Abstracts Submitted in the Biological Sciences (A-K)
Undergraduate Summer Research Symposium August 12, 2009

Ordered alphabetically by student's last name

DiSabatino Friedberg Harrison Kinard
Carter Dalecki Dvorzhinskiy Greenfield Hoffman King, D
Chinmaya Damkin Fascelli Haghighat Huynh King, M
Clarke DeBroux Foltz Hamilton Hyatt Kissig

Investigating the effects of TGF-β1 on the LNCaP Progression Model Within Bone Marrow Stroma

Christopher W. Ahmer, Fayth L. Miles,  Cindy Farach-Carson, and Robert A. Sikes
Department of Biological Sciences

Background. Survival of prostate cancer (PCa) cells that have spread to the bone marrow is the fatal step for cancer patients. Understanding the interactions of metastatic prostate cancer cells with the normally hostile bone marrow stromal cells in the bone environment is key to controlling the disease. Transforming growth factor β1 (TGF-β1), a cytokine that is highly elevated in the bone microenvironment, has a prominent role in regulating cell growth, yet is strongly correlated with advanced prostate cancer in vivo.  Previously we have shown, using the LNCaP progression model of genetically related, increasingly metastatic human prostate cancer cells, that TGF-β1 promotes apoptosis and inhibits cell growth.  Additionally, we have shown that conditioned medium (CM) from HS-5 bone marrow stromal cells originating from progenitors surrounding the bone microvasculature strongly induces apoptosis and neuroendocrine differentiation (NED) of prostate cancer cells.  Thus, we sought to determine if synergy existed between HS-5 CM and TGF-B1 signaling, enhancing these effects.   Methods. PCa cells were cultured with HS-5 CM, as well as CM from HS-27a bone marrow stromal cells derived from the endosteal surface of the bone, in the presence of TGF-β1.  Subsequently, live/dead assays were performed and morphology examined.   Results. TGF-β1 in combination with HS-5 CM enhanced apoptosis of PCa cells as compared to cells treated with HS-5 CM alone.  Furthermore, co-stimulation with TGF-β1 and HS-5 CM appeared to enhance NED, characterized by increased process length and intercellular extensions,  as well as the NED marker, neuron specific enolase (NSE).   Conclusions. these results highlight the significance of signaling in the activated microenvironment in determining the fate of PCa cells during colonization of bone. Summer Project was funded By the Stetson Scholarship Program.

DNA synthesis licensing explains human colonic stem cell quiescence and changes
in organization and dynamics of crypt cell populations during colon tumorigenesis

Koree W. Ahn2 Tao Zhang1, Daniel Relles1, Jeannie Seu1, Olaf A. Runquist2, Marc Brandt3,
Scott D. Goldstein4,  Gerald A. Isenberg4, Jeremy Z. Fields5, Bruce M. Boman1,6,7

1 Dept of Biologic Sciences, University of Delaware, Newark DE; 2Dept of  Chemistry, Hamline University, St. Paul, MN; 3Division of Colorectal Surgery, Rush University,  Chicago, IL; 4 Division of Colorectal Surgery, Thomas Jefferson University, Philadelphia PA;  
CA*TX, Inc., Gladwyne PA; 6 Thomas Jefferson University, Philadelphia, PA; 7 Helen F.  Graham Cancer Center, Christiana Care Health System, Newark, DE

We reported that initiation/progression of colorectal cancer is mediated by crypt stem cell (SC)  overpopulation, and that overpopulation is caused by dysregulation of two mechanisms: 1)  regulation, by cell-cycle processes, of the probability of proliferative cells being in S-phase; 2)  regulation, by cellular differentiation/apoptosis, of the proportion of proliferative cells. To  investigate how these mechanisms become dysregulated, we immunohistochemically mapped:  crypt cell populations expressing DNA synthesis licensing proteins (CDT1/geminin/MCM2),  which regulate cell transitioning through S-phase; markers for cell-cycle arrest (P21); and  apoptosis (TUNEL). In normal colonic epithelium, the proportion of cells expressing licensing  proteins was low at crypt bottom (where SC reside), high among proliferating cells, and nil in upper crypt (where terminally differentiated/apoptotic cells reside). This suggests that SC are not  licensed for DNA synthesis (consistent with their relative quiescence/low S-phase probability)  and that licensing mediates generation of proliferating cells from SC. Thus, regulation of  / licensing may control the size of SC and proliferating cell populations. In pre-malignant crypts,  the population of licensed cells was expanded and distributed farther up the crypt, while P21-  and TUNEL-positive cell populations in the upper crypt contracted. These changes in crypt cell  subpopulations suggest that, as cells migrate up the crypt, the transitions between cell  phenotypes — from stem (unlicensed) to proliferating (licensed) to terminally-differentiated  (cell-cycle arrested) to apoptotic cells — are delayed. Thus, during tumorigenesis, by delaying  cell maturation along the crypt axis, dysregulated licensing may contribute to expansion of SC  and proliferating cell populations, and their exponential increase in carcinomas.

Methylation analysis of the G-C Rich DMPK gene:
Causation for the severity  difference between adult-onset and congenital myotonic dystrophy?

