Abstracts Submitted from the Biological Sciences (L-Z)
Undergraduate Summer Research Symposium August 12, 2009

Ordered alphabetically by student's last name

Randel Shock Timothee
Schaefer Sloofman Torres Wilson
Scheehan Stull Toseef
Shelly Sullenberger Vent

Molecular Basis of Calcitriol and Cell Signaling Pathways: Protective Effects Against Cancer

Rachael Latshaw, Rachel Schaefer, Jeremy Bonor, Beth Bragdon and Anja Nohe
University of Delaware Department of Biological Sciences

Clinical research reveals an inverse relationship between serum levels of calcitriol, the biologically active form of vitamin D, and the risk of developing and surviving hormone-dependent cancers. Calcitriol’s role as a nuclear transcription factor allows it to regulate cell growth, proliferation, differentiation and apoptosis. The mechanisms by which calcitriol functions at the molecular level are unknown, leaving the reason this molecule improves cancer prognoses a question mark among physicians and scientists. With lacking knowledge on the relationship between cancer and calcitriol signaling pathways and calcitriol’s absorbance into cancerous and normal cells, research must focus on the mechanism of the vitamin D receptor (VDR) and cellular uptake of calcitriol. To begin this research, calcitriol must be successfully coupled to quantum dots (QDs), high luminescence particles with a narrow emission spectrum. QDs are fluorescent nanoparticles with a cadmium selenide core and zinc sulfide shell used to tag calcitriol for live cell imaging. Infrared analysis is used to verify coupling and show a shift where the polyacrylic acid coating on QDs reacts with a hydroxide on calcitriol to form an ester. A reporter gene assay confirms calcitriol is active and confocal microscopy provides live cell imaging of calcitriol absorbance through the plasma membrane of C2C12 mouse myoblastic stem cells.

Progesterone Receptor Regulation of Dopaminergic Cell Number of the Mouse Substantia Nigra Pars Compacta

Thaddeus G. Lehman and Princy  Q. Mennella
Delaware Technical and Community College and Delaware State University

Steroid hormones and their receptors can influence the development of the mammalian brain. For instance, the nuclear steroid hormone receptor for progesterone, the progesterone receptor (PR), may facilitate the development of sexually dimorphic structures. PRs may also regulate the development of non-sexually dimorphic structures. PR expression is present in the substantia nigra pars compacta (SNc) on the day of birth through postnatal day 7, but is absent by postnatal day 14. Consequently, the adult SNc lacks PR expression. This suggests PR has some developmentally specific role in the SNc. In the present study, we used immunocytochemistry for the rate-limiting synthetic enzyme for dopamine, tyrosine hydroxylase (TH), as a marker for dopaminergic cells. Manual counts of cells that displayed THir between mice lacking functional PR (PRKO) and wild type (WT) mice were performed. Preliminary results indicate that PRKO mice demonstrated a trend towards a greater number of THir cells compared to WT mice. It appears that PR in the developing SNc may be influencing the survival of dopaminergic neurons. However, follow up experiments, with larger sample sizes, would need to be conducted to replicate these findings. Supported by NSF EPSCoR, Grant EPS-0447610.

Cloning and expression of neural cell adhesion molecules for functional studies of prostate cancer metastasis

Nadia Lepori-Bui, Keith Jansson, and Robert A. Sikes
Center for Translational Cancer Research, Laboratory for Cancer Ontogeny and Therapeutics

Background:  Prostate cancer (PCa) is the most commonly diagnosed cancer in U.S. males. Metastatic PCa results in over 27,000 deaths annually and >85% involve metastasis to bone, particularly to the lumbar and sacral spine.  Current paradigms state that PCa metastasis is through the circulation and/or lymphatic system, but this does not explain the specific localization of PCa to bone and, more specifically, the lumbar spine.  We hypothesize that PCa metastasizes to the spine via adhesion to nerve and/or nerve-associated (Schwann) cells via the V-set immunoglobulin (Ig) domain of a novel cell adhesion molecule, SCN2B. Schwann cells express a potential cognate binding partner of SCN2B, called MPZL2 or Myelin-P0-like protein.  Tenascin-R (TnR) also is involved in neural tracking, but has domains repulsive to SCN2B.  Methods: Restriction enzymes were used to clone MPZL2 and the repulsive domain of TnR into mammalian expression vectors as fusion constructs with a small peptide tag included for purification. Amplification of fragments for cloning was accomplished using PCR-directed amplification, cloning into TA-vector intermediates followed by subcloning those inserts into pcDNA 3.1 myc-His and PLN1 mammalian expression vectors. Results: The full length cDNA of MPZL2-myc-His has been successfully subcloned and transfected into LN cells.  MPZL2-ectodomain and TnR were amplified successfully and cloned into the TOPO vector but not yet into mammalian expression vectors. Conclusions: The cloning of these domains will allow adhesion studies with SCN2B and other cellular adhesion molecules to determine their role in PCa metastasis.  Additionally, transfecting the full length isoforms of these CAMs into the LNCAP progression model will allow us to determine possible functional effects of constitutive overexpression.  

