Download april 21, 2016 - DeSales University

LEHIGH VALLEY MOLECULAR AND CELL
BIOLOGY SOCIETY
INAUGURAL MEETING
APRIL 21, 2016
HOSTED BY:
DESALES UNIVERSITY
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WELCOME STUDENTS AND COLLEAGUES TO THE
INAUGURAL MEETING OF THE LEHIGH VALLEY
MOLECULAR AND CELL BIOLOGY SOCIETY!
PROGRAM
4:30-5:00 P.M.
5:00-6:00 P.M.
6:00-7:00 P.M.
7:00-8:00 P.M.
REGISTRATION AND POSTER SET-UP
UNDERGRADUATE POSTER SESSION
DINNER
KEYNOTE ADDRESS
Understanding Breast Cancer
Jose Russo, MD, FACP
Fox Chase Cancer Center
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LVMCBS is a new organization created by two DeSales University faculty members to promote
open communication and facilitate collaboration between faculty and undergraduate students at
regional colleges and universities in Eastern Pennsylvania. Historically, there has been a lack of
molecular and cell biology focused local and regional conferences that allow students to present
their research and discuss science with their peers. This society seeks to fill that void with an
annual poster session and keynote talk on a topic of interest to the field. The goal of LVMCBS
is to promote student-driven research by providing a forum outside of their home institutions to
disseminate their data or research plan while simultaneously learning how others approach
scientific problems. Through LVMCBS, students will have an opportunity to present their work
and interact with students and faculty from other institutions.
We are looking to expand the event next year to include more colleges and universities. If you
are interested in joining our team or hosting a future meeting, please contact Dr. Joshua Slee
([email protected]) or Dr. Lara Goudsouzian ([email protected]).
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Undergraduate Research Poster Session
The Lehigh Valley Molecular and Cell Biology Society
DeSales University
April 21, 2016
ABSTRACTS
1. Investigating the Role of GCN5 in trichome development in Arabidopsis thaliana
Minnah Sheikh and Dr. Amy Hark
Muhlenberg College, Allentown, PA 18104
GCN5 is a histone acetyltransferase that influences the expression of genes that impact
developmental events. Arabidopsis thaliana plants with mutations in GCN5 show
abnormalities in leaf, flower, and trichome development. Differentiation of single-celled
trichomes has been well characterized in the literature. Our group has shown deviations from
the typical developmental pattern in gcn5 trichomes, most notably in branching. Based on
overlapping phenotypes, we hypothesize that GCN5 interacts with KAK, ZWI, and TRY,
which have also been shown to impact trichome branching. As a test of this hypothesis, we
are determining expression levels of these genes in rosette leaves of gcn5 mutants.
Experimental results suggest decreased expression of ZWI in gcn5-6 mutants and
significantly decreased expression of TRY in gcn5-1 plants. In gcn5-6 mutants TRY
expression varies in comparison to wildtype controls. We are also using qRT-PCR to assess
levels of GCN5 transcript in kak, zwi, and try mutants. GCN5 seems to be expressed at
slightly higher levels in zwi and try mutants. Altogether, our data suggests that there may be
some interaction between GCN5 and TRY. While the evidence may suggest interactions
between these genes, it is difficult to connect gene expression changes directly to the
branching phenotype observed.
2. A 96-Well Plate Crystal Violet Assay Protocol
Daniel Salovich, Michael Lawler, Taylor Gandy, Tyler Short, Bailey Semian, John Kilduff,
& Fr. Peter Leonard
DeSales University, Center Valley, PA 18034
We describe a 96-well crystal violet assay protocol that is validated for use with the MCF10F and MCF-10Fbs using a MultiScan plate reader. Well shape, lysing agent, stain type,
stain volume, and washing method are critical to obtaining reproducible results. The new
protocol will be used to measure the chemotherapeutic potential of polyamines on these cell
lines.
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3. In vitro binding studies between Gap Junction Protein, Connexin 43 (Cx43) and its
master-regulator, Zona Occludens -1 (ZO-1)
Aubrey Jones and Dr. Anastasia Thevenin
Lafayette College, Easton, PA 18042
Cell-cell communication is vital to maintaining cellular homeostasis and is accomplished
through Gap Junctions (GJs). GJs are made up of transmembrane proteins called connexins
(Cxs) that interact to form channels, allowing for passage of small molecules and ions.
