1. VASCULAR TISSUE ENGINEERING: ENDOTHELIAL CELL-MEDIATED EFFECTS
ON EXTRACELLULAR MATRIX SYNTHESIS BY ARTERIAL SMOOTH MUSCLE
CELLS IN CULTURE
Brophy MJ, Flanagan TC1
1
UCD School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4.
The mechanical strength of tissue-engineered vascular grafts has been promoted by methods that
enhance extracellular matrix (ECM) synthesis by seeded cells, including bioreactor
conditioning/growth factor supplementation. Endothelial cells are an important component of such
grafts due to their anti-thrombogenic properties, but there are conflicting data in the literature
concerning their effect on ECM synthesis by arterial smooth muscle cells (SMCs). The present study
aims to determine whether or not an overlying layer of endothelial cells, and/or their secretions,
enhances or inhibits ECM production by co-cultured SMCs, which may determine an optimal timepoint for graft endothelialisation.
For 2-D analysis, commercially-available human umbilical artery SMCs (HUASMCs) were cultured
either alone, in direct contact within human umbilical artery endothelial cells (HUAECs), or exposed
to secretions from HUAECs using co-culture well inserts. For preliminary 3-D analysis, HUASMCs
were embedded within fibrin gels and were cultured either alone, with a surface monolayer of
HUAECs, or in HUAEC-conditioned medium. All cultures were maintained for 12 days and ECM
development (type-I collagen, elastin, fibronectin) was analysed using immunofluorescence
microscopy. In addition, collagen and elastin production was quantified using spectrophotometric
assay.
In 2-D culture, it was observed that HUASMCs and HUAECs in direct cell contact produced an
increased amount of elastin (~7.6%) when compared to HUASMCs cultured alone, while collagen
production was significantly reduced (~29%). These results were corroborated by
immunofluorescence microscopy. Preliminary results from 3-D fibrin gel culture showed a 26.8%
increase in elastin production in the group containing HUASMCs and a surface monolayer of
HUAECs compared with HUASMCs cultured alone, while collagen production in 3-D gel constructs
was also significantly reduced in this group (~39%), mirroring the 2-D results. Strikingly, a clear
potent relationship between both cell types was observed, with significant contraction of gels in this
group alone to a fraction of their size after 3 days in culture.
The results of the present study indicate that seeding of ECs onto fibrin-based vascular grafts should
be completed toward the end of the in vitro conditioning period to allow for adequate deposition of
collagen and elastin proteins by seeded SMCs and to prevent premature tissue contraction.
Acknowledgement:
This project was funded by the National Children’s Research Centre. MJB also acknowledges
funding through a HRB Summer Student Scholarship.
Presenting Author: Ms Megan Brophy
Supervisor: Dr Thomas Flanagan
Co-Supervisor: Dr Nobue Itasaki
1
14. THE DEVELOPMENT OF DIAGNOSTIC ACCURACY AND SEARCH PATTERN
BEHAVIOR IN THE INTERPRETATION OF CHEST RADIOGRAPHS.
Kelly BS1, Toomey RJ1, Kavanagh EC2, Rainford LA1
1
Diagnostic Imaging, UCD School of Medicine and Medical Science, University College Dublin, Belfield,
Dublin 4.
2
Department of Radiology, Mater Misericordiae University Hospital, Eccles St, Dublin 7.
To investigate the development of radiological image interpretation skill through medical training by
measuring both diagnostic accuracy and eye movements during visual search.
Five Consultant Radiologists, deemed the reference expert group, four Radiology Registrars, five
Senior House Officers (SHOs) and six Interns formed four clinician groups. Participants were shown
30 chest radiographs, 14 of which had a pneumothorax and were asked to give their level of
confidence as to whether a pneumothorax was present. Receiver Operating Characteristic (ROC)
Analysis was carried out on diagnostic decisions. Eye movements were recorded by a Tobii TX300
eye tracker. Four Eye-tracking metrics were analysed. Variables were compared to identify any
differences between groups.
All data were compared using the Friedman non-parametric method. The average area under the ROC
Curve for the groups increased with experience (p=0.009). Statistically significant difference in
diagnostic accuracy was found between Consultants and Registrars (p=0.046). All four eye-tracking
metrics reduced with experience, this was statistically significant for Registrars compared with SHOs.
The total reading time reduced with experience; significantly for Registrars compared to SHOs
(p=0.046) and between SHOs and Interns (0.025).