Brian Barnette, Sarah Swain, Susan Kirwin, and Vicky Funanage
Alfred I. duPont Hospital for Children/Nemours Children’s Clinic

Myotonic dystrophy type I is an autosomal dominant genetic disease.  The disease arises from an expansion of a trinucleotide repeat, (CTG)n located in the 3’ untranslated (3’ UTR) region of the dystrophica myotonin protein kinase (DMPK) gene on chromosome 19.  Located proximal to this region is a CpG island; a region of DNA with a high concentration of CpG sequences that often associate with gene promoters in humans.  The purpose of this study was to determine the methylation status of this CpG island in individuals affected with DM1, notably congenital DM1, and to compare this methylation pattern with that seen in  individuals not affected by DM1.  The method of bisulfite-conversion was used to ascertain the methylation status of the CpG island.  This method converts all unmethylated cytosine residues into uracil residues.  The converted DNA is then amplified by means of PCR where the converted uracil residues are amplified as thymine residues.  This amplified DNA is then cloned and sequenced to compare the methylation status of the CpG island among individuals.  Currently, we are amplifying and sequencing this CpG island. These techniques will be used to explore a relation, if any, between methylation of the CpG island and the difference in expression of full-length DMPK transcript between adult-onset and congenital DM.

The role of Bcl-2 during neuronal migration and survival in the chick midbrain

Cory D. Bovenzi and Deni S. Galileo
Department of Biological Sciences, University of Delaware

In the developing brain, neurons are born in the ventricular zone and migrate outward via radial glia scaffolds. There also is an early period of widespread programmed cell death (PCD) in the brain. We believe there is a relation between these two processes whereby the cells that die do so because of anoikis: loss of contact with the necessary extracellular matrix produced by radial glia. Remaining neurons are those that attached to radial glial matrix and received survival signals through an intracellular signaling pathway. In the developing chicken midbrain, we believe this pathway involves fibronectin secreted by radial glia interacting with the α8β1 integrin receptors on the neuronal membrane, initiating a Focal Adhesion Kinase-mediated cascade eventually increasing expression of Bcl-2, an anti-apoptotic protein. Higher levels of Bcl-2 increase the likelihood that a cell will survive periods of widespread PCD. To study the role of Bcl-2 in brain development, this protein was ectopically expressed with a replication-incompetent retroviral vector in the embryonic chick optic tectum. An infected progenitor cell divides to produce a cohort of clonal progeny that all carry the marker lacZ gene. The number and distribution of lacZ + neurons in Bcl-2 overexpressing cohorts were counted and compared to control clones. Expression of another potential integrin involved (α5β1), whether contact with radial glia in culture facilitates neuronal survival, and whether greater Bcl-2 normally has an inverse relationship with cell death in the avian optic tectum were also investigated.  This project was funded by the Howard Hughes Medical Institute

MicroRNAs Associated with Environmental Stress in
(Second place oral presentation in Sigma Xi competition)

Rebecca S. H. Brown, Dong-Hoon Jeong, and Pamela J. Green
Department of Plant and Soil Science

Plants are limited in the number of ways that they can respond to environmental stress because they are sessile organisms.  Because of this, plants have developed sophisticated mechanisms that enable them to cope with adverse environmental conditions.  One way plants respond to environmental stress is by modifying their gene expression through the use of microRNAs (miRNAs).  miRNAs are noncoding small RNAs that regulate gene expression at the posttranscriptional level by base pairing with complementary messenger RNA (mRNA) molecules, causing either mRNA cleavage or translational inhibition.  To elucidate the roles of miRNAs in environmental stress responses, wild type plants and miRNA enriched mutant plants were subjected to various environmental stresses, such as drought, cold, submergence, salt, and combined stresses of submergence and salt.  Small RNA libraries from stress-treated seedlings and flowers were constructed and sequenced using deep sequencing technologies.  Computational analysis revealed differential expression of miRNAs between stress-treated plants and control plants with some miRNAs upregulated and some downreguled by environmental stress.  Additional analysis revealed potentially novel miRNAs in Arabidopsis as well.  These results suggest that miRNAs play important roles in stress responses, and that miRNA identification in Arabidopsis has not been saturated.  Future work will involve validating highly stress-regulated miRNAs and prospective new miRNAs, and examining their potential target genes for regulation.  Stress-regulated miRNAs will be incorporated into multi-network models and their biological roles will be hypothesized and tested through functional studies.  R.S.H.B. was supported by the Howard Hughes Medical Institute, and NSF grant MCB#0548569 to P.J.G. provided research support.

Molecular Characterization, Isolation, and Identification of Chicken Litter Bacteria

Shannon E. Carter1 , Thomas E. Hanson 2,  and Keka C. Biswas1
1Wesley College, Dover DE 19901, 2 University of Delaware, Newark DE 19716

The initial aim of this undergraduate research project was characterization and identification of bacteria present in the bedding litter from chicken farms that are capable of degrading manure-derived antibiotics and steroidal estrogenic hormones like  17-β estradiol and estrone. characterization of bacterial isolates through DNA extraction, colony PCR , sequencing to understand molecular mechanisms using enzyme isolation and protein purification techniques. 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. In this study, 16S rRNA sequencing was used to characterize bacterial isolates from chicken litter. The use of 16S rRNA sequencing to accurately identify bacterial isolates has become an essential operation in both clinical and environmental microbiology laboratories. There are many advantages in using the 16S rRNA sequencing including its presence in almost all bacterial genomes, and because the function of this gene over time has not changed. The second goal was to evaluate the bacteria’s ability to then degrade estrogenic hormone and steroid containing pharmaceutical drugs. This is to understand the microbiological degradation process for the fission of the steroid skeleton in the drugs and to document any relationship between the degradation  process and the presence of susceptible points (viz. functional groups, double bonds, side chains, etc.).  This project is supported by NSF-EPSCoR Grant EPS-0814251, NIH NCRR INBRE grant 2 P20 RR016472-08 and The Delaware Biotechnology Institute.