Previous research has shown that Connexin 43 (Cx43) GJ function is regulated by
phosphorylation at ~15 serine residues on Cx43 C-terminus. ZO-1 (Zona Occludens-1) is a
known binding partner of Cx43, and work in mammalian cells demonstrated that when ZO-1
is bound, GJ channels stay open. Upon ZO-1 release, channels close and eventually
internalize. It was shown that these events are regulated by Cx43 phosphorylation. To
understand how phosphorylation of Cx43 regulates its interaction with ZO-1 in a more
quantitative fashion, Cx43 C-terminus and ZO-1 were each expressed and purified from
BL21 E. coli cells. Preliminary binding studies were conducted using affinity
chromatography, taking advantage of different affinity tags present on purified WT Cx43 and
ZO-1. These binding experiments have demonstrated that this interaction is stable and easily
detectable by SDS-PAGE and coommassie staining. Current work is focused on obtaining
Cx43 phosphomimetic mutants for future binding experiments with ZO-1, as well as for
quantitative Kd measurements using backscattering interferometry (BSI).
4. bis(trifluorobutyl) & bis(trifluoropropyl) spermidine Effects on MCF-10F & MCF10Fbs
Daniel Salovich, Michael Lawler, Taylor Gandy, Tyler Short, Bailey Semian, Fr. Peter
Leonard, & Dr. Francis Mayville
DeSales University, Center Valley, PA 18034
The viability of MCF-10F & MCF-10Fbs cells were tested in the presence of 500 µM bis
(trifluorobutyl) and bis (trifluoropropyl) spermidine. Tumorigenic cells exhibited a higher
death rate, presumably from apoptosis, but cells with a less carcinogenic phenotype are also
affected and exhibit a slower growth rate compared to the control group. We propose that
future experiments will need to be performed to determine the effects of these agents at a 20
µM concentration, which is physiologically more relevant.
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5. A Promotor Element in The C. elegans NHR-67 Tailless Gene Mediates HLH2/Daughterless Regulation of Uterine Organogenesis
Shari Bodofosky and Dr. Bruce Wightman
Muhlenberg College, Allentown, PA 18104
The tailless family of nuclear receptors is highly conserved among animals. In humans, it
functions in regulating neuronal stem cell differentiation. The C. elegans tailless ortholog,
nhr-67, is expressed in a dynamic pattern in pre-uterine cells: initially in the 4 pre-VU cells
during the L2, then upregulated in the anchor cell (AC) in response to the lin-12/lag-2 Notch
reciprocal signaling system. During the L3 stage, nhr-67 expression is maintained at high
levels in the AC and at low levels in VU descendants that produce the adult ventral uterus.
nhr-67 is required for expression of the lin-12/Notch receptor in pre-VU and VU cells and for
multiple markers of AC identity, indicating that it functions in differentiation of both uterine
cell types. Deletion of a 276bp region of the nhr-67 promoter results in a loss of nhr-67
expression in pre-VU, AC, and VU cells. Expression of 276bp region::gfp shows the region
is necessary and sufficient for nhr-67 expression during ventral uterine development. The
region includes two E box sequences that we propose bind the HLH-2 transcription factor,
which functions in AC and pre-VU development. We have performed site-directed
mutagenesis to delete the E boxes and other conserved elements from the 276bp promoter
region and tested their functions directly in vivo. Our data demonstrate the primary role of
the E box sequences in regulating nhr-67 in the AC, pre-VU, and VU cells. Funded by the
NIGMS.
6. bis(trifluorobutyl) & bis(trifluoropropyl) putrescine Effects on MCF-10F & MCF10Fbs Cell Lines
Michael Lawler, Daniel Salovich, Taylor Gandy, Tyler Short, Bailey Semian, Fr. Peter
Leonard, & Dr. Francis Mayville.
DeSales University, Center Valley, PA 18034
The effects of bis(trifluorobutyl) and bis(trifluoropropyl) putrescine were tested to evaluate
the viability of MCF-10F and MCF-10Fbs human breast cancer cells. A crystal violet assay
was used to measure cell viability after exposure to either of the synthesized putrescine
derivatives. Data analysis suggests an inhibition in cell growth, possibly from apoptosis.
Further studies are necessary at a physiologically relevant concentration to assess potential
chemotherapeutic efficacy.
