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East Tennessee State University presents the 2008 Appalachian Student Research Forum April 2 – 3, 2008 Millennium Centre • Johnson City, TN Schedule of Events April 2 – 3, 2008 The Centre at Millennium Park Johnson City, TN Wednesday, April 2, 2008 11:00 am – 2:30 pm Poster Set-Up Ballroom 3:00 – 5:30 pm Poster Judging Ballroom Refreshments sponsored by: The Ronald E. McNair Program and The Center for Community Outreach and Family Services Thursday, April 3, 2008 8:30 am – 9:00 am Check-in and Continental Breakfast 9:00 am – 1:00 pm Poster Viewing Ballroom 9:00 am – 2:00 pm Vendor Exhibition 2nd floor 10:30 am – 12:00 pm Vendor Seminars Room 230 Shimadzu: “Fast LC with a Conventional HPLC System” Promega: “Maxwell(tm) 16: Automated gDNA, RNA, & HIS-Protein Purification” Millipore Life Sciences: “Mammalian Cell Culture: Selection and Application of Microporous Membrane Cell Culture Inserts” Continental Breakfast and Mid-Morning Break sponsored by: The Center for Community Outreach and Family Services 2008 Appalachian Student Research Forum Page 1 9:30 am – 12:00 pm 12:00 – 1:00 pm Oral Presentations Graduate Students Residents and Post-Doctoral Fellows Room 130 Auditorium 1st floor BUFFET LUNCH Lunch sponsored by: The Office of the Vice President for Health Affairs The School of Graduate Studies The Center for Nursing Research The College of Public Health 1:00 – 2:00 pm Keynote Address Auditorium “Proteinase Regulation in the Ovarian Tumor Microenvironment” M. Sharon Stack, Ph.D. Professor and Vice-Chair for Research Mulligan Endowed Professor of Cancer Research Department of Pathology & Anatomical Sciences University of Missouri, Columbia 2:15 – 3:00 pm Awards Ceremony Auditorium 3:00 – 4:30 pm Poster Viewing Ballroom ** Posters must be removed by 4:30 pm** 2008 Appalachian Student Research Forum Page 2 Keynote Presentation “Proteinase Regulation in the Ovarian Tumor Microenvironment” M. Sharon Stack, Ph.D. Professor and Vice Chair for Research Mulligan Endowed Professor of Cancer Research Department of Pathology & Anatomical Sciences University of Missouri, Columbia • • • B.S. Clemson University, Biochemistry 1981 Fulbright Scholar, Universitat Bonn, Germany 1982 M.S. Biomedical Science/Biochemistry, East Tennessee State University 1985 Ph.D. Biochemistry, University of Louisville1989 Post-Doctoral Research Associate, Duke University 1989-1991 Assistant Research Professor, Duke University 19911994 Assistant Professor, Associate Professor with Tenure, Professor, Northwestern University 1994-2006 Professor and Vice-Chair for Research, University of Missouri, 2007-present • • • • • ABSTRACT: The ability to invade host tissues and metastasize is the major cause of cancer-related death. During tumor invasion, metastasizing cells disrupt normal cell-cell and cell-matrix contacts and acquire a migratory, invasive phenotype. Subsequent alterations in cellular architecture mediated by modified extracellular matrix (ECM) attachments induce expression of proteinases that degrade ECM proteins, facilitating migration through the modified tissue to establish metastatic foci. Epithelial ovarian carcinoma is the leading cause of death from gynecologic malignancy, as 75% of women with this disease succumb to complications resulting from disseminated intra-peritoneal metastasis. The initiating events in the development of ovarian carcinoma are poorly understood. However, ovarian carcinoma has an unique metastatic mechanism requiring reversible modulation of cell-cell and cellECM contacts that involves shedding of cells from the primary tumor followed by subsequent intraperitoneal adhesion, invasion and proliferation. Unlike most highly metastatic tumors, the majority of women with advanced intraperitoneal disease have no clinically apparent lymphatic or hematogenous metastases, implying that a novel mechanism for metastasis is operative in ovarian cancer. Further, as metastases are largely confined to the peritoneal cavity, microenvironmental factors that modulate intraperitoneal adhesion, motility and invasion play a predominant role in ovarian pathobiology. Ongoing research utilizes an integrative approach involving examination of 2-dimensional (2D) and 3D tissue culture systems complemented by murine tumor models and analyses of human tumors. Understanding the molecular mechanisms by which tumor cells orchestrate multiple microenvironmental cues to regulate the expression and activity of these metastasis-associated proteinases is the major focus of the laboratory. 2008 Appalachian Student Research Forum Page 3 Forum Task Force Dr. Scott Champney, Chair Biochemistry & Molecular Biology Dr. Russ Brown Dr. Ranjan Chakraborty Psychology Health Sciences Dr. Jay Franklin Dr. Lee Glenn Sociology & Anthropology Professional Roles/Mental Health Nursing Ms. Margaret Goergen Dr. Gary Henson Sponsored Programs Physics, Astronomy & Geology Dr. Gary Kukulka Dr. Dhriendra Kumar Family Medicine Biological Sciences Ms. Amy Leonard Dr. Foster Levy Fisher Scientific/Coordinating Vendor Undergraduate Research/ Biological Sciences Dr. Cecilia McIntosh Dr. Louise Nuttle Graduate Studies/Biological Sciences Sponsored Programs Dr. Rebecca Pyles Dr. Mike Ramsey Honors College/Biological Sciences Kinesiology, Leisure& Sport Sciences Dr. Mitch Robinson Ms. Barbara Sucher Biomedical Graduate Program Continuing Medical Education Dr. Mick Whitelaw Dr. Michael Woodruff Physics, Astronomy & Geology Vice Provost for Research Ms. Jennifer Vanover Biomedical Graduate Student/Microbiology 2008 Appalachian Student Research Forum Page 4 2008 Judges Poster Presentations Division I – Undergraduates Division III – Grad Students – 2+ Yrs. Biomedical Sciences Biomedical Sciences, Natural Sciences & Dhirendra Kumar Mathematics, and Social & Behavioral Edward Michieka Onyango Sciences Natural Sciences and Mathematics Arpita Nandi Terry Elisabeth Lancaster Tricia Metts Thomas Jones Gary D. Henson Division IV – Medical Students Zulfiqar Ahmad Yue Zou Social and Behavioral Sciences Dennis DeFoe James Joseph Fox, III Tiejian Wu Division V – Residents and Post-Doc Fellows Judith A. Hammond Rob Wondergem Michelle Crimi Antonio Rusinol Division II – Graduate Students 1-2 Yrs Biomedical Sciences Oral Presentations Yu-Lin Jiang Bert C. Lampson Residents and Post-Doctoral Fellows Natural Sciences and Math Jay Mehta Thomas Laughlin Doris Hubbs Padmanabhan Rengasamy David Linville Behavioral and Social Sciences Andrea D. Clements Graduate Students Laraine Powers Beth Bailey Nathanial Fethke Chris Newell 2008 Appalachian Student Research Forum Page 5 2008 Forum Sponsors Philip D. Cooper Memorial Research Trust Fund, Inc. East Tennessee State University The Office of Research & Sponsored Programs The School of Graduate Studies The Center for Appalachian Studies and Services The Ronald E. McNair Post Baccalaureate Achievement Program The Office of the Vice President for Health Affairs The College of Arts & Sciences Office of the Dean Department of Biological Sciences Department of Computer & Information Services Department of English Department of Mathematics Department of Physics, Astronomy & Geology The College of Business & Technology Office of the Dean The College of Clinical & Rehabilitative Health Sciences Office of the Dean Department of Physical Therapy The Claudius G. Clemmer College of Education Office of the Dean Department of Human Development and Learning 2008 Appalachian Student Research Forum Page 6 The James H. Quillen College of Medicine Office of the Dean Department of Anatomy & Cell Biology Department of Biochemistry & Molecular Biology Department of Obstetrics/Gynecology Department of Pathology Department of Pediatrics Department of Pharmacology Department of Physiology Department of Psychiatry & Behavioral Sciences Department of Surgery The College of Nursing Office of the Dean Center for Nursing Research The College of Public Health Office of the Dean Special Thanks The 2008 Student Research Forum Task Force would like to recognize and thank The East Tennessee State University Center for Community Outreach and Family Services for their generous support of this year’s Student Research Forum, without which this event would not have been possible. 2008 Appalachian Student Research Forum Page 7 Exhibitors 2008 Appalachian Student Research Forum Page 8 Poster Presentations Division I – Undergraduates Biomedical Sciences OXIDATIVE DAMAGE TO THE SUGAR-PHOSPHATE BACKBONE OF DNA CAUSED BY CARBONATE RADICAL ANIONS Jeremy Brooks*, Lauren Brooks*, Laura Catron*, Amanda Kelly*, Lindsey McAmis*, Joe Powell*, Danna Sharp* and Yuriy Razskazovskiy† * The Talent Expansion in Quantitative Biology (TEQB) Program, ETSU † Dept. of Physics, Astronomy and Geology, ETSU The carbonate radical anion CO3•- is a biologically relevant oxidizing intermediate generated in seawater by sunlight and in cells through CO2-catalyzed decomposition of peroxynitrite anions ONOO-. This species has been shown to induce oxidative damage to guanine bases in DNA that makes it a source of potentially mutagenic DNA lesions. The focus of this study is the reactivity of CO3•- towards the sugarphosphate backbone of DNA, including its ability of inducing immediate and latent strand breaks through hydrogen abstraction from the deoxyribose moiety. For the purposes of this project CO3•- was generated by UV (λ>300 nm) photolysis of inorganic complex [Co(NH3)5CO3]NO3 in phosphate-buffered solution of calf thymus DNA, and DNA damage products were analyzed by HPLC. Product analysis showed the formation of heat-labile DNA lesions that released a characteristic low molecular weight product 5methylenefuran-2-one upon spermine-catalyzed thermal decomposition in phosphate-buffered solutions (pH 6.9). Based on this characteristic behavior we conclude that the carbonate radicals oxidize the sugarphosphate backbone primarily at the C1`-position of the deoxyribose moiety that leads to the formation of so-called oxidized abasic sites in the DNA structure. Quantitative estimates based on HPLC measurements show that about 8% of all carbonate radicals generated in the system react through this pathway. A COMPARITIVE STUDY OF THE HANDS OF LEMURS AND OTHER SUSPENSORY SPECIES. R.A. Deliz Dept. of Sociology & Anthropology, East Tennessee State University, Johnson City, TN The hands of prosimian primates play a major role in locomotor activities. The structure of the hands of extant sifakas exhibits characteristics indicative of leaping and clinging. Their grasping efficacy pales in comparison to the recently discovered extinct species, whose curved phalanges are convergent with slothlike suspensory positional behaviors or grasping styles of gibbons and siamangs. I have examined previous studies of the recently extinct species of the subfossil lemurs Paleopropithecus and Mesopropithecus and explored the morphological heterogeneity between these and modern sifakas, indris, sloths, gibbons, and siamangs. In addition, I have examined modern sloths and anteaters for comparative purposes. In each species I measured phalangeal curvature and length and identified skeletal markers of 2008 Appalachian Student Research Forum Page 9 locomotor adaptations, in comparison with the subfossils. Utilizing this data I have drawn inferences about the relationship between skeletal form and locomotor function in modern lemurs as well as primates and other suspensory species. ODD BEAR OUT? Milan Ganger and Terry Lancaster Dept. of Health Sciences, East Tennessee State University, Johnson City, TN Polar Bears are unique bears because they are semi-aquatic. Polar Bears tend to swim with their backs and neck out of the water. They also have webbed feet for swimming and this layer of waterproof fur over their black colored skin. This fur is so unique that humans used to hunt polar bears just for their fur to keep warm in the arctic. Not only do we look at the outer characteristics of the Polar Bear, but the inside as well. My question is due to their irregular way of swimming, do Polar Bears have some different distinctiveness along their spine? I will be looking at the transverse and spinous processes to determine if a Polar Bear is semi-aquatic from a given vertebrae. I will compare the vertebrae of the Polar Bear to other similar semi-aquatic species (i.e.: Northern Elephant Seal, Hooded Seal, South American Sea Lion, and the Australian Sea Lion) and two terrestrial bear species (i.e.: American Brown Bear and Black Bear) as well. Using sliding and spreading calipers, I will measure the length, width, and depth of the transverse processes and spinous processes of the cervical, thoracic, and lumbar vertebrae. I will compare all measurements of the Polar Bear with the Northern Elephant Seal, Hooded Seal, South American Sea Lion, Australian Sea Lion, the American Brown Bear, and the Black Bear. Along with these measurements, I will measure the length of each region of vertebrae (i.e. cervical, thoracic, and lumbar) and the curvature of the neck. With these measurements, I will be able to identify some distinct characteristics that will categorize the Polar Bear as either semi-aquatic or terrestrial by using Principal Component Analysis (PCA) to identify the semi-aquatic grouping and the terrestrial grouping of mammals and then attempt to “place” the Polar Bear in one of these groups by its morphology. SYNTHESIS AND EVALUATION OF GAMMA TOCOPHEROL LYSINATE AS AN ANTI-CANCER AGENT IN HUMAN PROSTATE AND COLON CANCER CELL LINES Bassam Lamale, Yu-Lin Jiang and Sharon Campbell Dept. of Chemistry, East Tennessee State University, Johnson City, TN Vitamin E is a fat-soluble vitamin represented by eight different forms classified as either tocopherols or tocotrienols which contain antioxidant properties. Alpha tocopherol is the most bioavailable among the isoforms in humans and protects cell membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction. Mediterranean cultures have a lower risk for cancers and cardiovascular disease due to diets that are rich in vitamin E. Campbell et al. showed that RRR-γtocopherol showed more antiproliferative activity on HCT-116 colon cancer cell lines compared with RRR-α-tocopherol. Vitamin E isoforms are insoluble in aqueous solution. An inability to absorb a sufficient amount of fat will result in vitamin E deficiency making the need for a water soluble vitamin E vital. Altering the chemical structure of RRR-γ-tocopherol (GT) with the addition of polar atoms may increase the solubility and has been shown to increase the antiproliferative activity in MCF-7 breast cancer cells. Further, redox silent analogues of Vitamin E that contain polar molecules added to the hydroxyl antioxidant site by ester or ether link have demonstrated an increase in antiproliferative activity over the Vitamin E from which it was derived. We began to look for biologically relevant polar molecules that could be added to the vitamin E and hypothesized that the addition of an amino acid to the 2008 Appalachian Student Research Forum Page 10 antioxidant site may enhance the antiproliferative activity of GT. We selected the amino acid lysine owing to its three polar groups. The organic synthesis of RRR-γ-tocopherol lysinate (GTL) was carried with the esterification of GT with protected lysine using dimethylaminopyridine as a catalyst and dicyclohexylcarbodiimide as a reagent in the first step. The resultant product was then deprotected with hydrogen chloride in ethyl acetate to the water soluble lysinate of GT. The structure of the product was validated using NMR. GTL was assayed for its ability to effect cell proliferation in the human cancer cell lines HCT-116 (colon) and PC-3 (prostate) using the MTT assay. The MTT assay shows that GTL has a higher percent growth inhibition compared with GT at physiological and pharmacological concentrations in both cell lines. Western blot analysis for the proliferation and apoptosis proteins PPAR γ, cyclin D1 and Poly (ADP-ribose) polymerase (PARP) was performed. Consistently in both cell lines, western blot analysis shows that PPARγ is upregulated, cyclin D1 is down regulated and PARP is cleaved to a greater extent with GTL than GT. These data demonstrate that the addition of the lysine group by an ester linkage at the antioxidant hydroxyl site improves the ability of the molecule to inhibit proliferation in two different cancer cell types. Further the genes that are regulated by this molecule are similar to those previously determined to be important in the GT-mediated cell death and apoptosis. The GTL reaction is faster than that of GT. Further work needs to be done to determine what makes the kinetics of the reaction faster with GTL when compared to GT. EXPRESSION OF HUMAN NEUTROPHIL ELASTASE USING SUMO FUSION TECHNOLOGY Jessica Pugh and David Johnson Dept. of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN Human polymorphonuclear leukocytes (neutrophils), which play a critical role in innate immunity, have three active serine proteases (Elastase, Cathepsin G and Protease 3) in their azurophilic granules that aid in bacterial killing. Human neutrophil elastase (HNE) has been linked to several diseases including pulmonary emphysema via degradation of lung elastin, and mutations in the HNE gene (Ela2) result in low neutrophil counts with susceptibility to bacterial infections. Recombinant HNE has never been produced and the enzyme isolated from neutrophils is missing 20 amino acids from its C-terminus. A method of expressing recombinant HNE would allow study of this unusual C-terminal processing and make site directed mutagenesis studies possible. SUMO (small ubiquitin-related modifier protein) fusions are known to improve recombinant protein solubility in E. coli. The SUMO fusion vector pETHSUL, which is also a ligation independent cloning vector (LIC), was obtained from Dr. Patrick Loll (Drexel University, COM). An image clone of HNE was obtained from American Type Culture Collection. The coding region of the HNE gene was amplified in a PCR reaction using Stratagene’s PfuUltra II Fusion HS DNA Polymerase using primers designed to generate LIC compatible products. The pETHSUL vector was cut with BseRI and then treated with TSAP thermosensitive alkaline phosphatase to remove the 5’ phosphates. The vector was then gel purified, and treated with T4 DNA polymerase in the presence of 1 mM dCTP to add a string of Cs. The HNE PCR product was made LIC ready by removing the 5’ phosphates, followed by adding a string of Gs using T4 DNA polymerase and 1 mM dGTP. A mixture of the LIC ready HNE PCR product and LIC ready SUMO fusion vector pETHSUL was added to BIOBlue Chemically Competent cells. The vector with its tightly bound HNE PCR product was taken up by the cells as if they had been ligated together. Transformed cells were screened on plates with ampicillin. The LIC process is very efficient because all eight colonies screened by PCR were all positive for the HNE insert and the sequence was correct and in frame from sequencing data. Rosetta-gami cells will be transformed with pETHSUL-HNE and pSUPER, which contains the SUMO protease gene which will be used to cut the SUMO off of the SUMO-HNE fusion protein. The Studier Method for Autoinduction of Protein Expression in E. coli using the T7 promoter will then be 2008 Appalachian Student Research Forum Page 11 used to express SUMO-HNE and the SUMO protease. Stable expression of HNE will be beneficial for further studies concerning the functions and diseases associated with this protease. Supported by National Heart, Lung and Blood Institute grant 1R15HL091770. A COMPARATIVE ANALYSIS OF LONG BONE FORMULAE TO ESTIMATE LIVING STATURE (HEIGHT) Michelle Wood, and Terry E. Lancaster Depts. of Criminal Justice and Health Sciences, East Tennessee State University, Johnson City, TN Stature estimation from long bones has been a primary focus of anthropological studies since the latter half of the 1800's. Most stature estimations are done using only a single long bone, such as the humerus, radius, ulna, femur, tibia, or fibula. The humerus, femur and tibia are the most widely accepted long bones used for these predictions, and they are rarely used combined in estimation formulae. In this study we developed our own series of formulae to predict the stature of an individual from the long bones of the skeleton. Using linear regression models we constructed a series of prediction equations based on length and midshaft diameter measurements of the humerus, radius, ulna, femur, and tibia using these bones singly and in combination to produce the best estimator of stature. We then divided our sample by sex and then by race and recalculated our prediction equations to isolate and identify the effect of these on stature. Natural Sciences & Mathematics CONSTRUCTION AND FUNCTIONAL ANALYSIS OF CHIMERIC PROTEINS FROM PUTATIVE FLAVONOID GLUCOSYLTRANSFERASE CLONES FROM GRAPEFRUIT Josephat Asiago, Daniel K. Owens and Cecilia A. McIntosh. Dept. of Biological Sciences, East Tennessee State University; Johnson City, TN Flavonoids are one of the major groups of plant natural products and many exist in glucosylated form. They are known for coloration, flavor, plant-plant interaction, plant-microbe interactions, plant-insect interaction, ultra violet protection and pollen maturation. Plant UDP- glucosyltransferases (UGT’s) are enzymes that transfer a glucose residue from the activated donor to an acceptor molecule. The transfer of glucose to the acceptor molecule helps to stabilize, detoxify and solubilize metabolites. Grapefruit is known for high levels of accumulation of flavanone 7-O-glycosides as well as flavone-7-O-glycosides, flavonol 7-O and 3-O glycosides. The majority of flavonoid GT’s characterized to date belong to the 3-OGT group (act on flavonols and anthocyanins). There are at least five different flavonoid glucosyltranferases that have been identified in grapefruit: flavonol 3-O-GT, flavonol 7-O-GT, chalcone GT, flavone 7-O-GT and flavanone 7-O-GT. This makes grapefruit a good model to look for different flavonoid GT’s for structure and functional analysis. Eight putative glucosyltranferases have been identified in our lab. PGT 7 has shown activity with quercetin and characterization is in progress. PGT4 has not shown any activity with flavonoid acceptors. This may be attributed to the low level of soluble protein or to the fact that this enzyme acts on another group of secondary metabolites. The amount of soluble protein in PGT4 is currently being optimized by using less rich medium during induction and collection of samples at different time points. French press instead of sonication will be used to burst open the cells in an effort to protect the protein from denaturation. The PGT4 protein from the burst cells will be purified using the HIS TAG-metal affinity column before re-testing for activity. Construction of 2008 Appalachian Student Research Forum Page 12 chimeras will provide important information on the active moieties and their location in PGT 7 and PGT4 and the activity of chimera will be compared to that of the parent proteins. Chimeric protein constructs were obtained from PGT7 and PGT4 as follows: PGT 4 was digested with restriction enzymes (Sac I and Xho I). These enzymes are internal to PGT4 and do not cut the vector. The C terminus of PGT7 was amplified using clone specific primers (CSP 58F and CSP58R) designed to incorporate Sac I and Xho I in the sense (CSP 58F) and the antisense (CSP 58R) primers respectively. Products were digested with Sac I and Xho I. The N-terminus section of PGT4 and the C-terminus section of PGT7 were gel purified. These pieces were ligated and cloned into expression vector. The full structure of chimeric construct will be confirmed by sequencing the chimeric clone. Once the primary structure is confirmed, the chimeric clone in expression vector will be transformed into BL21 (DE3), RIL E. coli cells and induced for protein expression. The expressed protein will be tested for flavonoid GT activity and compared to the parent proteins. BIOCHEMICAL IDENTIFICATION AND CHARACTERIZATION OF SABP2-INTERACTING PROTEINS BY AFFINITY CHROMATOGRAPHY Raj C. Bant, Kajal C. Patel and Dhirendra Kumar Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN Salicylic acid is required by plants to fight pathogen infection and is critical for inducing both local and long lasting systemic acquired resistance (SAR). SABP2, a 29 kDa plant protein, was identified in a screen to discover proteins which bind to salicylic acid (SA). In plants, SABP2 catalyzes the conversion of methyl salicylate (MeSA) into SA, which then activates defense proteins to mediate a resistance response. Recently, MeSA was identified as the long distance phloem mobile signal required for systemic acquired resistance (SAR). SABP2 receives this SAR signal in systemic tissue and converts it into SA. RNAi mediated silencing of SABP2 expression makes the transgenic tobacco plants more susceptible to TMV. Given the important role of SABP2 in mediating plant resistance it is important to identify cellular proteins which may interact and regulate its activity. Therefore, we are conducting a screen to identify and characterize its interacting partners in the cell. As a first step, high amounts of recombinant SABP2 was expressed in E. coli. This recombinant SABP2 was then bound to Ni-NTA agarose column and utilized as an affinity matrix to bind and purify SABP2 interacting proteins (SBP2IP). Bound proteins will be analyzed by SDS-PAGE followed by silver staining. Alternatively native SABP2 and its interacting proteins from tobacco plants could be purified using a affinity column containing anti-SABP2 antibodies linked to protein A/G agarose matrix. MOLECULAR MECHANISM OF ATP SYNTHASE- ROLE OF CHARGE IN THE CATALYTIC SITES Laura E. Brudecki and Zulfiqar Ahmad Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN F1Fo-ATP synthase is the smallest known biological nano motor, found from bacteria to man. It is the enzyme responsible for ATP synthesis by oxidative or photophosphorylation in membranes of bacteria, mitochondria, and chloroplasts. ATP synthase is the fundamental means of energy production in animals, plants, and almost all microorganisms. Synthesis and hydrolysis both are carried out at the catalytic sites of F1Fo ATP synthase. X-ray structures show the presence of αPhe-291 residue in the phosphate binding subdomain, in close proximity to the known Pi binding residue, βArg-246. In order to understand the role of charges in phosphate binding, we mutated αF291 to αF291D and αF291E. The choice of this residue 2008 Appalachian Student Research Forum Page 13 was based on its close proximity to the phosphate analogs AlF3 and SO42- , which are bound in catalytic sites of x-ray structures of bovine ATP synthase. Using Pi protection from NBD-Cl (7-chloro-4nitrobenzo-2-oxa-1, 3-diazole) inhibition of ATPase in membranes as the assay, we found that these mutations reduce the ATPase activity while retaining oxidative phosphorylation significantly. This mutation also led to the abrogation of Pi binding as seen earlier in case of βN243D mutation. We also found that in contrast to wild-type, ATPase of mutants was not inhibited by MgADP-fluoroaluminate, MgADP-fluoroscandium, or MgADP-azide. INVESTIGATING CLIMATE CHANGE IN THE MIDCONTINENT AND SOUTHEASTERN APPALACHIAN: SPELEOTHEM RECORDS FROM CAVES IN MISSOURI AND TENNESSEE Jessica Buckles, Yongli Gao, William Hill Dept. of Physics, Astronomy, and Geology, East Tennessee State University, Johnson City, TN Two speleothem samples were collected from Power Mill Creek Cave (PMCC), Missouri, for uraniumseries dating at the University of Minnesota and stable isotopic analyses at the University of Kentucky. The ages of the speleothem samples range between 53,170 and 660 years old. We obtained carbon and oxygen isotope data on one speleothem for a time period between 53 to 35 ka and reconstructed the regional climatic and vegetation changes. In general, climatic oscillations and vegetation changes in this region are consistent with the earlier study from speleothem samples of Crevice Cave, Missouri (Dorale et al., 1998). The midcontinental climate cooled and forest replaced grassland between 55 and 41 ka. Then, the temperature suddenly increased at 37 ka. Preliminary analysis of oxygen isotope oscillation shows that this warming around 37 ka was even more dramatic than that reported in the previous investigation at Crevice Cave. Recently, we started to process speleothems collected in eastern Tennessee. These speleothems were dated at different ages. No extended record of continental climate change exists for eastern North America going back through the past 11 marine isotope stages. The primary objective of this research project is to assemble a calibrated, highly-resolved composite record of decadal- to centennial-scale climate change from the mid-latitudes of eastern North America that extends across the last four interglacial/glacial cycles. ISOLATION OF THE FULL-LENGTH CLONES OF GRAPEFRUIT 181-1D AND CP1-D10, PUTATIVE FLAVONOID GLUCOSYLTRANSFERASES Patricia Campbell, Daniel Owens, and Cecilia McIntosh Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN This research centers within the topic of secondary metabolites produced in the Plant Kingdom, in particular flavonoids in Citrus paradisi. Flavonoid compounds are found naturally in many plants for such main purposes as aiding in reproduction and defense. This is accomplished through flavonoids being responsible for some of a plant's unique odor, taste, and color characteristics as well as having a role in pollen recognition. Flavonoids also protect leaves from UV light damage, fungal and microbial pathogens, and act as antifeedants. Flavonoids have been shown to possess antioxidant and possible anticarcinogenic characteristics in mammals. Our lab is focused specifically on the flavonoid glucosyltransferases. These are the enzymes whose job as a catalyst is to aid in the transfer of monosaccarides(sugars) to a receiving molecule, in this case, flavonoids. Glucosyltransferases are key enzymes in the synthesis of the flavnonoids actually found in plant tissues. There are many putative sequences for glucosyltransferases in databases, but unfortunately there is little functional characterization found in the literature. My focus of interest lies within two grapefruit putative GT clones (CP1-D10 and 2008 Appalachian Student Research Forum Page 14 181-1D) that have been found to only differ in a small repeat area so far. This research is designed to test the hypothesis that these are nearly identical clones with the major difference being confined to the currently identified repeat area, and try to test their functional similarities. This is being done through a series of experiments. First, clone specific primers were used in PCR to pull out the desired DNA fragment. Once amplified, the PCR product was then cloned into a Topo holding vector and transformed into E.coli competent cells which were then plated for colony growth on LB media plates. The colonies were tested, such as through PCR screening procedures, to confirm that they contain an insert of the expected length. DNA sequencing is being performed to determine which clone(s) has been obtained. Sequences will be analyzed to further assess the degree of identity of the clones. If results confirm the presence of two distinct putative glucosyltransferase clones with high identity, the clones will be migrated into a protein expression vector. If time permits, experiments will be conducted to optimize conditions for inducing protein production followed by assays in the detection of their activity for the structural and functional characterization to be carried out. SYSTEMATIC STUDY OF THE LATE NEOGENE NUTS OF CARYA (JUGLANDACEAE) FROM NORTHEAST TENNESSEE Graham Cooke, Yusheng Liu Dept. of Biological Sciences, East Tennessee State University, Johnson City TN The discovery of the Gray Fossil site, located in Gray Tennessee in 2000 lead to a series of discoveries lending additional information to the study of the North American-East Asia disjunct, this has laid the groundwork for comprehensive study of the paleo-climate at the time of the creation of the deposits in the late Miocene. The site has yielded an unusual amount of fossilized animalia and plantae, notably the presence of Pristinailurus bristoli, Tapirus polkensis, in addition the presence of Fruit, and or Pollen from families Juglandaceae, Fagaceae, Pinaceae amongst many other. The fossilized animal remains found in the deposit indicate the temperate subtropical environment currently found in the locality is far different to what was present in the late Miocene. Using a combination of techniques we hope to provide additional information to the reconstruction of the paleo environment, and utilize the currently recognized phylogeny of Juglandaceae to validate the characteristics used to identify the species of Juglandaceae present, and in turn their following evolution. PATTERN OF CALCIUM MOBILIZATION IN A VIVIPAROUS AUSTRALIAN SKINK, PSEUDEMOIA PAGENSTECHERI Santiago P. Fregoso, James R. Stewart, and Tom W. Ecay, Depts. of Biological Sciences and Physiology, College of Arts and Sciences, East Tennessee State University, Johnson City, TN In order to obtain calcium during embryonic development, oviparous lizards take advantage of their yolk and eggshell reserves. However, they do not exploit the eggshell until the last few stages of development. Viviparous lizards, on the other hand, do not possess an eggshell from which to extract calcium and have a proportionally smaller yolk. Many species rely heavily on placental provisioning of nutrients during embryogenesis. Previous studies have shown that highly specialized placental lizards receive most of their calcium from the placenta. This study aims to expand on that information by ascertaining the mobilization pattern of calcium provisioning across the placenta during development of a placental scincid lizard, Pseudemoia pagenstecheri. Samples of yolk and embryos were removed from females at specific intervals during development and analyzed for calcium using atomic absorption spectrometry. The timing 2008 Appalachian Student Research Forum Page 15 of calcium mobilization to embryos from placental transfer was similar to calcium recovery from the eggshell of oviparous species. However, oviductal calcium secretion occurs much later in development in this viviparous species. This difference suggests a timing shift from the ancestral mode and a possible nutritional advantage to placentotrophy. EMBRYONIC CALCIUM MOBILIZATION IN AN OVIPAROUS LIZARD, SAPROSCINCUS MUSTELINUS Courtney Garland, James R. Stewart, and Tom W. Ecay Depts. of Biological Sciences and Physiology, East Tennessee State University, Johnson City, TN Oviparous lizards provide calcium to their embryos through calcium-rich yolk and calcium deposited into the eggshell. Oviparous skinks receive between 30-50% of their embryonic calcium from the eggshell with the remainder coming from the yolk. Yolk calcium is deposited during vitellogenesis, while calcium in the eggshell is secreted during the shelling process in the oviduct. We studied the