Dietary fat may influence gut bacteria profile and diversity in
Mus musculus

Tianyu (Tom) Liu, Amy C. Vollmer, and Sarah Hiebert Burch
Department of Biology, Swarthmore College

Mammalian gut microbes have been implicated in a variety of functions including the regulation of energy intake and storage, the control of immune system development and activity, and the synthesis of novel chemicals. Diet is a powerful predictor of fecal bacteria profile across mammalian species, and obesity has been correlated with shifts in gut bacterial profiles and a decrease in bacterial diversity. The effect of oligosaccharides on gut bacterial profiles has been well documented but the effect of dietary fatty acid is poorly understood. Previously, groups of Mus musculus were fed different diets rich in saturated fatty acids, omega-3 polyunsaturated fatty acids, and omega-6 polyunstaurated fatty acids over a ten-week period. Mouse fecal pellets were collected before and after the study. In our investigations, bacterial DNA was extracted from the pellets and characterized by 16S rRNA analysis. Sections of the hypervariable V3 region of bacterial 16S rRNA sequence were amplified, cloned, sequenced, and classified to observe differences in ratios of bacterial phytotypes as a result of differing dietary fatty acid content. 16S rRNA sequences were also used to create phylogentic trees and rarefaction curves to compare bacterial diversity among the experimental groups. Results may elucidate the role of bacteria in how diet affects the body and suggest dietary methods to regulate human health.

Investigating the role of the gpa-14 gene in
C. elegans behavior

Rochelle S. McDonald, Rosaria Formisano, Tim Pierpont and Harb S. Dhillon
Department of Biological Sciences,  Delaware State University Dover, DE 19901

The similarity in the basic molecular processes between diverse organisms allows the use of the simple nematode, Caenorhabditis elegans, in understanding the molecular and cellular basis of learning and memory. The neurotransmitter dopamine has been linked to behavior in humans as well as other organisms. In C. elegans, dopamine has been implicated in associative learning as well as habituation behaviors. I am investigating the possibility of a relationship between the gene dop-2, which codes a dopamine auto-receptor and the G-protein alpha-subunit gene, gpa-14. We hypothesized that if the gene products, dop-2 and gpa-14 interact with each other, then we should see behaviorally similar phenotypes for mutants in these genes. We have characterized the gpa-14 knock-out mutants using a number of behavioral assays. Based on our chemotaxis and mechano-sensation assay results, we found that gpa-14 mutants are normal in their sensory-motor responses. Our chemotaxis-based conditioning assay results show they are also normal in their associative learning capabilities. But the habituation profile is abnormal; this was tested using a mechano-sensation based habituation assay. Interestingly gpa-14 mutants habituate faster than wild type, which has also been observed for other dopamine deficient mutants. We have now initiated work on engineering constructs in which gpa-14 and dop-2 are fused to different fluorescent protein genes, in order to study their physical interaction. This project was funded by the EPSCoR (NSF) Program.

Microbe-Mineral Interactions and their Influence on Selenium Transformations in Soil Environments

Allison M. Moran and Keka C. Biswas
Wesley College Dover, DE 19901

Selenium occupies a unique position in regards to its continuing conflicting aspects of its toxicological and nutritional significance in bacterial reduction of selenium (Se). Bacterial reduction of selenium (Se) oxyanions Se(VI) and Se(IV) to elemental Se is one of the major biogeochemical processes removing Se from agricultural drainage water and the depositing Se in the sediment. Indigenous bacteria have a significant role in the biogeochemical cycling of Se and may be stimulated by addition of a suitable organic source for Se reduction. While few bacteria have the capability of coupling the reduction of selenate or selenite to elemental selenium, many aerobic bacteria have a glutathione-based process of forming elemental selenium and several anaerobic bacteria use metal reducing systems to produce elemental Selenium. Upon our screening process,  Se(VI)-and  Se(IV)-reducing bacteria were isolated from bedding litter and groundwater and identified by amplification and sequencing of 16S rRNA. Bacillus strains appeared to be dominant in the bacterial assemblages active in Se(VI) and Se(IV) reduction. Using environmental bacterial isolates, we have followed the reduction of selenite to colloidal elemental selenium by pure cultures and the method detects elemental selenium even if elemental selenium is formed inside the cells.  Our research addresses a method for measuring colloidal red selenium in the presence of chemicals typically associated with soil or water containing toxic compounds.  Using environmental bacterial isolates, identified by colony PCR and DNA sequencing, we have followed the reduction of selenite to colloidal elemental selenium detected by AFM and Confocal microscopy techniques. This Project described was supported by NSF-EPSCoR Grant EPS-0814251 obtained through Delaware Biotechnology Institute.  

Synthesis of Silver Nanoparticles for Use in an Animal Model

Suranjit Mukherjee, Prasad Dhurjati, Vladimir Smorodin, and Anja Nohe
Department of Biological Sciences

Nanoparticles derived from silver are an ideal candidate for biological applications due to silver’s natural anti-bacterial, anti-inflammatory, and anti-platelet activity. However, not much is known about the accumulation of silver particles in vivo. The purpose of this study was to determine whether different sized and charged silver particles would selectively accumulate in different tissues within mice. To achieve this we attempted to synthesize silver nanoparticles in the sizes of 5, 10, and 20 nanometers capped with L-cysteine and MSA, inject them into mice, and track the particle distribution at the organ level over a course of 5 days. By tracking particle size vs. organ accumulation, we could potentially develop a quantitative model to allow for the use of silver nanoparticles for targeted drug delivery purposes. During the past 10 weeks, silver nanoparticles were synthesized via a reduction reaction of silver nitrate (AgNO3) with sodium borohyride solution (NaBH4) and DMF. Addition of L-cysteine and MSA as capping agents stabilized the particles and prevented aggregation. Separation of the the silver particles according to size was performed by centrifugation, gel chromatography, dialysis and TLC. Using gel chromatography, we successfully isolated silver particles of 4-15nm and another population approximately 100nm in range. Future work will include the injection of these particles into mice and determining their accumulation. Funding for this work was provided by HHMI.

