturally due to slight changes in the refractive index of the air. The stronger the atmospheric turbulence, the greater the density of phase singularities present at the receiver [1].
Fig.1. Single phase singularity with 2π radian discontinuity Fig.2. Schematic of experimental setup used in the detection of singularities
Phase singularities have been shown to be one of major cause of problems for adaptive optics (AO) systems [2] which attempt to correct for distortion caused by the atmosphere in line of sight free space optical communications over mid-to-long range horizontal paths (>1km). It is the 2π discontinuity in the phase of the wavefront which causes difficulties for common AO reconstruction techniques. To negate the effect of the phase singularities they must be detected and then taken into account in the wavefront reconstruction. An experimental set up has been built and is being used in the laboratory to examine the detection of phase singularities in atmospheric turbulence. This consists of a turbulence generator using a spatial light modulator (SLM) to mimic the atmosphere and a Shack-Hartmann wavefront sensor as the receiver. The branch point potential method for phase singularity detection, first proposed by LeBigot and Wild in 1999 [3], is then implemented in post processing to locate the position of the phase singularities. Phase singularity detection can then be practiced under different conditions in a controlled manner.
35
References [1] Voitsekhovich, V. V., et al. (1998) Density of turbulence-induced phase dislocations, Applied Optics, 37, 4525 – 45355. [2] Primmerman, C. A., et al. (1995) Atmospheric-compensation experiments in strong-scintillation conditions, Applied Optics, 34, 2081 – 2088. [3] LeBigot, E. O., & Wild, W. J. (1999) Theory of branch-point detection and its implementation, J Opt. Soc. Am. A, 16, 1724 – 1729.
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OPTICAL DESIGN OF UV SOURCE FOR CORNEAL COLLAGEN CROSS LINKING
Matthew Sheehan and Alexander Goncharov Applied Optics, School of Physics, National University Ireland Galway
We have designed and constructed a UV source for use in corneal collagen cross-linking procedures. Firstly we introduce a basic outline of the collagen cross-linking procedure and provide a background rational for treating keratoconus with collagen cross-linking. Then the optical features of our design are discussed in reference to the practical considerations for clinical use. The optical system compares favorabily with commercial systems and offers substantially more control and safety than designs relying on direct illumination. The prototype was relatively inexpensive to construct and includes a mounting unit and a optical power testing unit. The device is currently being used in an ophthlmology practice to treat forme fruste keratoconus.
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Radiobiological Modelling Impact of Organ Motion in Prostatic Tumours
S Walsh†, M Moore‡, W van der Putten‡ †School of Physics, National University of Ireland Galway, Ireland
‡Medical Physics and Bio-Engineering Dept, Galway University Hospital, Ireland Email: [email protected]
Abstract Appropriate radiobiological modelling has the promise to correctly predict radiobiological metrics such as tumour Control Probability (TCP) and Normal Tissue Complication Probability (NTCP). The work presented here is part of a larger study within the Medical Physics Research Cluster (MPRC) of models which take into consideration most variables which play a role in determining radiobiological response. The methodology for the modelling process was based upon percentage differential DVHs calculated by Oncentra Master Plan, the clinical treatment planning system utilised in GUHs (Galway University Hospitals). Five intermediate risk prostate cancer patients were randomly selected and anonymised from GUHs patient database. Two CRT plans, and three IMRT plans were executed. The dose distribution scored in each of the patient’s Computed Tomography data sets was then used as the dose input parameter for radiobiological assessment. The radiobiolgical assessment of the treatment plans is based upon the ‘Marsden Tumour Control Probability’ (TCP) and the ‘Lyman-Kutcher-Burman Normal Tissue Complication Probability’ (NTCP) models and their ability to accurately describe the clinical responses reported in the literature for prostate cancer patients undergoing external beam RT.
Recursive partitioning methods are a popular non-parametric alternative to the classical parametric and non-parametric models in regression, classification and survival problems. They have been recognised as a useful modelling tool as they produce a model that is very easy to interpret. The beauty of these methods lies in their simplicity and the relative ease in which the results of the analysis can be explained to a person with a non statistical background. From the statistical point of view, however, the criticism arises from the lack of statistical tests and the absence of p-values. In general, the aim of any statistical model is to explain the complicated inter-structure between a large number of explanatory variables and the response, in the simplest way possible, and to use the model to predict the outcome of interest when new observations are considered. Single trees are an excellent way to describe the structure of the learning data but their predictive power can be disappointing. In the last decade, many efforts have been made to overcome this problem. These methods are generally known as "ensemble methods" and they use a set of trees, created by bootstrapping the original data, in order to improve predictability. The price to be paid, however, is the absence of a singular tree. In this work, a data set of 1586 patients with cardiovascular disease will be analyzed. The primary endpoint was a cardiovascular composite endpoint, which included death from a cardiovascular cause or any of the cardiovascular events of myocardial infarction (MI), heart failure, peripheral vascular disease and stroke. Seventeen factors/covariates will be considered for development of a prognostic model and the results of different methods for growing survival trees will be compared.
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ANALYSIS OF AN OBSERVATIONAL STUDY IN COLORECTAL
CANCER PATIENTS
Authors – Cara Dooley, John Newell and John Hinde School of Mathematics, Statistics and Applied Mathematics,
The aim of the study was to compare survival of colorectal cancer patients in the whole population against the survival of patients in a sub-population who also had inflammatory bowel disease (IBD). All individuals who suffered from colorectal cancer were drawn from the entire Irish population using data from January 1994 to December 2005 provided by the National Cancer Registry of Ireland (NCRI). The control group contained many more observations (n>20000) when compared to the IBD group (n = 170). Given the number of control patients, there was large diversity in this group. In a conventional designed experiment or trial, patients entering the trial would be taken to be as similar as possible, with similar numbers in each group. Usually patients would be similar in age, health etc. As this was an observational study, there was no design prior to collecting the data. To compensate for this lack of design, each IBD patient is matched to the "closest" control patient. For each pair of IBD and control patients a distance is calculated and those two patients which have the smallest distance between them (and are so are the most similar) are matched. The distance used in this case is a Malanhobis distance based on ranks. The matching is carried out using the Optmatch Package in R. Following the matching, analysis was carried out on the new reduced data set to see if there was a difference in survival in patients who had colorectal cancer and those who also had the secondary disease (IBD).
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“BEST TECHNIQUES FOR TOLERANCE INTERVAL ESTIMATION, WITH AN APPLICATION IN SPORTS FITNESS TESTING AND CLINICAL DATA” Orla Doolin School of Mathematics, Statistics & Applied Mathematics, NUIG. [email protected] Supervisor: Dr. John Newell In elite sports, tolerance intervals can be employed to compare output of individual athletes fitness assessments to intervals deemed to capture the “typical” values of the target population of elite athletes. In a clinical setting, tolerance limits for diagnostic data facilitate the detection of “atypical” or extreme measurements. A simulation study is set up to generate both gaussian normal and non-normal univariate data comparing various tolerance limit approximations to their exact values, and to look at bias and variance characteristics of the quantile estimators. Out of this, a table ranked by best methods for tolerance interval calculation is shown. The proposed ‘best’ methods are then applied to a set of clinical laboratory values of glycosylated haemoglobin (HbA1c) and to fitness data on an elite squad of athletes. Readings of HbA1c, observed to have a non-normal positively skewed distribution, are recorded from a cohort of 383 patients with Type 1 diabetes. Readings from a fitness testing battery are from a squad of professional soccer players and deemed to have a reasonably normal distribution. With any statistical interval the purpose is to infer a region that bounds uncertainty for some scalar quantity from a sample that is reasonably representative of the target population. Tolerance intervals, the lesser known relatives of confidence and prediction intervals. They seek to locate the bulk of an underlying distribution of individual measurements with some specified confidence, as opposed to setting bounds on a particular parameter of interest (confidence interval) or on a particular individual (prediction interval). Tolerance intervals for large fractions β and confidence levels γ will typically be larger than corresponding 100* β% prediction intervals, as it is intuitively harder to bracket most future readings than to bracket a single future reading. Both parametric or a nonparametric approaches to univariate tolerance limits are compared in this study.
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Heritability and Genome-Wide Association (GWAS) Assay of MicroRNA Regulatory Efficacy
Paul Geeleher, Cathal Seoige and Aaron Golden. School of Mathematics, Statistics and Applied Mathematics
MicroRNAs are small non-coding RNA molecules that regulate the expression level of genes by binding to messenger RNA targets, which prevents these messenger RNA molecules from being translated into mature proteins. We have performed an analysis to investigate the genetic compont involved in the regulatory effect of microRNAs. The Regulatory Effect (RE) score [1] is used to measure the inhibitory effect of a microRNA in a sample by measuring the expression levels of the microRNA's messenger RNA targets, independent of the expression level of the actual microRNA; it is calculated from the the average difference in expression level of the targets versus non-targets, where the targets have been determined previously by target-prediction algorithms such as PITA [2]. The Hapmap project is an international collaboration which aims to catalogue the main sources of variation in the human genome with and between distinct geographic populations based on single nucleotide polymorphisms (SNP). The project members have used SNP microarrays to genotype groups of trios of individuals (two parents and one child) from several worldwide geographic regions. Other affiliated groups have used the cell lines produced by the project to perform gene expression studies that measure messenger RNA levels. Using this data and because these cell line samples are related by genetic lineage we have been able to ascertain that there is a heritable component to the regulatory effect of microRNAs. This seems to suggest that the entire miRNA processing machinery operates at a lower level of activity overall in a sub-population defined by specific genotype SNPs. We have performed a genetical genomics assay but have thus far been unable to identify expression quantatitive trait loci (eQTL) that show a statistically significant association with this altered RE-score. We are currently assessing the likely causative effects of such diminished microRNA regulatory efficacy. [1] Chao Cheng, Xuping Fu, Pedro Alves and Mark Gerstein: mRNA expression profiles show differential regulatory effects of microRNAs between estrogen receptor-positive and estrogen receptor-negative breast cancer. Genome Biology 2009, 10:R90 [2] Michael Kertesz, Nicola Iovino, Ulrich Unnerstall, Ulrike Gaul & Eran Segal: The role of site accessibility in microRNA target recognition: Nature Genetics 39, 1278 - 1284 (2007)
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COMPUTATIONAL HOMOLOGY WITH PERMUTAHEDRAL COMPLEXES
Fintan Hegarty School of Mathematics, Statistics and Applied Mathematics
[email protected] We develop a mathematical machinery for efficiently computing the homology of cellular spaces with a view to applications in image analysis. We discuss and implement a cubical approach to homology computation, and introduce and implement a new approach based on permutahedral complexes. Our guiding example is the analysis of medical data from computed tomography images.