oviparous scincid lizard Saproscincus mustelinus. We used atomic absorption spectroscopy to estimate the quantity of calcium in yolk, eggshell and the embryo at specific time points during incubation. We found that yolk calcium is mobilized gradually throughout the early stages of embryonic development and uptake begins to increase during the later stages as embryonic growth accelerates. Calcium from the eggshell is also mobilized during the later stages while embryonic growth rate is high. We found that Saproscincus mustelinus receives 54% of its calcium from the eggshell, showing the heavy reliance of the developing embryo on the eggshell as well as the yolk. The percentage of recovery from the eggshell is higher than most other oviparous lizards. Therefore, we hypothesize that Saproscincus mustelinus has a more specialized mechanism for retrieving calcium from the eggshell than most oviparous lizards. IMROVEMENT OF THE MOTOR FUNCTION OF ESCHERICHIA COLI ATP SYNTHASE BY SELECTION PRESSURE Johnny J Grindstaff, Prasanna K Dadi and Zulfiqar Ahmad, Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN ATP synthase is responsible for ATP synthesis by oxidative or photophosphorylation in membranes of bacteria, mitochondria, and chloroplasts. It is the fundamental means of energy production in animals, plants, and almost all microorganisms. Escherichia coli ATP synthase in its simplest form contains eight subunits namely α3ß3γδεab2c10-14. The α/β interface has a total of six sites, three of which are catalytic and three non catalytic. Synthesis and hydrolysis both are carried out at the catalytic sites which are mainly contributed by the β subunits in the α/β interface of F1 sector. In earlier studies, our lab has shown that βR246 is involved in phosphate binding and mutation of βR246A leads to the abrogation of Pi binding. Phosphate binding is an essential part in the formation of ATP from ADP+Pi. A powerful way to understand the molecular mechanism of ATP synthase and how ATP is synthesized is the knowledge of the suppressors (genetic revertants) for mutants which interfere with the Pi binding. The most suitable mutation appeared to be βR246A. The growth yield of βR246A was tested on minimal glucose and on non fermentable carbon source succinate. Testing on thousands of succinate plates along with wild-type as positive control and pUC118 as negative control gave us 23 putative candidates. After further analysis of the 23 candidates on limiting glucose and 20mM succinate media, we were able to identify six clones which appeared to be the genetic revertants of β R246A. The 4th colony named GR4 showed maximal growth on succinate plates which grew better than wild-type, suggesting that the motor function might 2008 Appalachian Student Research Forum Page 16 have improved. This improvement was further confirmed by the growth on minimal succinate media. Currently, we are in the process of sequencing the plasmids obtained from those six Genetic Revertants. THE SIERPIŃSKI SQUARE GRAPH AND SOME OF ITS PROPERTIES Elizabeth Harris, Joshua Berry, Dr. Anant Godbole Dept. of Mathematics, East Tennessee State University, Johnson City, TN We generalize the well-studied Sierpiński Triangle and define the Sierpiński Square, a structure obtained by forming, iteratively, progressive Sierpiński Triangles and an inner square. SQ1 is the unit square, and SQn, n>1, is obtained from the first n iteration of the above process. Most researchers in graph theory study a particular property for several families of graphs. Here we take the other, less common, approach, namely to study a large set of properties for one family of graphs, namely SQn. Properties studied include the order, size, chromatic number, total edge length, diameter, domination number and Hamiltonicity of SQn. SA-METHYL TRANSFERASE INVOLVEMENT IN DISEASE RESISTANCE SIGNALING Tazley Hotz and Dhirendra Kumar, Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN Salicylic acid (SA) has been shown to play an important role in plant defense response against microbial pathogens. SA produced is lipid immobile and cannot pass through the chloroplast membrane to enter into the cytoplasm to mediate the disease resistance signaling. It has been suggested that SA synthesized in the chloroplast is converted into lipid mobile methyl salicylate (MeSA) by an enzyme, salicylic acid methyl transferase (SAMT). To investigate the role played by SAMT in SA-mediated disease resistance signaling, we examined the expression of SAMT in resistant tobacco’s response to tobacco mosaic virus (TMV). To compare SAMT expression, we also studied tobacco plants susceptible to TMV expression and a transgenic tobacco plant (NahG) which does not accumulate SA. Expression studies were performed using semi-quantitative RT-PCR. To investigate the role of SAMT in plant immunity, we are in the process of making transgenic tobacco by either over expressing SAMT or by knocking out its expression using RNAi. These transgenic lines will be infected with viral and bacterial pathogens to determine the role of SAMT in plant defense. Understanding the biochemical pathways involved in plant defense response against pathogens will help to develop crop plants showing better resistance to everevolving pathogens. THE EFFECTS OF GALAXY INTERACTIONS ON STAR FORMATION AND MORPHOLOGY Sabrina Hurlock, Beverly J. Smith, and Mark Hancock, Dept. of Physics, Astronomy, and Geology, East Tennessee State University, Johnson City, TN We have analyzed SARA (Southeastern Association for Research in Astronomy) optical images of several interacting galaxy systems selected from the Arp Atlas, and compared with GALEX (Galaxy Evolution Explorer) UV images, Spitzer infrared (3.6 µm and 8.0 µm) images, and Sloan Digital Sky Survey images. Using SARA, we have observed the galaxies in visible wavelengths including B, V, and 2008 Appalachian Student Research Forum Page 17 R broadband filters and narrowband redshifted H-alpha filters. We used the Image Reduction and Analysis Facility (IRAF) program to remove the bias and dark current from the images, flat-field the data, and to align and combine the images. In order to obtain a pure H-alpha image, we first subtracted out the continuum, or starlight, using the R band image. These continuum subtractions allow us to view areas of intense star formation that we would otherwise not be able to distinguish due to the light from the older stars. With this multi-wavelength data set, we are able to determine the effects that interaction has on the structure of the galaxy including the formation of bridges and tails, and where new star formation is occurring. MODELING MICROPARASITIC DISEASES McKayla Johnson, Tashauna Gilliam, and Istvan Karsai Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN Worldwide about 1,415 human pathogens are known, of which over half are microparasites. An infection begins when a virus or bacterium enters the host and starts to replicate. If the immune system fails, the host becomes sick and will infect other individuals with the pathogen. Three main properties that control the transmission and life cycle of the virus are infectiousness, duration of the disease, and the chance of recovery of the host. Each parameter has the ability to lead to a continuation or an extermination of the disease within the population. The interplay of the parameters will predict how the disease may affect populations, and knowing this, we can plan preventive steps that hinder and stop the infection in populations. Infectiousness, duration, and chance of recovery are used as main parameters in this study. Each one has an effect on the viability dynamics of the host and the microparasite. The infectiousness concerns the ease with which a microparasite can infect its host. The chance of recovery concerns how strong the virulence is in relation to the ability of the host in recovery and the development of immunity concerning the same strain of disease in the future. The duration of the disease determines how long a person with the disease may infect others before death or recovery. The three of these parameters commonly evolve to produce an equilibrium-like situation. If a disease is too aggressive, with little to no chance of recovery and has a high infectious rate, the hosts will die out, thus eliminating the virus. On the other hand, if a disease has a long duration and moderate infectiousness, the possibility of the disease remaining in the population without exterminating itself is higher. The interactions of these three important parameters were studied via a technique called parameter sweep in the model called Virus (Wilensky (1998) in the NetLogo 3.1.3 software. In this agent based model, the individuals move randomly throughout the environment, allowing random contact among the individuals. Our results show that the more infectious a disease becomes, the more likely it is to extinguish itself. As the disease becomes more infectious and the duration lengthens, immunity will reach its maximum and then decrease for each following duration. Regardless of infectiousness with a chance of recovery at 50%, a disease causes a minimum in healthy individuals if it has the duration of 50 weeks. Results such as these conclude that several parameters can be combined to model the viability of a disease within a population, predicting its toll on the population as well as on its own viability. 2008 Appalachian Student Research Forum Page 18 ALLENE-INOC REACTION Rachel Keith, Dr. David Young Dept. of Chemistry, East Tennessee State University, Johnson City, TN The goal of the set of experiments is to synthesize an allene to be used in an intramolecular nitrile oxide cycloaddition, abbreviated INOC. The reaction has already been published using simple alkenes, however, we propose to perform the experiment with allenes. Progress on our route to an allene to be used in this reaction will be reported. DEVELOPMENTAL EXPRESSION OF CARBONIC ANYHDRASE II IN THE EXTRAEMBRYONIC MEMBRANES OF THE CORN SNAKE, PANTHEROPHIS GUTTATUS Maleka Khambaty1, James R Stewart2, Tom W Ecay1. Dept. of Physiology, East Tennessee State University, Johnson City, TN 2 Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN 1 The eggshell provides 28% of the calcium in a hatchling corn snake, with the remainder coming from yolk. The chorioallantois contributes to calcium homeostasis during embryogenesis by mediating the transport of shell calcium to the embryo. Our objective is to understand how the embryo regulates dissolution of mineralized shell calcium and its transport during development. We hypothesize that cells of the chorioallantois secrete acid onto the eggshell thereby dissolving calcium carbonate crystals. Acid production is catalyzed by carbonic anhydrase II (CA II). Immunoblotting demonstrates an increase in CAII expression throughout development in the chorioallantois but no detectable expression in the yolk sack. Increased CAII expression is coincident with increased calbindin-D28K expression, a marker for calcium transport. Histochemical staining identifies CA positive cells at the outer surface of the chorioallantois adjacent to the eggshell. CAII and calbindin-D28K immunohistochemisty are being used to determine if acid secretion and calcium transport occurs in the same or different cells of the chorioallantois. (Supported by the NSF and an APS Undergraduate Summer Research Fellowship.) SYNTHESIS AND CHARACTERIZATION OF DNA BASE RECEPTORS Bassam Lamale1, Lee M. Daniels2, and Yu Lin Jiang1 1 Dept. of Chemistry, East Tennessee State University, Johnson City, TN 2 Rigaku Americas Corporation, 9009 New Trails Drives, The Woodlands, TX Two DNA base binding receptors 1 and 2 have been synthesized by three-step reactions, including Suzuki arylation of dihaloarenes, followed by metallization with n-butyl lithium and carboxylation with carbon dioxide. Crystals of the receptors were obtained from slow cooling of hot solution of the compounds in acetonitrile. The crystal structures of the compounds have been determined using X-ray crystallography. Both receptors form dimers through binding of one carboxylic group to the other. The structure of 1 shows that carboxylic group is in the same plane as the phenyl group; however, the pyrenyl group is perpendicular to the phenyl group. The structure of 2 shows that both carboxylic and pyrenyl groups are parallel to each other but perpendicular to the naphthyl group. The molecular recognition of DNA base 9butyl adenine has also been carried out. The binding of 9-butyladenine by 1 results in the decrease of 2008 Appalachian Student Research Forum Page 19 pyrene fluorescence intensity, but the binding of the 2 by 9-butyladenine results in increase of pyrene fluorescence. O OH OH O 1 2 ENVIRONMENTAL FACTORS CONTRIBUTING TO COLONY SUCCESS IN A SOCIAL SPIDER SYSTEM Brent J. Linville and Thomas C. Jones Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN Population density is affected by an organism’s survival and reproductive success in a particular area, which in turn are affected by environmental factors. Population densities of the northern social spider, Anelosimus studiosus, vary significantly among seemingly suitable habitats within its range in east Tennessee. For example, the spider occurs in high densities at Bays Mountain Lake, but not at all at Watauga Lake. We hypothesize that this variance in density can be attributed to several environmental factors including prey density, temperature, and relative humidity. To test this, we surveyed populations at six lake locations in east Tennessee that vary in population density. We also placed experimental colonies at each of these sites to quantify their survival and reproductive success at each site. Over the course of the active season (May to October) we monitored temperature, relative humidity and prey density, as well as colony survival. At the end of the season we collected the nests for a final census of the number of juveniles. We will present results of nest survival and success with regard to natural population density and environmental data. STOICHIOMETRIC DETERMINATION OF HYDROGEN-PEROXIDE DECOMPOSITION VIA COPPER CATALYSIS Christopher Moore and Dr. Jeffrey Wardeska Dept. of Chemistry, College of Arts and Sciences, East Tennessee State University, Johnson City, TN Hydrogen peroxide has a decomposition reaction that is generally known by the stoichiometric equation of 2H2O2 Æ 2H2O + O2. Using two imidazole adducts of the tridentate-copper catalyst, [CuSaLAD]2, we were able to catalyze the decomposition of hydrogen peroxide within minutes. Our research focused on the stoichiometric decomposition of H2O2 by the copper-imidazole catalysts. To obtain this stoichiometric decomposition directly, we measured the O2 formed from the catalysis reaction in a closed system. By injecting the H2O2 into the system, a pressure change due to the formation of O2 from the catalysis reaction was calculated. Subtracting the pressure measured from a blank run, the pressure of O2 was then applied to the Ideal Gas Law to obtain a molar quantity for O2. The stoichiometric relationship obtained was not of the general equation mentioned above. In order to further investigate the reaction’s mechanism we used kinetic studies, cyclic voltammetry, and a chemical test for copper(I) formation. By allowing the closed system under high pressure to remain closed, a decrease in pressure over time was obtained. From this data collection, rate constants were calculated for equimolar and excess copper (Cu : 2008 Appalachian Student Research Forum Page 20 H2O2) solutions while H2O2 remained constant. The rate constants were compared for each copperimidazole catalyst resulting in no copper-copper bridging through the imidazoles. The comparison also displayed that no steric effect was observed between the placements of the methyl group. The cyclic voltammetry provided evidence of Cu(I) formation as a new peak on the oxidative scan. This peak soon disappeared, resulting in the possible reversible oxidation of Cu(I). Finally, to assure that the peak from the voltammetry experiment was from the formation of Cu(I) when H2O2 is catalytically decomposed, a chemical indicator was used. The 2,2-biquinoline indicator was added to the copper-imidazole catalyst prior to the addition of H2O2 resulting in a pink color after addition and the evidence of Cu(I) formation. The data collected from all experiments have been compiled to compare to previous research and mechanisms in order to develop the best possible mechanism. Once the mechanism has been determined, the stoichiometric determination of catalytically decomposed H2O2 can be shown. ROLE OF SABP2 IN THE INDUCTION OF SYSTEMIC ACQUIRED RESISTANCE IN RESPONSE TO BACTERIAL PATHOGENS IN PLANTS Cory N. Phillips, Kajal C. Patel, Raj C. Bant, Elizabeth P. Huntley and Dhirendra Kumar Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN Plants do not possess an adaptive immune system like animals; however, they can deploy various defenses, performed and induced, that are used to combat pathogen invasions. One compound that triggers these defenses against pathogens is a plant hormone called salicylic acid (SA) which is made in the chloroplast of plant cells via the shikimate pathway. SABP2 is a 29 kDa protein which binds to SA with very high affinity and is critical for both local and systemic acquired resistance. SABP2 converts the methyl salicylate (MeSA), synthesized by plants in response to pathogen infection into active SA, which then activates defense proteins. Recent studies identify methyl salicylate as the long distance phloem mobile signal required for onset of systemic acquired resistance (SAR). SABP2 is required in the uninfected systemic leaves to receive and process this SAR signal. All the host resistance response studies involving SABP2 have been conducted using viral tobacco pathogen, tobacco mosaic virus (TMV). The role of SABP2 in SAR induced responses to bacterial and fungal pathogens has not been investigated. Bacterial diseases cause great losses of agricultural crops, which are important for food security. Therefore, we are investigating the role of SABP2 in SAR and local resistance induced by virulent, avirulent, and non-host bacterial pathogens. These results will not only have biological importance, but also have major implications for farming in the future by reducing dependence on harmful pesticides and insecticides. They may even provide a break away from pesticides to a more sophisticated solution, such as enhancing plants’ own natural defenses. THE INCIDENCE AND PREVALENCE OF BATRACHOCHYTRIUM DENDROBATIDIS IN TEMPORARY WETLAND POPULATIONS OF AMBYSTOMA MACULATUM IN THE HOLSTON RIVER VALLEY Mehdi Pourmorteza and Tom Laughlin, Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN Chytridiomycosis is an emerging infectious disease affecting amphibians worldwide. It is caused by the chytrid fungus Batrachochytrium dendrobatidis, a fungus causing sporadic morbidity and mortality in amphibian populations. The fungus infects the surface layer of the frog’s skin, causing damage to the keratin layer. Many researchers believe that this infection inhibits the frog’s ability to respire and osmoregulate. To better understand the dynamics of the chytrid fungus, the incidence and prevalence of 2008 Appalachian Student Research Forum Page 21 Chytridiomycosis will be measured in salamanders in three different populations; one in Shady Valley where water is polluted with cattle waste, and the other two in the South Holston River floodplain and at Bays Mtn. Park where water conditions are relatively unpolluted. DNA from salamanders has been extracted from two different populations thus far using Qiagen DNeasy tissue kits. The isolated DNA will be used for PCR reactions using primers previously developed for this species. OSTEOCRANIAL DEVELOPMENT IN THE OVIPAROUS CORN SNAKE, PANTHEROPHIS GUTTATUS (SERPENTES: COLUBRIDAE) Elizabeth K. Price1, Rebecca A. Pyles1, James R. Stewart1 and Tom W. Ecay2 Dept. of Biological Sciences and 2Dept. of Physiology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 1 Study of osteocranial development in vertebrates serves as an important source of evidence for functional, systematic, evolutionary, and developmental questions. Osteocranial development has been studied extensively in fishes, amphibians, birds and mammals; however, such studies are particularly lacking for squamate reptiles---the lizards and snakes. Most previous studies of squamate skeletal development have been based on small sample sizes, have not reported variation, and have utilized embryo or postnatal size to compare specimens. Because size varies considerably among species and with incubation temperature, results of these studies are not comparable. This study was designed as a baseline study of osteocranial development in an egg-laying (oviparous) snake, Pantherophis guttatus, commonly called the corn snake. Data from this study are intended to provide a ‘developmental clock’ describing the uptake and use of calcium for ossification of the embryonic skeleton. Eighty embryos and young were identified to developmental stage utilizing a standard table (Zehr, 1962) and size of a skeletal element (Meckel’s cartilage, with or without investing bones; to nearest 0.05 mm) used as a relative measure of growth. Specimens were cleared and double-stained (Hanken & Wassersug, 1981; Taylor & van Dyke, 1985) to reveal both developing cartilage and bone in whole-mount. In the cranium, first evidence of ossification was a palatal bone (palatine, stage 29—31). During stage 32, ossification of the mandible (prearticular, surangular) and one jaw suspensory bone (pterygoid) proceed quickly, while the first upper jaw and skull roofing bones show only initial centers of ossification. Ossification of particular bones show variation across developmental stages, but a relatively consistent pattern can be identified. By stage 37, just prior to hatching, most bones of the cranium are ossified, except for the parietals and occipital complex of the skull roof, and the basisphenoid of the floor of the braincase. Interestingly, the earliest indication of calcium in the embryonic cranium appears as calcium-containing deposits sequestered in endolymphatic fluids within the inner ear (otic capsules). These deposits are present in the earliest developmental stages observed (stage 27), and well before ossification of any skeletal bone begins, and they persist through all later stages or until superficial ossification of the otic capsules obscure observations. Comparison of our results, based on our interpretation of comparable developmental stages, to ossification patterns reported for Python and Naja (cobra) indicate that substantial variation in ossification has been significantly underrepresented in previous studies. However, a generally consistent pattern of ossification is proposed in which palatal and jaw bones ossify early and braincase ossifies late. The presence of calcium in endolymphatic fluids very early suggests that mobilization of calcium to the embryo occurs well before skeletal ossification begins during development. Results of this study provide a model approach to enable direct comparison of skeletal development among different species. 2008 Appalachian Student Research Forum Page 22 THE INCIDENCE AND PREVALENCE OF RANAVIRUS IN TEMPORARY WETLAND POPULATIONS OF AMBYSTOMA MACULATUM IN THE HOLSTON RIVER VALLEY Joel Waddell and Tom Laughlin Dept. of Biology, East Tennessee State University, Johnson City, Tennessee Frog virus 4 (FV4), a ranavirus in the family Iridoviridae, is an infectious disease of recent concern because it has been associated with morbidity and mortality in wild and commercial amphibian populations on several continents. Recent evidence suggests that environmental degradation can impair amphibian immune systems. This project will investigate the incidence and prevalence of FV4 in three Ambystoma maculatum populations in the Holston River Valley. I will screen DNA samples for the presence of FV4 from approximately 100 individuals from each of three different populations in Sullivan County, Tennessee characterized by varying degrees of human impact. The extracted DNA will be screened using PCR to amplify a~500 base pair region of the major capsid protein of FV4. If FV4 in present, I expect to find a direct correlation between poor water quality and an increased incidence and prevalence of this ranavirus. MOLECULAR WIRES: CURRENT RESEARCH AND FUTURE APPLICATIONS J. Ryan McLain and Jeffery Wardeska Dept. of Chemistry, East Tennessee State University, Johnson City, TN Molecular wires are going to revolutionize technology in much the same way the microchip did in the 20th century. They will allow us to create molecular circuits, which are circuits consisting of individual molecules; this is significant due to the size ratio allowing for so many technological leaps. Currently the focus is on organic molecules, but this research is shifted towards the inorganic. It is based upon multinuclear mixed-valence iron complexes. Fe(CN)6 and FeHEDTA were used to create different ratio complexes which could be built upon to chain out and create a full molecular wire. FT-IR, UV-Vis, Cyclic Voltammetry, and Magnetic Moments were the analysis techniques employed in order to test the complexes. These showed that using different ratios when mixing the compounds, would in fact, lead to different ratios within the complexes. This technology is brings us a few steps closer to that of nano-scale electronic devices. PICTURE THIS! USING 2D IMAGES AS A MEANS OF MEASURING SKELETAL ELEMENTS Chris Swartz Dept. of Computer and Information Sciences, East Tennessee State University, Johnson City, TN For several reasons, having a method of measuring bones or other specimens without actually traveling to the collection is quite beneficial. For researchers this reduces or even eliminates travel costs, especially if one is traveling just to see one or two specimens. For curators this alleviates the loaning of even one or two valuable specimens. A database of images and measurements would benefit both of these situations and in the case of very large bulky items that are difficult to maneuver or small delicate items that should be handled as little as possible, it could eliminate the headache of handling these specimens all together for both parties. For my project, I was asked to develop a means of taking measurements from 2008 Appalachian Student Research Forum Page 23 photographs of skeletal elements. I used a digital camera to take the photographs and measured the specimens from the photos using SigmaScan Pro ™. These measurements were compared against actual physical measurements taken from the same specimens using calipers and an osteometric board. This was done to determine if the photographic measurements were a comparable means of data collection. I wanted a variety of skeletal elements to work with, so the skeletal data collection consists of extinct and extant mammals such as seals, sea lions, bears, whales, Eocene whales, lemurs, sloths, great apes, anteaters, walruses, and humans using data collected by my sponsoring professor and fellow students in her lab. These specimens were collected from the Smithsonian Institution: National Museum of Natural History. The current results of this project show that my measurements taken from a digital photo are acceptable within a 95% confidence range when compared to measurements taken manually from the actual bones. The major errors in measurement from digital photos come from improper placement of scales and the distance ratio of the element being measured to the camera. The beauty of this method is threefold in that 1) it works for the skeletal elements I was asked to originally make it work for; 2) it has the potential to work for more than just bony elements, such as pottery, pelts, or any materials that can be photographed as long as the guidelines are observed; and 3) despite the simplicity of this method, further study has shown a potential for 3D imagery and measurements from this method. This third aspect of my technique is part of my continuing research in this area. Social & Behavioral Sciences DRIVING IN APPALACHIA Breana Chappell, Paul Nease, Robin Leonard, Ben Martin, Dr. Chris Dula Dept. of Psychology, East Tennessee State University, Johnson City, TN. Appalachia has historically been referred to as a regionally unique area. Previous research has shown certain populations, especially low income populations, are at an increased risk for involvement in collisions. Additionally, research indicates that as personal income increases, individual risk for involvement in traffic collisions decreases (Keeler 2003). Some research attributes these decreases to ability to afford safer automobiles and increased traffic safety education. Little research has been conducted that focuses specifically on low income areas of the United States, such as Appalachia. Safety belts and their usage may have gone largely ignored by some populations of this area. Also, the number of lifetime crashes could be related to the non-usage of seatbelts by drivers in Appalachia. The authors hypothesize that people who identify themselves as ‘Appalachian’ are less likely to use safety belts while driving than those who do not identify themselves as being Appalachian. The authors additionally hypothesize that those who identify themselves as Appalachians would report having been involved in more traffic collisions than non-Appalachian participants. A survey was given using an online participant pool to a sample of college students at a regional southeastern Appalachian university. The survey was completed by 225 students, 156 females and 69 males. The participants mean age was 22.26 years with a standard deviation of 6.13 years. In the survey the participants were asked if they considered themselves to be Appalachian. Additionally using a 5 point Likert Scale, they were asked how often they used seatbelts, how many collisions they had been involved in during their entire life, whether or not they were the driver. A Chi-Square Test was conducted to ascertain any relationship between seat belt usage and Appalachian self-identification. The analysis showed a relationship but failed to reach significance, p= .059. An independent t-test was conducted to determine what relationship, if any, exists between lifetime crashes and Appalachian self-identification. This relationship was found to be statistically significant, t= 2.499, p= .013. Although not statistically significant, a relationship was determined to exist between seat belt usage and Appalachian self-identification. 2008 Appalachian Student Research Forum Page 24 PERCEPTIONS OF TATTOING ON ONESELF AND OTHERS Matthew A. Dorton, Benjamin A. Martin., Sonia L. Coney, Chris S. Dula Dept. of Psychology, East Tennessee State University, Johnson City, TN In social psychology there are many terms to incorporate the aspects of the self and it’s interactions with society. Understanding of the concepts regarding self-awareness, social self, and the executive self is an essential part to the correct perceptions of the self’s identity within its society. Previous research has found prevalence of tattooed individuals within subpopulations (Edgerton & Dingman, 1963) and stereotypes about personalities as deviant (Howell, Payne, & Roe, 1971). Discovering the impact that tattoos have on society may be understood through the analysis of social comparisons, intrinsic and extrinsic motivations, self-esteem, self-presentation, action identification; as the connection between principles and actions, self-regulation, and self-defeating behavior (Baumeister &Newman; 1994; Deci, 1971; Ferrari, 2001; Festinger, 1954; Jones & Berglas, 1978; Locke & Kristof, 1996; Shoda, Mischel, &. Peake, 1990; Tangney, Baumeister, & Boone, 2004; Vallacher & Wegner, 1985, 1987). Previous literature has made some attempts at categorizing the reasons for getting a tattoo (Garcia, 1987). However, existing categories lacked a complete definition. Data was collected from two open-ended demographic questions: “why did you get a tattoo” and “why do you think others get a tattoo.” The positive reasons and themes are 1. Religious/Beliefs (Garcia, 1987) 2. Remembrance/Commemoration/ Sentimental (Bezaury & Moron, 1983; Garcia, 1987) 3. Art Collection/Decorative/Aesthetic (Garcia, 1987) 4. Social group identification/Popularity (Garcia, 1987; Roberts, Koch, & Armstrong, 2006) and 5. Self-Statements – including: Self-Knowledge/Concepts, and Self-Agency (Executive Decisions) (Bezaury et. al., 1983; Millner & Eichold, 2001; Rubio, 2003). For the negative reasons and themes the categories include: 6. Military/Gang/Prison/Sexuality (Bezaury et. al., 1983; Garcia, 1987) 7. Aggression/AntiSocial/ Death or “Dark” themes/Pain enjoyment (Bezaury et. al., 1983) and 8. Impulsivity/Alcohol Related Reasons/Peer-Pressure (Roberts et. al., 2006). Through the use of the theory of symbolic interactionism which describes human relations through language and symbols which have acquired a conventional significance within the individual’s society; predicts that tattoos in society will continue to become more acceptable and widespread as their fashion and popularity continue to grow (Atkinson 2003; Irwin, 2001; and Pitts, 2003). Participants were 210 undergraduate students aging from 18 to 62 years of age (142 women, 68 men, M=20.5, SD=4.99) completed the survey for a modest amount of extra credit. A cross- tabulation was performed to detect differences in answers between the categories and a chi-square analyzed whether or not having tattoos affected the answers to the demographic questions. Results 3.9% were identified with impulsivity, aggression, & pain enjoyment as their reason for getting a tattoo. 96.1% identified with various positive reasons such as: religious beliefs, remembrance/art, aesthetic, popularity, self statement & other executive decisions. 5.2% participants speculated that people get tattoos for negative reasons. 94.8% of participants speculated that people get tattoos for positive reasons. Results from this study may provide strength to the theory that, instead of an impulsive behavior, tattooed individuals indeed make conscious decisions regarding their tattoos. Future research should focus on possible relationships between theses results, knowledge of tattoos, and stigma against tattoos. 