Reader performance increased with experience. The level of experience at which there was a
statistically significant difference was higher for diagnostic accuracy than for eye-tracking metrics.
This data would suggest that specific training is needed to improve radiology expertise and that the
development of an “expert” search pattern predates the development of “expert” levels of diagnostic
accuracy.
Presenting Author: Mr Brendan Kelly
Supervisor: Dr Louise Rainford
Co-Supervisor: Dr Rachel Toomey
2
24. IDENTIFICATION OF A GENE CAUSING EPILEPTIC ENCEPHALOPATHY IN A
SINGLE IRISH FAMILY
Nealon J1, Conroy J1, McCreary D1, McGettigan P2, Crushell E3, Lynch SA4, Ennis S1
1
UCD School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4.
UCD School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield,
Dublin 4.
3
Children's University Hospital Temple Street, Temple street North, Dublin.
4
The National Centre of Medical Genetics Ireland, Our Lady’s Hospital for Sick Children, Crumlin, Dublin.
2
Epileptic encephalopathies (EE’s) are rare but devastating forms of epilepsy, which present as
frequent and severe seizures with cognitive and behavioural disturbances. The cohort in this study was
a single family consisting of two sisters with idiopathic EE in addition to an unaffected mother, father
and brother. The objective was to identify the potential genetic cause for this familial form of EE
using next-generation sequencing.
Exome sequencing was performed on DNA samples of each family member at Atlas biolabs
(http://www.atlas-biolabs.com/home). Following bioinformatics analysis and rare/novel variant
identification, the resulting data was analysed based on three patterns of inheritance. These were
recessive, compound heterozygous and de-novo models. The list of rare variants was compared to a
list of epilepsy-linked genes. The resulting candidates were screened in control populations (Irish
exome control database, Seattle exome database, and 1000 genomes project). In silico investigations
to predict mutation effects were undertaken using SIFT, Polyphen and MutationTaster. Literature
reviews and further analysis were performed using OMIM, Pubmed, UCSC and Orphanet. High
priority candidate variants were confirmed by Sanger sequencing.
Eight variants were identified in five candidate genes (1 recessive, 3 compound heterozygous and 1
EE gene). Additional variants of unknown effect were also present. Currently these cannot be ruled
out as EE candidates. Furthermore, it is possible that the disease causing mutation is not present in the
exome and therefore cannot be identified using this technique.
Although potential candidate variations have been reported, further work is required to confirm these
as disease causing mutations.
Acknowledgements:
We sincerely thank the families for the use of clinical information and genetic samples. We would
like to thank Dr Ellen Crushell and Dr Sally Ann Lynch for their collaboration in this project. This
project was funded by The Children’s Fund for Health - Children’s University Hospital, Temple
Street and the Pathological Society of Great Britain and Ireland Undergraduate Bursary.
Presenting Author: Mr John Nealon
Supervisor: Dr Sean Ennis
Co-Supervisor: Dr Judith Conroy
3
27. ROLE OF HYPOXIA INDUCIBLE TRANSCRIPTION FACTORS HIF-1 AND HIF-2 IN
MACROPHAGE DIFFERENTIATION TOWARDS PRO-INFLAMMATORY M1 OR PRORESOLUTION M2 PHENOTYPES
Uwadiae H1, Godson C1,2, Higgins D1,2
1
Diabetes Complications Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research,
University College Dublin, Belfield, Dublin 4.
2
UCD School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4.
Hypoxia occurs when oxygen supply is insufficient for cellular demand. Hypoxia inducible
transcription factors (HIF-1α and HIF-2α) play a primary role in mediating cellular adaptation to
hypoxia and inflammation through regulation of a number of genes. The HIF-α subunit binds HIF-β
forming functional HIF-1 or HIF-2 transcription factors. Recent studies have shown evidence of
differing roles between HIF-1 and HIF-2 in hypoxic and inflammatory process.
The aim of this study was to determine whether macrophage differentiation towards either proinflammatory M1 or pro-resolution M2 is influenced by HIF-1 and HIF-2, which may impact
progression or regression of inflammation in renal injury. A number of strategies were employed to
optimise transfection-efficiency of THP-1 monocytes and THP-1 derived macrophages with either
HIF-1 or HIF-2. Transfection-efficiency was monitored using a reporter luciferase assay for HIFtranscriptional activity. Effect of over-expression of HIF-1 and HIF-2 was determined by
monitoring changes in expression of M1 marker MCP-1 and the M2 markers TGF and IL-10 using
ELISA and quantitative real time PCR analysis.