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ANALYSIS OF GRAZING BIFURCATIONS WITHIN A DISCONTINUITY-GEOMETRY FRAMEWORK
N.Humphries and Dr P.T.Piiroinen*
School of Mathematics, Statistics and Applied Mathematics, NUI Galway *[email protected]
A periodically-forced impact oscillator (PFIO) is a mathematical model that can be applied in a wide range of real-world applications, such as rattle in gear systems. This model has two fundamental components, a differential equation that models the system in free-flight between impacts and a discrete reset rule to model the behaviour of the system at impact. The discontinuities introduced into the model by the reset rule mean that any solution to the differential equation with appropriate initial conditions now only applies up to the next impact, whereupon the reset rule defines initial conditions for a new free-run. A PFIO is an example of a piecewise-smooth system, and in such a system conventional analytic and numeric techniques do not always give a full picture of the qualitative changes in behaviour under parameter variation.
If the impacting surface of a PFIO is sufficiently far from the natural centre of the oscillation there will be no interaction between the surface and the dynamics of the system, and thus the only motion will be periodic non-impacting orbits. However, as the impacting surface is brought ever closer to this centre, eventually impacts will start to occur and periodic orbits with impacts may appear. The first point of contact between the non-impacting orbit and the impact surface is often referred to as a grazing bifurcation. The exact dynamic behaviour born at this bifurcation will depend on the other parameters of the system.
This overall study is exploring the use of a discontinuity-geometrical framework, which is based on the form and properties of a number of system-specific geometric objects within a three-dimensional representation space. Firstly we will give a brief introduction to this framework, with definitions of the relevant geometric objects and examples of how individual impact-to-impact trajectories can be represented. After this we will use the discontinuity geometry to explore and explain some of the different dynamic behaviours manifested by a damped harmonic oscillator around grazing bifurcations.
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A STUDY OF ITEM SELECTION USING PRINCIPAL COMPONENT ANALYSIS AND CORRESPONDENCE ANALYSIS
Nur Fatihah Mat Yusoff
School of Mathematics, Statistics and Applied Mathematics National University of Ireland, Galway
This study investigates the dimension-reduction techniques in psychometric testing by using Principal Component Analysis (PCA) and Correspondence Analysis (CA). Psychometric research is one of the fields of social science study that is interested in the theory and techniques of education and psychological measurement. Researchers in this area are frequently concerned with the construction and validation of measurement instruments. Theoretically, PCA is a mathematical algorithm that transforms a number of possibly correlated variables into a smaller number of uncorrelated variables by performing a covariance analysis between variables. The PCA concept is closely related to Factor Analysis (FA) which aims to detect structure in the relationships between variables. It is a common technique that has been used by social science researcher in conducting validity and reliability analysis of their study. The CA can be considered as a factor method for the categorical variables and is often linked with producing a low-dimensional graphical display of variables and units. Simple CA is a technique designed to analyse a two-way table, while Multiple Correspondence Analysis (MCA) is an extension of simple CA in that it is applicable to a large set of variables. The result will provide information which is similar in nature to those produced by principal component analysis, and allows us to explore the structure of the categorical variables included in the table.
This study is concerned with reducing the dimension, or number of variables, in an instrument by using the data from a pilot study on personality traits. The original instrument was developed by Oliver P. John and Sanjay Srivastava from University of California, Barkeley in 1999. The pilot survey was conducted at the University Malaysia Sarawak, Malaysia where 80 students from second year and above were randomly selected as respondents. In the original instrument, there are 44 items to assess five personality traits or the big five dimensions. We believe that some of the items, or even dimensions are not relevant in the Malaysian context. At the end of this study, our aim is to produce the best instrument that can represent all of the variables that we are interested in for subsequent use in structural equation modelling of student achievement.
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Computing In Finite Groups
Liam Naughton
School of Mathematics, Statistics and Applied Mathematics [email protected]
The table of marks of a finite group G is a matrix whose rows and columns are labelled by the conjugacy classes of subgroups of G and where for any two subgroups of G, H and K, The (H,K) entry in the matrix is the number of fixed points of H on K in the transitive action of G on the right cosets of K in G. The table of marks characterises all permutation representations of G and is sometimes called the supercharacter table of G. It also provides a compact description of the subgroup lattice of G and enables us to perform calculations in the Burnside ring of G. In this research we are developing new methods to calculate tables of marks.
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MODELS FOR GLYCEMIC REGULATION
Liam O’Callaghan and Petri Piiroinen* School of Mathematics, Statistics & Applied Mathematics, NUIG
*petri.piiroinen @nuigalway.ie
Glycemic Regulation, or the control of a person’s blood sugar level, is an important aspect of patient care in a hospital’s intensive care unit. Patients who have been injured or have just undergone an operation require constant monitoring to ensure that the level of glucose in their blood remains within its normal range. The body has a highly complex system by which blood sugar is regulated, but it is often the case that, in critically ill patients, this system fails or becomes inadequate, and must be augmented by other means.
The mechanisms by which blood sugar is regulated (by a complex process involving many hormones, including insulin and glucagon) are quite well understood. The dynamics of the system, however, are not. Essentially, a practitioner administering insulin would have an idea of the chain of events that, following the drug’s administration, result in lowering a patient’s blood sugar level. However, the magnitude of such an effect can at present not be predicted with a significant level of accuracy.
Mathematical models exist that attempt to describe the glucose regulatory system in greater detail. For example, models have been developed that predict certain features of the system, or explain the results of certain tests. These models have the advantage of being small and manageable, but when taken out of context and applied to the case of a patient in intensive care, they don’t perform so well. More work, therefore, needs to be made to advance these models to the stage where they are clinically useable
We present an analysis of a “minimal model” which was recently developed for glycemic control in the critically ill. A minimal model is essentially the smallest possible model that expresses the features that are required. The analysis consists of looking for blood glucose values to which the model will move towards, or move away from, and also the nature of this motion. Whether or not the predictions made by this model are realistic is obviously of critical importance.
The prospects for future advancement and the development of a full-scale description of the glucose regulatory system will also be presented. In particular, the binding process between insulin and its corresponding receptors needs to be effectively modelled, and the subsequent signalling which results in the lowering of blood glucose explained, if the effect of insulin is to be properly quantified.
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Convolutional Codes from Group Rings
Jessica OShaughnessy Supervisor: Prof. Ted Hurley
School of Mathematics, Statistics, and Applied Mathematics NUI, Galway
Convolutional codes have many applications. The original uses were in deep space communications. More recently they have also been used in digital signals, GSM, and ATM networks2. Finding convolutional codes that have desirable properties and are easy to decode is often a challenge. An original group ring construction for convolutional codes was proposed in 20093. This group ring construction used an isomorphism between group rings and group ring matrices to construct generator and control matrices. This construction is extended to a second group ring construction. The second group ring construction focuses on units in the group ring GC2 , where G is typically a cyclic group. This second construction has several interesting free distance properties. Additionally, it can be used to construct LDPC convolutional codes, systematic convolutional codes, and some other optimal convolutional codes. Algebraic constructions of convolutional codes allow for ease in encoding and decoding. An algebraic understanding of convolutional codes has been considered4 as well as several algebraic constructions5. However, the majority of known convolutional codes have been found through computer search rather than by algebraic means. Further algebraic constructions open up many possibilities for convolutional codes and give more opportunities for them to be used in practice.
2 Costello Jr, D.J. and Forney Jr, G.D, Proceedings of the IEEE , 2007, 1150-1177. 3 Hurley, T., International Journal on Pure and Applied Mathematics, 2009, 50, 431-463. 4 McEliece, R.J, Handbook of Coding Theory , 1983, 1065-1138. 5 Gluesing-Luerssen, H. and Schmale, W, Acta Applicandae Mathematicae, 2004, 183-237.
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MODELLING OF DRUG DIFFUSION FROM THE POLYMER COATING STENTS
Vo Thi Ngoc Tuoi
Supervisor: Martin Meere Department of Applied Mathematics
The effect of polymer swelling on drug diffusion from polymer coating stents is investigated. The polymer swelling modelling will lead onto work in the modelling of thermoresponsive polymers, such as Poly(N-isopropylacrylamide) (PNIPAm). This polymer is hydrophilic below the lower critical solution temperature (LCST) C, but hydrophobic above C, a property that can be exploited to act as an on/off switch to control drug delivery by varying temperature. Above the LCST, collapsed polymer prevents drug release. Below the LCST, the drug diffuses when the polymer absorbs fluid and swells. In addition, the effect of burst release is observed. Burst effect is the immediately release of an initial large of drug after activation. Initially, there is a good agreement between numerical solutions of the model and experimental datas.
032032
Figure: A polymer swelling controlled drug delivery system containing dissolved and dispersed drug. The polymer absorbs fluid and swells; moisture content in polymer can affect the drug mobility significantly. [1] B. Narasimhan, R. Langer, Zero-order release of micro- and macromolecules from
polymeric devices: the role of the burst effect, Journal of Controlled Release 47 (1997) 13-20.
[2] J. Siepmann, F. Siepmann, Mathematical modelling of drug delivery, International Journal of Pharmaceutics 364 (2008) 328-343.
[3] S. Mcginty, S. Mckee, R.M. Wadsworth, Modelling drug eluting stents, Mathematical Medicine and Bilology: A Journal of the IMA (2009).
[4] X. Huang, C.S. Brazel, On the importance and mechanisms of burst release in matrix-controlled drug delivery systems, Journal of Controlled Release 73 (2001) 121-136.
