The 14^th Annual

NC State University
Undergraduate Research Symposium

Biological Sciences Abstracts

Abstracts are listed in alphabetical order by the last name of the corresponding author.

Student Author(s):	Abrams, Joshua M.
Department(s):	Zoology
Research Mentor(s):	Craig V. Sullivan/Zoology
Title of Presentation:	Microsatellite Marker Evaluation for the Hybrid Striped Bass Industry

Farming of hybrid striped bass (HSB) is a major part of the U.S. aquaculture industry. From its inception in 1987, aquaculture of HSB grew rapidly to satisfy market demands for fresh or live fish. Growth of this industry has now ceased due to high production costs which impede product entry into broader retail markets. The HSB industry has identified selective breeding as a promising means for improving production efficiency and lowering production costs. Breeding programs for other species have demonstrated the utility of microsatellites, 1-6 base pair DNA repeat loci, as markers for tracking parentage of offspring during progeny testing. Use of microsatellites will allow HSB breeders to communally rear and test multiple progeny groups, greatly reducing space requirements and costs associated with single family rearing. To support development of a breeding program for HSB, I assessed microsatellite marker loci isolated from a striped bass cDNA library to identify polymorphic (>3 alleles per locus) markers. Polymorphism was examined using a geographically diverse screening panel of DNA samples from 15 striped bass, six white bass, and two HSB. Of 79 microsatellite loci tested, 72 (91%) amplified successfully in at least one of the two parent species, and most amplified successfully in HSB. Fifty-four markers were polymorphic in striped bass with 21 having >7 alleles. In white bass, only 23 markers were polymorphic with two having >7 alleles. Of the 72 markers successfully amplified, 28 loci were “perfect” repeats (e.g., CA)n and 44 were “imperfect” repeats [e.g., (CA)n(GTG)n(CA)n]. In striped bass, 43% of perfect repeat markers had >7 alleles while only 25% of imperfect markers had >7 alleles. Consideration of repeat type may speed identification of markers with greater polymorphism in striped bass. The 54 new polymorphic microsatellite markers identified in this project will support selective breeding efforts for the HSB industry.

Student Author(s):	Anderson, Shannon E.
Department(s):	Animal Science
Research Mentor(s):	Joe P. Cassady/Animal Science
Title of Presentation:	Phenotypic Correlations Among Measurement of Behavior, Loineye Area, Backfat and Growth in Pigs

The objective of this research is to study relationships between aggressive behavior and growth traits. Selection for improved production efficiency may result in a correlated response in behavior. Pigs in this study were housed at the Lake Wheeler Road Field Laboratory Swine Educational Unit. Data were collected on 20 litters and 147 pigs. Measures of behavior were the total attempts to struggle during two backtests (BTS) and total attacks during two resident-intruder tests (RIS). Growth traits analyzed were average daily gain from birth to weaning (ADGf), average daily gain in the nursery (ADGn), average daily gain on-test (ADG), days to 104 kg (D104), weaning weight (WWT), birth weight (BWT), backfat depth (BF), and longissimus muscle area (LMA). Data were analyzed using the GLM procedure of SAS, and a model including fixed effects of BTS, RIS, sex, and damid. All two way interactions were tested, found not to be significant, and were dropped from the final model. Finishing pen and nursery pen were included as fixed effects in the model when appropriate. Off-test weight was entered as a fixed regression covariate for LMA and BF. BTS tended to affect birthwt, ADGn, and LMA (P < 0.1). RIS affected ADGn (P < 0.05) and tended to affect D104 (P < 0.1). Damid was significant for BWT, ADGn, D104, ADG, BF, and LMA. Damid tended to have an effect on WWT. Npen had a significant effect on ADGn. Sex was significant for ADG and BF. Finpen had a significant effect on D104. Off-test weight was significant for BF and LMA. Pigs that were heaviest at birth and reached D104 faster were more likely to attack. We concluded that a phenotypic relationship exists between pig behavior and growth. Selection for increased lean gain in pigs may result in an increase in aggressive behavior.

Student Author(s):	Ansari, Sofia Harrington Ford, Collins A.
Department(s):	Botany
Research Mentor(s):	Eric Davies/Botany
Title of Presentation:	Gene Expression in Wounded Plants: Changes in Polysomes in Pea Roots

Gene expression involves the use of genes (DNA) to code for messenger RNA (mRNA) in the nucleus (transcription), transport of the mRNA into the cytoplasm where ribosomes attach to it (initiation), move along it (elongation) and are released (termination) decoding the mRNA to make a protein (translation). Several ribosome traversing the same mRNA is called a polyribosome or polysome.Earlier work from this laboratory has shown that when aged pea stems are wounded, they exhibit an increase in olysomes, but a decrease in protein synthesis. This is caused by the inhibition of movement of ribosomes along the messenger RNA. Here we are studying the effects of wounding on pea roots and show: a) the response to wounding is exceedingly rapid; b) aged (basal) root tissue responds the same way as aged stem tissue, but c) apical (tip) root tissue responds by decreasing in polysomes. We are currently conducting experiments with NaF, an inhibitor of initiation, to determine whether this decrease in polysomes is caused by a decrease in initiation.

Student Author(s):	Ballance, Christinia M.
Department(s):	Animal Science
Research Mentor(s):	Kimberly D. Ange-van Heugten/Animal Science Matt Poore/Animal Science Barbara Wolfe/North Carolina Zoo
Title of Presentation:	Endophyte Infested Tall Fescue and its Relationship to Mandible Lesions and Lowered Reproductive Performance in Thompson’s Gazelles and Impalas

Fescue is a hardy grass typically used by zoos and farmers to feed grazing animals. Despite heat stress tolerance and low maintenance needs, fescue is often infected with a toxic fungus (endophytes). Traditional livestock that consume infected fescue can experience numerous side effects including low productivity and reproductive problems. At The North Carolina Zoo, the Thompson’s gazelles and impalas, that graze on fescue have developed mandibular lesions and decreased reproductive performance. Determining the cause of these issues is crucial to better the quality of life for the animals as well as protecting the future of each species. It was hypothesized that the zoo fescue was endophyte infected and potentially responsible for the health issues. To test this hypothesis, the pasture was divided up into seven sections (A-G) and the animals were observed to see where they primarily grazed. Thirty samples were collected from each pasture section and sent to the NC Department of Agriculture and Consumer Services for endophyte infestation testing (Table 1). It was found that Thompson’s gazelles and impalas graze in the most highly affected areas (Table 1). Despite the fact that other hoofstock graze on the same pasture, mandible lesions and reproductive problems are uncommon. Although all pasture areas indicate endophyte concern levels, it is interesting that the gazelles and impalas graze on the areas of the pasture with the highest percentage of infestation. Due to these results, future research is taking place to ascertain whether specific blood parameters within exotic hofstock differ among grazing locations.

Table 1. Pasture areas frequented by selected NC Zoo hoofstock and the percent endophyte infestation of each pasture area.

Pasture area

Percent infested

100

Species

Impala

Thompson’s Gazelle

Defassa Waterbuck

Nile Lechwe

Student Author(s):	Barick, Elham C.
Department(s):	Zoology (Biological Sciences)
Research Mentor(s):	Sarah Ash/Animal Science (Husbandry)
Title of Presentation:	Cost Difference between Healthy and Less Healthy Snack Choices in NCSU C-Stores

It is clear that a nutritious diet will lead to better health, but the economics of nutrition is often not incorporated into the advice people receive about their diet. A possible reason for not buying more healthy foods is the high cost associated with them. This study looks at the cost differences of healthy and less healthy snacks on campus at NC State University. A survey of prices and nutrition of the snacks available in campus C-Stores was used to determine differences and to create a pamphlet to present the information to students. On both a cost per item and cost per serving scale, healthy snacks were not more expensive than less healthy snacks. Student feedback about the pamphlet helped to point out its effectiveness. Cost, but also nutrition and taste, is an important factor in student’s snack choices. More time and research is needed to deepen and broaden the topics covered in this study.

Student Author(s):	Barton, Katie D.
Department(s):	Microbiology
Research Mentor(s):	Michael Sikes/Microbiology
Title of Presentation:	Regulation of V(D)J Recombination and the Roles of Promoters and Enhancers

The human immune system is able to defend against a wide variety of infectious agents because its B and T lymphocytes are able to make receptors that can specifically recognize each infectious agent that they encounter. The process responsible for the diversity of antigen receptors is called V(D)J recombination. In this process, clusters of variable (V), diverse (D), and joining (J) gene segments which code for the domain of the receptor that is responsible for antigen recognition are rearranged in each developing lymphocyte. As a result, each lymphocyte makes a unique antigen receptor. Without proper recombination, the immune system would lack the antibody diversity that it needs to protect our bodies from infection. Our previous research using a TCRβ minilocus transfected into the TDR-19 recombinase-inducible B cell has shown that transcriptional promoters and enhancers in one antigen receptor locus, TCRβ, contribute to the regulation of D to J recombination. Subsequent V to DJ recombination is expected to have similar regulation. However, V to DJ recombination does not normally occur in B cells. This block is partially due to the presence of the non-TCRβ constant region (Cµ) elements. The minilocus is not helpful in studying V to DJ recombination as it is. Rather, to study V to DJ recombination in the TDR 19 cell line, the existing TCRβ minilocus must first be altered. We have isolated the Cβ from thymus DNA through PCR and constructed a rearrangement substrate by inserting the Cβ into the TCRβ minilocus using DNA ligase. To this construct, we have added the κ enhancer by a similar strategy. We have also inserted into the minilocus an unrearranged D-J segment from the TCRβ locus. This construct will be transfected into the TDR 19 cell line in order to compare rearrangement in TCRβ constructs with Cβ to TCRβ constructs with Cµ.

Student Author(s):	Bond, Alysia Mayhew, Cassandra
Department(s):	Biological Sciences
Research Mentor(s):	Fred Corbin/Crop Science Yvonna Sheldon/Crop Science
Title of Presentation:	The Confirmation of The Stability of a Soybean Cytochrome P450 cDNA in Transgenic Nicotiana tabacum

Soybean cDNAs encoding cytochrome P450 monooxygenases were isolated previously and the gene product of one (CYP71A10) was very specific for catalyzing the demethylation of selected phenylurea herbicides. The specificity of the enzyme was further defined by expression in a tobacco line. The purpose of this study was to determine the stability of the transgene and to confirm that the metabolizing properties of the cytochrome P450 had been retained through seven generations of transgenic tobacco. In addition, bioassays to confirm the stability of the cytochrome P450 cDNA were conducted by planting seeds on a sterilized Murashige and Skoog medium that contained selected rates of the phenylurea herbicide, linuron. Transgenic seedlings were completely tolerant and grew normally. By contrast, the wild-type checks became chlorotic and did not grow beyond the cotyledon stage when compared to untreated checks. In the metabolism experiments, the radiolabeled herbicides diflufenzopyr, sulfentrazone and prosulfuron and metabolic compounds were separated by thin-layer chromatography. Following herbicidal uptake and metabolism, the 14C data that were quantified by liquid scintillation spectrometry and confirmed that detoxification in transgenic seedlings exceeded that recorded for wild-type checks.