(First Place in the Sigma Xi Oral Presentations)
Pathogenicity Islands Are Discrete and Ancient Integrative Elements: An Evolutionary and Functional Analysis

Michael G. Napolitano, Salvador Almagro-Moreno, and E. Fidelma Boyd
Department of Biological Sciences

The evolution and emergence of pathogenic bacteria is tightly linked to the horizontal transfer of pathogenicity islands (PAIs) and bacteriophages encoding a range of virulence factors. The current theoretical framework is that PAIs and other genomic islands (GEIs) are a combination of diverse integrative elements such as bacteriophages, integrative conjugative elements (ICE), or plasmids. In this work, we describe how integrases from phages and pathogenicity islands form distinct phylogenetic groups. In addition, we show that island-encoded integrases are not evolutionarily related to a range of integrative elements. These findings do not support the view that pathogenicity and genomic islands are a broad group of integrative elements derived from other elements. Using VPI-2 as a model for pathogenicity islands, we determined the role of the VPI-2-encoded integrase (intVPI-2) and two putative excisionases (vefA and vefB) in excision. We examined VPI-2 excision levels in V. cholerae mutant strains (delta)intVPI-2, (delta)vefA and (delta)vefB and also in strains that overexpressed these genes. We found negligible levels of excision in the (delta)intVPI-2 strain whereas overexpression of intVPI-2 showed a marked increase in excision. In the (delta)vefA or (delta)vefB strains the excision levels of VPI-2 remained the same, however, overexpression of vefB (VC1809) showed a 2-fold increase in excision. Overexpression of vefA (VC1785) in the wildtype causes complete excision of the island, however complimenting vefA with the deleted strains (delta)vefA and (delta)vefB leads to only a minor increase in excision above basal levels.

Hyaluronic Acid Receptor Proteins and Their Roles in Prostate Cancer Metastasis

Ngoc T. Nguyen, Lisa A. Gurski, and Mary C. Farach-Carson
Department of Biological Sciences and the Center for Translational Cancer Research,  University of Delaware

Hyaluronic acid (HA) is a polysaccharide that is distributed ubiquitously throughout the extracellular matrix (ECM) of all tissues and is especially prevalent in connective tissues such as bone and cartilage. HA plays a role in cell motility and invasion, processes involved in metastasis, through engagement with cell surface receptors, such as cluster of differentiation 44 (CD44) and receptor for hyaluronan-mediated motility (RHAMM). The purpose of this project is to study the expression of these HA receptor proteins and to determine if more motile, invasive cells show increased expression of HA receptors. We utilized the lymph node-derived cancer of the prostate (LNCaP) progression model of human prostate cancer (LNCaP, C-4, C4-2, and C4-2B), cell lines derived from a common lineage that differ in their ability to form distant metastases. This model progresses from non-metastatic LNCaP cells to metastatic, highly aggressive C4-2B cells. Through Western blotting, we have discovered that CD44 expression does not significantly increase among the cells in the progression model, suggesting that invasiveness and CD44 expression were not directly correlated in prostate cancer. Western analysis did, however, demonstrate a significant increase in RHAMM expression from LNCaP cells to the more invasive lines, conveying that RHAMM expression was important for prostate cancer aggressiveness and metastasis. Furthermore, we have employed immunofluorescence to determine protein localization. This project is funded by the Howard Hughes Medical Institute.

Genotype Phenotype Correlations in Hereditary Non-polyposis Colorectal Cancer

Kathryn Orio, David Smith, Mary C. Farach-Carson, and Zohra Ali-Khan Catts
Department of Biological Sciences, Helen F. Graham Cancer Center, and Center for Translational Cancer Research,  Newark, DE. 

Hereditary non-polyposis colorectal cancer (HNPCC) is the most common hereditary colorectal cancer syndrome accounting for approximately 3-5% of all colorectal cancer cases.  Most colorectal cancers occur sporadically, but 5-10% are due to a genetic predisposition.  The pedigrees and test results for nineteen families who tested positive for HNPCC found in the Familial Cancer Risk Assessment Program at the Helen F. Graham Cancer Center were collected and analyzed for genotype phenotype correlations.  Those positive for a mutation in MLH1 (MutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)) were found to have classic HNPCC (most families meeting Amsterdam criteria) and a predominance of young colon cancers.  When endometrial cancer was seen, the age of diagnosis was also early. Those who tested positive for MSH2 (mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)) were found to have a more significant history of renal and pancreatic cancers, a presence of breast and/or ovarian cancer, and variable colon cancer diagnostic ages that were not consistent with mutation type or location.  One could hypothesize that modifier genes may impact the variability seen within these families.  Those testing positive for MSH6 (mutS homolog 6 (E. coli)) had no predominance of colon cancer and later age of onset for all cancers.  In addition, this is a very small sample of HNPCC families, so these results may not be extrapolated to general population.  Additional research including more families and specific mutation types may be needed to achieve statistically significant conclusions.