Biochemistry No. ID Number Surname Name Year/Programme 31. 04404653 Colleran Christine 2PS1 32. 02559447 Conroy Pauline 2PS1 33. 06125263 Dantas Tiago 3PS1 34. 06124453 Deepti Ayswaria 4PS1 35. 02392198 Foley Sarah 4PS1 36. 08232114 Frizzel Aisling 2PS1 37. 03379230 Gallagher Kathleen 3PS1 38. 06106170 Hamilton Danielle 1PS1 39. 06239011 Kumar Ramesh 4PS1 40. 03025063 O’Donoghue Yvonne 3PS1 41. 02747812 Prendergast Áine 4PS1 42. 08232931 Sokol Anna 2PS1 Botany No. ID Number Surname Name Year/Programme 43. 07233822 Eschmann Christine 3PS1 44. 09232177 Pielach Anna 1PS1 Earth and Ocean Sciences No. ID Number Surname Name Year/Programme 45. 06239022 Durcan John 3PS1 46. 06131395 Gilligan Martin 2PS1 47. 02797127 Guihen Damien 4PS1 48. 03783481 Lynch Edward 3PS1 Microbiology No. ID Number Surname Name Year/Programme 49. 08233229 Barczynska Alicja 2PS1 50. 00621056 Cunningham Claire 2PS1 51. 04435451 Feehily Conor 1PS1 52. 04031717 Finn Rebecca 2PS1
53
53. 05128005 Flannery John 1PS2 54. 08233162 Gill Aileen 2PS1 55. 04010566 O’Boyle Nicholas 2PS1 56. 08233197 Rajko-Nenow Paulina 1PS2 57. 03081028 Twomey Marcus 3PS1 58. 08233026 Utratna Marta 2PS1 Zoology No. ID Number Surname Name Year/Programme 59. 05363527 McEvilly Paul 1PS1 Environmental Science No. ID Number Surname Name Year/Programme 60. 04392841 Staunton John 2PS1
54
UBF, a master regulator of ribosome biogenesis
Christine Colleran, José-Luis Prieto and Brian McStay* Centre for Chromosome Biology, National University College Galway, Galway
The nucleolus is the most prominent subnuclear compartment whose main function is ribosome biogenesis. Ribosome biogenesis involves transcription of the ribosomal genes (rDNA) by the dedicated RNA polymerase I machinery to generate an rRNA precursor transcript that is processed, modified and assembled with ribosomal proteins to generate functional ribosomal subunits. The rate of ribosome biogenesis is intimately linked to cell growth and proliferation, thus ribosome biogenesis is up regulated in cancer cells and down regulated in differentiating cells. Nucleoli assemble around tandem arrays of ribosomal genes termed nucleolar organizer regions (NORs) located on the short arms of the five human acrocentric chromosomes. Transcriptionally active NORs appear as regions of under-condensed chromatin on metaphase chromosomes termed secondary constrictions. Upstream binding factor (UBF) is necessary for efficient rDNA transcription and correlates with the undercondensed chromatin state of ribosomal genes at mitotic NORs. NORs in which the rDNA repeats have not been transcribed lack UBF binding, appear fully condensed during metaphase, and lack a secondary constriction. The level of UBF in a cell correlates directly with the proportion of actively transcribed rDNA repeats. For example, differentiating cells have reduced UBF levels and a reduced number of active rDNA repeats resulting in down regulation of ribosome biogenesis.
To study the regulatory role of UBF in ribosome biogenesis we analyzed the effect of depleting UBF using a stable cell line expressing human UBF shRNA. As UBF levels decrease, ribosomal genes condense causing NORs to move to the periphery of the nucleolus and eventually dissociate from nucleoli (see figure). These results show that UBF is responsible for the under-condensed chromatin state of active rDNA repeats and that it is a key determinant in controlling the rate of ribosome biogenesis.
References Mais et al (2005) Genes and Development 19, 50-64. Prieto and McStay (2007) Genes and Development 21, 2041-2054. McStay and Grummt (2008) Annual Review of Cell and Dev. Biology 24, 131-157
Genome instability and aneuploidy are common characteristics of many tumours. Supernumerary centrosomes are also observed in many tumour cell lines, leading to the model that during mitosis, cells with multiple centrosomes fail to divide properly. This may result in progeny with an unbalanced complement of chromosomes. One model for how centrosome amplification occurs is that centrosomes amplify during an extended cell cycle arrest following DNA damage. We wished to investigate mechanisms by which centrosome/centriole amplification can occur and the outcome of such amplification. A range of candidate centrosomal genes were also screened by quantitative RT-PCR to investigate possible differences in transcriptional levels after DNA damage. The mRNA levels of several candidates altered following ionising radiation. We will examine how these candidates affect centrosome activity after ionising radiation. Inhibition of CDK1 leads to an extended delay in G2 phase, as cells cannot enter mitosis. Following CDK1 inhibition by the small molecule RO3306 in untransformed hTERT-RPE1 cells, we observed up to 26% of cells with more than two centrosomes after 30 hours. After RO3306 washout, ~20% of cells quickly re-entered the cell cycle into mitosis, with many of the cells with multiple centrosomes forming multipolar spindles when released from the cell cycle block. Immunoblot analysis of phospho-H2AX levels following RO3306 treatment indicated that DNA damage had not occurred. In addition, we have generated an inducible PLK4 allele in human osteosarcoma U2OS cells. PLK4 overexpression induces rosettes of daughter centrioles around a single mother centriole. These centriole rosettes are able to form a bipolar spindle leading to a seemingly normal cell division. However, the subsequent cell division is not so organised. Multipolar spindles form, or bipolar divisions with multiple centrosomes occur leading to a disorganised cell division. Using live cell imaging, we aim to follow the fate of cells in which we have caused multipolar spindles and determine if and how they can complete mitosis. We also aim to determine what death mechanisms are involved in the cells which fail to complete mitosis.
Dissecting the Roles of Centrin Isoforms in a Vertebrate Cell Line Tiago J. Dantas1, Yifan Wang1, Catherine Liptrot2, Peter Dockery2 and Ciaran G. Morrison1* 1Centre for Chromosome Biology, School of Natural Sciences and 2Anatomy, School of Medicine, National University of Ireland Galway, Galway, Ireland Aneuploidy results from chromosome mis-segregation and has been suggested to contribute to tumour development. In higher eukaryotes, accurate segregation of the chromosomes depends on the correct assembly of the mitotic spindle. Centrins are small highly conserved proteins and are associated with the centrosomes which constitute the main microtubule organising centre. Previous studies using siRNA in human HeLa cells showed the requirement of Centrins for centrosome duplication and microtubule anchorage. However, a recent study using the human U2OS cell line showed conflicting results. Here we use the hyper-recombinogenic chicken DT40 cell line to dissect the roles of the various Centrin isoforms. All 3 chicken isoforms, encoded by Cetn1, Cetn2 and Cetn3, are expressed in DT40 cells. We have successfully targeted all three Cetn loci, generating single, double and triple mutants of the Centrin genes in DT40 cells. RT-PCR, immunoblot and immunofluorescence microscopy have confirmed the absence of the Centrins. Unexpectedly, Centrin-deficient cells undergo normal cellular division with no obvious cell cycle defects. Light microscopy analysis of the cells did not reveal any significant difference in centrosome composition or the cellular microtubule network. Additionally, no perceptible abnormalities were found when analysing the centrosome ultrastructure using electron microscopy. However, we have observed a moderate increase in sensitivity to UV irradiation and 4-nitroquinoline 1-oxide (4-NQO) treatment in the Cetn2 null cells. Taken together, our results show that Centrins are not essential for centrosome duplication in DT40 cells but suggest their involvement in the repair of UV and 4-NQO-induced DNA lesions.
Sulfated fucans, such as fucoidan, are strongly anionic polysaccharides found in marine organisms, in particular in marine brown algae and echinoderms. Their structures vary among species, but the major features are conserved within phyla. Sulfated fucans are found in marine brown algae and echinoderms. Ascophyllum nodosum is brown seaweed, which is closely related to Fucus vesiculosus. A. nodosum is available in large quantities in Irish coastal waters and provides the raw material for production of human foods, cosmetics, fertilisers, and for the extraction of industrial gums and chemicals. These marine carbohydrates are reported to exhibit an assortment of biological activities. Reported effects include anti-coagulation, anti-inflammatory and anti-viral activity. One aspect of particular interest is the ability of fucoidans to inhibit proliferation and induce apoptotic cell death in a variety of tumour cell lines. In this project, Ascophyllum nodosum has been utilised as the source of fucoidan for extraction and characterisation studies. Fucan sulfates from marine brown algae usually have complex and heterogeneous structures. Many factors can affect the composition of fucoidan including the type of seaweed, harvesting season, climate and extraction methods. Here we describe a method for extracting fucoidan using water and ethanol. Initial structural characterisation indicates that a high molecular weight fucoidan (larger than 100 kDa) has been obtained, which has many features of A. nodosum fucoidans reported to-date. Preliminary NMR data shows a large, intense peak at 1.3 ppm which is indicative of L-fucose, a major monosaccharide component of fucoidans. Subsequent to characterisation, a panel of human cancer cell lines were treated with varying concentrations of fucoidan (0- 1000 µg/ ml) and viability was assessed using an XTT-based cell proliferation assay. These assays performed to date have shown cells exhibit sensitivity to fucoidan, and differential responses from each cell line. Annexin V (an apoptotic cell marker) binding assays were performed, using the conditions above, and analysed by flow cytometry. Results obtained reveal a potent apoptotic effect in HCT-15 (colorectal adenocarcinoma) and HCT 116 (colorectal carcinoma) cell lines after fucoidan treatment for 48 hours. Following promising initial data, apoptotic mechanistic dissection will be carried out using cell lines with specific gene over-expressions or gene knockouts related to intrinsic and extrinsic apoptotic pathways. This work aims to provide a mechanism by which fucoidan induces its apoptotic effect within a cancer cell.
HUMAN REPLICATION AND REPAIR PROTEINS THAT MODULATE THE RISK OF DNA TRINUCLEOTIDE REPEAT EXPANSIONS
Aisling Frizzell* and Robert Lahue
Centre for Chromosome Biology, School of Natural Sciances, National University of Ireland, Galway. *Email: [email protected]
Background: Expanded trinucleotide (TNR) tracts are causative for at least 15 inherited neurological diseases such as Huntington’s disease. Somatic expansions in patients occur in neurons and in glial cells, but the molecular mechanism of expansions in glial cells is largely unknown. The aim of this investigation is to determine the role of key cellular proteins that modulate TNR instability in a human astrocytic (glial) cell line, SVG-A. We hypothesise that candidate DNA replication and repair proteins affect expansion risk, because new DNA must be synthesised to expand a TNR. If so, siRNA knockdown of those proteins should alter expansion frequencies. Here we report results with two such proteins; FEN1, a flap endonuclease involved in replication and base excision repair, and SPAR1, a DNA helicase that helps control homologous recombination. Experimental approach: Cultured human astrocyte cells, SVG-A, were treated with siRNA against either FEN1 or SPAR1, with scrambled siRNA as a control. Knockdown efficiencies were determined either by western blot (for FEN1) or by quantitative RT-PCR (for SPAR1). Treated cells were also tested for their ability to expand CTG repeats introduced on a plasmid by transient transfection. Expansion frequencies were quantified by novel genetic assays, developed in the Lahue lab and further refined in this work. Expansions were verified by PCR. Results: 80% knockdown of FEN1 in SVG-A cells was achieved. Loss of FEN1 appears to have no effect on CTG expansions in SVG-A cells, as the expansion frequency was not significantly different for that of scrambled siRNA control cells. Knockdown of SPAR1 resulted in 75% loss of SPAR1 mRNA as determined by real-time RT-PCR. SPAR1 knockdown cells have an expansion frequency approximately 15-fold higher than that of scrambled siRNA control cells, suggesting that SPAR1 is important for reducing expansion risk in glial cells. However, SPAR1 knockdown has only been performed once, so the expansion data are preliminary. Confirmatory experiments are ongoing. Conclusions: The role of FEN1 in expansions in mammalian cells is controversial. No expansion of a (CTG)n·(CAG)n repeat was observed in a DM1 knock-in mouse model. Furthermore, the huntingtin triplet-repeat locus was stable under long-term FEN1 knockdown in a HeLa cell derivate. However, an increase in expansions in mice with Fen1 haploinsufficiency was observed. The data presented in this study is agreement with the suggestion that FEN1 does not play a role in expansions. The initial SPAR1 data outlined in this study suggests that SPAR1 plays a similar role to its yeast homolog Srs2 in preventing expansions. Srs2 has been shown to prevent expansions in yeast by unwinding aberrant secondary DNA structures that are intermediates in the TNR expansion process. Further investigation is needed to determine if this is also the case for SPAR1.