Function and Expression of Compatible Solute Transporters in
Vibrio parahaemolyticus

Nikhil B. Chinmaya, Seth Blumerman, & E. Fidelma Boyd
Department of Biological Sciences

Bacteria living in varying salt concentrations are under constant danger of death from the loss of the delicate osmotic equilibrium within their cells. Vibrio parahaemolyticus is a moderate halophile that is an emerging human pathogen known to cause gastroenteritis. V. parahaemolyticus lives in estuaries and oceans around the world in wide ranging salinities and osmotic conditions. Like most bacteria, V.  parahaemolyticus has evolved a system of osmoadaptation that uptakes compatible solutes to protect and preserve the enzymatic activity of the bacterial cell. V. parahaemolyticus has four single component compatible solute transporters we took the previously cloned genes of these transporters designated VP1456, VP1723, VP1905 all located on chromosome I, and VPA0356 located on chromosome II. Whose function we have little to no understanding of in the bacterial cell. Therefore, to understand the function of these transporters We examined their function by cloning the genes and transforming them into pBBR1MCS creating pVP1456, pVP1723, pVP1905, and pVPA0356, a vector in a E. coli strain MKH13 (Transporter deficient strain of E. coli). We determined that some of these transporters are able to uptake glycine betaine but were not able to take up proline. To get a comprehensive understanding of the compatible solute transporters, and how they behave in their natural environment we also tested V. parahaemolyticus under specific conditions to observe the levels of gene expression of the particular transporter genes, to see a correlation.

Probing the Unknown: Development and Testing of PCR Assays for Viral Genes in Environmental Samples

Jennifer E. Clarke1, Mara Hyatt2, William Kress, Rachel Marine, Michael Dumas, Sanchita Jamindar, Shawn Polson, K. Eric Wommack
1Linconln University, Lincoln University PA,  2Delaware Technical and Community College

Polymerase chain reaction (PCR) enables researchers to produce millions of copies of a specific DNA sequence from minute quantities of a starting template. Applied to environmental samples, PCR provides a means to access and explore the diversity of genes within viruses and microorganisms. The central goal of this project was to explore the environmental occurrence and diversity of predicted open reading frames (ORFs) from two single stranded DNA (ssDNA) viral metagenome libraries. Prior to development of PCR assays, predicted ORFs were clustered by homology, and ranked according to the number of member ORFs.  Primer sets were developed for three of the top twenty ORF clusters.  A four step procedure was used in the development of the assays. First, primer sets were tested against a sample known to contain the gene of interest. Second, temperature gradient PCR was used to discern the optimal annealing temperature (Tm °C) for each of the primer sets. In each case the optimal Tm°C was higher than the theoretical prediction. Third, a positive PCR control was developed by isolating DNA of a specific PCR amplicon from a Chesapeake Bay sample. Finally, a serial dilution of the positive control DNA was tested against each corresponding primer set. Testing of the primer sets against samples from White Clay Creek and soil from the Newark Agricultural Experiment Station revealed negative results even after the samples were spiked with a positive control, suggesting inhibition of the PCR by the environmental sample. For the soil samples it is hypothesized that humic acid is causing this inhibition. This research was funded by the National Science Foundation.

Phosphorylation Modification of the Survival Motor Neuron Protein

Amelia Curtis and Wenlan Wang
Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, DE
 Department of Pediatrics, Thomas Jefferson University, Philadelphia, PA

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by the degeneration of motor neurons in the spinal cord and atrophy of the proximal muscles of the limbs and trunk. SMA is a genetic disease caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The encoded SMN protein is a ubiquitous protein that plays a role in the assembly of snRNPs. Complete loss of SMN is lethal but reduced levels of SMN cause selective death of motor neurons. The goal of my research is to purify SMN protein and then identify the phosphorylation sites of this protein. Our lab has previously overexpressed wild type SMN protein in a baculovirus system. Here, I have expressed a small scale of GST-tagged SMN protein in insect Sf9 cells through baculovirus infection and then purified it by glutathione-agarose affinity purification. The identity of the purified protein was confirmed by Western blotting analysis and the concentration was determined by SDS-PAGE using BSA as the standard. Phosphorylation of purified GST-SMN from the baculovirus was determined by Western blotting using antibodies against phosphor-serine, phosphor-threonine, and phosphor-tyrosine. Potential phosphorylation sites identified by bioinfomatic approaches were further investigated by in vitro kinase assays using lysates or purified kinases followed by mass spectral analyses. 

Characterization of Synovial Mesenchymal Stem Cells
by Immunohistochemistry, Cell Differentiation, and Cytokine Level Analysis

Belynda Dalecki, Revital Herrmann, and Paul Fawcett1
1Nemours Biomedical Research,  A.I. duPont Hospital for Children

Juvenile Idiopathic Arthritis (JIA) is an autoimmune disease characterized by inflammation of joints in pediatric patients under the age of 18. Lyme disease is caused by a bacteria, Borrelia burgdorferi, carried by deer ticks and can be classified as Lyme Acute (LA), with duration of symptoms less than 6 months, or Lyme Chronic (LC), with duration of symptoms longer than 6 months. Lyme Chronic arthritis occurs in about 10% of pediatric patients who, despite treatment with antibiotics, continue to show symptoms of inflammation.  Synovial fluids were obtained for analysis from patients undergoing arthrocentesis as part of their clinical care. Cells were centrifuged, counted and morphologically identified as leukocytes (neutrophils, lymphocytes, and monocytes). Other small round cells in the fluid that, unlike leukocytes, have exhibited an ability to grow and proliferate in culture were previously identified as synoviocytes.  In this study, we wanted to determine whether the synoviocytes are mesenchymal stem cells (MSCs). MSCs have the capability to differentiate into several cell lineages including bone, fat, cartilage, and muscle. Using a sample obtained from a LC patient, we tested the synoviocytes’ ability to undergo transformation through Osteogenesis and Adipogenesis. Transformation was followed by Enzyme-linked immunosorbent assay (ELISA) (R&D Systems) and protein micro array (RayBiotech) to determine the transformation effect on cytokine production by the cells. Finally, Immunohistochemistry was used to identify mesenchymal stem cell markers on cultured cells obtained from JIA, LA, and LC patients. This project was supported by grant number 2 P20 RR016472-09 under the INBRE Program of the National Center for Research Resources (NCRR), National Institutes of Health (NIH).   