P2 Receptors and ATP enhances the migration of prostate cancer cells

Wachen Peters, Christine Maguire, Randall L. Duncan, and Robert A. Sikes
Department of Biological Sciences

Background: Purinergic signaling stimulates many biological processes such as cell proliferation, differentiation, and apoptosis. Two classes of purinergic receptors, GPCR (P2Y) and gated channels (P2X), have been identified that bind ATP as a ligand.  Similarly, activation of these receptors by ATP can promote cell migration, a critical component of wound healing. ATP also has been shown to have an anticancer effect on in vivo.  Herein, we describe the effect of ATP on the migration of an isogenic progression series of prostate cancer (PCa) cell lines on three different extracellular matrices, Collagen I (bone), Matrigel (basement membrane), and Schwann cell-derived (Nerve support cells) in order to mimic growth on these common sites or routes of metastasis.  We hypothesize that ATP treatment activates purinergic receptors that increases cell migration on all three extracellular matrices. Methods:  RT-PCR was used to determine the mRNA expression profile of P2 receptors and ecto-nucleotidases. 0.1nM ATP or 2U/ml apyrase was added to PCa cell lines cultured on three different matrices in vitro. Migration was examined using wound healing assays by photomicroscopy for four days, as well as RT-PCR to determine effect of interaction with each ECM on the mRNA profile.  Results: ATP increased the migration of LNCaP cells on Collagen I to a higher degree than on either Schwann and matrigel over vehicle alone. Peak wound closure occurred after 48 hours, while treatment of cells with apyrase alone inhibited cell migration.  Differential expression of P2 receptors and ectonucleotidases was seen when comparing growth of cells on the matrices. Conclusions:  ATP is necessary for PCa migration on collagen I, matrigel and Schwann cell matrix.  The differential expression of P2 receptors is speculated to be responsible for variable wound healing on extracellular matrices. Fund: Department of Defense HBCU/MI Undergraduate Research Training Grant, PC080950

The SPAFF as a predictor of marital satisfaction

Lauren C Pulinka, Rachael Greenberg, Brittany Moriarty, Elana Graber and Jean-Philippe Laurenceau
Department of Psychology

Previous research has shown that the success of marriages can be predicted by examining positive and negative emotions during conflict tasks (Gottman & Levenson 2000). The Specific Affect Coding System (SPAFF) was developed and refined for coding a range of positive and negative affects predictive of marital outcomes (Coan & Gottman 2007). Based on observing marital interactions, Gottman and Levenson (2000) used SPAFF codes to predict early divorce based on the presence of negative affect and later divorce based on the absence of positive affect. The current study utilized behavioral observation of couple interactions to examine emotions that may be predictive of relationship outcomes (e.g., relationship satisfaction, divorce) two years later. Participants consisted of 120 heterosexual newlywed couples (married < 6 months) from Miami-Dade, Florida. Couples were videotaped participating in a warm-up task, two conflict tasks, and a love task, which are in the process of being coded using the SPAFF. They completed this process at three time points.  Most research of this kind has focused on positive and negative affects in the context of conflict interactions, whereas the current study will also focus on the presence of positive and negative affects in a positive (i.e., love) task.  We are currently coding the love tasks of the first time point and expect to replicate the previous findings.

Osteoarthritis and Fruit Flies:
Studying the role of heparan sulfate proteoglycans in essential and homologous signaling pathways

Rachel Randell, Richard Wittmeyer, and Erica Selva
Department of Biological Sciences

In humans, degradation of articular cartilage leads to osteoarthritis.  Here, the Drosophila wing imaginal disc is used to model the extracellular environment during signaling events required for the maintenance of articular cartilage.  In both human articular cartilage and in the fly wing disc, the extracellular matrix contains heparan sulfate proteoglycans (HSPGs), which are known to modulate signaling pathways (including Wnt, Hedgehog and BMP).  In Drosophila, HSPGs promote extracelluar ligand movement and are responsible for distinct patterns of signaling pathway activation in the wing disc.  In humans, the same signaling pathways maintain cartilage homeostasis and integrity.  To study the effects of HSPGs on signaling, flies have been generated in which wild type tissue and proteoglycan mutant “clones” lay side by side in the same wing disc.  Immunostaining of these discs for signaling ligands and activation targets reveals the importance of HSPGs for these essential signaling pathways.  Our results will determine the role of HSPGs with the hope of identifying targets for osteoarthritis therapeutics. 