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7. FAX-1 and UNC-42 Transcription Factors Regulate Developmental Arrest in C.
elegans
Susanna Birnbaum and Dr. Bruce Wightman
Muhlenberg College, Allentown, PA 18104
The fax-1 nuclear hormone receptor and unc-42 homeobox gene control interneuron
identities in C. elegans. fax-1 is the ortholog of unfulfilled in Drosophila and PNR/NR3E3 in
vertebrates, where it functions in the development and function of mushroom bodies and
photoreceptors, respectively. The fax-1 and unc-42 transcription factors function in
specifying the identities of an overlapping subset of nematode interneurons, including the
command interneurons AVA and AVE, which function in coordinated movements. Both
genes are required for the expression of neuron-specific genes, including glutamate receptors
subunits, and axon pathfinding. Mutations in both fax-1 and unc-42 cause an incompletelypenetrant slow-growth phenotype that arises from temporary arrest after hatching at the L1
stage. L1 arrest has been shown to be controlled by the insulin-like signaling pathway that
also controls dauer formation and longevity. The daf-2 insulin receptor is a primary mediator
of insulin signaling in C. elegans. Strong daf-2 mutations cause L1 arrest, while weak daf-2
mutations cause dauer-arrest. Both fax-1 and unc-42 mutations cause a fully-penetrant L1
arrest in combination with a weak daf-2 mutation. The L1 arrest can be reversed by a
mutation in the daf-16 forkhead transcription factor, which functions downstream of daf-2.
These observations indicate that the fax-1 and unc-42 transcription factors may function in a
pathway that controls developmental progression. Current genetic data support a role for fax1 and unc-42 in a TGF-beta signaling pathway that is parallel to the insulin pathway. Given
that both genes are required for the development of a limited set of interneurons, these
experiments suggest a previously unappreciated role for interneuron function in regulating
developmental temporal progression and arrest. Supported by NIGMS.
8. bis(trifluorobutyl) & bis(trifluoropropyl) spermidine 20µM Effects on MCF-10F &
MCF-10Fbs Cell Lines
Daniel Salovich, Michael Lawler, Tyler Short, Bailey Semian, Taylor Gandy, Fr. Peter
Leonard, & Dr. Francis Mayville
DeSales University, Center Valley, PA 18034
The viability of MCF-10F & MCF-10Fbs cells were tested in the presence of 20 µM bis
(trifluorobutyl) and bis (trifluoropropyl) spermidine. Tumorigenic cells exhibited a higher
death rate, presumably from apoptosis, but cells with a less carcinogenic phenotype are also
affected and exhibit a slower growth rate compared to the control group.
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9. Denopamine’s Actions on the Developing Vertebrate Four-Chambered Heart
Yennifer Arguello, Joanna Haddad and Dr. Jacqueline McLaughlin
Penn State Lehigh Valley, Center Valley, PA 18034
Denopamine is a β-adrenergic receptor agonist and a positive inotropic agent with a modest
effect on heart rate. It is used in the treatment of angina (chest pain due to a reduction of
blood flow to the heart). This research utilized the chick embryonic heart as a model system
to examine the effects of Denopamine on the heart rate (chronotropic) and contractility
(iontropic) of the developing vertebrate four-chambered heart. It was hypothesized that
Denopamine at low dosages would cause the in vitro heart rate of both 5 and 6 day isolated
chicken hearts to slightly increase and the force of contractions in all chambers to do the
same. It was further reasoned that arrhythmias would ensue at high dosages. The results
showed increased heart rate with normal sinus rhythm and enhanced force of atrial and
ventricular systole at low doses. As the concentration of drug was increased arrhythmias
followed that were consistently seen through repeated trials: forceful contractions with
tachycardia, atrioventricular (AV) block, and atrial flutter. Results suggest that Dopamine’s
actions on the developing chicken heart mirrors those found in the developed human four
chambered heart when used at low dosages, and that its induces dangerous heart arrhythmias
at high doses. Based on experimental data gathered and review of the FDA Drug Safety
Categories during pregnancy, it appears that there is evidence of possible fetal risk.
10. The effect of Putrescine and bis(trifluoroButyl) Putrescine on Bovine Aortic
Endothelial Cells
Janalyn Frederick, Dr. Francis Mayville, Fr. Peter Leonard, Dr. Joshua B. Slee
DeSales University, Center Valley, PA
Putrescine, a natural polyamine, has not been shown to hinder cancer growth. However,
modifications in the physical structure of the molecule have been known to cause cancer cell
death. As a potential cancer therapy drug, death of healthy cells is something that should be
avoided. Preliminary tests on non-cancerous Bovine Aortic Endothelial Cells show that one
type of modified putrescine, bis (trifluoroButyl) Putrescince, was deadly to these cells. The
naturally occurring Putrescine was not lethal to the non-cancer cells. The cell death observed
could be attributed to the large dosage of the modified polyamine being used. Future studies
will be performed to determine if the high concentration of the modified polyamine was the
cause of the death of healthy cells. Other future studies would include the determination of
whether new modified polyamines could be potential anti-cancer agents.