Transfection-efficiency was highest in THP-1 monocytes transfected prior to differentiation into
macrophages using Fugene HD (Invitrogen) and HiPerfect (Qiagen) transfection reagents. Overexpression of HIF-1 enhanced MCP-1 mRNA expression whereas over-expression of HIF-2
increased TGF- mRNA expression. While MCP-1 protein levels were not significantly changed with
HIF-1 or HIF-2 over-expression, HIF-2 significantly increased expression of the M2 marker IL10 in THP-1-derived macrophages.
The data support a role for the promotion of an M1 macrophage phenotype via HIF-1 mediated
processes whereas HIF-2 promotes an M2 macrophage phenotype.
References:
1. Schofield, C.J. and P.J. Ratcliffe, Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol,
2004. 5(5):343-54.
2. Takeda, N., et al., Differential activation and antagonistic function of HIF-{alpha} isoforms in
macrophages are essential for NO homeostasis. Genes Dev, 2010. 24(5): 491-501.
Acknowledgements:
Biochemical Society Summer Vacation Studentship
Irish Health Research Board Translational Medicine Fellowship
Presenting Author: Mr Harmony Uwadiae
Supervisor: Dr Deborah Higgins
Co-Supervisor: Prof Catherine Godson
4
40. BIOINFORMATICS ANALYSIS TO IDENTIFY PROCESSED PSEUDOGENES IN
BREAST CANCER
Ew JV1, Freitas I2, Tarrant F2, O’ Gaora P2
1
UCD School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4.
UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin
4.
2
Pseudogenes are mutated copies of genes which are generally considered non-functional. However,
new evidence shows that pseudogenes play a role in tumour biology. Processed pseudogenes (PPGs)
arise from reverse transcription of mRNA and transposition into the genome. In this pilot study, breast
cancer genomic data were subjected to a bioinformatics workflow to detect potential PPGs.
Whole exome sequencing data from 5 normal breast tissue samples and 5 breast cancer tumour
samples were downloaded from The Cancer Genome Atlas repository. Reads were aligned to the
human genome using the splice-aware aligner STAR. Uniquely aligned reads which aligned across
exon-exon junctions (splitreads) were identified for further processing. The splitread counts in tumour
samples were compared to those in normal tissue to identify significant differences in potential PPGs.
Hierarchical clustering analysis showed clear differentiation of normal and tumour samples. From
23,230 genes in the annotated genome, 2,597 were found to harbour aligned splitreads. Using
negative binomial distribution testing implemented in DESeq, 30 genes were found to show
statistically significant elevations or depressions in the tumour samples (p<0.05).
This study identifies 30 genes as potentially important PPGs in breast cancer genomes. Nevertheless,
further lab work needs to be performed to confirm the presence of pseudogenes. Furthermore,
increased numbers of samples will provide greater statistical power for detection of PPGs.
Pseudogene signature profiles may eventually be correlated to tumour subtypes and disease outcome.
In conclusion, this study is the first step to understanding breast cancer pseudogene biology.
Acknowledgement:
With thanks to the Pathological Society of Great Britain and Ireland for funding this project.
Presenting Author: Ms Ju Vern Ew
Supervisor: Dr Peadar O’Gaora
Co-Supervisor: Mr Finbarr Tarrant
5
49.THE ROLE OF SRF IN EPITHELIAL TO MESENCHYMAL TRANSITION (EMT) IN
ADVANCED PROSTATE CANCER.
Dilworth R1, Hanrahan K1, Watson RWG1,2 , Prencipe M1,2
1
UCD School of Medicine and Medical Sciences, University College Dublin, Belfield, Dublin 4.
UCD Conway Institute of Biomedical and Biomolecular Research, University College Dublin, Belfield, Dublin
4.
2
Prostate cancer is the most frequently diagnosed cancer (excluding non-melanoma skin cancers) in
Irish males, with incidence rates in Ireland among the highest in Europe. 1 Epithelial to Mesenchymal
Transition (EMT), a process through which cells lose their epithelial characteristics and acquire a
mesenchymal phenotype, has been associated with cancer cell migration, invasion, and therefore
metastasis. Recently, Serum Responsive Factor (SRF) was identified as a key transcription factor
involved in the development of castrate resistant prostate cancer.2 Given the involvement of SRF in
cellular differentiation and cytoskeleton organisation, it was hypothesized that SRF plays a role in the
induction of EMT in prostate cancer cells.
