John K. Koeppe
Spring 2014
Department of Biology
Undergraduate Research Symposium
University of North Carolina at Chapel Hill
John K. Koeppe Undergraduate Research Symposium
Department of Biology
University of North Carolina at Chapel Hill
April 4, 2014
215 Coker Hall
The following Biology majors/2nd majors will give an oral presentation of their biology research
in addition to completing their Senior Honors Thesis in Biology. They will receive a designation
of either Distinction of Highest Honors in Biology or Distinction of Honors in Biology on their
UNC transcript.
8:30 AM
Welcome Remarks
Dr John Bruno, Biology Honors Program Chair
Dr Victoria Bautch, Department of Biology Chair
8:45 AM
Zachary Blom
The roles of Canoe and Polychaetoid (homologues of mammalian ZO-1 and
Afadin) in Drosophila development.
9:00 AM
Dylan Carroll
Understanding the Causes of Coloration Plasticity in a Population of Spea
bombifrons
9:15 AM
Hanna Labiner
Determining the Role of Castor in Heart Development
9:30 AM
Break – refreshments in Coker hallway
9:45 AM
Raymond Barry
Investigating the mechanism that regulates DNA dynamics upon double-strand
break in budding yeast
10:00 AM
Jennifer E. Neal
Characterization of the Dual PHD Domains of the Rco1 Protein in Rpd3S
Complex Function
10:15 AM
Chris Rota
Evaluating the Role of rtel in Synthesis-dependent Strand Annealing in D.
melanogaster
10:30 AM
Keith J. Murphy
Investigating the role of FoxA1 and FoxA2 as Transcriptional Regulators of Sox9
10:45 AM
Break – refreshments in Coker hallway
11:00 AM
Varun Gulati
The Role of Corticotropin-Releasing Factor (CRF) Signaling in the Ventral
Tegmental Area in the Regulation of Binge-Like Alcohol Consumption
11:15 AM
Andrew Parker Kendle
The Role of the Amygdala in the Neurobiological and Behavioral Responses to
Chronic Ethanol Exposure, Stress and Withdrawal
11:30 AM
James Dunville
The Effects of α-Flupenthixol on Habit-Like Ethanol Seeking and SelfAdministration
11:45 AM
Break – refreshments in Coker hallway
12:00 PM
Cameron Wood
Probing the Structure of mChe-12’s TOG Domains to Understand Their Role in
Regulating Microtubule Dynamics in vitro
12:15 PM
Andrew Hyde
Metagenomic Analysis of Subsurface Archaea from the
Miscellaneous Crenarchaeotal Group
12:30 PM
Aimee D. Wilde
Regulation of Hmp and Ldh1 in Straphylococcus aureus
12:45 PM
Aidan J. Berry
Nitric Oxide Efficacy as a Function of Bacterial Physiology
1:00 PM
Break – refreshments in Coker hallway
1:15 PM
Danny Clancy Trotier
The role of Sox4 in normal intestines and colorectal cancer
1:30 PM
Kerry Cheek
Examining the Amount of DNA Obtained from Saliva Samples in Relation to the
Pharmacogenetics of Kidney Transplantation
1:45 PM
Patrick Short
Tumor Microenvironment RNA Expression Analysis in Human Xenograft Mice
2:00 PM
Kinnari Buch
A Decreased Endogenous IFNβ Biological Effect May Contribute to the
Development of the Autoimmune Response in Patients with Relapsing-Remitting
Multiple Sclerosis
2:15 PM
Byeong Wan (Danny) Kim
The Importance and Potentiality of Collagen on Human Health
2:30 PM
Break – refreshments in Coker hallway
2:45 PM
Neelesh Ratan Dewan
Decrypting an achiasmatic chromosome segregation pathway in Arabidopsis
thaliana
3:00 PM
Nguyen Huynh An Markus Le
Dependence of Fungal Plant Pathogen Host Breadth on Plant Characteristics
3:15 PM
Scott Marshall Lewis
Temperature Dependent Increase in Cross Over Frequency in Arabidopsis
thaliana
3:30 PM
Break – refreshments in Coker hallway
3:45 PM
Casey Clements
Spatial Learning Using Acoustic Signals in the
Túngara Frog (Physalaemus pustulosus)
4:00 PM
Caroline Rauffenbart
Characterizing Behavioral Phenotypes of Fragile X Syndrome in Mice
4:15 PM
Sarah Taylor
Dopamine Depletion in the Nucleus Accumbens of Rats Induced by a
Phenylalanine- and Tyrosine-free Amino Acid Mixture
4:30 PM
Sakib Huq
Role of the Dynorphin/KOR System in Mediating the Stress Response in Mice
4:45 PM
Closing Remarks
Dr John Bruno
Research Commendation
The following Biology seniors prepared a poster to showcase their research. Their posters were
presented on April 2, 2014 in the Genome Sciences Building. They will receive the following
notation on their UNC transcript: Commendation for Undergraduate Research in Biology.
Sarah Bradford
Kevin Currin
Charles Czysz
Elizabeth Detmar
Justin Dizon
Natalie Dunlop
Kaitlyn Ferguson
Michael Huynh
Jalaal Khan
Laralee Lynch
Nadia Nagy
Investigating the mechanism that regulates DNA dynamics upon double-strand break in
budding yeast
Raymond Mario Barry
Research Advisor: Kerry Bloom
Cells must maintain genomic integrity despite constant exposure to sources of DNA
damage. The most genotoxic form of damage is the double-strand break and its improper repair
results in genomic instability and cancer predisposition. An undamaged homologous sequence
can serve as the template for repair of a double-strand break, and increased mobility may be
required to facilitate an efficient homology search. Chromatin exhibits increased mobility upon
DNA damage, but the physical mechanism remains unknown. To explore this mechanism, we
employ in vivo single-particle tracking of tagged loci in haploid yeast. We find that cytoskeletal
actin contributes to increased nuclear motion upon damage, reflecting its role in meiosis to
promote homolog pairing by nuclear mixing. This presents a new model for increasing chromatin
mobility to facilitate homology search in the DNA damage response.