Effect of proteasome inhibition on the DNA damage response in cisplatin-
treated human cells lacking DNA polƞ Kathleen Gallagher, Severine Cruet-Hennequart, Michael P. Carty
DNA Damage Response laboratory, Biochemistry, School of Natural Sciences, National University of Ireland, Galway.
.
The chemotherapeutic platinum-based drug cisplatin acts through the formation of
platinum-DNA adducts, intrastrand crosslinks and interstrand crosslinks, blocking DNA
replication. Human cells express a number of specialised polymerases, including DNA
polymerase ƞ (polƞ), capable of carrying out DNA replication in the presence of damage.
DNA replication arrest activates the DNA damage response, involving post-translational
modifications of downstream effector proteins. Among the major types of post-
translational modifications are protein phosphorylation and protein ubiquitination.
Phosphorylation, in particular by the PI-3 kinase-related protein kinases (PIK kinases)
ATM, ATR and DNA-PK plays an important role in the cellular response to DNA
damage. Protein ubiquitination includes monoubiquitination, which usually modifies
protein function but does not lead to degradation, and polyubiquitination, which targets
proteins for proteasome-mediated degradation. To provide insight into the interplay
between phosphorylation and ubiquitination in the response of human cells to cisplatin,
the effect of proteasome inhibition on cisplatin-induced protein phosphorylation and
ubiquitination was investigated in pol ƞ-deficient XP30RO cells. XP30RO cells were
treated with 0-5µg/ml cisplatin, in the presence or absence of the proteasome inhibitor
MG-132. The levels and post-translational modifications of three key DNA damage
response proteins, RPA2 and FANCD2 were investigated using western blotting and
phosphospecific antibodies. This analysis demonstrates that (i) the proteasome inhibitor
MG132 alters the pattern of cisplatin-induced RPA2 phosphorylation at a number of sites
in the N-terminal; (ii) compared to normal cells, cisplatin-induced FANCD2
monoubiquitination is increased in cells lacking DNA polƞ. This data demonstrates that
cisplatin-induced modification of individual DNA damage response proteins by protein
phosphorylation and ubiquitination can be modulated by proteasome inhibition.
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THE ROLE OF NBS1/XRS2 BRCT REPEATS IN THE DNA DAMAGE RESPONSE
Agnieszka Rupnik, Danielle Hamilton, Muriel Grenon and Noel Lowndes Genome Stability Laboratory, Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Ireland [email protected] Abstract In response to genotoxic stress, the cell has developed stringent mechanisms termed cell cycle checkpoints to facilitate the repair and prevent the propagation of DNA damage to subsequent generations. An essential component of this pathway is the Mre11/Rad50/Nbs1 (Xrs2 in budding yeast) or MRN (MRX) complex. This complex is responsible for the recognition of DNA double strand breaks and the propagation of damage signals to the checkpoint machinery. Hypomorphic mutations of Nbs1 are associated with the cancer prone condition, Nijmegen Breakage Syndrome, however it still remains the most poorly characterised component of the complex. The recent discovery of a second BRCT motif in Nbs1, as well as equivalent tandem BRCT motifs in its budding yeast functional homologue, Xrs2, prompted us to investigate the possible roles of these phospho-peptide binding motifs in the DNA damage response. In order to assess the functions of the BRCT tandem repeats we generated novel Nbs1 and Xrs2 BRCT mutants in both DT40 and budding yeast cells. We assayed the requirement for these motifs in checkpoint activation following DNA damage in addition to their remaining putative roles. To examine more closely the structure of the MRN complex, we also performed preliminary analysis of MRN complex hydrodynamics using gel filtration chromatography. In particular we looked at changes in the human MRN complex conformation and/or size following treatment with Ionizing Radiation or Ultra Violet light in cells that lack functional Nbs1. The results presented here suggest that the BRCT tandem motifs of Nbs1 may have a previously unidentified role in the DNA damage response and shed new light on the dynamics of the interactions between the components of the MRN complex.
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MULTIPLE POTENTIAL Cdk PHOSPHORYLATION SITES OF S. cerevisiae Rad9 ARE REQUIRED FOR Chk1 ACTIVATION. Ramesh Kumar, Carla Abreu, Karen Finn, Aisling O’Shaugnessy, Noel F Lowndes and Muriel Grenon. Genome stability laboratory, Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland. DNA damage checkpoints are triggered in response to DNA insults. Once activated, these pathways prevent replication and segregation of the damaged DNA. In addition to cell cycle delays, they regulate repair, transcription and the apoptotic response to DNA damage. Budding yeast RAD9 was the first checkpoint gene identified. The checkpoint activity of this protein has been reported in all phases of the cell cycle. Loss of this gene impairs checkpoint-activated cell cycle arrests and increases genomic instability. Rad9 is phosphorylated during a normal cell cycle and hyperphosphorylated in response to DNA damage. Genetic analyses indicate that cell cycle phosphorylation of Rad9 is Cdc28/Clb dependent. The protein contains 20 potential Cyclin dependent kinase (Cdk) phosphorylation sites (S/T-P). This work will present the generation and analysis of novel rad9 mutants designed to understand the role of Rad9 cell cycle phosphorylation in its functions. In particular the role in regulating Chk1, a downstream checkpoint effector, will be investigated. [email protected]
63
IMMUNE RESPONSE TO THE GASTRIC PATHOGEN HELICOBACTER PYLORI: MICROARRAY ANALYSIS OF
GASTRIC CD2+ CELLS
Yvonne O’Donoghue1, 2, Kaveh Mashayekhi2, Ali Moshfegh3, S. Akbar Zulquernain4, Joan O’Keeffe5 and Anthony P. Moran2*
1Biochemistry and 2Microbiology, School of Natural Sciences, National University of
Ireland, Galway 3Department of Oncology and Pathology, Karolinska Institutet, Cancer Center
Karolinska, University Hospital, Stockholm, Sweden 4Department of Gastroenterology, University Hospital, Galway
5Department of Life and Physical Sciences, School of Science, Galway-Mayo Institute of Technology
Helicobacter pylori is a prevalent, human gastric pathogen which causes peptic ulcers
and is a co-factor in gastric cancer development. This bacterium induces an immune
response involving classical T cells but potentially also unconventional T cells and
Natural Killer (NK) cells (collectively CD2+ cells). To further understand the host-
pathogen interaction in the gastric mucosa following H. pylori infection, transcriptomic
analysis using cDNA microarrays was performed to determine gene expression of CD2+
cells in infected versus uninfected mucosae. Single-cell suspensions from the epithelial
(EP) and lamina propria (LP) layers of gastric biopsies from H. pylori-positive (HP+) and
-negative (HP-) individuals were prepared, CD2+ cells were then positively selected, and
RNA isolated from each population resulting in four groups (EPHP-, EPHP+, LPHP- and
LPHP+) for microarray experiments. After bioinformatics analysis, 54,675 responsive
genes were identified; 12,311 of which were significantly up-regulated in EPHP+
(compared with EPHP-) whereas 3,536 genes were up-regulated in LPHP+ (compared
with LPHP-); 4,863 genes were significantly down-regulated in EPHP+ and 1,079 genes
in LPHP+. Significant changes in gene expression of markers of cytotoxicity, cell
proliferation, cytokines, cellular receptors, CD1 and signal transduction were observed.
In summary, significant responsiveness of these immune cells to H. pylori infection was
noted. Although not extensively studied previously, based on the present results, gastric
CD2+ cells require closer examination in the mucosal immune response to H. pylori.
64
Cisplatin and -irradiation-induced DNA damage responses in human mesenchymal stem cells.
Á.M. Prendergast1*, S. Cruet-Hennequart1, F. Barry2 and M.P. Carty1
1DNA Damage Response laboratory, Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland, Galway and 2Regenerative Medicine Institute (REMEDI), National
Human mesenchymal stem cells (hMSCs) are a key component of the stromal compartment of bone marrow, and differentiate into other cell lineages including osteoblasts, chondrocytes and adipocytes. Since hMSCs play a role in maintaining proliferation of haematopoietic stem cells, hMSCs may be important in leukaemia development. hMSCs are also recruited to the stroma of solid tumours, including breast tumours. Cancer therapies such as cisplatin and radiotherapy act by damaging DNA and compromise bone marrow function. Genome damage to the hMSC progenitor population as a result of cancer treatment could contribute to the development of secondary cancers in, for example, the bone. However, relatively little is known about the response of hMSCs to DNA damage, and an improved understanding of the effects of DNA damaging anti-cancer agents on hMSCs could provide new insights into cancer treatment. In the present study we have investigated the effects of two DNA damaging agents, cisplatin and -irradiation, on cell cycle progression and activation of DNA damage response pathways in hMSCs. The effect of cisplatin and -irradiation on cell cycle progression in hMSCs was determined by measuring bromodeoxyuridine incorporation by immunofluorescence and by flow cytometry. In untreated hMSCs in culture, the percentage of cells undergoing DNA replication decreases from 11% to 2% in normal growth media after 48 hours. Treatment with 10g/ml cisplatin induces S-phase delay and arrest in G2 and M phases. In contrast, exposure to -irradiation (10 Gy) induces prolonged G1-phase arrest for up to 48 hours. Consistant with the induction of cell cycle arrest, both cisplatin and -irradiation increase the level of the tumour suppressor protein p53, and of p21, a p53-inducible inhibitor of cell cycle progression. To provide evidence for induction of DNA damage in hMSCs by cisplatin and -irradiation, we examined phosphorylation of histone H2AX on serine 139, which generates characteristic -H2AX nuclear foci that can be detected by immunoflourescence. Both cisplatin and -irradiation induce -H2AX foci, but the kinetics of focus formation is different for the two agents. Following -irradiation, the peak of -H2AX focus formation is observed between 2 and 6 hours, and between 12 and 24 hours following cisplatin treatment. This is consistent with direct induction of DNA strand breaks by ionising radiation, while cisplatin-induced breaks result from processing of DNA damage. To further investigate the role of specific signalling pathways in the response of hMSCs to DNA damage, we have used small molecule inhibitors to target the PI-3 kinase-related protein kinases (PIKKs), DNA-PK and ATM. Using this approach, in combination with western blotting using phosphospecific antibodies against known PIKK substrates, we show, for example, that the 34kDa subunit of the single-stranded DNA-binding protein replication protein A (RPA) is phosphorylated in a DNA-PK-dependent manner following cisplatin treatment. This provides evidence that both cisplatin and -irradation induce DNA damage in hMSCs, leading to activation of key DNA damage response pathways, including PIKK signalling. Thus, cancer treatments may affect genome stability in the human mesenchymal stem cell pool.