The Unwinding Efficiency of Simian Virus 40 T Antigen is affected by the Amount of Core Origin DNA Present

Kyle Damken, Weiping Wang, and Daniel Simmons
Department of Biological Sciences

Simian Virus 40 (SV40) is a small tumor virus that serves as an excellent experimental model for eukaryotic DNA replication.  T Antigen (T Ag) is a virally encoded protein necessary for the initiation of SV40 DNA replication at its single core origin.  Certain host cellular proteins bind to T Ag, forming the initiation complex and allowing DNA replication to begin.  The SV40 core origin (64 bp) consists of 3 distinct regions, the central region is called Site II, where T Ag binds, the early palindrome (EP) region, where the DNA is melted and the AT rich track, where the DNA is untwisted.  The purpose of this research project was to determine which sequences of the core origin are important for DNA unwinding.  A series of unwinding assays were performed using varying lengths of P-32 labeled origin DNA containing different amounts of the EP region.  The results showed that T Ag was efficient at unwinding DNA with just Site II, but as base pairs from EP were added unwinding activity dropped significantly.  Unwinding was restored when the entire EP region was present.  These data can be explained by a model describing the mechanism of unwinding by T Ag.  To compare these results with the unwinding of circular DNA, DNA minicircles will be generated containing different amounts of the core origin, and will be subjected to unwinding assays with T Ag and Topo 1 to determine the importance of the EP region and the AT tract for efficient SV40 DNA unwinding.  This research will help to show more clearly which parts of the core origin are needed for DNA unwinding.

Investigating  the function and development of Motor Neurons In Wild Type and SMA Spinal Cord Models

Bianca DeBroux, Rashida Williams, and Melissa Harrington
Delaware State University, Biological Sciences

Electrophysiology, Calcium imaging, and Genotyping were used to measure and understand the development of motor neurons in both normal and SMA spinal cord models. These experiments used two mouse models one of which had the SMA mutant gene and the other which did not. Electrophysiology with the Med 64 multielectrode recording system measure the neural activity within a network of motor neurons. Calcium imaging was used to measure intracellular communication, which supports the results with the Med 64 dishes that measure electrical motor neuron activity. The last technique used was genotyping of the different mouse models. Knowing the genotype of these model helps differentiate between the 3 different type of mouse pups that we can obtain in a litter. Knowing specially if these mice are homozygous wild type or homozygous for SMN knockout or heterozygous will allow us to relate the exact difference between the mutant and wild type model motor neuron activity to genotype. This experiments thus far have allow us to obtain electrical signal on med 64 dishes, we were not yet able to determine the significance of motor neuron activity recorded. With genotyping we saw bands for both the mutant and wild type gene products. 

A Comparative Analysis of the Activation of RhoA and RhoC GTPase and Subsequent Cellular Activity

DiSabatino, S. and Kenneth van Golen
Department of Biological Sciences

RhoA and RhoC are GTPase enzymes that function as molecular switches within the cell that control the actin cytoskeleton. When bound to GTP, RhoA and -C are in an active state and are able to participate in various signal transduction pathways within a stimulated cell. One of the main actions of active RhoA and -C is the triggering of cytoskeletal rearrangement, a process readily observed within invasive and motile cancer cells. Considering RhoA and -C are over 90% homologous in structure at the protein level, it has been assumed that they function identically. Data from our laboratory and others demonstrates this is not case. The two GTPases could potentially display temporal and/or spatial differences in their activations. Using mouse fibroblast NIH3T3 cells as a model, we hope to distinguish which membrane receptors, G-protein coupled receptors or tyrosine kinase receptors, are predominantly responsible for the activation of RhoA and RhoC and in what timeframe these respective activations occur. The data gathered from these experiments will provide a more specific knowledge of the relationship between RhoA and RhoC as they relate to cell motility and potentially to cancer progression. The practical applications of such knowledge could be potentially far reaching in both the research and medical communities. This summer’s work has been funded by the Charles Peter White Grant.

Efflux Pump Upregulation in Salmonella Resistant to Dodecyltrimethylammonium Chloride

Aleksey Dvorzhinskiy, Megan Kautz, Natalie Stevenson, and Diane Herson
Department of Biological Sciences

Salmonella spp. are gram-negative pathogens estimated to cause 1.4 million cases of food poisoning annually in the United States and are increasingly becoming resistant to disinfection in natural, clinical, and laboratory environments. In this study Salmonella enterica 4931 strains were developed which were able to grow in the presence of 500-600 ppm Dodecyltrimethylammonium chloride (DTAC), a quaternary ammonium compound.  These strains with reduced susceptibility to DTAC (SRS) also showed a cross resistance to penicillin.  Later, the SRS strains were passaged in the absence of DTAC to mimic environmental conditions after exposure to DTAC. The minimum inhibitory concentration (MIC) of DTAC for these DSRS strains did not vary appreciably from the parental level for fifty passages. Reduced susceptibility to DTAC has been reported to be correlated with an increase in the amount of transcription of acrB, an efflux pump which has been linked to multi-drug resistance. The results from our research support this finding. This study showed that all SRS and DSRS strains in the presence of the efflux pump inhibitor Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) showed a greater sensitivity to DTAC than the parental strain. This is possibly another physiological indication of a greater reliance of all resistant strains on efflux pumps for defense against anti-microbial compounds. The discovery of efflux pump inhibitors such as CCCP could lead to advances in combating SRS strains in clinical and natural environments. This research was generously funded by the Howard Hughes Medical Institute.