Nuclear Signaling Pathways Involving Vitamin D3:
Calcitriol’s Effect on the Regulation of Genes Involved in Promoting or Preventing Cancer

Rachel J. Schaefer, Anja G. Nohe, Rachael A. Latshaw, Jeremy C. Bonor, and Beth C. Bragdon1
University of Delaware, 1University of Maine

The active form of vitamin D, calcitriol or vitamin D3 regulates the expression of many genes in the nucleus.  Calcitriol enters the cell and binds to a VDR, or vitamin D receptor.  The VDR binds to a RXR, or Retinoid X Receptor to form a heterodimer, which interacts with a VDRE, or vitamin D response element with co-activators or repressors to up-regulate or down-regulate a gene with a VDRE.  Calcitriol controls the expression of several genes that influence cell proliferation and differentiation in healthy cells and prevent it in cancerous cells.  It is expected that calcitriol will up-regulate genes that support cell proliferation and differentiation and down-regulate genes that promote apoptosis, or programmed cell death in healthy cells and have the opposite affect in cancerous cells.  Since it is works as a cancer preventative in controlling such functions, the gene expression and nuclear pathways involved are being viewed using calcitriol coupled quantum dots.  Since calcitriol is present throughout the entire gene expression pathway, the VDRE involved in the expression can be viewed under a confocal microscope by labeling calcitriol with quantum dots and allowing the C2C12 cells to take up the labeled calcitriol.  Quantum dots are nanoparticles that give off a fluorescent glow under UV light.  The stability of this binding to calcitriol using the chemical cross linker DCC is measured through FT-IR at three different temperatures.  Once the calcitriol coupled quantum dots are properly bound, they are used to view the gene expression of the stem cells used to represent normal, healthy cells.  After the results of this experiment are confirmed, the gene expression pathways involving calcitriol in the nucleus will also be viewed in cancerous cells.  

The Effects of Histone Modifications on Lens Cell Denucleation

Robert P. Sheehan, Yan Wang, and Melinda K. Duncan
Department of Biological Sciences

The lens of the eye contains two different types of cells, epithelial cells and fiber cells, with fibers cells deriving from the equatorial epithelium. When these cells differentiate, cell organelles are broken down, including the nucleus. However, small fragments of DNA from the nucleus remain. This led to interest in studying this denucleation, to learn why these pieces of DNA remained instead of being degraded. Our lab had previously studied the dynamic role epigentic factors played in lens development, which sparked the idea that similar epigenetic factors could be involved in this denucleation. We thus began to study histone modifications in the lens, specifically the trimethylation of Lysine 9 on histone H3. This modification causes a compact, less accessible form of chromatin that has no active gene transcription and may be less easily broken down. We compared the presence of this modification to opposing ones such as the acetylation of Lysine 9 on histone H3 and acetylation of Lysine 8 on histone H4 which result in a loosely configured, accessible euchromatin. In examining the lenses of humans and mice, we found a pattern of expression of H3 Lys 9 trimetheylation in the transition zone and fiber cells. This corresponded with fragments of DNA that were expected as remnants of denucleation. However, there appeared to be no acetylation of either histone studied. This suggests that remaining fragments of the nuclei are entirely composed of heterochromatin, as their highly compacted state prevented them from being broken down along with the rest of the nucleus. Supported by Howard Hughes Medical Institute and the National Eye Institute.

Expression and localization of human sperm Plasma Membrane Calcium ATPase 4b (PMCA4b)
which is required for normal motility in mouse sperm

Katharine E Shelly, Rolands Aravindan, Patricia A Martin-DeLeon
Department of Biological Sciences

Sperm motility defects (asthenozoospermia) accounts for a large percentage of cases of male infertility.  Two distinct types of motility are required of sperm to reach and fertilize an egg.  Progressive motility assists sperm in reaching the fertilization site, while hyperactivated motility serves to free sperm from the lining of the oviductal wall in order to enable them to effect fertilization.  While both are dependent on Ca2+ and ATP, the latter utilizes more ATP than progressive motility (1). In mice, hyperactivated motility and fertility are lost in the absence of Plasma Membrane Calcium ATPase isoform 4b (PMCA4b).  This ATP-driven enzyme is the principal calcium efflux pump in sperm. Thus PMCA4b plays an important role in murine sperm function.  The objective of this investigation is to characterize the presence of PMCA4b in human sperm by Western blotting, as well as to determine its subcellular localization by immunocytochemistry (ICC).  Flow cytometric analysis will also be used to confirm the presence of PMCA4b. Detection of PMCA4b was attempted by Western blotting of solubilized proteins from 3 human samples using PMCA4 polyclonal antibody in concentrations of 1:500 and 1:833.  Immunocytochemistry and flow cytometry were performed using various antibody concentrations (1:100, 1:250 and 1:500) for specific staining, compared to IgG controls for non-specific staining.  This work was supported by the Charles Peter White Fellowship and the NIH-COBRE grant #5P20RR015588-07.