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CHARACTERISATION OF THE ROLE OF TRANSLESION SYNTHESIS BY DNA POLYMERASE η AND DNA DAMAGE
SIGNALLING IN THE CELLULAR RESPONSE TO CISPLATIN AND CARBOPLATIN
A.M. Sokol1, J. Bianco2, S. Cruet-Hennequart1, P. Pasero2 and M.P. Carty1
1DNA Damage Response Laboratory, Centre for Chromosome Biology, School of Natural
Sciences, National University of Ireland, Galway, Ireland 2Department of Genome Dynamics, Institute of Human Genetics, CNRS UPR 1142,
Treatment with platinum-based chemotherapeutics, successfully used in cancer therapy, leads to the formation of lesions in the DNA which block cell cycle progression and ultimately result in cell death. Human cells lacking the translesion synthesis protein, DNA polymerase η (polη) are more sensitive to killing by cisplatin and carboplatin, emphasizing the role of polymerase η in the response to these drugs. In this study, the response to cisplatin and carboplatin was analysed in XP30RO cells lacking polη and in a derivative cell line, TR30-2 that expresses polη from a POLH transgene. To directly determine whether polη is required for DNA synthesis following DNA damage by cisplatin and carboplatin, the length of individual nascent DNA fibers in cells lacking polη or expressing polη was analysed using the technique of DNA combing (Fig.1). This demonstrated that following drug treatment, nascent DNA strands are shorter in cells lacking polη than in cells expressing polη, consistent with a role for polη in bypass of platinum-induced lesions during DNA replication (Fig.1). To investigate the effect of replication arrest in cells lacking polη on downstream signalling pathways, we focused on replication protein A (RPA), which is the main single-stranded DNA binding protein in mammalian cells, and is involved in most aspects of normal DNA metabolism including replication, recombination and repair. The 32kDa subunit of RPA (RPA2) is phosphorylated on a number of N-terminal sites by cdks in a cell cycle-dependent manner, and by PIK kinases in response to DNA damage. Cisplatin- and carboplatin-induced hyperphosphorylation of RPA2 on Ser4/Ser8 was studied in detail as well as the relationship between RPA2 Ser4/Ser8 hyperphosphorylation and Rad51, a key player in the repair of DNA strand breaks by homologous recombination. It was observed that RPA2 hyperphosphorylation is significantly increased following treatment of XP30RO cells with an equitoxic dose of cisplatin or carboplatin, but this event occurred later in cells treated with carboplatin. Analysis of hyperphosphorylated RPA2 and Rad51 by immunofluorescence showed that in response to platinum-based chemotherapeutics, individual XP30RO cells stain positively either for phospho-Ser4/Ser8 RPA2 or for Rad51 foci, but rarely for both events. This data provides insight into the specific requirements for DNA replication and DNA damage signalling in human cells following
66
TR30-2 XP30RO Individual DNA fibers obtained using
the technique of DNA combing
Cisplatin 0.5µg/ml
Mock-treated
Carboplatin 50µM
treatment with platinum-based chemotherapeutics. Fig. 1 Representative, individual DNA fibres visualized by the technique of DNA combing. XP30RO and TR30-2 cells were mock-treated or treated with indicated doses of cisplatin and carboplatin for 24h.
67
The Need for Improved Sustainable Management to Protect Biodiversity (Ascophyllum nodosum)
WP4: Natural Resource Exploitation and Global Change
Christine Eschmann, Sorcha Ní Longphuirt & Dagmar Stengel Botany, School of Natural Sciences, National University of Ireland, Galway
[email protected] Climate Change is driven by natural forcing factors and human influences that cause global warming. The possible effects on vegetation and habitats are discussed widely. Intertidal plants are exposed to highly variable, sometimes very rapidly changing, and potentially stressfully environmental conditions like desiccation, solar irradiance, temperatures, osmotic stress and the availability of nutrients. The brown seaweed Ascophyllum nodosum (L.) Le Jolis (Phaeophyceae) is one of the dominant mid-intertidal species on Ireland’s sheltered rocky shores. Dense stands of A. nodosum provide substrate and habitat for other algae and animals. It is ecologically important as primary producer and has been used as a model organism to evaluate environmental impacts, to investigate experimentally and to assess responses to climate change. Global warming and therefore increasing seawater temperatures will affect the vegetation of A. nodosum –beds in composition, distribution and coverage as its growth rate is sensitive to water temperatures and positively correlates with mean annual seawater temperatures. Underwater, but also in the intertidal zone, UV-radiation is often regarded as an important factor for zonation of the algal vegetation. There are also human originated threats to A. nodosum like eutrophication and harvesting. As a result of A. nodosum harvesting, the environment of the harvested intertidal area is changed dramatically - some effects are the decrease of biomass, the increase of solar irradiation (higher PAR and UV), more extreme temperatures and more severe desiccation. This project focuses on the effects of global change and resource exploitation on the important intertidal keystone primary producer A. nodosum. The main objectives are to develop monitoring protocols that allow a fast and reliable assessment of biodiversity change as a consequence of global change in the future, and to protect this already vulnerable habitat. This should lead to predictions of species or community responses to climate change and to ensure future sustainable exploitation. The overall aim is to evaluate the potential for A. nodosum to fulfil a role as biological indicator for global change and to consider its possible role as a carbon sink.
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THE IMPACT OF HEMI-PARASITE YELLOW RATTLE (RHINANTHUS MINOR) ON THE FLORAL DIVERSITY OF EAST
CONNEMARA GRASSLAND AND SELECTED HOST GRASS SPECIES
Anna Pielach, Zoë Popper Botany and Plant Science, School of Natural Sciences, National University of Ireland,
BACKGROUND As a result of intensive grassland regimes, vigorous species of grasses dominate in areas which once used to be occupied by hay meadows with a high diversity of flowering plants. Yellow rattle (Rhinanthus minor) is a conspicuous annual dicotyledonous herb of semi-natural grassland including traditionally managed hay meadows and nutrient-poor open environments of gravely esker ridges. Yellow rattle is a hemi-parasite known to infect and weaken other plant species, particularly grasses and legumes. Rhinanhus minor infiltrates the root system of the host plant using a highly specialised structure called a haustorium, enabling it to absorb nutrients from the host plant. When sown into intensively managed grassland, yellow rattle can reduce the vigour of grasses and facilitate colonisation by less competitive flowering plants. Yellow rattle has therefore been practically applied in restoration of meadow biodiversity. FIELD WORK
The West Corrib limestone landscape consists of small fields, poor pastures and meadows contrasted with areas of bog and the Killanin Esker which stretches from Roscahill to Tullykyne. Fieldwork is carried out in the grounds of Brigit’s Garden, located in the heart of the area (53°23’09 N, 9°12’42 W), and along the esker. Yellow rattle grows locally and is found on the esker and a nearby cutover bog (53°22’54 N, 9°11’57 W). Brigit’s garden is managed for biodiversity and yellow rattle seeds collected from the esker have been sown into the garden meadows. The field work undertaken so far has revealed a high diversity of grassland habitats within Brigit’s Garden (lowland hay meadow, esker meadow, wet rushy meadow, grazed grassland and regularly mown amenity grassland). These differ in their topography, management, plant communities and abundance of yellow rattle. FUTURE WORK
Within each meadow in Brigit’s Garden, permanent quadrats will be arranged allowing vegetation to be sampled and compared between locations with different abundances of the yellow rattle. The mechanisms used by the parasite to facilitate the infection process by modifying host cell wall will be investigated in lab-grown and field-collected plants. Histological and immunocytochemical techniques that specifically label plant cell wall components will enable visualization of modifications to cell structure and cell wall biochemical composition facilitating an improved understanding of the infection process.
Arsenic occurs naturally as both organic and inorganic species in marine waters (c.2 µg L-1)[1] and sediments (c.40 mg kg-1)[2] and is incorporated into the food web by a range of marine biota. Seaweeds have a strong tendency to accumulate arsenic, among other metals, and as a result the concentrations of total arsenic in seaweeds may be over one hundred times greater than maximum permissible concentrations applied to other foods under Irish legislation. The majority of marine biota consumed by humans have complex uptake and metabolic systems which concentrate, but also detoxify, arsenic to the extent that the predominant form (arsenobetaine in fish) is considered to be virtually non-toxic. However, this natural safeguard mechanism operates to a lesser extent in primary producers, and as a result the speciation of arsenic in seaweeds differs greatly to that in animal tissues. Arsenosugars account for a large proportion, but inorganic arsenic may predominate in some seaweeds. In this project, a number of red, green and brown algae were selected with a view to identifying those species with elevated proportions of inorganic arsenic, as well as estimating the potentially toxic load from the species most commonly consumed in Ireland. Seasonal and intra-specific variation in the levels of the brown alga Ascophyllum nodosum which is commercially harvested and currently used in animal feeds were also investigated. Total and inorganic arsenic were assessed using fully validated methodologies (HPLC-HG-AFS). A novel data set for arsenic levels in a range of economically valuable Irish seaweed species is presented. 1. Smedley, P.L. and D.G. Kinniburgh, United Nations Synthesis Report on Arsenic
in Drinking Water: Chapter 1: Source and Behaviour of Arsenic in Natural Waters, in Water and Sanitation, W.H.O., Editor, WHO.
2. Francesconi, K.A. and J.S. Edmonds, Arsenic in the sea. Oceanographic marine biology annual review, 1993. 31: p. 111-151.
Investigating the cause of rapid temperature shocks at Tisler Cold-Water Coral Reef; coupling large scale climate with benthic ecological change
Damien Guihen & Martin White
Dept. Earth and Ocean Sciences, NUI, Galway, Ireland [email protected]
Continuous long-term measurements of bottom temperature, salinity and current velocity profiles, have been recorded at a regular interval for almost 4 years at the Tisler Reef. The Tisler Reef is a 2 km long cold-water coral reef on the sill of the Koster Fjord, in the northeastern Skagerrak, Norway. The reef is comprised principally of the sceractinian coral Lophelia pertusa, at depths between 70 and 160m, and is an important habitat for sponge, crustacean and fish species including a number of species of commercial importance. Measurements were made using a bottom mounted RDI Workhorse 300kHz ADCP with an attached Seabird Instruments MicroCAT SBE 37 TS logger. The ADCP was deployed between 110 and 140 meters depth and sampled at thirty-minute intervals. Analysis of the current velocity data has identified a number of features, such as topographic steering and seasonal reversals. These reversals in flow direction are often associated with significant changes in bottom temperature. During the autumn months of 2006 and 2008, a series of large and rapid increases in bottom temperature were observed, followed by a steady decline lasting several months. The occurrence of the 2006 and 2008 events correspond with the observation of mass mortality in the long-lived sponge Geodia baretti. Historical temperature records from the region suggest that these temperature shocks are distinct. On two occasions, 2006 and 2008, a pulse of warm, low saline water from the Baltic Sea, entered the Skagerrak via the Kattegat following the collapse of the Skagerrak Front. The warm water was advected North in the cyclonic surface circulation of the Skagerrak and was mixed downwards subsequent to the deepening of the thermocline due to wind stress. Wind driven flow reversal drove the warm water back over the reef, rapidly raising temperature at the seabed by 4C. Unusually warm sea surface temperatures in the southern Skagerrak have been observed by satellite in the late summer of both 2006 and 2008 indicating that global climate trends may have a swift and negative impact on the cold-water coral reef ecosystems of the Skagerrak. The influence of large-scale regional atmospheric conditions on the reef will also be considered.