Characterization of Adult Human Neural Progenitor Cell Differentiation In Vitro and In Vivo

Michele Fascelli, Deni S. Galileo, and Grace Yang
Department of Biolgical Sciences

Adult human neural progenitor cells (AHNPs) can be isolated from the adult hippocampus and cerebral cortex.  Others showed they can differentiate into neurons and astrocytes in vitro and when injected into mouse brains, while our lab had similar results in chick embryo brains.  AHNP studies have future applications for regenerative medicine and understanding brain tumor carcinogenesis.  I injected GFP-expressing AHNPs into E5 chick embryo midbrain and hindbrain ventricles in order to visualize their motility and development.  Approximately 10 days after injection, brains were dissected, sectioned at 200 microns on a vibratome, and screened for green cells using a dissecting microscope with epifluorescence.  Sections will be immunostained for cell type-specific markers and analyzed by confocal microscopy.  Currently, different methods are being evaluated for facilitating antibody penetration into the sections.  Western blot analysis showed full-length L1 protein expression in AHNP cells; immunocytochemistry confirmed the expression of membrane surface L1 (L1 is a transmembrane protein that has a different function when proteolyzed from the surface).  Pax-6 transcription factor also was detected by western blot analysis; however, immunostaining for Pax-6 did not reveal expected nuclear localization.  Future work will be done with co-cultures of AHNPs and chick brain cells as an in vitro¬ model for AHNP cell differentiation.  These will be characterized for differentiated neurons, astrocytes, and oligodendrocytes.  This project was funded by the Howard Hughes Medical Institute.

Abnormal Protein Expression in SMA Motor Neurons derived from Embryonic Stem Cells

Steven Foltz1, Dosh Whye2, and Wenlan Wang2,3
1University of Delaware, Newark, DE, 2Nemours Biomedical Research, AI DuPont Hospital for Children, Wilmington, DE;
 3Department of Pediatrics, Thomas Jefferson University, Philadelphia, PA

Spinal Muscular Atrophy (SMA) is a neuromuscular disease characterized by degeneration of spinal motor neurons accompanied by muscle paralysis.  Deletions or mutations of the survival motor neuron 1 (SMN1) gene have been cited as the cause of this devastating genetic disorder.  Despite the highly specific tissue degeneration observed in SMA patients, the SMN protein encoded by the SMN1 gene is ubiquitously expressed.  Thus far, conclusive evidence as to why loss of a ubiquitously expressed protein would result in the highly selective loss of motor neurons remains to be established.  Here, murine embryonic stem cells derived from control and SMA mice were differentiated into motor neurons by retinoic acid and small molecule agonist of sonic hedgehog.  Abnormal protein expression in SMA motor neurons was analyzed by proteomic and Western blotting analyses. These studies revealed that several stress-related proteins are upregulated in SMA motor neurons differentiated from embryonic stem cells.  Current work is devoted to elucidating the specific pathway(s) that are affected in SMA motor neurons, with the ultimate goal of identifying SMA biomarkers that can be used in clinical trials and the revelation of pathways that can be regulated to treat SMA.

The effect of 1H7 and 3B7 treatment on molecular signaling through the IGF-IR in prostate cancer

Brett Friedberg, Erica Dashner, and Kenneth L. van Golen
Department of Biological Sciences

Prostate Cancer (PCa) affects one out of every six American men and is the second leading cause of cancer related deaths among males.  Advanced PCa easily metastasizes to bone which causes unrelenting pain. Current chemotherapies do not help in advanced PCa. The humanized antibody, 1H7, has been used in other cancers to reduce tumor size but it has not been tested in PCa. 1H7 and another antibody, 3B7, binds the growth- and metastasis-promoting Insulin-like Growth Factor 1 receptor (IGF-1R). Currently, we are looking at signaling effects of these antibodies on PCa cells at different stages of the disease, studying what effects these antibodies might have on the activation of certain proteins in PCa cells. By performing western blots on protein extractions from LNCAP, C4-2, and PC3 cells we were able to see changes in the activation levels of certain proteins caused by treatments with IGF-1 and the antibodies 1H7 and 3B7. Cells treated with 1H7 and 3B7 expressed higher levels of activated Akt, and lower activation levels when treated with IGF-1. Phosphorylated tyrosine residues on IRS-2 were only seen in cells treated with 1H7 and 3B7 whether or not IGF-1 was added. Phosphorylated serine residues on IRS-2 varied greatly in all cell lines. Treatment with 1H7 and 3B7 appears to alter molecular signaling through the IGF-1R.

Investigation of the Presence of Purinergic Receptors in Cancer Cell Lines

Julia Greenfield, Christine Maguire, Robert A. Sikes and Kenneth L. van Golen
Lincoln University

Once thought to be only involved in the metabolic process, knowledge of the complete function of the ATP molecule has expend and with it the concept of purine nucleotides and nucleosides as extra cellular messengers.  These messenger molecules constitute what is known as the purinergic signaling system, which plays a role in the signaling of many cellular functions from neurotransmission to cell differentiation[1].   Previous literature has shown that ATP signaling mediated by P2 receptors has anticancer effects.   However, other studies have demonstrated a role of purinergic signaling in cancer progression leading to metastasis.  We hypothesize that certain purinergic molecules are present on prostate cancer cells and dictate their ability to metastasize. Previous work in our laboratory has shown a distinct pattern of purinergic receptor expression on the mRNA level in the LNCaP series of isogenic cell lines.  In addition, these cells produce and release ATP into the extracellular environment. To illuminate which of the eight P2 receptor are expressed on the protein level, immunoblots were preformed, selecting for a number of the P2Y receptors.  Currently we have demonstrated expression of the P2Y1, P2Y6, and P2Y14 receptors which have the strongest signal.  Expression of the remaining receptors need to be determined.

