Final year project example posters 2013 2014

Example Posters from 2013-14
Dr Peter Bush.
School of Pharmacy and Biomolecular Sciences, University of Brighton

Repair vs Regeneration
The Future of Regenerative Medicine
------------------ supervised by Dr. Liz James
Objective
• Comparing the effectiveness of
different biomaterials as E.C.M.
Scaffolds aiding regeneration.
• To understand the viability of these
scaffolds after they are exposed to
certain stresses.
• Method of storage so the scaffold is
kept viable while it is not being use.
Method
• A variety of scaffold types will be tested
on mice, the test should provide
information to which scaffold is the
most effective .
• Each test will have a different
parameter, this will give data showing
what effects the tissue regeneration.
• After several weeks the mice will be
checked for any marks or scars and any
significant difference provides
quantitative data to the regeneration of
the biomaterial.
Biomaterials have long been a focal
point of tissue engineering due to the
advancement In our understanding of
the functions of the extracellular
matrix and the mechanism of
dedifferentiation.
http://ec.europa.eu/research/press/2000/pr2703en-an.html
Hyaluronic Acid based
Scaffold (Hydrogel)
http://openi.nlm.nih.gov/detailedresult.php?i
mg=2426767_nano0101-15-03&req=4

Studying the impact that e-learning has on university science students in
the medical field by comparison of various published articles detailing it’s
effects.
----------------------. Jacqueline Elsom
Introduction
The use of e-learning has become an ever increasing alternative to traditional study in the 21st
centaury, this study has taken several already published articles and research papers detailing
effects that the various forms of e-learning has on students, particularly students within the
medical field. Effects that e-learning has upon students is measured by examination results in
an intervention group who received e-learning teaching methods compared to control group
who did not receive e-learning teaching methods. Studies where then compared to each other
to analyse trends, This study opens many questions as to where the educational system is
moving in terms of teaching styles, and the relevance of conventional teaching methods.
Being a comparative study it was essential to first pool a large collection of literature
to which would be analysed, the literature being collated would have also had to fit
the criteria that the literature were peer reviewed published articles. The data of
these articles also had to be of similar nature (i.e. Contain test results of students in
the same field of study with and without the aid of e-learning techniques.) With
databases containing peer reviewed journals and articles to be used as search engines
to locate articles, named databases were “PubMed” “PubMed Central” “Google
Scholar” “Brighton University” “Science Direct” using search terms with a
combination of terms such as “E-learning” “distance learning” “Biomedical science
students” “medical students” to narrow down the search and to find appropriate
results. The collection of articles will then have to be scrutinised further removing any
which the author finds does not fit the study (at their discretion.) The extraction of
key data from the literature will manually have to be done and manipulated if need be
to stylise the data in a mutual order (i.e. Average grade Percentages). The use of
statistical analysing programmes such as Microsoft Excel and Minitab are important
in configuring the data and for analysis. Test of heterogeneity using Chi-sq, ANOVA
test of variance and Tukeys plot analysis are analytical comparison made.
Materials and Methods
Hypothesis
H1=E-learning does provide a significant positive effect in the teachings of students studying in the
medical field
H0=E-learning does not provide a significant positive effect in the teachings of students studying in
the medical field
*In this study the term “positive effect” is defined by increase in test score results
Tukey Pairwise Comparisons
Grouping Information Using the Tukey Method and 95%
Confidence
Factor N Mean Grouping
Mean results test 9 76.92 A
Mean Results control 9 65.50 B
*Different grouping indicates statistically different*
Table 1. Summary table for all trials used and the data extracted from used trials
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
ScorePercentage
Trial
Comparison between mean test score percentages with and without the aid of e-learning in various
published trials
Mean results Intervention %
Mean Results Control %
Fig.1 Bar chart showing the mean percentage test scores in various studies in the intervention group compared to a control
Test pop
Test pop
Control pop
Control pop
Fig.2 Showing the 95% CI interval for StdDev of means from control and intervention groups
Fig.3 Boxplot comparing intervention and control score data
RESULTS
CHI-SQ
N DF Chi-Sq P-Value
589.52 8 20.1277 0.010
Table 2. Chi.Sq of values
Table 3. Tukey comparison of intervention and control
Table 4. Pair t-test results of intervention compared with control
Discussion and Conclusions
Results have indicated that e-learning has had a positive effect on the intervention groups and have shown that
its use does contribute to a statistically different (higher) test score result when compared with test scores
achieved in its absence. The comparative bar chart shows consistently in all 9 trials that the intervention group
means are higher than the control. Tukeys grouping have shown that the intervention means are statistically
higher. Paired t-test results show a p-value of 0.003 which allows the rejection of the null hypothesis. E-learning
has shown to be a practical and useable method of alternative teaching for students in the 21st centaury .
References
Y.Morgulis, R.K.Kumar and G.M Velan, (2012), Impact on learning of an e-learning module on leukaemia: a randomised controlled trial
C.S.Silva, M.B.Souza, R.S.Silva Filho, L.M.Medeiros, P.R.Criado, (2011), E-learning program for medical students in dermatology.
M.Boeker, P.Andel and A.Frankenschmidt , (2013),Game-Based E-Learning Is More Effective than a Conventional Instructional Method: A Randomized
Controlled Trial with Third-Year Medical Students
R.Abdelhai, S.Yassin and U.G.H.Fors, (2012), An e-learning reproductive health module to support improved student learning and interaction: a prospective
interventional study at a medical school in Egypt
C.Cuca, P.Scheiermann and R. Breitkreutz,(2013) Assessment of a New E-Learning System on Thorax, Trachea, and Lung Ultrasound
D.Zhang, L.Zhou, R.O.Briggs, J.F.Nunamaker Jr,(2006),Instructional video in e-learning: Assessing the impact of interactive video on learning effectiveness
A.Phadtare, A.Bahmani and R.Pietrobon ,(2009), Scientific writing: a randomized controlled trial comparing standard and on-line instruction
N.Mehrdad, M.Zolfaghari, N.Bahari and S.Eybpoosh, (2010), Learning Outcomes in Two Different Teaching Approach in Nursing Education in Iran: E-Learning
versus Lecture
F.Moazami, E.Bahrampour, M.Moattari, (2014), Comparing two methods of education (virtual versus traditional) on learning of Iranian dental students: a
post-test only design study

Construction of a road tunnel at a migratory road used by common toads
(Bufo Bufo) will decrease road mortality of the common toads.
----------------------- and Dr. Inga Zeisset
Introduction
Common toads have been declining in the last 30 years and a major reason of declining is the mortality in the roads. This project
will test the hypothesis of a significant decrease of road mortality of the common toad with the construction of a road tunnel at a
common toad migratory road. There have been a few similar conservation projects in USA, UK and other countries but most of
them lacked sufficient planning. The study will propose methods for volunteers to collect road-kill data (24hrs per month) and
will also propose an optimal spatial and temporal study design. Also the rainfall and temperature data sets will be included in the
data analysis. The results of the project will help the decision in undertaking further work or improvements on the tunnels.
Temporal and Spatial Study Design
The data of the project should have replicated spatial (different segments
of study area) and temporal design (day, month, year).
The temporal design of this project should include:
 A duration of 5 years.
 Daily monitoring when possible3.
 Monitoring will start at around 5a.m.
 The monitoring should last for 6 months (March-April and July-
August ) (as shown in figure 1.).
The project include a BACI (Before-After-Control-Impact) study
design which is data taken before, after construction at impact sites and
at control sites. Also the replicated control and impact sites should be
equally spaced4 all over the ‘road effect’ area, will depend on the amount
of roads that are present in the study area (around the minimum of 6
roads).
The control sites5 need to be selected by the factors that should be
similar between the control and impact sites which are:
 the presence of common toads
 the abundance of the toad populations
 the type of land use by humans
 common toad habitat characteristics
Methods
The project will use a minimum of 3 trained volunteers each day who will be
walking around the ‘road effect’ area (the maximum migration area of toads)
and use GPS systems to locate the road-kills and map them on the area map.