Student Author(s):	Boulter, Leah D.
Department(s):	Biological Sciences-Concentration Nutrition
Research Mentor(s):	Jill A. Barnes/Molecular Biomedical Sciences
Title of Presentation:	Effectiveness of the Chemotherapeutic Agent 17-AAG in Breast Cancer Cells Overexpressing Heat Shock Protein 70 (Hsp70)

Breast cancer is the most frequent cancer and the second leading cause of cancer death among women. Heat shock proteins (Hsps) are highly expressed in cancer cells and thus, these proteins are targets for anticancer activity. The Hsp90 inhibitor, 17-AAG, has recently entered clinical trials. An attractive feature of Hsp90 as a drug target is that it is required for the stability and function of a wide range of oncogenic proteins. Hsp70, which is also a protein chaperone, can protect cancer cells from the effects of chemotherapeutic agents. Therefore, this study was conducted to address whether or not overexpression of Hsp70 alters the toxic effects of 17-AAG in breast cancer. Doxycycline-regulated Hsp70 human MCF-7 breast cancer cell lines were used in this study. “Hsp70 On” cells demonstrate an overexpression of Hsp70 while “Off” cells express control levels. Hsp70 On and Off cells were treated with 0 (control), 50 nM or 100 nM of 17-AAG and harvested at 48, 72 and 96 hours after treatment. Hsp70 protein levels were determined using Western analysis, cell growth and viability was assed using trypan blue and flow cytometry was used to determine cell cycle distribution. Hsp70 protein levels were significantly induced in the Off cells following treatment with 17-AAG. In contrast, Hsp70 On cells showed minor induction of Hsp70. Results from cell growth/viability studies demonstrated that Hsp70 On cells showed significantly higher cell counts as compared with Off cells after 96 hours. Flow cytometry indicated G2/M cell cycle arrest in the Hsp70 Off cells after exposure to 17-AAG. However, no cell cycle arrest was observed in the Hsp70 On cell groups. These data suggest that breast tumors expressing high levels of Hsp70 may not respond as effectively to treatment with 17-AAG and could significantly impact how clinicians make treatment decisions for their patients.

Student Author(s):	Zach Bradford
Department(s):	Biology
Research Mentor(s):	Nina Allen/Botany Yue (Jeff) Xu/Botany
Title of Presentation:	A Web-interfaced Database for the Scanning Ion-Selective Electrode Technique (SIET)

It is important to understand the electrophysiological parameters of cells in organisms. The Scanning Ion-selective Electrode Technique (SIET) non-invasively measures both ionic concentrations and ionic fluxes in an aqueous medium with a spatial resolution of less than 10 micrometers and with picomolar sensitivity. The SIET measures the extracellular fluxes moving in and out of living cells. Ca2+, H+, Cl-, K+, Cd2+, Al3+, Mg2+, O2 and NO are some of the ions/molecules that can be measured. The aims of this study were: 1) to design and implement a web interfaced database which holds about 3,000 references related to SIET and to make it available on the web to SIET users allowing them to retrieve relevant information efficiently and conveniently 2) to create a foundation for future development of an Artificial Intelligence or Expert System which can help SIET users design experiments quickly, easily and with great reliability. An Entity to Relationship (E-R) model has been developed as the first step to build the database followed by table conversion. PHP server side scripting language, which can generate dynamic web pages, and the MySQL database server were adopted to implement the whole system. A web interface is being developed, which allows researchers to retrieve relevant references by either typing in the authors' name(s) or keywords of the titles.

Student Author(s):	Catalfamo, Caroline K.
Department(s):	Animal Science
Research Mentor(s):	Joseph P. Cassady/Animal Science
Title of Presentation:	The Phenotypic Relationship Between Pig Behavior and Growth in Pigs

The objective of this research was to test the relationship among two measurements of pig behavior and growth. Coping ability in stressful situations may be related to overall lean gain. Pigs (n = 147) were tested using the backtest and resident- intruder test and each test was performed twice. During the backtest a pig was placed on its back in a supine position and gently restrained for 60 seconds. Backtest score (BTS) was the number of bouts of struggling by the pig. The resident intruder test was done when pigs were between 28 and 40 d of age. A solid divider was placed down the center of the resident pig’s pen. The resident pig was placed on one side of the divider away from its penmates. An intruder pig of the same sex and smaller size was placed into the pen. When an attack occurred pigs were immediately separated, the test was terminated, and the resident intruder test score (RIS) was a 1. If after 5 minutes no attack occurred, the test is terminated (RIS = 0). RIS and BTS were uncorrelated. Total pounds of fat-free lean was affected by RIS (P < 0.04) and tended to be affected by BTS (P < 0.08). Pounds of fat-free lean gain per day tended to be affected by RIS (P < 0.07). Piglets with RIS = 2, showing more aggression, were leaner than those with RIS = 1 or 0 (P < 0.05). There was only a tendency for RIS to affect adjusted loin muscle area at 114 kg (P < 0.1). Increased backtest and resident intruder test scores were associated with increased kilograms of lean. Thus, past selection for increased lean growth in pigs may have increased aggression among pigs.

Student Author(s):	Chamberlain, Rachel E.
Department(s):	Biological Sciences
Research Mentor(s):	Peter L. Mente/Biomedical Engineering
Title of Presentation:	Type II Collagen Deterioration in Porcine Cartilage after Induced Damage

Articular cartilage degradation leads to the onset of Osteoarthritis (OA), a degenerative disease that has no cure. Type II collagen is the most abundant component of articular cartilage. Type II collagen has a triple helix structure that gives cartilage strong tensile strength and helps it function in weight bearing and joint movement. This study was conducted to examine what type of impactions causes breakage of type II collagen so that it may become possible to predict what type of injuries lead to OA. Damage to type II collagen was induced by impacting porcine patella. The tissue was analyzed immunohistochemically using the COL2-3/4m mouse monoclonal antibody that recognizes the denatured type II collagen triple helix. Human OA tissue was used as a positive control and non-impacted porcine cartilage was used as a negative control. No qualitatively significant staining was seen in the negative control. The experimental tissue showed dark brown staining near the edge of the tissue verifying that there was type II collagen damage. Porcine OA tissue was also stained and it yielded an unexpected pattern of stain with one edge darkly stained. The collagenase-treated tissue yielded a dark brown stain throughout the tissue. Dark brown staining was seen around the cracks of the tissue that was impacted multiple times and the Human OA tissue.

Student Author(s):	Coffman, Christine E.
Department(s):	Biological Sciences
Research Mentor(s):
Title of Presentation:	Coronary Calcification and Computed Tomography

A new indicator for heart disease, coronary calcification is detectable by a Computed Tomography scan. At Watson Clinic in Lakeland Florida the Cardiologists have started using the CT on patients with risk factors for heart disease. The patients involved were in the “intermediate risk group.” This research was conducted to see if more common tests for heart disease would give a patient a better evaluation of their cardiac risk. A total of eighteen different statistics including gender, age, smoking preference, family history, height, weight, body mass index, blood pressure, triglyceride level, cholesterol, blood sugar level and CT score were evaluated. All together, 239 patients’ data was taken. Next, the Framingham Risk score was evaluated, and the data was analyzed. Fifty-four percent of the men and 41% of the women had a positive CT score, meaning they had some calcification of their coronary arteries. Fifty-six percent of the patients with a positive CT score had mild calcification, 26% had moderate calcification, and 18% had significant coronary calcification. Because of this test, the patients, who would not have normally received medication, were alerted to their condition. Sixty-seven people out of the 239 tested began taking medication, while 40 modified theirs in some way. Forty-four patients went on for further testing, with two of the patients eventually receiving open heart surgery. The number of risk factors a patient had was averaged by their CT score range. It was shown that the number of risk factors had no correlation to the CT score. A patient with less than a 10% Framingham 10-year risk is considered to be in the low risk group for a cardiac event. Forty-three percent of the patients with a Framingham risk of less than 10% had a positive CT score.

Student Author(s):	Collier, Timothy S.
Department(s):	Chemistry Marine, Earth, and Atmospheric Sciences
Research Mentor(s):	Mary H. Schweitzer/ Marine, Earth and Atmospheric Sciences Jennifer L. Wittmeyer/ Marine, Earth and Atmospheric Sciences
Title of Presentation:	Characterization of Ratite Protein Biochemistry as a Baseline for Comparison to Theropod Fossils

Birds, the extant taxon most closely related to dinosaurs, have often been used to test hypotheses about aspects of dinosaurian

biology, including growth rates, biomechanics and physiology. Ratites are the most primitive avian taxon, and therefore may share more

characters with dinosaurs than more derived groups. However, ratites are less well studied than other, more commercially valuable taxa.

In particular, the reproductive tissue unique to birds, medullary bone, has not been figured or studied in the literature, although it is well

characterized chemically and morpologically in other birds. Medullary bone is a gender-specific, highly vascular, well mineralized tissue

that is deposited on the endosteal surface of long bones only during ovulation. In galliformes (chickens, quails) medullary bone is

biochemically distinct from surrounding compact bone. The goal of this study is to characterize chemical differences between medullary

and compact bone tissues in ratites. We present the first biochemical analyses performed to characterize these distinctions in ratites

(emus, ostriches). Utilizing methods such as nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and

high-iron diamine (HID) staining, we will characterize unique components in the reproductive bone tissues of ratites. The presence of soft

tissues in dinosaur bone has opened the door to the possibility that we may retrieve biochemical data with which to compare non-avian

theropods to living birds. Our data will establish a baseline for the comparison of ratite and theropod biochemistry.

Student Author(s):	Cooper, Ryan J.
Department(s):	Biochemistry Microbiology
Research Mentor(s):	Paul Wollenzien/Biochemistry
Title of Presentation:	Role of Metallothionein in Lead Toxicity

Lead is one of the most toxic metals known to man, and is widely recognized as an environmental pollutant that poses risks to humans. The most prevalent systems affected by exposure to lead include the kidneys, liver, central nervous system, hematopoetic system, endocrine system and reproductive system. A distinctive pathomorphologic characteristic associated with lead poisoning is the presence of inclusion bodies, which are frequently nuclear, roughly spherical and composed of a lead-protein complex. Although the mechanism of inclusion body formation has not been definitively established, studies show metallothionein-null (MT-I/II knockout) cells, which do not make the metal-binding protein, MT, are unable to form inclusion bodies, and show an increased sensitivity to lead toxicity. In further testing of the possible role that MT may play in susceptibility to lead toxicity, wild type (WT) and MT-I/II knockout mouse fibroblast cells were exposed to lead (200 mM) over a varying time course (4, 12, 24, 48 hours), and critical subcellular organelles and fractions were isolated and tested for lead content (nuclear, mitochondrial, microsomal, and cytosol) by atomic absorption spectroscopy. Analysis of the various cellular components showed a substantial increase in lead accumulation in the MT-I/II knockout cells in comparison to the WT cells. In particular, the MT-I/II knockout cells showed an increased accumulation of lead in the mitochondria, which is considered a key target of lead toxicity. These results further demonstrate an important role for MT expression in both resistance to lead toxicity, and accumulation of lead within cellular components.

Student Author(s):	Craft, Sarah Margaret Smith, Arianna L.
Department(s):	Animal Science
Research Mentor(s):	Audrey O'Nan/Animal Science Joe Cassady/Animal Science Melissa Ashwell/Animal Science Chris Ashwell/Poultry Science
Title of Presentation:	Evaluation of Candidate Genes Affecting Adipose Delipidation in Mice Fed Conjugated Linoleic Acid Using Real-Time PCR

The incidence of obesity is rapidly increasing in the United States. Due to this arising problem, trans-10 cis-12 conjugated linoleic acid (CLA) has gained much attention for its potential delipidative abilities. In our previous study oligo microarray were used to investigate gene expression in epididymal fat when CLA was fed to an obese line of mice. Epididymal fat tissues were collected at days 5 and 14 from 185 mice and RNA was extracted. Results from the microarray experiments indicated that over 100 genes were differentially expressed in adipose tissue. Here we have used Real- Time PCR to verify the findings for 8 of the genes. PCR primers were designed for adiponectin, adipsin, leptin, interleukin-6, resistin, cytochrome-c, B-cell Lymphoma-2, and tumor necrosis factor-α using Molecular Probe’s Beacon software. Amplification was optimized for each gene and gene expression from pooled samples was measured for adiponectin, leptin, adipsin, cytochrome-c, and resistin, using the Bio-Rad iCycler and the iQ SYBR Green Supermix kit. Real-time data were normalized using beta-actin as the reference housekeeping gene. Adiponectin, leptin, adipsin, and resistin expression levels decreased and cytochrome-c levels increased with CLA treatment and the pattern of induction or suppression was concordant with microarray findings. The magnitudes of the effects were different between the two methods but may be due to combination of day 5 and day 14 samples in data analysis and pooling of samples. Preliminary evidence indicates that these genes may play a role in adipose delipidation and current studies are underway to evaluate individual animals at all 8 genes.