Study of unique microRNA expression signatures in normal colonic epithelium and colon cancers

Sepehr Sedigh Haghighat, Greg Gonye1, Tao Zhang, and Bruce Boman
Department of Biological Sciences, University of Delaware and 1Thomas Jefferson Medical University

Dysregulation of crypt cell  proliferation and differentiation has been implicated in the development of colorectal cancer (CRC). As part of our investigation of regulatory factors involved in the stem cell origin of CRC, we investigated the role that microRNAs (miRNAs) might play in colon carcinogenesis. miRNAs are RNAs 20-24 nucleotides long that were recently shown to modulate many cellular signaling pathways through post-transcriptional regulation of messenger RNA (mRNA) levels and thus protein synthesis. Our previous micorarray analysis (368 gene chip) showed increased expression of 37 miRNAs in CRCs compared to normal colonic epithelium. Here we further evaluated miRNA expression in CRC versus purified colonic epithelium by quantitative PCR (QPCR). Total RNA was immediately isolated from tissue by the TrizolTM method. RNA was transcribed into first-strand complementary DNA (cDNA) through reverse transcription (RT). cDNA was then amplified through QPCR. Statistical analysis and plots of expression data for miRNAs were done to find miRNA genes that are differentially expressed. We found, using QPCR, four miRNAs to be differentially expressed in CRCs versus normal colonic epithelium including mir-25 and mir-198. Identification of miRNAs specifically expressed in normal colonic crypts and changes in their expression in CRCs will provide important information to help understand mechanisms of colon tumorigenesis.

Testing natural compound extracts for anti-cancer properties

Jeneice Hamilton and Kenneth VanGolen
Department of Biological Sciences

Five natural compound plant extracts (spices) that are suspected to have anti-cancer properties were obtained from India. The purpose of this experiment is to provide scientific evidence to confirm whether or not the spices possess such properties. Each of the compounds, namely: Zingiba officinale, Tinospora, Ccucuma Longa, Piper Betel and Piner Longum, was tested on human PC-3 cells. A thousand cells were placed in each well of a 96 well plate. The following concentrations (in microliters) of spices dissolved in DMSO was added to the cells: 1:10, 1:50, 1:100, 1:250, 1:500, 1:1000 and 1:2500. Controls were 1:500 and 1:1000. Readings were taken at different time points, that is, every day for four consecutive days and the final reading was taken on the seventh day. At the end of each time point, cells were treated with MTT and incubated for two hours. A MTT assay was then ran and the data was collected and graphically presented showing absorbance against time.  Based on these data, it was observed that higher concentrations of each spice adversely affected the cells and the effects are mostly noticeable at a later time point. Compound  Zingiba officinale effect on cells is only noticeable by day four however, the change was not significant. Compound Tinospora  effect on the cells was barely noticeable on day three, but fell on day four and increased significantly on day seven at the four highest concentrations. Compounds Piper Betel  and Piner Longum showed quite a significant effect on cells by day four. Compound Ccucuma Longa is still undergoing evaluation and the final readings for the last four compounds are set for the next 24 hours. It is however predicted that absorbance of cells at the higher concentrations will be much less than at the lower concentrations. This implies that the extracts are either inhibiting the growth of the cells or killing them.

The Role of Extracellular Matrix on ATP release by Prostate Cancer Cells in Bone Metastasis

Dominick Harrison1, Randall Duncan, Robert Sikes, Mary Boggs, and Christine Maguire
1Delaware State University

The Effect of Extracellular Matrix on ATP release by Prostate Cancer Cells in Bone Metastasis /  / Prostate Cancer is one of the most common types of cancer found in men affecting one in six in the United States, but it is the metastasis of this cancer to bone and pain associated with this metastasis that dramatically impacts the quality of life of the patient. Unfortunately, the mechanism of how cancer cells metastasize is unclear. Recent studies have suggested that tumor cells produce abundant ATP and our lab postulates that the release of ATP from these cells, and the subsequent binding to purinergic receptors regulates metastasis. As the cancer cells metastasizes, it encounters and binds to different types of extracellular matrix (ECM) proteins that we believe will increase the release of ATP and enhance metastasis of the cancer. To test this hypothesis, we used the LNCaP (Lymph Node Carcinoma of the Prostate) progression model of Prostate Cancer. This model consists of four cell lines at various stages of prostate cancer progression from relatively benign (LNCap) to highly aggressive and metastatic to bone (C4-2B). In this project, I grew C4, C4-2, C4-2B cells on different ECM proteins and determined if the release of ATP from these cells was dependent on the attachment of these cells to the ECM. Cells were grown on either tissue culture plastic, collagen I or neural ECM proteins produced by primary culture of Schwann cells. Basal release of ATP was measured by removing an aliquot of medium from the cells grown on these ECM proteins and indirectly measuring ATP using the luciferin-luciferase assay. My lab has shown that ATP release is regulated by extracellular calcium entry, so we added either ionomycin (calcium ionophore) or high KCL (depolarization to active voltage sensitive calcium channels) to the medium to determine if cells grown on this different ECM’s had different amounts of ATP available for release. Department of Biological Sciences, Funded by Department of Defense