Volunteers will be handed equipment, by the project supervisors, to use
during the monitoring the start of the monitoring sessions. The materials
include: gloves, GPS, magnifying glass and waterproof clothing.
The amphibian
tunnels had no effect
or reduced the
mortality of common
toads.
Inform the local
people around the
tunnel area about the
aims and goals of the
tunnels.
The local people will
support the project
and will keep the
tunnels clean and
safe.
Inform the world’s
public and scientific
community on the
data and results of
the project.
The conservation and non-
conservation projects will
enable the use the data and
results of the project to
undergo future more
rigorous scientific projects.
Background
Common toads breed and spawn in permanent ponds and they migrate to
terrestrial environments to hibernate1.
Several characteristics of the common toads make the species vulnerable to
road mortality and these characteristics include the migration of the young
and adult individuals to and from the ponds that they were born and the
appearance of the species in meta-populations1.
The recommended mortality mitigation measure2 of common toads is an
underpass which could be a box shaped tunnel and should be spaced at
every 50-60m between each tunnel.
References
1. Armstrong B., Baker J., Carson G. et al. (2011) Common toads and
roads: Guidance for planners and highways engineers in England
Amphibian and Reptile Conservation.
2. Iuell B., Bekker G.J., Cuperus R., Dufek J., Fry G., Hicks C., Hlavac V.,
Keller V., Rosell C., Sangwine T., Torslov N., Wandall B. (2003) COST 341
Wildlife and Traffic: A European Handbook for Identifying Conflicts and
Designing Solutions. KNNNV Publishers, Brussels.
3. Santos S. M., F. Carvalho, and A. Mira. (2011) How long do the dead
survive on the road? Carcass persistence probability and implications for
road-kill monitoring surveys. PLOS ONE 6
4. van der Grift E.A., van der Rodney R. and Fahrig L. et al. (2012)
Evaluating the effectiveness of road mitigation measures. Biodivers.
Conserv. Published online
5. Roedenbeck I.A., Fahrig L., Findlay C.S., Houlahan J.E., Jaeger J.A.G.,
Klar N., Kramer-Schadt S. and van der Grift E.A. (2007) The
Rauischholzhausen agenda for road ecology. Ecol. Soc. 12(1):11.
Figure 1. The figure shows the life cycle of
the common toads and seasonal activity
Key Milestones

SB
+
R
es
+
H2O
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[150μM
]
SB
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H2O
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[300μM
]
R
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H2O
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[150μM
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R
es
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H2O
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[300μM
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H2O
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H2O
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SB203580
Resveratrol
Vehicle
D
M
SO
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10
8
6
4
2
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Treatment
%Cytotoxicity
4.7780
8.0373
3.1953
11.1110
4.3043
7.3540
-0.5413
2.9233
0.6817
Mean % Cytotoxicity
Methods
Immortalised human corneal endothelial cells (HCEC), known as Zante were grown in culture to provide an in vitro model of the
eye. The p38 MAPK inhibitor SB203580 was included in some treatment conditions in order to investigate if resveratrol acts via
this pathway. Two different concentrations of hydrogen peroxide (H2O2) were used to simulate moderate and severe oxidative
stress in culture. Cells were treated in triplicate with one of the following treatment conditions:
• Resveratrol at 40μM
• SB203580 at 10μM
• H2O2 at 300μM
• H2O2 at 150μM
• Resveratrol + H2O2 at 300μM
• Resveratrol + H2O2 at 150μM
• Resveratrol + SB203580 + H2O2 at 300μM
• Resveratrol + SB203580 + H2O2 at 150μM
Cytotoxicity was measured using the Pierce™ LDH Cytotoxicity Assay kit and a 96-well plate reader at 492nm and 690nm
wavelengths. Maximum and Spontaneous LDH activity was also analysed along with a vehicle control (DMSO).
Resveratrol - friend or foe, for protection against oxidative damage in the
eye?
Author: ----------------- Supervisor: Dr Angela Sheerin
What is Resveratrol?
Resveratrol (3,5,4′-trihydroxystilbene) is a naturally occurring
polyphenol found in the skin of grapes and has been attributed to
providing some of the cardiovascular benefits associated with moderate
red wine drinking3. It has also been shown to mimic some of the
beneficial effects of calorie restriction including decreased production of
reactive oxygen species (ROS) and consequently protection against
oxidative stress 4. However, resveratrol exhibits many pro-apoptotic
properties and has been shown to induce apoptosis and halt proliferation
in many different cell types, particularly cancers 5.
References
1. Wakamatsu, TH., Dogru, M., Tsubota, K. (2008) Tearful relations: oxidative stress, inflammation
and eye diseases. Arq Bras Oftalmol. 71, 72-79
2. Prevention of blindness and visual impairment, Priority eye diseases, World Health Organisation
(2014), accessed May 2014.
3. Siemann, EH., Creasy, LL. (1992) Concentration of the phytoalexin resveratrol in wine. Am. J.
Enol. Vitic., 43, 49-52
4. Nakata, R., Takahashi, S., Inoue, H. (2012) Recent advances in the study on resveratrol. Biol.
Pharm. Bull., 35(3) 273-279
5. Aggarwal, BB., Bhardwaj, A., Aggarwal, RS., et al. (2004) Role of resveratrol in prevention and
therapy of cancer: preclinical and clinical studies. Anticancer research. 24, 2783-2840
Oxidative Damage and Eye Disease
Oxidative damage has been implicated in the development and exacerbation of many eye diseases including glaucoma, cataract
and age related macular degeneration 1. These are the top three leading causes of blindness worldwide according to the World
Health Organisation 2. Protecting the eye against excess oxidative stress could decrease cases of degenerative eye disease
worldwide. The aim of this study was to investigate whether resveratrol can provide protection against oxidative damage and
reduce cell death in an in vitro model of the eye. Hypothesis: resveratrol will have a protective effect against oxidative damage.
Results
Absorbance data across 5 weeks of repeat experiments was gathered. 3
sets of data were chosen for statistical analysis and converted into %
cytotoxicity for each treatment condition. The mean % cytotoxicity
across the 3 data sets is presented in figure 1. According to the two-way
ANOVA calculated using Minitab 16, there is no significant difference in
% cytotoxicity between cells treated with just H2O2, those treated with
resveratrol and H2O2 and those treated with resveratrol, SB203580 and
H2O2 at either the higher or lower concentration. Figure 2 is a boxplot
showing the variation in % cytotoxicity across the 3 data sets.
Conclusions and Discussion
• The presence of resveratrol at 40μM in cultured Zante cells does not significantly affect the
cytotoxicity of H2O2 at either 300μM or 150μM.
• Resveratrol does not appear to be using the p38 MAPK signalling pathway in this cell line to a
significant extent.
• The boxplot comparison shows that the data is spread unevenly across the different treatment
conditions, with a range of results across the 3 data sets. This suggests some inconsistencies
between the repeat experiments. The range in % cytotoxicity is large, suggesting there could be
issues with the repeatability of the experiment or with the experimental compound stock solutions.
Figure 1
SB
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H2O
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[150μM
]
SB
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[300μM
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H2O
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H2O
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]
SB203580
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esveratrol
Vehicle
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M
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15.0
12.5
10.0
7.5
5.0
2.5
0.0
Treatment
%Cytotoxicity
Boxplot of % Cytotoxicity
Figure 2

Evaluation of Polymeric Micelles for Drug Delivery
Author:-------------- Supervisor: Dr JPSalvage
Nowadays developing a biocompatible drug system has become a huge interest, especially research into targeting and delivery of diagnostic [1]. Drug delivery systems are being designed to achieve cell based delivery to increase drug selectivity and
drug efficacy [2] as well as function to act as a reservoirs for the drug themselves [1].