Student Author(s):	Cranford, Lauren C.
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	Frank C. Church/Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill Yolanda M. Fortenberry/Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill
Title of Presentation:	Essential Thrombin Residues for Inhibition by Plasminogen Activator Inhibitor-1 in the Absence and Presence of Heparin and Vitronectin

The serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1) is a major regulator of fibrinolysis and coagulation due to its ability to inhibit thrombin. We used 55 recombinant thrombin mutants in which solvent accessible residues are replaced with alanine to determine their effect on thrombin, PAI-1, heparin, and vitronectin (VN) combinations. Results from this study identified thrombin residues that either increased or decreased thrombin inhibition by PAI-1. First, we confirmed that Glu25 (39-loop) had an enhanced rate of inhibition by PAI-1 in the presence and absence of heparin and VN. Also, thrombin residues Asn216/Asn217 (203-206 loop) and Lys145/Thr147/Trp148 (autolysis loop) showed increased rate of inhibition by PAI-1. This suggests that these three thrombin residues contribute to the slow rate of thrombin inhibition by PAI-1. Second, we identified two exosite-1 mutants, R68A and Y71A, that showed decreased rates of inhibition by PAI-1. These results suggest that Arg68 and Tyr71 are potential PAI-1 interacting residues. Third, we identified four exosite-2 mutants that are resistant to PAI-1-heparin accelerated inhibition, which implies that the thrombin residues important for anti-thrombin-heparin inhibition are also involved in PAI-1-heparin inhibition. Conversely, these exosite-2 thrombin mutants are not as resistant to VN-accelerated PAI-1 inhibition. Lastly, active site and sodium binding site mutants were very resistant to PAI-1 inhibition in the absence and presence of heparin and VN. Considering that thrombin, PAI-1, and VN are localized in the atherosclerotic arterial vessel wall, our results illustrate the importance of various thrombin domains for PAI-1 inhibition with and without heparin and VN.

Student Author(s):	Crawford, Thomas R., Jr.
Department(s):	Environmental Technology Program, Department of Forestry and Environmental Resources
Research Mentor(s):	E.G. Nichols/Environmental Technology Program, Department of Forestry and Environmental Resources
Title of Presentation:	Is Glomalin Present in Vegetated Refinery Waste?

This research focuses on the extraction of glomalin from non-vegetated and vegetated sediments contaminated with petroleum hydrocarbons from a refinery waste site and a fuel-oil impacted inland canal. Arbuscular mycorrhizal fungi (AMF) are associated with the majority of higher terrestrial plants. These obligate biotrophes, living on plant roots, produce an uncharacterized glycoprotein called glomalin. Considered a “super glue,” glomalin is pervasive in soil organic matter (SOM) and is involved in the creation of soil aggregates. When AMF hyphae are degraded, they shed glomalin from their cell walls into the soil where it binds to inorganic and organic matter forming aggregates. Glomalin may be significant to carbon cycling in soils as well as contaminant C cycling in contaminated soils and sediments. The relationship of AMF to vascular plants and the mass amounts of glomalin detected in numerous soils suggest that glomalin should be present in vegetated-contaminated media as well. Vegetated and non-vegegetated sediment samples from a petroleum refinery waste pit and fuel-oiled shoreline sediments were analyzed for glomalin content. The presence of glomalin in contaminated sediments was determined by two sodium citrate extraction methods: 1) an Easily Extractable Glomalin (EEG) and 2) a Total Glomalin (TG) extraction. Humic acid and humin were also extracted from sediment samples using sodium hydroxide. Both glomalin and humic fractions were dried and tarred to obtain mass amounts of recovery from sediments and submitted for stable carbon isotope analyses (13C and 14C). TG was present in all samples, while EEG was present in five of seven samples. The presence of vegetation in contaminated sediments increased the mass amount recoveries of glomalin. Mass amounts of TG and EEG compared to mass amounts of humic fractions resulted in six of seven samples demonstrating higher levels of glomalin present.

Student Author(s):	Criveanu , Beatrice
Department(s):	Microbiology
Research Mentor(s):	James Brown/Microbiology
Title of Presentation:	A Bacterial Predator of Bacillus?

The genus Bacillus is comprised of rod-shaped, Gram positive, aerobic, spore-forming bacteria that are

commonly found in soil. It produces one spore per cell and the spores are resistant to extreme environments. Spores

can be problematic because they release toxins that cause medical complications. So far the only way to treat these

medical conditions has been with antibiotic therapy. In this research, Bacillus cereus was isolated from a soil sample obtained

from Pullen Park, Raleigh, NC. The rod shaped cells appeared to have large storage granules that comprised most of the cell and

contained very small, motile microorganisms inside the cell. 16S rRNA sequence analysis was performed on the sample along with

electron microscopy to better understand their extraordinary cellular morphology. The electron microscopy revealed small enveloped

organisms present in the periplasmic space of the Bacillus cells and an absence of spores; presently there are no known bacterial

predators of Bacillus. These small organisms might be similar in their life cycle to Bdellovibrio, small bacterial predators that infect Gram

negative bacteria. The future goal of this research is to determine whether these small bacteria are predators of Bacillus. The

discovery of these small parasitic bacteria that inhabit the periplasmic space of Bacillus could have a tremendous impact on the way

anthrax and other spore related conditions are treated and prevented.

Student Author(s):	Doss, Jennifer F.
Department(s):	Biological Sciences Botany
Research Mentor(s):	Mariana E. Kirst/Botany Rebecca S. Boston/Botany
Title of Presentation:	Use of a Bacterial Cloning and Expression System to Produce Maize Secretory Proteins

Bacterial cloning and expression plasmid vectors provide a method for mass-producing proteins to be used in further applications such as characterization of protein activities and antibody production. In this study I was interested in producing antigens for production of antibodies against proteins in the maize secretory pathway. I chose two maize cDNAs, one encoding mannose-1-phosphate guanylyltransferase and the other encoding a heat shock 70 (HSP70) binding protein. Initially primers were designed that flanked the coding region of cDNA sequences. This region was then amplified using optimized PCR conditions and ligated into a pGEM cloning vector that would allow efficient amplification of the plasmid in E. coli. DNA was isolated from putative transformants and cleaved with restriction enzymes to verify that the fragment of interest had been cloned and to allow purification of this fragment for subsequent movement into an expression vector. The fragment of interest was then ligated into a pRSET expression vector containing a 6-His tag; this tag will bind to an immobilized nickel affinity column, allowing for protein purification. The resulting transformants were analyzed by DNA sequencing to verify that the DNA was present in the correct reading frame for proper protein translation within the cell. Work is ongoing to optimize production and purification of the recombinant protein.

Student Author(s):	Easter, David S. Daniels, Ashanti B. Good, Haley J.
Department(s):	Food Science
Research Mentor(s):	Lynn Turner/Food Science Kevin Keener/Food Science Matt Poore/Animal Science
Title of Presentation:	Economically Reducing Sweet Potato Waste Load

Moody-Dunbar, a North Carolina sweet potato processing company, generates approximately 21,600 tons of solid waste per year. Approximately $250,000 per year is spent on land application of solid waste. This company desired reduction of the solid waste generated and the investigation of alternative processing methods compared to land application. Alternatives were desired due to the increase in transportation costs, decrease in land availability for waste disposal. Sweet potato waste contains approximately 90% moisture. Moisture removal will result in a decrease in the cost of waste disposal. Water removal processes investigated included centrifugation, filtration, static compression (pressing), each without chemical modification, and acid hydrolysis. The goal of this project was to evaluate three potential waste treatment alternatives: further land application (fertilizer), animal feeding, and a value-added human food ingredient. Sweet potato waste used as human food is infeasible since the system as designed does not provide a food grade material. Additional research of each alternative resulted in focusing on the sweet potato waste being used as animal feed. The product was centrifuged at 4,000 rpms (1,912 g) to remove at least 50% of the waste weight. Natural fermentation (lactic acid) occurs in the product. A desired pH of approximately 4.5 provides a product that can be successfully used as a component of a mixed feed ration for beef cattle. The product was then thermally processed to stop fermentation (pH of approximately 4.5).

These results provide Moody-Dunbar with a mode of reusing the sweet potato waste as cattle feed, thus reducing their waste disposal cost and making it possible for the processing plant to profit by using the sweet potato waste as animal feed.

Student Author(s):	Eguiluz, Sitka
Department(s):	Genetics
Research Mentor(s):	Gregory C. Gibson/Genetics
Title of Presentation:	Molecular Evolution of a Potential Vaccine Target in the Hookworms, A. caninum and N. americanus

Infection with the hookworms Necator americanus and Ancylostoma caninum remains a burden on the health and economies of many developing nations. We have conducted a study designed to assess the level of population structure in these hookworms and to assess the variability of a candidate vaccine target gene. Sequence analysis of the mitochondrial protein-coding gene cytochrome oxydase (cox)-1 and the 3’ region of the nuclear gene that codes for the Ancylostoma Secreated Protein (ASP)-1 was performed for the canine hookworm, Acylostoma caninum from North Carolina and Maryland, and for the human hookworm Necator americanus from Bombay and Papua New Guinea. The 3’ region of the ASP-1 sequences was characterized for individual nematodes, and displays high levels of genetic variation within each population. The sequences for the cox-1 gene also vary within and between populations of A. caninum, but the degree of divergence between the two genera is less than for ASP-1. According to the findings presented in this study, there is significant variation in the 3’ region of the ASP-1 gene in A. caninum. Due to such variation, this region would not make a good target for vaccine development. Moreover, comparison with the control mitochondrial cox-1 data indicates that the variation in ASP-1 is widespread across the species and not strongly restricted geographically.

Student Author(s):	Ewing, Kayla L.
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	Clay Clark/Molecular and Structural Biochemistry
Title of Presentation:	An Investigation of the Global Structure and Protein Function of Procaspase Mutants: D3A, D3E, D3N, D175E, and D175N

Caspase-3 is an enzyme involved in apoptosis, which is the process of programmed cell death. In order to make an active caspase-3 enzyme, a proenzyme called procaspase-3 must first be cleaved at 3 specific aspartate residues within the protein. These sites include the D175, D28, and D9 positions. One current topic of investigation for this proenzyme is what particular positions are important for the protein to fold and function correctly. In order to investigate this topic, different mutations were made in the protein at the 9th, 28th and 175th positions. At each of these positions, a conserved aspartate residue was substituted with an alanine, glutamate or asparagine. The mutatants studied included the two triple mutants, D3E and D3N, as well as the two single mutants, D175E and D175N. The latter only had a single substitution at the 175th position. These particular positions were chosen because it is hypothesized that these are critical positions for caspase-3 activation. After using site directed mutagenesis to make these mutants, the mutants were over expressed and harvested in E. coli cells, and then purified. The proteins were then examined using global fluorescence assays. From these assays, it was found that the 175th position probably plays a vital role in quiescence of the enzyme. In addition, residues in the prodomain are likely important for stabilizing the dimer. Further studies should be conducted which directly investigate the effects of the 9th and 28th positions on the protein’s folding and function.