Mathematical Modeling of Colon Stem Cell Dynamics

Jennifer M. Hoffman, Bruce M. Boman1, and Gilberto Schleiniger2
1Department of Biological Sciences at the University of Delaware, Center for Translational Cancer Research, Helen F Graham Cancer Center ,
2Department of Mathematical Sciences, University of Delaware

Previous research has proven that the overpopulation of colonic stem cells is the ultimate driving mechanism in tumor growth, which may result in colorectal cancer.   For this reason, a mathematical model is necessary in order to examine how altered colonic epithelial dynamics give rise to colonic stem cell overpopulation.  A reaction-diffusion model consisting of a system of partial differential equations were created to replicate the normal proliferation, differentiation, and diffusion of the stem cells in the colonic crypt.  This model was then coded and solved using Matlab.  Also, this model can be used to explore how parameter changes can simulate changes in normal tissue and to fit biological data for premalignant and malignant colon tissues.  Therefore, in the future, these new model parameters can be determined and the differences between the normal tissue model parameters and the perturbed tissue model parameters may provide an explanation for how altered epithelial dynamics lead to colon cancer stem cell populations that drive tumor growth.  This project was funded by the University of Delaware’s Undergraduate Research Program.

The Presence of JAM-A in Sperm Proves Essential for Normal Sperm Motility: Progressing from Mice to Humans

Jean Huynh, Rolands Aravindan, and Patricia A. Martin-DeLeon
Department of Biological Sciences

There are a number of factors that lead to male infertility, and among the most important ones is sperm motility. Junctional Adhesion Molecule - A (JAM-A) has been recently shown to be a protein that is essential for normal sperm motility. In mouse sperm, it localizes within the plasma membrane overlying the head, the midpiece, and the proximal principal piece of the flagellum. Western analysis of human sperm has revealed the presence of JAM-A, suggesting that it may also play a role in human sperm motility. Thus, it is important to extend the analysis of JAM-A to determine its role in human sperm function. This project is designed to expand our understanding of JAM-A in human reproduction. Immunocytochemistry (ICC) was used to localize the expression of JAM-A in human sperm, which is present throughout the tail and head of human sperm. Western blots revealed the presence of an ~32 kDa JAM-A band in human sperm proteins. Lastly, Flow cytometry was conducted to confirm the Western blot data. Once JAM-A has been fully characterized in human proteins, the next step will be to co-localize proteins associated with JAM-A. The localization and characterization of JAM-A in human sperm is expected to increase our understanding of human male infertility and subertility, and will be a means of laying the groundwork for assisting a subset of couples with fertility issues.Funded by NIH-COBRE grant #5P20RR015588-07.

Global whereabouts of three recently discovered single-stranded DNA viral genes

M. Hyatt1, J. Clarke2, W. Kress3, R. Marine4, M. Dumas4, S. Jamindar4, S. Polson6, K.  E. Wommack4,5,6
1Delaware Technical and Community College, Newark, DE; 2Dept. of Biological Science, Lincoln University, Lincoln University, PA;
3Dept. of Civil and Environmental Engineering, University of Delaware, Newark, DE; 
Dept. of Biological Sciences, University of Delaware, Newark, DE; 5Dept. of Plant and Soil Science, University of Delaware, Newark, DE; 6Delaware Biotechnology Institute, University of Delaware, Newark, DE.

Phages are remarkably abundant and play critical roles in bacterial population dynamics and biogeochemical cycling.  Studying phage genetics in different environments can provide clues about their ecological importance and diversity.  Predicted open reading frames (ORFs) from two single-stranded DNA (ssDNA) viral metagenome libraries were clustered by homology and ORF clusters were ranked by the number of member ORFs.  Primer sets were developed for three ORFs in the top twenty clusters. Viruses within water samples were concentrated using tangential flow filtration.  Soil viruses were extracted using sonication, centrifugation, and filtration with potassium citrate as an eluant. Using PCR, each primer set was tested on seventeen viral concentrates from various points around the globe including the Chesapeake Bay from which two of the clusters were derived, Bergen, Norway, and Santa Barbara, California.  When positive results were obtained, gel bands were extracted and purified, cloned and sequenced.  Genes that were originally discovered in viral metagenomes from the Chesapeake Bay and Dry Tortugas were present in other sites in Delaware, but not in Santa Barbara, California or Bergen, Norway.  Study of single-stranded DNA viruses have been often overlooked when examining the virome of an environment.  This research has shown that it is possible to selectively target unique ssDNA genes and test for their presence in multiple environments.  Study of these highly abundant and often unknown genes will help elucidate what kind of impact ssDNA viruses have on their hosts and potentially give researchers an assay for detection of specific viruses in new environments.  This research was funded by the National Science Foundation.

Investigation of the Mechanism Underlying Radiation Induced Bone Mass Loss

Dan King and Anja Nohe
Department of Biological Sciences

During space flight astronauts have been reported to lose up to 2% bone mass per month. Because radiation exposure is a constant threat in space, the effect of radiation on bone must be better understood. In order to explain why they lose bone mass, our goal is to investigate how radiation affects Bone Morphogenetic Protein (BMP) signaling. BMPs are a group of growth factors known to be initiators of osteoblast differentiation through activation of various signaling pathways. It has been shown that radiation reduces BMP signaling, alkaline phosphatase activity (ALP), and the Smad signaling pathway. This result is not due to the down regulation of BMP receptors or any change in the proliferation of cells. We investigated the localization of BMP receptor type Ia (BRIa) on the plasma membrane and its interaction with Caveolin 1 alpha, a scaffolding protein found specifically in caveolae, and Casein Kinase 2, a protein kinase believed to be a regulator of BRIa movement. C2C12 cells were stimulated with BMP-2 for 30 minute, 2 hour, and 18 hour increments then fixed and labeled for BRIa, CK2, and Cav-1 alpha in order to observe the signaling dynamics through Image Cross Correlation Spectroscopy. Cells were also stimulated with peptides which were developed to affect CK2-BRIa binding. Results show that after stimulation with BMP-2, BRIa and CK2 are located together with an increased aggregation after 30 minute and 2 hour stimulation. After 18 hour simulation with BMP-2, BRIa-CK2 interaction is reduced and dispersion is observed. These results show that the receptors are either endocytosed or BRIa is reshuttled to different regions on the membrane. A deeper understanding of cellular signaling is required in order to determine the effect of the protein aggregation on osteoblast differentiation.