Nitric Oxide Efficacy as a Function of Bacterial Physiology
Aidan J. Berry
Principal Investigator: Dr. Mark Schoenfisch (Analytical Chemistry Department)
Graduate Research Mentor: Katelyn Reighard
Biology Sponsor: Dr. Ann Matthysse
Nitric oxide (NO), a reactive free radical, acts as a broad-spectrum antimicrobial agent
via mechanisms of nitrosative and oxidative stress that negatively impact the integrity of the
bacterial membrane. These multiple bactericidal mechanisms prevent the development of
resistance, which makes NO particularly promising as a therapeutic for combating a variety of
bacterial infections. This study investigates the impact of bacterial physiology (i.e. growth phase
and gram type) on the efficacy of NO. To determine the role of bacterial growth phase, the
concentrations of NO-releasing G1-PO dendrimers that were bactericidal for mid-log and
stationary phase bacteria were compared. The role of gram type on NO efficacy was examined
by comparing minimum bactericidal concentration (MBC) assays using gram-positive
(Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa) strains. For both species,
bacteria in mid-log phase were most susceptible to NO-treatment. P. aeruginosa was more
susceptible to NO than S. aureus. To determine if these susceptibility differences were primarily
caused by membrane thickness and stability, assays were performed using G1-hexyl dendrimers,
alternative membrane-disrupting agents. Similar trends were observed, which suggests that
membrane integrity is a key factor in the antibacterial efficacy of NO.
The roles of Canoe and Polychaetoid (homologues of mammalian ZO-1 and Afadin) in
Drosophila development.
Zachary Blom
Research Advisor: Dr. Mark Peifer
Most complex organisms begin life as a single cell that must extensively divide and organize
through morphogenesis to become a completely developed organism. For this process to
correctly function, cells must dynamically adhere and move in a strictly regulated manner. The
purpose of my research was to study the morphogenetic role of two Drosophila proteins: Canoe
(Cno) and Polychaetoid (Pyd), which have been previously implicated in Drosophila regulatory
processes. I tested the hypothesis that these proteins have a vital role in morphogenesis, and
mutants will show abnormal phenotypes. I looked at whole cell and cellular junction
organization, as well as other features to characterize phenotypes of mutant flies. Our results
suggest polarity disruptions, epithelial cell disorganization, and increased lethality,
demonstrating the vital role of these proteins. These results help establish new understanding of
how these proteins (and their mammalian homologues) function in morphogenesis and provide a
basis for future studies.
Behavioral Syndromes in Crickets
Sarah Bradford
Research Advisor: Dr. Karin Pfennig
Research Mentor: Emily Schmidt
Recently, there has been a shift in behavioral ecology from studying the evolution of
independent behaviors to studying the evolution of correlated suites of behaviors. Such suites are
known as behavioral syndromes, or personalities. While evidence for behavioral syndromes has
been found in a number of species, there are still many species in which behavioral syndromes
have not been studied or identified. In this study, we aimed to determine if behavioral syndromes
exist in the house cricket, Acheta domesticus. In this experiment, male and female crickets
underwent a series of tests to measure three personality traits: boldness, exploration, and
sociability. Our goal was to determine whether domestic crickets have a behavioral syndrome
involving two or all three of these personality traits. We also sought to compare males and
females in regards to their average levels of each personality trait, as well as the correlations
between these traits. Overall, this study will improve our understanding of behavioral syndromes
in crickets.
A Decreased Endogenous IFNβ Biological Effect May Contribute to the Development of the
Autoimmune Response in Patients with Relapsing-Remitting Multiple Sclerosis
Kinnari Buch
Research Advisor: Dr. Silva Markovic-Plese
Biology Sponsor: Dr. Corey Johnson
Interferon-Beta (IFNβ), a cytokine, or glycoprotein, which is released by virtually all cell
subsets in response to viral and bacterial pathogens, has been used as a therapy to suppress
multiple sclerosis (MS) disease activity for approximately 20 years. However, studies have
mainly focused on the mechanisms of action of the therapeutically administered recombinant
IFNβ-1a. This study aimed to characterize the role of endogenous IFNβ in the development of
the autoimmune response in this disease by evaluating its biological activity in the serum of
patients with relapsing-remitting (RR) MS in comparison to healthy controls (HC). The results of
a sensitive cell-based bioassay showed reduced expression of interferon-inducible genes,
myxovirus resistance 1 (MX1) and protein kinase RNA regulated protein (PRKR), in RRMS
serum-treated WISH cells, suggesting a deficient endogenous IFNβ biological activity in RRMS
patients in comparison to HCs. This study will enable physicians to identify patients with lower
endogenous IFNβ levels who will optimally respond to IFNβ-1a treatment.
Plasticity during development can lead to genetically similar organisms displaying drastically
different phenotypes in response to environmental cues. This research examined the genus
Spea, a clade of spadefoot toads, which displays remarkable developmental plasticity that
heavily affects their morphology as tadpoles. A specific population of spadefoot toads exists in
which tadpoles are able to develop lighter coloration in response to the environmental factors
of the pond in which they live. I manipulated the sand coloration and light intensity in which
tadpoles were raised to gain insight into possible environmental cues to which the tadpoles
could use to create this plastic response. Substrate coloration was found to influence the
coloration of spadefoot tadpoles and metamorphs, as those raised on a lighter substrate
developed a lighter coloration than those raised on a darker substrate. Furthermore, this
plasticity was found to be indeterminate, as juvenile toads not subjected to substrate or light
treatments for a month lost the coloration differences found between them at the tadpole and
metamorph stages. By showing how this population of spadefoots responds to substrate
coloration, we can now look into the pathways these tadpoles use to respond to this
environmental cue. The results of this experiment could help display an instance in which
developmental plasticity rapidly evolved to respond to a novel environmental cue and indicate
plasticity's role in aiding the way organisms adapt to novel environments.