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11. The Effects of LED Lighting on the Growth of Chlorella vulgaris
Amanda Geis, William Sampson, Dr. Jacqueline McLaughlin, and Dr. Tai-Yin Huang
Penn State Lehigh Valley, Center Valley, PA 18034
Species of microalgae have the potential to double their biomass within 24 hours during the
exponential phase of their growth curve and to contribute hydrocarbons for biofuel
production. In order for microalgae to reach maximum growth, optimal light conditions must
be present within the narrow band of the visible light spectrum that is associated with the
specific photopigment(s) within each species light harvesting system. The use of LED lights
may promote microalgae optimal growth conditions due to their potential to provide a single
wavelength, lower heat dissipation, lower power consumption, a longer-lifetime, and high
efficiency of electricity conversion. A study was carried out wherein the green algae,
Chlorella vulgaris, was cultured under red LED lighting at 625nmλ at 5,000 mcd or
traditional full spectrum grow lights. The results showed that a higher concentration of algae
with a smaller cell size can be obtained over a seven day period using the red LED light than
algae grown under a traditional grow light source. The data suggest that using LED lighting
is a more efficient method of cultivation of C. vulgaris. Future work includes investigating
lipid production from the C. vulgaris, after its growth dynamics (cell concentration) and cell
size are optimized using varied LED wavelengths.
12. Localization of Cofilin in Bovine Aortic Endothelial Cells as a Result of Cell Stress
Melody Dillee and Dr. Joshua B. Slee
DeSales University, Center Valley, PA 18034
Cofilin, a member of the actin depolymerizing factor (ADF) family of proteins, is required
for the reorganization of actin filaments. Immunofluorescent microscopy of cofilin indicates
that phosphorylated cofilin (serine-3) increases in the nucleus and decreases in the cytoplasm
during fluid shear stress (FSS). In addition, it has been observed that when placed under
conditions that cause cell stress, such as oxidation and ATP depletion, cofilin becomes
dephosphorylated and locates to the nucleus. Further research will investigate the localization
of cofilin in response to cell stress through treatment with H2O2 and TNF-α in bovine aortic
endothelial cells (BAOECs).
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13. Investigation of the therapeutic response of a spontaneously-forming 3D in vitro
cancer model
Nelisa Bechtel and Dr. Maria Theodoraki
Arcadia University, Glenside, PA 19038
Inflammatory breast cancer (IBC) is an aggressive and highly metastatic cancer without
definitive prognosis or successful treatment. MARY-X is an IBC model that, in vitro, forms
tight, compact aggregates of cells called spheroidsMARY-X. The spheroidsMARY-X mimic
in vivo tumor emboli as they contain proliferative cells on the periphery and
dormant/hypoxic cells in the center. This makes them valuable in drug development as they
allow successful translation of anticancer therapeutics. MAD28, a drug in development,
induced total dissolution of the spheroidsMARY-X displaying complete response, whereas
FDA-approved paclitaxel (PTX) failed to elicit a response. The purpose of this study is to
determine the mechanism of action of MAD28 with respect to its ability to target the
pathophysiological gradients within the spheroid model and to determine the mechanism of
cell death upon treatment. To comprehend the specific population targeted by each drug, the
spheroidsMARY-X were suspended in a matrigel scaffold and then treated with appropriate
drug concentrations. The treated samples were sectioned and labeled with Ki-67 or cleaved
caspase-3 to assess for proliferation and induction of apoptosis, respectively. To determine
the minimum concentration of MAD28 that induces apoptosis in spheroidsMARY-X, we
performed western blot analysis using caspase-7 and cytochrome c. Overall, our results
indicate that MAD28 targets both proliferative and hypoxic/dormant cells of the
spheroidsMARY-X and induces apoptosis more efficiently than PTX. Future studies will
focus on analysis of colocalization between caspase-3 and Ki-67 and time dependent
induction of apoptosis.
14. Localization of BMP6 in vascular endothelial cells treated with heparin
Kelsey Elliott1, Dr. Linda J. Lowe-Krentz2, and Dr. Joshua Slee1
1
DeSales University, Center Valley, PA, 18034
2
Lehigh University, Bethlehem, PA, 18015
Bone morphogenetic protein 6 (BMP6) has been found to bind heparin and influence the
activity of bone morphogenetic proteins, which are members of the transforming growth
factor- (TGF-) superfamily of proteins that regulate proliferation, differentiation, pattern
formation, and apoptosis. It can be predicted that BMP6 upregulation plays a role in the
anticoagulant functions of heparin, but not much is known about the heparin antiinflammatory mechanisms. There is a known link between BMP6 and smooth muscle cell
physiology, but it is unclear how BMP6 upregulation affects physiology. A microarray
completed during previous research indicated a 1.899-fold increase in BMP6 for vascular
smooth muscle cells treated with heparin in comparison to untreated cells. Current research
looks to validate these findings by observing localization and expression changes of BMP6 in
response to heparin treatment.