Fibronectin in the Lens of the Eye

Matthew R. King, Melinda K. Duncan, and Yan Wang
Department of Biological Sciences

Although once thought to be either absent or non-essential to the lens, the recent finding that fibronectin (FN) knock-out zebra fish develop cataract has sparked interest into FN’s role in lens biology.  FN, like collagen IV, is a common protein in extra cellular matrices (ECM’s) and is present in posterior lens capsule: basement membrane surrounding lens cells. Immunofluorescent staining revealed that that FN concentrations progressively increase as the back of the posterior capsule is approached. Staining embryonic lenses with FN proved capillary and capsular FN indistinguishable because during development FN containing endothelial capillary cells engulf the lens.  Even double staining with a capillary specific antibody-PECAM and FN revealed too much overlap for a pattern to be observed.  RT-PCR investigation showed high concentrations of FN mRNA in the epithelial cells as compared to fiber cells.  Additional RT-PCR analysis into the two major splice variants of FN (EDA and EDB) showed that epithelial cells have a higher amount of EDB FN than fiber cells.  Inversely, fiber cells displayed EDA FN while epithelial cells did not.  The finding that FN is more concentrated in the epithelium suggests FN’s origin to be there; contrary to pervious speculation that FN’s origins are extraneous.  Also since FN (specifically the EDA splice variant) is associated with wound healing, it is important to understand the distribution of the two variants in normal lenses in order to see how this distribution changes in wound healing responses such as post cataract surgery clouding.  Funded by CPW endowment.

The Effects of Apoptosis in Metastatic Cancer

Monique N. Kinard and Cynthia M. van Golen
Delaware State University

Apoptosis is a regulated physiological process leading to cell death also known as ‘cell suicide’. Apoptotic processes have been an issue of exploration within a variety of diseases especially cancer.  In cancer, we usually expect apoptosis to be down or prevented. Therefore, the rate of apoptosis becomes essential because a lack of apoptosis can cause cells known to become tumors to go through unchecked growth. In this study, neuroblastoma, the most common cancer occurring in infancy and childhood is used to examine proteins involved in apoptosis. Prostate carcinoma, the second most common cancer induced in males was also examined. The main focus was to study apoptosis, which is regulated by numerous proteins including the Bcl and Caspase families. Both diseases were analyzed specifically because of their ability to metastasize to other parts of the body, specifically bones, and both have an up-regulation of the IGF system which plays a role in tumorigenesis and initiates anti-apoptotic signals. Therefore, preventing IGF signaling may induce apoptosis in neuroblastoma and prostate cancer. Neuroblastoma cell lines, IMR32, SK-N-AS, SK-N-SH, and SY5Y, were used to evaluate the expression proteins of Bcl and Caspase proteins; the prostate carcinoma cell lines, C42, LNCap and PC3 were also used. Each cell line was incubated in CO2 at 37 degrees Celsius. Cells were incubated, analyzed and some treated with a neutralizing antibody against the IGF-1R.  Bcl and caspase protein levels were analyzed used Western immunoblotting. Several Bcl and Caspase proteins showed differences in expression corresponding to differences in tumorigenicity.

Role of Junctional Adhesion Molecule-A (JAM-A) in the Progression of Non-alcoholic Fatty Liver Disease (NAFLD)

Megan E. Kissig and Ulhas P. Naik
Department of Biological Sciences

Non-alcoholic fatty liver disease (NAFLD) is characterized by an abnormal amount of fat accumulation in the liver, specifically more than 5% fat by weight.  Little is known about why the fat accumulates in the liver but it has been found that intestinal permeability due to leaky tight junctions may be a contributing factor.  In our lab we study junctional adhesion molecule-A (JAM-A), a protein located at the tight junctions of epithelial and endothelial cells.  My aim was to find what effect JAM-A has on weight gain and the eventual development of NAFLD. To analyze this relationship, groups of Jam-A (+/+) and Jam-A (-/-) mice were put on either a high fat or low fat diet for 20 weeks.  During this time the mice were weighed every two weeks and blood samples were taken every four weeks to test for plasma levels of cholesterol and triglycerides.  At the end of the 20 weeks, the mice were sacrificed and the livers and fat pads were removed, sectioned, and stained to find further effects of the diet, including any progression of NAFLD.  There was a significant variation between the high-fat and low-fat Jam-A (-/-) groups, whereas there was little difference between the Jam-A (+/+) groups.  Also, the high-fat Jam-a (-/-) mice had significantly higher LDL-cholesterol and total cholesterol levels in the plasma than the other groups.  It was also found that the livers of the high-fat Jam-A (-/-) mice showed significantly more fat droplet accumulation than the high-fat Jam-A (+/+) mice.  This suggests that JAM-A regulates weight gain, cholesterol levels, and ultimately NAFLD development.  This project was funded by the Beckman Foundation.

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 15 August 2009 by Hal White
Copyright 2009, University of Delaware