2008 Appalachian Student Research Forum Page 25 THE ROLE OF MODELING IN RELATION TO SEQUENTIAL TOUCHING James Anthony Garland Dept. of Psychology, East Tennessee State University, Johnson City, TN The sequential touching task is a popular nonlinguistic procedure used to tap into infant and toddler’s categorical understanding. Although children’s performance on the task demonstrates convergent validity with linguistic measures of categorical understanding, the task also suffers from low reliability. Although not usually documented, users of the sequential touching procedure are well aware of the difficulty involved in scoring infants touching behaviors. The purpose of this study is to investigate whether modeling targeted actions within the sequential touching procedure would result in a more reliable assessment of infant and toddler categorical understanding. It is suggested that a simple modification of the procedure would result in the ability to magnify the distinction between categorizing and noncategorizing infants. Participants include 27 infants with a mean age of 15 months. Specifically, it is expected that children who are explicitly shown distinctions between two categories presented in a standard sequential touching task, will be better able to demonstrate their categorical understanding as compared to their initial baseline performance and as compared to their peers in a non-modeling control group. IMPACT OF DRUGS AND ALCOHOL ON MANNER OF DEATH BY SEX AND AGE AMONG AUTOPSY CASES PERFORMED AT THE UPPER EAST TENNESSEE FORENSIC CENTER IN 2007. Caleb Harrell1, Paul Benson2, Teresa Campbell2, Emily Lemieux1, David Lisenby2, Laura Parsons2, Kathy Bryant1, Debbie Bailey1, Kim Hughes1, and Kenneth Ferslew1 1 Toxicology and 2Forensic Pathology , Upper East Tennessee Forensic Center, East Tennessee State University, Johnson City, Tennessee. The Upper East Tennessee Forensic Center performs autopsies on the questionable and medicolegal deaths which occur in the eight counties of the First Tennessee Development District. Toxicological evaluations of specimens collected at autopsy are used to determine if drugs and/or alcohol are involved in determining the cause and manner of death. A descriptive database was established defining all parameters and data pertinent in each case (age, sex, cause/manner of death, and toxicological results). The purpose of our research was to determine descriptive statistics on the impact of drugs and/or alcohol by manner/cause of death, age, and sex in the autopsies performed in 2007. Specimens (blood, gastric contents, urine, vitreous humor, and bile) from the autopsies were analyzed for drugs and alcohol using multiple analytical toxicological procedures (colorimetric, TLC, immunochemistry, GC, GCMS, and LCMS). Toxicological results were compiled in an electronic database (Microsoft Access) to allow for analysis and interpretation. Results indicate that out of 277 total cases 66% were male, 34% were female, 85% were positive for drugs, 27% were positive for alcohol, 23% were positive for both drugs and alcohol, and 12% had neither drugs nor alcohol. Analysis of the distribution of cases positive for drugs, alcohol, and drugs/alcohol revealed that males had a greater percentage of cases involving alcohol alone as well as cases positive for drugs/alcohol than females. Acute drug overdoses accounted for 34% of total cases with no substantial sexual differentiation. Of the 94 acute overdose cases, 4 (≈4%) were intentional (suicides) and 90 (≈96%) were accidental. Autopsies were performed on all age groups (percentage of cases/ years of age): 5% <14, 4% between 15 and 19, 8% between 20 and 24, 14% between 25 and 34, 22% between 35 and 44, 16% between 45 and 49, 13% between 50 and 54, 10% between 55 and 64, 6% between 65 and 74, and 2% ≥75 years of age. The distribution of positive drug 2008 Appalachian Student Research Forum Page 26 cases closely mirrored the distribution of cases by age groups. Manner of death analysis revealed (of total cases) that 47% were accidental, 27% were natural, 15% were suicides 6% were homicides, and 5% were undetermined. No appreciable disparity in distribution of manner of death was found between the sexes. Analysis of the results of the toxicological evaluations revealed there were a large number of cases in which opiates (100), alcohol (75), benzodiazepines (110), sedatives (18), and/or stimulants (14) were identified. Review of these results leads to the conclusion that drugs and alcohol have a significant impact in the questionable and medicolegal deaths occurring in Upper East Tennessee. CELL PHONE USAGE: CHANGING THE DYNAMIC OF INTERPERSONAL SPACE Kelley Marie Hatch Dept. of Communication, East Tennessee State University, Johnson City, TN Newspaper and magazine article headlines are the harbingers of public sentiment: “Cell Hell,” “Cell Phone Follies” and “A Call for Cell Phone Etiquette” are a sampling of recent news items expressing public outrage over the misuse of cell phones. Cell phone usage in public has struck a dissonant chord. Most people have a cell phone horror story: a romantic anniversary dinner interrupted by an inconsiderate cell phone user, overhearing someone’s embarrassing medical problem etc. The purpose of this research is to ascertain the impact of cell phone usage on the dynamics of interpersonal space. For this study, “interpersonal space” is defined as the spatial relations between individuals in terms of what is public space versus what is private space. The portability and accessibility of cell phones changed perceptions of what was once considered public space into private space for the intent of individual conversations. No longer does it appear that individuals feel the need to take a phone conversation away from other people in order to maintain a semblance of privacy. This begs the question, what has occurred in society as catalyst for this change? At a time when our human experience is inundated with technology: laptop computers, Mp3 players, DVDs, GPs, electronic billboards, talking cars – fast paced, fast moving machinery --- our desire for human contact is greater than ever. We are for the first time in a position to need to daily negotiate our humanness amongst technology. Cell phone technology helps by bridging the divide between technology we must live with, and human contact which we cannot live without. As we are renegotiating space and technology, the research question then becomes, has cell phone usage changed what we talk about in public spaces erasing lines between public and private? To test this research question 400 respondents from 22 states and with an average age of 33 years, found that people were always aware of their surroundings when placing a cell phone call, and reported being comfortable placing a call in a public place. Respondents were asked to divulge not only the topics of their conversations in public space, but also topics overheard in a public space, and specifically any found to be shocking. Survey results in conjunction with existing literature justify the conclusion that individuals making or answering a cell phone call in a public space retreat into a personal cell cocoon or PCC, an oblivion to their immediate surroundings. 2008 Appalachian Student Research Forum Page 27 DEPRESSION AND ALCOHOL ABUSE: ETHNIC DIFFERENCES IN THE MODERATING ROLE OF HOPE AND HOPELESSNESS Angela Jones, Jameson Hirsch, Preston Visser, Dept. of Psychology, East Tennessee State University, Johnson City, TN Elizabeth Jeglic, John Jay, College of Criminal Justice, Dept. of Psychology, New York, NY Depression is often complicated by the co-occurrence of alcohol abuse and dependence; nearly one-third of individuals with major depressive disorder also have a substance abuse disorder. Ethnic differences in comorbidity exist. Whites have a higher rate of alcohol abuse than Blacks, Hispanics, and Asians; however, the rates of alcohol abuse have risen sharply for adolescent Blacks and Hispanics. Although the relationship between depression and alcohol abuse is bi-directional, a cognitive framework suggests that depressive symptoms may lead to alcohol use as a maladaptive coping strategy. Not all individuals who are depressed, however, resort to abuse of alcohol, utilizing more adaptive means of problem resolution instead. Such differentiation in problem-solving may be the result of an ability (hope) or inability (hopelessness) to identify future oriented goals and conceptualize pathways toward goal achievement. Hopeful individuals may be able to find alternative solutions to stressful situations, whereas hopeless individuals may be unable to do so, possibly resorting to abuse of alcohol instead. We examined the moderating role of trait hope and hopelessness on the relationship between symptoms of depression and severity of alcohol abuse, in an ethnically diverse sample. We recruited an ethnically diverse sample (Black, 25%; Hispanic, 41%; Caucasian, 18%; Asian, 6%) of 385 college students (69% female), with a mean age of 19.61 years old (SD=3.12), from an urban, Eastern university. Participants provided written, informed consent and completed measures of depressive symptoms, trait hope, hopelessness, and a screener for alcohol abuse and dependence. Multivariate, hierarchical regressions and moderator analyses were conducted, statistically controlling for age and gender. Initial models assessed moderator status of trait hope; secondary models also examined hopelessness as a moderator. Analyses were conducted for the entire sample and for ethnically stratified groups. With only trait hope included in the model, hope moderated the association between depressive symptoms and severity of alcohol abuse for the entire sample, suggesting that, in the context of depression, individuals who are more hopeful report less severe symptoms of alcohol abuse. In ethnically stratified analyses, trait hope neared significance as a moderator only for Whites (p=.06). With hopelessness included in the model, neither was significant for the overall sample. In stratified analyses, hopelessness was a moderator for Hispanics (p<.05) and neared significance for Blacks (p=.08), indicating that depressed individuals who are also hopeless may manifest more severe alcohol abuse, whereas trait hope was a moderator for Whites only (p<.05). Increased abuse of alcohol in the context of depression is not inevitable; indeed, this relationship appears dependent, in part, on one’s outlook toward the future. Ethnic differences suggest that sociocultural characteristics may influence the moderating effect of hope and hopelessness, and that these constructs may not be operationally equitable across ethnicities. Limitations include unknown generalizability to other recruitment settings and demographic subgroups, and the potential for biased self report of psychiatric symptoms. Our findings highlight the need for more comprehensive, future research, and suggest that strategies designed to help young adults identify meaningful and attainable personal goals could be an important component of substance abuse programs. 2008 Appalachian Student Research Forum Page 28 DEVELOPMENTAL HISTORY OF INDOOR TANNING IN HIGH SCHOOL FEMALES Leslie King and Joel Hillhouse, Ph.D. Dept. of Public Health, East Tennessee State University, Johnson City, TN There are over a million new cases of skin cancer diagnosed annually in the US with the incidence of melanoma, the most lethal form of skin cancer, rising faster than any other cancer. It is important to develop effective interventions that can possibly reduce the future occurrence of and mortality from melanoma and non-melanoma skin cancers. High school represents an ideal focus for intervention implementation. There is evidence that high school indoor tanning initiation is associated with later longterm habitual indoor tanning behavioral patterns which are highly resistant to change. This research study is using focus groups to look at personal, social and family factors related to initiation and maintenance of indoor tanning behavior in high school students. Efficacious health promotion interventions depend on strong theoretical modeling to be successful. The focus group information collected in this study is the typical first step in developing these essential models. This research could lead to more effective interventions to substantially reduce the future occurrence of melanoma and non-melanoma skin cancers in this population. While the relationship of modern tanning devices is still under investigation, a number of case-controlled studies have reported significant positive associations between indoor tanning and melanoma morbidity with increased risk associated with early and regular use. A recent report out of the International Agency for Research on Cancer concluded that exposure to sunbeds before 35 years significantly increases the risk of melanoma and squamous cell carcinoma. In spite of these facts, indoor tanning is gaining in popularity, especially among the youth. The research study is being carried out at ETSU. It will include 40 high school females between the ages of 14 and 16 years old. Eight to ten students will meet with a facilitator in a private group setting. The discussions will last about 50 minutes per group. Each student will be asked questions relating to their personal beliefs and attitudes regarding indoor tanning (e.g. perceived susceptibility to skin aging, tanning alternatives attitudes, etc.). They will also be guided to elaborate on factors contributing to tanning initiation and personal tanning history (e.g. special events, social influence, etc.). The participants will also be asked to review a brochure designed for a previous American Cancer Society grant in hopes of a possible adaptation that this age group can better identify with. The resulting data will be subjected to qualitative analyses to identify key and emerging themes of indoor tanning initiation, maintenance as well as reasons for quitting. This data will be the first step in developing conceptual models of indoor tanning decisions in high school student populations. Review of the brochure may warrant further revision and have the potential to be implemented in future interventions. There is currently no published research addressing the indoor tanning related decision-making in this age group. The models that emerge from the focus group results will lead to more thorough assessment and intervention approaches toward high school indoor tanning. This will ultimately lead to substantially reducing indoor tanning occurrences. NICOTINE-CONDITIONED HYPERACTIVITY IN ADOLESCENT MALE AND FEMALE D2-PRIMED RATS. Lehmann J, Sheppard AB, Amine L, Brown RW. Dept. of Psychology, East Tennessee State University, Johnson City, TN The aim of this study was to determine the ability of a nicotine-conditioned context to elicit locomotor hyperactivity in a neonatal quinpirole animal model of psychosis, and whether this conditioned hyperactivity could be blocked by the D2 antagonist eticlopride. Sprague-Dawley rats were treated with 2008 Appalachian Student Research Forum Page 29 either saline or the dopamine D2 receptor agonist quinpirole from postnatal days (P) 1-21 to create priming of the dopamine D2receptor, a phenomenon that we have shown persists throughout the animal's lifetime. Beginning on P33, animals were injected i.p. with either nicotine (0.5 mg/kg), the D2 antagonist eticlopride followed by nicotine, or saline and placed into the arena 10 min after injection every other day through P49. A non-paired group was included. Results showed that, D2 priming blocked the typical initial hypoactivity produced by nicotine, and these animals also sensitized to nicotine more rapidly than controls. Eticlopride blocked sensitization to nicotine in both D2-primed and non D2-primed rats. On P50, a portion of these animals were administered a drug-free test in which rats were given saline before being placed into the locomotor arena. Interestingly, control animals administered nicotine demonstrated conditioned hyperactivity on the drug free test that was blocked by eticlopride, but eticlopride did not affect nicotine-conditioned hyperactivity in D2-primed rats. This result indicates priming of the D2 receptor was able to overcome D2 receptor blockade in adolescent rats. Further studies are analyzing the role of nicotinic receptors in this phenomenon, as collaborators have shown that D2 priming produces alpha7 nicotinic receptor upregulation. DURATION OF BREASTFEEDING PREDICTS DIMENSIONS OF INFANT TEMPERAMENT Kellye L. Lingerfelt and Dr. Andrea Clements Dept. of Psychology, East Tennessee State University, Johnson City, TN The benefits of breastfeeding and the duration of breastfeeding required to achieve those benefits have long been debated. The American Association of Pediatrics (AAP) has noted benefits to the infant in such areas as increased immunity to infectious disease, improved neurodevelopment, reduced rates of infant mortality, as well possible benefits much later in life included reduced risk of obesity and diabetes. Other studies have suggested cognitive benefits of breastfeeding. As of 2001, the reported rate of the initiation of any breastfeeding at all in the US was 70% (46% breastfeeding exclusively—no formula supplementation), with 33% (17% exclusively) still breastfeeding at 6 months. The purpose of this study was to evaluate temperament differences among children who differed in breastfeeding history. To our knowledge no one to date has investigated the relationship between breastfeeding and dimensions of infant temperament. The data were collected through two studies being conducting by the Program for the Study of Infancy at East Tennessee State University. One is a study in which mother-infant pairs are brought into the lab for a one hour temperament, language, and mother-infant interaction procedure, and the other is an ongoing collection of temperament, language, and child history from a university laboratory school childcare program. The participants in these studies included 94 rural Appalachian children (83% White). When participants are first enrolled in these studies, parents are asked to fill out a Prenatal Birth Stress Inventory which included pregnancy and birth history variables and a temperament measure (Infant Behavior Questionnaire [IBQ], Early Childhood Behavior Questionnaire [ECBQ], or Child Behavior Questionnaire [CBQ]). In our study, 71.6% of mothers reported breastfeeding for 3 months or more and 46.1% reported continuing to breastfeed for 6 months or more. We did not ask about exclusive versus supplemented breastfeeding. After statistical analysis, it was found that several dimensions of temperament differed in relation to duration of reported breastfeeding. Focused attention was unexpectedly lower in children who were breastfed for six months or more (n = 31; mean=3.69, SD=.88) than in children that were breastfed six months or less (n = 32; mean=4.56, SD=.85; F(1,61)=6.78, p=.012). Expected differences were found in other temperament dimensions. Frustration (F(1,53)=6.68, p = .013) and sadness (F(1,61) = 8.97, p = .004) were found to be significantly lower in infants that were breastfed six months or more. The ability to be soothed was rated significantly higher (F(1,61) = 5.03, p = .029) for the children who were breastfed six months or more than for children breastfed less than six months. Differences were found for the same variables in the same directions when comparing ratings of children breastfed 3 months or more compared to children breastfed less than three months. 2008 Appalachian Student Research Forum Page 30 A LOOK AT THE DEVELOPMENT OF THE MARTIN STIGMA AGAINST TATTOOS MEASURE Benjamin A. Martin, Paul Nease, Heidi E. Mason, Ashley D. Dickson, and Christopher S. Dula Dept. of Psychology, East Tennessee State University, Johnson City, TN, Tattoos are becoming very popular in today's society, but empirical research lacks a way to measure the stigma that is held against tattooed individuals. Although previous studies have documented and discussed views of the tattooed and non-tattooed, little is known about the subject. This prior research, however useful it may be, primarily consists of qualitative data collection methods (e.g. quasi-interview methods), subtracting from the overall objectivity and generalizability of the results. In order to better analyze stigma against tattooed individuals, an instrument was developed to measure this phenomena in a quantitative fashion. The current study details the development of the Martin Stigma Against Tattoos Survey Measure (MSATS) to measure the stigma against those with tattoos. Undergraduates (n=241,175 women & 66 men) were administered a set of surveys via an online survey system called Sona Systems to assess social stigma against tattoos. Psychometric analysis was performed and stigma was calculated by summing the responses. In addition to strong face-validity, extraction of one higher-order factor provided evidence of a single overall construct. Exploratory factor analysis exhibited one factor, interpreted as social stigma against tattoos. Confirmatory factor analysis supported the structure of the scale and suggested coefficient reliability. Cronbach's alpha was .89 for the original 17-item instrument and was .92 for the modified version, which provided evidence of internal consistency reliability. The MSATS was found to be reliable and valid with a college age sample of individuals with and without tattoos. The Martin Stigma Against Tattoos Survey Measure will help reduce negative stigmatizing behavior against those with tattoos and allow researchers the opportunity to examine stigma against tattooing in various environments or situations. The MSATS could be useful in helping to both identify and reduce inappropriate stigmatizing behavior. This scale could be applied in future research to gauge whether a stereotype placed on tattooed employees by biased employers has any merit. This may be helpful in breaking current actual and perceived employment barriers and allow previously stigmatized persons to function at a higher occupational level. Another potential benefit of the MSATS could be to assess managerial candidates within a company to help screen for unnecessary bias within a company toward tattooed employees. NICOTINE SENSITIZATION IN ADOLESCENT BETA ARRESTIN-2 KNOCKOUT MICE: CORRELATIONS WITH BDNF. Noel DM1, Correll JA1, Thompson KN2, Longacre ID,1 Yin D3, Brown RW1. Dept. of Psychology , East Tennessee State University Johnson City, TN 2 Dept. of Anatomy and Cell Biology, Quillen College of Medicine, East Tennessee State University Johnson City, TN 3 Dept. of Internal Medicine, Quillen College of Medicine, East Tennessee State University Johnson City, TN 1 Beta Arrestin-2 is a protein that regulates hydrolysis of the G-protein and is co-localized with the dopamine D2 receptor. In this study, 3-4 week old adolescent BA-2 KO and wild type C57/B6 mice were administered either nicotine tartarate (s.c, 0.5 mg/kg) or saline 10 min before being placed into the locomotor arena on each of seven (Experiment 1) or 14 (Experiment 2) consecutive days. A drug-free abstention period of seven days followed nicotine sensitization in each experiment, at the end of which animals received a nicotine challenge (0.5 mg/kg free base). Experiment 1 results showed that BA-2 KO 2008 Appalachian Student Research Forum Page 31 mice were slightly hypoactive and did not demonstrate sensitization by day 7, whereas wild type controls did not demonstrate an increase in activity as compared to saline-treated wild types by day 7, but did show an increase in activity over the 7 days of nicotine treatment. On the nicotine challenge, BA2 KO blocked expression of nicotine sensitization. In Experiment 2, BA-2 KO mice demonstrated sensitization although not the levels of the controls. On the challenge, beta arrestin-2 again blocked expression of sensitization. Brain tissue was taken in both experiments for analysis of Brain-derived neurotrophic factor (BDNF) in the nucleus accumbens, and showed that nicotine produced a significant increase in BDNF which was blocked by the knockout of beta arrestin-2. These results show that beta arrestin-2 plays a more important role in expression as compared to induction of nicotine sensitization, and blocks nicotineinduced increases in BDNF in the nucleus accumbens. RELIGIOSITY, SPIRITUALITY, FUNDAMENTALISM, AND ACTIVELY CARING BEHAVIORS Robert B. Russell & Chris S. Dula. Dept. of Psychology, East Tennessee State University, Johnson City, TN Current research on the helping behaviors exhibited religious individuals is inconclusive. While religious individuals overwhelmingly identify themselves as frequently committing altruistic acts (Mattis et al., 2000), few researchers have found religion to be a mediating factor on helping behaviors in controlled settings (Jackson & Esses, 1997). Religious fundamentalism--the degree to which one believes their religion to be unquestionably true--has ironically been shown to negatively correlate with caring behaviors, particularly those outside one’s own religious group (Jacson & Esses, 1997). Spirituality--a feeling of purpose, connection to others, and one’s general interest in existential queries (Hodge & McGrew, 2006), however, has been consistently found to positively correlate with both self-reported and verifiable altruistic acts (e.g. Greenwood & Harder, 2003). Geller (1991) theorized that several factors play into one’s propensity to ‘actively care’ for others, including high self-esteem, a sense of belongingness, high self-efficacy, an optimistic attitude, and a sense of self-control. The purpose of this study was to determine if an individual’s level of spirituality, religiosity, and religious fundamentalism relate to those factors which influence actively caring. It was hypothesized that spirituality would show a positive relationship with the actively caring factors; also, religious fundamentalism was expected to negatively correlate with these factors. A total of 204 undergraduate psychology students (61 Men, 143 Women) ranging in age from 18 to 55 years of age (Median = 20) completed the Inventory of Religiousness (IoR; Rayburn, 1997), Religious Fundamentalism Scale (RFS; Altemeyer & Hunsberber, 1992), Inventory of Spirituality (IoS, Rayburn & Richmond, 1999), and the Actively Caring Survey (ACS; Geller, 1995) as measures of religiosity, religious fundamentalism, spirituality, and actively caring behaviors, respectively. Because self-reported altruistic behavior differed from those observed in experimental settings, the Marlowe-Crowne Social Desirability Scale (MCSDS; Crowne & Marlowe, 1960) was included as a measure of desirable responding bias. Data was collected anonymously by use of an online survey administration software, and all participants received a modest amount of extra credit as compensation for their time. Data analysis consisted of computing correlation coefficients among the IoR, RFS, IoS, and all subscales of the ACS, while the MSCDM was held as a constant to lessen the effect of social desirable responding on the results. Because the number of correlations conducted was significantly large, the Bonferroni method was used to control for Type I errors, requiring a p value of less than .0023 (.05/21) for significance. Seven of the 21 correlations were significant at this level: Spirituality was positively correlated with the Actively Caring, Self Efficacy, Optimism, Belonging, and Personal Control subscales of the ACS, along with the total ACS score (.54, .42, .35, .33, .34, & .48, respectively), supporting the first hypothesis. Religious fundamentalism was found to negatively correlate with only the ACS self-esteem subscale (-.29); as such, the second hypothesis was not strongly supported. These findings may indicate that one’s level of spirituality is more directly related with altruistic and helping 2008 Appalachian Student Research Forum Page 32 behaviors than religious affiliation. Limitations and suggested directions for future research will be discussed. AMBIVALENT SEXISM AND SEX ROLE IDENTITY AS FACTORS IN SEXIST HUMOR APPRECIATION Jennifer Snyder and Dr. Peggy Cantrell Dept. of Psychology, East Tennessee State University, Johnson City, TN Overtly prejudiced remarks in public are often shunned in modern society; however, the reactions to and appropriateness of humor involving potentially sensitive topics, including gender roles and stereotypes of women, remain mixed and controversial. The possible relationships, if any, among sexist humor and other variables as well as any potential consequences of the presence of such humor in public or private domains have been under empirical investigation within psychology. The current study examined ambivalent sexism, gender, and gender role identity in a college sample (N = 254) for any possible relationships with sexist humor against women. Funniness ratings and repeating behaviors of 10 sexist internet jokes were compared. Results show that men were more likely to rate sexist humor as funny and more likely to repeat sexist humor than women. Ambivalent sexists significantly rated sexist humor as funnier and were more likely to repeat sexist humor than benevolent sexists and non-sexists; no significant differences were found between hostile sexists and any other sexism category. No significant differences were observed among gender role identity classifications. This study provides additional evidence for the complementary nature of hostile and benevolent sexism in the justification of prejudice. Results are discussed in light of Prejudiced Norm Theory (Ford, 2004). CORRELATIONS BETWEEN THE BIG FIVE PERSONALITY TRAITS AND SUSCEPTIBILITY TO SOCIAL INFLUENCE Lolita D. Turner, Robert B. Russell, & Chris S. Dula Dept. of Psychology, East Tennessee State University, Johnson City, TN Social influence can be defined as influence exerted by an outside person or group(s) to alter an individual’s behavior or actions. This influence can be exerted both subtlety and overtly, and plays a role in everyday interpersonal interactions. Susceptibility to this social influence and its varying forms has been shown to remain stable over the course of an individual’s life (Dula, Lehman, & Geller, under review), and may be related to numerous other facets of personality; however, no research has been conducted which examines a possible relationship between this susceptibility and the Big Five traits of personality. The aim of this study was to examine this possible relationship. Because of findings in the current literature, it was hypothesized that an individual’s level of Conscientiousness, Agreeableness, and Extraversion would positively correlate with susceptibility to social influence. A total of 204 undergraduate psychology students (61 Men, 143 Women) ranging in age from 18 to 55 years of age (Median = 20) completed the NEO short-form (NEO-FFI; Costa Jr. & McCrae, 1992) and the Social Influence Scale (SIS; Dula, Lehman, & Geller, under review) as self-reported measures of Big Five personality characteristics and susceptibility to six types of social influence, respectively. Data was collected anonymously by use of an online survey administration software, and all participants received a modest amount of extra credit as compensation for their time. Data analysis consisted of computing correlation coefficients among all subscales of the NEO-FFI and the SIS. Because the number of correlations conducted was significantly large, the Bonferroni method was used to control for Type I errors, requiring a p value of less than .0017 (.05/35) for significance. Eleven of the 35 computed 2008 Appalachian Student Research Forum Page 33 correlations were significant at this level: Extraversion was positively related with Reciprocity, Ingratiation, Scarcity, and Total SIS scores (.37, .28, .39, & .44, respectively), Openness and Scarcity were positively correlated (.30), Agreeableness correlated positively with Reciprocity, Ingratiation, and Total SIS Score (.35, .26, & .26, respectively), and Conscientiousness was significantly positively related to Reciprocity, Ingratiation, and Total SIS score (.33, .36, & .32, respectively). All three major hypotheses were confirmed, suggesting that susceptibility to social influence effects/is effected by an individual’s level of Extraversion, Agreeableness, and Conscientiousness. Unfortunately, no directionality can be derived from the current results: As the study consisted of correlational statistical procures, it is unknown how personality and social influence interact with one-another. While personality differences may moderate one’s level of social-influence susceptibility, susceptibility to social influence may contribute to defining one’s personality. Because so little research has been conducted on the topic, more research is needed before any definitive conclusions are reached. Limitations and directions of future research will be discussed. ADVENTUROUSNESS AND IMPULSIVE BEHAVIOR: IS THERE A RELATIONSHIP? Benjamin Martin, Audrey Watkins, Jessica Williamson, Robin Leonard, Chris Dula Dept. of Psychology, East Tennessee State University, Johnson City, TN The amount of adventure that a person seeks is often an indicator of how that individual views risk taking behaviors. As stated by Zuckerman and Neeb in 1980, high sensation seekers are likely to participate in risky activities such as driving fast and recklessly or acquiring body-art and/or engaging in body modification. In fact, tattooed persons are considered to regularly engage in behavior that is risky, immoral, non-conforming, and/or unstable (Forbes, 2001). When these behaviors were examined in non confined populations, Forbes found that tattooed individuals conform less to social expectations and perform risk-taking behaviors (2001). Most participants reported that they had thought about their tattoos for at least a month before going to get it (Forbes, 2001). The evidence provided by Zuckerman and Neeb suggests a possible positive correlation between an individual’s level of adventure/sensation seeking and prominence of impulsivity. According to Moore and Rosenthal in 1993 there is a strong correlation between adventure/sensation seeking and impulsivity. The purpose of this study is to assess whether there is a relationship between adventurousness and impulsivity within tattooed and non-tattooed populations. It is hypothesized that individuals that seek adventure will have higher levels of impulsivity. It is also hypothesized that individuals with tattoos will be more adventurous and impulsive than individuals with tattoos. Participants were 175 undergraduate students (55 men and 120 women). Their ages ranged from 18 to 62 years of age (M = 20.5, SD = 4.99). Participants completed survey through Sona Systems, an online participant pool at a regional southeastern university. Extra credit was awarded for participation. Barratt Impulsivity Scale (BIS). Demographic Questionnaire including questions about the number of tattoos an individual has, if any. It is hypothesized that individuals that seek adventure will have higher levels of impulsivity. It is also hypothesized that individuals with tattoos will be more adventurous and impulsive than individuals with tattoos. Results did not suggest impulsivity varies between tattooed and non-tattooed populations. Thus it may be that tattooed individuals are unfairly believed to be highly impulsive. Tattooing is viewed by many as an impulsive and irresponsible behavior (Eksteen & Jankowski, 2006) 2008 Appalachian Student Research Forum Page 34 AWARENESS OF RACISM AMONG SELF-IDENTIFIED NEGROS AND MULATOS IN ECUADOR Meagan Stark, Kenneth Mijeski, and Scott Beck Depts. of Political Science and Sociology & Anthropology, College of Arts and Sciences, East Tennessee State University, Johnson City, TN In the fall of 2004 an interesting and unique survey of over 8,000 adult Ecuadorians was conducted in regard to racism and related topics. The interview items were developed by a team at SIISE, a semigovernmental research organization, and were attached to the employment and income survey conducted by the Ecuadorian Census Bureau (INEC). In our research we use a number of measures from this survey that focus on awareness of, and sensitivity to, issues of racism, prejudice and discrimination in Ecuador. In Ecuador there are four major culturally defined ethnic groups: White, Mestizo (mixed with Spanish and Indigenous ethnicity), Indigenous, and Afro- Ecuadorian. Survey items dealing with the respondents’ ethnic self-identification allow us to compare and contrast the level of racial awareness across ethnic groups. In Ecuador, and much of Latin America, those of African decent are often classified as either Negro (black) or Mulato (mixed). This study is particularly interested in measuring the degree of racial awareness, or the awareness of discrimination and stereotypical racist attitudes, among those who selfidentified as Negros and Mulatos. Other independent variables of interest are the level of education attained and the region in which the respondent resides, as well as whether they live in an urban or rural area. Also, by utilizing 2001 Census data on the racial composition of the parroquia (parish) in which the respondents reside, we attempt to determine whether those living in more racially heterogeneous areas are more aware of prejudice and discrimination. The results will be discussed. 