Dylan Carroll
Examining the Amount of DNA Obtained from Saliva Samples in Relation to the
Pharmacogenetics of Kidney Transplantation
Kerry Cheek
Research Adviser: Dr. Tim Wiltshire
ABSTRACT
The recent application of pharmacogenetics to the kidney transplantation process has
provided a new method for creating drug dosage regimes that are specific to each patient. These
regimes are based on each patient’s genotype, which is usually obtained from a blood sample.
The purpose of this study was to determine whether saliva samples could be used for examining
genes of interest in transplant patients, as a less invasive alternative to blood samples. To test this
hypothesis, the amount and purity of DNA was measured from saliva samples. Following this,
the DNA was run with a set of 13 primers for genes commonly tested in pharmacogenetic studies
to determine if the DNA had potential for use. The results suggest that saliva samples produce
an amount of DNA useful for testing – though the resulting product contains more impurities
than the DNA obtained from blood – and that the DNA can be used for further pharmacogenetic
testing. These results imply that saliva samples could possibly be used as an alternative to blood
samples in the future, making it more likely that patients would agree to participate in trials.
Spatial Learning Using Acoustic Signals in the Túngara Frog (Physalaemus pustulosus)
Casey Clements
Research Advisor: Dr. Sabrina Burmeister
Spatial navigation is critical to an animal's procurement of food, mates, and territory. As
a result of the importance of the visual system in an animal's survival, the majority of previous
studies have investigated how animals associate visual cues with spatial locations. Because the
sexual behavior of the túngara frog (Physalaemus pustulosus) relies heavily on the use of
acoustic signals, I hypothesized that they would be able to use acoustic cues to learn spatial
locations. To test this hypothesis, I presented each individual with two arbitrary sounds, a tone
and a recording of a mating call that had been reversed. The tone was initially associated with the
correct door of the maze, which led to a shelter. Once an individual learned the association, I
switched the sounds so that the reversed mating call was now associated with the correct
door. My results indicate that túngara frogs are able to associate an arbitrary sound with a
location and to reverse those associations. This study is an important contribution to research on
anuran cognition because it is the first to demonstrate the use of acoustic signals during spatial
learning, as opposed to the predominant visual studies.
The Impact of RNA Covariation on Linkage Disequilibrium
Kevin Currin
Research Advisor: Alain Laederach
Base pairs that are vital for the structural formation of RNA often exhibit structure-preserving
covariation. Selection for these structure-stabilizing pairs may produce linkage disequilibrium
(LD) by favoring allele pairs that are more conducive to pairing. The goal of the current study is
to determine the extent to which RNA covariation produces LD in human 3’ untranslated
regions. Allele pairs in high LD that consist of canonical base pairs, as well as the frequent G-U
noncanonical pair, obtained from the 1000 Genomes Phase 1 project will be compared to
potential pairs determined by RNA structural predictions to identify pairs of loci that may exhibit
both RNA covariation and LD. Comparisons of RNA structural predictions between pairs of
major and minor alleles will be carried out to further test the occurrence of RNA covariation at
these loci.
Annotation of eQTLS with Respect to RNA Regulatory Sites
Charles Czysz
Research Advisor: Alain Laederach
RNA mis-regulation has been implicated in many human diseases. The prevaling view is that
diseases are caused by errors in protein-coding regions, but RNA diseases result from mutations
in untranslated regions. A class of mutations which result in altered RNA levels are called
expression quantitative trait loci, eQTLs. My goal is to find eQTLs which affect RNA posttranscriptionally by eliminating those which act pre-transcriptionally. I will do this by
overlapping eQTLs with regulatory sites, starting with a type called DNaseI-Hypersensitivity
Sites, which indicate regions of open chromatin. A subset of DH sites have been correlated with
altered RNA levels, analagous to eQTLs, so I started with these regions. However, SNPs do not
occur in isolation, but are inherited as “blocks” as a consequence of genomic proximity and
ancestry. Each eQTL is linked to other SNPs, one of which might be causitive. By developing
scripts in the Bash, Python, Perl, and R programming languages, I constructed linkage blocks
and overlaped eQTL blocks with DH sites, finding 22% of the eQTLs were within or linked to
DH sites. By incorporating more regulatory sites, I will be able to further narrow down eQTLs
which don't act pre-transcriptionally.
Regulation of Non-Muscle Myosin II by protein RN-tre in D. melanogaster
Elizabeth Detmar
Research Advisor: Dr. Stephen Rogers
Research Mentor: Dr. Derek Applewhite
Non-muscle myosin II is a phosphorylatable protein that binds to actin to form a kinetic system
that controls cellular contractility, adhesion, and migration. To better understand myosin
regulatory pathways we performed a screen for proteins in D. melanogaster that disrupt myosin
localization during interphase; the screen yielded a single hit, RN-tre (related to the N-terminus
of tre). RN-tre is thought to be a Rab-specific GTP-ase activating protein implicated in cell
membrane trafficking, thus a role in the regulation of myosin is potentially novel. Our data
suggests that RN-tre depletion by RNAi results in inhibition of cellular contraction and abnormal
rates of actin and myosin retrograde flow. Experiments aim to characterize the pathway and
mechanism of RN-tre’s interaction with myosin in order to further understand cytoskeletal
regulation.
Decrypting an achiasmatic chromosome segregation pathway in Arabidopsis thaliana
Neelesh Ratan Dewan
Research Advisor: Dr. Gregory Copenhaver
Research Mentor: Daniela Muñoz
Meiotic recombination is the process by which homologous chromosomes exchange
genetic material during gamete production by “crossing over.” This shuffling of parental
chromosomes is not only thought to be important in increasing genetic diversity in a population,
but also a necessary step in segregating chromosomes appropriately during meiosis. Plant
breeders may find it desirable to preserve advantageous gene combinations in their crops through
generations and, in this context, manipulation of recombination can have a significant impact in
breeding methods. Most eukaryotes, including humans and plants, require at least one crossover
(CO) per chromosome to avoid non-disjunction and resulting genetic disorders associated with
chromosomal mis-segregation. Nonetheless, evidence of crossover-independent meioses has
been observed in many organisms including insects such as Drosophila melanogaster males,
mammals, and plants.