Possible Novel Mobile Genomic Elements and Vibrio Pathogenecity Islands in
Vibrio mimicus

Patrick Shock, Salvador Almagro-Moreno, Michael G. Napolitano, and E. Fidelma Boyd
Department of Biological Sciences

Vibrio cholerae is the causative agent of the diarrheal disease cholera. V. cholerae has known mobile genetic elements some of which are pathogenecity islands, such as VPI, VPI-2, VSP-I, and VSP-II, that may represent the initial genetic factor causing pandemic cholera. Moreover, another Vibrio species, V. mimicus has a very similar phenotype and genome as V. cholerae; however V. mimicus can only cause gastroenteritis, symptoms similar to non-pandemic V. cholerae. The similarity between the species phenotypes suggest the possibility that the pathogeneticy islands found in V. cholerae might transfer between these two genomes. Essentially, V. mimicus might act as a reservoir for the mobile genetic elements present in V. cholerae. Our first approach was to compare the genome of an environmental strain of V. mimicus MB451 to the genome of  V. cholerae N16961. There were six regions in the V. mimicus MB451 genome that were not present in the V. cholerae N16961 genome. The six regions had G/C contents below the normal G/C content of 47.03 for V. mimicus MB451. Also, these regions encode an integrase, which suggest the possibility of horizontal gene transfer. The second approach was performing PCR assays on DNA of different V. mimicus strains to determine the presence of pathogenecity islands. In the PCR assay, two of three environmental strains and five of seven clinical strains of V. mimicus showed presence of VPI by positive PCR bands for the integrase (VC0847).

FTIRI and AFM analyses of cartilage and mineral tissues in HIP/RPL29-deficient mice

Laura G. Sloofman, Padma P. Srinivasan, Elizabeth L. Adams, Kostas Verdelis,
Lyudmila Spevak, Mary C. Farach-Carson, Adele L. Boskey, and Catherine B. Kirn-Safran

Department of Biological Sciences

Ribosomal proteins (RPs) are necessary for the maintenance of a normal protein synthetic rate.  A short stature phenotype associated with increased bone fragility is observed in HIP/RPL29 null mice.  We hypothesized that HIP/RPL29-null mutants exhibit changes in the relative amount of bone/cartilage constituents leading to structural imperfections at the molecular level.  To understand the effects of HIP/RPL29 absence on mineralized tissue quality, microspectroscopy was performed on thin sections of growth plate and bone of tibiae in null and control mice of the C57BL6/J background.  Using Fourier Transform Infrared Imaging (FTIRI), we showed that a decrease in collagen crosslinking during the development of HIP/RPL29 null bone precedes an overall enhancement in the relative extent of mineralization in adult bones and growth plate cartilage. Spectroscopic profiles and geometric data were also obtained for HIP/RPL29-null teeth, another mineralized tissue, via FTIRI and quantitative micro-CT.  Like bone, HIP/RPL29-null teeth exhibited reduced geometric properties coupled with relative increases in the mineral densities of both dentin and enamel.  Therefore HIP/RPL29 disruption induces defects in the relative distribution and/or structural organization of the mineral and organic phases, resulting in hypermineralization of bones and teeth.  Cortical bone properties are being modeled using homogenization theory and elemental finite differences techniques.  Additionally, we are developing a protocol to evaluate changes in the material property of articular cartilage in mutant mice using atomic force microscopy (AFM). Preliminary data suggest that HIP/RPL29 deletion does not alter articular cartilage elasticity.  Because progenitor cells participating in articular cartilage formation are distinct from those contributing to underlying growth plate cartilage and shaft, it is possible that compensatory mechanisms exist in synovial joints and restore normal elasticity in articular cartilage of mutant bone.  This project was funded by the Howard Hughes Medical Institute and COBRE NIH P20-RR016458.

The Role of the Unfolded Protein Response in Cataract Development of Connexin50 Mutants

Jaime Stull, Melinda Duncan, and Zeynep Firtina
Department of Biological Sciences

Cataract is the leading cause of blindness worldwide. Although the majority of cataracts are age-related, they may also be caused by a heritable mutation. In humans and mice, congenital cataract development occurs in Connexin50 (Cx50) mutants. Our study involves two different Cx50 mutants, Cx50-S50P and Cx50-G22R, that both express abnormal lens phenotypes- microphthalmia, unorganized fiber cell structure, and cataract. While this observation is not surprising since Cx50 is a gap junction component responsible for supporting proper cell communication in the lens, it does not explain why the observed lens abnormalities are more severe when connexins are mutated compared to when the genes are absent in Cx50 null models. We hypothesized that when cells attempt to make Cx50 from a mutated gene, the resulting protein does not fold properly, inducing a cell-stress mechanism called the Unfolded Protein Response. Upregulation of molecular folding chaperone BiP supported our proposal of UPR activation in the lens and allowed for expansion of our investigation to include heterozygous mutants. We found that at 13.5 dpc and 15.5 dpc, Cx50 homozygous mutants expressed high levels of UPR activation, while Cx50 heterozygous mutants did not demonstrate a substantial upregulation of UPR markers. Supported by Howard Hughes Medical Institute, Fight for Sight, and the National Eye Institute. 

Regulation of RNA under Abiotic Stress in Arabidopsis

Matt Sullenberger, Linda Rymarquis, and Pamela Green
Department of Plant and Soil Sciences, Delaware Biotechnology Institute, University of Delaware

Plants must deal with biotic and abiotic stress on a daily basis in order to survive.  One of the ways plants deal with stress is to alter the expression levels of microRNAs (miRNAs). miRNAs are ~21 nucleotides RNA molecules, which regulate gene expression by directing protein complexes to cleave messenger RNA (mRNA) “targets” in a sequence-specific manor (Sunkar et al. 2004).  This research was designed to test the hypothesis that high temperature and salinity influence gene expression in plants by altering miRNA-mediated decay in the model plant Arabidopsis thaliana.  To evaluate changes in gene expression, stresses were added to seedlings for 8 hours before being harvested for RNA extraction.  A method known as Parallel Analysis of RNA Ends (PARE) was then used to capture 5’ monophosphorylated and polyadenylated decay products for deep sequencing.  Analysis of the data potentially will reveal novel miRNA targets or known targets whose rate of miRNA-mediated decay is altered under stress conditions.  Identifying novel or abiotic stress-regulated miRNA targets will add to the understanding of how plants respond to abiotic stress and lead to improvements in plant resistance. This work was supported by NSF EPSCoR, Grant EPS-0447610 and DOE (FGO02-91ER20021).