2008 Appalachian Student Research Forum Page 35 Division II – Graduate Students 1-2 Years Biomedical Sciences DETERMINING SECONDARY PRODUCT GLUCOSYLTRANSFERASE EXPRESSION DURING Citrus paradisi GROWTH AND DEVELOPMENT Jala Daniel and Cecilia McIntosh Depts. of Biological Sciences and Physiology, East Tennessee State University, Johnson City, TN Flavonoids are secondary metabolites that are not involved directly in cellular respiration and photosynthesis but have significant roles in plant defense and human nutrition. Glucosyltransferases (GT) transfer sugars from high energy sugar donors to other substrates. More than 5000 flavonoids result from this type of reaction and other modifications. Several different kinds of flavonoid glucosyltransferases exist in the tissues of grapefruit, making it a model plant for studying their structure and function. The focus of the McIntosh laboratory is to understand how flavonoid GT’s influence metabolism as well as elucidating structure and function. This has led to the isolation of 8 putative flavonoid GT clones. One GT bears 98% homology to a limonoid (terpenoid secondary metabolite) GT and another has been found to have flavonoid GT activity. The roles of the other six are currently being studied by other laboratory members. The goal of this investigation is to determine the expression patterns of the 8 putative GT’s during the grapefruit growth and development by quantifying mRNA expression levels in the roots, stems, leaves. This research is designed to test the hypothesis that these 8 GT’s are expressed constitutively. Alternatively, one or more could be expressed in a tissue-specific manner or developmentally regulated. Five growth stages have been defined. The first stage focuses on emerging roots; stages 2-4 focuses on leaves, stems, and roots; and stage 5 focuses on comparison of older and younger leaves. RNA was isolated using a Qiagen RNeasy Plant Mini Kit. Unique primers for each putative GT have been designed that are at least 20 nucleotides in length with similar annealing temperatures. RT-PCR will be used to semi-quantitate mRNA expression. This will include determining the linear range of amplification, so that all of the 8 GT’s can be compared in a semi-quantitative analysis. RNA will be reversed transcribed using oligo dT primers and the cDNA will be amplified with gene specific primers using a Titanium One-Step RT-PCR Kit. In order to carry out a semi-quantitative analysis, a limited number of PCR cycles (5-20 cycles) will be run for each sample so that the amount of cDNA made will be proportional to the amount of each specific mRNA species in the extract. Ribosomal 18s RNA will be used as an internal control to ensure that there are equal starting amounts of RNA. This method of RT-PCR analysis was chosen because small amounts of RNA can be used (as low as 20 ng) for each reaction and it is efficient for determining gene expression levels for multiple RNA samples. When trying to establish the biological function of these putative glucosyltransferases, part of the whole picture is establishing the reaction that is catalyzed and knowing when and where these GT’s are present. The results of this study will make a significant contribution to the understanding of plant secondary metabolism by linking biochemical function and gene expression patterns in order to help elucidate the metabolic and physiological impact of these gene products. 2008 Appalachian Student Research Forum Page 36 URINARY EXCRETION OF FUNGAL (1-3)-β-D-GLUCAN PHOSPHATE FOLLOWING IV ADMINISTRATION Debra K. Head, Janet W. Lightner, Tammy Ozment-Skelton, David L. Williams and Peter J. Rice Depts. of Pharmacology (DKH, JWL, PJR, DLW) and Surgery (TO-S, DLW), James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN (1-3)-ß-D-Glucans are polysaccharide components of cell walls in various fungi and bacteria. These fungal and/or bacterial glucans are pathogen associated molecular patterns (PAMPs) that circulate during infection and interact with pattern recognition receptors, including Dectin-1 and scavenger receptor A, to modulate innate immunity. Our earlier studies established the pharmacokinetics of intravenously administered glucans and demonstrated that glucans are absorbed following oral administration. In this study, we investigated the renal excretion of (1-3)-ß-D-glucans following intravenous administration in rats. Fluorescently-tagged glucan phosphate was prepared from Saccharomyces cerevisiae and administered (1 mg/kg, IV) to adult male rats anesthetized with isoflurane. Plasma samples were collected from the tail vein at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5 and 6 h, and urine was collected prior to and for 6 hours after glucan administration. Urine samples were passed through an AKTA HiTrap desalting column (5K MWCO) for analysis and to separate free fluorescence. The glucan fractions were combined and lyophilized, and the molecular mass of the excreted glucans was determined by gel permeation chromatography/multi-angle laser light scattering (GPC/MALLS.) Fluorescent-tagged glucan phosphate showed a half-life of approximately 3 hours following intravenous administration. Peak urine concentrations occurred within 3 h. Analysis by AKTA and fluorescence spectroscopy showed 71±3% of fluorescence remained in the >5K MWCO fraction. GPC/MALLS analysis of this fraction showed a minor peak with a molecular mass (171±11K) corresponding to injected glucan (~150K). Most glucan detected by GPC was of lower molecular mass (13±8.5K). Our results indicate that while a small fraction of glucan phosphate may be excreted unchanged in the urine, most (1-3)-ß-D-glucans are excreted by the kidneys as smaller polysaccharides. The presence of urinary glucans may be an important indicator of fungal infection. THE APPALACHIAN REGION: INCIDENCE RATES OF MELANOMA CASES COMPARED TO NON-APPALACHIAN REGIONS Lana McGrady, Shashank Jain, Leslie King, Sunil Swami, James Anderson, Ph.D, Joel Hillhouse, Ph.D. and Tim Aldrich, Ph.D., Dept. of Public Health, East Tennessee State University, Johnson City, TN. Cancer is the second leading cause of death in The United States. In Appalachian region cancer incidence and mortality rates have been found to be higher than rates for the rest of the country. The Appalachian region [410 counties] is divided into three sections: Northern, Central, and Southern. The focus of this study is the Central and Southern Appalachian regions where research has shown that the incidence rates of skin cancer are higher compared to the national average (22%, 21.8%, and 20% respectively). This is a region that is considered to be medically underserved especially in its rural areas. Higher rates of health disparities and poverty contribute to this problem. The US National Statistics report that the incidence rate of melanoma has increased by 18% since 1995. Cancer related mortality is also increasing nationally as a result of this rise for melanoma. Melanoma, the most lethal form of skin cancer has shown to have strong positive associations with exposure to UV rays through outdoor sun exposure and the use of indoor tanning beds. This is important as melanoma is the most frequent cancer in women 25 to 29 years of age. For men, the incidence rate has increased three-fold among ages 45 to 64. The overall incidence rate for melanoma is increasing faster than for any other cancer. This is sufficient reason for public health concern. This study sought to gather more comprehensive information about the incidence and mortality 2008 Appalachian Student Research Forum Page 37 rates resulting from melanoma in the Central and Southern Appalachian region. This includes identifying regional patterns and specific population sub-groups that have a higher risk of melanoma. The Skin Cancer Prevention Laboratory at East Tennessee State University began by requesting state-wise crude and age-adjusted incidence and mortality rates of melanoma for seven states that encompass the majority of Central and Southern Appalachia. Data was received for each state representing rates from the most recent five years available. We began analyses with Tennessee data and the bordering states including: Kentucky, Virginia, North and South Carolina, Georgia and Alabama. Data was recoded to differentiate between Appalachian and Non-Appalachian counties in each state. Preliminary analysis revealed significantly higher incidence rate of melanoma within some states. For example, North Carolina’s rate ratio (RR) = 1.6 and Georgia’s RR=1.24. When the data for all states is aggregated, the age-adjusted incidence rates will be compared to the national average of 18.5 per 100,000 men and women per year. These initial findings strongly advocate further research on this subject within the Appalachian region. The need for efficacious interventions is imperative given that melanoma mortality may be prevented if treated in the initial stages. Such interventions could include an increase in patient screening and raising awareness about melanoma. This could ultimately result in a decrease in mortality rates and an improvement in the quality of life within the region. CANNABINOID RECEPTOR (CB2) DEFICIENCY IS ASSOCIATED WITH REDUCED LESIONAL APOPTOSIS IN LOW-DENSITY LIPOPROTEIN RECEPTOR-NULL MICE Courtney Netherland, Alicia Bales, Theresa Pickle and Douglas P. Thewke Dept. of Biochemistry, East Tennessee State University, Johnson City, TN Introduction: CB2 is the primary macrophage cannabinoid receptor and is found in atherosclerotic lesions; however, its role in atherosclerosis is poorly understood. A CB2 agonist, Δ9-/tetrathydrocannabinol, induces lesion regression in ApoE mice and some macrophage processes linked to atherosclerosis are regulated by CB2 in vitro. Recently, we found that isolated CB2-deficient peritoneal macrophages are resistant to induction of apoptosis by oxidized low density lipoproteins. As macrophage apoptosis is a key process in the pathophysiology of atherosclerosis, we hypothesized that CB2 influences atherosclerosis, in part, by modulating lesional apoptosis. To test this hypothesis, we examined the effect of CB2 gene deletion on lesion formation and lesional apoptosis in Ldlr null mice. Methods: Ldlr-/- mice genetically deficient in CB2 were generated by crossing CB2-/- and Ldlr-/- mice. Sex matched groups (n ≥ 8) of 8-week old CB2-/-/Ldlr-/- (CB2-/-) and CB2+/+/Ldlr-/- (WT) mice were fed a Western diet for 8 or 12 weeks. Serum cholesterol and triglyceride levels, as well as lipoprotein profiles, were evaluated by standard methods. Lesion area and apoptosis was quantified in proximal aorta cross sections by oil red-O staining and in situ TUNEL analysis, respectively. Results: No differences were seen in plasma cholesterol and triglyceride levels or in lipoprotein profiles between CB2-/- and WT groups. No statistical difference was found between the mean lesion area of the CB2-/- and WT groups after 8 weeks (113,907 ± 32,263 vs 115,377 ± 35,457 μm2, p=0.93, for males) or 12 weeks (185,546 ± 30,098 vs 197,616 ± 40,900 μm2, p=0.47, for males; 215,254 ± 44,016 vs 201,718 ± 39,448 μm2, p=0.49, for females) on the Western diet. However, a significant decrease (~50%) was found in the mean number of apoptotic cells in CB2-/- lesions compared to WT lesions (27.2 ± 9.3 vs 50.5 ± 16.1 apoptotic cells per mm2, p <0.001, for males; 55 ± 25.7 vs 113 ± 37.5 apoptotic cells per mm2, p<0.001, for females). Conclusions: Systemic CB2 deficiency in Ldlr null mice does not affect lesion formation, but does significantly lessen lesional apoptosis. These results are consistent with our hypothesis and imply that CB2 may be a novel target for therapies aimed at decreasing plaque vulnerability. 2008 Appalachian Student Research Forum Page 38 RELATIONSHIP OF PEAK ISOMETRIC STRENGTH TO RATE OF FORCE DEVELOPMENT AMONG COLLEGIATE TRACK AND FIELD ATHLETES. K. Painter, A. M. Swisher, J.M. Kraska, M.W. Ramsey, C. Nelson, M.E. Stone and M.H. Stone. Sport Performance Enhancement Consortium, East Tennessee State University, Johnson City, TN Explosive strength, a function of rate of force development (RFD), is a primary determinant for the amount of peak force that can be produced in critical sport performance time periods (e.g. foot contact time). Evidence indicates that maximum strength and RFD are correlated. However, the characteristics of this relationship are not well established among athletes. This study examined the relationship of peak isometric force (IPF) and RFD from a mid-thigh pull, among male and female track and field athletes. Athletes were 12 sprinters (S), 10 jumpers (JP) and 12 cross-country runners (XC). Force-time curve analysis was conducted for each (2 trials) isometric pull and averaged for analyses. IPF and RFD from 0 – 200 ms were determined from appropriate curves. IPFs were normalized using an allometric (IPFa) scaling equation: absolute force/ (body mass (kg)0.67). ICCs were previously shown to be > 0.9. Relationships were established with Pearson’s r; statistical differences with a Bonferroni adjusted t-Test. IPF, IPFa and RFD were greater in males than females for S and JP but not for XC. IPF, IPFa and RFD were greater for S and JP compared to XC. Similar relationships of IPF to RFD were noted among groups S(r = 0.85), JP (r = 0.87), XC (r = 0.88). Among males, n = 19, the relationship of IPF to RFD was r = 0.89 and was somewhat higher compared to females, n = 15, (r = 0.78). Overall (n = 34) relationship between IPF and RFD was r = 0.90. These data indicate a strong relationship between maximum strength and “explosiveness” that is independent of sport training method. TOWARD EXPRESSION OF ACTIVE HUMAN CATHEPSIN G IN PICHIA PASTORIS Eliot T. Smith and David A. Johnson Dept. of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN Human cathepsin G (CatG) exhibits dual specificity for substrates of both trypsin-like and chymotrypsinlike proteases. Stored in cytoplasmic granules of human mast cells and neutrophils, CatG participates in bacterial killing and inflammatory responses of the immune system. Given the difficulty and high cost of purification, generation of a more efficient method for isolating adequate quantities of human CatG would enable more thorough characterization. Initial attempts to express CatG in P. pastoris met with limited success due to its highly basic character (pI ≈ 11.0). The modified strategy incorporates the gene for human serum albumin (HSA), a more acidic protein that P. pastoris successfully secretes, into the design. Target nucleotide sequences recognized by the restriction endonucleases EcoRI, KpnI, and SacII have allowed successful generation of a vector encoding a fusion protein of HSA and CatG in pPICzα. This contains a form of the CatG gene optimized for the codon usage of P. pastoris due to the yeast’s complement of tRNAs; however, the process does not alter the amino acid sequence. PCR amplification with extended primers allowed incorporation of EcoRI and KpnI sites onto the 5’ and 3’ ends, respectively, of the HSA gene for subsequent insertion via restriction digestion followed by ligation. The next step integrated a KpnI site and an enterokinase cleavage recognition sequence (DDDDK) onto the 5’ end as well as a SacII site onto the 3’ end of a CatG gene by the same methods. Sequencing data analyses confirm the successful assembly of this construct as well as its proper positioning within the target plasmid. Work will proceed with transfection of the plasmid construct into P. pastoris followed by induction of expression and secretion. Purification will use a chromatography column with specific affinity for HSA and enterokinase cleavage at the DDDDK site should remove the HSA fusion and yield 2008 Appalachian Student Research Forum Page 39 active CatG. Cleavage of synthetic peptide thiobenzyl ester substrates will verify the enzymatic activity by producing a color change. A yeast expression system for recombinant human CatG will facilitate further studies of this proteolytic enzyme such as its unusual dual specificity. Supported by the National Heart, Lung and Blood Institute via grant 1R15HL091770. THE RELATIONSHIP OF PEAK ISOMETRIC STRENGTH TO PEAK AEROBIC POWER AND 3000 M PERFORMANCE IN CROSS-COUNTRY RUNNERS A. M. Swisher, J.M. Kraska, M.W. Ramsey, K. Painter, C. Gooden, A. Layne, M.E. Stone and M.H. Stone. Sport Performance Enhancement Consortium, East Tennessee State University, Johnson City, TN Certain variables: peak aerobic power (PAP), running economy, and lactate threshold, act as limiting factors for endurance running. However, all of the mechanisms underlying high-level endurance running are not completely clear. Alterations in maximum and explosive strength have been shown to effect positive changes in endurance performance, likely by altering running economy. If strength related factors affect running economy, then this should be evident in the running performance of long-distance runners and perhaps PAP. The purpose of this study was to examine the relationships between peak isometric strength (IPF), explosive strength (rate of force development, RFD), PAP and 3 k performance (3KT) among 5 female and 7 male (n = 12) X-country runners. Force-time curve analysis was conducted for each (2 trials) isometric pull and averaged for analyses. IPF and RFD from 0 – 200 ms were determined from appropriate curves. IPFs were normalized using an allometric (IPFa) scaling equation: absolute force/ (body mass (kg)0.67). ICCs were previously shown to be > 0.9. Relationships were established with Pearson’s r; statistical differences with a t-Test (p < 0.05). IPF, IPFa and RFD were not different between sexes. PAP was statistically greater in males. Correlations were: PAP vs 3KT (r = -0.92); IPF vs PAP (r = 0.58), 3KT (r = -0.66); IPFa vs PAP (r = 0.45), 3KT (r = 0.45). RFD vs PAP (r = 0.58), 3KT (r = -0.65). Results indicate that strength characteristics correlate moderately to strongly with PAP and 3KT. TOLL-LIKE RECEEPTOR MODULATION OF DENDRITIC CELL: NATURAL KILLER CELL INTERACTION Stacie N. Woolard, S. Nandakumar, Evelyn Kurt-Jones*, and U. Kumaraguru Dept. of Microbiology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN * Program in Immunol. And Virology, U Mass Medical School, Worcester, MA-01655 Toll-like Receptors (TLRs) are pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPS) usually associated with bacteria and fungi. This early recognition results in a rapid initiation of innate immune response that sets the course for subsequent adaptive immune response. Herpes Simplex Virus (HSV) is one of the very few viruses that can trigger a TLR response. The induction of Dendritic Cells (DCs) maturation by TLR ligands is considered as a pivotal link between innate and adaptive immune responses. This renders the DCs capable of directing the immune response. Natural Killer (NK) cells also express some of the TLRs expressed by DCs. It is our hypothesis that HSV mediates the interaction and early signaling events of NK cell and DC via TLR. To study TLR induced initiation between NK and DC, the DCs and NK cells were stimulated with various TLR-ligands; HSV stimulation and un-stimulated DCs served as positive and negative controls, respectively. The DCs and NK cells were then allowed to incubate overnight. The impact of TLR ligands on NK cells was analyzed by the up-regulation of activation markers (CD25 and CD69) and the secretion of cytokines (IFN- ). The enhancement of the function of DCs was studied by measuring the up-regulation of co-stimulatory 2008 Appalachian Student Research Forum Page 40 markers (CD80, CD86, MHC class I and II, CD40 etc.). To further understand the role of TLRs in NK:DC interaction, DCs and NK cells were generated from MyD88 knock-out mice. MyD88 is the adaptor protein used by most TLRs and its absence hampers signal transduction negating the effect of TLR engagement. Our results indicate that Viral TLR-ligands are able to modulate the NK:DC interaction and this is one of the many mechanisms used by these two innate immune cells to initiate a potent acquired immune response. Natural Sciences & Mathematics MOLECULAR MECHANISM OF FERRICSIDEROPHORE TRANSPORT VIA OUTER MEMBRANE RECEPTORS Jennifer K. Cooke, Ranjan Chakraborty Dept. of Health Sciences, East Tennessee State University, Johnson City, TN Iron is an essential nutrient for life and growth in most single-celled and multicellular organisms. Although it is abundant in the natural environment, iron exists mostly as insoluble iron-oxyhydroxide. Bacteria are able to overcome this problem by several mechanisms, such as the secretion of small organic molecules called siderophores. Siderophores chelate iron from the environment with high affinity and transport it into the cell via complex transport system. In E. coli, an outer membrane receptor protein known as FhuA transports ferrichrome, a fungal siderophore produced by Ustilago sphaerogena. The crystal structures of FhuA and several such receptors have been determined; the structure consists of a 22stranded beta barrel and a plug which occludes the pore. Opening of the FhuA channel will either require dislodgement of the plug domain or a conformational change within the plug domain to form a channel. Our data indicates that during transport, the plug domain does not leave the barrel; rather it undergoes a conformational change to form a channel. We used site-directed mutagenesis to change four amino acids in the plug and barrel to cysteine in order to form disulfide bonds and tether the plug in place, preventing it from being dislodged during transport. The conformational change may involve partial or complete unfolding of central β-strands. In order to check whether the plug unfolds, the double-cysteine mutant (104/149C) was constructed to form a disulfide bond connecting the β2 and β4 strands, preventing the unfolding of the plug. This mutant shows normal binding with completely diminished transport, indicating that this region has an important structural role. There is an arginine which is conserved in all the receptors and in FatA, it has been shown that mutation of this arginine to alanine reduces transport without affecting binding. In FhuA, we mutated this residue to alanine and glutamine and checked its effect on in vivo binding and transport using radiolabeled iron. These mutants have been overexpressed in pET21b plasmid for purification, crystallization, and structural analysis. THE PATHWAY LEADING TO THE SYNTHESIS OF SALICYLIC ACID IN PLANTS RESISTING PATHOGEN INFECTION Alexander Eddo and Dhirendra Kumar Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN Salicylic acid (SA) is a well known plant signaling molecule that accumulates upon plant-pathogen interaction. This accumulation consequently corresponds to the plant being resistant to infection without which it is susceptible. Pathogenesis-related (PR) gene expression and the synthesis of defensive compounds associated with both local and systemic acquired resistance in plants require SA. The goal of this study is to identify molecules that might be directly targeted to cause the SA accumulation. Primers 2008 Appalachian Student Research Forum Page 41 of genes involved in the normal biosynthesis of SA were used to perform semi-quantitative RT-PCR in order to compare their expression levels in susceptible and resistant tobacco plants challenged with Tobacco Mosaic Virus. Since SA does not accumulate in susceptible plants, differential gene expression between the two lines could be responsible for the increased levels of SA. SA synthesis shares a common substrate, Chorismate with the synthesis of aromatic amino acids. HPLC was used to measure amino acid levels to see if the increase in SA could be due to a decrease in amino acid levels. Preliminary results showed that genes involved in amino acid synthesis are not down-regulated although there was a decrease in amino acid levels in both plant lines. This decrease might correspond to an increase in chorismate levels made available for SA synthesis. We are determining the Chorismate levels to confirm this hypothesis. Study in this area can help in the creation of resistant crop plants which can lower our dependence on harmful herbicides and pesticides. THE DETECTION OF POSSIBLE OUTER MEMBRANE RECEPTOR, 'FEPA', PROTEIN FROM RHIZOBIUM LEGUMINOSARUM STRAIN IARI 312 D. Hammond Jr., B. Clarke, E. Storey, A. Windham, R.N. Chakraborty Dept. of Health Sciences, East Tennessee State University, Johnson City, TN Iron is an essential element for the majority of cell types, with only few exceptions. While the fourth most abundant metal on the earth’s crust, in normal physiological conditions (biological pH and aerobic conditions) iron is primarily found in insoluble oxyhydroxides making it unavailable to microorganisms. The maximum amount of free unassociated iron concentration is 10-17 µM while the amount needed to sustain microbial life is 1 µM. To combat the iron limiting environment, many gram-negative organisms produce siderophores. Siderophores are small molecular weight molecules that scavenge ferric ions and transport them across the cell membranes into the cell via multicomponent transport systems. Iron is important because it serves as a cofactor for the components of the electron transport chain and a variety of oxidoreductases. Siderophore mediated iron transport is one of the ways in which microorganisms scavenge iron from the environment. Rhizobia form a symbiotic relationship with its host plant and fix atmospheric nitrogen, making it available to the plant in the form of ammonia. Siderophores have been described in a variety of Rhizobium species and are thought to play an important role in the nodulation process. This is because the enzyme, nitrogenase, which fixes atmospheric nitrogen, contains many atoms of iron. We have already characterized the siderophore by R. leguminosarum IARI 312. Previous data has shown the presence of both a catechol and hydroxamate siderophore produced by IARI 312. The Electron Spray Mass Spectroscopy (ESMS) data for the catechol-type siderophore produced by IARI 312 are consistent with the known readings of enterobactin, a siderophore produced by Escherichia coli. SDS-PAGE analysis of the outer membrane of IARI 312 compared to FepA gene in pET17b expressed by E. coli strain BL21 (DE3) shows the presence of a protein, expressed only in low iron conditions, with a molecular weight equivalent to FepA. From this data, the present research is focusing on amplification of the ‘FepA’ receptor in IARI 312. Once the gene is amplified, cloning, sequencing, and expression of the DNA will further confirm the presence of ‘FepA’ or a homologue in the genome of IARI 312. It is important to characterize siderophore mediated iron transport systems in Rhizobia for their agricultural significance as well as to understand in general the mechanism of iron transport in gram-negative organisms. 2008 Appalachian Student Research Forum Page 42 EFFECTS OF PERSULFATE OXIDATION ON MICROBIAL POPULATION Kawalpreet Kaur, Lisa Gallagher and Michelle Crimi Dept. of Environmental Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN The improper disposal of hazardous wastes has resulted in the contamination of air, water and soil and poses a threat to human health. In Situ Chemical Oxidation (ISCO) is a rapidly growing technique for the cleanup of contaminated soils and groundwater involving the injection of a strong chemical oxidant in the contaminated zone to destroy the targeted contaminants. Sodium persulfate (Na2S2O8) is an emerging oxidant that is being used. Persulfate when activated by heat, elevated pH and transition metals like Fe, generates sulfate radicals (SO4-.), which are strong oxidants. Due to heterogeneities and low-permeability layers common to soil, complete removal of the contaminants is not always achieved, leading to rebound of contaminant concentrations in groundwater. Both intrinsic and engineered bioremediation applications have the potential to degrade lower concentrations of contaminants in groundwater following an initial reduction in contaminant mass. Therefore coupling ISCO with bioremediation can potentially effectively remediate contaminated sites. However, there are concerns regarding the compatibility of ISCO with persulfate and bioremediation because effects of persulfate on natural microorganisms that participate in bioremediation are unknown. Persulfate ISCO effects, like generation of free radicals, low or high resulting pH, and altered redox potential can affect the microbial population. Microbes like sulfatereducing bacteria (SRB) can use the sulfate produced as a byproduct of persulfate oxidation as an electron acceptor and can degrade contaminants under anoxic conditions. Therefore batch studies were conducted to investigate the impact of activated persulfate on soil microbial populations. Six treatment solutions were used, including groundwater (as a control), tryptic soy broth (TSB-Microbial Supplement), low pH (≤3 as control for low pH that can result from acidic persulfate) persulfate/Fe-EDTA, persulfate/Fe-CitricAcid and persulfate/alkaline pH (≥10.5). These treatment solutions were selected because they are commonly used in field to activate persulfate. Soils from a petroleum contaminated site were mixed with treatment solutions in 50ml centrifuge tubes for 48 hours and were centrifuged. Supernatant was then removed to perform the analyses described below. In the second set of samples additional volumes of TSB were added to the supernant and were shaked for 7 days allowing the treatment solutions to reequilibrate. This was done to study if microbial populations that are adversely affected by the persulfate treatment will rebound when background conditions are reestablished. Three analyses, including Acridine Orange Direct Count (AODC- which estimates biomass), Biolog® Ecoplates (which assesses the community level physiological profile based on carbon utilization pattern) and Winogradsky columns (microcosm analysis for SRB populations) were used on the treated soil. Results show that biomass was not altered significantly by persulfate treatment. However, the community carbon utilization pattern was changed, indicating that the community has shifted or that different metabolic pathways are utilized post ISCO. SRB were present and active indicating the presence of anaerobic conditions. Results of the second set of samples show that microbial populations rebound upon return to background conditions. This work will be useful in coupling persulfate ISCO with bioremediation processes, which can mitigate the cost and can be the most efficient method of remediation of contaminated sites. 