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15. Distribution of Phosphorylated Connexin 43 in Gap Junctions: Implications for Gap
Junction Internalization
Rachel Margraf and Dr. Matthias Falk
Lehigh University, Bethlehem, PA 18015
Gap junctions (GJs) are transmembrane protein complexes that form channels between cells
and permit passage of small molecules. These channels are composed of protein subunits
called connexins (Cxs), the most well studied being Cx43. Clusters of GJ channels form
arrays known as GJ plaques. Specific phosphorylation events on the C-terminal tail of
connexins decrease intercellular communication, ultimately leading to internalization into
annular gap junctions (AGJs) and degradation. Previous studies have identified the
phosphorylation (p) of Cx43 on Serines (S) 255, 262, 279/282, and 368 to be associated with
decreased GJ intercellular communication (Thévenin et al. 2013), but the order of these
events is not well understood. We hypothesize that pS368 occurs first, followed by p279/282,
and that the later phosphorylation will occur in more discrete regions towards the center of
the plaque. Using antibodies specific to pS279/S282 and pS368, I examined their distribution
within the GJ plaque and within AGJs via fluorescence microscopy. I found pS279/S282
Cx43 located in discrete regions of GJ plaque centers, while pS368 Cx43 was more widely
distributed in the plaque. Additionally, AGJs contained both pS279/S282 and pS368. These
results suggest that pS368 occurs first, followed by pS279/S282, and these events aid in
signaling GJ internalization.
16. Efficacy of Botanical and Synthetic Disinfectants
Amanda Yanisch, Casey DeStasio, and Dr. Joshua Slee
DeSales University, Center Valley, PA 18034
The debate of all-natural products versus synthetic products in cleaning has become a very
prevalent issue in present day. The “Go Green” movement has encouraged many people to
use botanical-based disinfectants instead of harsher synthetics ones. This ideology arises
from the belief that plant-based disinfectants are equally as effective as synthetic
disinfectants without the concern of potentially harmful ingredients. The purpose of this
research is to identify the effectiveness of all-natural disinfectants against synthetic
disinfectants. In this study, synthetic disinfectants labeled as “A” and “D”, and botanical
disinfectants labeled as “B” and “C” were used. The disc diffusion test of disinfectant
efficacy was used and demonstrated all disinfectants tested except for disinfectant “D,”
showed considerable zones of inhibition indicative of S. aureus death. None of the
disinfectants tested showed distinguishable zones for E. coli. The data suggest that the
botanical-based disinfectants were as effective as the synthetic ones.
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17. The effect of temperature on the gene expression of UDP-N-acetylglucosamine
pyrophosphorylase-2 in Tribolium castaneum during development
Christie Ellis and Dr. Marilyn Baguinon
Kutztown University, Kutztown, PA 19530
Tribolium castaneum, a common insect pest found in cereal grains, has two genes that
encode for proteins similar to UDP-N-acetylglucosamine pyrophosphorylase (UAP). In
Drosophila melanogaster, UAP is required in the formation of chitin, the major component of
the exoskeleton. This research is conducted to gain insight into the roles of TcUAP2 in T.
castaneum. Specifically, we are investigating the effect of culture incubation temperatures on
the expression of the TcUAP2 gene during key developmental stages. Three sets of 1-liter
cultures of red flour beetles were incubated at 25˚C, 30˚C, and 37˚C. After a specified period,
the larvae, pupae and adult insects were collected. Total RNA was extracted, the TcUAP2
mRNA was isolated and the corresponding cDNA synthesized using primers specific for the
TcUAP2 gene. Our results have shown that the TcUAP2 gene was expressed in the larva,
pupa and adult stages, in all the three culture incubation temperatures. The sequences of the
cDNA samples will be determined to confirm the identity of the expressed gene.
18. Towards the Expression of His-tagged farnesyl diphosphate synthase from
Thermoplasma volcanium
Lucie Loftus, Ryan Vignogna, and Dr. Julie Himmelberger
DeSales University, Center Valley, PA 18034
Farnesyl diphosphate synthase (FPPS) is an important enzymatic catalyst for the formation of
farnesyl diphosphate (FPP), a precursor of a family of fifteen-carbon containing natural
products, known as the sesquiterpenes. In recent years the structure of many FPPS homologs
have been analyzed, but the homolog of FPPS from the archaeon Themoplasma volcanium is
not yet known. Our goal is to study the structure of this FPPS homolog to determine how it
any structural adaptations for the thermophilic environment. Current work involves cloning
the FPPS gene into a pET28b plasmid with an N-terminal His-tag, followed by expression
and purification of the FPPS for protein crystallization trials.