In all sexually reproducing species studied thus far, including plants, the topoisomeraselike protein SPO11 facilitates the required double-strand break (DSB) to initiate crossing over.
Thus in the absence of SPO11 recombination does not occur, resulting in a profoundly sterile
phenotype. My project seeks to find a SPO11 mutant that maintains fertility in the absence of
recombination through a forward genetics mutagenesis screen.
The Role of Melanocortin-1 Receptor (Mc1r) Expression in Color Plasticity of Spea
bombifrons
Cyril Justin Dizon
Research Advisor: Dr. Karin Pfennig
Research Mentor: Antonio Serrato
Variation in color has an important role in the fitness of an organism whether the color is used
for sexual selection, warning predators, thermoregulation, or crypsis. Field observations suggest
that S. bombifrons display color matching with their background substrate. In lizard species
located in White Sands National Monument, the activity of the Mc1r gene is known to dictate
their coloration. High activity of Mc1r will produce a dark pigment whereas low activity will
produce a light pigment. Previous research revealed that in amphibians, the degree of
melanocyte production is not affected by variation in the coding sequence of Mc1r. With these
findings, perhaps differences in expression levels of Mc1r in S. bombifrons are related to the
color variations in the tadpoles. To perform the experiment, I assigned color values to tadpoles
in the lab. Then I extracted their RNA and performed qPCR comparing expression to the color
values.
Biomechanical Characterization of the Periodontal Ligament: Orthodontic Tooth
Movement
Natalie Dunlop
Research Advisor: Dr. Ching-Chang Ko
Biology Sponsor: Dr. Tyson Hedrick
The periodontal ligament (PDL) is the connective tissue between each tooth and the
alveolar bone that controls the tooth’s movement and response to pressures exerted upon it. None
of the existing attempts to quantify the response of the PDL accurately represent its viscoelastic
nature. This research aims to define a viscoelastic model to represent the PDL’s response to
mechanical strain placed on the tooth, with the end goal of using the elastic modulus value
obtained for input into computer simulation of orthodontic tooth movement. The simulation will
then more accurately predict tooth movement as a result of specific orthodontic forces placed on
a tooth. The testing will use a DMA (dynamic mechanical analysis) machine to perform a
uniaxial stretch on in vitro bovine PDL segments, generating a stress-strain graph for
mathematical analysis that will create the model to define the elastic modulus value of the PDL.
The Effects of α-Flupenthixol on Habit-Like Ethanol Seeking and Self-Administration
Holton James Dunville
Research Advisor: Dr. Donita L. Robinson
Inflexible, habitual drug seeking despite negative consequences is a key component of
alcoholism. Habitual behavior requires the dorsolateral striatum (DLS) in rodents, while flexible
behavior requires the dorsomedial striatum (DMS). Lesion of dopamine inputs to the DLS can
prevent habit formation; however, the role of dopamine to reverse an existing alcohol-seeking
habit is unknown. We therefore tested the hypothesis that blocking dopamine in the DLS will
initiate a reversion to flexible behavior in which the DMS is active. Rats were trained to selfadminister sweet solutions (S) or sweet solutions with ethanol (S/E) until they expressed habitlike seeking. Electrodes were surgically implanted in the DMS to measure neuronal activity.
Behavioral flexibility was then assessed using a contingency degradation procedure with and
without bilateral DLS infusions of the non-specific dopamine receptor antagonist α-flupenthixol
(FLU). Following contingency degradation, both S and S/E rats showed a reduction in reinforcer
seeking after FLU administration but not saline. Preliminary results showed a decrease in DMS
neuronal firing rate over the course of the first degradation session for S/E rats, which predicted
reduced S/E seeking behavior. Our results suggest that a reduction in DLS dopamine facilitates
reversal of habitual reward seeking, and changes in DMS neuronal activity may precede the
resurgence of flexible behavior.
Analysis of Spadefoot Tadpoles: Examining a Predisposition for Nonalcoholic Fatty Liver
Disease
Kaitlyn Ferguson
Research Advisor: Dr. Karin Pfennig
Spadefoot toads suffer from fatty liver disease, and are potentially a model for this disease in
humans. The goal of my work is to understand how different diets affect liver morphology and,
possibly, disease risk. Spadefoot tadpoles are unusual in that they produce two types of tadpoles:
an omnivore form, which feeds on detritus (like most frog tadpoles), and a carnivore form, which
has a highly specialized protein diet. I am comparing the liver morphology of omnivore and
carnivore tadpoles to determine whether differences in liver morphology emerge early in
development on these different diets. Such work is important, because such differences, if any,
might affect subsequent risk of liver disease later in life. Moreover, I am also comparing liver
morphology across different populations and species, to evaluate what factors might affect
disease risk. Ultimately, this work will lend insight into the ecological factors that might enhance
risk for fatty liver disease, including among human populations.
The Role of Corticotropin-Releasing Factor (CRF) Signaling in the Ventral Tegmental
Area in the Regulation of Binge-Like Alcohol Consumption
Varun Gulati
Research Advisor: Dr. Jennifer Rinker
Binge drinking is a pattern of excessive alcohol consumption related to numerous health
problems, notably alcohol dependence. Corticotropin-releasing factor (CRF) signaling within the
ventral tegmental area (VTA) is known to regulate dependence-induced alcohol consumption. It
remains unclear which receptors regulate binge-like consumption. This study utilized the
drinking-in-the-dark design to examine how intra-VTA CRF-1 receptor (CRF-1R) antagonism
and CRF-2R agonism influenced binge-like alcohol drinking. Blockade of CRF signaling, by
CRF-1R antagonism or CRF-2R autoreceptor activation, reduced binge-alcohol consumption. It
was also unknown whether CRF-mediated effects were the result of local VTA interneurons or
projection neurons from other brain regions. Transgene expression of eGFP in the VTA of CRFCre mice confirmed the presence of local CRF interneurons. By confirming the role of CRF
receptors within the VTA in binge-alcohol consumption and establishing the presence of local
CRF neurons in the VTA, this study clarifies the CRF-mediated effects on binge-like alcohol
consumption and identifies a potential new mechanism mediating alcohol consumption.