2008 Appalachian Student Research Forum Page 43 MAPPING THE SKIN PATTERN OF SPOTTED SALAMANDERS, AMBYSTOMA MACULATUM Lok Raj Pokhrel, Istvan Karsai, Thomas Laughlin, and Rebecca Pyles Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN Animals pigment patterns are one of the most obvious traits of animals and serve a variety of functions. Pigment patterns of adult salamanders typically are produced by melanophores, xanthophores and iridophores. In spotted salamanders (Ambystoma maculatum), the dorsal spot pattern development begins during the transformation of the larvae to metamorphs as they leave the water. Morphological color patterns, especially in spotted salamander, has been of great interest since the species is visually striking with bilaterally arranged orange to yellow spots throughout the dorsum. We are studying the spotted salamanders (A. maculatum) as a model for “spot patterns” and making an effort to understand if the variations in the dorsal spots pattern reflect the stability of the developmental processes in its biphasic life history. We collected 23 specimens of spotted salamanders at Osceola Island: recreational area, near south Holston Dam, in Sullivan County, TN and took their snout-vent-length (SVL) and total length (TL). The salamanders were photographed digitally to capture all dorsal spots, then the spots were counted and spot area measured via ImageJ (Image analysis software). Statistical analyses were conducted to test the null hypothesis that there is no significant relationship between left-right spot patterns; which showed that there is significant relationship between the left-right spots patterns with P<0.05 (P=0.0001). This result signifies that there should be some common developmental pathways for the generation of the dorsal spot pattern which is concordant with the basic developmental mechanism of pigment pattern formation known today. Secondly, the ratio of variances of left spot area to right spot area is not significantly greater than 1.0 (ratio=1.74) suggesting developmental stability in the population sampled. This could also mean that this population of spotted salamanders did not encounter significant levels of stressors during their metamorphosis, and thus were not affected to show significant variation in spot pattern on their dorsum. Thus, understanding the dorsal pigment pattern would help us understand developmental stability and the habitat quality for the sustenance of the amphibian populations. CHARACTERIZATION OF A MUTANT STRAIN OF R. ERYTHROPOLIS THAT OVERPRODUCES SIDEROPHORE Melanie Pratt and Bert C. Lampson Dept. of Health Sciences, East Tennessee State University, Johnson City TN Iron is abundant in the Earth’s crust, but in the presence of oxygen and at physiological pH, the iron is in a form that is not soluble and therefore unavailable to the bacterial cell. Through the use of siderophores, microorganisms are able to obtain this otherwise unusable form of iron, transport it back into the cell and reduce it to a useful form. Rhodococcus is an environmentally important genus of the actionobacteria and is potentially useful in bioremediation, the production of chemicals, and the removal of sulfur from oil. R. erythropolis is known to produce a mixed ligand siderophore called heterobactin. To determine the uptake and transport of this siderophore, I have used the plasmid pTNR to induce random mutagenesis in the chromosome of this bacterium. The plasmid pTNR contains a transposon that can act as an insertional mutagen when transferred into Rhodococcus cells by electroporation, randomly inserting into the chromosome. Following mutanagenesis, mutants will be screened on CAS (Chrome Azurol S) plates to detect the production of siderophore. DNA from non-producing and over-producing mutants will then be extracted and fragments of chromosomal DNA will then be cloned. To do this, a Southern Blot will be performed with DNA from over-producing strains. Genes that carry the transposon will then be analyzed and cloned so that the DNA from the clones can be sequenced for possible transport protein genes. We hypothesize that over-producing strains will be deficient in uptake and transport of the siderophore. To 2008 Appalachian Student Research Forum Page 44 test this, DNA recovered from the mutant strain will be sequence and analyzed to detect genes that may code for siderophore transport proteins. Interruption of a transport gene by the transposon presumably causes the bacteria to over-produce siderophore to compensate for a transport defect as siderophores are important for iron acquisition and bacterial cell survival. ANALYSIS OF SIDEROPHORE PRODUCING ABILITY OF CLINICAL AND COMMENSAL ISOLATES OF STAPHYLOCOCCUS AUREUS E. Presswood1, A. K. Reynolds1,S. Shukla2, R. Chakraborty1; 1 Dept. of Health Sciences, East Tennessee State University, Johnson City, TN, 2 Marshfield Clinical Research Foundation, Marshfield, WI Staphylococcus aureus is a common cause of skin infections, pneumonia, sepsis, and other diseases in humans. S. aureus infections are a growing problem, both in and out of the hospital. With many strains now resistant to multiple drugs, it is necessary to find new targets and strategies for drug design. One possible target for drug design is the siderophore mediated iron acquisition systems in bacteria. Iron is a vital element for the growth of most microorganisms and has been correlated to the virulence of pathogenic bacteria, including S. aureus. Many bacteria, including pathogens, produce small organic molecules called siderophores under iron restricted conditions, that specifically chelate ferric ions and transport them to the cell via a complex transport system. Within the human body, the availability of soluble iron is restricted to 10-9 to 10-18 M due to the presence of several iron binding proteins, such as hemoglobin, transferrin, and lactoferrin, used for storing and transporting iron throughout the body. This concentration is not sufficient for the growth of pathogens. The goal of present research is to analyze a large number of clinical and commensal isolates of S. aureus for their ability to produce siderophore(s). S. aureus has been reported to produce at least three different types of siderophores: staphyloferrin A, staphyloferrin B, and aureochelin. In this study we have analyzed the siderophore mediated iron acquisition systems in 40 hospital associated, methicillin-resistant strains of S.aureus (MRSA), 30 community associated MRSA strains, and 30 commensal isolates. The majority of the isolates have been found to produce one or more types of siderophores on Chrome Azurol S (CAS) medium. These isolates have also been analyzed for their ability to hemolyze and acquire iron from heme. The chemical characterization of siderophores is also under progress. Our long-term goal is to evaluate the suitability for using the siderophore mediated iron acquisition system as a target for drug design. SKELETAL MORPHOLOGY AND SYSTEMATICS OF LATE PLIOCENE ALLIGATOR SP Jeremy B. Stout Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN The American alligator (Alligator mississippiensis) is one of only two species of alligator in the modern world. It is only distantly related to the other extant species (A. sinensis), separated geographically by approximately 11 000 km and up to 25 million years of evolution. Much closer relatives to A. mississippiensis are known from the geologic past of North America. A disparity exists, though, in the fossil record between A. mississippiensis and its closest relative, the late Miocene – early Pliocene A. mefferdi. While A. mississippiensis is well known from the early Pleistocene and later, no Alligator was known from the middle and late Pliocene until the discovery of Haile 7G, a new late Pliocene (~ 2 Ma) site from Alachua County, Fl. North American fossil Alligator from this gap in the geologic record will undoubtedly provide invaluable clues to the evolution and historical biogeography of the modern American alligator lineage. This research seeks to examine the available Alligator material from this site 2008 Appalachian Student Research Forum Page 45 and apply it to the diagnostic skeletal (primarily cranial) character matrix established by Brochu, 1999 and 2004*. This will afford the specimens species-level identification and confirm one of the three possible hypotheses: a.) that the Haile 7G alligators represent the modern A. mississippiensis, b.) that they belong to a known fossil taxon (eg. A. mefferdi), or c.) that the specimens cannot be easily placed within known taxa, and therefore will either become the holotype of a new species or exhibit a suite of characters from more than one species. The character data will then be applied to a cladistic program (PAUP 4.0) to place the fossil alligators into a phylogenetic perspective. At present, one large skull is on loan to the ETSU Museum of Natural History and is being prepared by the author. Based on the preliminary findings of this specimen, the available character data suggests A. mississippiensis, though the alternate hypotheses cannot yet be ruled out. * Poster and/or oral presentation(s) provide complete citation. INITIAL INDENTIFICATION OF THE GENES INVOLVED IN THE BIOSYNTHESIS AND TRANSPORT OF SCHIZOKINEN, A DIHYDROXAMATE TYPE SIDEROPHORE PRODUCED BY RHIZOBIUM LEGUMINOSARUM IARI 917 W. Wright, D. Hammond, E. Storey, B.C. Lampson, R.N. Chakraborty; Dept. of Health Sciences, East Tennessee State University, Johnson City, TN Siderophores are small organic molecules produced by many microorganisms under iron deficient conditions. They scavenge ferric ions and transport them across the cell membranes into the cell via multicomponent transport systems. With a few exceptions, iron is a vital element for the survival of most living cells. This is due to the fact that iron serves as a cofactor for the components of the electron transport chain and a variety of oxidoreductases. Iron is present in abundance in earth’s crust; however, under physiological pH and aerobic conditions it is not available to the microorganisms as it forms insoluble ferric oxyhydroxide polymer. Siderophore mediated iron transport is one of the ways in which microorganisms scavenge iron from the environment. Rhizobia form a symbiotic relationship with its host plant and fix atmospheric nitrogen, making it available to the plant in the form of ammonia. Siderophores have been described in a variety of Rhizobium species and are thought to play an important role in the nodulation process. This is because the enzyme, nitrogenase, which fixes atmospheric nitrogen, contains many atoms of iron. We have already characterized the siderophore by R. leguminosarum IARI 917 to be ‘schizokinen’; in the present study we have characterized the binding and transport kinetics of ‘schizokinen’ and have also tried to identify the genes involved in its biosynthesis. Random mutations of strain IARI 917 were performed using mini Tn5 mutagenesis. Siderophore overproducers and non-producers were detected on CAS (chrom azurol S) plates. DNA sequence analysis of a non-producer mutant carrying the transposon revealed a gene similar to an alpha keto acid reductase enzyme, which is already reported to be involved in siderophore biosynthesis besides amino acid biosynthesis. The sequence analysis of other mutants have shown similarities to amino transferases (may be involved in biosynthesis of schizokinen as it does contain amino groups). DNA sequence of an overproducing mutant is presently being analyzed. The hypothesis is that the overproducer may have a defective repressor mechanism, a faulty siderophore receptor, or a mutation in the internalization system. It is important to characterize siderophore mediated iron transport systems in Rhizobia for their agricultural significance as well as to understand in general the mechanism of iron transport in gramnegative organisms. 2008 Appalachian Student Research Forum Page 46 Social & Behavioral Sciences THE VALIDITY OF SELF-REPORTED WEIGHT AMONG FEMALE COLLEGE STUDENTS Suzanne Allen, Charlena Lau, Kristen Carlosh, & William T. Dalton, III Dept. of Psychology, East Tennessee State University, Johnson City, TN Investigating the validity of the self-reported values of weight allows for the proper assessment of studies using questionnaire-derived data. The study examined the accuracy of self-reported weight in a sample of female adults. The effects of age, race and ethnicity, depression, defensiveness, and eating disordered behavior on the degree of discrepancy (the difference between self-reported weight and measured weight) were considered. Methods. The analysis used data from fifty-four college age females. Self-reported and measured weights were abstracted and analyzed according to sex, age, measured weight, self-reported weight, and body mass index (BMI). Results. Significant differences were found among one’s reported weight and actual weight. The weight discrepancy was positively associated with age. Eating disordered behaviors, depression, and defensiveness may play predictive roles in gauging how inaccurate or accurate one’s self-reported weight actually is. Conclusions. Self-reported weight is often used due to ease in data collection. However, because of the inaccuracies posed by self-reported weight, researchers should precede using self-report anthropometric measures with caution. Future research directions should focus on the inclusion of attitudinal scales and other self-report data in improving prediction of actual weight. DETECTING DIETARY RESTRAINT: A REVIEW OF THE DIS AND RRS Suzanne Allen, Kristen Carlosh, Charlena Lau, and William T. Dalton, PhD Dept. of Psychology, East Tennessee State University, Johnson City, TN There has been much controversy regarding the validity of the Revised Restraint Scale and the Dietary Intent Scale with regards to identifying restrained eaters. The current study examines whether these two scales yield parallel results pertaining to binge eating behaviors and body mass index. Introduction. Both the Revised Restraint Scale (RRS) and the Dietary Intent Scale (DIS) were created in an effort to identify a restrained eater, one who attempts to restrict their intake of food in general, and/or of “fattening” foods in particular. For example, the following questions can be seen on the Revised Restraint Scale (Herman & Polivy, 1980) “Do you have feelings of guilt after overeating?” and “How conscious are you of what you are eating?” On the basis of previous research and for the purposes of this study, those individuals scoring 15 or higher on the Revised Restraint Scale will be classified as restrained eaters and those scoring lower than 15 will be classified as unrestrained eaters. The Dietary Intent Scale (Stice, 1998) is a nine-item scale measuring dietary restraint over the last six months. Based on a five-point scale ranging from “Never” to “Always”, participants answered items coordinating with the frequency of their behavior. Such questions included on this scale are “I often skip meals in an effort to control my weight” and “I eat low-calorie foods in an effort to avoid weight gain.” Because both scales were created to identify dietary restraint among individuals, it is presumed that an individual would yield parallel scores on both questionnaires. Also, if both scales measure the same construct, similar correlations should be found on other related scales (e.g., Binge Eating Scale). Thus, the current study examined whether the Revised Restraint Scale and the Dietary Intent Scale yielded similar results in defining dietary restraint and correlating with other co-morbid characteristics (i.e., binge eating, body mass index). Design. Fifty-four female undergraduate students were given both the DIS and RRS, along with the Binge Eating Scale (BES). Additionally, height and weight measurements were taken for each participant. Pearson’s correlations were then used to determine the relationship among aforementioned 2008 Appalachian Student Research Forum Page 47 variables. Of the 54 participants measured for height and weight, 8 were in the range of obesity, while 11 were considered overweight, 33 were considered of normal weight, and 2 were considered underweight for their height. More importantly for this study, 24 of the participants were categorized as restrained eaters by the RRS, while 20 were categorized by the DIS. Results. Significant differences were found between the RRS and DIS. The RRS correlated positively with the BES. However, such relationship was not found with the DIS. Perhaps more intriguingly, when compared to the participants’ BMI, the RRS was positively correlated, while the DIS had a negative correlational distribution. This may indicate that the two scales measure two different and distinct groups. Further implications will be discussed. VOICE ONSET TIME AS A CLINICAL INDICATOR OF HYPOFUNCTIONAL VOICE DISORDERS Amanda Arnold, Lisa Phillips, Lindsay Pickler, Whitney White, and Amanda McCamey Dept. of Communicative Disorders, College of Clinical and Rehabilitative Health Sciences, East Tennessee State University, Johnson City, TN The purpose of this study was to measure and compare the voice onset times (VOTs) of healthy individuals using a normal and breathy voice in an effort to determine if VOT can be used as a noninvasive clinical indicator of laryngeal function. Recordings were made of 20 adults between the ages of 20-48 with normal laryngeal function, each using a normal (Group 1) and breathy voice (Group 2). The participants’ productions were designed and collected in such a manner to control for speaking rate, vowel context, pitch, and loudness; all of which have been shown to influence VOT. A mixed analyses of variance showed that hypofunctional productions demonstrated longer VOTs across all stop consonants when compared to normal productions. Within the stops, a significant difference between the voiced and voiceless stops was noted, although no gender differences were found. It was concluded that VOT can be used as an indirect clinical indicator of laryngeal function. TOUCHING, ORAL, AND PENETRATION: DOES OFFENSE TYPE MAKE A DIFFERENCE? Brandon C. Bogle, Chris S. Dula, Robin L. Leonard Dept. of Psychology, East Tennessee State University, Johnson City, TN. There is an extensive body of literature in the field of sex offender research. However, there is a lack of research examining the attitudes of the general public toward sex offenders and their offenses. Current literature indicates that the attitude of the general public towards sex offenders is highly negative (Nelson, Herlihy, and Oescher, 2002). Along with these negative attitudes, are misconceptions that have been generalized about these offenders. Federoff and Moran (1997) identified 9 falsely held myths commonly generalized to all sex offenders. If the public holds staunch and unwarranted negative attitudes toward sex offenders, there is little hope for released sex offenders to successfully reintegrate into society and thus, less informal monitoring of former offenders and little incentive to prevent re-offending. However, there is little information as to what aspects of sex offenders are most stigmatized or to what degree prejudices might vary toward sex offender. The current project is a continuation of the research examining the public’s attitudes toward sex offenders and sex offenses. The primary objective of the current project is to determine if the type of sex offense has an effect on the attitudes toward sex offenders on three distinct constructs: punishment, perversity, and severity. Data were subjected to a Wilks’ Lambda repeated measures MANOVA. F (2, 418)= 31.789, p<.001, ηp2= .288. Least Significant Difference Post Hoc Test (Pairwise Comparisons) yielded significant interaction effects between Touching, Oral, and Penetration 2008 Appalachian Student Research Forum Page 48 offenses on punishment ratings F(6, 204)= 48.871, p<.001, ηp2= .590. The results of this study indicate that offense type does indeed make a difference in attitudes toward the offense. The three types of offense, touching, oral, and penetration are distinct, yet touching seems to differentiate from the other two offenses. It may be that oral and penetration offenses are intrusive in nature whereas the touching offense is generally of an external nature and is therefore not viewed as negatively as the other two offenses. The correlation coefficients between the three aspects of attitude, punishment, perversity, and severity are so high that they seem to indicate co-linearity, which may indicate that they are not distinct constructs and couch a single construct. The use of traditional college students may limit generalizability to the rest of the general population. In addition, the sample was taken from a culturally unique region of the U.S. Future research will examine the effects of various constructs on attitudes toward sex offenders and sex offenses. These factors include gender, age, victim consent, offender status, victim status, and offender attractiveness. The authors also plan to further validate the Bogle Sex Offender Scale (BSOAS). AMPHETAMINE SENSITIZATION IN A RODENT MODEL OF PSYCHOSIS. Zackary A. Cope1, A. Brianna Sheppard1, Ian D. Longacre1, Marla K. Perna1, Kimberly N. Thompson2, David S. Roane3, Russell W. Brown1 1 Dept. of Psychology, College of Arts and Sciences, East Tennessee State University, Johnson City, TN 2 Dept. of Anatomy and Cell Biology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 3 Dept. of Pharmaceutical Sciences, College of Pharmacy, East Tennessee State University, Johnson City, TN This study was designed to analyze the effects of amphetamine on locomotor sensitization in a rodent model of psychosis developed in our laboratory. Past studies have shown neonatal quinpirole (dopamine D2/D3 agonist) produces a significant increase in dopamine D2 receptor sensitivity that persists into adulthood, a phenomenon known as D2receptor priming. An increase in D2receptor sensitivity is consistent with several behavioral disorders, including schizophrenia. Rats were administered quinpirole (1 mg/kg) or saline from postnatal days 1-11 and raised to adulthood (postnatal day 60). Beginning in adulthood, rats were administered d-amphetamine sulfate (1 mg/kg) or saline every other day for 14 days, resulting in a total of seven exposures to the drug. Approximately 10 min after drug injection, rats were placed in a locomotor arena and overall activity was analyzed. Results showed that D2-primed rats receiving amphetamine demonstrated a significant increase in locomotor activity across all seven days of testing relative to all other groups. Controls receiving amphetamine also demonstrated a significant increase in activity over days, demonstrating sensitization. Interestingly, D2-primed rats given saline also demonstrated sensitization to the locomotor arena, but this is consistent with past observations in our laboratory and suggests a lack of ability in these animal to habituate to the environment. Seven to fourteen days after locomotor sensitization testing was complete, cerebrospinal fluid samples were taken via microdialysis from the nucleus accumbens core to be analyzed for dopamine levels, and these samples are currently being assayed. Supported by NIH grant 1 R15 DA 020481-01 to RWB 2008 Appalachian Student Research Forum Page 49 THE IMPACT OF GRAMMATICAL COMPLEXITY ON THE PRODUCTION OF SENTENCE DISRUPTIONS Tessie Burke, Carol Davidson, Karen Mumpower, Katie Sims, and Kerry Proctor-Williams, PhD. CCCSLP Dept. of Communicative Disorders, College of Clinical and Rehabilitative Health Sciences, East Tennessee State University, Johnson City, TN Research suggests that as grammar develops the frequency and type of sentence disruptions a child produces will change as a reflection of grammatical complexity (Rispoli, 2003). Types of sentence disruptions include stalls and revisions. Stalls are sentence disruptions that include repetitions, fillers, and pauses that reflect glitches in performance. Stalls buy the speaker time to produce the utterances. Revisions are sentence disruptions that reflect self-correction due to increased language complexity. Revisions are corrections of utterances produced that do not match the speaker’s intention. The rate of stalls should remain stable or slightly increase, and the rate of revisions should increase as sentence length increases for the child in the midst of grammatical development. Our study investigates the relationship between utterance length and complexity and sentence disruption among preschool children. It examines the spontaneous language samples collected during story re-tell and story generation tasks from 10 typically developing children ages 3;2 to 4;11 (years; months). The analyzed samples were controlled for length to include only utterances of 4, 5, 7, and 8 words in length. The results show that, overall, stalls occurred more often than revisions regardless of utterance length. It was also found that there was no main effect for length; stalls and revisions were not more likely to occur in longer utterances. However, there was a trend in this direction. Further, there were no observed correlations between measures of language development and rate of disruptions. These results supported previous findings that children produce sentence disruptions while grammatical complexity develops, in contrast to earlier studies, however, their level of language development did not predict their disruption rate. This reveals that the relationship between production of disruptions and complexity is intricate. The current findings suggest that the frequency of disruptions produced during grammatical development peaks and then begins to level off as language development reaches a certain point. Once the child has reached a comfort level with longer and more complex sentences, disruptions even out and the relationship is less predictable. RURAL ELEMENTARY AND MIDDLE SCHOOL PERSONNEL PERCEPTIONS OF STUDENT NUTRITION, PHYSICAL ACTIVITY, AND OBESITY Lauren LaBounty and Karen Schetzina M.D., MPH, Dept. of Pediatrics, College of Medicine, East Tennessee State University, Johnson City, TN Background: The percentage of children with obesity and type II diabetes in the United States has increased over the past two decades, particularly in the rural Southeast. Schools are a proposed setting for prevention initiatives. Purpose: The purpose of this study was to gain a better understanding of school personnel perceptions of child obesity and diabetes prevention. This understanding will facilitate a unique collaboration between a regional university, a hospital system, community organizations, and schools in making healthy changes in policy, curricula, and environments in elementary and middle schools. The approach will be modeled after the Coordinated School Health Program (CSHP). Methods: The study was approved by the university Institutional Review Board. Key personnel from six school systems in Northeast Tennessee were invited to participate in a written survey that included questions about nutrition, physical activity, and obesity. Results: All school systems participated, with a total of 32 administrators and teachers completing the survey. Sixty-five percent of respondents believed that the amount of student physical activity at their school was too little. All respondents felt that to be healthy, 2008 Appalachian Student Research Forum Page 50 students needed a total of 30 minutes of physical activity at school each day in every grade level. Eightyfour percent of respondents thought that the nutritional quality of foods and drinks consumed by students at school was fair, good, or very good. Most teachers did agreed that schools should not sell unhealthy snacks as fundraisers and that classroom parties should include mostly healthy foods and drinks. Eightytwo percent of personnel were either extremely or very concerned about child obesity in their school community. Over ninety percent agreed that schools should do more to promote physical activity and healthy nutrition among both children and teachers. All respondents felt that parents needed to be more involved in this effort with their children. Conclusion: School personnel from this Northeast Tennessee sample were concerned about child obesity and seemed willing to implement changes in their schools to promote physical activity and healthy nutrition. The next phase of this study will test whether a program modeled after and implemented in conjunction with Tennessee CSHP is effective in producing changes in school policies, curricula, and environments that will promote physical activity and healthier nutrition. FAF RESEARCH Dr. Vijayakumar Guntupalli, Jenny Gilbert, B.A., Lauren Laethem, B.A.A., Lashondra Phillips, B.A., Cody Robinson, B.S., Angie Travers, B.A. Dept. of Communicative Disorders, East Tennessee State University, Johnson City, TN The purpose of this study was to determine the effect of frequency-altered auditory feedback (FAF) on the frequency of stuttering events during conversation. Two male children (ages 11 and 10 years) who stutter participated in this study. An ABAB time-series design was used. Each participant held spontaneous conversation under both non-altered auditory feedback (NAF) and FAF conditions. During FAF condition, participant’s speech was routed through a digital signal processor that shifted the frequency of speech signal either up or down by half octave before being presented to participant ears via insert ear phones. Frequency of stuttered syllables and number of syllables produced per minute were recorded during baseline and treatment conditions. Both participants exhibited significantly more disfluencies under NAF conditions when compared to FAF. Participant 2 showed a significant decrease in stuttering under the second interval of NAF conditions compared to 1. Other research and theories about the relationships between FAF and NAF on stuttering related to this treatment are discussed. 2008 Appalachian Student Research Forum Page 51 Division III – Graduate Students 2+ Years Biomedical Sciences, Natural Sciences & Mathematics, and Social & Behavioral Sciences COMPARISON OF THE ACTIVITY AND EXPRESSION OF THE ZMPSTE24 ENDOPROTEASE IN CELLULAR QUIESCENCE Christina Bridges and Antonio Rusiñol Dept. of Biochemistry and Molecular Biology, College of Medicine, East Tennessee State University, Johnson City, TN The nuclear lamins are a family of intermediate filament proteins that form a thin, fibrous scaffold lining the inner nuclear membrane, thereby providing structural rigidity to the nucleus (Goldman RD 2002). One member of this family, the 72 kDa Lamin A, is initially synthesized as a 74kDa precursor, prelamin A. This precursor undergoes an unusual maturation pathway in which two endoproteolytic cleavages occur (Beck 1990). The first cleavage can be carried out by the endoproteases Zmpste24 or Rce1. The second cleavage is unique to lamin A in higher vertebrates, and is specifically carried out by ZMPSTE24 (Corrigan DP 2005). Two human genetic diseases, Hutchinson-Gilford Progeria Syndrome (HGPS) and Restrictive Dermopathy (RD), arise from defects in this endoproteolytic processing pathway. Though previous work from our laboratory has provided the insight into the processing pathway of prelamin A, as described above, the functional significance of the pathway remains puzzling. Related literature (Ukekawa R 2007, Gruber J 2005) indicates a role for accumulation of Prelamin A in quiescence, where ZMPSTE24 is down-regulated (Ukekawa R 2007, Gruber J 2005), and thus not available to process the Prelamin A to maturation. Additionally, cells that do not express A/C lamins (Mattia E 1992, Stewart C 1987, M. B. Broers JL 1997) lack a quiescent state. Cells that do express the A/C lamins are typically able to enter quiescence under conditions of serum starvation or contact inhibition. To compare the activity of Zmpste24 with cell cycle stage, we induced quiescence in HeLa cells by serum starvation and measured the rate of Zmpste24 activity compared to the rate of DNA synthesis. For this comparison, we labeled cells with [3]H-Thymidine and monitored incorporation as an indicator of the rate of DNA synthesis over a serum starvation time course of 30-32 hours, and measured the rate of Zmpste24 cleavage of its Prelamin A substrate over the same time course. The results demonstrate a rapid posttranslational down-regulation of activity of Zmpste24 in quiescence that mimics the pattern of downregulation of DNA synthesis as cells enter quiescence, as well as a rapid increase in activity as serum is returned and DNA synthesis levels increase. We also measured the expression levels of Zmpste24 by Western Blot and Northern Blot over a 5 day serum starvation time course, and found levels to be relatively unaltered until the fifth day. These results together suggest that there is a rapid posttranslational down-regulation of activity of Zmpste24 in quiescence that is independent of the slower regulation of its synthesis. To further investigate, we overexpressed Zmpste24 by transfection into fibroblast cells, plated at high density to induce quiescence, and monitored BrdU incorporation as an indicator of DNA synthesis. The results demonstrate a likely ability to bypass the quiescent state when ZMPSTE24 is overexpressed. This implies the potential that down-regulation of Zmpste24 is, thereby, required to enter or maintain quiescence. 