Polymeric micelles in recent years have received increase in science interest [3], due to their ability being able to offer a stable micelle and targeted drug delivery inside the body. Block copolymers self assembles to from stable micelle in a selective
solvent, which is a good solvent for one block but a poor solution for the other block [5]. The micelles consist of a hydrophobic core and a hydrophilic outer shell, the hydrophobic core which create a potential for the micelle to encapsulate hydrophobic
drug within its core during the formation of the micelles and can act as a non-aqueous reservoirs for the drug.
Micelles are formed from phosphorylcholine (PC) and Poly 2 diisopropylamino ethyl methacrylate (DPA).PC has a similar property to phospholipids; it contains a positive and negative charge end known as zwitterionic, this hydrophilic outer shell
provides excellent biocompatibility this will therefore minimize the micelle being rejected by the body. Meanwhile DPA; a hydrophobic polymer which is very to sensitive pH, is insoluble above pH6 but it will completely dissovles when pH drops
below 6.
The ratio of the copolymer used in this experiment were MPC30-DPA60 ,the micelles were formed in PBS solution to mimic the physiological pH of the human body to ensure that the diblock copolymer will self-assemble when injected to human body.
In this study . This study investigates the effect of different concentrations of MPC-DPA diblock copolymer on cell cytotoxicity in order to find out the critical micelle concentration and in addition to that the rate of uptake of the micelles at critical
micelle concentrations are determined.
Materials and Methods:
Cell Culture:
3T3 cells were were grown in Dulbecco's Modified Eagle Medium (DMEN) with 10% Fetal Bovine Serum (FBS) at 500µl
per well. Cells were allowed to grow in the well for 24 hours at 37oC in 5% CO2.
Preparation of MPC-DPA diblock copolymers:
400mg of the polymer were weighted out and was dissolved in 10ml of methanol (40mg/ml) , 100µL were pipette out and
injected into 9.9ml of phosphate buffered saline (PBS) giving a solution of micelles at 0.4mg/ml. Micelles will self-assemble
when the polymer are injected to PBS as the hydrophobic core will be surrounded by hydrophilic outer shell of MPC. 1.5mg
of Courmarin-6(CM6) were weighted out and dissolved into 5ml of polymer solvent to give a dye concentration of
0.3mg/ml. When this solution are injected into PBS micelleation and encapsulation of the dye will take place.
Particle characterization by Photon Correlation Spectroscopy(PCS):
The effect of decreasing polymer concentration on micelles properties (Z-averages , Kcps, Pdi) were investigated using
PCS. Measurements were taken at 25oC, 5minutes equilibrium using a Zetasizer NANO Z590 by Malvern Instrument Ltd.
Cell cytoxcity:
Cytoxcity of polymer, dye and solvents were carried out using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide(MTT) assay, with the addition of acidic isopropanol (IPA). Reduce of tetrazole to insoluble formzzan is initiated by
acidic isopropanol (IPA), this reduction only occurs in viable cells mitochondria.
Cellular Uptake::
After acknowledgment of critical micelle concentration cells were seeded in a 12 wells plate at 5000cells and 10000 cells and
were exposed to polymer which contains the dye for 1,4 and 24 hours to investigate rate of cellular uptake.
Statistics analysis:
• One way anova
• Turkey multiple comparison post-test
Cellular uptake result
Brightfield Fluorescent Overlay Control
1Hour24Hours
Fig2 shows flucrosence confocal laser scanning microscopy images of 3T3 cells
incubated with polmeric micelles encapsulated with dye at critial micelle
concentration
PCS and Cytotoxicity Results
0
10
20
30
40
50
60
70
80
90
0.8 0.4 0.2 0.1 0.05 0.025
Meannanopraticlessizeinnanometers/nm
Concentration of polymer mg/ml
Week 1
Week2
Week3
0
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0.15
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Week1
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Intensity(kcps)
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0 0.1 0.2 0.3 0.4 0.5
Percentagecellsurvival
Concentration of samples mg/ml
Polymer/So
lvent
Polymer/So
l/Dye
MeOH
PBS
Fig 1.2 A graph showing effect of different concentrations
of polymer on the polydispersity index with standard error
bars in each weeks result showing the data's standard
error.
Fig 1.1 Above a graph showing the effect of different
concentrations of polymer solution on mean
nanoparticles size(Z-average) with standard error
bars. Each week results are shown individually.
Fig1.3 A graph showing the intensity of the
polymers in kilocount per second (kcps).
Each week's results are presented
individually according to the polymer
concentration.
Fig1.4 A graph showing cell percentage survival
with different concentrations of samples from
normalized data where percentage cell survival were
calculated with each weeks respective control. Error
bars with standard error of the data are shown in the
graph.
Conclusion:
The micelles appeared to be stable at 25o C however the experiment was conducted at 25o C therefore for this polymeric
micelle to serve as a drug carrier experiments should be done at 37o C. Statistical test suggested that the type of sample the
cells were exposed to had no significant difference on the cell viability . The micelles showed the ability of encapsulating
CM6 dye and results from confocal images has shown that cell uptake of micelle are very efficient. Although results from
this experiment may not be promising for pharmaceutical application but it is certainly a promising future for live cell
imaging.
Reference:
1. Cot'i, K., Belowich, M., Liong, M.,
Ambrogio, M., Lau, Y., Khatib, H., Zink,
J., Khashab, N. and Stoddart, J. (2009).
Mechanised nanoparticles for drug
delivery. Nanoscale, 1(1), pp.16--39.
2. Pierige, F., Serafini, S., Rossi, L. and
Magnani, M. (2008). Cell-based drug
delivery. Advanced drug delivery reviews,
60(2), pp.286--295.
3. Salvage JP, Rose SF, Phillips GJ, Hanlon
GW, Lloyd AW, Ma IY, Armes
SP, Billingham NC, Lewis AL
4. Novel biocompatible phosphorylcholine-
based self-assembled nanoparticles for
drug delivery. J Control Release. 2005 May
18;104(2):259-70
5. Loh, W. (2002). Block copolymer
micelles. Encyclopedia of surface and
colloid science, pp.802--813
6. Giacomelli, F., Step'Anek, P., Giacomelli,
C., Schmidt, V., J"Ager, E., J"Ager, A.
and Ulbrich, K. 2011. pH-triggered block
copolymer micelles based on a pH-
responsive PDPA (poly [2-
(diisopropylamino) ethyl methacrylate])
inner core and a PEO (poly (ethylene
oxide)) outer shell as a potential tool for
the cancer therapy. Soft Matter, 7 (19), pp.
9316--9325.
7. Licciardi M , Craparo EF, Giammona G,
Armes SP, Tang Y , Lewis AL (2008)
In vitro biological evaluation of folate-
functionalized block copolymer micelles
for selective anti-cancer drug delivery.
Macromol Biosci. 2008 Jul 7;8(7):615-26

Is the Survival of Klebsiella Pneumoniae, from the stomach to the GI tract,
indicative of changes in specific virulence factors?
Author: --------------- Supervisors: Dr Angela Sheerin & Dr Ian Cooper
Introduction:
Over the years more and more pathogenic bacteria are causing infections resulting in diseases world wide. One such bacterium that has become an
important pathogen in recent years is Klebsiella pneumoniae , this pathogenic bacteria has been increasing mortality and morbidity in a plethora of
communities and countries. It has been considered generally opportunistic in causing nosocomial infections in those who are immunocompromised but in
the last decade cases have been seen in those who are considered healthy especially in Southeast Asia and is becoming important as it is starting to track
west i.e. to Europe[1]. This enterobacterium is accountable for up to 8% of urinary tract infections, pneumonia and soft tissue infection as well as having
resistance to multiple antibiotics-causing dilemma. Thus a comprehensive understanding of the physiological process of this pathogen seems necessary to
reduce infection process.