Student Author(s):	Fields, Ashley B.
Department(s):	New York University School of Medicine, Departments of Medicine and Pathology
Research Mentor(s):	Leslie I. Gold/Medicine and Pathology
Title of Presentation:	Pigmented Epithelium Derived Factor (PEDF) Inhibits Normal Endometrial Epithelial Cell Growth

Endometrial cancer is the most common cancer of the female reproductive tract, and is currently the fourth most common cancer that affects women in the United States. As a result of the frequency of the cancer, substantial research has been performed in order to understand the developmental process of this cancer with TGF-b. Previous studies have indicated that another growth factor, pigmented epithelium derived factor (PEDF) possibly plays a substantial role in prevention of angiogenesis and hyperplasia, which are predispositions to endometrial cancer development. Other studies have also shown the significance of this growth factor through PEDF-null experiments using the endometrium of mice; mice lacking the PEDF coding gene, Serpinf1, who suffered from hyperplasia and angiogenesis. Here, we observed the expression levels of this growth factor in the endometrial tissue of malignant and non-malignant endometrial tissue after treatment with PEDF. In addition to this, we also observed the inhibition rates of PEDF in a dose-dependent manner following treatment with PEDF over a four, six, or seven day period. Our results suggest that PEDF plays a role in the inhibition of malignant endometrial cell growth and PEDF expression is decreased in malignant endometrial tissue, thus suggesting that the pathogenesis of endometrial cancer may be due to decreasing levels of PEDF.

Student Author(s):	Fischer, Patrick
Department(s):	Entomology Biochemistry
Research Mentor(s):	Christina M. Grozinger/Entomology
Title of Presentation:	Neural Processing of Multi-component Pheromones

Pheromones are chemical signals used by many species to influence behavior. Pheromones generally consist of blends of chemicals working in specific ratios, which usually function synergistically. However, the molecular mechanisms by which they work are unknown. Queen Mandibular Pheromone (QMP) is released by the queen honeybee to her workers, and consists of five chemicals. Behavioral bioassays have shown no effect of the separate components; however, these assays are limited in their sensitivity. It has been shown in previous experiments that expression levels of certain genes are regulated by QMP. Thus, gene expression levels may be a more sensitive measure of the neuronal processing of each component than behavioral assays. The expression of these “marker” genes, or lack thereof, will demonstrate the effect of the individual components of QMP. Young bees were exposed to QMP and four of its major components. Relative mRNA levels of two marker genes were measured by quantitative real-time PCR. Based on the results from the behavioral assays, it was expected that only the full blend, and not the individual components, would affect gene expression. However, it was found that the individual components of QMP do significantly affect gene expression, with possible variation among different genotypes of bees. This shows that the neural processing of pheromones is more complex than previously thought. Brain gene expression is highly sensitive method for monitoring neuronal processing, and a much more direct than behavioral bioassays. Using genes as markers allows more subtle yet significant changes to be measured definitively.

Student Author(s):	Goudy, Chad J.
Department(s):	Microbiology
Research Mentor(s):	Wei Shi/ Soil Microbiology
Title of Presentation:	Microbial Profiles of Two Contrasting Wetland Soils Based on Biomass Carbon, Nitrogen, and Phosphorus Content/Ratios

Wetland soils have been acknowledged for their ability to decontaminate pollutants in agricultural runoffs, such as nitrogen and phosphorus. While soil microorganisms are the primary bio-agents that regulate element transformations and thus govern the fate of pollutants, their ecological attributes in wetland soils have not been well understood, particularly pertaining to microbial population dynamics. This study examined soil microbial biomass and soil chemical properties under contrasting soil environments. A 30-day laboratory microcosm experiment was conducted with a 2 × 2 factorial design: two soils (a mineral and an organic soil) and two soil conditions (aerobic and anaerobic). The carbon, nitrogen and phosphorus contents of microbial populations were determined with chloroform fumigation extraction; soil inorganic nitrogen and phosphorus were measured with potassium sulfate extraction. Results are expected to show a higher total biomass in the organic over the mineral soil and in the aerobic over the anaerobic conditions. The relationships of microbial biomass carbon to biomass nitrogen and to biomass phosphorus would also provide the information regarding microbial community composition. The changes in microbial population size are likely associated with the changes in soil inorganic nitrogen and phosphorus concentrations.

Student Author(s):	Halloran, Rachel E.
Department(s):	Animal Science
Research Mentor(s):	Joe Cassady/Animal Science
Title of Presentation:	The Effect of the Age of Castration on the Beef Cattle Production

The objective of this research is to determine the effect of castration at birth, 3 months of age, or at weaning on growth and carcass value. Commercial Angus (at least 75% Angus) bull calves (n = 33) were randomly assigned to treatment groups. The first treatment group was castrated at birth, the second group was castrated when the group averaged three months of age and treatment group 3 was castrated when they were weaned at an average age of 7.5 months of age. Growth traits analyzed with adjusted 205 day weight, adjusted 550 day weight, average daily gain from birth to weaning, and average daily gain from weaning to slaughter. Carcass traits analyzed with yield grade, quality grade, ribeye area, percentage kidney, heart and pelvic fat (KPH), and carcass value in dollars. The main effect of treatment was not significant for traits analyzed with the exception of adjusted 205 day weight. Adjusted 205 day weight differed among treatments (P < 0.06). Calves castrated at weaning were 22 kg heavier (P < 0.05) than those castrated at 3 months of age. All of the treatments resulted in at least a quality grade of Choice. While statistically significant differences were not found among treatments for the other traits there was a consistent pattern in that calves castrated at weaning were heavier with leaner carcasses and less marbling. Castrating calves at 3 months of age would not be recommended. At that age there is not enough testosterone being produced to stimulate growth, and calves experience stress due to castration. Thus, castration at birth when stress is expected have less of an impact or castration at weaning after calves have enjoyed some increase in growth due to testosterone is recommended.

Student Author(s):	Heyder, Gunther P.
Department(s):	Biological Sciences
Research Mentor(s):	Marianne Niedzlek-Feaver/Zoology
Title of Presentation:	Possible Advantages to Adults Overwintering in Schistocerca americana

Schistocerca americana is the only genus in the Southeast that overwinters as an adult. All other species of grasshopper overwinter as eggs or nymphs. There are two different advantages that may be proposed for this unusual life history, which has to be relatively costly in terms of energy requirements for maintenance. 1. If females also mate in the Fall, storing sperm over the winter, eggs can be fertilized and oviposited as soon as weather permits in the Spring. Since substantial amounts of protein are transferred with the sperm, females may even be able to begin to yolk the eggs during the winter. Schistocera americana is known as the bird locust and more than other species is preyed upon by birds and so faces heavy predation pressure in the spring. The quicker a female can ready eggs for laying the greater the probability she will lay those eggs before being eaten by a bird. 2. Schistocerca americana is one of the largest species of grasshopper found in the United States. They may molt into adults and overwinter, so they can finish maturing in the spring in sufficient time to find a mate and yolk eggs before Spring’s end. If they waited until Spring to develop, they could well be the last species to mate and oviposit late in the summer when conditions are not as favorable for these activities. Schistocerca americana individuals were collected at bi weekly intervals (weather permitting from September to December. The condition of the reproductive organs was evaluated, spermathecae (sperm storage organs) were then extracted, embedded in paraffin, sectioned and stained using Feulgen’s technique (dye specific for DNA and one of the few stains that will stain sperm). The presence of sperm and mature follicles favor the first advantage proposed, the lack of such the second.

Student Author(s):	Hill, Lisa M.
Department(s):	Biological Sciences
Research Mentor(s):	Joan Eisemann/Animal Science
Title of Presentation:	Porcine Hepatocyte In Vitro Response to Treatments of Differing Concentrations of Growth Hormone

Growth hormone promotes growth of lean muscle mass and partitions nutrients within the body. The liver plays a major role in the response of the body to growth hormone via the production of Insulin-like Growth Factor I (IGF-I), which directly affects the growth of the body tissues. The direct effects of growth hormone on the liver and on nitrogen metabolism in liver were explored in vitro. It was hypothesized that cultured porcine hepatocytes subjected to increasing concentrations of growth hormone would respond with production of less nitrogenous waste (urea) and increasing amounts of secreted IGF-I. The cells of two livers from healthy nursery pigs were isolated, plated, and cultured with five treatments containing different amounts of growth hormone. The cells were harvested after three days in culture. The cellular material was assessed for crude amounts of protein and DNA, and the culture media was assessed for secreted amounts of urea, protein, and IGF-I. Data obtained from these assays included urea synthesis over 3 hours, urea synthesis over 24 hours, protein to DNA ratios, and secreted IGF-I. With increasing growth hormone concentrations, the data showed that urea synthesis decreased and IGF-I secretion increased. The effects of growth hormone on in vitro liver cells support the experimental hypothesis, with the exception that concentrations of growth hormone above 500 ng/mL were shown to have a negative effect on cells via decreased levels of IGF-I secretion.

Student Author(s):	Holder, Katharine L.
Department(s):	Clinical Sciences
Research Mentor(s):	Anthony T. Blikslager/ Clinical Sciences
Title of Presentation:	Effects of Meloxicam on the Permeability of the Equine Jejunum after Ischemia

Horses have traditionally been treated with Flunixin (Banamine) for colic, which is an analgesic and non-steroidal anti-inflammatory drug (NSAID). It inhibits the COX enzymes, which are responsible for the synthesis of prostaglandins. These prostaglandins are formed in response to injury, but are also associated with normal physiologic function. In addition, prostaglandins are linked to pain perception within the nervous system. By blocking COX, the production of prostaglandins is inhibited, thus producing anti-inflammatory and analgesic effects. Different forms and functions of the COX enzyme are associated with distinct isoforms of the enzyme. For example, COX-1 is associated with the positive physiologic effects of prostaglandins, such as protection of the gastrointestinal mucosa and facilitation of the repair of ischemic intestinal tissue. COX-2, however, is associated with prostaglandins that increase blood flow and neutrophil chemotaxis as a component of inflammation. Flunixin is non-selective for COX enzymes, which means it inhibit all of the COX isoforms to decrease concentrations of prostaglandins in the tissue. However, Flunixin retards the healing of injured intestine, as well as preventing pain and inflammation. Meloxicam was chosen to test as a possible alternative to Fluxinin because it is a COX-2 specific inhibitor. Our research hopes to prove that by selectively inhibiting COX-2, desirable anti-inflammatory and analgesic properties will be retained without preventing repair of injured intestine by COX-1. The project consists of three separate trials where groups of six horses undergo surgery to simulate colic. Each group was given butorphanol to control pain. The first group served as the control group and did not receive any additional medication. The second group received Flunixin, and the third group will receive Meloxicam. Their pain was monitored using a pain score chart through the night. The following morning, their gut was collected and evaluated for permeability. I assisted with the first trial in assessing post-operative pain. This data will be used to compare to the following two groups.

Student Author(s):	Hollowell, Lucy D.
Department(s):	Animal Science
Research Mentor(s):	Sarah Ash/Animal Science
Title of Presentation:	Developing Material for an Alternative Pork Production and Marketing System

North Carolina is the second leading state in hog production in the US, but the majority of the operations are owned by vertically integrated companies, making it difficult, if not impossible, for independent producers to continue to be competitive. The NC CHOICES program, funded by the W.K. Kellogg Foundation, was created to help give independent producers a chance to begin or continue to raise hogs in North Carolina by providing them with the resources needed to raise pork in nontraditional means, such as without antibiotics, or organically. The program aims to connect the producers with the consumers by following a model similar to a CSA in which the consumer buys a share of a hog directly from the farmer at the start of the growing season, thus minimizing his economic risk. In order to do this, the consumers will view information about the products and the farms via a website. The objective of this Honors project is to gather information from the farmers to be used on the website and also to help give project coordinators information regarding the constraints faced by the farmers involved in the program. The methods used to gather this information include interviewing the participating farmers and taking pictures of them and/or their farm to be included on the website. The information gathered in the interview will be transcribed and condensed into a short article used to give consumers information about the farmer following approval by the NC CHOICES steering committee. As a result of this project, I hope to provide information to consumers that will increase their interest in the products, increasing farmer profitability. The information will also be used to examine the constraints that affect the producers in order to give the program information that will help it enlist more farmers in the future.