Role of the Dynorphin/KOR System in Mediating the Stress Response in Mice
Sakib Huq
Research Advisor: Dr. Thomas Kash
Biology Sponsor: Dr. Catherine Lohmann
Alcohol and drug abuse represent two of today’s greatest unsolved public health problems. The
dynorphin/kappa opioid receptor (KOR) system has been shown to mediate the increased
rewarding effects of drugs of abuse and the increased anxiety and stress seen during alcohol
withdrawal. In the present study, we examined the role of the dynorphin/KOR system in the Bed
Nucleus of the Stria Terminalis (BNST) in stress related behaviors using an animal model. We
stereotaxically injected mice with an inducible caspase construct to selectively destroy dynorphin
cells in the BNST and tested mice using the elevated plus maze (EPM), open field, forced swim
stress (FSS), and fear conditioning behavioral testing paradigms. Our results show that at basal
conditions, ablating dynorphin does not produce significant differences in behavior but that
dynorphin and the KOR may play an important role in behavior following a stressful event
(FSS). Our preliminary results, along with ongoing work replicating these experiments using
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), may provide novel
insight for the role of the dynorphin/KOR system in mediating the stress response.
Identifying Interactions Between RBA1 and the TIR Domains of Arabidopsis thaliana
Using A Yeast-Two Hybrid Screen
Michael Huynh
Research Advisors: Dr. Marc T. Nishimura, Dr. Jeffery L. Dangl
The Toll/interleukin-1 receptor (TIR) domain is an important contributor to the innate immune
response of both plants and animals. RBA1 is a TIR-only protein that contains a TIR domain, but
lacks the NBS and LRR domains typically required for TIR-NBS-LRR R protein function.
Despite its unusual domain structure, RBA1 is capable of recognizing the presence of the plant
pathogen effector protein HopBA1 and triggering downstream defense responses. Since TIR
domains are known to dimerize, one compelling hypothesis is that RBA1 dimerizes with another
full-length TIR-NBS-LRR protein. In order to test for physical interactions between TIR
domains, we have cloned a comprehensive set of the 146 TIR domains found in Arabidopsis
thaliana. To begin testing for interactions between the TIR domains and RBA1, the TIR domains
will be recombined into yeast-two hybrid vectors and then assayed for protein-protein
interactions. These studies will provide better understanding of how dimerization of the TIR
domains regulate defense and how heterodimerization can lead to the expansion of immune
receptors.
Metagenomic Analysis of Subsurface Archaea from the
Miscellaneous Crenarchaeotal Group
Andrew Hyde
Research Advisor: Andreas Teske
Biology Sponsor: Ann Matthysse
Research Mentor: Cassandre Lazar
The subsurface biosphere may account for one-third to one-half of the total biomass on Earth;
however, we have only recently begun to explore this frontier. The life found in the subsurface is
largely bacterial and archaeal; of these, most remain unstudied. Among phylotypes found in
marine sediments, the Miscellaneous Crenarchaeotal Group (MCG) of archaea emerge as
dominant players. Despite its apparent ubiquity, hardly anything is known about this very diverse
group of microbes. In this work, I have analyzed several complete archaeal genomes belonging
to the MCG group from the subsurface of the White Oak River estuary. I found genes indicating
that the MCG subgroups analyzed here have the ability to reduce nitrite and other oxidized
nitrogen compounds. These results suggest that our understanding of marine biogeochemical
cycling may need to be modified to account for the significant role these organisms may play.
The Role of the Amygdala in the Neurobiological and Behavioral Responses to Chronic
Ethanol Exposure, Stress and Withdrawal
Andrew Parker Kendle
Research Advisor: Dr. Darin J. Knapp
Stress during abstinence from alcohol worsens anxiety and may contribute to relapse. Treatment
options for abstaining alcoholics are limited, in part due to a lack of understanding of how stress
and chronic alcohol alter relapse-associated behaviors. Current research is focused on the role of
the amygdala in the “priming”, or progressive worsening with each withdrawal, of stress-induced
anxiety. To investigate this role I injected a vasopressin receptor-1b antagonist, SSR149415, and
an oxytocin agonist, WAY 267464, bilaterally into the central amygdala of rats during each
withdrawal of a cycled alcohol treatment. Measurement of anxiety by way of social interaction
tests showed that only the vasopressin manipulation returned stress-induced anxiety responses to
levels typical of unprimed animals, suggesting vasopressin receptor-1b in the central amygdala
mediates a necessary step in priming. After this data was obtained, I began a similar protocol
with a vasopressin agonist to see if this circuitry was also sufficient to induce priming of stressinduced anxiety after withdrawal. These data along with ongoing optogenetic research into this
neurocircuitry may pave the way for treatments that help prevent alcoholic relapse and alcohol
abuse.
Analysis of the LKB1 Heterotrimeric Complex through Homogeneous Time Resolved
Fluorescence
Jalaal M. Khan
Research Advisor: Prof. William Janzen
Biology Sponsor: Dr. Stephen Rogers
Research Mentors: Chatura Jayakody and Catherine Simpson
The purpose of this research project is to develop an assay for the LKB1 protein kinase
heterotrimeric complex to discover chemicals that could be future candidates for the treatment of
cancer or research tools. Mutations in the LKB1 gene are known to cause Peutz-Jeghers
syndrome in which individuals suffer from tumors in the gastrointestinal track and are more
likely to develop other forms of cancer. Similarly, an overproduction of the LKB1 kinase is able
to restrict cells to the G1 checkpoint, preventing the cells from future division. Thus, LKB1 acts
as a tumor suppressor. LKB1 is activated through the attachment of the pseudokinase STRAD 
and MO25  , a protein that connects to the C-terminal of STRAD  to help secure STRAD  to
LKB1 to form a heterotrimeric complex. In order to analyze LKB1, I plan to develop and
validate an assay and perform high-throughput screening of LKB1 to discover possible activators
and inhibitors of the kinase.