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GREISEN STEW: SALTS, BRINE, VAPOUR AND VOLATILES IN A MINERALIZED GRANITE ROOF ZONE, NEWFOUNDLAND
Edward P. Lynch1, Martin Feely1 and Derek H. C. Wilton2 1Department of Earth and Ocean Sciences, National University of Ireland, Galway
2Department of Earth Sciences, Memorial University, St. John’s, NL, A1B 3X5, Canada [email protected]
Mineral deposits associated with granite intrusions are known to form in high-level apical zones where a volatile-rich residual melt may evolve to concentrate and subsequently deposit metal-bearing minerals. Mineralization may be accompanied by metasomatic alteration of the host granite to form a silicified carapace termed a greisen. These late-stage plutonic events are facilitated by the exsolution and circulation of magmatic-hydrothermal fluids, leaving the granite to somewhat ‘stew in its own juices’. This study is investigating the nature of the mineralizing fluids responsible for base metal deposition and associated greisen alteration within the roof zone of a granite batholith. The Devonian Ackley Granite in southeastern Newfoundland is a large (c. 2500 km2) composite batholith comprised of seven geologically distinct subunits. The granite exhibits a north-south tilt, with the southernmost units representing the roof of the intrusion. An irregular zone of quartz-topaz greisen and associated metal sulphide-oxide mineralization is developed along the granite’s southern margin. Evidence of the hydrothermal regime that facilitated Ackley roof zone alteration and mineralization is preserved in microscopic fluid inclusions (FIs) within greisen and vein quartz (Fig. 1). The characterisation (petrography) and semi-quantitative analysis (microthermometry, Raman spectroscopy) of inclusion fluids provide an estimate of the physico-chemical conditions that prevailed during hydrothermal activity. Petrographical examination of greisen quartz has identified an assemblage of four fluid types responsible for mineralization and alteration. Type 1 FIs enclose a complex, multiphase (liquid, vapour, solid), hypersaline fluid (> 26.3 eq wt% NaCl) of the general composition NaCl-KCl-CaCl2-H2O±CO2. A variety of precipitated salts and trapped minerals are found. Combined microthermometric and Raman spectroscopic analysis of the solids has identified halite, sylvite, hematite, topaz, pyrite and gypsum crystals. Type 2 and Type 4 FIs are vapour-dominated fluids containing a mixture of H2O-N2-CH4- and CO2-H2O±CH4 species, respectively. These represent the higher temperature fluids (450°C to 300°C) operating in the system. Type 3 FIs contain a liquid-rich aqueous brine of the general composition H2O-NaCl-KCl. The saline fluids (Type 1 and 3) indicate a period of lower temperature hydrothermal activity operating between 350°C to 190°C.
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THE STUDY OF CAMPYLOBACTER BIOCIDE RESISTANCE USING A CHEMOSTAT MODEL
Alicja Barczynska, Cyril V. Carroll
Department of Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
[email protected] Campylobacter spp. is Gram-negative bacilli with a single polar flagellum at one or both ends of its spiral cells. Campylobacter have been recognized as a major cause of human food-born enteritis, which can lead secondary complications such as meningitis, appendicitis, spontaneous abortion, urinary tract infections and Guillain-Barré Syndrome (GBS). Cases of campylobacteriosis are often associated with handing raw poultry or eating raw or undercooked poultry meat. The consumption of unpasteurized milk or contaminated water, pork, beef, lamb and seafood can also cause illnesses connected with that bacteria. Unlike other foodborne pathogens, Campylobacter spp. are apparently fragile organisms that are unable to multiply outside the animal host and are highly susceptible to a number of environmental conditions as they lack most of the stress response factors common in other enteric bacteria. Presently, little is known about the genetic or physiological basis of disinfectant tolerance in Campylobacters. This work used an in vitro chemostat model to study resistance development in Campylobacter to the food industry biocide (Citric acid) and development of cross resistance to other biocides (lactic acid, acidified sodium chlorite, trisodium phosphate) and to antibiotics (Tetracycline, Nalidixic acid and Ciprofloxacin). Campylobacter jejuni 2124GF – a natural food isolate (Citric acid MIC - 0.125%) was grown in chemostat culture (dilution rate:0.025h-1) over 30 generations and exposed to increasing increments of Citric acid. With every doubling of Citric acid concentration in the chemostat (every 3 days), culture samples were taken to evaluate resistance development and for gene expression analysis using RT-PCR. The Citric acid adopted strain (MIC 0.25%) showed no increased resistance to the other biocides or antibiotics tested. However, virulence genes expression increased to 400%, for CmeA and to 500% for FlgS and CdtB when the strain was adapted to Citric acid compared to non-adapted strains.
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IVD REGENERATION USING TARGETED GENE DELIVERY
Claire M. Cunningham1,2,3, Sibylle Grad4, Mauro Alini4, Abhay Pandit2,3 and J.
Gerard Wall1,2,3
1Microbiology, 2Network of Excellence for Functional Biomaterials, 3National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
Degeneration of the intervertebral disc (IVD) has been identified as a leading causative factor of low back pain. While current therapies temporarily alleviate the associated pain, they do not restore disc function.
The aim of this project is to deliver a biodegradable nanocapsule, containing an encapsulated therapeutic gene and a surface-tethered, cell-specific scFv antibody fragment for targeting, to nucleus pulposus cells in the IVD region in order to stimulate regeneration.
Microarray and RT-PCR analysis of patient samples led to identification of the multi-domain NCAM protein as a suitable cell target for NP cell delivery. Overlap PCR was used to clone the terminal NCAM domain with N-terminal ompA and C-terminal hexahistidine tags for periplasmic expression and purification, respectively. Following optimisation of expression in E. coli, periplasmic NCAM was extracted, purified using immobilised metal affinity chromatography and used to pan a naïve human scFv phage display library. After 4 panning rounds, polyclonal phage ELISA and restriction digest analysis identified significant clonal enrichment, leading to monoclonal phage screening and isolation of E. coli clones producing NCAM-binding scFv fragments. Further clone screening and characterisation of a number of these scFvs is underway to identify binders with the highest affinity.
High affinity anti-NCAM scFvs will be conjugated to available nanocapsules for initial cell targeting and gene delivery studies in vitro. It is anticipated that this work will lead to enhanced gene delivery to NP cells for targeted IVD regeneration.
75
A PATHWAY FOR THE REMOVAL OF INTRACELLULAR γ-AMINO BUTYRIC ACID (GABA) FROM LISTERIA MONOCYTOGENES
Feehily C., Karatzas K.A.G., O'Byrne C. Bacterial Stress Response Group, Dept. of Microbiology, School of Natural Sciences,
Listeria monocytogenes is a human pathogen that is of major concern to the food industry. As the initial site of infection for L. monocytogenes is the intestinal lumen, it must traverse a range of environmental stresses including osmotic stress, temperature fluctuations and most importantly the low pH environment of the stomach. Previous studies have identified the GAD system as a major mechanism of acid tolerance in this bacterium. This system comprises a glutamate/GABA antiporter system and a glutamate decarboxylase which prevent the drop of the intracellular pH to lethal levels. Here we demonstrate that under acidic conditions L. monocytogenes accumulates intracellular GABA. The production of intracellular GABA is independent from the antiporter-based system and probably derives from the decarboxylation of intracellular pools of glutamate. We show that intracellular GABA is removed following the reintroduction of the cells in neutral pH. We also investigate the metabolism of GABA using an enzyme-based assay and the results suggest that GABA may not only be exported via the transporter system but may also be converted to succinate semialdehyde and eventually to succinate. This pathway, which involves the activity of a putative succinate semialdehyde dehydrogenase (Lmo0913), may substitute for the bacterium's lack of a complete tricarboxylic acid cycle.
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THE EFFECT OF Vibrio parahaemolyticus ON HUMAN MAPK SIGNALLLING CASCADES AND HOST CELL RESPONSE.
Rebecca Finn1, Ksenia Matlawska-Wasowska1, Eleanor Coffey2, Alan Baird3, Aoife Boyd1
1Pathogenic Mechanisms Laboratory, Discipline of Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
2Turku Centre for Biotechnology, Åbo Akademi and Turku University, Turku, Finland. 3School of Agriculture, Food Science & Veterinary Medicine, Veterinary Science Centre, University
Infection with Vibrio parahaemolyticus following consumption of contaminated seafood poses a huge health risk. Infection is characterised by widespread destruction of the intestinal mucosa with epithelial cell damage and inflammation. This study will investigate the mechanisms by which V. parahaemolyticus causes activation of human MAPK signalling pathways and how this activation promotes and stimulates host responses. V. parahaemolyticus contains two Type Three Secretion Systems, TTSS1 and TTSS2. These TTSS enable the bacteria to secrete various effector proteins into the cytosol of the host epithelial cells and in this way cause infection. Three of the main effectors shown to interfere with host cell signalling pathways are VP1686, VopT and VopA (VopP). The effect of V. parahaemolyticus TTSS effector proteins on the MAPK signalling cascades was assessed by analysing Caco-2 cells following co-incubation with the bacteria. Immunoblotting was carried out to determine which MAPK proteins are activated in response to various V. parahaemolyticus mutants and wildtype bacteria. We observed that all three MAPKs (p38, p42/44 and JNK) are affected following co-incubation and the TTSS play an important role in this process. TTSS1 mutants (ΔVscN1) did not lead to MAPK activation following co-incubation with Caco-2 cells. This suggests that TTSS1 is vital for V. parahaemolyticus induction of MAPK activation. Immunoblotting was carried out on samples obtained from Caco-2 cells treated with various MAPK inhibitors. The inhibitors target either the MAPK directly or proteins located upstream in the signalling cascade. In the future these inhibitors will be employed to determine the ability of V. parahaemolyticus to influence epithelial function.
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ASSESSING THE IMPACT OF WASTE WATER TREATMENT PLANT EFFLUENT ON NOROVIRUS CONTAMINATION IN
SHELLFISHERIES John Flannery*, Paulina Rajko-Nenow, Sinéad Keaveney, Vincent O’Flaherty, Bill
Doré.