Two isogenic prostate cancer cell lines were used to investigate this hypothesis: the LNCaP parental
cell line and their castration-resistant LNCaP Abl sub-cell line. Both lines were transfected with SRFtargeting siRNA and scramble (control) siRNA to knock down SRF. The effectiveness of SRF
knockdown and the expression of epithelial and mesenchymal protein markers were assessed by
Western blotting. Cell migration was assessed by the scratch assay, and cell invasion was assessed
using Matrigel™-coated inserts.
SRF siRNA significantly knocked down SRF protein expression. Invasion assays showed
significantly decreased cellular invasion in the parental cell line after SRF knockdown. Western
blotting showed upregulation of E-cadherin, an important epithelial marker, after SRF knockdown in
both parental and Abl cells.
Our findings demonstrate that SRF plays a role in inducing EMT-like processes in prostate cancer
cells. SRF represents a promising therapeutic target for reducing the metastatic potential of advancing
prostate cancer.
References:
1. National Cancer Registry Ireland. Cancer in Ireland 2013: Annual Report of the National Cancer
Registry.
Cork:
National
Cancer
Registry;
2013.
Available
from: http://www.ncri.ie/publications/statistical-reports/cancer-ireland-2013-annual-report-nationalcancer-registry
2. Prencipe M, Madden SF, O’Neill A, O’Hurley G, Culhane A, O’Connor D, Klocker H, Kay EW,
Gallagher WM, Watson RW (2013) Identification of transcription factors associated with castrationresistance: is the serum responsive factor a potential therapeutic target? Prostate 73(7):743-753
Presenting Author: Ms Rachel Dilworth
Supervisor: Dr Maria Prencipe
Co-Supervisor: Prof R William G Watson
6
59. ANALYSIS OF CAESAREAN SECTION RATES IN GESTATIONAL DIABETES:
USE OF THE ROBSON GROUPS TO ALLOW MEANINGFUL EXAMINATION OF
DATA
Courtney W, Carroll C, Courtney D, Higgins M, Robson M, McAuliffe F1, Foley Mq
1
UCD Obstetrics and Gynaecology, UCD School of Medicine and Medical Science, University College
Dublin, National Maternity Hospital, Holles Street, Dublin 2.
This was a prospective study examining patients who attended this large tertiary level obstetric
unit from January 2005 to December 2012. It aimed to examine the rate of caesarean section
(CS) in gestational diabetes (GDM) patients as classified by the Robson Criteria, an
internationally recognised mode of analysis.
Over this time, 61,116 women delivered infants greater than 24 weeks gestation in this unit, of
which, 1126(1.8%) were diagnosed with gestational diabetes.
During this time, the overall hospital CS rate was 19.6%, compared to 32.5% rate in the GDM
population. Nulliparous women undergoing induction of labour (IOL)(Group 2a) had a 63%
increase in CS delivery rate over the non-GDM population; multiparous women (Group 4a) had
a 20% increase over the non GDM population. By comparison, those with GDM who went into
spontaneous labour incurred a 19% (nulliparous) and 1.2% (multiparous) increase in CS rates
over the non-GDM population. The majority of women with a previous CS delivered by repeat
CS (71%).
Overall, the GDM cohort had a 65% increased rate of delivery by CS compared to the general
population (p<.0001). The highest rate of CS was amongst women with a previous CS. Those
undergoing IOL (groups 2a and 4a) were more likely to need CS than those who went into SL.
This data supports a policy of thoughtful IOL in women with GDM and realistic counselling of
GDM women, particularly those in the nulliparous category, regarding the possible outcomes of
IOL. In this instance, elective CS could be meaningfully discussed.
Contd.
7
Robson Group
Diet GDM
Rate (%)
CS Insulin GDM CS Overall CS
Rate (%)
Rate (%)
1
Nulliparous,
single cephalic 28/153
>37 weeks in (18.3)
spontaneous
labour
2
13/65
(20)
1176/16421
(7.2)
18/34 (52.9)
2869/8619
(33.6)
17/33 (51.52)
(2323/8046)
28.87
1/1 (100)
573/573
(100)
0/109 (0)
220/18321
(1.2)
17/45 (37.8)
11/43 (25.6)
765/6139
(12.5)
8/36 (22.22)
7/39 (17.9)
312/5696
(5.5)
9/9 (100)
4/4 (100)
443/443
(100)
109/159 (68.5)
78/102 (76.5)
3364/5735
(58.6)
7/7 (100)
6/6 (100)
1177/1273
(92.5)
5/5 (100.)