The Importance and Potentiality of Collagen on Human Health
Byeong Wan (Danny) Kim
Research Advisor: Dr. Mitsuo Yamauchi
Biology Sponsor: Dr. Maria Servedio
Collagen is the most abundant and perhaps the most important structural protein in vertebrates,
including humans. One of the best ways to study the importance of collagen is to investigate how
various changes to collagen affects cancer metastasis. Recent research in the Yamauchi lab found
that specific collagen post-translational modifications may play important role in cancer
migration. The purpose of my research was to understand the role of collagen and discover the
significance it has on the human body. Specifically, I answered the question, “what important
roles does collagen play that may critically relate to an individual’s health?” My results indicate
that collagen plays crucial role by providing bone mineralization and supplying for the collagen
highway system for cancer metastasis. A better appreciation of collagen’s capabilities and its
correlation to cancer metastasis could suggest powerful tools for combating cancer.
Determining the Role of Castor in Heart Development
Hanna Labiner
Research Advisor: Dr. Frank Conlon
Research Mentor: Kerry Dorr
Castor (Casz1) is a zinc finger transcription factor that has been shown to be required for
heart development in Xenopus. Casz1 has also been linked to high blood pressure and
hypertension in humans through a recent Genome Wide Association Study. The purpose of this
research was to determine the role of Casz1 in cardiac development and how Casz1 fits into the
cardiac transcription program. A mouse model was used because of the high genomic
conservation between mice and humans. To determine the spatial and temporal expression of
Casz1 mRNA during murine heart development, I utilized in situ hybridization. In addition, to
confirm that CASZ1 protein is present in the developing heart, I performed
immunohistochemistry. My studies showed that Casz1 is expressed in the atria and left ventricle
of the developing heart. To determine the role of Casz1 in cardiac development, I examined the
cardiomyocyte mitotic index of wild type embryos and Casz1 mutant embryos and demonstrated
that cardiomyocytes lacking Casz1 over proliferate. This indicates that Casz1 regulates
cardiomyocyte proliferation. Future studies are aimed at identifying genes that Casz1 regulates,
providing further insight into the cardiac gene program. These studies hold implications for
understanding congenital heart defects by giving us further insight into the molecular
mechanisms that regulate cardiac development.
Dependence of Fungal Plant Pathogen Host Breadth on Plant Characteristics
Nguyen Huynh An Markus Le
Research Advisor: Dr. Charles Mitchell
Over time, travel between continents has become easier, allowing many diseases and
pests to leave their native habitats, sometimes with disastrous results. Famous examples include
the chestnut blight and woolly adelgids, which have greatly harmed ecosystems in the United
States. I investigated the factors that allow these invasive species to flourish in non-native
environments by examining how a plant’s susceptibility to pathogens depends on its phylogeny,
traits, and native geographic range. To do so, I used grasses that varied widely across these three
factors in an observational field study and in vitro inoculation experiment. Collected
susceptibility data was compared to traits like leaf mass per area, growth rate, and photosynthetic
rate. Results indicate significant susceptibility differences among different grass species, and
significant correlation between susceptibility and leaf mass per area. The results will help
illuminate what allows pathogens to infect new hosts, allowing planning for the prevention and
mitigation of species invasion.
Temperature Dependent Increase in Cross Over Frequency in Arabidopsis thaliana
Scott Marshall Lewis
Research Advisor: Dr. Gregory P. Copenhaver
Meiotic recombination is crucial for generating genetic diversity in gametes and while the
necessity is understood its regulation is not. Previous work with Arabidopsis thaliana
demonstrated recombination frequency was greater in plants grown in elevated temperatures. In
order to determine the molecular basis of these additional cross overs we will test the hypothesis
that additional cross overs originate from the interference insensitive pathway. We will utilize
Atmsh4 and Atmus81mutant lines of Arabidopsis to assess if the increase in cross overs is
attributable to either the interference sensitive (Type I) or interference insensitive (Type II)
pathway. Recombination frequency will be visually assayed by pollen tetrad analysis which
utilizes fluorescent transgene markers. We do not expect to see an increase in cross overs for
plants that have a mutation in AtMUS81 grown at elevated temperature confirming Type II
origin.
Morphological Differences in Spea Hybrids
Laralee Hagan Lynch
Research Advisor: Dr. Karin Pfennig
Research Mentor: Emily Schmidt
Spea bombifrons and S. multiplicata have become a model system for answering questions in
developmental biology. S. multiplicata, facultative omnivores, and S. bombifrons, carnivores,
have morphological traits which complement their foraging patterns, while hybrid offspring
exhibit a range of intermediate phenotypes. We tested whether there were significant
developmental and morphological differences in S. bombifrons and Spea hybrids from two
populations. We found no significant difference in mass, condition, survival, or fluctuating
asymmetry of pure bred and hybrid tadpoles, though there was some difference in morphology.
Morphological differences were driven by two characters, S. bombifrons had more developed
mouthparts, while hybrids had more labial teeth. S. bombifrons were also significantly more
carnivorous. Developmental differences were significant for tadpoles of only one population,
suggesting population differences are driving results. I will use linear mixed effects models to
analyze our data, and determine the role of population in our results.
Investigating the role of FoxA1 and FoxA2 as Transcriptional Regulators of Sox9
Keith Murphy
Research Advisor: Gidi Shemer
Research Mentor: Scott Magness
Two stem cell states have been identified within the crypts of the small intestine: ‘quiescent’ and
‘active’. SOX9, a transcription factor encoded from the Sox9 gene has been hypothesized to be
the master regulator between these ‘active’ (low expression of SOX9) and ‘reserve’ (high
expression of SOX9) intestinal stem cell states. Therefore, putative cis-regulatory sequences
upstream of the SOX9 promoter have been analyzed through TRANSFAC© to determine
potential candidates for up-regulation of SOX9 in quiescent and active stem cells. Preliminary
results indicate that FOXA1and A2 represent potential transcriptional regulators of the SOX9
gene. This hypothesis has been supported by immunohistochemistry, qRT-PCR analysis, and
ChIP-sequencing demonstrating that FOXA1/2 co-expresses in cells that express high levels of
SOX9 within the intestinal crypt. These studies provide further understanding of how stem cell
identity is normally regulated in the cells of the intestinal crypt.