Student Author(s):	Holtzhausen, Alisha
Department(s):	Biochemistry
Research Mentor(s):	John Cavanagh/Biochemistry
Title of Presentation:	The Interaction between Domains in the Response Regulator Spo0A

Sporulation of Bacillus subtilis is instigated by a phosphorelay mechanism that involves various proteins and response regulators. An external signal activates the phosphorylation of the kinases KinA, KinB, KinC, KinD and KinE, which then phosphorylates Spo0F, which then phosphorylates Spo0B and lastly Spo0A. Spo0A is a two-domain response regulator with an N-terminal regulatory domain that is eventually phosphorylated as well as a DNA binding C-terminus. Upon phosphorylation of the N-terminus the C-terminus binds to the AbrB gene. Spo0A is the negative regulator of AbrB and repression of AbrB initiates sporulation. In this study NMR has been utilized to study the interactions between the N- and C- termini of Spo0A. By isotopically labeling the C-terminus, it is possible to determine its response to the presence or absence of the N-terminus. The response of the C-terminus to both the phosphorylated and unphosphorelated N-terminus is studied in order to determine whether the two domains interact differently when one domain is inactive.

Student Author(s):	Horne, Krystle S.
Department(s):	Lab of Signal Transduction, RTP and Microbiology, NCSU
Research Mentor(s):	Alyson Scoltock / National Institute of Environmental Health Sciences, RTP
Title of Presentation:	UV-C Modulation of Glucocorticoid Receptor is a Phosphorylation Event

It is well documented that ultraviolet (UV) induces multiple actions in a variety of cells via MAPK signaling. In particular, we examined the human hepatoma cells, Hep G2, to investigate UV-induced effects on the glucocorticoid receptor (GR), another important stress signaling molecule. Interestingly, we observed that the GR protein experienced an up shift in mobility on a SDS polyacrlyamide electrophoresis 1-2 hours after treatment with UV irradiation. UV-C is known to stimulate stress-induced MAP kinases in cells; we examined whether UV-C causes an activation of JNK and P38 kinases in HepG2 cells using phospho-JNK and P38 antibodies. Western blot analysis shows UV-C activates both JNK and P38. To investigate the possibility that the shift is due to phosphorylation of the GR, we utilized (L929 cells) a mouse fibroblast cell line with wild-type GR and a mutant of this which contains GR unable to be phosphorylated (A8). After UV irradiation a shift was shown in the wild-type GR and not in the phospho-mutant GR. This data suggest that the GR is in fact phosphorylated in response to UV irradiation. In conclusion, the data demonstrate that the shift in size of GR caused by UV-C is a phosphorylation event possibly due to UV activation of JNK or P38 kinases.

Student Author(s):	Houpe, Jamal S.
Department(s):	Life Sciences
Research Mentor(s):	Chamindrani Mendis-Handagama/Veterinary Science
Title of Presentation:	Genotyping Mice To Detect Anti-Mullerian Hormone Overexpressing Transgenics

Anti-Mullerian Hormone (AMH) is a 140 kDa protein produced by the Sertoli cells in the developing testis and causes the regression of the female genital duct system (Mullerian ducts) in the male fetus. AMH is also present in the postnatal male and has a function in regulating the male hormone secretion by the testis. The purpose of the present study was to genotype a litter of male rat pups to detect which pups carry the anti-Mullerian Hormone (AMH) overexpressing gene, to conduct a research project on male puberty and contraception. Phenotypically, the AMH positive and negative male pups are identical and therefore, identification of the transgenic mice by their appearance is impossible. C57BL6 female mice (normal) were mated to AMH overtransgenic male transgenic mice (heterozygous) to produce pups. At three weeks of age pups (n=8 pups) were sexed observing the external genitalia to choose the males. This step was followed by obtaining a sample of tail clips from each male pup to extract DNA and ear tagging each mouse for identification. DNA extraction was performed using a Qiagen Dneasy Tissue kit according to the manufacturer’s protocol. The isolated DNA samples were amplified by using polymerase chain reaction (PCR). Those 8 samples were subjected to agarose gel electrophoresis with 100bp ladder (standards) and known DNA samples from AMH positive & negative rats. Results showed that 3 out of 8 DNA samples taken from male pups were positive for the gene that overexpress AMH. In conclusion, this research showed that genotyping is essential and aided in detecting the AMH overexpressing transgenic mice to be used in our research project to unveil the mechanisms that control male puberty and contraception.

Student Author(s):	Hoverstad, Eric
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	Clay Clark/Molecular and Structural Biochemistry
Title of Presentation:	The Effect of Different Amino Acids on Procaspase-3 Activity and Maturation

Caspases, cysteinyl aspartate-specific proteases, are critical enzymes that play a role in programmed cell death, or apoptosis. Three sites within the procaspase-3 enzyme are cleaved during maturation, aspartate 9, 28, and 175. At aspartate 175, the procaspase-3 enzyme is cleaved, between its large and small subunit, as the procaspase-3 zymogen forms the active caspase enzyme. The enzyme now serves as a destructive protease, cleaving structural proteins, DNA repair enzymes and other substrates necessary for cell viability. In our study, we changed the aspartate 175 residue into glutamate and asparagine. Alanine was also studied. Using site directed mutagenesis, mutant DNA was created. Mutant procaspase-3 enzymes were expressed through transformation into competent cells. Protein purification was used to collect the protein for analysis. The activities of these enzymes were studied using a fluorescence assay. It was demonstrated that alanine, in procaspase-3, replacing aspartate at 9, 28, and 175, was active. However the activity was 200 fold less than that of the wild type procaspase-3 zymogen. The glutamates and asparagines did not appear to have any measurable activity. Single mutants using glutamate and asparagine at aspartate 175 did not show activity. Activity in the D3A mutant may be due to an inability to hydrogen bond, thus keeping the active site open. The capability of glutamate and asparagine to hydrogen bond may cause the inactivation the zymogen by closing the active site. In any substitution, maturation of the Procaspase-3 enzyme did not occur. Through this study, we can understand how activity is affected at this specific amino acid site. This can be used for potential drug targets to inhibit or activate the caspase enzyme. Controlling the activity of this enzyme opens the door to prohibiting harmful cellular processes caused through diseases and infections.

Student Author(s):	Huening, Jessica J.
Department(s):	Psychology
Research Mentor(s):	Jane Lubischer/Zoology
Title of Presentation:	Does Excessive Axonal Sprouting Cause Terminal Disruption in Rat Motoneurons?

Motoneurons innervate muscles through branches, called terminals, at the end of their axons. These terminals match the pattern of acetylcholine receptors present in the muscle fiber membrane. When a muscle is partially denervated (i.e., some motoneurons are removed) the remaining motoneurons grow terminal sprouts to compensate for this loss. Partial denervation of muscle occurs in some diseases, such as Lou Gehrig's disease (ALS). In developing muscles, motoneurons appear to lose terminal branches after partial denervation, even while they grow sprouts from along the length of their axons. One hypothesis is that this loss of terminal branches, or terminal disruption, results from excessive growth of sprouts elsewhere along the axon. The current study tests this hypothesis by inducing axonal sprouting using a manipulation other than partial denervation. We induced axonal sprouting by cutting nerves that branch from the rat soleus nerve (at 14 days of age) while leaving the soleus nerve intact. To view motoneuron terminals and acetylcholine receptors in the soleus muscle we used specific antibodies to label terminal branches and bungarotoxin to label acetylcholine receptors. Terminals were considered to be disrupted if they no longer fully covered the underlying acetylcholine receptors. We found that these experimental nerve cuts resulted in significant terminal disruption (compared to sham operated controls). Interestingly, this effect appeared to be stronger in females than in males. Future work will directly test this possible sex-difference in terminal disruption. We will also quantify the amount of axonal sprouting induced by our manipulation in order to correlate the extent of sprouting with terminal disruption.

Student Author(s):	Hunt, Priscilla K. .
Department(s):	Botany
Research Mentor(s):	Wendy F. Boss/Botany
Title of Presentation:	Characterization of T₃ Arabidopsis Plants Overexpressing Lipid Kinases

Phosphatidylinositol-(4,5)-bisphosphate (PtdIns(4,5)P₂) is one of the main components in phosphoinositide (PI) signaling and a precursor of the second messenger inositol-(1,4,5)-triphosphate (InsP₃). PtdIns4P 5-kinase (PIP5K) catalyzes the synthesis of PtdIns(4,5)P₂, which in turn is cleaved to form InsP₃. InsP₃ is an important second messenger that triggers the release of calcium from intracellular stores. Because the production of PtdIns(4,5)P₂ by PIP5K has been considered the rate limiting step in the release of intracellular calcium stores, we are trying to increase the activity of PIP5K by overexpressing the gene in Arabidopsis thaliana. Our hypothesis is that by overexpressing this kinase we will be able to alter the way a plant responds to many external signals, such as drought or gravity. We transformed Arabidopsis plants by Agrobacterium tumefaciens mediated transformation with an Arabidopsis PIP5K1, AttPIP5K10, and a type I human PIP5K α. The plants were grown in the NCSU Phytotron and the third generation transformants selected and used for morphological and RNA analysis. Specifically, we used RT-PCR to confirm the expression of the Arabidopsis and human kinases. To assess potential phenotypic differences, we measured root lengths of seedlings grown on agar and examined leaf structures of adult plants. The plants overexpressing human PIP5K had shorter primary roots and smaller leaves than the plants overexpressing the Arabidopsis PIP5Ks. Plants overexpressing AtPIP5K1 had no differences in root length but had wider, lobed leaves. AtPIP5K10 plants had similar morphologies to wild-type plants.

Student Author(s):	Job, Lauren E.
Department(s):	Animal Science
Research Mentor(s):	William L. Flowers/Animal Science
Title of Presentation:	Spermatogenesis and Reproductive Capabilities of Boars Exposed to Varied Nursery Conditions

Previous observations have suggested that the key developmental stage for boars occurs within the first three weeks of life. This study was designed to test reproductive characteristics of boars (n=36) exposed to lactation environments (the conditions during the first three weeks of life) of 6 or less piglets nursing each sow (low litter size)or greater than 10 piglets nursing each sow (high litter size). The two treatment groups, one consisting of male piglets farrowed in the spring (n=18) and one consisting of male piglets farrowed in the fall (n=18), were randomly placed with either a low litter or high litter, the high litter being the control because it mimics standard production practices (n=9/treatment/season). Post weaning, the boars were raised in identical conditions. The boars were trained for collection at 6 months of age. After training, the boars were collected every week for a 36 month period. The semen collected from each boar was analyzed for motility, morphology, acrosome activity, acrosome morphology, and number of sperm cells present per ejaculate. Tests for the presence of two fertility proteins in the semen were conducted using 3-D Gel methods; and heterospermic insemination and the use of DNA-fingerprinting was used to isolate the boars that were more likely to father piglets.

An interaction between treatment and season was observed (p<0.05)for most of the semen characteristics. Boars reared in small litters had more spermatozoa of higher quality than boar raised in large litters. However, the advantage of the boars raised in small litters was much greater for those born in the spring than their counterparts born in the fall. These data indicate that being raised in a small litter during lactation enhances adult reproductive characteristics in boars.

Student Author(s):	Johnson, Meredith C.
Department(s):	Animal Science
Research Mentor(s):	Theo van Kempen/Animal Science
Title of Presentation:	The Effect of Dietary Fiber on the Perceived Odor of Swine Manure

An odor test was conducted with swine farmers, rural, and city residents to evaluate their response to the odor of swine manure. Fifteen barrows were fed three different diets that varied in their fiber levels. Their manure was collected and half was immediately frozen and half was left undisturbed to age for three weeks. Once half the samples had aged, volunteers differing in their age, sex, and exposure to swine rated the samples on their intensity, irritation, and pleasantness. The participants also tried to differentiate the unlike sample in groupings of three samples, then specified whether the different samples smelled better or worse than the other two. This method of testing allowed both the fiber levels of the pigs’ diets and the freshness of the manure to be evaluated. An identical set of manure samples were sent to Duke University and were evaluated by a professional odor panel. The trained panel showed that there were significant effects of diet composition and manure age on odor. However, the non-trained panel failed to detect such differences. The most important piece of data gathered from this trial was the observation of the human participants’ reaction to the waste. People appeared to smell with their emotions and not their noses, so even if there were significant differences in the smell of the waste from the different diets, the people were unable to detect them because they couldn’t get past the fact that they were smelling manure. These data thus suggest that altering the diet of pigs to reduce the irritation and the intensity of their waste may not be the most reliable way to reduce odor complaints.