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19. The effect of ultraviolet radiation on the gene expression of UDP-Nacetylglucosamine pyrophosphorylase-1 during development in Tribolium
castaneum
Alyssa Boswell and Dr. Marilyn Baguinon
Kutztown University, Kutztown, PA 19530
Tribolium castaneum, a common insect pest of cereal grains, carries two genes that have
nucleotide sequences similar to the gene that encodes for UDP-N-acetylglucosamine
pyrophosphorylase (UAP). The UAP gene is found in all species but most organisms carry
only one copy of the gene. In Drosophila melanogaster, previous research had shown that the
UAP enzyme is essential in the formation of chitin, a major component of the exoskeleton of
arthropods. In T. castaneum, the specific functions of the two enzymes are still unclear. To
gain insight into the specific roles of TcUAP1, we are investigating the expression of the
gene under ultraviolet radiation during larvae and pupae stages of development. Larvae and
pupae were exposed to ultraviolet radiation at different periods. Following the UV exposure,
the insects were incubated at 37˚C for 24 hours. Total RNA was then extracted, the mRNAs
isolated and the cDNAs synthesized using DNA primers specific to TcUAP1 gene.
Preliminary results have shown that young larvae exposed to UV radiation for 30 minutes
still expressed the TcUAP1 gene. Additional results on other developmental stages and
longer UV radiation exposures will be presented. The cDNA samples will be sequenced to
confirm the identity of the expressed gene.
20. The extraction, isolation, and antioxidant analysis of annonacin from fruit of the
North American Pawpaw (Asimina triloba)
Ellie Charamut, Christy Kovaleski, and Dr. Francis Mayville
DeSales University, Center Valley, PA 18034
Soxhlet extractions using ethanol as a solvent were performed to extract Annonaceous
acetogenins (Annonacin) from the fruit of the North American Pawpaw (Asimina triloba).
Extracts were then analyzed using Ultraviolet/ Visible Spectrometry (UV-Vis) to identify
Annonacin and any other acetogenin like compounds present in samples. Annonacin present
was then quantified using High Pressure Liquid Chromatography (HPLC), as well as
Ultraviolet/ Visible Spectrometry (UV-Vis). Determined concentrations of Annonacin
present in fruit samples were then analyzed and compared to known antioxidants through
formation and detection of reactive oxygen species.
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21. Overexpression of klf-2 in the intestine of Caenorhabditis elegans
Macy Decker, Kaleb Davis, and Dr. Christopher Brey
Marywood University, Scranton, PA 18509
Krűppel-like transcription factors (klfs) are one of the most common transcription factors
found in all living organisms. One of their key roles is in regulating fat metabolism. In
humans, there are 17 klf genes, whereas in Caenorhabditis elegans there are only 3, making it
the ideal model organism to study fat metabolism. In this study, we focused on the specific
gene klf-2 since the expression localization of klf-1 and klf-3 is already known to occur in
the intestine. To determine the localization of klf-2 expression, we created a construct
pJS1.promklf2 which contained approximately 1,000 bp of the klf-2 promoter and 23 bp of
the first exon fused to gfp. This expression construct was co-microinjected with pRF-4, a
marker which allows for selection based on the worm’s mutant roller phenotype. Upon
screening, klf-2 is found to express in the lower intestine of the worms. These results are
consistent with klf-1 and klf-3 expression localization. By studying the expression of klf-2,
insight into the role this transcription factor plays in fat metabolism can be applied to our
understanding of metabolic diseases in humans.
22. The Synthesis of Several Spermidine Analogs in a 100% Ethyl Alcohol as Possible
Growth Inhibitors of Breast Cancer Cells.
Joann Almocherki, Thomas Salim, and Dr. Francis Charles Mayville Jr.
DeSales University, Center Valley, PA 18034
This investigation involves comparative syntheses of two spermidine analogs in 100% ethyl
alcohol. These nucleophilic substitution reactions with alkyl halides produce
bis(trifluoropropyl), and bis(trifluorobutyl) spermidine. The use of ethyl alcohol as a reaction
solvent allowed for synthesis of the polyamine analogs to proceed in a greener manner.
Alcohols are preferred solvents as they are more environmentally friendly, can be reclaimed
or recycled, and reactions typically run at lower temperatures; this is not the case for
traditional volatile organic solvents. The data in this study supports the previous study’s
hypothesis that the use of 100% ethyl alcohol as the solvent, would produce a greater yield
than using 95% ethyl alcohol with less toxicity of other alcohol solvent such as 1-butanol.