4/4 (100)
685/815
(84.1)
13/20 (65)
4/5 (80)
654/1066
(60.7)
2/2 (100)
4/4 (100)
220/220
23/29 (79.3)
2a
Nulliparous,
single cephalic 21/27 (77.8)
>37 weeks in
induced labour
2b
Nulliparous,
2/2 (100)
single cephalic
CS before labour
3
Multiparous,
single cephalic
2/282 (0.71)
>37 weeks in
spontaneous
labour
4
4a
Multiparous,
single cephalic
>37 weeks in
induced labour
4b
Multiparous,
single cephalic
>37 weeks CS
before labour
5
Previous
CS,
single cephalic
>37 weeks
6
All nulliparous
breeches
7
All multiparous
breeches (incl.
previous CS)
8
All
multiple
pregnancies
(incl.
previous
CS)
9
8
Table 1:
Ten
All abnormal lies
(incl.
previous
CS)
10
All
single,
cephalic, <=36 9/27 (33)
weeks
(incl.
previous CS)
Total (%)
29.49
(100)
13/25 (52)
882/2546
(34.6)
38.04
19.67
Robson
Group
Caesarean Section Delivery rates in Diet controlled GDM vs. Insulin Controlled GDM vs. General
Population
Presenting Author: Mr William Courtney
Supervisor: Prof Michael Foley
Co-Supervisor: Dr Mary Higgins
9
77. EZH-2 EXPRESSION IS INHIBITED IN PROSTATE CANCER CELL LINES BY
MICRORNA MIR-124 OVEREXPRESSION
Gaffney B1, Ma AH2, Shi XB2, Xue L2, Devere White RW2
1
2
UCD School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4.
Department of Urology, University of California, Davis, School of Medicine, Sacramento, CA, USA.
Prostate cancer (CaP) continues as the second leading cause of cancer deaths in males in the western
world . The mechanism of the switch from androgen dependent CaP to castration-resistant prostate
cancer (CRPC) remains unclear and has been further complicated by the involvement of microRNA
(miRNA) [1]. These small non-coding RNAs are able to regulate gene expression by binding to the
3‘UTR of target genes [1].
The aim of this study is to confirm that the miRNA miR-124 binds to the enhancer of zeste homolog 2
(EZH2) gene through a suggested binding site on the 3’UTR and downregulate its transcriptional
level in prostate cancer cell lines. Recent work in the lab found adding a miR-124 mimic in animal
and cell models was found to inhibit tumor cell growth, while additional evidence shows miR-124 is
downregulated in clinical CaP samples [2]. EZH2 was identified as a target of miR-124. The
expression of EZH2 has been correlated with CaP progression, especially CRPC [3]. It is thought that
miR-124 is downregulated in CRPC allowing the EZH-2 to negatively regulate the apoptotic pathway
as well as increasing invasion and proliferation pathways of cancer cells.
The EZH2 3’UTR fragments with and without the predicted binding site for miR-124 were cloned
into a reporter plasmid. A luciferase reporter assay was used to quantify experimental and control
reporters. The results of this study show that miR-124 appears to be binding to the suggested binding
site on the 3‘UTR of the EZH-2 gene and downregulating its expression. This downregulation is
significant in both PC3 and C4-2B cell lines. Further work needs to identify the functional
significance of this finding but treatment of CRPC with miR-124 continues to represent a possible
therapeutic approach.
References:
1. Yu, J.J. and S.J. Xia, Novel role of microRNAs in prostate cancer. Chin Med J (Engl), 2013.
126(15): p. 2960-4.
2. Shi, X.B., et al., Tumor suppressive miR-124 targets androgen receptor and inhibits proliferation
of prostate cancer cells. Oncogene, 2012.
3. Xu, K., et al., EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycombindependent. Science, 2012. 338(6113): p. 1465-9.
Presenting Author: Mr Brian Gaffney
Supervisor: Dr Ai-Hong Ma
Co-Supervisor: Mr Ralph W deVere White
10