2008 Appalachian Student Research Forum Page 52 GENETIC SUSCEPTIBILITY TESTING FOR BERYLLIUM: WORKER KNOWLEDGE, BELIEFS, AND ATTITUDES James Gorniewicz, Dr. Ken Silver, and Dr. Gary Kukulka. Depts. of Psychology, Environmental Health, and Family Medicine, East Tennessee State University, Johnson City, TN Since the 1940s, thousands of nuclear energy workers have been exposed to beryllium, a metal which causes a potentially fatal autoimmune mediated disorder known as chronic beryllium disease (CBD). Genetic testing may provide exposed employees with better information about their individual chances of developing CBD and thus assist them in making informed choices regarding current employment and future medical treatment. Collecting genetic information in the workplace raises a variety of legal, ethical, and social issues. Many individuals fear genetic testing due to concerns over confidentiality, employability, and insurability. Therefore, we wanted to gain a better understanding of what nuclear energy workers thought regarding these important issues and genetic testing. Specifically, we wanted to know more about workers’ beliefs and attitudes concerning ideal ways of collecting genetic information in the workplace so as to ensure confidentiality and avoid the possibility of genetic discrimination from employers or insurers. We conducted three focus groups with former and current energy workers in northern New Mexico. Each group was comprised of five to eleven individuals who were exposed to beryllium, diagnosed with CBD, or tested positive for beryllium sensitivity. Open-ended questions were asked on a variety of topics. Focus group sessions were audio taped, transcribed, and then independently analyzed using grounded theory methodology and NVivo7 qualitative data analysis software. Results of this work identified: 1) specific domains where increased worker education may be beneficial (genetic testing, genetic susceptibility, etc.), 2) numerous worker concerns relating to confidentiality, and 3) worker recommendations for the successful implementation of future genetic testing programs. Understanding the unique insights provided by energy workers could lead to improved employee education as well as improved health outcomes for workers and their families. Findings from this study will be used to suggest priority issues in the development of policies regarding genetic testing and will be presented to organizations that represent the interests of energy employees. Findings from this study could be used to better educate primary care clinicians regarding the recognition and management of CBD and patient concerns - particularly on complex issues related to the advisability and interpretation of genetic tests. Results of this study may also be used to develop an in-depth questionnaire for a larger subsequent study of genetic testing. VITAMIN E (TOCOTRIENOLS) AND PROSTATE CANCER: A PROTEOMICS APPROACH Christian Mbangha Muenyi Dept. of Chemistry, East Tennessee State University, Johnson City, TN Proteomics is the large scale study of proteins in cells or organisms. The purpose of this study was to characterize the proteomic alterations occurring in a prostate cancer (LNCaP) cell line after treatment with delta-tocotrienol (a form of vitamin E not very prevalent from most dietary sources). We found that both gamma- and delta-tocotrienols induced time and concentration dependent growth inhibition and programmed cell dead (apoptosis) in LNCaP cells. Secondly, we used two-dimensional gel electrophoresis (2-DE) to characterize changes in protein expression levels associated with this treatment. Our results show that a specific set of proteins are regulated at both early and late times following treatment with delta-tocotrienol and these proteins have been characterized by their apparent molecular weights and isoelectric points. The alteration observed at early time points are particularly interesting 2008 Appalachian Student Research Forum Page 53 because these changes are likely to reflect the underlying molecular mechanisms for triggering cancer cell death. A CANNABINOID (WIN 55,212-2) INDUCES APOPTOSIS IN PANCREATIC TUMOR CELLS VIA CB1/CB2-DEPENDENT AND INDEPENDENT MECHANISMS Theresa Pickle and Douglas Thewke Dept. of Biochemistry and Molecular Biology, East Tennessee State University, Johnson City, TN Pancreatic cancer is the fourth leading cause of cancer related deaths. In 2008, an estimated 37,170 Americans were diagnosed with cancer of the pancreas and an estimated 33,370 Americans died of pancreatic cancer. The mean survival rate is ~6 months, and only 4% of patients survive five years. Despite extensive testing, only one chemotherapy agent (gemcitabine) has been found to produce any benefit to these patients, unfortunately, the clinical response rate to gemcitabine is less than 10% with life prolongation being only 6 weeks on average. Cannabinoids, such as Δ9-tetrahydrocannabinol (THC), the active agent of Cannabis sativa, exhibit anti-tumor properties. Cannabinoids produce their biological effects by engaging specific receptor-mediated signaling pathways. To date, two cannabinoid-specific receptors, designated CB1 and CB2, have been cloned and characterized from mammalian tissue. The current study was conducted to investigate the potential use of cannabinoids as an anti-tumor treatment in pancreatic cancer. Using RT-PCR we found evidence for expression of CB1 and CB2 in three human pancreatic cancer tumor cell lines (AsPC1, CaPan1, and Mia-PaCa2). Cell viability analysis revealed that treatment of these pancreatic tumor cell lines with Win 55,212-2, a potent CB1/CB2 agonist, results in a significant reduction in cell viability. When AsPC1 cells were subjected to Win 55,212-2 in combination with gemcitabine or 17-AAG, an hsp90 inhibitor, a cumulative decrease in cell viability was observed after 48 hours. Treatment of Mia-PaCa2 and CaPan1 cells with Win 55,212-2 in combination with gemcitabine or 17-AAG did not result in additive growth inhibition. Caspase-3 activity assays revealed that the growth inhibitory effects of Win 55,212-2 involve induction of apoptotic mechanisms. The induction of caspase-3 activity by Win 55,212-2 in Mia-PaCa2 cells was prevented by a CB1 receptorspecific antagonist, AM251, but not by a CB2 receptor-specific antagonist, SR144528, suggesting the mechanism of apoptotic induction in Mia-PaCa2 cells is mediated by CB1. However, AM251 had no effect on the induction of caspase-3 activity in AsPC1, but there was a reduction in caspase-3 activity in the presence of SR 144528, suggesting the mechanism of apoptotic induction in AsPC1 cells is mediated by CB2. In CaPan1, Am 251 and SR144528 had no effect on the induction of caspase-3 activity. In conclusion, the results presented here demonstrate that several pancreatic tumor cell lines express cannabinoid receptors and that a cannabinoid, Win 55,212-2, potently inhibits the viability of these pancreatic tumor cells, at least in part, by inducing apoptosis, via CB1/CB2 receptor dependent and independent mechanisms. THE HOST ADHERENS JUNCTION MOLECULE NECTIN-1 IS DEGRADED BY CHLAMYDIAL PROTEASE/PROTEASOME-LIKE ACTIVITY FACTOR (CPAF) IN CHLAMYDIA TRACHOMATISINFECTED GENITAL EPITHELIAL CELLS Jingru Sun, Robert V. Schoborg Dept. of Microbiology, College of Medicine, East Tennessee State University, Johnson City, TN Chlamydia trachomatis, an obligate intracellular human pathogen, is one of the leading causes of sexually transmitted disease and the most common cause of preventable blindness worldwide. Previous studies in 2008 Appalachian Student Research Forum Page 54 our laboratory have demonstrated that accumulation of the host cell adhesion molecule nectin-1 is significantly decreased in C. trachomatis serovar E-infected cervical epithelial cells. This downregulation requires active C. trachomatis protein synthesis and replication. RT-PCR analyses indicate that chlamydial infection does not alter accumulation of any nectin-1 transcripts, demonstrating that nectin-1 is reduced at a post-transcriptional level. In the present study, Western blot analyses show that nectin-1 down-regulation is C. trachomatis concentration-dependent. In addition, the half life of nectin-1 is much shorter in C. trachomatis-infected epithelial cells than that observed in mock-infected cells, indicating nectin-1 is likely down-regulated post-translationally. The chlamydia-secreted protease CPAF degrades several important host proteins, such as USF-1, RFX5, Keratin 8 and pro-apoptotic BH3-only proteins. Our data have demonstrated that the timing of CPAF expression within infected cells correlates with the infection time-dependent cleavage of nectin-1. Previous studies by Zhong et al. (2000) have shown that lactacystin inhibits the proteolytic activity of CPAF while MG132 does not. Our in vivo inhibition experiments using these two inhibitors have demonstrated that down-regulation of nectin-1 is inhibited by lactacystin but not by MG132. Finally, a cell-free cleavage assay has shown that nectin-1 is degraded by the functional recombinant GST-CPAFwt protein but not by the inactive CPAF mutant GST-CPAFD502A. Degradation of nectin-1 in vitro by recombinant GST-CPAFwt is also inhibited by lactacystin, as was previously observed in vivo. Taken together, our results suggest that the host adherens junction molecule nectin-1 is degraded by CPAF in C. trachomatis-infected cells. Since nectin-1 is crucial for formation of adherens junctions, tight junctions and cell adhesions, nectin-1 down-regulation by CPAF could weaken contacts between an infected genital epithelial cell and adjacent, uninfected cells. This would allow release of the intact, infected cell into genital tract lumen, facilitating chlamydial dissemination and causing chronic, ascending genital tract infections. INVESTIGATING NECTAR RHYTHMS IN SQUASH (CUCURBITA PEPO): EFFECTS ON HONEYBEE (APIS MELLIFERA) FORAGING BEHAVIOR Sam Boyd, Jennifer N. Johnson, Byron N. Van Nest, Darrell Moore, Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN It has been shown that honeybees are able to learn and anticipate the time in which flowers present nectar. Foragers are able to anticipate the time and location of the sucrose reward after only one day of training. We have discovered that when the food reward is removed, this time-memory for individual foragers shows an apparent extinction over several days. However, following a rain event, a greater than expected number of foragers return to the feeding station, showing that extinction is delayed. The present study investigates whether this apparent delay in extinction is due to an adaptive response of the bees to an increase in nectar quality (concentration and/or volume) following a rain event. To test this hypothesis, we used squash plants (Cucurbita pepo) grown in a greenhouse at ETSU (Johnson City, TN) under controlled watering conditions. The plants show a remarkably consistent daily rhythm in nectar secretion: the sugar concentration is highest early in the day, typically 37% (6:00 AM), and diminishes to about 18% just before the flowers close (12:00 PM). Nectar volume, on the other hand, increases during this period. Volume starts at less than 2ml (6:00 AM) but reaches 22ml by noon. Based on these findings, the overall objective of this work is to test the effect an increase (mimicking a rain event) or decrease (mimicking drought) in water availability will have on the nectar rhythm for volume and concentration. We hypothesize that (1) a simulated rain event will cause an increase in the nectar volume produced, and (2) the rain event will cause an increase in nectar concentration. Both improvements in nectar quality increase the profitability of the nectar for the honeybees, therefore increasing foraging intensity. Two greenhouse experiments (GH1 and GH2) were conducted. GH1 consisted of 3 days of controlled watering (baseline) before a simulated rain event (2x baseline), followed by three additional days of baseline watering. GH2 included an 8-day simulated drought immediately preceding the rain event. In both experiments, nectar was collected daily, in 90-minute intervals, while the flowers were open. At 2008 Appalachian Student Research Forum Page 55 each sample time, nectar volume and concentration for 6-8 randomly chosen flowers were recorded. These values, as well as time of day and experimental day were used to perform a GLM statistical analysis (using experimental day to represent watering amount). The results of GH1 show that an increase in water did not cause a change in volume but did cause a decrease in the concentration on the day following the simulated rain day with respect to the first three days. The only significant changes for GH2 were between the volume of the first day and the last three days, possibly due to the deteriorating health of the plants. Interestingly, both the volume and concentration rhythms were maintained throughout both experiments, despite the drastic changes in water, indicating the robust nature of the daily nectar rhythm. However, these results do not explain the delay in honeybee time-memory extinction following a rain event. Further studies using other plants are needed. APPLICATION OF MULTIVARIATE STATISTICAL ANALYSES TO MICROBIAL WATER QUALITY PARPAMETERS IN FOUR GEOGRAPHICALLY SIMILAR CREEKS IN NORTHEAST TENNESSEE TO IDENTIFY PATTERNS ASSOCIATION LAND USE TO FECAL POLLUTION K.K. Hall, B.G. Evanshen, K.J. Maier and P.R. Scheuerman, Dept. of Environmental Health, College of Public Health, East Tennessee State University, Johnson City, TN The addition of surface water bodies to impaired waters (303D) lists for pathogen impairment and the need to address these through the Total Maximum Daily Load (TMDL) process has resulted in increased research to find methods that effectively and universally identify fecal pollution sources. A fundamental requirement to identify such methods is understanding the land use, and microbial and chemical processes that influence fate and transport of fecal indicators from various sources to receiving streams. To better understand current water quality and predict water quality changes as development occurs, we initiated a water quality monitoring program. Four creeks within the Watauga River watershed in Northeast Tennessee are routinely monitored using a targeted sampling approach to determine the extent and sources of fecal pollution. These creeks are monitored based on the existence of pathogen TMDLs or the expectation that they will require pathogen TMDLs. Boones, Cash Hollow and Sinking Creek are listed on the State of Tennessee’s 303D list due to pathogen loading, but only Cash Hollow and Sinking Creeks have TMDLs. Buffalo and Sinking Creek have similar patterns of fecal coliform loading, suggesting that TMDL development may require multi-year data using a targeted sampling approach instead of a 30-day geometric mean. Boones and Cash Hollow Creek have elevated fecal coliform concentrations (1115 and 733 CFU/100ml, respectively) but different land use patterns. Agricultural land use impacts Boones Creek, while surface runoff and land development impact Cash Hollow Creek. Previous multivariate statistical analyses have demonstrated that the microbial and chemical parameters influencing water quality differ between creeks, further suggesting that land use patterns influence microbial contamination. The objective of this research is to update results of previous reports and apply multivariate statistical techniques to identify common patterns associating land use to monitored water quality parameters and various pollution sources. Canonical correlation and canonical discriminatory analyses demonstrate that sources of impairment are related to land use patterns in the watershed. The application of multivariate statistical methods to water quality data has helped to identify common patterns associating land use to monitored water quality parameters and various pollution sources. Combined with a targeted water quality monitoring program, this data analysis approach is a useful method to identify sources of impairment and to identify Best Management Practices that will prevent and remediate the effects of rapid urbanization in the Watauga River watershed. 2008 Appalachian Student Research Forum Page 56 HETEROLOGOUS EXPRESSION AND ELUCIDATION OF BIOCHEMICAL FUNCTION OF TWO PUTATIVE FLAVONOID GLUCOSYLTRANSFERASE CLONES (PGT2 AND PGT3) FROM CITRUS PARADISI M.V.K.P Siddhartha, Daniel K. Owens and Cecilia A. McIntosh Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN Flavonoids are phenolic compounds found naturally in plants and are one of the major classes of plant secondary metabolites. To date there are 6000 naturally occurring flavonoids in the plant kingdom and they play a variety of roles in protecting plants against UV light, acting as pigments, for instance anthocyanins a class of flavonoids, impart red and blue colors to fruits such as strawberries, grapes, blueberries, and apples and some provide characteristic flavors. Flavonoids are also known for their antioxidant and antimicrobial properties. Flavonoids have 9 major subclasses synthesized through a core pathway. At every branch point of flavonoid biosynthesis, flavonoids can either be converted into other flavonoid classes via the core biosynthetic pathway or can be derivatized to give the distinct compounds found in plants. Derivatization reactions include hydroxylation, methylation, sulfation, acylation, methoxylation, glycosylation, and prenylation and Glycosylation is a predominant modification reaction which includes addition of sugars to flavonoids and these reactions are catalyzed by glucosyltransferases (GTs). Glycosylation evident in citrus paradisi where 40-70% of the dry weight of young leaf and fruit tissues is composed of the bitter flavanone diglycoside, naringin.The research focus of our lab is on regulation of flavonoid metabolism specifically on glycosylation and the GTs catalyzing these reactions. Citrus paradisi was chosen as a model system as it is known for accumulation of glycosides of several flavonoid subclasses including flavonone 7-O-glycosides, flavones 7-O-glycosides, flavonol 3-Oglucosides, and chalcone glycosides, flavonol 7-O-glcuosides and the enzymes that are responsible for these reactions. Current efforts are focused on cloning, expression and characterization of putative glycosyltransferases from grapefruit. Currently we have 8 putative GTs in hand. The research described here is designed to test the hypothesis that grapefruit PGT2 and PGT3 clones are flavonoid glucosyltransferases. PGT2 and PGT3 clones have been cloned into pCD1 vector and transformation has been achieved using BL21 cell lines. Heterologous expression will facilitate the expression of soluble proteins. We present here the strategies that are being used for optimizing the expression of soluble protein such as testing media composition, culture temperatures, and induction duration. Once the expression of soluble protein is optimized, expressed protein will be screened for flavonoid GT activity using different substrates. If activity is found, the expressed enzymes will be further characterized. Characterization will include elucidation of the reaction products, substrate specificity, optimum pH, activators, inhibitors, isoelectric point, as well as kinetic parameters. Information on efforts to obtain additional putative GT clones from grapefruit will also be presented. NORMATIVE SPEECH ACCURACY MEASURES OF CHILDREN 24-30 MONTHS OF AGE Marie Fillers, B.S., Amanda Cox, B.A., Amanda Mershon, B.S., and Nancy Scherer, Ph.D. Dept. of Communicative Disorders, East Tennessee State University, Johnson City, TN The availability of normative guidelines for early speech development is limited. One measure of speech accuracy that has gained popularity is Percent Consonants Correct-Revised (PCC-R); however, there is limited normative data for children under three years of age. Campbell, Dollaghan, Janosky, and Adelson (2007) used a curve fitting approach to interpret PCC-R data from some existing studies, although the data at the early ages was limited. The purpose of this descriptive study was to compare PCC-R of typical 24 and 30 month olds to the performance curves developed by Campbell et al. Method: Participants – The sample included 28 typically developing children, 14 (7 males and 7 females) in the 24-month age group and 14 (8 males and 6 females) in the 30-month age group. Mean MLU scores for the 24-month group 2008 Appalachian Student Research Forum Page 57 was 1.8 while the 30-month group was 2.5. Procedures – A 100-utterance spontaneous speech sample was collected using three theme-based play sets containing a controlled number of consonants in initial and final word positions. Each sample was video recorded, transcribed phonetically by two graduate students of Speech-Language Pathology at East Tennessee State University, and analyzed for PCC-R and consonant inventory. Inter-rater transcription reliability on 17% of the sample, was calculated to be 90.5%. Results: Consonant inventory and PCC-R scores were compared between the 24 month and 30 month age groups using t-tests. The consonant inventories and PCC-R scores were statistically larger at 30 months with p<0.05. The expansion in consonant inventories indicated that, by 24 months, nasals (e.g., m, n), glides (e.g., w, j), and stop consonants (e.g., p,b,t,d,k,g) have generally been mastered. By 30 months inventory increases consisted of primarily fricatives (e.g., f, s, z, sh) with an increase from 3.1 to 4.5 at 30 months of age. PCC-R scores from this study were then compared to the Campbell et al. performance curve using t-tests. The data collected in the present study were not statistically different from the Campbell et al. performance curves (24 months, df= 13, t =-1.5, p=0.15, and 30 months df = 13, t =-0.56, p=.59). The data in this study support the validity of the performance curves at both 24 and 30 months of age. Discussion: This study provided validation of PCC-R performance curves developed by Campbell et al. at two age points under three years of age. This finding provides additional support for the use of the performance curves in interpreting speech accuracy measures during speech assessment of children under three years of age. While more data should continue to be collected to validate other age ranges, the performance curves appear to provide accurate normative guidelines for comparison of speech impairments that will be helpful for speech-language pathologists during their assessments of very young children. These norms may facilitate early identification of children with speech impairments and subsequent referrals for early intervention. 2008 Appalachian Student Research Forum Page 58 Division IV – Medical Students EFFECTS OF CIGARETTE SMOKE EXTRACT ON CYTOKINE PRODUCTION FROM IL-1BETA-ACTIVATED MAST CELLS Thomas Soike, Ta-Chang Lin, Kenton Hall, Zong Doa Wu, Yu-Yin Liu, Jonathan Moorman, Guha Krishnaswamy, and David S. Chi Dept. of Internal Medicine, East Tennessee State University, Johnson City, TN, 37614 Dept. of Environmental Engineering, National Cheng Kung University, Tainan, Taiwan Human mast cells are multifunctional cells capable of a wide variety of inflammatory responses and associated with allergy, asthma, and atherosclerosis. Our previous studies have shown that IL-1β activates human mast cells to produce selected inflammatory cytokines. We examined effects of cigarette smoke extract (CSE) on the production of inflammatory cytokines from IL-1β-activated human mast cells. Mainstream (Ms) and Sidestream (Ss) cigarette smoke were collected onto fiber filters and extracted in RPMI-1640 medium. Two ml of HMC-1 at 1 x 106 cells / mL were cultured with Ms and Ss CSE at various concentrations in the presence or absence of IL-1β (10 ng / mL) for 24 hrs. The supernatants were harvested and assayed for IL-6, IL-8, and MCP-1 by ELISA. The Ms and Ss CSE (32.5 to 500 g / mL) alone induced either no or trace amounts of cytokines from HMC-1. However, both Ms and Ss CSE significantly increased IL-6 and IL-8 production (all p < 0.001) from IL-1βactivated HMC-1 in a dose dependent fashion, except Ms CSE at 500 g / mL, which significantly decreased IL-6 production (p < 0.0001). Contrarily, both Ms and Ss CSE at concentrations from 62.5 to 500 g / mL, but not 32.5 g / mL, significantly inhibited MCP-1 production (all p < 0.05) by IL-1βactivated HMC-1 in a dose dependent fashion. The effects of Ms CSE appeared to be more potent than that of Ss CSE. The results show that CSE had differential effects on the production of inflammatory cytokines from IL-1β-activated mast cells, which may partially explain why cigarette smoke contributes to lung and cardiovascular diseases. (Supported by the Ruth R. Harris Endowment and RDC of ETSU) BONE MINERAL DENSITY AND CONTENT OF COLLEGIATE THROWERS: INFLUENCE OF MAXIMUM STRENGTH J. M. Whittington, E. J. Schoen, L. L. Labounty, J. A. Gentles, J. M. Kraska, A. M. Swisher, J. E. Keller, M. E. Stone, M. W. Ramsey, R. C. Hamdy, and M. H. Stone Sport Performance Enhancement Consortium, East Tennessee State University, Johnson City, TN Bone is a plastic tissue, changing in density and size with different levels of stress. . Furthermore, it appears that BMD is altered in a site specific manner. However, BMD has not been studied extensively in all types of athletes, particularly well trained strength-power athletes, such as throwers. The purpose of this study was to examine the BMD of USA Division I collegiate throwers (shot put, discus, etc.). BMD was compared to normative data and to different athletes. Measures of whole body maximum strength and throwing performance were correlated with BMDs. Potential right/left side and sex differences were examined. Athletes were 4 males, 3 females age 19.9 ± 0.9 years. BMD was measured with a DEXA Maximum isometric strength was measured using a mid-thigh pull standing on a force plate. Force timecurves were generated during the strength tests. Peak force (PF) and normalized peak force (PFa) were correlated with BMDs. Comparison indicates throwers have denser bones compared to normative data and compared to other types of athletes. Male throwers tend to have greater total body BMD than female throwers (p < 0.05). Dominant arm showed slightly greater BMD compared to non-dominant (p < 0.05). Furthermore, BMD is related to PF (r = 0.68) and PFa (r = 0.56). Throwers have greater BMD’s than non- 2008 Appalachian Student Research Forum Page 59 athletes or most other types of athletes. However, throwers showed only a small indication of sidedness. These observations likely stem from their training program (whole body heavy loading). CHILDHOOD OBESITY AND TELEVISION: A CORRELATION IN JOHNSON COUNTY, TN Wanda Couch, Megan Cruze, Jessica Jones, Kyle Little, Kristie Markland, Andrew McCormick, Mary McCormick, Katherine Rochelle, Ryan Ward, Dr. Jim Shine and Dr. Judith Anderson. Rural Primary Care Track Dept. of Family Medicine, Quillen College of Medicine, East Tennessee State University, Johnson City, TN Whether you are in rural Appalachia or in a major city, obesity is an escalating problem in our society today. The growing problem of obesity in Johnson County, Tennessee, led the Health Council to specifically identify childhood obesity as an area of concern in their community. Because of this increased emphasis on learning about this trend and a desire to decrease it throughout Johnson County, a group from ETSU was approached regarding studying and implementing a program to hopefully reverse this epidemic. The project that was implemented by the group from ETSU was based on the results from the data collected in the spring semester of 2006. The group hoped that through our project two goals would be accomplished. The first goal of the project was to inform the community about the appropriate amount of time that doctors recommend children spend watching television, 2 hours. The second goal of the project was to suggest and inform the children of the many alternative activities to watching television that are available in their community. The project enacted by the ETSU group was loosely based on a project started in California that encouraged children to reduce the amount of time they watched television. From this, the slogan “Break Free from T.V.” was used throughout our projects duration. In order to evaluate the effect of the educational program, an initial survey was distributed to children asking three basic questions: 1) Do you believe watching TV makes you gain weight, 2) How much TV. is ok to watch each day, 3) Please list 3 things you do instead of watching TV. or playing video games. After collecting this survey, our group began trying to inform everyone of the community about the doctor recommended amount of time a child should spend watching TV. This information was distributed through fliers, newspaper articles, skits, morning announcements, and talking with people of the community. At the conclusion of the project, the survey was redistributed to the same children, and the answers were compared to the initial survey. The results from the analysis of the surveys indicated that the goals of the project had been accomplished. The children surveyed appeared to be more knowledgeable about the recommended amount of time to watch TV. Through the course of this project, it was realized that informing children about the relationship between TV and gaining weight was an obtainable goal. While this conclusion was good for the project, the next step will be accessing the feasibility of continuing this project in the future with the hope of decreasing the obesity problem in the Johnson County community. 2008 Appalachian Student Research Forum Page 60 ACCESS TO OBSTETRIC CARE IN RURAL NORTHEAST TENNESSEE: ASSOCIATION WITH BIRTH OUTCOMES Heather N. Wright, B.S., Beth A. Bailey, Ph.D., Laura K. Jones Cole, M.S., M. A. Tennessee Intervention for Pregnant Smokers (TIPS) Dept. of Family Medicine, East Tennessee State University, Johnson City, TN Background A nationwide downward trend in the number of rural obstetric providers has created considerable barriers to prenatal care for pregnant women residing in underserved areas. This limited access to prenatal and delivery services, compounded by socioeconomic, environmental and cultural factors, has the potential to limit health information pregnant women receive, complicate pregnancy and delivery, and adversely affect birth outcomes. This study sought to illuminate differences in birth outcomes between two Northeast Tennessee counties with disparate access to obstetric care. Methods Birth records from the two delivery hospitals in Washington County, TN were used to compare birth outcomes of patients who resided in Johnson County (identified by the state of Tennessee as a medically underserved area, with no obstetric providers or delivery services) and Washington County (a recognized urban area with multiple obstetric practices and delivery hospitals). T-tests and chi-square analyses were used to compare the women from the two counties on the birth outcomes of interest, including newborn birth weight, birth length, gestational age, and incidence of low birth weight and preterm birth. Results During the two year study period (2006-2007), complete delivery records were available for 2220 women from Washington County, and 114 women from Johnson County. Compared to those from Washington County, babies born to women from Johnson County had significantly lower birth weights, decreased birth lengths and shorter gestational durations. In addition, Johnson County babies were twice as likely to be low birth weight and more than four times as likely to be extremely preterm. There was no significant difference in smoking status between the two counties. Discussion Consistent with current literature, these results suggest that ready access to prenatal care may play an important role in birth outcomes in Southern Appalachia. Low birth weight and preterm delivery are high priority public health concerns and are associated with increased risk of infant mortality and long-term health impairments. Efforts to ameliorate these problems in rural areas of the South should address issues related to local availability of obstetric and delivery services. 2008 Appalachian Student Research Forum Page 61 Division V – Residents / Post-Doc Fellows EOSINOPHILLIC GRANULOMA OF THE LUNG "A MIMICKER OF MALIGNANCY" Adel M. EL-Abbassi, Ryland P. Byrd, and Thomas M. Roy Dept. of Internal Medicine, East Tennessee State University, Johnson City, TN Eosinophilic granuloma , pulmonary histicytosis X, Lanerhans cell granulomatosis are the synonyms for an uncommon interstitial lung disease. We are presenting a case of 52 year old man with 40 pack-year history of cigarette smoking presented to the ER for progressive shortness of breath on exertion, occasional dry cough, chest tightness, weakness and easy fatigability. CT scan of chest was done and confirmed multiple small scattered masses in both lungs. PET scan showed increased uptake in the lung nodules. Bronchoscopy was performed and Cytological analysis of BAL yielded no Diagnosis. FNA was done as a next step but was not conclusive. Patient underwent wedge resection of the LUL and biopsy was compatible with Langerhans cell histiocytosis. This case represents and highlights the association of smoking to eosinophilic granuloma, and the presentation which can mimic malignancy with positive PET scan. A NOVEL PELVIC SURGICAL SIMULATION MODEL Foulk, Brooke E.; Herrell, Howard E.; Hemphill, William K.; Sims, J. Paul; Eason, Martin J.; and, Olsen, Martin E. Dept. of Obstetrics and Gynecology and Center for Experiential Learning, Quillen College of Medicine, East Tennessee State University, Johnson City, TN Pelvic surgery can be very challenging to accurately and safely teach. Procedures take place in a small and deep cavity, which can be difficult for learners to accurately view and understand the correct steps in surgical technique. The anatomy in the female pelvis is also very intricate and complex, making dissection very complex to novice surgeons with limited experience. Even to the expert gynecologic surgeon, pelvic surgery can be complicated. For this reason, attempting to teach pelvic surgeries to students and residents can be dangerous using living patients. Our purpose in creating a novel highfidelity pelvic surgical simulator was to make this process safe, teachable and reproducible, with the ability for teachers to easily monitor and critique students. Material scientists along with our simulation experts and obstetrics and gynecology faculty have devised a high-fidelity pelvic model, specifically for surgical simulation. This novel model fits into our established patient simulator and represents a realistic pelvis, including the female anatomic organs, layers of the abdominal wall, an anatomically correct circulation system which can be adjusted to flow at different rates and pressures, and a system to monitor and record learners performing procedures in real-time. By developing a simulation that has realistic anatomy, including gynecologic organs, a circulatory system with actual perfusion, the layers of the abdominal wall including skin, subcutaneous adipose tissue, fascial layers, muscle, peritoneum and other anatomic landmarks, the instructor is able to teach a step-wise approach to many gynecologic surgeries. The model has a realistic look and feel, making the learning experience much more believable and accurate. A video output worn by the learner records what the learner is viewing and allows teachers to observe each move made by the surgeon and critique both correct and incorrect maneuvers. This process has enabled students and residents to “get their hands dirty” without having to step foot in a real operating room, accruing operating time and costs, and risking harm to living patients. With this pelvic surgical model, we are able to simulate multiple pelvic surgeries for various etiologies, including ectopic 2008 Appalachian Student Research Forum Page 62 pregnancy, different types of ovarian cysts, abdominal and vaginal hysterectomy, tubal ligation, oophorectomy, and salpingectomy. This allows endless numbers of surgical possibilities, patients, and time. Learners can avoid the pressures of operating room with living patients and can be closely monitored and instructed without the constraints of time, money, and their limited experience. Procedures are easily reproducible, with adjustments made as needed for each learner’s specific needs. This novel, high-fidelity pelvic simulator model provides endless possibilities for gynecologic surgical training. A WEB-BASED ELECTRONIC FETAL MONITORING TUTORIAL Howard Herrell, Brooke Foulk, Martin Eason, Imran Quraishi Dept. of OB/GYN and the Center for Experiental Learning, Quillen College of Medicine, East Tennessee State University, Johnson City, TN Interpretation of electronic fetal monitoring has traditionally been taught to learners through a formal, lecture-based didactic, with examples of fetal tracings presented in the form of static images showing a representative portion of a tracing. To date, there have been no non-commercial, web-based attempts at modernizing this aspect of obstetrical education. We sought to develop a web-based tutorial integrating video recordings of tracings, interactive teaching modules, case-based learning, and interactive quizzes. We acquired more than one hundred video recordings of fetal tracings of normal and interesting cases from the archives of the Johnson City Medical Center related to obstetrical cases from 2003–2006. These videos served as the basis for the development of core teaching modules, quizzes, and cases. A narrative was constructed discussing the history of fetal monitoring, its physiologic basis, and methods of interpretation based on currently accepted guidelines. These were constructed into a website (http://www.ob-efm.com) and improvements were continuously made based on feedback from peer reviewers and medical students/residents who were asked to trial the website. Learners have provided individual feedback that they prefer this web-based, interactive approach over our previous traditional lecture-style didactic. Research is currently underway to objectively assess short- and long-term retention of subject matter by novices exposed to either the traditional didactic or to the website. We have observed more uniformity with the fetal monitoring terminology guidelines suggested by the National Institute of Child Health and Human Development and better communication between providers. We are currently working to validate these observations. We propose that this pedagogic method, which relies on the learner to self-direct through the material at his or her own pace and ties the material with real-to-life examples of the material being studied, better prepares the learner to interpret fetal tracings, document the interpretation, and intervene where necessary as compared to a traditional lecture. We have observed better initial and long-term retention of the material. Additionally, this format can free-up traditional lecture time and give the learner instant feedback in the form of online quizzes. Research is underway to validate these conclusions. This resource has also been directly tied to our lab-based simulation program and representative fetal tracings/cases have been used as a back-drop for obstetric simulations. HIGH-FIDELITY SIMULATION AS AN ADJUNCT TO PHYSICIAN IMPAIRMENT TRAINING Howard E, Herrell, Martin J. Eason, and Martin E. Olsen. Dept. of Obstetrics and Gynecology and the Center for Experiential Learning, College Of Medicine, East Tennessee State University, Johnson City, TN We sought to develop a high-fidelity simulation of an obstetric or gynecologic