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23. BPA affects diverse endocrine and metabolic pathways during development in
Drosophila melanogaster
Jennifer Nguyen and Dr. Sheryl T. Smith
Arcadia University, Glenside, PA 19038
Bisphenol A (BPA) is a high-production industrial monomer used world-wide in the
manufacture of polycarbonate plastics and epoxy resins. BPA is an Endocrine Disrupting
Chemical (EDC) that can disrupt hormonal pathways central to proper development as well
as metabolic pathways related to obesity and type II diabetes. Our group previously reported
that Drosophila can serve as a useful model for studying the effects of BPA on development.
Specifically, we found that BPA caused an increase in larval growth that resulted in an earlier
onset of pupariation. Growth and maturation in Drosophila are governed in part by the
Insulin/Insulin growth factor (IIS)/Target of Rapamycin (TOR) signaling network and the
Ecdysone signaling pathway. In this study, we sought (1) to test whether BPA can affect
IIS/TOR signaling in Drosophila larvae by assessing fat deposition, (2) to test whether BPA
exposure during larval development can affect endoreduplication of polytene chromosomes,
and (3) to identify gene expression changes associated with the activation of specific
pathways in response to BPA. We found that BPA exposure during larval development
resulted in an increased size of Drosophila fat body cells, suggesting that BPA can affect
IIS/TOR signaling. Our study also showed that the ecdysone-dependent process of
endoreduplication of polytene chromosomes is suppressed in third instar larvae treated with
BPA. From the RNA-sequencing study, we found a downregulation of prophenol oxidase 2
(PPO2) and Cytochrome P450-4c3 (Cyp4c3), belonging to gene families implicated in BPA
detoxification.
24. The Synthesis of Two Putrescine Analogs in 100% Ethanol as Possible Growth
Inhibitors of Breast Cancer Cells
Emily Brown, Alexa Beaumont, Elizabeth Phillip, and Dr. Francis C. Mayville Jr.
DeSales University, Center Valley, PA 18034
This investigation will involve the synthesis of several putrescine analogs produced in 100%
ethanol as the solvents. These nucleophilic substitution reactions with alkyl halides will
produce bis (3,3,3-trifluoropropyl), and bis (4,4,4-trifluorobutyl) putrescine. In this study
100% ethanol was used as the greener reaction solvent, which allowed for production of the
polyamine analogs to proceed in a less toxic environment. There are many advantages for
using alcohols over traditional volatile organic solvents in these synthetic reactions. Alcohols
are preferred solvents as they are more environmentally friendly, can be reclaimed or
recycled, and reactions are run at lower temperatures. It was also found, in this work that the
use of 100% ethanol increased the product yield dramatically when compared to the more
toxic solvents used in previous studies in our laboratory.
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25. BioS368 Cell Biology Laboratory at Lehigh University: Cell Culture and
Fluorescence Microscopy
Dr. Matthias Falk
Lehigh University, Bethlehem, PA 18015
Cell culture and fluorescence staining/labeling techniques that allow observing organelles,
proteins and nucleic acids in cells are important techniques that are applied by Cell Biologists
all over the world. The Cell Biology Laboratory at Lehigh is a unique experience to learn and
apply state-of-the-art fluorescence microscopy techniques including imaging proteins in
living cells. The course has four main sections: (1) Students thoroughly learn how to culture
immortalized cell lines, (2) to stain sub-cellular structures in fixed and living cells using
specific probes and antibodies (including double and triple color labeling), (3) to express and
observe proteins tagged with fluorescent protein probes (GFP and derivatives, RFPs) in
living cells, and (4) to interfere with cellular processes using specific drugs. Pursued
experiments are not standard experiments available commercially in kit form, but are based
on actual, unique research projects pursued in the instructor’s laboratory that have been
adapted to the classroom. Students maintain their own cells during the entire course and grow
cells in dishes and on cover slips for experimental manipulation and microscopic
observation. The course is designed to give students a hands-on experience in cell biological
experimentation. Interested? All cell images presented here were acquired by previous
BioS368 students.
26. High Temperatures Diminish Telomere Position Effect at Saccharomyces cerevisiae
Telomeres
Ellen Weidle, Rachel Jesiolowski, and Dr. Lara Goudsouzian
DeSales University, Center Valley, PA 18034
Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes. In
addition to protecting the ends of chromosomes from DNA double-strand break repair
pathways, telomeres solve the end replication problem that is common to all linear
chromosomes. A gene placed adjacent to a Saccharomyces cerevisiae telomere shows
reversible transcriptional repression, an effect known as Telomere Position Effect (TPE).
This alteration of genetic expression is not seen when the same gene is moved to a position
more internal to the chromosome. High temperatures have been shown to diminish telomere
length. We assayed for the ability of a yeast telomere to silence a URA3 gene under different
temperature conditions. We showed that TPE was greatest at 23°C and 30°C, but was
diminished at 37°C.