Marine Institute, Rinville, Oranmore, Co Galway Ireland Tel. 353(0)91 387240; Fax:
The deep marine environment is largely unexplored and vastly under-sampled partly due to the extreme nature of the environment and the significant challenges associated with collecting samples. Understanding the abundance and diversity of marine prokaryotic communities represents the first step in elucidating the enormous contribution they make towards the biogeochemical cycles of planet Earth. Bacterial abundance and distribution was investigated by epiflourescence microscopy at three study areas; Galicia Bank (NE Atlantic), Western Mediterranean and Eastern Mediterranean. In addition, bacterial community structure in the water column (300 m to 5100 m) was studied by means of Denaturing Gradient Gel Electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes at a number of sites in the Central Mediterranean Basin. Bacterial abundance was found to decrease with depth at all sites. No significant differences in distribution with depth were found between the three study areas despite hydrological differences. Cluster analysis revealed that bacterial communities were more closely related by depth than by geographic location. This suggests that abiotic factors related to depth may play a more important role in structuring bacterial communities than other factors.
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DETERMINATION OF THE MOLECULAR MECHANISMS OF CELLULAR ADHERENCE AND INVASION UTILISED BY THE
GASTROINTESTINAL PATHOGEN Vibrio parahaemolyticus
Nicky O’ Boyle and Aoife Boyd*. Pathogenic Mechanisms Laboratory, Discipline of Microbiology, School of Natural
Sciences, National University of Ireland, Galway, Ireland. [email protected]
V. parahaemolyticus is a food borne pathogen which is capable of altering host cell
signalling pathways by introducing effector proteins via two Type Three Secretion
Systems. This results in remodelling of the actin cytoskeleton, disruption of
gastrointestinal epithelial architecture and host cell cytotoxicity. Intimate association
between the bacterium and the host cell is required in order for this process to occur. We
have shown using the gentamicin protection assay that V. parahaemolyticus is capable of
invading epithelial cells. The association between V. parahaemolyticus and epithelial
cells was examined by visualising green fluorescent protein and red fluorescent protein
tagged bacteria co-incubated with stained Caco-2 cells using fluorescent microscopy.
This study also illustrates a means of identification of the adhesins and invasins utilised
by the bacterium during pathogenesis. A random genomic library was constructed using
the pWEB-TNC™ cosmid cloning kit. V. parahaemolyticus DNA fragments of
approximately 40 kb were introduced into the non-invasive E. coli HB101. Library clones
were subsequently screened for the ability to invade Caco-2 epithelial cells by incubation
with Caco-2 cells followed by the gentamicin protection assay. Insert DNA from invasive
clones will be sequenced, revealing V. parahaemolyticus genes which play a role in the
adhesion and invasion process.
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CHARACTERISATION OF NOROVIRUS CONTAMINATION IN A SHELLFISHERY USING REAL-TIME QRT-PCR AND SEQUENCE
ANALYSIS
Paulina Rajko-Nenow*, John Flannery, Sinéad Keaveney, Vincent O’Flaherty, Bill
Norovirus (NoV) is commonly known as the “winter vomiting bug” and is the single most important agent of food-borne viral gastroenteritis worldwide. Bivalve shellfish grown in areas contaminated with human waste may become contaminated with human pathogens including NoVs. Shellfish, particularly oysters, may subsequently be eaten raw and can cause illness. A study was undertaken to investigate NoV contamination in a shellfishery over a 24 month period from September 2007 until September 2009. Oysters (Crassostrea gigas) were collected monthly. Real-time quantitative reverse transcription PCR (RT-PCR) was used to determine the levels of human NoV genogroups (GI and GII). NoV GII was detected in 21 samples; 15 of these samples also contained NoV GI. The highest levels of NoV were detected during the winter months. To examine the genetic characteristics of NoV GI and GII, nested RT-PCR analysis was undertaken to amplify a partial region of the RNA polymerase gene (~113bp). The resulting sequence data was analysed using the genotyping tool available on the Food-borne Viruses in Europe network website. Whereas most community outbreaks are associated with NoV GII.4 genotype, multiple NoV genotypes were identified in many of the shellfish samples during this study. The dominant genotype was NoV GI was I.4 which was detected in 13 samples. The data highlights the diversity of NoV genotypes found in contaminated shellfish and highlights the potential role shellfish play as a vector for infections in humans involving multiple NoV genotypes.
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COMPARATIVE GLYCAN ARRAY ANALYSIS OF GUILLAIN-BARRÉ SYNDROME- AND ENTERITIS-RELATED
CAMPYLOBACTER JEJUNI ISOLATES
Marcus E. Twomey and Anthony P. Moran*
Microbiology, School of Natural Sciences, National University of Ireland, Galway *Senior author: [email protected]
Glycan array technology has emerged as a valuable tool in the investigation of protein-
carbohydrate interactions, such as those involved in microbial adherence to host cells.
Campylobacter jejuni, the primary cause of bacterial gastroenteritis globally, is also the
predominant antecedent infection in development of the paralytic disease, Guillain-Barré
syndrome (GBS). Molecular mimicry of peripheral nerve gangliosides by the
lipooligosaccharide of C. jejuni can induce cross-reactive antibodies leading to GBS
development. Nevertheless, since ganglioside mimicry is not only limited to GBS-
associated isolates, additional mechanisms including adherence and cellular invasion may
contribute to disease severity. We carried out glycan array analysis of 6 clonally-related,
genetically and serologically indistinguishable C. jejuni isolates, which were associated
with different clinical outcomes, GBS (4 isolates) or enteritis-only (2 isolates). The array
was spotted with 67 human and milk-related oligosaccharides, as well as 21
glycosaminoglycans and polysaccharides, including heparin, hyaluronan, chondroitin,
and carrageenan. Binding of fluorescently labelled bacteria to a wide range of terminal
galactose-, glucose-, mannose-, fucose- and sialic acid-containing glycans was observed.
Noteworthy, C. jejuni GBS-associated isolates bound at a higher rate than enteritis-
related isolates to the ganglioside-related structure Gal1-3GalNAc. High rates of
binding to the glycosaminoglycans and carrageenans was also observed. Additionally, the
binding data from glycan array analysis was confirmed using a tissue culture (Caco-2)-
lectin inhibition assay. The study demonstrated the power of glycan array technology to
characterise C. jejuni-carbohydrate binding and has given new insights into the
pathogenesis of GBS-associated C. jejuni strains.
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INVESTIGATION OF THE CONDITIONS THAT TRIGGER THE ACTIVATION OF THE ALTERNATIVE SIGMA FACTOR σB
IN LISTERIA MONOCYTOGENES
Marta Utratna, Conor O’Byrne* Bacterial Stress Response Group, Department of Microbiology,
School of Natural Sciences, National University of Ireland, Galway, IRELAND
Barnacle adhesive cement is renowned for its strength, durability and ability to form in aqueous environments. Barnacles are notorious biofoulers on manmade structures such as buoys and slow moving ships. They have the ability to adhere to a vast variety of flotsamic materials, such as wood, plastic, metal, organic matter. The pedunculate buoy barnacle Dosima fascicularis (Ellis and Solander, 1786) is unique in that it produces a buoyant float surrounding the site of attachment. This implies the cement proteins of D. fasicularis are unique from the protein secretions in all other barnacle species. As of yet, the cement apparatus of D. fascicularis has not been studied morphologically or histochemically. We have characterised the cement apparatus, including the cement glands, cement canals and sites of secretion, of the pelagic barnacle D. fascicularis. We found that it responded more strongly to stains for proteins with components within the pH range of 2.5 to 6.0. Using histological and histochemical methods the location and properties of the cement producing glands were defined. These techniques both displayed the gross morphology of the cement apparatus and allowed more detailed analyses of the cement apparatus to be carried out. The procedures included a number of stains for a variety of functions e.g. structure, pH, lipid content, mucosubstances, plus basic and acidic protein composition. The cement apparatus includes a paired delivery system to the exterior environment, draining from numerous small unicellular glands. These glands are interspersed within the ovarioles of the peduncle. These globular cement glands range from 100 – 200µm in diameter. The cytoplasm is primarily proteinaceous, containing mucosubstances of a pH between 2.5 and 6.0. The glands are connected by a complex network of canals, including intracellular and intercellular canals. The cement secretion travels through these canals prior to collection in the principle canal and release through the adhesive pores. These results are the beginning of the complete characterisation of D. fasicularis cement. A thorough understanding of the structure and chemistry of the cement apparatus will aid the extraction of the cement substance prior to secretion from the animal. This in turn will be subjected to electrophoresis and protein analyses in order to define the number and structure of proteins present. It is hoped that this research will, in the future, allow the synthesis of strong, natural adhesives that will have a wide array of applications in wet environments such as surgery or in assisting the production of novel surface coatings which may help prevent barnacle biofouling.
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The use of hydrological parameters and invertebrate bioindicators to assess the implications of infilling of wetlands in County Galway with construction
and demolition (C & D) waste. John Staunton1,2, Mike Gormally1, Tiernan Henry2
1 Applied Ecology Unit, Centre for Environmental Science, NUI, Galway. 2 Earth and Ocean Science, NUI, Galway.
Wetlands are important components of watersheds and provide useful ecosystem services including the transfer and storage of water, plant and animal production, organic decomposition, habitat provision, flood control, filtering and cleansing of water, erosion control and recreation. Despite these valuable ecosystem services, many wetlands, particularly those in the west of Ireland, have been or are currently being infilled with C & D waste. Very little is known about the impact of infilling on wetland ecosystem function in Ireland and this provided the incentive for this project.
Galway County Council provided a list of all licensed C & D waste sites in the county and suitable sites were selected using the following criteria:. a) the site was originally a wetland prior to infilling; b) at least part of the site had no infill and wetland vegetation still existed on site; c) the site was large enough to accommodate systematic surveys of plant and invertebrate communities; d) the site had adequate and safe access for surface and groundwater sampling; e) permission to access the sites was granted by landowners. Eight sites in total, located north of Galway city and east of lough Corrib were selected comprising of 5 cutover bog sites, 2 reed and large sedge swamp sites and a marsh site. Aerial invertebrates were collected using pan traps placed on infill and wetland vegetation during Summer / Autumn 2009 with further sampling planned for 2010. Particular emphasis is being given to marsh flies (Diptera: Sciomyzidae) which are proven indicators of change in other wetland types including turloughs and callows. Plant species composition around the pan traps was also assessed using quadrats. All sites were mapped using a trimble GPS to create a detailed digital model of site topography for the purposes of determining surface water flows. In addition, pizometers were set up (3 per site) to establish the direction of groundwater flow, and to take samples, both up-gradient and down-gradient of the infill. These samples will be tested for a number of indicator parameters (temperature, electrical conductivity, pH, benzene, CaCO3, Fe, etc) to assess if the chemical makeup of the groundwater is affected by overlying C & D waste.