Klebsiella pneumoniae as with all bacteria have a pre determined ability to cause disease which is its pathogenicity (likelihood of causing the disease) which
is quantitative and can be measured, this is defined as virulence. Pathogens such as K.pneumoniae possess virulence factors which contribute to their
virulence. Virulence factors refer to properties for example gene products, that permit an microorganism such as K.pneumoniae to inaugurate itself on or
within a host consequently potentiating its ability to cause disease[2]. Such factors include cell surface proteins which moderate attachment of bacteria, cell
surface carbohydrates and proteins that protect the bacterium and bacterial toxins, hydrolytic enzymes, capsular polysaccharide (CPS), lipopolysaccharide
(LPS) and biofilm formation to name a few[3]. In addition bacteria such as K.pneumoniae possess virulence genes which aid in its survival and contributes to
the pathogenesis and thus differential serotype. Two important genes are magA and rmpA. The magA gene is usually associated with the
hypermucoviscosity phenotype and the rmpA gene confers a mucoid phenotype which is regulated by the availability of iron i.e. siderophores.
Hypermucoviscosity has been identified as a virulence factor among clinical bacteraemia isolates of K. Pneumoniae[4]. Expression of these virulence factors
can be modified depending upon its surroundings, for example the acidic environment of the stomach can directly influence what genes get turned on thus
altering the phenotype of the bacteria to cope with the new adjacent conditions. These virulence factors allow colonisation, immunoevasion,
immunosupression, entry in and out as well as obtaining nutrition from host. Colonisation of the gastrointestinal(GI)tract is the first port of call in an
Klebsiella pneumoniae infection, however before this it must be able to survive the transit from stomach to GI tract which acts as the barrier for defence
against infection. The harsh acidic environment (pH1.5-2.5) of the stomach should in effect “kill” the bacteria, however some survive and ultimately colonise
GI tact which can lead to sepsis. It can then be proposed that this survivability is inherently due to the virulence factors it possesses[5].
The underlying hypothesis is that Klebsiella pneumoniae that survives the stomach is most likely a result of certain virulence factors being
expressed. The use of caco2 cells will be used to test cytotoxicity to see if any products of the bacterium leeched out into environment which can
result in enhanced virulence and a string test to test for mucoviscosity(magA).
0
0.5
1
1.5
2
2.5
3
0 15 30 45 60
Length(mm)
Time interval (minutes)
Hypermucoviscosity comparison between Nutrient Broth and
Gastric Broth Colonies
NB pH7
GB pH2
Linear (NB pH7)
Linear (GB pH2 )
-20
0
20
40
60
80
100
120
140
160
15 30 45
NumberofColonies
Time Interval (minutes)
Average difference between nutrient and gastric colony counts
at 15,30 and 45 minutes
NB pH7
GB pH2
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Controls 24hr SN NB pH7 24hr SN GB pH2.5 24hr SN GB pH2
OpticalDensity(540nm)
Average Cytotoxicity comparison via MTT assay
Methods and Materials:
K.pneumoniae culturing ,broth set up ,serial dilution, supernatant and cell pellet collection. Cultures of Klebsiella pneumoniae spp. ozoaenae were donated
from the National Collection of Type Cultures and supplied by the University of Brighton Microbiology department. For control; Overnight cultures (ONC) were
made and 1ml pipetted into 100ml nutrient broth these were then incubated for 37 °C and timed for a maximum of 45 minutes to which at every 15 minute
interval a serial dilution was performed and plated and incubated at 37 °C for 24 hours. At 45 minutes, equal volumes of inoculated broth are centrifuged at
3000g for 10 minutes using a Heraus centrifuge by thermoelectron. Cell pellet and supernatant separated and frozen. For gastric experiment . 1g of mince beef
is autoclaved and mixed with 10ml of an ONC and left on the orbital shaker for 1 hour. Following this contents of universal are empty into gastric both prepared
by Microbiology staff. And steps aforementioned are repeated. All steps carried out in sterilised conditions near Bunsen flame. API 20e was also carried out.
Viable count , string test and CFU. Colonies are counted using a Fisher Scientific colony counter. String test to test hypermucoviscosity. Single colonies also
scooped up using inoculating loop and frozen in 1ml nutrient broth +10% glycerol. CFU/ml is worked out using ;average number of colonies x (1/volume
plated)x dilution factor.
Tissue culturing, seeding density and plating. Caco2 cells in Gibco’s DMEM from life technologies were routinely passsaged and trypsinised(GE healthcare)
from T75 flask. Seeding density is calculated with aid from haemocytometer. Appropriate number of cells plated onto12 well plate and incubated for 24 hours at
37 °C in a Hera cell incubator. All steps carried out in ESCO class II BSC safety biological hood, whilst wearing latex gloves.
Co-Culturing, and Cytotoxicity (MTT assay). Supernatant from both gastric and control experiment are cultured together with Caco2 cells in 12 well plate. In
addition cell pellets are co cultured. NOTE these are cell pellets from the colonies scooped and placed in 1ml nutrient broth and 10% glycerol, these are re-
suspended in an ONC and separated to get cell pellet and then pipetted into 12 well plates. In addition supernatant and cell pellet combined are pipetted into
wells in 12 well plate. Cell pellets are cultured for 4 hours and supernatant 24 hours. MTT assay is carried out and results are read on a Thermofisher plate
reader from a 96 well plate.
Development of methodology:
Multiple methods were carried out before a working model was found which was
sufficient to distinguish survivability in an environment that simulated stomach
conditions . Initially no ONC’s were done and gastric broth had a pH of 1.5. Slight pH
changes and addition of certain factors such as meat as well as inoculation volume
resulted in a working model.
Figure 1:
Shows example
of optimising
meat
experiment ,
from left to
right, 1g meat
with 1ml, 5ml
and 10ml
inoculates in
pH2 gastric
broth.
Statistical Analysis:
One way ANOVAs
Tukeys pair wise Comparison test
P value< 0.05 significance level
Results:
Figure 2. Results show good percentage identification from meat experiment
therefore we know that it is Klebsiella pneumoniae in the culture/meat and a
small percentage that it may be another bacterium i.e. Citrobacter freundi.
Figure 3. Shows graphical representation of difference in number of colonies at each time interval. We can see that there is huge
difference between the broths at all intervals between gastric and nutrient (control) broth. At each time interval for the gastric broth we
can see a depreciation in the number of colonies from 15 to 45 minutes.
Figure 4. Shows results from string test which . The values of the average length of colonies for those grown from nutrient broth have all
very similar results. Increasing from 15 to 45 minutes the length from the string test of the colonies from gastric broth increase at each
interval exponentially.
Conclusion:
Colonies that were subjected to gastric broth behaved differently to those that interacted to the nutrient broth. From figure 3 we can see that there is
a significant kill off rate from just viewing the graph. The transit time from stomach to GI tract is approximately 30 minutes. We can see that those
that survived at 45 minutes are most likely to be the ones that go on to colonise the GI tract, this survival can be proposed to virulence factors being
expressed which enhance the pathogenicity of the pathogen. The Klebsiella pneumoniae that was plated (45mins) showed an increase in length the
longer it remained in the broth, showing characteristics of magA expression Therefore there is a link between CFU and mucoviscosity at 45 minutes,
although colonies are fewer they are more mucoviscous. Statistical analysis was carried out on the gastric broth (no bacterium) and control (just
caco2) in the form of an ANOVA it gave a p value >0.05 therefore showing no significant difference, therefore the cell killing is down to the bacterium
being cultured. P value<0.05 for comparison between nutrient broth supernatant and gastric broth supernatant indicates significant difference. In
addition Tukeys pair wise comparison carried out on experimental supernatant of control, and experimental supernatant of pH2.5 and pH2 gastric
broth showed that there is no statistical difference between the gastric broth in cytotoxicity however there is a difference compared to control, which
indicates that a stressed environment results in virulence factors being expressed leading to expression of different phenotype able to cope with
hostile surroundings.
It can therefore be said that the pathogenicity of a bacteria depends on the aptness to employ virulence factors which are then localised to the cell
surface, released into the extracellular milieu or injected directly into cells of the host.
Further Work: Looking at biofilm formation as well cytokine response could be looked at in the future.