Protein Kinase C Mediates Purinergic Receptor Induced Contraction in MC3T3-E1 Osteoblasts

Patricia Timothee, Victor Fomin, Kirk Czymmek and Randall L. Duncan
Department of Biological Sciences
Osteoblasts respond to mechanical load with a rapid release of ATP that, in turn, binds to two classes of purinergic receptors (P2 X and P2Y). Our lab has reported that P2X7 receptor activation is essential to mechanotransduction in osteoblasts. We have recently observed that activation of this receptor results in a rapid change in osteoblast morphology and induces cellular contraction. We hypothesize that activation of P2X7 receptors during mechanical stimulation activates two distinct pathways, RhoA GTPase and Protein Kinase C, that lead to the contraction of the osteoblast. Here, we examined the changes in MC3T3-E1  preosteoblast morphology and contraction using the Zeiss 5LIVE rapid confocal microscope during activation of the P2X7 receptor and how these changes were affected by inhibition of specific sites in the RhoA GTPase and PKC pathways.  BzATP, a known agonist of the P2X7 receptor, was added to MC3T3-E1 cells and changes in cell area following BzATP stimulation were quantitated using Differential Interphase Contrast (DIC) microscopy.  Addition of 0.5mM BzATP to MC3T3-E1 cells induced a 34.14% reduction in cell area while inhibition of the P2X7 receptor with a blocking antibody significantly reduced the BzATP contraction.  These data suggest that the P2X7 receptor mediates this response.  Inhibition of PKC with the non-specific inhibitor, GF109203X, attenuated the BzATP-induced contraction however blocking Rho Kinase with LYXXX had little effect on BzATP induced contractions..  Finally, we were able to completely attenuate contraction by removal of calcium from the extracellular media.   This data suggests an interactive role between PKC and the P2X7 receptor, as well as the importance of extracellular calcium entry to potentiate contraction.  However, these data do not limit the potential role of other P2X receptors during BzATP induced cell contraction.(supported by INBRE 2P20RR016472-09 and NIH/NIDDK R01 DK058246). 

The Role of alg10 in N-glycosylation during Drosophila development

Jessica Torres, Carly Dominica, Evan Lebois, and Erica Selva
Department of Biolgical Sciences

In order for proteins to function properly in extracellular signaling, they may undergo several post-translational modifications, such as Asparagine (N) linked-glycosylation.  Within this process, the alg10 gene encodes a glycosyltransferase that adds the terminal glucose onto the dolichol-oligosaccharide complex prior to its en masse transfer to nascent polypeptides.  In Drosophilia melanogaster, mutations in alg10 are embryonic lethal, resulting in severe and pleiotropic defects.  Mutations in human homologs of other genes required to build the dolichol-oligosaccharide complex result in a group of disorders, congenital disorders of glycosylation, that cause infantile death.  This study aimed to determine the pathway through which the alg10 mutation disrupts normal development.  To achieve this end, alg10 mutant embryos and imaginal eye discs were stained with various molecular markers to define developmental defects in mutant tissues.  Indicators of the unfolded protein response (UPR) and of apoptosis revealed elevated levels of both processes in alg10 mutant tissues.  Due to improper folding, proteins accumulate in the ER, triggering UPR.  The unfolded protein response, however, is not able to accommodate the large excess of unfolded proteins and as a result the cell undergoes apoptosis.  This apoptotic mechanism accounts for the rough eye phenotype observed in mutant eye discs and perhaps the ventral open CNS phenotype observed in mutant embryos.  Continuing investigation into the CNS defects will be focused on the specific pathway through which alg10 acts, with recent data supporting a defect in axon pathfinding.

Temporal specification of progesterone receptor expression
in the lower rhombic lip of embryonic rats

Tayyaba Toseef  and Princy Q. Mennella
Delaware State University

Brain development is susceptible to influences of steroid hormones, such as progesterone. The presence of progesterone receptors (PRs) in any cell confers sensitivity to progesterone. Thus, we can examine the influence of progesterone during neural development by studying the distribution of PRs in the brain. Previous research has shown that PR expression is present at high levels in the lower rhombic lip (LRL) on the day of birth. The LRL is a developmentally transient brain structure which gives rise to diverse types of brainstem neurons. The high level of PR expression in the LRL suggests that progesterone may be playing an important role in the functions of this structure. However, the initial expression of PR in the LRL has yet to be determined. The goal of this research was to determine the onset of PR expression in the LRL and to trace the pattern of PR expression over fetal life. Fetal rat brains were collected on embryonic days (E) 18, 20, and 22. Immunocytochemistry (ICC) was conducted to visualize PR expression in the LRL. Results indicate that PRimmunoreactivity (PRir), although faint, was present in the E18 LRL. The intensity and number of cells expressing PRir increased dramatically between E18 and E20 and persisted into E22. These findings suggest that PRs may play an influential role in the functions of this structure. Future experiments will include an ICC analysis on rat brains on E16 and E17 and an investigation of potential sexual dimorphism of PR expression in the LRL.