National Centre for Biomedical Engineering Science (NCBES)
90
STUDENT POSTER PRESENTERS
NCBES No. ID Number Surname Name Year/Programme 63. 06116167 Prasad Babu R. 3PS1
91
EFFECT OF QUANTUM DOTS ON DIFFERENTIATED PHEOCHROMOCYTOMA CELLS
Babu R. Prasad1, Aaron Tonery1, Natalia Nikolskaya1, David Connolly1, Terry J. Smith1, Stephen J. Byrne2, Yurii K. Gun’ko2, Yuri Rochev1*
1National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland; 2CRANN and The School of Chemistry, Trinity College Dublin, Dublin
Quantum Dots (QDs) have the potential to use as simultaneous multiple colour labels due to its unique optical characteristics. In view of developing new neuro-therapeutic and neuro-prosthetic strategies, QDs have been modified to create active cellular interfaces with nerve cells [1] and for imaging of neuronal and glial interactions called synaptic clefts, or between an astrocyte process and a neuron [2]. Little work has been undertaken in observing the neuron cell response over long time scales upon exposure to QDs. So we focussed on the analysis of Pheochromocytoma 12 (PC12) cells exposed to QDs which have the ability to be differentiated into neurons upon treatment with nerve growth factors (NGF). We used water soluble Cadmium Telluride Thio-Glycolic Acid capped QDs of ~2-5 nm size. Using Confocal Microscopy, we observed the localisation of QDs distributed heterogeneously. There is no inhibition of neurites growth due to QDs. No evidence of degradation of neurites until 12 days. After that there is sharp increase in degradation of neurites and the cells started to die with QDs still be seen in the dead cells. In another experiment, neurites were destroyed when PC12 cells are exposed to higher concentrations of QDs due to toxicity. References:
1. Gomez N, Winter JO, Shieh F, Saunders AE, Korgel BA, Schmidt CE: Challenges in quantum dot-neuron active interfacing. Talanta 2005, 67:462-471.
2. Pathak S, Cao E, Davidson MC, Jin SH, Silva GA: Quantum dot applications to neuroscience: New tools for probing neurons and glia. J Neurosci 2006, 26:1893-1895.
College of Arts, Social Sciences and Celtic Studies
College of Medicine, Nursing Studies and Health Sciences
Postdoc Researchers
Visiting Students
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STUDENT POSTER PRESENTERS
College of Arts, Social Sciences and Celtic Studies No. ID Number Surname Name Year/Programme 64. 04446933 Ó Catháin Padraig 2PA1 College of Medicine, Nursing Studies and Health Sciences No. ID Number Surname Name Year/Programme 65. 04317262 Duffy Michelle 2PM1 66. 03493547 O’Flynn Lisa 2PM1 Postdoc Researchers No. ID Number Surname Name Year/Programme 67. n/a Ní Longphuirt Sorcha Botany, Natural
Sciences
Visiting Researchers No. ID Number Surname Name Year/Programme 68. n/a Gao Chunxia n/a
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HADAMARD MATRICES FROM DIFFERENCE SETS
Padraig Ó Catháin School of Mathematics, Statistics and Applied Mathematics
Quite recently K.J. Horadam posed a question about the automorphism groups of Hadamard matrices arising from twin prime power difference sets. The author and Richard M. Stafford have shown that these automorphism groups act intransitively on the rows of the matrix, thus answering that question. The author is currently working to extend this result to the other known families of difference sets.
95
MESENCHYMAL STEM CELLS (MSCs) POTENTLY INHIBIT THE PRIMARY INDUCTION OF T-HELPER 17 (Th17) CELLS THROUGH CELL-CELL CONTACT.
Authors – Michelle Duffy and Matthew Griffin Regenerative Medicine Institute, National Centre for Biomedical Engineering Science,
Corresponding author: [email protected] Introduction MSCs are immunosuppressive with the potential to treat autoimmune/inflammatory disease. Pro-inflammatory Th17 cells are pathogenic in some autoimmune diseases. In this study the interactions between mouse MSCs and CD4+ T-cells undergoing primary Th17 induction in vitro were examined. Materials and Methods Th17 induction consisted of stimulation with anti-CD3 and autologous APCs in the presence of IL-6, TGFβ1, anti-IFNγ and anti-IL-4. Analyses included ELISA, surface/intracellular flow cytometry, cell division analysis by CFSE dilution and RT-PCR. In some experiments, MSC effects on flow-sorted naïve (CD4+/CD62Lhi/CD25-) and memory (CD4+/CD62Llo/CD25-) T-cells were separately examined as was indomethacin or a DMSO vehicle control. Results MSCs potently inhibited primary Th17 induction in co-culture at T-cell:MSC ratio as low as 400:1. This effect was characterised by preserved T-cell viability, reduced proliferation, suppression of CD25 up-regulation and decreased production of IL-17 upon re-stimulation in the absence of MSCs. Th17 inhibition occurred when either naïve or memory T-cells were induced in the presence of MSCs. Skewing to other T-helper phenotypes was not observed although an IFNγ-secreting component was preserved among memory T-cells. Th17 suppression was not mediated by fibroblasts. The effect was maintained when APCs were replaced by antiCD3/CD28-coated beads. Transwell experiments and conditioned MSC medium had minimal suppressive activity however conditioned medium from MSC/Th17 co-cultures transferred the inhibitory effect. The inhibitory effect was reversed when indomethacin was introduced to the co-culture to block prostaglandin production. Discussion and Conclusions In the mouse, the capacity of MSCs to modulate T-cell activation extends to primary Th17 induction through a contact- or proximity-dependent mechanism related to the COX pathway. Of interest, the effect is quite potent in vitro and applies to both naïve and memory-phenotype responders.
Fig.1. MSC inhibition of Th17 cells was characterised by decreased production of IL-17.
In organ transplantation the ultimate goal is tolerance, this refers to the state in which a recipients’ immune system is reprogrammed to accept an allograft without the need for further immunosuppression. In this respect, tolerance achieved by the application of cell-based immunotherapy in allotransplantation has been investigated. This study aims to examine bone marrow derived dendritic cells (BMDCs) and their potential for genetic modification for the prevention of corneal allograft rejection. BMDCs were propagated from rat bone marrow precursors cells in a medium supplemented with recombinant rat granulocyte macrophage colony-stimulating factor (GMCSF) and interleukin-4 (IL-4) (5ng/ml). In-vitro generated BMDCs were characterized by their expression of CD11b/c, MHCII and the costimulatory molecule CD86 using flow cytometry. BMDC function was tested in allogeneic mixed lymphocyte reactions and in antigen specific reactions. Results show that BMDCs express levels of MHC II sufficient to present antigen and subsequently activate the T cells, illustrated by their expression of CD25 and OX40. However, the in vitro generated BMDCs are unable to fully stimulate the T cells and although activated the T cells do not proliferate. Further experiments have illustrated that BMDCs are efficiently transduced with lentiviral vectors. This study therefore demonstrates that BMDCs generated in-vitro might have the ability to induce tolerance and have potential for further modification by lentivirus transduction. Future experiments will investigate the potential of over-expression of immunomodulatory molecules by lentiviral gene transfer on the induction of transplantation tolerance by cellular immunotherapy.
97
Macroalgal carbon budgets and potential impacts of Ocean Acidification Sorcha Ni Longphuirt and Dagmar Stengel Botany and Plant Science, School of Natural Sciences, MRI, NUI Galway Macroalgaeare a highly productive component of coastal waters and contribute significantly to the carbon cycle in both subtidal and intertidal regions of the oceans. They are also structurally important as they form habitats for associated floral and faunal species. Predicted future climate change will result in higher CO2 levels in the atmosphere, and as a result of absorption, dramatic changes to the carbonate system of the world’s oceans. Such changes will result, firstly, in an increase in the availability of carbon for photosynthesis during submersion and exposure periods and, secondly, in a decrease in pH levels with untold implications for the functioning of macroalgal species. The significance of these changes will depend on the capacity of macroalgae to utilise the different forms of carbon available to them and their ability to adapt to future conditions. Added to this, future climate changes will involve alterations to a range of environmental parameters including temperature and UV light. The synergistic impacts of these factors need to be considered to determine the possible future changes to the photosynthesis and primary production of macroalgal species. Within the scope of current research programmes at NUI Galway the carbon budgets and photosynthesis of two intertidal macroalgal species, the indigenous Ascophyllum nodosum and the invasive Sargassum muticum are being studied over the entire tidal cycle (exposure and submersion). Determination of such seasonal carbon budgets will allow us to elucidate their contribution to the overall cycling in coastal zones. Laboratory studies using infra red gas spectrophotometry are being carried out to determine the short term influence of increased atmospheric CO2 on the primary production of these species. Mesocosm studies are currently underway which will allow the assessment of the influence of projected increases in pCO2 concentrations (approx 800 ppmv) and temperatures (+3°C) over the medium term (2-3 months). These will be expanded to further investigate the combined effects of increased pCO2 and UV light on the functioning of the seaweed.
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MOLECULAR SIMULATION STUDIES OF ESTROGEN RECEPTOR ALPHA AND TAMOXIFEN METABOLITES
Estrogens exert their physiological effects by binding to the estrogen receptor (ER). In this way, they primarily regulate the growth and differentiation of the reproductive organs. Over- expression of estrogens will cause several diseases, of which the most notable is breast cancer. Tamoxifen (TAM; Figure 1) is an anti-estrogenic drug that is widely used for the treatment thereof. It is an antagonist of ER that competes with estrogens in binding to the ER ligand-binding domain (LBD). Through metabolization of cytochrome P450 (CYP) enzymes, Tamoxifen is converted to 4-hydroxy-TAM and other active metabolites, and it is thus regarded as a prodrug. According to substantial data derived from in vivo and in vitro experiments some of these metabolites have higher anti-breast cancer efficiency than TAM, for example, 4-hydroxy-TAM and endoxifen; whereas others display effects similar to TAM, for example N-desmethyl-TAM and N-didesmethyl-TAM; in addition, there is even a metabolite that induces hepatic tumorigenesis, alpha-hydroxy-TAM. Using the crystal structure of 4-hydroxy-TAM bound the ERα (PDB code: 3ERT), we refined the LBD of ERα, and then docked the other metabolites into this site, followed by molecular dynamics (MD) simulations in water solvated environments. Finally we analysed the conformational changes in the C-terminal Helix of ERα-LBD (Helix-12), which is associated to the agonist, antagonist, or partial agonist activity of the ligand. From the results we can see that all the metabolites are pure antagonists for ERα, even the toxic alpha-hydroxy-TAM does not induce any unfavourable conformations. Indeed, the bulky side chain moiety of TAM and its metabolites serves to prevent the formation of an active agonistic tertiary structure of helix 12, a property that can be used for designing new anti-breast cancer drugs. Furthermore, we calculated corresponding interaction energy of each metabolite in their Z and E isomers, giving a satisfactory binding affinity correlation between experimental and computational data. One of the major challenges in computational approaches to drug design is the accurate prediction of binding affinities to biomolecules, and thus molecular simulations as presented in this work may be used as a tool in the development of new lead compounds against breast cancer.