Student Author(s):	Johnson, Serrin R.
Department(s):	Animal Science
Research Mentor(s):	Matthew H. Poore/Animal Science
Title of Presentation:	Castration and Concentrate Feeding Influence Performance and Carcass Characteristics of Meat Goats

The recent growth in the meat goat industry demands standards for optimal production practices. This project studied the post-weaning growth of buck or wether kids fed orchardgrass hay only (Hay; 10.6% CP, 39.9% ADF, 0.4% Ca, 0.3% P) or hay plus Cooperative Research Farms Pelleted Meat Goat Ration (17.9%CP, 15.8% ADF, 1.2%Ca, 0.61% P). The concentrate was either hand-fed at 2% of body weight (HF) or fed free-choice (FC). The goats (at least 75% Boer) were born at the NCSU Small Ruminant Unit and were randomly selected to be bucks or wethers. After weaning, 18 bucks (initial wt 19.7 kg) and 18 wethers (initial wt 17.0 kg) were divided evenly among the 3 treatments in a 2x3 factorial arrangement. The goats were fed for 85 days and harvested on day 86, at approximately 6 months of age. There were few interactions so only main effects are presented. Bucks gained faster (P<.01) than W (0.138 vs 0.105 kg/d) and rate of gain for all 3 diets differed (P<.05; 0.048, 0.109, and 0.207 kg/d for H, HF, and FC, respectively). Total DMI was higher (P<.02) for B than for W (800 vs 685 g/d), and total DMI for all 3 diets differed (P<.05; 503, 731, and 992 g/d for H, HF, and FC, respectively). Bucks had a higher gain:feed (P<.02) than W (0.158 vs 0.142) and the gain:feed for all 3 diets differed (P<.05; 0.096, 0.148, 0.208 for H, HF, and FC, respectively). Hot carcass wt (CW) was higher (P<.01) for B than W (15.7 vs 13.0 kg) and CW for all 3 diets differed (P<.05; 8.98, 14.3, 19.7 kg for H, HF, and FC, respectively). The study showed bucks outperformed wethers and that FC produced the highest level of performance and the most desirable carcasses.

Student Author(s):	Johnston, David A. Pope, William W.
Department(s):	Food Science
Research Mentor(s):	Tyre Lanier/Food Science Brad Wright/Food Science
Title of Presentation:	The Gel Forming Ability of Calpains

Gelation of proteins is important to texture development in many food products. Most muscle protein gels are prepared by heating after solubilizing the protein by addition of 2-3% NaCl.

Calpain is a Ca2+ dependent cysteine protease which can degrade the muscle proteins (desmin, titin, and nebulin) that bundle the myosin fibers together in the sarcomere. Once calpain has cleaved these ultrastructural proteins, the myosin molecules are freed to better disperse. The hypothesis of this work is that by treating myofibrils with calpain the increased dispersability will allow for better gel forming ability.

Crude enzyme was extracted from fresh king mackerel dorsal muscle. The sarcoplasmic proteins were collected and dialyzed. The dialyzed material was purified with column chromatography and fractions were tested for activity using UV spectrophotometry. The crude enzyme was then injected into isolated myofibrils from minimally processed chicken breast muscle and incubated overnight. Gels were made with 2% salt (78% moisture) and cooked. The corresponding gels were subjected to torsion testing to measure stress and strain values at fracture as an indication of gelling ability. Statistical evidence (p=0.05) showed that gels treated with the enzyme extraction were stronger and more elastic than gels prepared from untreated meat.

Student Author(s):	Jolley, Laura J.
Department(s):	Zoology
Research Mentor(s):	Nick M. Haddad/Zoology
Title of Presentation:	Does the Presence of Men in HIV Prevention Classes Hinder Women’s Ability to Learn about HIV Prevention in Sub-Saharan Africa?

Since the first publicized emergence of the HIV virus in the early 1980s, AIDS has claimed more than 30 million lives. Today it is estimated that 45 million people are infected worldwide. Over half of those infections come from Sub-Saharan Africa. Currently, women are making up the highest number of new infections; this is especially true for new HIV infections in Sub-Saharan Africa. Biological as well as cultural factors put women in developing nations at a much higher risk of becoming infected. The primary goal of this research is to determine whether a co-ed or an all female environment is more effective in teaching women of a developing nation about HIV prevention and education. Two HIV prevention classes were taught at Junior Secondary Schools in Ghana, West Africa. The female students of the classes were evaluated on the amount of HIV prevention knowledge gained during the class. The presence of the course significantly increased the knowledge base and understanding of the female students in both the co-ed and all-female class. However, the young women in the all-female class improved significantly more than the women in the co-ed class. Therefore, it can be concluded that the presence of males in the class did significantly hinder the females learning ability in this study. Further research can be completed to determine the generality of these findings by using a larger sample size and a simple random sample from several schools in the area, and then comparing the knowledge gain of the young women. Since the young women in a segregated environment gained significantly more knowledge than when their male counterparts are present, recommendations include incorporating more segregated HIV prevention programs into Sub-Saharan Africa.

Student Author(s):	Jones, Christopher I.
Department(s):	Biochemistry
Research Mentor(s):	Paul F. Agris/Biochemistry
Title of Presentation:	Modification-assisted and Mg2+-dependent Folding of Mitochondrial tRNAs

RNA transcription followed by sizing, modification and folding generates the mature, functional form of a tRNA. Whereas

transcripts of cytoplasmic tRNAs fold into the familiar L-shaped tertiary structure in vitro, mitochondrial tRNAs (mtRNA) transcripts are

known to improperly fold. Mis-folding of mtRNA transcripts may be due to their adenosine and uridine rich nucleoside composition and

lack of posttranscriptional modifications. In addition, many mtRNA have a truncated or missing dihydrouridine (D) stem and/or loop which

in cytoplasmic tRNAs is involved in tertiary structure folding interactions of hydrogen bonding and Mg2+ bridging with the ribothymidine (T)

stem and loop domain. Using gel mobility shift analyses, and UV, circular dichroism and NMR spectroscopy and aminoacylation of the

tRNAs with cognate synthetase, we investigated the folding interactions of chemically synthesized and site-specifically modified

mitochondrial and cytoplasmic tRNAs. We have determined that Mg2+ is critical to the folding interaction of the truncated D-domain of

human mtRNAMet with the tRNA’s T-domain. Contrary to the expectation that Mg2+ stabilizes RNA folding, the mtRNAMet D-domain

structure was unfolded and relaxed, not stabilized, in the presence of Mg2+. Mg2+ had little to no affect on the folding interaction of a

mtRNALeu, nor of a cytoplasmic tRNALeu. The functional folding of all three tRNAs was enhanced by the conserved, but different, modified

nucleosides, pseudouridine and dihydrouridine. Because the D-domain is transcribed prior to the T-domain, we conclude that mtRNAMet

mis-folding of the 5'-half of the molecule is abrogated in the presence of Mg2+ and that conserved modifications facilitate formation of

mtRNA native tertiary structure, in general.

Student Author(s):	Jones, Lindsey C.
Department(s):	Biochemistry and Microbiology
Research Mentor(s):	Barbara Sherry/Molecular Biomedical Sciences
Title of Presentation:	An Investigative Study of Reovirus Induction of Interferon-alpha in Cardiac Myocytes

The purpose of this study is to examine the role of interferon-a (IFN-a) in the cardiac immune response to viral infection. Viral myocarditis is an important disease to characterize because of the myocyte’s inability to regenerate. Evidence from previous studies suggests that the loss of myocytes due to infection is a permanent condition, while fibroblasts can continue to divide and differentiate. Due to the destruction of cardiac myocytes, it is important to understand their ability to protect themselves from viral attack via an immediate IFN response. Other studies have suggested that IFN-β is an important defense mechanism for cardiac myocytes; however, no studies have shown that IFN-α is also up-regulated. The characterization of IFN-α induction was done through the use of Real Time PCR amplification. Primer sets were designed for each known IFN-α subtype and their validity was assessed using L cell DNA. The primer sets were then used in a series of RT-PCR reactions in order to compare IFN-α in mock-infected myocytes and reovirus-infected myocytes. It was determined that viral induction of IFN-α was in fact up-regulated for two IFN-α gene subtypes, IFN-α 4 and IFN-α 2. These results are important because previous studies have indicated that myocytes may not be able to generate IFN-α as a defense mechanism against viral attack. While the results of this experiment suggest that IFN-α induction by some IFN-α gene subtypes is possible, further investigation is necessary to solidify these findings. These results are significant in that this is the first time that IFN-α induction has been seen in cardiac myocytes due to infection with any virus.

Student Author(s):	Kaminski, Brian E.
Department(s):	Biomedical Engineering
Research Mentor(s):	Kimberly Raiford/Molecular Biomedical Sciences Linda D. Martin/Molecular Biomedical Sciences
Title of Presentation:	Interleukin-13 Induces Changes in the Phosphorylation State of Cofilin in Human Airway Epithelial Cells

Injury and repair of the airway epithelium is an on-going process in allergic asthma. Interleukin-13 (IL-13), a central mediator of allergic asthma, may play a role in this process as it induces proliferation of airway epithelial cells via the autocrine action of transforming growth factor alpha (TGF-alpha) (Booth et al. Am J Respir Cell Mol Biol 25:739-743, 2001). This autocrine loop occurs when IL-13 activates the movement of intracellular stores of TGF-alpha to the apical surface of airway epithelial cells. This study seeks to determine whether IL-13 induces changes in the actin cytoskeleton of airway epithelial cells, as polymerization of actin would likely be needed for movement of TGF-alpha. Specifically, effects of IL-13 on the phosphorylation state of cofilin were examined in primary human airway epithelial (NHBE) cells and in the human bronchial epithelial cell line, HBE1, grown in air/liquid interface culture (differentiated) or submerged (undifferentiated). (Cofilin is dephosphorylated to the active form, increasing the rate of actin depolymerization.) IL-13 induced proliferation and release of TGF-alpha in HBE1 cells (10ng/ml; 24 hrs), events observed previously in differentiated NHBE cells. NHBE and HBE1 cells were then exposed to IL-13 (0.1; 10ng/ml) for 15, 30, 60, or 120 min, with total cellular proteins examined by Western blot to determine the phosphorylation state of cofilin. IL-13, in a concentration-dependent manner, induced dephosphorylation of cofilin by 15 min only in differentiated NHBE or HBE1 cells. Phosphorylation returned to control levels by 60 min. This dephosphorylation of cofilin occurred just prior to the observed increase in TGF-alpha release (maximal at 1 hr). In conclusion, IL-13 can induce dephosphorylation of cofilin in airway epithelial cells, suggesting that TGF-alpha release likely requires cytoskeletal changes involving actin. Furthermore, since this dephosphorylation occurs only in differentiated cells, epithelial cells at different stages of repair may respond differently to IL-13 present in inflamed airways.