emergency that combines both knowledge of the diagnosis and management of the emergency with education regarding physician 2008 Appalachian Student Research Forum Page 63 impairment in order to emphasize the core competencies in our curriculum. Using high-fidelity mannequins in our simulation laboratory, we developed an obstetric and a gynecologic emergency simulation that could be combined with our physician impairment curriculum. The obstetric emergency involves the learners being presented with sustained fetal bradycardia remote from delivery, necessitating emergency cesarean delivery, while the gynecologic emergency requires the learner to diagnose ruptured ectopic pregnancy while instituting medical resuscitation and ordering emergent surgical management. In each scenario, after the learner has decided that the patient needs to proceed to the operating room, a participant arrives at the scenario who acts as an impaired attending physician. This impaired physician attempts to disrupt patient care and block the learners’ attempts to take the patient to the operating room. Realism is added through scripted irrational behavior, dishevelled appearance, and whiskey dabbed on the shirt collar as an olfactory queue to the learner. The learners are required to (1) diagnosis, manage, and understand the severity of the emergency; (2) identify physician impairment; and (3) counteract the impaired physician and call for other supervisory assistance in less than ten minutes. In the subsequent debriefing, the learners meet with an expert in physician impairment and learn the signs and types of physician impairment and strategies to prevent it; they are educated about programs to assist and monitor impaired physicians; and they receive training in due process and the rights and responsibilities afforded to impaired physicians. As a secondary objective, the management of the obstetric or gynecologic emergency is reviewed. Variants of this simulation have been used to train and evaluate OB/GYN residents, clerks, and prospective resident applicants. Learners at all levels have reported their confidence bolstered in the management of the emergency and their knowledge greatly enhanced with the “surprise” secondary goal of physician impairment training. High-fidelity simulation of obstetric and gynecologic emergencies provides an excellent opportunity to train residents and students in the management of secondary learning objectives involving the core competencies not previously thought adaptable to highfidelity simulation. Physician impairment is only one possibility and a variety of other scenarios involving professionalism, interpersonal and communication skills, systems-based practice, practice-based learning and improvement, as well as medical knowledge and patient care have been successfully tied to highfidelity simulation in this manner at our institution. POST-PRIMING MANIPULATION OF DISCORDANT ANTIVIRAL T CELLS Subhadra Nandakumar1, Akaike, T.2, Mege, R.M.3 and Uday Kumaraguru1 1 Dept. of Microbiology, College of Medicine, East Tennessee State University, TN 2 Dept. of Biomolecular Engineering, Institute. of Technology, Yokohama, Japan, 3 INSERM, Institut du Fer a Moulin, Paris, France. Memory T cells that have encountered the antigen earlier are maintained in a proliferative state and are subjected to aging limitations of proliferating cells and eventually undergo “replicative senescence”. Conditions for such accelerated senescence of T cells are more conducive in latent infection with HSV that periodically reactivates stimulating a T cell response every time. The key features of such T cells are their oligoclonality, functional impairment and acquisition of new phenotype. Their accumulation results in contraction of repertoire, loss of co-stimulatory molecule, decrease or change in cytokine production and strongly correlates with severe manifestations of diseases. Hence, we propose that understanding the mechanisms regulating their ontogeny, change in phenotype, acquisition of novel function and means to rejuvenate will be pivotal to development of future therapies. Our analyses, using in vitro generated antiHSV senescent T cells and in vivo generated T cells from latently infected and periodically reactivated mice have shown phenotypic changes different from normal memory T cells. This includes among others increased expression of NK inhibitory receptors (KLRG1), MHC unrestricted killing, aberrant cytokine production, and reduced proliferative ability. Cadherins described recently as the ligand for KLRG1 are also down regulated in chronic viral infections. Our investigations besides characterizing the functional defect in the responding T cells also looks at modulation of KLRG1hi T cells with their cognate ligands 2008 Appalachian Student Research Forum Page 64 the N and E cadherins. The results from our study seem to suggest that the responding T cells have changed their phenotype, are less poly-functional, and lose their MHC restricted behavior and becoming potentially hazardous leading to self destruction. Interventionist manipulations and signaling through the KLRG1 or other inhibitory receptors with their ligand can help in modulating the functional behavior and result in the rescue of dysfunctional T cells. IDENTIFICATION, RECOMBINANT EXPRESSION, AND BIOCHEMICAL CHARACTERIZATION OF A FLAVONOL 3-O-GLUCOSYLTRANSFERASE FROM CITRUS PARADISI Daniel K. Owens1 and Cecilia A. McIntosh1,2 1 Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN 2 School of Graduate Studies, East Tennessee State University, Johnson City, TN Flavonoids are a diverse group of natural products that are ubiquitous in higher plants with over 6000 naturally occurring compounds having been identified. These compounds are associated with a number of essential physiological roles in planta and have agriculturally and pharmacologically significant activities. Glycosylation is a predominant modification reaction in flavonoid biosynthesis affecting the solubility, stability, and subsequent availability of compounds. Glycosylation of flavonoids also has an impact upon the quality of foods and food products. This is of particular interest in citrus species where flavonoid glycosides convey taste characteristics making flavonoid glucosyltransferases interesting targets for biotechnology applications. In this work, a C. paradisi (grapefruit) glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from C. sinensis (orange). The encoded C. paradisi protein is 51.2 KD with a predicted Pi of 6.27 and is 94.5% identical to the C. sinensis homologue. The enzyme glycosylated only the flavonol aglycones Quercetin (Km=67 µM; Vmax=20.45 pKat/µg) , Kaempferol (Km= 12 µM; Vmax= 11.63 pKat/µg), and Myricetin (Km= 33 µM; Vmax= 12.21 pKat/µg) among a number of tested compounds representing various flavonoid subclasses. Glycosylation was shown to occur at the 3 position within the flavonoid backbone based on HPLC and TLC analysis with certified reference compounds. The optimum pH was determined to be 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme is inhibited by the metal ions Cu2+, Fe2+, and Zn2+ as well as UDP (Ki = 69.5 µM) which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thoroughly established biochemical functions and characteristics for this C. paradisi flavonol 3-O-glucosyltransferase add to the growing base of flavonoid glucosyltranferase knowledge, and will be utilized to further investigate structure and function relationships for these types of enzymes. WHAT IS THE BEST TREATMENT OF CHRONIC FECAL RETENTION? Zia Rahman, M.D., and Fereshteh Gerayli, M.D., Johnson City Family Medicine Residency Program, Johnson City, TN Chronic Functional Constipation (CFC) with or without encopresis is a common pediatric problem, which is distressing to the child and family. High quality randomized controlled trials ( RCTs) on the management of CFC are lacking. The objective of this study was to identify the best available evidence on the management of this chronic condition. A structured search of Medline, BMJ Clinical Evidence, Cochrane Library and National Guideline Clearinghouse between 1966 and Feb 2007 was performed by 2008 Appalachian Student Research Forum Page 65 the authors and the medical school librarian. Our search located seven relevant RCTs and two relevant systematic reviews (SRs.). The Best available evidence for treatment of chronic functional constipation in children is on use of osmotic laxatives). Increased fiber is the most commonly recommended first step in the management of CFC, however, there is only limited evidence to support this approach (SOR: B, based on one RCT). There is insufficient evidence on the use of stimulant laxatives (SOR: A, based on a systematic review). There is no evidence on use of bulk forming laxative. Minimal evidence supports use of mineral oil as a stool softener. There is limited evidence to support the use of behavioral intervention and no evidence that biofeedback or anorectal manometry adds any benefit to conventional treatment in the management of CFC in children with fecal incontinence. Increased fluid intake is usually suggested, but there is limited evidence to support this dietary modification. In conclusion, the best evidence for treatment of chronic functional constipation (CFC) in children is with osmotic laxatives (Strength of Recommendation [SOR]: A, based on six fair to good quality randomized controlled trials [RCTs]). EVALUATION OF INFLUENCE OF FLUORIDE IN DRINKING WATER OR AS A DIETARY SUPPLEMENT OF PREGNANT WOMEN RECEIVING PRENATAL CARE AT ETSU OB/GYN ON DENTAL HEALTH OF THEIR OFFSPRING AND IT’S IMPACT ON INCIDENCE OF PRETERM LABOR. Olga Sarkodie , Martin Olsen, Kevin Breuel, Dept. of Obstetrics and Gynecology, College of Medicine, East Tennessee State University, Johnson City, TN The United States spends more than twice as much on healthcare as the average of any other of developed nations. Yet more than 41 million Americans have no health insurance, and even more have no dental insurance. In theory, primary teeth could benefit from prenatal exposure to fluoride. There are some encouraging trends in existing data, but administration of dietary fluoride supplements to pregnant women is not recommended at this time. Topical fluoride in tooth-paste is one of methods that are responsible for a significant drop in the level of cavities since 1960. According to ADA (American Dentistry Academy), systematic fluorides such as community water fluoridation and dietary fluoride supplements are effective in reducing tooth decay. These fluorides provide topical as well as systemic protection because of presence of fluoride in saliva. Fluoride can prevent tooth decay by strengthening teeth to block acid from penetrating the enamel. The most commonly recommended level of fluoride in drinking water is 0.7-1.2 parts fluoride per million parts water. Water fluoridation has been proven to reduce inexpensive means of obtaining the fluoride necessary for optimal tooth decay prevention, but not everyone lives in a community with a centralized, public, or private water source that is fluoridated. Of the roughly 513 registered water districts in Tennessee about 155 choose not to fluoridate. Although in modern research there are controversies regarding necessity of fluoridation CDC (United States Center of Disease of Control and Prevention) recognizes the fact that public water fluoridation is one of 10 great health achievements of the 20th century. We feel that current standards of providing pregnant women with dental care are sometimes not sufficient. We think there is a connection in between dental health of pregnant women and their future children as well as it (dental health) may influence on outcome of pregnancy such as preterm labor.We started this project anticipating to clarify the role of prenatal exposure to fluoride in drinking water or as a supplement in incidence of preterm labor and dental health of offspring. Our study failed to show statistically significant difference between two groups of patients (group A—Patients who delivered prematurely, and group B--those who delivered full term.), with regards of use of Fluoride. Further research is needed to clarify the importance of dental health on the outcome of pregnancy and dental health of offspring. 2008 Appalachian Student Research Forum Page 66 AN UNUSUAL CAUSE OF HYPOTENSION IN A PATIENT WITH ANTICOAGULATION Gerson David Valdez Figueroa, M.D., Gerson Teran, MD, Roger Dean Smalligan Dept. of Internal Medicine, East Tennessee State University, Johnson City, TN Objectives: 1- Identify spontaneous rectus sheath hematoma as an unusual complication of anticoagulation in elderly patients. 2- Recognize Carnett’s sing as a simple clinical finding helpful to distinguish intrabdominal causes of abdominal pain than abdominal wall pathology. CASE : A 77 year old female with past medical history of HTN , DM presented to the ER with an hypertensive crises, vomiting and confusion on initial workup sodium : 124, troponin I : 0.21 which prompted the administration of aspirin , metoprolol and anticoagulation with enoxaparin after blood pressure stabilization, on second day of hospitalization cardiology was consulted and adenosine stress test was scheduled , during the stress test patient BP : dropped from 161/51 to 70/50 mm Hg ,patient became confused ,and complained of nausea and diffuse abdominal pain ,PE: BP:70/50 , HR: 68 x’ , RR:22 x’, T: 97.2 F, General alert oriented , in acute distress , HEENT unremarkable, neck supple no JVD no bruits, lungs clear ,CVRR no murmurs rubs nor gallops, abdomen supple tender over the RLQ, a 12 x5 cms mass over the same area , Carnett’s sing was present ,BS present no organomegaly, EXT: pulses symetrical, neurological exam no focal patient was placed of IV fluids , BP medications holded as enoxaparin , ECG showed no abnormalities ,and cardiac troponin I :0.02 ng/ml , CBC showed HB : 7.1 , HT:31%( 10 the day before) , WBC: 19800,N:70 % ,PLT : 306000 , patient was started on IV fluids and a CT scan of the abdomen showed a right rectus sheath hematoma and pelvic hematoma , patient received 3 Units PRBC , surgery was consulted and they opted for conservative management , patient was monitored closely , her hemoglobin stabilized after transfusion and serum sodium returned to normal limits after NS infusion patient had and uneventful clinical course.DISCUSSION:This case depicts an unusual but life threatening condition ; rectus sheath hematoma it is a rare condition, linked to age ,systemic anticoagulation, intrabdominal injections, abdominal wall strain, minor trauma , pregnancy as risk factors, our patient had received anticoagulation for 24 hrs ,and developed an intrabdominal mass within hours , clinical manifestations include: abdominal wall pain( 84%) and abdominal mass( Fothergill’s sing) , decrease in hematocrit, ecchymosis 69% , Carnett’s sing ( increase in the abdominal pain when patient lift the head and shoulders off the examination table) can help in the diagnosis, the diagnosis is confirmed by abdominal USG or CT scan of the abdomen, Treatment is mostly expectant, including blood transfusion and pain management ,some cases require intravascular embolization or surgery ,Although outcome is usually good ,there are reports of fatal cases .Physicians must be alert of this rare but life threatening condition specially in elderly patients with anticoagulation. METASTATIC ADRENOCORTICAL CARCINOMA PRESENTING WITH DIPLOPIA. Gerson David Valdez, M.D. and Gerson Ariel Teran, M.D., Roger D Smalligan M.D., MPH Dept. of Internal Medicine, East Tennessee State University, Johnson City, TN Objectives : 1-To remind internist useful clinical findings that can point an unusual cause of Cushing’s syndrome such as adrenocortical carcinoma . 2- Remind an unusual presentation of an unusual neoplasm . 2008 Appalachian Student Research Forum Page 67 CASE: A 43-year-old woman with a history of hypertension and diabetes mellitus presented with 5months of amenorrhea, acne , and hirsutism, 3 months of abdominal pain with a 9 pound weight loss, and 2 weeks of diplopia. PMH: HTN 2 years, DM 14 months. Family/social history: unremarkable and negative for smoking, alchohol or illicit drugs. Medications: metformin, lisinopril, glipizide, furosemide. PE: BP: 161/89, HR 96, RR 18, alert/oriented, cushinoid facies, neck with buffalo hump, abdomen obese with striae, no hepatosplenomegaly, skin with abundant acne and hair over thorax, neurological exam showed left sided sixth cranial nerve palsy. Laboratory Hgb 14.5, WBC 11,000, creatinine 0.9, sodium 136, potassium 4.4, calcium 9.1, random cortisol 39 (5-22), AM cortisol 37 (5-22), urine cortisol 193 (<45), ACTH 7 (10-46), TSH, DHEA 15.3 (1.9-7.6), DHEA SO4 872 (35-430), testosterone 237 (10-70), normetanephrine 0.31 (<0.5), metanephrine 0.2 (<0.9), serum aldosterone 28 (<31), prolactin 6.5 (2.8-29), FSH 0.3 (3-33.4), LH 0.1 (1.5-9.3). Abdominal CT revealed a left adrenal mass (11.4x7.4cm) and hypodense nodules on the liver and extensive retroperitoneal and mesenteric lymphadenopathy. Head MRI showed a 1.2x0.8x1.2cm mass in the cavernous sinus and adjacent to the pituitary gland compatible with metastases. Liver biopsy showed adrenocortical cells by with specific stains compatible with adrenocortical carcinoma. The patient received radiation therapy for the brain mass and palliative chemotherapy with Mitotane. Ten days later the patient requested comfort care after developing intractable nausea/vomiting and prerenal acute kidney injury and she died shortly afterwards. DISCUSSION: Adrenocortical carcinoma is a rare neoplasm with an incidence of 0.02-2 cases per million per year. In 53 % cases the tumor is functional with the most common presentation being Cushing’s syndrome, occasionally along with virilization (10%)as in this case. Our patient presented with a cranial nerve palsy secondary to a cavernous sinus metastasis . The fact that she paradoxically lost weight and had associated virilization raised suspicions of adrenocortical carcinoma as the underlying cause of her hypercortisolism. Thirty-four percent patients with adrenocortical carcinoma have metastasic disease at the time of the diagnosis with the most frequent sites being liver, lymph nodes, lungs and rarely the bones. To our knowledge this is the first case reported of intracranial metastasis producing an isolated sixth cranial nerve palsy. Our patient received radiotherapy as a palliative measure. Further palliative measures have not been standardized in this setting . Our patient developed a gastric hematoma, nausea, vomiting and prerenal acute kidney injury. Physicians should remember to consider hypercortisolism in cases of intractable hypertension and diabetes and also to consider adrenocortical carcinoma if there is an unexplained weight loss in this setting. NON-PHARMACOLOGICAL THERAPEUTIC OPTIONS IN TREATMENT OF INSOMNIA Umesh Vyas, M.D., Hetal Brahmbhatt, M.D. Dept. of Psychiatry and Behavioral Sciences, James H Quillen College of Medicine, East Tennessee State University, Johnson City, TN Educational Objectives: At the conclusion of this presentation, the participant will be able to know different non-pharmacological therapeutic options available for use in their practice especially for chronic insomnia. Introduction and Hypothesis: Insomnia is the most frequent sleep disorder. It is a symptom, and is defined as “chronic inability to obtain the amount of sleep needed for optimal functioning and wellbeing”. Prevalence estimates indicate one third of adult population reports insomnia. Prevalence is higher among women, elders and patients with medical or psychiatric disorders. Insomnia is said persistent if it lasts from one to six months, and chronic if it lasts more than six months. Untreated insomnia is associated with significant morbidity and mortality. Thorough assessment of insomnia is essential in choosing the most appropriate strategy for management. If a cause of insomnia is identified, initial treatment should be directed at specific factor. Since insomnia is a chronic condition, long-term and safe treatments are warranted. Non-pharmacologic options have been available for decades, but lack of physician awareness and training, difficulty in obtaining reimbursements and questions about efficacy have limited their use. These therapies offer the greatest potential for long term gains in preventing 2008 Appalachian Student Research Forum Page 68 recurrence in insomnia. Pharmacological options are most useful for acute insomnia. They are commonly used but long term use of hypnotics can become complicated by drug tolerance, dependence or rebound insomnia. Method: Pubmed.gov was searched by using pre-determined key word: Insomnia. Results: Non-pharmacological interventions produce reliable and durable clinical benefits in the treatment of primary insomnia, insomnia associated with medical or psychiatric conditions and insomnia in elders. Additional research is still needed to develop and validate treatment algorithms that would optimize outcomes and reduce morbidity. Conclusion: Non-pharmacological therapies should always be a component in treatment of Insomnia. 2008 Appalachian Student Research Forum Page 69 Oral Presentations Graduate Students A TEMPORAL AND SPATIAL ANALYSIS OF NORTH AMERICAN AVIAN REDISTRIBUTIONS IN RESPONSE TO CHANGES IN CLIMATE Kenneth W. McDonald1,3, M. Phillip Nott2,3, Justin T Peyton1,3, Thomas C. Jones1,3, Tashauna A. Gilliam1, Mckayla B. Johnson1, & Laura L. Catron1 1 Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN 2 The Institute for Bird Populations, Point Reyes Station, CA Understanding the response of organisms to climate variables is important to assessing the impact of projected changes in climate. Our analysis of North American bird banding records of select avian species over a known period of climatic change demonstrates that the distribution and change of avian morphological characters and population parameters correspond over space and time to perturbations in climate variables indicative of overall changes in climate. Furthermore we suggest hypotheses regarding the redistribution and evolution of avian species relative to current models of forecasted global climate change. LONG-TERM GEOCHEMICAL IMPACTS FROM PERMANGANATE OXIDATION: FIELD-SCALE ASSESSMENT Kelly Moore and Michelle Crimi, Ph.D. Department of Environmental Health, East Tennessee State University, Johnson City, TN In-situ chemical oxidation (ISCO) using permanganate is a remediation technology that has been used under a wide range of geological conditions for the destruction of hazardous contaminants in soil and ground water. The most common contaminants treated by permanganate ISCO are chlorinated solvents (i.e. tetrachloroethylene (PCE), trichloroethylene (TCE), dichloroethylene (DCE), and vinyl chloride (VC)). Soluble manganese, a byproduct of the oxidation reaction, is a concern post-treatment because it can exceed the US EPA secondary drinking water standard of 0.05 mg/L. Another concern is chromium, found as a co-contaminant or naturally-occurring at a site, which can be oxidized by permanganate to a more toxic form (hexavalent chromium). Permanganate oxidation can also cause significant increases or decreases in groundwater pH and also leads to significant increases or in oxidation-reduction potential. Often, large quantities of permanganate are injected into the subsurface without considering the possible long-term effects on ground water quality. While theoretical information exists (guidance documents), no comprehensive and integrated review of field case study data has been conducted to assess the role residual manganese has on groundwater quality. A comprehensive review was conducted of data from 30 sites where permanganate has been injected. Data sets include pre- and post-oxidation measurements of metal concentrations (manganese and chromium), pH, and ORP, along with the remediation design approach (i.e. oxidant concentration, mass delivered, and injection method). Data were normalized based on monitoring date and distance from the target treatment zone (TTZ). A two-way ANOVA was used to compare pre- and post-ISCO values over time and distance. Several correlation analyses were run to identify relationships between manganese concentrations and other parameters and with the design approach. Results show that in most cases manganese concentrations decrease with distance from TTZ and with time. Concentrations return to pre-ISCO levels by 48 weeks post-ISCO. Results also show that hexavalent chromium increases significantly after injection of permanganate. However, after 2008 Appalachian Student Research Forum Page 70 approximately 6 to 20 weeks, concentrations return to pre-ISCO levels. Other results indicate that permanganate ISCO can increase or decrease pH levels depending on site characteristics (i.e. media type). These levels usually return to normal by 32 to 52 weeks post-ISCO. ORP levels increase after permanganate injection. After 20 to 32 weeks, the levels begin to decrease and by 48 to 52 weeks the levels are very negative. The levels usually remain more negative than the pre-ISCO levels. Manganese and chromium concentrations and ORP levels appear to be correlated with the mass and concentration of permanganate delivered. Whereas, pH levels seem to be more positively correlated with the site characteristics. The results of this research have several important benefits. One benefit is the providing of field-scale evidence to support guidance based theoretical recommendations. Another benefit is the determination of impacts that manganese can have on ground water quality. This knowledge will help ISCO practitioners make more informed decisions related to system design and implementation. This will in turn reduce the likelihood of adverse effects to public and environmental health. DEVELOPMENT OF A GIS PROTOCOL TO PRIORITIZE WATER QUALITY IMPROVEMENTS IN RESERVOIR EMBAYMENTS IN TENNESSEE T. Shannon O’Quinn, Yongli Gao, and Jessica Buckles Dept. of Physics, Astronomy, and Geology, East Tennessee State University, Johnson City, TN To simplify water quality improvement in reservoirs, it has been suggested that efforts should be focused on smaller and more manageable units such as reservoir embayment areas. Embayments are prime locations to locate marinas, parks, beaches, and residential homes. Current data and information on reservoir embayments in Tennessee will be assembled into a GIS-based database. GIS-based data models have been developed to identify specific characteristics of embayments that influence water quality. Embayments of 11 main reservoirs have been mapped and digitized in ArcGIS. Initial characterization criteria include water quality, embayment area/watershed area ratio, embayment area/reservoir area ratio, flux between embayment and main reservoir, and stream influence on embayments. A characterization process has been developed for Chickamauga and Boone reservoirs to identify and prioritize embayments that are most likely to be affected by watershed restoration efforts. This protocol is being expanded to prioritize water quality improvements in reservoir embayments in Tennessee. ETSU HEALTH CORPS Mihir Patel, Department of Public Health, East Tennessee State University, Johnson City, TN The aim is to deliver preventive health care to undeserved rural Appalachian population by ETSU medical students, student nurses, medical residents and medical fellows under the supervision of ETSU medical faculty. The project is designed to respond to the original mission of ETSU Quillen College of Medicine (Teague/ ranston Act, i.e., federally supported medical schools for medically undeserved areas.) The project intends to evaluate a model of rural preventive health care. Special emphasis is placed on a trans–generational social system (Health Corps Concept) in which young professionals come to the rescue of elderly medically undeserved individuals living in the Appalachian countryside. The viability of the project depends on voluntarism and philanthropy. Important support will be sought from the state (ETSU), local business (ambulances supported by automobile dealerships, school districts, churches, drugstore chains (Walgreens, CVS), department stores (Wal-mart pharmacies). medical device industry and Pharma industry. Because preventive medicine is the crucial component of a viable national health care system, it is hoped that grant support at eh national level will be achievable. Although we will 2008 Appalachian Student Research Forum Page 71 initially obtain philanthropic support , this project will be critically evaluated for its medical economic viability independent of any grant support. CONTROL OF MnO2 PARTICLES DURING PERMANGANATE-ISCO THROUGH USE OF CHEMICAL STABILIZATION AIDS Mark Quickel1, Michelle Crimi2, Saebom Ko3 Dept. of Environmental Health, East Tennessee State University, Johnson City, TN In situ chemical oxidation (ISCO) is a remediation technique used in the treatment of soils that contain organic contaminants. Permanganate (MnO4-) is a common oxidant used in the ISCO process. During remediation, manganese dioxide (MnO2) particles form as a by-product of the reduction of permanganate. These particles have been shown to deposit in and around permanganate injection wells. The deposition of MnO2 over time may alter the flow of permanganate, preventing effective oxidant distribution and ultimately reducing contact with contaminants. Based on laboratory experimentation, groundwater chemistry is known to impact the behavior of MnO2, thus their transport and deposition in the subsurface. Soil composition is also expected to affect their transport and deposition via surface chemistry and filtering effects. The goals of this experimentation are to (1) better understand the influence of various media types on transport and retention of MnO2 and (2) to improve transport and inhibit deposition of the particles through the use of stabilization aids. To test the effects of different porous media compositions, the following media types were employed in these studies: sand, sand with clay (montmorillonite), sand with organic matter, and sand with iron oxides (goethite). The stabilization aid, sodium hexametaphosphate (HMP), was identified in batch experimentation as an effective additive to prevent settling, stabilize particles in solution, and reduce overall particle size. It is presumed that HMP will facilitate the transport of MnO2 particles in porous media. 