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LEHIGH VALLEY MOLECULAR AND CELL BIOLOGY SOCIETY INAUGURAL MEETING APRIL 21, 2016 HOSTED BY: DESALES UNIVERSITY 1 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 2 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]). 3 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. 4 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. 5 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. 6 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. 7 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. 8 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). 9 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. 10 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. 11 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. 12 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. 13 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. 14 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. 15 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. 16 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. 17 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. 18 19