Figure 5. Shows the difference in cytotoxicity between the control and nutrient broth and two gastric broth experimental supernatants
References:
[1] Groopman, J (2008-08-11), "Superbugs", The New
Yorker, retrieved 2013-07-07, "The new generation of resistant
infections is almost impossible to treat.“
[2] Chen LH, Xiong ZH, Sun LL, Yang J and Jin Q, (2012). VFDB
2012 update: toward the genetic diversity and molecular
evolution of bacterial virulence factors. Nucleic Acids Res. 40 (1),
pD641-D645. (doi: 10.1093/nar/gkr989)
[3] Yuen-Fun LH. (2007). Identification of Endogenous
Mechanisms That Affect Klebsiella pneumoniae Growth in the
Murine Host . PhD dissertation. University of Michigan, P1.
[4] Yu VL, Hansen DS et al. (2007). Virulence characteristics of
Klebsiella and clinical manifestations of K. pnemoniae
Bloodstream infections. Emerging Infectious Diseases. 13 (7),
p986-991
[5] Coudeyras S, Nakusi L, Charbonnel N, Forestier C. (2008). A
Tripartite Efflux Pump Involved in Gastrointestinal Colonization
by Klebsiella pneumoniae Confers a Tolerance Response to
Inorganic Acid. Infection and Immunity. 76 (10), p4633-4641

Investigation of the gliaprotective potential of N-acetylcysteine amide in an in vitro model of
hydrogen peroxide-induced oxidative stress in diabetic neuropathy
Author: ----------- Project Supervisor: Dr Jimi Adu
Introduction
It is estimated that over half of all diabetic patients develop symptoms associated with diabetic neuropathy, which encompasses a
variety of nervous system disorders affecting the peripheral nervous system[2]. Typically, disturbances in peripheral nervous system
conduction produce sensory and motor difficulties, causing pain, paresthesia (pins and needles, numbness, burning and prickling
sensations) of limbs, loss of temperature, pain and pressure sensation, muscle weakening, wasting, ,recurrent infection of the lower
limbs and feet, and a host of cardiac, gastrointestinal, urogenital and ocular problems that are a huge cause of morbidity and
mortality for the diabetic population[2,3]. The current lack of effective treatment options means many sufferers struggle to manage
their symptoms, and lead a poor quality of life.
The pathogenesis of diabetic neuropathy is well understood, and oxidative stress is of principal concern in the development of
diabetic neuropathy complications[4]. Oxidative stress arises due to an imbalance in the production of reactive oxygen species (highly
reactive oxygen molecules with an unpaired electron) and the efficacy of the mechanisms that exist to prevent their damage[4]. The
link between hyperglycaemia and overproduction of reactive oxygen species is now well-established, and thus, the diabetic
population are at increased risk of their detrimental effects. Beneficial actions of reactive oxygen species include regulation of cell
division, apoptosis, inflammation and phagocytosis, but at increased concentrations they contribute to the loss of neurotrophic
factors necessary for neuronal development, promote apoptosis of neurons and glial cells, and induce peroxidation of DNA, lipids
and proteins[4]. The mechanisms that contribute to reactive oxygen species overproduction are complex, but in all cases
hyperglycaemia is the underlying stimulus[5].
The most effective preventative measure of diabetes complications is tight glycaemic control, but the burden associated with this
ultimately leads to problems[5]. The use of antioxidants, the biological molecules that inhibit oxidation by scavenging free radicals, as
therapeutic agents for diabetic neuropathy has had partial success in improving both sensory and motor nerve conduction, reducing
the abundance of reactive oxygen species, catabolizing autoxidative species, and preventing the extent of lipid peroxidation in
hyperglycaemia-induced rats[6]. However, a suitably effective antioxidant therapeutic is still far away, and further investigation is
needed to address the alarming increase of diabetic complications that is resulting from the increasing prevalence of diabetes
mellitus[1].
The purpose of this study was to investigate the gliaprotective potential of N-acetylcysteine amide (AD4) on RT4-D6P2T rat Schwann
cells exposed to hydrogen peroxide to model the oxidative damage that occurs in diabetic neuropathy. AD4 has previously been shown
to replenish intracellular thiols of red blood cells to reduce the extent of oxidative damage, and this identifies it as a potential effective
therapeutic agent in diabetic neuropathy[7]. The majority of studies investigating treatment options in diabetic neuropathy have
focused their efforts on neuronal involvement, but the ability for hyperglycaemia to demonstrate a cytotoxic effect in neurons has
historically proven difficult[8]. For this reason, the involvement of Schwann cells has been investigated in this study, as the interplay
between these cell types is vital in nervous communication, yet the involvement of glial cells in diabetic neuropathy is relatively
unexplored.
Maintenance of rat RT4-D6P2T Schwann cells: RT4-D6P2T cells were maintained in 1x Dulbecco’s modified eagle’s medium (DMEM)
in 5% heat inactivated foetal bovine serum (FBS) at 37°C in a 5% CO2 incubator.
Preparation of cell culture dishes: Poly-L-ornithine (PORN) was prepared in 0.5M borate buffer, in hydrogen water at a pH of
8.4, and 1ml was added to 35mm Nunclon dishes and left to dry overnight. Dishes were aspirated and washed three times with cell
culture grade water, and dried. 20µl of 20µg/ml laminin was added to 980µl DMEM and mixed, and 70µl of this was added
to the centre of each dish. Dishes were maintained at 37°C in a 5% CO2 incubator for three hours, before being washed twice with
1ml DMEM, and once with 1ml DMEM containing 5% FBS. Dishes were replaced in the incubator.
Determination of RT4-D6P2T cell density: Cell density concentrations of 5x103 cells/ml, 1x104 cells/ml, 1x105 cells/ml and 1x106
cells/ml were seeded in prepared dishes, and incubated overnight. Cellular viability was assessed using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and absorbance values measured at 540nm with a spectrophotometer using
Digiread. Absorbance values were calculated where
% cellular viability = (absorbance of treated cells/absorbance of untreated cells) x 100.
Determination of hydrogen peroxide concentration: A hydrogen peroxide concentration range was established, and cellular viability
assessed using the MTT assay. Prepared dishes with seeded cells were exposed to 1mM, 10mM, 50mM and 100mM hydrogen
peroxide for 24 hours and 48 hours, to determine the most effective time period to achieve a desired 50% reduction in cellular
viability.
Assessment of the protective potential of AD4: AD4 was administered at concentrations of 1mM, 10mM, 50mM and 100mM in co-
treatment and pre-treatment schedules. For co-treatment, cells were exposed to hydrogen peroxide and AD4 simultaneously, and
cellular viability was assessed 24 hours later. For pre-treatment, cells were exposed to AD4 and incubated for 24 hours prior to
addition of hydrogen peroxide.
Statistical analysis: All statistical analysis was performed using Minitab 17. Data are expressed as mean ± standard error of the mean.
One-way analysis of variance was performed to test for significant differences between population means. Tukey’s method was used
to determine the nature of statistically different data. In all cases of statistical testing, a significance level of p < 0.05, indicating
statistical difference, was used. * indicates statistical significance.
0.25 0.25
0.89
2.38
0
0.5
1
1.5
2
2.5
3
1.00E+03 1.00E+04 1.00E+05 1.00E+06
Meanabsorbanceat540nm
log10 Cell density concentration (cells/ml)
Figure 1. Mean absorbance values at varying RT4-D6P2T cell density concentrations As
the MTT assay is only effective within the linear range of the graph, an appropriate cell
density concentration within this range was chosen. The chosen cell density was 4x105
cells/ml.
Results
86
45
39 42
100
93
37
28 28
0
20
40
60
80
100
120
Control 1 10 100 1000
Meancellularviability(%)
Hydrogen peroxide concenration (mM)
24 hours
48 hours
Figure 2. Mean cellular viability of RT4-D6P2T cells following 24 hours and 48 hours
exposure to hydrogen peroxide There was no significant difference between the mean
cellular viabilities at hydrogen peroxide concentrations of 1mM and 10mM between 24
hours and 48 hours, The mean cellular viabilities were significantly greater after 48
hours at concentrations of 100mM and 1000mM. A concentration of 10mM after 24
hours was chosen, so to reduce cellular viability by approximately 50%.