Localization of the Survival Motor Neuron and SIP1 in PC12 Cells

Amanda Vent1, Ilsa Gomez-Curet2, Wenlan Wang2,3
1University of Delaware, Newark DE; 2Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, DE;
 3Department of Pediatrics, Thomas Jefferson University, Philadelphia, PA

Spinal Muscular Atrophy (SMA) is a neurological disease which occurs in 1 in 6000 live births caused by deletions or mutations of the Survival Motor Neuron 1 (SMN) gene. All patients have at least one copy of the highly homologous gene, SMN2, which allows some function of motor neurons but ultimately does not compensate for the loss of SMN1. The SMN protein often associates with other proteins known as Gemins which aide in the assembly of small nuclear ribonuclear proteins (snRNPs). This experiment studies the localization of the SMN protein and one of these Gemins called the SMN Interacting Protein 1 (SIP1), in the nucleus and cytoplasm of PC12 cells. Previous studies have shown a high degree of localization between SMN and SIP1, as well as the loss of function of SIP1 upon separation from SMN. Transfection of GFP was performed using Amaxa Nucleofection, DharmaFECT Transfection, and Lipofectamine 2000 in order to determine the most efficient method of transfection with the highest cell viability. It was determined that the use of 1.5 μL DharmaFECT reagent 4 was the most efficient method, however repeated experiment failed to provide a successful transfection of differentiated PC12 cells. Lipofectamine 2000 and DharmaFECT reagents 1 and 3 were ultimately used to transfect 293T and NIH 3T3 cells in order to observe the localization of the fluorescent tagged wild-type and patient-derived mutant plasmid constructs. /  / Leptomycin B, an antibiotic used to retain substrates within the nucleus which have been marked for export by exportin1, was also used to study the localization of SMN and SIP1 in the nucleus. Final results showed that 5-10 ng/mL of Leptomycin B was unsuccessful in keeping the proteins in the nucleus of naïve PC12 cells. A repeated experiment using differentiated PC12 cells and 20 ng/mL showed a localization of both SMN and SIP1 in the nucleus.

Biodegradation of Estrogenic Hormones by Poultry Litter Isolates

Crystal J. Weesner and Keka C. Biswas
Wesley College Dover, DE-1990

It is known that a variety of bacteria have the unique capability to use sterols as a carbon source to degrade both the steroid skeleton and the side-chain. Characterization of these bacteria and their extra cellular enzymes will have a wide range of applications, especially in studies involving environmental degradation of veterinary antibiotics and steroidal hormones. Research efforts were aimed at increasing our understanding of the fate of endocrine destructing hormones from poultry excreta in soil and subsurface environments. The goal was to develop predictive models and guidelines related to land application of poultry manures to agricultural lands. Elevated concentrations of estrogen [e.g. estradiol and its primary metabolite, estrone] have been observed in soil, groundwater, and surface waters impacted by runoff from agricultural fields where manure has been applied [4]. The initial aim of this undergraduate research project was the characterization and identification of  bacteria present in the bedding litter from chicken farms that are capable of degrading manure-derived steroidal estrogenic hormones.  The second goal is to evaluate the bacteria’s ability to then degrade steroid containing drugs. Bacterial isolates from the chicken house and fresh litter were used to determine degradation and hormone metabolism and degradation products were analyzed by gas chromatography. /  / This project described was supported by NSF-EPSCoR RII, Grant Number EPS-0814251 obtained through Delaware Biotechnology Institute. We would also like to acknowledge NIH-NCRR-INBRE Grant number 2 P20 RR016472-09.

Construction of the TOL-2 5’ 5Kb zCNR1p GAL4-VP16 plasmid and an investigation of its ability to assist
in the visualization of the endocannibinoid system in a developing zebrafish embryo

Maxwell Z. Wilson, Jonathan A. Raper, and Alison L. Dell
University of Pennsylvania

The development of an organism’s nervous system is highly regulated by the widespread utilization of G protein coupled receptors.  Yet exploration into the role of the endocannabinoid system, one of the most extensive groups of G protein-coupled, neuromodulatory lipids and their receptors, in development has hardly occurred.  In this investigation the cannabinoid receptor’s, abbreviated CB1, role in zebrafish axon guidance was evaluated by genetically linking it to a fluorescent protein, citrine, via the UAS-GAL4 system.  Preliminary results indicate that the 5 Kb upstream region to the CNR1 gene, which codes for the CB1 receptor, does in fact contain the promoter for the CNR1 gene and that some expression of the CB1 receptor is visible in the 24 and 48 hour developing zebrafish embryo. 

Links: Summer 2009 Undergraduate Research Symposium, Symposium Abstracts from other Colleges and Departments,

2009 Undergraduate Research Summer Enrichment ProgramUnversity of Delaware Undergraduate Research Program, Howard Hughes Undergraduate Program.
Created  6 August 2009. Last up dated 21 August 2009 by Hal White
Copyright 2009, University of Delaware