Student Author(s):	Karns, Gabriel R. Snow, Robert H. Anderson, Carolyn J. Horne, Michael B.
Department(s):	Wildlife Sciences
Research Mentor(s):	Richard Lancia/Wildlife Sciences
Title of Presentation:	Avian Population, Feeding Trend, and Hierarchial Studies of the Improved Turner House

We placed bird feeders in the front and backyards of Turner House (Headquarters of the NCSU Fisheries and Wildlife Program) to assess bird use of the feeders and to document change in bird species and abundance while the backyard habitat was re-created by planting native plant species. We developed and installed a digital camera monitoring system to record bird use of the feeders, and we conducted timed visual observations. We also banded eight goldfinches (along with 4 other bird individuals of other species) on 17 March so we could determine whether the same or new individuals were using the feeders. We monitored the feeders from early February until early April, 2005, and found a large and diverse bird population present at Turner House. We observed 1,250 individual visits to the bird feeders by 19 species. We also determined seed preferences. Of 626 observed visits by American goldfinches, all but four of those visits were made to the thistle tube feeder. On the other hand, common grackles were seen making visits to four of the five feeders. We calculated total bird visits and charted the activity from February through April. The "sightings per hour rate" was largely random, and the two peaks that we saw were not easily attributed to an identifiable factor. Thus, bird use did not seem to change over this time period. Based on observations per hour, 50% of the population was composed of American goldfinches, 22% house finches, and 11% were Yellow-rumped warblers. The remaining 17% of the observations were from 16 species on our diversity list. Thus, even in an urban environment with little wildlife cover because the new plantings were just getting established, we observed a diversity of bird species using the feeders.

Student Author(s):	Karriker, Brian D.
Department(s):	Photobiology/Photochemistry Department, NIEHS/NIH
Research Mentor(s):	Colin Chignell/ Photobiology/Photochemistry Department, NIEHS/NIH Piotr Bilski/ Photobiology/Photochemistry Department, NIEHS/NIH
Title of Presentation:	Quenching of Singlet Molecular Oxygen in Protein Milieu: Contributions from Separate Amino Acid Residues

We have investigated how protein environment may affect ¹O₂ production and reactivity using rose bengal, methylene blue and perinaphthenone as photosensitizers. Proteins account for most of the dry mass in cells and tissues, thus cellular oxidation associated with oxidative stress must occur predominantly in a protein environment. It is plausible that this environment can modulate the reactivity of singlet molecular oxygen (¹O₂). Initially we measured the quenching rate constants of ¹O₂ by all individual amino acids (AA) found in proteins. While simple aliphatic amino acids were very poor ¹O₂ quenchers, quenching increased for sulfur containing AA and was most efficient for aromatic AA. As model proteins, we chose albumin, immunoglobulin (IgG’s), and histone. If produced by added photosensitizers, ¹O₂ can be detected by its infrared phosphorescence in the protein environment, and ¹O₂ reactivity can be accurately assessed. The quenching rate constants for ¹O₂ quenching in D₂O by proteins were small: 8.7x10³ (mg/ml)^-1s^-1 for histone, 7.6x10³ (mg/ml)^-1s^-1 for IgG, and 5.5x10³ (mg/ml)^-1s^-1 for albumin. The ¹O₂ production and quenching reflected the heterogeneous nature of proteins and types of AA residues present in the vicinity of ¹O₂ production. We hope our approach will help to better model and understand ¹O₂ reactivity in protein environments.

Student Author(s):	Kim, Sarah N.
Department(s):	Microbiology
Research Mentor(s):	Jonathan Olson/Microbiology
Title of Presentation:	Characterization of Factors Influencing Cell Shape in Campylobacter jejuni

Campyobacter jejuni is a gram negative spiral bacteria that is the major cause of bacterial food poisoning in

the United States.  While the physiology of C. jejuni experiencing rapid growth has been studied extensively,

little effort has been directed at studying the physiological processes that occur when the bacterium is found

in sub-optimal growth conditions.  When C. jejuni senses environmental stress or starvation, it quickly

transforms to a metabolically inert form, marked by a loss of spiral cells and the appearance of non-motile

coccoid cells. In order to study the molecular basis of the transition,  different methods of analysis were

conducted.  First, a culture was grown for an extended period of 27 days, and the optical density and the

number of Colony Forming Units (CFU) were recorded daily.  There was a result of a steady decrease in

CFU from day one, which correlated to a switch from the spiral to the coccoid morphology.  Proteomic

analysis was used to identify proteins that are expressed under conditions that promote the spiral to coccoid

transmission.  Two-dimensional gels were used to identify protein spots that are differentially expressed

under the various growth conditions.  We are also characterizing MreB, a protein shown to influence cell

shape in other bacteria such as Escherichia coli.  The mreB gene was amplified by polymerase chain

reaction (PCR) and cloned into an E. coli vector.  We plan on disrupting the coding region with a

Campylobacter specific antibiotic resistant cassette with the aim of producing a C. jejuni mreB mutant.

Student Author(s):	Kirkley, Benjamin G.
Department(s):	Biological Sciences Spanish Language
Research Mentor(s):	Larry Grand/Plant Pathology Caroline Vernia/Plant Pathology
Title of Presentation:	Evaluation of Large Trees for Wood Decay on Campus: Case Study of Selected Old Growth Oak Trees On NCSU Main Campus

The NC State campus has numerous populations of trees, and among them a very abundant oak population. The goal of the project was to determine and evaluate the occurrence of wood decay in large oak trees in selected areas on the north campus of NC State University. The nineteen oak trees selected were at least 24 inches in diameter at breast height (dbh) and had either current fungal structures or potential for fungal development. Each tree was evaluated and designated on a campus map found below. The fungi present were photographed, identified and examined to determine the specific wood decay present. Based on decay characteristics and damage estimation, an assessment of the viability of each tree was made as well as a recommendation for future protocol concerning the tree (i.e., measures to be taken to remove any danger or restore the stability and fracture-safety of the tree).

Student Author(s):	Lamb, Kristy L.
Department(s):	Microbiology
Research Mentor(s):	Michael L. Sikes/Microbiology
Title of Presentation:	Engineering a Recombinase Inducible T Lymphocyte Line

B and T lymphocytes must be able to express a wide repertoire of antigen receptors in order to effectively recognize the enormous diversity of unique antigens encountered. To create such range, B and T lymphocytes undergo highly regulated rearrangement of variable (V), diversity (D), and joining (J) segments of their antigen receptor genes. The final result of this DNA splicing process is a unique VDJ segment that encodes the variable portion of an antigen receptor. Recombination is dependent upon the dimeric recombinase enzyme RAG (recombinase activating gene-1 and –2). RAG binds recognition sequences (RSs) flanking each V, D, or J segment but these RSs must be made accessible to the RAG proteins. Evidence suggests this accessibility is regulated by transcriptional enhancers in each antigen receptor gene. By using a scaled-down TCRβ locus inserted into the chromatin of a recombinase-inducible B cell, our lab previously showed that transcriptional promoters must also be active for D to J rearrangement of the TCRβ antigen receptor gene to occur. A similar role for promoters in regulation of Vβ to DJβ, however, is not currently known. In order to investigate the potential roles of promoters and enhancers in V to DJ rearrangement, we first sought to construct a recombinase-inducible T cell line that would allow us to measure RAG-1 expression and subcellular localization separately from that of RAG-2. These observations are made possible by fusing fluorescent tags to the N-terminus of the RAG genes. We have previously created a Green Fluorescent Protein (GFP)-RAG2 fusion vector. My creation of the Destabilized Red (DSRed)-RAG1 construct and subsequent stable transfection of the construct into our current T cell line provides a platform off which investigation of the role of promoters and enhancers in the V to DJ recombination can now begin.

Student Author(s):	Landreth, Emilie V.
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	John Cavanagh/Molecular and Structural Biochemistry
Title of Presentation:	The Inter-Domain Interaction in Bacillus subtilis Spo0A

Sporulation of Bacillus subtilis is initiated by a complex signal transduction pathway referred to as a multi-component phosphorelay. Following a signal indicating environmental hostility, one (or more) of five kinases (KinA, KinB, KinC, KinD and KinE) becomes phosphorylated. The kinase then phosphorylates Spo0F, which then passes the phosphate group to Spo0B. Spo0B then phosphorylates the master control regulator protein Spo0A. SpoOA is the negative regulator of the protein AbrB, which is responsible for an array of processes in the cell at this time. Repression of AbrB ensures the sporulation cycle is successfully entered. Comparative genomic studies have shown that this phospohrelay is present in Bacillus anthracis-the bacterium responsible for anthrax spores and the causative agent of anthrax infection. Here, the interactions between the N- and C-terminal domains of Spo0A are studied. Spo0A has an N-terminal regulatory domain that gets phosphorylated and a C-terminal output domain which is a DNA binding domain. Upon phosphorylation of the N-terminus the C-terminus binds to the AbrB gene. Sizing techniques and NMR methods are used to help identity the location of the inter-domain interface in Spo0A. This study addresses questions concerning the influence of one domain over the other in two-domain response regulators and whether isolated domains exhibit characteristics of the full length protein as well as the exchange of information between domains and therefore the mechanism of activation.

Student Author(s):	Lassiter, Christopher L.
Department(s):	Microbiology
Research Mentor(s):	Lisa Lyford/ Biotechnology Program
Title of Presentation:	Development of a Laboratory Course in Molecular Site-Directed Mutagenesis

The rapidly changing field of Biotechnology requires new courses that address ever-emerging new technologies. A new course (BIT 495E/595E Mutagenesis) in the Biotechnology Program was developed to teach practical methods of mutagenesis. Site-directed mutagenesis is powerful tool for altering DNA molecules at specifically targeted sites. Two current and widely used methods, Inverse Polymerase Chain Reaction (PCR) and Overlapping Extension PCR, were developed into laboratory modules. We used these methods to create mutations in two different genes encoding the alpha7 nicotinic receptor, and the inward rectifier potassium channel (IRK1). We designed mutgenic oligonucleotide primers to incorporate specific mutations in these genes. In the IRK1 gene, the amino acid arginine at position 218 was mutated to a stop codon (nonsense mutation). In the alpha7 gene, the amino acid phenylalanine at position 123 was mutated to cysteine (missense mutation). In each case, diagnostic restriction sites were added or removed in the process of creating the mutation. We used restriction digestion with restriction endonucleases to confirm that the mutations were correctly introduced. The techniques learned in this course can be applied in a variety of academic or industrial research projects.

Student Author(s):	Lee, Cyrus A.
Department(s):	Biochemistry
Research Mentor(s):	Steve Guest/UNC Dental School Gregory Essick /UNC Dental School
Title of Presentation:	Parotid Salivation Evoked by Thermal Stimulation of the Tongue

Parotid salivation has been shown to be influenced by the temperature of liquids held in the mouth. Here we investigated the role of a dry thermal stimulus, positioned statically on the anterior tongue, in changing the resting rate of salivation. Methods: Parotid saliva was collected using a Lashley Cup from 9 adequately-hydrated subjects. Thermal stimuli were delivered using a copper tube through which temperature-controlled water flowed. During separate trials, the tube stimulus was 10, 22, or 44ºC, or the resting temperature of the tongue or hypothenar of the hand (control site). Temperature either increased or decreased from trial to trial during each session. During each trial, the resting salivation rate was measured for 6 minutes while the subject remained seated with the mouth closed. Subsequently, salivation was measured for 6 minutes during application of the thermal stimulus. The stimulus device was then removed for 3-6 minutes. The trial was repeated for the remaining three test temperatures. The two body sites were investigated on separate days. Results: Resting salivation rate averaged 0.06 grams/minute, and prior to thermal stimulation did not vary with the body site (p > .24) or temperature (p > .43) that was subsequently tested. However, the resting salivation rate changed upon thermal stimulation, with temperature and body site having an interactive effect (p < .05). On the tongue the 10ºC stimulus increased salivation by 194% (p < .03). The mouth-temperature stimulus decreased salivation by 55%, although this decrease did not attain statistical significance (p < .11). None of the thermal stimuli applied to the hand significantly altered salivation (all four p-values > .18). Conclusion: These results demonstrate that temperature-evoked changes in parotid salivation do not require the unique spatiotemporal dynamics of a liquid wetting the oral mucosa. Supported by student research funds at the Dental Research Center, School of Dentistry, University of North Carolina at Chapel Hill.