100
66
88 89*
96* 101*
91*
0
20
40
60
80
100
120
Control CH2O2 CAO 1 10 50 100
AD4 Concentration (mM)
Figure 3. Mean cellular viability of RT4-D6P2T cells following 24 hours exposure to
hydrogen peroxide and co-treatment with AD4 All concentrations of AD4 produced a
significantly greater mean cellular viability compared to CH2O2. There was no significant
difference in mean cellular viability between concentrations of AD4.
100
75
52
84
102* 97*
91* 95*
0
20
40
60
80
100
120
Control CH2O2 C100H2O2 CAO 1 10 50 100
AD4 concentration (mM)
Figure 4. Mean cellular viability of RT4-D6P2T cells after 24 hours pre-treatment with
AD4 prior to 24 hour exposure to hydrogen peroxide All concentrations of AD4
produced a significantly greater mean cellular viability compared to CH2O2. There was
no significant difference between concentrations of AD4. C100H2O2 produced a more
appropriate reduction in cellular viability towards the desired 50% reduction.
89
96 101
91
102
97
91
95
0
20
40
60
80
100
120
1 10 50 100
AD4 concentration (mM)
Cotreatment
Pretreatment
Figure 5. Mean cellular viability or RT4-D6P2T cells in both co-treatment and pre-
treatment schedules There was no significant difference in the mean cellular viabilities
at equivalent concentrations between co-treatment and pre-treatment schedules.
100 100
88
84
0
20
40
60
80
100
120
Cotreatment Pretreatment
Treatment schedule
Control
CAO
Figure 6. Mean cellular viability of Control and CAO groups in co-treatment and pre-
treatment schedules In both the co-treatment and pre-treatment schedules, there was
no significant difference in the mean cellular viability between the Control and CAO
groups, suggesting no cytotoxic effect of AD4 at concentrations of 100mM.
Conclusions
The data show that in both co-treatment and pre-treatment schedules, all concentrations of AD4 produce a significantly greater mean cellular viability compared to the
untreated CH2O2, and suggest that AD4 is effective in protecting RT4-D6P2T cells from hydrogen peroxide-induced oxidative stress. The data show that both co-treatment and
pre-treatment schedules were equally effective in their protective potential, and there was no significant difference between the concentrations of AD4 used. Additionally, the
data suggest there was no cytotoxic effect of AD4 at concentrations of 100mM compared to the Control population. This study has corroborated the antioxidant potential of
AD4 confirmed by others, but also identifies its potential use as a therapeutic agent in diabetic neuropathy[8]. This is a promising finding in the fight against complications of
diabetes mellitus, where current treatment options are lacking, and thus, affected individuals lead a poor quality of life. The results of this study generate potential future
directions for study in the assessment of AD4 as an effective treatment option for delaying or preventing the extent of cellular damage resulting from oxidative stress in
diabetic neuropathy.
References
[1] Shaw, J. E., Sicree, R. A. and Zimmet, P. Z. (2010) Global estimates for the prevalence of diabetes for 2010 and 2030, Diabetes Res Clin Pr 87(1): 4-14
[2] Said, G. (2007) Diabetic neuropathy – a review, Nat Clin Pract Neurol 3(6): 331-340
[3] Vinik, A. I., Maser, R. E., Mitchell, B. D. and Freeman, R. (2003) Diabetic Autonomous Neuropathy, Diabetes Care 26(5): 1553-1579
[4] Sytze van Dam, P. (2002) Oxidative stress and diabetic neuropathy: pathophysiological mechanisms and treatment perspectives, Diabetes Metab Res 18(3):
176-184
[5] Figueroa-Romero, C., Sadidi, M. and Feldman, E. L. (2008) Mechanisms of disease: The oxidative stress theory of diabetic neuropathy, Rev Endocr Metab Dis
9(4): 301-314
[6]Bravenboer, B., Keppelle, A. C., Hamers, F. P. T., van Buren, T., Erkelens, D. W. and Gispen, W. H. (1992) Potential use of glutathione for the prevention and
treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat, Diabetologia 35(9): 813-817
[7] Grinberg, L., Fibach, E., Amer, J. and Atlas, D. (2004) N-acetylcysteine amide, a novel cell-permeating thiol, restores cellular glutathione and protects human
red blood cells from oxidative stress, Free Radic Biol Med 38(1): 136-145
[8] The Diabetes Control and Complications Trial Research Group (1993) The Effect of Intensive Treatment of Diabetes on the Development and Progression of
Long-Term Complications in Insulin-Dependent Diabetes Mellitus, New Engl J Med 329(14): 977-986
Control: H2O2 -; AD4 - CH2O2: 10mM H2O2; AD4 - C100H2O2: 100mM H2O2; AD4 - CAO: H2O2 -; 100mM AD4

Male Mosquitoes Use Wing Beat Frequency to Establish
Hierarchy of Dominance
----------------- Supervisor Dr Ian Russell
Methods
Results
Conclusions
References:
1. Gibson G, Warren B, Russell IJ. Humming in tune: sex and species recognition by mosquitoes on the wing. Journal of the Association for Research in Otolaryngology :
JARO. 2010;11(4):527-40.
2. Cator LJ, Harrington LC. The Harmonic Convergence of Fathers Predicts the Mating Success of Sons in Aedes aegypti. Animal behaviour. 2011;82(4):627-33.
3. Beckoff, M. Quantitative studies of three areas of classical ethology: social dominance, behavioral taxonomy and behavioral variability. In Quantitative Methods in the
Study of Animal Behavior: 1977;p1-46.
Landaus index of linearity (LID): EXP 1: 0.8, EXP2:
0.95, EXP3: 0.725
(It has been suggested that a LID of over 0.9 is
considered a strong linear hierarchy)[3].
Dominance index (DI): was calculated for every
mosquito in each experiment and showed that in
most cases there were males that always
increased in frequency and males that always
decreased in frequency this
can also be seen in the interaction tables.
Setup for male-male interactions:
Mosquitoes were tethered and allowed
to fly in circles within min distance 5cm
and max distance 13 cm apart from each
other. Microphones were placed 5 cm
away from the centre of each flight path.
Microphones were placed in such a way
so that WBF’s from each mosquito can
be isolated.
Experiments were carried out with five
male mosquitoes and each were put
against each other once. Recordings
were converted into log spectrums and
analysed in Excel (Microsoft).
Setup for female choice
experiment: Two male mosquitoes
are tethered and allowed to fly in
circles at two opposite sides of a
30cm3 netted cube with equal sides.
Microphones were placed net side
5cm away from the centre of their
flight paths. Virgin female
mosquitoes are allowed to free fly
around the cube and allowed to
interact with the males. This
experiment was repeated 6 times
with different males and virgin
females.
In the Male-Male interactions there were four main findings, they are:
• Male mosquitoes interact with other male mosquitoes by changing their own WBF’s in order to avoid sharing a
common frequency.
• Male mosquitoes that initially fly at a higher frequency tend to increase frequency when interacting with a male of a
lower initial frequency.
• Landaus index of linearity shows a moderate to strong hierarchy of dominance which suggests that wing beat
frequency could be a mechanism of dominance in mosquitoes.
• Dominance index shows there tends to be at least one male that always increases in WBF and one male that
decreases in WBF in every interaction, possibly the winners effect.
For the Female choice experiment there wasn't enough data to come to a conclusion but a clear description of the
female – male interactions could be produced. Also the females seemed to prefer the males with a higher frequency
but no statistical significance could be done . This research provides a platform for further research into WBF
matching as a dominance trait.
Spectrogram showing the fundamental and 2 harmonics
from two males interacting with each other. They both start
at a similar initial frequency and the higher frequency male
increases in frequency and the lower frequency male
decreases.