1-Dimensional (1-D) transport experiments were carried out in two stages to evaluate the control of MnO2 particles with permanganate ISCO. The first stage involved the characterization of the movement of MnO2 through the various media types. The 1-D columns were wet packed with the selected media. Trichloroethylene (TCE), a common organic contaminant, was injected at the influent end of the columns, and simulated groundwater was passed through the columns. Permanganate was then introduced and passed through the columns to generate and transport MnO2 in situ. Samples of the effluent were collected and analyzed for total solids, total dissolved solids, and total suspended solids. An extraction was carried out on both the effluent and sections of the column. The extraction allowed for the separation of water extractable and barium extractable Mn, and dissolved any retained MnO2 for quantification. All conditions were then repeated with the addition of sodium hexametaphosphate. With the addition of HMP, it was found that particle retention was decreased for each media type. Particle retention, directly in the NAPL source zone, decreased by 85% in sand + goethite media, 53% in sand + clay media, 47% in sand + organic carbon media, and 25% in sand media. The decrease in particle retention can lead to better oxidant distribution and contact time with the contaminant, which translates to more efficient ISCO treatment. SKELETAL MORPHOLOGY AND SYSTEMATICS OF LATE PLIOCENE ALLIGATOR SP Jeremy B. Stout Dept. of Biological Sciences, East Tennessee State University, Johnson City, TN The American alligator (Alligator mississippiensis) is one of only two species of alligator in the modern world. It is only distantly related to the other extant species (A. sinensis), separated geographically by approximately 11 000 km and up to 25 million years of evolution. Much closer relatives to A. 2008 Appalachian Student Research Forum Page 72 mississippiensis are known from the geologic past of North America. A disparity exists, though, in the fossil record between A. mississippiensis and its closest relative, the late Miocene – early Pliocene A. mefferdi. While A. mississippiensis is well known from the early Pleistocene and later, no Alligator was known from the middle and late Pliocene until the discovery of Haile 7G, a new late Pliocene (~ 2 Ma) site from Alachua County, Fl. North American fossil Alligator from this gap in the geologic record will undoubtedly provide invaluable clues to the evolution and historical biogeography of the modern American alligator lineage. This research seeks to examine the available Alligator material from this site and apply it to the diagnostic skeletal (primarily cranial) character matrix established by Brochu, 1999 and 2004*. This will afford the specimens species-level identification and confirm one of the three possible hypotheses: a.) that the Haile 7G alligators represent the modern A. mississippiensis, b.) that they belong to a known fossil taxon (eg. A. mefferdi), or c.) that the specimens cannot be easily placed within known taxa, and therefore will either become the holotype of a new species or exhibit a suite of characters from more than one species. The character data will then be applied to a cladistic program (PAUP 4.0) to place the fossil alligators into a phylogenetic perspective. At present, one large skull is on loan to the ETSU Museum of Natural History and is being prepared by the author. Based on the preliminary findings of this specimen, the available character data suggests A. mississippiensis, though the alternate hypotheses cannot yet be ruled out. * Poster and/or oral presentation(s) provide complete citation. ETHNIC DIFFERENCES IN RISK AND PROTECTIVE FACTORS FOR DEPRESSION Preston Visser1, Jameson Hirsch1, Angela Jones1, and Elizabeth Jeglic2 Dept. of Psychology, East Tennessee State University, Johnson City, TN. 2 Dept. of Psychology, John Jay College of Criminal Justice, New York, NY. 1 Risk and protective factors for depression are well-documented; however, less is known about risk factors and protective characteristics among ethnically and racially-diverse groups. There is a particular lack of research on the function of protective factors across ethnic groups. We examined the association between positive psychological characteristics, including trait hope and religiosity, and depressive symptoms in an ethnically diverse sample of college students (N= 407; Black = 24.5%, Hispanic = 39.2%, White = 17.4%, Asian= 5.4%). We also investigated anger and hopelessness, which are more traditional risk factors for depression. Participants completed the Beck Depression Inventory II (BDI-II), the Beck Hopelessness Scale (BHS); Trait Hope Scale, the Multidimensional Anger Inventory (MAI) and the Brief Multidimensional Measure of Religious/Spirituality (BMMRS). We hypothesized that higher scores on measures of hopelessness and anger and lower scores on measures of religiosity and trait hope would be related to increased levels of depressive symptoms. Data were analyzed using correlation techniques and a series of multivariate linear regressions, statistically controlling for age and gender. In correlation analyses, greater levels of hopelessness and anger were significantly associated with increased depressive symptoms, and higher levels of trait hope were significantly negatively associated with depressive symptoms (for all models, p<.001). In a multivariate model examining all cases, regardless of ethnicity, hopelessness, anger and trait hope were significant predictors of depressive symptoms; anger and hopelessness predicted increased depressive symptoms, whereas trait hope was associated with decreased symptoms of depression. We also stratified our sample by ethnicity. Anger was significantly related to depressive symptoms in Blacks (β= .237), Hispanics (β= .312), Whites (β= .497), and Asians (β= .803; all ps<.001). With the exception of Asian participants, trait hope was negatively related to depressive symptoms. In Blacks and Hispanics hopelessness was a significant risk factor for increased depressive symptoms (β= .460 & .382, respectively). In Black individuals, a marginally significant trend toward significance existed for the association between increased religiosity and decreased depressive symptoms 2008 Appalachian Student Research Forum Page 73 (p= .059); this relationship warrants further examination. Our findings have implications for future research and therapeutic interventions. Researchers should more closely examine how potential etiological influences of depression may vary across ethnicities. For example, it may be insightful to determine why hopelessness in Blacks and Hispanics is strongly predictive of depression while trait hope strongly predicts depression in Whites. Investigation of cultural characteristics and how they relate to the development of depressive symptoms may help researchers to better understand the complex interactions between risk and protective factors of depression. In the treatment of depression, clinicians should promote protective cognitive and emotional characteristics while reducing the impact of traditional risk factors. Although risk and protective factors for depression are largely similar across racial and ethnic groups, differences that exist may be of therapeutic usefulness. UP-REGULATING DEATH RECEPTOR 5 VIA TUNICAMYCIN ADMINISTRATION INCREASES ANTI-HSV IMMUNE RESPONSE Stacie N. Woolard, Subhadra Nandakumar, and Uday Kumaraguru Dept. of Microbiology, College of Medicine, East Tennessee State University, Johnson City, TN Tunicamycin is a naturally occurring antibiotic that induces endoplasmic reticulum stress; thus enhancing susceptibility to TNF-Related Apoptosis Inducing Ligand (TRAIL) mediated apoptosis. In previous tumor research, Tunicamycin has been implicated to improve cancer therapies by sensitizing tumor cells to apoptosis. Tunicamycin in a dose-dependent manner up-regulates DR5, one of the two death receptors for TRAIL; enabling the control of malignant cell growth. However, the cooperative relationship between Tunicamycin and TRAIL has yet to be examined in the course of Herpes Simplex Virus (HSV) immune induction. Preliminary data from our laboratory points to the involvement of TRAIL in antigen presentation. Dendritic Cells (DCs), the professional antigen presenting cell, will present antigen to T cells to induce an immune response. However, upon infection, dermal DCs migrate to peripheral lymph nodes to transfer the viral antigen to the professional APC, which is lymph node resident. Within the lymph nodes, Natural Killer cells expressing TRAIL induces apoptosis in the antigen bearing DCs; allowing the apoptotic transfer of viral antigen for CD8 T cells cross-priming. We propose that the expression of DR5 can be manipulated by administering a controlled concentration of Tunicamycin in a viral infection as well. Cloned Dendritic Cells (DC2.4) and splenocytes were co-cultured with various concentrations of Tunicamycin and analyzed every eight hours to determine the optimal concentration and time point of DR5 up-regulation. Using this kinetics information, we determined the optimal timing and dosage of Tunicamycin requirement for the up-regulation of DR5 on DCs in vivo. With this information, injections of Tunicamycin can be administered in vivo to modulate the quality of CD8+ T cell function. Concluding, Tunicamycin is able to up-regulate the death receptor for TRAIL-mediated antigen transfer elevating the quality of an anti-viral response. 2008 Appalachian Student Research Forum Page 74 Medical Residents and Post-Doctoral Fellows TRACHEAL METASTASIS OF LUNG CANCER: A RARE COMPLICATION Venkataramanan Gangadharan MBBS, Muhanad Al Hasan MD, Ryland P. Byrd, Jr. MD Pulmonary/Critical Care Division, Dept. of Internal Medicine, East Tennessee State University, Johnson City, TN A 77 year-old man complained of shortness of breath on moderate exertion and hemoptysis about 2-3 times per week measuring to less than a teaspoon at each instance. He was diagnosed with squamous cell cancer of the lung one year prior to these complaints and had undergone successful resection of the tumor. His stage at surgical resection was Stage 1B (T2N0M0). The surgical margins and lymph nodes were free of malignancy. He had no adjuvant chemotherapy or radiation treatments. The patient was afebrile and his vital signs were normal. His physical examination was significant only for a post-thoracotomy scar. There was no wheezing, stridor, or other airway noises present on auscultation of the chest. His chest radiographs demonstrated post-surgical changes with no new findings. Computerized tomography of his chest documented a small pleural effusion and tracheal mucosal irregularities. Fiberoptic bronchoscopy demonstrated variable sized masses in the trachea but no bronchial abnormalities. On further examination it was noted these masses were friable in nature and easily bled on manipulation. Histopathology examinations of biopsies obtained at fiberoptic bronchoscopy were returned as squamous cell carcinoma. The patient was subsequently referred to undergo brachytherapy. Lung cancer is one of the most commonly diagnosed malignancies and is the most common cause of cancer related death in the United States. Its presentation varies from small asymptomatic lung nodules discovered serendipitously, to symptomatic masses, to symptoms related to metastatic disease or paraneoplastic syndromes. As with many malignancies, metastasis from lung cancer is possible. Lung cancer typically metastasizes to the brain, bone, and adrenal gland. Endobronchial metastasis from a primary lung cancer is unusual with a reported incidence of 2-18%. Metastasis to the trachea from a primary lung cancer is even rarer. The reported incidence is approximately 0.6%. Symptoms from tracheal metastasis include: cough, dyspnea, hemoptysis, and respiratory distress from tracheal obstruction which may mimic asthma. A careful history and thoracic examination is essential in order to provide appropriate and timely investigations and interventions to reduce morbidity. This report should, therefore, alert health care providers to the possibility of tracheal metastasis. It is hoped that early recognition of tracheal metastasis may afford the patient better and more effective treatment. A CASE OF VENTRICULAR TACHYCARDIA TERMINATED BY CAROTID MASSAGE Sumit Kalra, MD, Romsford Brenya, MD, Rajendra Kadari, MD, Terry Forrest, MD Department of Internal Medicine, East Tennessee State University, Johnson City, Tennessee Initial evaluation of a broad complex tachycardia includes a differentiation between ventricular tachycardia and supraventricular tachycardia with aberrant conduction. Vagal maneuvers are a time honored method for this differentiation based on the fact that supraventricular tachycardias are slowed or terminated by enhanced vagal tone whereas ventricular tachycardias are not affected. We present an unusual case of ventricular tachycardia which was terminated by carotid massage. An 88-year-old Caucasian male with a past medical history of chronic bronchitis, diabetes, hypertension and hypothyroidism was admitted with respiratory tract infection. On telemetry, he was noticed to have a broad complex tachycardia at a rate of 140 beats/minute. The patient denied any chest pain or shortness of 2008 Appalachian Student Research Forum Page 75 breath associated with this heart rhythm and was hemodynamically stable. His physical examination was normal and his laboratory profile did not reveal any electrolyte abnormality. He denied any past history of coronary artery disease, angina, syncope, aborted sudden death or any cardiac arrhythmia. The differential at this point included ventricular tachycardia Vs supraventricular tachycardia with aberrant conduction. On carotid massage, his rhythm converted to sinus rhythm but changed back to broad complex tachycardia as soon as the carotid massage was stopped. A 12 lead EKG revealed broad complex tachycardia with AV dissociation, fusion beat, and capture beat all pointing towards a diagnosis of ventricular tachycardia. A carotid massage was attempted again and the rhythm cardioverted back to sinus rhythm again only to revert back to ventricular tachycardia on stopping the carotid massage. He was admitted to the Cardiac Care Unit and successfully cardioverted with intravenous amiodarone. Later, an adenosine myoview stress test and echocardiogram were performed and were reported as normal. A diagnosis of idiopathic ventricular tachycardia was made in the absence of ischemic heart disease and a structurally normal heart. About 10% of ventricular tachycardias originate in structurally normal hearts and are idiopathic. A majority of these are believed to originate in the outflow tracts of the heart (left or right ventricular outflow tract ventricular tachycardias, OTVTs) and can be recognized based on the 12 lead EKG. It has been proposed that the mechanism of these OTVTs may be cyclic AMP (cAMP) mediated. The beta adrenergic receptor stimulation activates the myocardial cell adenylate cyclase thus increasing the intracellular cAMP concentration. cAMP then activates protein kinase A leading to an increased intracellular calcium concentration which subsequently causes an increase in the inward sodium current (delayed afterdepolarizations). When a delayed afterdepolarization reaches a threshold, it generates an action potential and leads to a triggered ventricular tachycardia. Acetylcholine (through vagal maneuver) is an inhibitor of adenylate cyclase and acts on M2 muscarinic receptors on the myocardial cell to decrease the concentration of cAMP. This in turn attenuates the afterdepolarization through the mechanism described above and leads to the termination of triggered activity. Our patient had an EKG most consistent with Outflow tract ventricular tachycardia (Subtype: Aortic Cusp Ventricular outflow tract tachycardia) based on previously published EKG criteria. We believe that the OTVT in our patient was probably cAMP mediated and therefore terminated by vagal stimulation as described above. Our case reminds internists that although vagal maneuvers are a useful tool in the differentiation between supraventricular and ventricular origin of broad complex tachycardias, they are by no means diagnostic. AN EXCEPTIONAL VARIANT OF SINGLE CORONARY ARTERY ANOMALY, PRESENTING WITH ACUTE ST SEGMENT ELEVATION MYOCARDIAL INFARCTION Georges El Khoury and Lucien Abboud Depts. of Internal Medicine and Cardiology, College of Medicine, East Tennessee State University, Johnson City, TN Congenital coronary artery anomalies are infrequently seen during coronary angiography occurring in 0.06 % to 1.3 %. Some variants are totally benign and are discovered incidentally in the catheterization laboratory, however others may present with sudden cardiac death. Single coronary artery is one subtype which is often associated with other congenital cardiac malformations most of which are benign. We report a 75 y o male patient with an exceptional variant of single coronary artery anomaly with right coronary artery arising from the distal left circumflex system who presented with ST Elevation Myocardial Infarction of the anterolateral wall secondary to Left Anterior Descendant artery occlusion. The patient has no previous cardiac history. Would the patient has survived if he had the lesion involving the Left circumflex artery or would it be a case of undiagnosed coronary artery anomaly presenting with sudden cardiac death? 2008 Appalachian Student Research Forum Page 76 ELEVATED IGE, EOSINOPHILIA AND PERSISTENT AIRWAY OBSTRUCTION Christopher W. T. Miller and Guha Krishnaswamy Dept. of Allergy and Immunology, College of Medicine, East Tennessee State University, Johnson City, TN The prevalence of Strongyloides parasitosis in the East Tennessee belt is strikingly high, and may be masked by atypical clinical or laboratory features. A 67-year-old female patient presented with a recent worsening of her baseline intermittent cough and dyspnea, associated with a number of nonspecific complaints (rhinorrhea, nasal congestion, and sneezing). Pertinent past medical history included severe chronic obstructive pulmonary disease, diabetes mellitus type 2, and a history of non-small cell lung cancer treated with radiation therapy 13 months before the current presentation. Physical exam was essentially unremarkable apart from some mild wheezing bilaterally. Work-up yielded a normal white blood cell count with an elevated eosinophil percentage varying from 16% to 21%, along with an elevated C-reactive protein level of 70.7. Allergic assays were essentially negative for major offenders and stool studies were repeatedly negative for ova and parasites. Serum immunoglobulin assays revealed an extremely elevated IgE level of 4998.5. The patient was assessed by several specialists without reaching a definitive etiology despite extensive work-up (including bone marrow biopsy, chest computed tomography, HIV, hepatitis screen, autoimmune markers, heavy metal screen, TSH and cortisol levels). A presumptive diagnosis of hyper-IgE syndrome was established. However, a serum immunoglobulin G assay for Strongyloides returned positive at 14.7, leading to a diagnosis of strongyloidiasis. The patient was treated with three courses of ivermectin (15 mg single-dose regimens over 3 months). The patient’s respiratory symptoms greatly improved, with a corresponding decrease in laboratory findings to nearnormal values. This case reiterates the need for a high level of suspicion in patients with suggestive symptoms of strongyloidiasis even in the face of negative stool assays. MOLECULAR LINK BETWEEN TOLL LIKE RECEPTORS, OBESITY AND RISK OF CANCER Subhadra Nandakumar, Stacie Woolard and Uday Kumaraguru Dept. of Microbiology, College of Medicine, East Tennessee State University, Johnson City, TN According to the National Cancer Institute statistics, the incidence of new cases of cancer in 2007 is about 1,444,920 and it was estimated that there were about 559,650 cancer related deaths in the US. This number is much above the average cases in the developing and under-developed countries. Recent studies propose a link between obesity and susceptibility to cancer. Oddly obesity is a major problem affecting 66% of the American population as reported by CDC. Hence, it is tempting to hypothesize that obesity and cancer development may correlate with exposure pattern to infection that engages Toll like receptors (TLRs). We tested our hypothesis in C57Bl6 mice. These mice were housed in two different environments, one an ultra-clean environment, where cages, bedding, food and water needed for the mice were sterilized. The other was the dirty environment, which was simulated by administering the mice with cocktail of TLR ligands (containing antigenic components of virus, bacteria and fungi) periodically. These mice were later subjected to tumor challenge and were analyzed for various immunological parameters. Our findings show that the mice growing in “dirty” environment had significantly lower weights and were able to resist tumor challenge much better than those growing in “clean” environment. Based on the outcome of this study we can conclude that TLR activation by its ligands results in an increased lipid metabolism leading to the prevention of obesity and heightened immune preparedness. This increases the ability of the host to resist the tumor challenge. We demonstrate that the activated phagocytic ability, mediated by key cytokines as the mechanism of this resistance. 2008 Appalachian Student Research Forum Page 77 PHILADELPHIA-CHROMOSOME POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA: LONG-TERM SURVIVAL IN AN ADULT JEHOVAH’S WITNESS Vivekananda Pattabiraman, MD, Harsha G. Vardhana, MD, Stephen J. Smith MD, and Koyamangalath Krishnan, MD Dept of Internal Medicine, Quillen College of Medicine, East Tennessee State University, Johnson City, TN Key words: Philadelphia-positive ALL (Ph+ ALL), Stem Cell Transplantation (SCT), Tyrosine kinase Inhibitor (Imatinib) . Background: Adult Ph+ ALL has a poor prognosis. We present a case of Ph+ ALL who belongs to the Jehovah’s faith and exhibited prolonged survival with modified conventional chemotherapy along with long-term treatment with a tyrosine-kinase inhibitor. Case Report: A 31 yr old man who belonged to the Jehovah’s faith was diagnosed with Ph+ ALL. His religious beliefs precluded blood transfusion and hence he was not considered suitable for SCT. He had anemia and thrombocytopenia at diagnosis. and was therefore treated with attenuated doses of induction chemotherapy (along with supportive treatment with GM-CSF, Erythropoietin, amino-caproic acid) followed by usual doses of consolidation and maintenance chemotherapy for 2 years.Imatinib was started along with maintenance chemotherapy and continued indefinitely. We have been successful in achieving 80month survival (until Feb 2008) along with clinical remission of 60 months. Conclusion: Adult Ph+ ALL is known to be a malignancy with poor prognosis. SCT is regarded as the treatment of choice but survival rates are far from satisfactory. Conventional chemotherapy along with Imatinib is capable of inducing long-term clinical, hematological and molecular remission and also better survival when compared to SCT. Tyrosine Kinase inhibitors are a promising therapeutic option for patients unable to undergo SCT. References: Garcia et al: The hyper CVAD regimen in adult ALL: Hematology Oncology clinics Of North America 14,1381-1386 Thomas DA et al: Treatment of Ph+ ALL with Hyper-CVAD and imatinib mesylate: Blood 2004 Jun 15;103(12):4396-407 EFFECTS OF VITAMIN E ISOTYPES ON KG-1 LEUKEMIC CELLS GENE EXPRESSION, CELL GROWTH AND PROLIFERATION Nakhle Saba, Sarah G. Whaley, Koyamangalath Krishnan and Sharon Campbell Depts of Internal Medicine and Medical Oncology, College of Medicine, East Tennessee State University, Johnson City, TN Background: Vitamin E is a fat-soluble vitamin that exists in eight different isoforms: four tocopherols (alpha-, beta-, gamma- and delta-), and four tocotrienols (also alpha-, beta-, gamma- and delta-). Studies are underway to determine whether vitamin E, through its ability to limit production of free radicals, might help prevent or delay the development of cancer. Other studies suggest that vitamin E may modulate signal transduction pathways to prevent or reverse the effects of cancer. In our study, we examined the effect of vitamin E isotypes on some gene products involved in acute leukemia such as HOXA9 (Homeobox A9), PBX1 (Pre-B cell leukemia transcription factor 1), E2A-PBX1, HOXB6, CDX2, NUP98, CYCLIN D1 and CYCLIND3, as well as on in-vitro leukemic cell growth and differentiation. HOXA9 is a transcription factor with a central role in both hemopoiesis and leukemia. A high level of HOXA9 is a characteristic feature of acute myeloid leukemia (AML) and may be sufficient to cause this disease. PBX1 is involved in a form of pre-B-cell acute lymphoblastic leukemia (B-ALL). E2A-PBX1 is a protein resulting from a chromosomal translocation t(1;19)(q23;p13.3) which involves PBX1 and E2A genes. E2A-PBX1 transforms cells by constitutively activating transcription of genes 2008 Appalachian Student Research Forum Page 78 regulated by PBX1 or by other members of the PBX protein family. NUP98 encodes for a 98 kilodalton (kDa) O-linked glycoprotein, a component of the nuclear pore complex; Translocation t(7;11) (p15;p15) in AML fuses the genes for NUP98 and class I homeoprotein HOXA9. NUP98-HOXA9 fusion protein may promote leukemogenesis. CDX2, a member of the caudal-type homeobox family is aberrantly expressed in most cases of acute myeloid leukemia and promotes leukemogenesis. Monoallelic expression of CDX2 is observed in 90% of patients with AML and may perturb hematopoiesis by affecting HOX gene expression. HOXB6 Collaborates with other genes in generation of AML. Methods: KG-1 cells were treated with 10 and 20 micromolar (μM) gamma tocotrienol (γT3) and delta (δT3) tocotrienol, and 25 and 50 μM gamma tocopherol (γT) and delta tocopherol (δT) for 72 hours. Western Blot analysis was performed on the cell lysates using HOXA9, PBX1, E2A-PBX-1 HOXB6, CDX2, NUP98, CYCLIN D1 and CYCLIND3 antibodies. MTT assays were done separately using concentrations starting from 5 μM up to 80 μM of all four isoforms (γT, δT, γT3 and δT3). Results: δT3 and γT3 have downregulatory effect on HOXA9, PBX1 and CDX2 genes when used at 10 and 20 μM concentrations for 48 hours treatment; γT and δT have downregulatory effect on HOXA9, PBX1, E2APBX-1, HOXB6, CDX2, CYCLIN D1 and CYCLIND3, and an upregylatory effect on NUP98 when used at 50 μM concentration for 72 hours treatment. MTT assay showed that all four isoforms (δT3, γT3, γT and δT) have a potent inhibitory effect on KG-1 leukemic cell growth and proliferation. ELICITING PATIENTS’ CANCER STORIES AND HEALTH DISPARITIES WITH AN INTEGRATED PATIENT-CENTERED STORYTELLING INTERVIEW, AND THE USE OF THESE VIDEO RECORDED STORIES TO EDUCATE AND TRAIN HEALTH CARE PERSONNEL IN EFFECTIVE PATIENT PHYSICIAN COMMUNICATION Harsha G. Vardhana, MD, Forrest Lang, MD, Joseph Sobol, PhD, Fred Tudiver, MD, and Koyamangalath Krishnan, MD Depts. of Internal Medicine and Family Medicine, East Tennessee State University, Johnson City, TN In Southern Appalachia, a number of factors result in high rates of cancer incidence and mortality. We decided to explore and document the factors responsible for this disparity through a qualitative research method. The project involves a set of patient interviews conducted over time with cancer patients that focus on key oncology-care transition points. The interviews integrate patient centered communication (an emphasis on the patient’s ideas, concerns and expectations) with elements of the story-telling tradition. The stories they communicate will be video recorded and distributed to medical audiences through a module. In the initial phase of this American Society of Clinical Oncology (Young Investigators Award) and Appalachian Center for Translational Research Disparities (ACTRID) funded study the investigators interviewed and analyzed a pilot cohort of cancer patients. The initial beta testing began by interviewing six patients, each selected from the University cancer center. The recorded, interviews lasted between 1.5 to 2.5 hours. At this point in our beta trial, we will present and discuss our interview template that incorporates important elements of oncology care, patient-centered communication skills and the story telling tradition, initial “stories and perspectives” (in the form of videos) from the beta test that identify potential sources of factors influencing health care disparities and common themes which have evolved in our interviews highlighting the similarities and differences in the experiences and preferences of cancer patients. From our Patient-Centered/Storytelling Beta trial we are beginning to identify certain emerging themes. The themes provide moving video-audio records of patients’ experience and reactions to a set of common communication issues in cancer care. Examples include: 1) Jargon and Medical Obfuscation. Patients preferred that their physicians not talk to them in medical terms; 2) Understanding of prognostic communications. Appalachian patients prefer not to make decisions in regards to their cancer care based on numbers given in terms of response rates and overall survival from their oncologist; 3) Nursing support roles. Nurses played a crucial role in terms of offering emotional support to cancer patients; 4) Examples of insensitive communication about bad news. Patients 2008 Appalachian Student Research Forum Page 79 expressed concerns that health care providers appeared emotionally distant during the communication of bad news; 5) Emotion handling-the good the bad and the ugly. Patients have provided vivid stories in which physicians and nurses have ignored patients’ feelings, and also stories of great human compassion. 6) Role of spirituality and religion in dealing with cancer. In addition to publishing our data, we will develop and distribute a module to help practicing oncologists to communicate more effectively with patients. This will enable, physicians to reach common ground with their patients regarding cancer treatment plans. By achieving better patient-physician relationship and integration of the patient’s personal and cultural beliefs with evidence based cancer treatment we hope to reduce cancer related health care disparity. REM SLEEP BEHAVIOR DISORDER (RBD) AS AN EARLY MARKER FOR DEVELOPMENT OF NEURODEGENERATIVE DISEASES: A LITERATURE REVIEW Umesh Vyas MD, PGY III, Dept. of Psychiatry and Behavioral Sciences, James H Quillen College of Medicine, East Tennessee State University, Johnson City, TN Introduction and Hypothesis: RBD is a parasomnia characterized by intermittent loss of Electromyographic atonia normally present during REM sleep and emergence of purposeful complex motor activity associated with vivid dreams, and there is enactment of dreams. Normally, a diffuse hypotonia of muscles occur during REM sleep which inhibits motor enactment of dreams. This condition was first described by Schenck CH et al in 1986. It usually affects older males, and can be either idiopathic or symptomatic of various underlying disorders, in particular neurodegenerative disease; in later case RBD may be prodromal symptom of neurological disease. RBD can be acute which is almost always induced by medications (Multiple medications can induce or aggravate RBD symptoms and should be avoided in these patients), or chronic which can be idiopathic but most patients will eventually exhibit signs and symptoms of variety of degenerative neurologic disorder. Sleep disorders have a high prevalence especially in patients with Parkinson’s disease (PD) of particular interest is RBD. An idiopathic form may predict later development of Parkinson’s disease or other neurodegenerative disorders. Prevalence of RBD in general population is 0.5%. Its prevalence is 38% for Parkinson’s disease, 90% for Multiple System Atrophy, and 95% for Dementia with Lewy bodies. Early diagnosis of RBD is of paramount importance because of existence of effective treatment and for preventing injuries to patient and bed partner. A Polysomnogram (PSG) is necessary to diagnose RBD, showing absence of REM sleep atonia and related abnormal behavior. Method: Author has performed review of articles in pubmed.gov by using key words: REM sleep Behavior Disorder. Conclusion: RBD often antedates development of a neurodegenerative disorder. Evidence of RBD in PSG can be used to prescreen high risk patient for development of neurodegenerative disorders, especially in high risk patient population. Close follow up of patients with idiopathic RBD could enable early detection of neurodegenerative disease. This finding may be of great interest when early effective treatment strategies and neuroprotective agents become available. 2008 Appalachian Student Research Forum Page 80 2007 Award Winners Division I – Undergraduates Biomedical Sciences First Place: Regina Phillips, ETSU. Faculty Sponsor – Dr. Sharon Campbell, Dept. of Internal Medicine. Natural Vitamin E Isoforms, Gamma and Delta Modulate TGFβ in PC-3 Prostate Cancer Cell Lines Second Place: Jonathan Calloway, ETSU. Faculty Sponsor – Dr. Michael Ramsey, Dept. of Kinesiology, Leisure & Sport Sciences. Relationship Between Collegiate Throwers' Ability to Produce Maximum Force and Their Ability to Potentiate Natural Sciences & Mathematics First Place: Leslie Epling, ETSU. Faculty Sponsor – Dr. Cecilia McIntosh, Dept. of Biological Sciences. Cloning PGT-4 for Expression and Characterization as a Putative Glucosyltransferase in Citrus Paradisis (Grapefruit) Second Place: Connie Blalock, ETSU. Faculty Sponsor – Dr. Debra Knisley, Dept. of Mathematics. Fermat's Last Theorem, A Combinational Approach Social & Behavioral Sciences First Place: Ian D. Longacre, ETSU. Faculty Sponsor – Dr. Russell Brown, Dept. of Psychology. Nicotine Sensitization in Adolescent and Adult Rats D2 Receptor Primed as Neonates: Gender Differences and Neurotrophic Factors Second Place: Andrew B. Hughes, ETSU. Faculty Sponsor – Dr. Russell Brown, Dept. of Psychology. Methylphenidate (Ritalin) Exposure to Adolescent D2-Primed Rats Eliminates the Initial Hypoactive Response to Nicotine in Adulthood Third Place: Leslie King, ETSU. Faculty Sponsor – Dr. Joel Hillhouse, Dept. of Psychology. The Future Implications of Indoor UV Tanning at a Young Age Fourth Place: Matthew F. Lazenka, ETSU. Faculty Sponsor – Dr. Russell Brown, Dept. of Psychology. Age and Gender Differences in the Virtual Morris Water Maze from 12 - 25: A Fresh Look at Strategies 2008 Appalachian Student Research Forum Page 81 Division II – Graduate Students, 1-2 Years Natural Sciences & Mathematics First Place: David Hammond, ETSU. Faculty Sponsor – Dr. Ranjan Chakraborty, Dept. of Health Sciences. Characterization of Binding & Transport & Initial Identification of the Genes Involved in the Biosynthesis & Transport of Schizokinen, A Dihydroxamate Siderophore Produced by Rhizobium Leguminosarum IARI 917 Second Place: Tande Jacob Fosso, ETSU. Faculty Sponsor – Dr. Scott Kirkby, Dept. of Chemistry. A Computational Chemistry Study of Spin Traps Arts & Humanities/Social & Behavioral Sciences (combined) First Place: Jessica Brown, ETSU. Faculty Sponsor – Dr. Kerry Proctor-Williams, Dept. of Communicative Disorders. Typically Developing Children Display Two Prelinguistic Acts Pre Minute Before First Words Second Place: Zachary A. Cope, ETSU. Faculty Sponsor – Dr. Russell Brown, Dept. of Psychology. Amphetamine Sensitization in a Rodent Model of Psychosis Third Place: Brandon Phillips, ETSU. Faculty Sponsor – Dr. Christopher McCrea, Dept. of Communicative Disorders Voice Onset Time as a Clinical Indicator of Hyperfunctional Voice Disorders Division III – Graduate Students, 2+ Years First Place: Michelle Chandley, ETSU. Faculty Sponsor – Dr. Barney Miller, Dept. of Psychiatry. Identification of a Possible Functional Cre-Element Found in the Promoter of the α7 Nicotinic Receptor Subunit and its Association with Schizophrenia Second Place: Jennifer Vanover, ETSU. Faculty Sponsor – Dr. Robert Schoborg, Dept. of Microbiology. HSV-2 CO-Infection Stimulates Chlamydia Trachomatis Persistence via a Novel Mechanism Division IV – Medical Students First Place: Christopher L. Cropsey, ETSU. Faculty Sponsor – Dr. Alok Agrawal, Dept. of Pharmacology. Binding of C-Reactive Protein to Immobile Immune Complexes Second Place: Scott Cook, ETSU. Faculty Sponsor – Dr. Kevin Breuel, Dept. of OB/GYN. Relationship of Matrix Metalloproteinases with Marker of Fetal Lung Maturity Division V – Residents/Post-Doctoral Fellows First Place: Rachna Bharti, MD, ETSU. Faculty Sponsor – Dr. Burt Banks, Dept. of Family Medicine. Review of Literature on Proteinuria in Pre-Eclampsia Second Place: Umesh Vyas, MD, ETSU. Faculty Sponsor – Dr. Hetal K. Brahmbhatt, Dept. of Psychiatry. Risk of Breast and Other Cancers due to Hyperprolactinemia Caused by Antipsychotics (Neuroleptics) or Other Medications: A Literature Review 2008 Appalachian Student Research Forum Page 82 Division VI – Case History First Place: Avani Prabhakar, ETSU. Faculty Sponsor – Dr. Hetal K. Brambhatt, Dept. of Psychiatry. A Case of Ceftriaxone Induced Neutropenia Second Place: Raja S. Vadlamudi, MD, ETSU. Faculty Sponsor – Dr. Mark F. Young, Dept. of Internal Medicine. Enteroscopic Retrieval: A Possible Treatment Modality for Displaced Esophageal Stent Post-Baccalaureate Oral Presentations First Place: Stacie N. Woolard, ETSU. Faculty Sponsor – Dr. Uday Kumaraguru, Dept. of Microbiology. Involvement of TNF Receptor Apoptosis Inducing Ligand (TRAIL) in NK Cell Mediated Anti-HSV Adaptive Immune Response Second Place: Carrie Jolly, ETSU. Faculty Sponsor – Dr. J. Russell Hayman, Dept. of Microbiology. Yeast Two-Hybrid Analysis of Protein-Protein Interactions Involving a Microsporidia Adam (A Disintegrin and Metalloprotease) Protein Second Place: Alexander Eddo, ETSU. Faculty Sponsor – Dr. Dhirendra Kumar, Dept. of Biological Sciences. Characterization of the Pathway Leading to the Synthesis of Salicylic Acid in Plants Under Attach by Pathogen Resident/Post-Doc Oral Presentations First Place: Ahmad Halawa, MD, ETSU. Faculty Sponsor – Dr. Ryland Byrd, Dept. of Internal Medicine. Wegener's Granulomatosis and Purulent Pericarditis: The First Case Report 2008 Appalachian Student Research Forum Page 83 2007 Student Choice Awards Division I – Undergraduates Andrew B. Hughes, ETSU. Faculty Sponsor – Dr. Russell Brown, Dept. of Psychology. Methylphenidate (Ritalin) Exposure to Adolescent D2-Primed Rats Eliminates the Initial Hypoactive Response to Nicotine in Adulthood Division II – Graduate Students, 1-2 Years Zachary A. Cope, ETSU. Faculty Sponsor – Dr. Russell Brown, Dept. of Psychology. Amphetamine Sensitization in a Rodent Model of Psychosis Division III – Graduate Students, 2+ Years Jennifer Vanover, ETSU. Faculty Sponsor – Dr. Robert Schoborg, Dept. of Microbiology. HSV-2 CO-Infection Stimulates Chlamydia Trachomatis Persistence via a Novel Mechanism Division IV – Medical Students Scott Cook, ETSU. Faculty Sponsor – Dr. Kevin Breuel, Dept. of OB/GYN. Relationship of Matrix Metalloproteinases with Marker of Fetal Lung Maturity Division V – Residents/Post-Doctoral Fellows Umesh Vyas, MD, ETSU. Faculty Sponsor – Dr. Hetal K. Brahmbhatt, Dept. of Psychiatry. Risk of Breast and Other Cancers due to Hyperprolactinemia Caused by Antipsychotics (Neuroleptics) or Other Medications: A Literature Review Division VI –Case History Avani Prabhakar, ETSU. Faculty Sponsor – Dr. Hetal K. Brahmbhatt, Dept. of Psychiatry. A Case of Ceftriaxone Induced Neutropenia 2008 Appalachian Student Research Forum Page 84