Redox Control of Hypoxia-Induced Excessive Autophagy Leads to Increased Myocardial
Damage
Nadia Nagy
Research Advisor: Dr. Hagir B. Suliman
Research Sponsor: Dr. Kevin Slep
Oxidative damage is associated with mitochondrial dysfunction that may result in energy
depletion, accumulation of cytotoxic mediators, and cell death. Understanding the interface
between stress adaptation and cell death is important for understanding both redox biology and
disease pathogenesis. Recent studies indicate that one major sensor of redox signaling at this
interface is autophagy. In this study, we hypothesized that chronic hypoxia induced oxidative
stress particularly in mice lacking Superoxide Dismutase 3 (SOD3) would increase autophagy in
the heart with myocardial damage. We then exposed wild-type (WT) and SOD3 knock-out (KO)
mice to hypoxia for 14 days, mice were sacrificed, and the hearts were snapped frozen in liquid
nitrogen until further analysis. Gene and protein expression for cardiac integrity and autophagic
pathway were determined by real time RT-PCR and western blot assay. The inflammasome
marker IL-18 was significantly increased in KO mice after hypoxia compared to WT. Sharp
increases in the autophagic proteins Atg-5, Atg 9 and Beclin was detected in KO mice with
decrease in the pro-survival autophagy marker LC3-II and increase in TUNEL staining. Thus
lack of SOD3 during hypoxia promoted inflammation and lead to cell death via activation of
autophagosome formation and accumulation.
Characterization of the Dual PHD Domains of the Rco1 Protein in Rpd3S Complex
Function
Jennifer Neal
Research Advisor: Dr. Brian Strahl
Biology Sponsor: Dr. Gidi Shemer
Research Mentor: Stephen McDaniel
Chromatin, the structure of DNA and histone proteins, permits compaction of genetic
material and variable availability of DNA for processes such as transcription. An improved
understanding of chromatin dynamics could lead to new cancer treatments. Changes to
chromatin occur in response to chemical modifications applied to and removed from histones by
protein complexes. Rco1 is a protein in the Rpd3S histone deacetylase complex. The aim of this
research was to determine the roles of the two PHD domains of Rco1 in Rpd3S function. I made
point mutations of evolutionarily conserved bases within each domain, and studied the PHD
mutants using genetic and biochemical assays. My results indicate that both PHD domains may
be involved with Rpd3S function and, specifically, Rco1 binding to chromatin. This research
represents the first investigation of the role of the second PHD domain, and contributes to
characterization of chromatin dynamics.
Characterizing Behavioral Phenotypes of Fragile X Syndrome in Mice
Caroline Rauffenbart
Research Advisor: Dr. CJ Malanga
Biology Sponsor: Dr. Karin Pfennig
Fragile X Syndrome (FXS) is the most prevalent inherited form of autism and affects
approximately 18,000 people in the United States. This condition is due to inappropriate
silencing of the Fmr1 gene on the X-chromosome and subsequent loss of Fragile X Mental
Retardation Protein (FMRP), an RNA-binding protein that represses synaptic protein synthesis.
Clinical features of FXS include cognitive impairments, increased seizure susceptibility and
altered social behaviors. FXS has been modeled in Fmr1-KO male mice to yield phenotypic
learning, memory, social exploration and spontaneous activity impairments. In these studies, I
will use the marble burying, open-field, acoustic startle/prepulse inhibition (PPI), social
exploration and nose poke behavioral assays to further characterize phenotypic differences
between Fmr1-KO and WT mice. These findings will help to provide a basis for future preclinical mouse studies that will attempt to correct behavioral and cellular abnormalities
associated with FXS using pharmacological interventions during different stages of
development.
Evaluating the Role of rtel in Synthesis-dependent Strand Annealing in D.
melanogaster
Chris Rota
Research Advisor: Jeff Sekelsky
Synthesis-dependent strand annealing (SDSA) is a DNA repair process closely
associated with the generation of non-crossover outcomes during homologous
recombination. Based on previous studies, Blm, the Bloom’s syndrome helicase
gene, is thought to be an essential regulator of SDSA. The purpose of my research
is to evaluate another candidate regulator called rtel, the Drosophila ortholog of
human RTEL-1, which is hypothesized to play a non-essential but significant role
in SDSA repair. My study utilizes a novel genetic construct to measure levels of
SDSA repair occurring in the brains of Blm and rtel mutant larvae through
changes in cellular fluorescence. Successful SDSA repair of double-strand breaks
in the construct result in a visible change in cell color from red to green. So far,
results support the initial hypothesis and the assay has been found to be a useful
in-vivo method of studying SDSA in Drosophila.
Tumor Microenvironment RNA Expression Analysis in Human Xenograft Mice
Author: Patrick Short
Research Adviser: Dr. Chuck Perou
Biology Faculty Adviser: Dr. Corbin Jones
ABSTRACT
While genomic analysis of tumor cells is a mainstay in cancer research, there is growing interest
in the characterization of the tumor microenvironment, comprised of nearby healthy somatic
cells, most notably fibroblasts and invading immune cells. Studying the RNA expression profile
of the tumor microenvironment provides a way to analyze local response to tumor growth and
ultimately to better characterize bodily response to different stages or genetic subsets of cancer.
The purpose of this research was to develop a tool that efficiently separates tumor sequence data
from human xenograft mice (mice with genetically human tumors) into separate
microenvironment and tumor expression profiles. While this separation was previously done by
physically excising healthy tissue under a microscope using laser capture microdissection,
performing this separation in silico allows for rapid analysis of hundreds of samples. Further,
using this tool, we can re-examine tumor expression profiles after filtering out ‘contaminating’
microenvironment sequence, resulting in a more accurate RNA expression profile.