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27. Genome-Wide Expression Analysis of 3 Mutant Yeast Strains
Rebecca Lukasak and Dr. Lisa Antoniacci
Marywood University, Scranton, PA 18509
The yeast nuclear envelope protein Mps3 functions in several aspects of chromosome
metabolism such as sister chromatid cohesion, telomere clustering, and DNA damage repair.
In addition, Mps3 physically and functionally interacts with the histone variant Htz1. Htz1
was previously identified as a histone variant that prevents DNA from forming silent
heterochromatin as a way to regulate transcription. Because of the involvement of both of
these genes in chromosome metabolism, it is hypothesized that both of these genes may also
function in regulating gene expression. In order to assess genome expression wild type yeast,
2 Mps3 mutants (mps3-3 and mps3-5) and an htz1∆ strain were examined for genome wide
expression levels in asynchronously growing cells. The data generated will be analyzed for
both up-regulation and down-regulation of gene expression in comparison to the wild type
strain. Identification of expression differences between the wild type and mutant strains
would suggest a function for Mps3 and Htz1 in gene expression.
28. Ethanol Influences Telomere Position Effect in S. cerevisiae
Thomas Basil, Alison Z. Dyszel, Angeline Lonardi, and Dr. Lara Goudsouzian
DeSales University, Center Valley, PA 18034
Telomeres are protective sequences of heterochromatin DNA found at the ends of linear
chromosomes. Telomeres are necessary for protecting DNA from the end-replication
problem, which causes ends of DNA to shorten with each round of replication. Telomere
shortening and lengthening can lead to many changes in cell division and gene expression.
While telomere shortening causes aging and damage to DNA, telomere lengthening can lead
to unlimited rounds of replication and ultimately, cancer. Also, changes in telomere length
cause changes in gene expression; we call this the telomere position effect (TPE). We
propose that the principles behind the telomere position effect can help determine how
certain substances and chemicals impact telomere length in human cells. In this experiment,
we tested the effects of ethanol on gene expression in Saccharomyces cerevisiae, our model
organism. In this experiment, we grew yeast with and without the presence of 5% ethanol on
selective YC-FOA plates to see how ethanol affects the expression of URA3, a gene which
produces uracil. The FOA plates enable us to determine how ethanol affects the expression of
URA3 gene, whose gene product, ODCase. The FOA plates enable us to see changes in
URA3 gene expression because the URA3 gene product ODCase reacts with FOA to create a
toxin that decreases cell growth. If the 5% ethanol changes the expression of the URA3 gene,
thereby changing the growth of the yeast cells, we can determine ethanol’s impacts on
telomere length. Our research shows that the presence of 5% ethanol leads to an increase of
yeast cell growth on FOA plates. This is due to the increase of silencing of the URA3 gene
present near the VII-L telomere. Because our TPE results correlate with the increase of
telomere length seen in the ethanol grown cells (Romano et al. 2013), we propose the use of
this assaying in screening a variety of chemicals and drugs for their effects on telomere
length before the use of telomere blots.
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29. Perfluorooctanoic acid (PFOA) modulates its effects through the Tor signaling
pathway in Drosophila melanogaster
Emily Ng and Dr. Sheryl T. Smith
Arcadia University, Glenside, PA 19038
Perfluorooctanoic Acid (PFOA) is an industrial, synthetic compound found in numerous
products, to which humans experience a constant exposure. PFOA may cause dysregulation
of nutrient sensing pathways, including the highly conserved Insulin/insulin growth factor
signaling (IIS) and the Target of rapamycin (Tor) signaling network in Drosophila. This
study examines PFOA-induced growth and tissue developmental defects dependent upon
TOR signaling and determines the concurrent effects of PFOA and amino acid restriction on
survival, and whether this is Tor signaling dependent. We assessed larval growth and
survival, salivary gland morphology, cuticle pigmentation, and survival during amino acid
deprivation in wild-type and TorΔP (null mutant) backgrounds. We present evidence that
PFOA administered orally at concentrations from 0.05mM to 5mM resulted in a nonmonotonic dose response with respect to growth. We examined salivary gland morphology
and found evidence of autophagy and cell death, as well as decreased cuticle pigmentation, in
animals treated with 1mM PFOA, suggesting that PFOA can exert its effects in a TORdependent, cell/tissue autonomous manner. The effects of protein deprivation on survivability
were significantly mitigated in TorΔP mutant larvae exposed to PFOA, compared to wildtype PFOA-treated controls. These findings suggest complex mechanistic interactions occur
between PFOA and the Tor signaling pathway.
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