Mosquitoes of the Culex quinquefasciatus species, were reared in the lab for 10 days until adult. Using temperature
induced narcosis (produced by freezing for approx. 30 seconds) the mosquitoes were tethered to 100μm stainless
steel wire using super glue (Loctite Henkel Limited) by their dorsal thorax. The steel wire was placed in a glass
capillary mounted on a micropositioner to allow the mosquito to fly in circles around it. Experiments were
performed in a sound proof booth during the day (between 9:30 and 16:30). Between experiments flight was
prevented using a small square of tissue paper placed on the mosquito’s legs and initiated by removing the tissue
paper. Flight tones were recorded using two particle velocity microphones (Knowles NR-23158-000) which were
placed 5cm from the flight path center of each mosquito. The stereo output from the microphones were amplified
before being digitalized using a Fireface UC audio interface (RME-Audio). The audio signals were recorded using
Spectrogram 16 (Visualization Software)(INSERT RATE). All recordings were stored on a laptop PC (adapted from
Gibson and Russell) 2006).
The tables show WBF increase and decrease from three sets
of 5 male mosquitoes interacting separately in pairs. Red
down arrows signify a decrease in WBF when interacting
with another male and a green up arrow signifies a increase
in WBF.
Spectrogram showing an
interaction between a male
and a virgin female. The female
moves close to the male
increasing WBF until her third
harmonic is within 10 Hz of
males second harmonic. The
point when the females signal
is the loudest is the point of
frequency matching.
Male – Male interactions:
The results show that in 92.6% of the interactions
the male mosquitoes with the higher initial
frequency increased their WBF when interacted
with a mosquito with a lower initial frequency.
During the experiment there were very
limited number of interactions due to
problems with getting the females to fly.
Three interactions were noted and 2/3 of
the interactions were with the higher WBF
male and the interaction with the lower
ended with the male increasing and
maintaining a higher WBF.
Female Choice experiment
Summary
• Mosquitoes detect periodic air displacement associated with tones created by wing beats of
nearby mosquitoes using an incredibly sensitive mechanoreceptor called the Johnston
organ.
• Species and sexual recognition is believed to be achieved through wing beat frequency
(WBF) matching of a male and females wing beats[1].
• WBF matching is also believed to be implicated in mate selection by the female and has
been correlated with mating success(2).
• In this study we focus on WBF matching as a form of dominance in male – male interactions
and secondly female WBF preference.

Population structure and genetic diversity of the
natterjack toad.
Student: ------------------------ Supervisor: Dr Inga Zeisset
The natterjack toad (Bufo/epidalea calamita) (Fig1) is of
conservation concern due to its declining numbers in
northern Europe1. This decline is accompanied by a low
genetic diversity - a further cause for concern2,3. In
contrast, populations in southern Europe show both
greater numbers and greater genetic diversity2,3.
Factors contributing to these differences across the
species’ geographic range include: differences in
population connectivity due to differing habitats,
phylogeographic history, and northerly populations’
position at the species’ range edge2,3.
Introduction
In this study, comparisons were made between
several natterjack toad populations from northern
and southern Europe - namely the United Kingdom
(UK) and Iberian Peninsula (Portugal and Spain),
respectively. Fig2 shows population locations.
The purpose of the study was to compare genetic
diversity, population structure, and migration
within these populations.
Aims:
Compare populations in the UK and Iberian
Peninsula with regards to:
• Levels of genetic diversity.
• Levels of genetic differentiation
between populations/evidence for
connectivity.
• Levels of migration between
populations, and relate levels of
migration to distance between
populations.
Objectives
Figure 2 Sampling locations
UK
Table 1 Measures of genetic diversity
Results
Figure 3 Proportional membership of individuals to
genetic clusters
Conclusions
References
 Iberian natterjack toad populations showed
greater genetic diversity than UK populations.
 Contrary to expectations some (limited)
evidence found for:
o Greater differentiation and less
connectivity between Iberian
populations than UK populations.
o Greater levels of migration between
UK populations.
 Increasing the number of sampling locations
would clarify assessments of migration and
population connectivity, and help to explain
unexpected results.
Site n Mean no.
alleles/locus
Mean Allelic
Richness
Mean He Mean Ho
Southerness 40 2.13 1.86 10.30 10.63
Dunnerholme 40 3.00 2.38 13.94 13.38
Sandscale 40 2.88 2.34 14.35 15.63
Algarve 33 9.75 6.32 22.76 17.75
South Doñana
Park
13 8.88 6.93 9.43 8.13
North Doñana
Park
37 10.13 6.32 24.82 17.75
UK (all) 120 2.19 2.67 12.86 13.21
Iberia (all) 83 6.53 9.58 19 14.54
Data collection
8 microsatellite loci genotyped by
PCR after sample collection. Fig2
shows sampling locations.
Data quality
Data checked for: Hardy-Weinberg
equilibrium and linkage equilibrium
- GENEPOP 4.25. Scoring errors and
null alleles - MICROCHECKER 2.2.36.
Genetic diversity
Analysis of: allelic richness,
observed heterozygosity (Ho),
expected heterozygosity (He),
alleles per locus - FSTAT 2.9.37.
Population structure
Analysis of differentiation - FST and
DEST - GeneALEx 6.58.
Cluster analysis and identification of
migrants - STRUCTURE 2.3.49.
Statistical tests
Mann-Whitney U tests - Minitab 17.
Means are across all loci and individuals n: sample number, He: expected
Heterozygosity, Ho: observed heterozygosity
(a) FST DEST FST DEST
Southerness Dunnerholme
Dunnerholme 0.122 0.114 - -
Sandscale 0.131 0.128 0.010 0.004
(b) FST DEST FST DEST
Algarve South Doñana Park
South Doñana Park 0.075 0.486 - -
North Doñana Park 0.049 0.310 0.054 0.319
Table 2 Measures of population differentiation
Pairwise estimates of FST and DEST (a) UK populations (b) Iberian Populations. Estimates
are significantly different from zero (p=0.05) except for the comparisons for
Dunnerholme with Sandscale.
Genetic diversity
Table 1 shows measures of genetic diversity. All
measures except mean Ho were significantly lower
(p=<0.05) for the combined UK data than the
combined Iberian data.
Q= proportional membership to cluster, K= population/cluster number,
vertical bars represent individuals, different colours of bars correspond to
different clusters, black vertical lines separate prior population (sampling
location) information. (a) UK data (b) Iberian data
Population structure –
clustering analysis
Dunnerholme and Sandscale represent
a single population, with Southerness
being distinct (Fig3a).
The Algarve represents a distinct
population, whilst the sampling
locations in Doñana National Park
represent distinct clusters but show
interconnectivity and admixture
(Fig3b).
1. IUCN-The World Conservation Union. (2013) IUCN Red List of Threatened
Species 2013.
2. Beebee TJC, Rowe G. (2000) Microsatellite analysis of natterjack toad Bufo calamita
Laurenti populations: consequences of dispersal from a Pleistocene refugium. Biol J
Linn Soc 69(3):367-81.
3. Oromi N, Richter-Boix A, Sanuy D, Fibla J. (2012) Genetic variability in geographic
populations of the natterjack toad (Bufo calamita). Ecol Evol 2(8):2018-26.
4. The Amphibian and Reptile Trust, Accessed from: http://www.arc-trust.org/ on
16/04/2104
5. Raymond M, Rousset F. (1995) Genepop (version-1.2) - population-genetics software
for exact tests and ecumenicism. J Hered 86(3):248-9.
6. Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P. (2004) MICRO-CHECKER:
software for identifying and correcting genotyping errors in microsatellite data. Mol
Ecol Not 4(3):535-8.
7. Goudet J. (1995) FSTAT (Version 1.2): A computer program to calculate F-statistics. J
Hered 86(6):485-6.
8. Peakall R, Smouse PE. (2012) GenAlEx 6.5: genetic analysis in Excel. Population
genetic software for teaching and research-an update. Bioinformatics 8(19):2537-9.
9. Pritchard JK, Stephens M, Donnelly P. (2002) Inference of population structure using
multilocus genotype data. Genetics 155(2):945-59.