Dopamine Depletion in the Nucleus Accumbens of Rats Induced by a Phenylalanine- and
Tyrosine-free Amino Acid Mixture
Sarah Taylor
Research Advisor: Dr. Donita Robinson
Dopamine is an important neurotransmitter modulating neuronal mechanisms of human
and animal behavior. In humans, one way to study the role of dopamine in neuronal mechanisms
is by temporary depletion of dopamine synthesis and release. The depletion can be accomplished
via administration of an amino acid (AA) mixture lacking the precursors to dopamine:
phenylalanine (Phe) and tyrosine (Tyr). By using the same methodological approach in animal
models, this study examined the neurobiological consequences of this AA depletion on
dopamine concentrations in the nucleus accumbens (NAc). Specifically, we used fast scan cyclic
voltammetry (FSCV) to test whether the Phe-/Tyr- AA mixture reduced real-time dopamine
transient activity in the NAc of rats. I hypothesized that administration of the mixture would
reduce dopamine synthesis and lead to less dopamine available for release. Thus, I predicted that
the Phe-/Tyr- AA mixture would decrease both the frequency and amplitude of dopamine
transients in the NAc of rats. Preliminary data (n=13) indicate that systemic administration of
Tyr-/Phe- AA mixture, but not a control mixture containing Phe and Tyr, decreased both
amplitude and frequency of dopamine transients in the NAc. We conclude that Phe-/Tyr- AA
mixture decreases dopamine release in the NAc due to reduced dopamine synthesis.
The role of Sox4 in normal intestines and colorectal cancer
Danny Trotier
Research Advisor: Dr. Scott Magness
Biology Sponsor: Dr. Gidi Shemer
Research Mentor: Dr. Adam Gracz
Sry-box containing (Sox) factors are versatile regulators of cell fate and proliferation in a
wide range of tissues, and recent research has demonstrated that Sox4 is expressed in the stem
cell zone of the small intestinal epithelium. To examine the mechanistic role of Sox4 in the
epithelium, we utilize a conditional knockout mouse model to demonstrate that intestinal
epithelial-specific loss of Sox4 leads to an increase in markers of proliferation and downstream
targets of the Wnt pathway. Sox4 knockout animals exhibit intestinal crypt hyperplasia in vivo,
which is consistent with constitutively active Wnt signaling. We also observed secretory lineage
allocation defects in the intestines of Sox4 knockout animals, which exhibit an increase in Paneth
cell numbers and a decrease in enteroendocrine cells relative to wild type controls. Together,
these results suggest that Sox4 contributes to maintaining the balance of proliferation and
differentiation in the intestinal epithelium, and plays a role in the regulation of intestinal stem
cells. In the tumor context, Sox4 may be playing an alternate role by promoting EpithelialMesenchymal-Transition in a mouse model of colorectal cancer. Preliminary data suggests Sox4
is aberrantly activated in tumors and is promoting an EMT program that may facilitate tumor
progression and metastasis.
Regulation of hmp and ldh1 in Staphylococcus aureus
Aimee Wilde
Research Advisor: Anthony Richardson
Methicillin Resistant Staphylococcus aureus (MRSA) has developed as a prevalent cause
of community acquired disease. S. aureus’s effectiveness is due to its exceptional resistance to
host immunity, including nitric oxide (NO). Full NO resistance requires activation of a gene
subset including hmp and ldh1. Hmp converts reactive nitric oxide into non-toxic nitrate. Ldh1
relieves redox imbalance incurred during NO-stress. Although preliminary work implicated
regulatory gene SrrAB for controlling Hmp expression, the regulation is incomplete, implicating
additional regulators. Ldh1 is stimulated by glucose, however the exact regulatory mechanism is
unknown; my research goal is to identify the precise regulatory mechanisms for both genes. To
find these regulators, I conducted a screen of mutants in every non-essential regulatory S. aureus
protein by transducing reporter plasmids for each gene into each mutant. Growth curves
measuring fluorescence upon NO-stress to find atypical induction of hmp or ldh1 indicated
possible regulators, including SigB and HrcA.
Probing the Structure of mChe-12’s TOG Domains to Understand Their Role in Regulating
Microtubule Dynamics in vitro
Cameron Champion Wood
Research Advisor: Dr. Kevin Slep
The study of proteins with microtubule organizing function is important in order to better
understand mechanisms of the cytoskeleton. The protein Che-12 is one such conserved protein
that affects microtubule dynamics in cilia development. How Che-12 carries out this function,
however, remains unknown. Following sequence analysis, it was hypothesized that Che-12
contains four TOG domains which are known to regulate microtubule polymerization. The
purpose of my in vitro study of mChe-12 (mouse) was to characterize mChe-12’s predicted TOG
domains and their interactions with microtubules. I have purified multiple constructs containing
these predicted domains and have used these purified proteins for both structure determination
using X-ray crystallography and tubulin polymerization assays using light scattering techniques.
Based on these experiments, I have helped validate the presence of TOG domains within mChe12 and begun to determine the role of mChe-12’s TOG domains in dictating tubulin
polymerization. Thus, we have been able to characterize the Che-12 family as a new class of
TOG domain containing proteins which are conserved in ciliated and flagellated organisms.
Special thanks to the following for making the 2014 Biology Research Symposium a success:
Participating Research Advisors, Sponsors, & Mentors
Dr John Bruno, Biology Honors Chair
Dr Kevin Slep, Biology Honors Co-Chair
Dr Gidi Shemer, Biology Undergraduate Research Director
Susan Whitfield, Biology Visual Arts Specialist
Brian Nalley, Biology Visual Arts Specialist
Hinar Polczer, Biology IT Director
Trisden Coughlin, Biology IT Technician
Brandon Lam, Biology IT Assistant
Volodymyr Siedlecki, Biology IT Assistant
Dr Christina Burch, Biology Website Director
Paula Lloyd, Biology Accounting Technician
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Best wishes to our outstanding Biology research students!
Thank you for your contributions to science.
Good luck in your future endeavors.