Image from The Amphibian and Reptile Trust4
Figure 1 The natterjack toad
Population differentiation - UK
Table 2 shows differentiation
measures. The data suggests
interconnectivity between the
neighbouring populations of
Dunnerholme and Sandscale and
isolation by distance from Southerness
(see Fig2).
Population differentiation -
Iberian Peninsula
See Table 2. A clear relationship
between distance and differentiation
of populations is not seen.
The estimates of DEST may suggest
greater differentiation between the
Iberian populations than the UK
populations.
Iberian Peninsula

 Ageing of the enteric nervous system (ENS), which is responsible for the innervation of the gastrointestinal (GI)
tract, is frequently associated with the development of chronic constipation resulting in slow transit and faecal
impaction in sufferers[1-3].
 Age-related changes within the GI tract may be due to alterations in neuronal signalling processes underlying
mucosal reflexes.
 Serotonin (5-HT) is an important neurotransmitter and paracrine signalling molecule involved in the initiation and
regulation of colonic mucosal reflexes. It is released from enterochromaffin (EC) cells located in the mucosal layer in
response to chemical or mechanical stimulation[4,5].
 It has been proposed that mucosal 5-HT activates 5-HT3 receptors on the mucosal endings of intrinsic primary
afferent neurons (IPANs). This results in the activation of ascending excitatory interneuron causing them to release
acetylcholine (ACh) which then binds to nicotinic receptors on excitatory motor neurons.[4-7]
 Activated motor neurons release ACh and tachykinins (TKs) which activate M2/M3 and NK2 receptors, respectively,
on smooth muscle cells to induce smooth muscle contraction oral to stimulation site (Fig. 1).[6,7]
Figure 1| Neuronal signalling pathways involved in
mediating smooth muscle contraction (as well as
relaxation) during mucosal reflexes.[5]
5. Results
Figure 3| Age-related changes on the pattern of evoking mucosal reflexes. Stroke-response curves of mucosal reflexes in (A) the
proximal colon and (B) the distal colon from animals aged 3, 12 and 18 months. Data shown as mean ± SEM, n=3-7. *p<0.05,
**p<0.01, ***p<0.001 vs 3 month group; † p<0.05, †† p<0.01 vs 12 month group.
Figure 4|The effects of age on mucosal reflexes in the distal colon. Bar graphs illustrating the effects of age on the (A) area, (B)
amplitude and (C) duration of mucosal reflexes evoked by 1 stroke in the 3, 12 and 18 month old animals. Data shown as mean ±
SEM, n=5. *p<0.05 ,**p<0.01 vs 3 month group.
The aim of this study was to investigate the effects of age on colonic mucosal reflexes in male C57BL/6J
mice and to determine whether alterations neuronal signalling contributes to this process.
Animals: C57BL/6J male mice aged 3, 12 and 18 months were studied.
Tissue Preparation: An incision was made along the mesenteric border of the whole colon and openings were pinned
flat to bath floor. With mucosal surface facing uppermost, two transducers were connected to the colon; one in the
proximal region and one in the distal region. The preparation was left to perfuse in warm buffered Krebs solution for
40 minutes, prior to recordings.
Evoking Mucosal Reflexes: A fine paint brush (size 2/0) was used to stoke the central region of the mucosal surface in
an oral to anal direction. The mucosa was stroked 1-5 times.
Pharmacology: Tissue initially perfused with 1 µM scopolamine (muscarinic antagonist) for 20 minutes before evoking
mucosal reflexes. The preparation was subsequently left to perfuse for a further 20 minutes with both scopolamine
and 1 µM GR159897 (NK2 antagonist) before mucosal stimulation. In separate experiments, the colon was perfused
with 1 µM ondansetron (5-HT3 antagonist) for 20 minutes before mucosal stimulation.
Figure 2| Mucosal reflexes generated in (A) the proximal and
(B) distal colon. Parameters measured (C) when analysing
mucosal reflexes.
Proximal Colon
Distal Colon
Figure 5| Pharmacological characterisation of mucosal reflexes with age. Bar graphs illustrating the effects of age and drug
treatment on the area of mucosal reflexes evoked by 1 stroke in the proximal colon of (A) 3 month, (B) 12 month and (C) 18 month
old animals. Similar graphs for the distal colon of (D) 3 month, (E) 12 month and (F) 18 month old animals. Data shown as mean ±
SEM, n=4-6. *p<0.05,**p<0.01 vs control. Treatments = Scopolamine (SCOP) and GR159897. No treatment = Control.
Investigation of the effects of age on colonic mucosal reflexes and associated neuronal
signalling pathways in a mouse model
Presented by ------------------------, Project Supervisor: Dr Mark Yeoman
School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, BN2 4GJ
D E F
A B C
A B C
A B
 Typical sample trace of the ascending contractile
response generated in the proximal (Fig. 2A) and
distal (Fig. 2B) colon during a mucosal reflex.
 The fast contractile response of a mucosal reflex
(Fig. 2A) is mediated by ACh.
 The tachykinins mediate a slower contractile
response that is usually seen at a higher number of
strokes (Fig. 2B).
 The area (g.s), amplitude (g) and duration (sec)
were the three parameters measured (Fig. 2C) to
assess the effects of age on mucosal reflexes.
A B C
2. AIM
1. Introduction
REFERENCES
[1] Rao S, Go JT. Update on the management of constipation in the elderly: new treatment options. Clinical Interventions in Aging. 2010; 5: 163-171. [2] Saffrey MJ. Ageing of the enteric nervous system. Mechanisms of Ageing and Development 2004;125(12):899-906. [3] Patel BA, Patel N, Fidalgo S, Wang C, Ranson RN, Saffrey M, et al. Impaired colonic motility and reduction in tachykinin signalling in the aged mouse. Experimental Gerontology. 2014; 53: 24-30. [4] Bertrand PP, Bertrand RL. Serotonin release and uptake in the gastrointestinal
tract. Anatomical Neuroscience: Basic and Clinical 2010; 153:47-57. [5] Dickson EJ, Heredia DJ, Smith TK. Critical role of 5-HT1A, 5-HT3, and 5-HT7 receptor subtypes in the initiation, generation, and propagation of the murine colonic migrating motor complex. Am J Physiol Gastrointest Liver Physiol 2010; 299(1):G144-157. [6] Galligan JJ.Pharmacology of synaptic transmission in the enteric nervous system. Current Opinion in Pharmacology 2002;2:623-629. [7] Grider J. Neurotransmitters Mediating the Intestinal Peristaltic Reflex in the Mouse.
JPET 2003;307(2):460-7.
6. Conclusions
P
4. Mucosal Reflexes & Analysis
 Mucosal reflexes in the proximal colon were evoked in an all-or-nothing manner irrespective to age and stimulus intensity. However, mucosal reflexes evoked in the distal colon showed a curvilinear pattern with the 12 month old animals generating
stimulus-dependent responses, whilst the 3 and 18 month old animals did not.
 The area of the mucosal reflex response decreased with age in the distal colon and this correlated with a decrease in the amplitude and increase in the duration of the response. This suggests that the functioning of the mucosal reflexes is impaired as
the distal colon ages.
 In the proximal colon, the addition of scopolamine reduced the contractile response in the 3 and 18 month animals. Thus, it appears that cholinergic signalling is not adversely affected with age in the proximal colon.
 The addition of scopolamine in the distal colon reduced the mucosal reflex response in the 3 month animals but was without effect in the 12 and 18 month animals. This suggests that alterations in cholinergic signalling arise as the distal colon ages.
 In the proximal and distal colon, the sequential addition of GR159897 following scopolamine standalone treatment failed to abolish the remainder of the mucosal reflex response in any of the age groups. This suggests that NK2 receptorsare not
involved in mediating mucosal reflexes at a low stimulus intensity.
 Mucosal reflexes were still generated in the presence of ondansetron in all age groups thus suggesting that 5-HT3 receptors are not required for the initiation of mucosal reflexes.
TKs
3. Methods

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