Student Author(s):	Luk, Francis C.
Department(s):	Biochemistry
Research Mentor(s):	Jay J. Cheng/Biological and Agricultural Engineering Melanie A. Head/Biological and Agricultural Engineering
Title of Presentation:	Intermittent Aeration of Anaerobically Pretreated Swine Wastewater

The swine industry is a major enterprise that has several waste-treatment challenges. Swine wastewater contains a high concentration of nutrients that can be used for plant growth but can become a pollutant when over applied. One of the potential pollutants present in anaerobically pre-treated swine wastewater is ammonia. Under aerobic conditions, ammonia can be converted to nitrate by nitrifying bacteria, a process called nitrification. To convert nitrate to nitrogen gas, which is harmless to the environment, denitrification must occur. Denitrification occurs only in the absence of melecular oxygen and in the presence of an organic carbon source. Intermittent aeration was used in this experiment to remove nitrogen from wastewater. In intermittent aeration, an air supply is turned on and off for a defined amount of time. During the aeration period, oxygen is pumped through the wastewater for a certain period of time to increase concentrations of dissolved oxygen. The aeration period is followed by a non-aeration period where no aeration is applied. This creates aerobic and anaerobic conditions that accommodate the microorganisms involved in nitrification and denitrification, respectively. In this study, various aeration to non-aeration(ANA) ratios were tested in the removal of nitrogen from anaerobically pretreated swine wastewater. In the reactor with a cycle of 1 h:1 h, an average of 98.8% of ammonia nitrogen was removed, but nitrate nitrogen accumulated, indicating that denitrification was not sufficient. In the 1:6 and 1:7 ANA reactors, the levels of ammonia nitrogen did not significantly change, which indicates the failure of nitrification. For reactors with 1:4 and 1:5 ANA, the amount of ammonia nitrogen in the effluents was significantly lower than the other reactors, and total nitrogen removal was 72% and 79.4%, respectively. This indicates that ANA ratios of 1:4 and 1:5 are the most effective for the removal of nitrogen from anaerobically pretreated swine wastewater.

Student Author(s):	Matheson, Allison B.
Department(s):	Botany
Research Mentor(s):	Jenny Xiang/ Botany
Title of Presentation:	Phylogenetic Relationships of Begonia with Emphasis on the Chinese Species–Evidence from ITS Sequences

Begonia is one of the most diversified genera in angiosperm, with ca. 1400 species in 65 sections in Africa, Asia, and America. About 150 species in 9 sections occur in China. Phylogenetic analysis of 156 ITS sequences representing 148 taxa from 36 sections (70 taxa in 7 sections from China) from all continents was conducted to elucidate the phylogenetic relationships within the genus. The resulting trees show that the African taxa form a series of basal branches and the Asian species are divided into several unequal clades successively diverged after the African taxa. Species from America and S. Africa form a clade sister to a large, poorly resolved Asian clade containing most species from sections Platycentrum, Diploclinium, Reichenheimia, Sphenanthera, and Leprosae. The Chinese species do not group according to their sections. None of the more well-sampled sections in China is suggested to be monophyletic. Possible rapid diversification in mainland Asian Begonia is inferred. The results suggest an African origin of Begonia and an Asian origin of the American-S. African clade. Re-evaluation of infrageneric classification is desirable.

Student Author(s):	McCormick, Kaitlin E. Sauer, Kathryn H.
Department(s):	Chemical Engineering Biomedical Engineering
Research Mentor(s):	Kevin Keener/Food Science
Title of Presentation:	The Effects of Carbon Dioxide Cooling on Lysozyme Activity

Previous research has shown that cryogenic carbon dioxide cooling of shell eggs slows bacterial growth compared to traditional cooling. It is suspected that carbon dioxide cooling enhances egg albumen (white) antimicrobial activity. In this study, the effect of carbon dioxide on purified egg white lysozyme activity was investigated. Lysozyme activity was varied over a pH range of 4.5 to 8.0 with carbonate and non-carbonate buffers at temperatures of 5 oC and 24 oC. A standard microbial cell assay consisting of micrococcus luteus was used. Solution absorbance was measured at 450 nm in a spectrophotometer and recorded every 30 seconds for a time period of nine minutes. The spectral absorbance linearly decreased with the time. The spectral absorbance rate (slope) was normalized to the standard assay conditions of pH 6.5 in a phosphate buffer at 24 oC.Results from these experiments found that lysozyme activity at 24 oC in a phosphate buffer assay (pH 6.5) had 38% greater antimicrobial activity than a phosphate buffer assay enhanced with carbonate. At 5 oC, the phosphate buffer assay had 18% of the lysozyme activity of the standard assay (pH 6.5, 24 oC), but the carbonate enhanced phosphate buffer had 51% of the lysozyme activity of the standard assay. This suggests that carbonate (carbon dioxide) enhancement at 5 oC and pH 6.5 increases lysozyme activity. At pH of 4.5 there was a decrease of 20% at room temperature in lysozyme activity due to carbon dioxide enhancement and also a decrease of 30% at 24 oC. At pH 8 and 24 oC there was 34% greater lysozyme activity with carbon dioxide than the standard assay. These results suggest that carbonate (carbon dioxide) addition to shell eggs during rapid cooling may increase lysozyme activity, which in turn may improve the egg safety.

Student Author(s):	Miller, Theodore R., Jr.
Department(s):	Biology
Research Mentor(s):	Lisa D. Parks/Zoology
Title of Presentation:	Studying the Effects of Varied Plant and Animal Research Experiments

This research developed out of a desire to create additional experiments students could complete over several weeks in an undergraduate biology lab. My main focus was testing plant interactions with different stimuli. One area of research studied the effects of a symbiotic relationship of mycorrhizae and rhizobium on legumes like peas, clovers, and beans. Much research had been done on these topics individually, but I was more concerned with the combined outcome. Rhizobium, a Gram negative bacilli bacterium, exists naturally in the soil. The bacteria form partnerships almost exclusively with legume roots; by which rhizobium fixes nitrogen from the atmosphere and soil, benefiting plant and bacterium. Correspondingly, mycorrihzae are symbiotic soil fungi which attach to plant roots. Instead of nitrogen fixation, this fungus helps plant roots absorb water and minerals. Another main area of research studied the results of ethylene (C2H4), a naturally occurring ripening gas from plants, on the growth and development of budding plants. This gas is highly effective and damaging to plants in low concentrations. Such consequences are deformed flowers, fruits, and leaves; stunted growth; and leaf and bud abscission. Plant species, temperature, duration of exposure, and stage of development all shape the results. A third area which was tested was squirrel location feeding preferences. Besides student traffic, campus is constantly being built up, torn down, and renovated. I placed several food sources around campus and monitored the consumption. Overall the students will be able to research many different facets of these experiments. They can focus on different areas of specific research for varied plants, dosages, or locations.

Student Author(s):	Miller, Thomas C.
Department(s):	Zoology
Research Mentor(s):	John Godwin/Zoology
Title of Presentation:	Comparing Neurosteroidogenic and Serotonergic Hypotheses for Prozac (fluoxetine) Effects on Aggression and Arginine Vasotocin mRNA Abundance in a Coral Reef Fish

A variety of neural systems play a role in affecting aggression. Fluoxetine (FLX), a widely prescribed anti-depressant (tradename Prozac) is a selective serotonin reuptake inhibitor that reduces aggression in many vertebrate animals. Previous work in the Godwin laboratory showed that combined R,S-FLX reduces aggression and AVT mRNA expression in the bluehead wrasse (Thalassoma bifasciatum), a Caribbean coral reef fish capable of functional sex change. Bluehead wrasse terminal phase (TP) males normally show territorial aggression towards conspecific TP males or initial phase (IP) males. These heightened levels of aggression in TP males compared to IP males and females are correlated with increases of arginine vasotocin (AVT) levels in the preoptic area of the hypothalamus. Interestingly, FLX can also affect steroid hormone production by the brain. An enantiomer of FLX (S-isomer) that has neurosteroidogenic and serotonergic effects is more effective at reducing aggression in male mice than an enantiomer (R-isomer) that only has serotonergic effects. This study contrasted the effects of R and S FLX on aggression and AVT mRNA levels in TP male wrasses in separate experiments. In the first experiment TP males in pools were given injections of R or S FLX (10 ug/g body weight) or saline as a control. Aggression towards a conspecific TP male was measured 60 minutes later. The S-isomer reduced aggression, while the R-isomer had no effect. In the second experiment TP males were given chronic, daily injections over two weeks of R, S, a mixture of R,S-FLX, or saline as a control. Following two weeks treatment, the brains were taken, fixed in paraformaldehyde, coronally cryosectioned, and processed by in situ hybridization. AVT mRNA levels in the hypothalamus will be measured and the results presented. These findings suggest neurosteroidogenic mechanisms of fluoxetine modulate aggression in the bluehead wrasse as in mammals.

Student Author(s):	Moffitt, Stephanie D.
Department(s):	Animal Science
Research Mentor(s):	Scott C. Whisnant / Animal Science
Title of Presentation:	The Effects of Tumor Necrosis Factor Alpha on Bovine Embryo Development

Mastitis is the most common and costly disease for dairy producers. Even with the best sanitation programs some outbreaks occur. One consequence of the infection is reduced pregnancy rates. However, we do not understand what causes the reduction. Cytokines are chemical messengers released by the cells of the immune system to signal other cells as part of the immune response to mastitis and other diseases. Three cytokines known to be increased during mastitis are tumor necrosis factor alpha (TNFa), interleukin-1beta (IL-1b) and interleukin-6 (IL-6). Our hypothesis is that the cytokines have a negative effect on embryo development. The objective of this experiment was to test the effects of tumor necrosis factor alpha (TNFa) on bovine embryos in vitro. Other research has shown that prostaglandins are inhibitory to bovine embryo development. Therefore we also added Indomethacin a prostaglandin synthesis inhibitor to the culture media for some embryos. Bovine ovaries were collected from a local abattoir. Bovine embryos were produced by IVF and embryos were cultured in media containing either 0 ng/mL of TNFa, 25 ng/mL of TNFa, or 25 ng/mL of TNFa plus 1 mg/mL of Indomethacin at 18 hr post insemination. On days 7 and 9 of culture, the stage of development of the embryos was evaluated and placed into the following categories: compact morula, early blastocyst, mid blastocyst, expanded blastocyst, and hatching blastocyst. Embryos from control media had 29% blastocyst development rate while those in TNFa had 19%. Adding indomethacin with the TNFa increased the blastocyst development rate to 30%. Results indicate that TNFa inhibits bovine embryo development and that Indomethacin blocks this inhibition.

Student Author(s):	Moll, Lori A.
Department(s):	Biological Sciences Botany
Research Mentor(s):	Dean Hesterberg/Soil Science
Title of Presentation:	Physical Properties of Renourished and Natural Beach Sands of Bald Head Island, NC

Beach renourishment is important for protecting North Carolina’s coastline. However, if the renourished sand has different properties than natural sand, then the beach ecosystem may suffer. Of particular interest is the effect of renourishment on loggerhead sea turtle nesting. To preserve the beaches of Bald Head Island (BHI), the U.S. Army Corps of Engineers pumped sand from the Wilmington Harbor onto three miles of beach in 2001. Our objective was to determine if physical properties of sand used for renourishment on BHI were consistent with the natural beach sand and met the NC Division of Coastal Management’s (NCDCM) proposed criteria for renourished sand. We hypothesized that renourished sand would contain more coarse-sized particles (>4.76mm) and more finer-sized particles (<0.074mm) than natural sand. Sand samples were taken from BHI between renourishment operations in 2001 and 2004. Core samples were collected at depths of 0-15cm, 15-30cm, and 30-46cm and analyzed for differences in water content, bulk density, and grain size distribution. We found the following trends: average bulk density was greater in renourished sand at all depths, water content increased with depth, and renourished sand contained more coarse-sized particles than natural sand, particularly at greater depths. Nevertheless, the particle size fractions >4.76mm and <0.074mm met the proposed criteria set by the NCDCM for beach nourishment. These criteria state that the average percentage by weight of the fine-grained sediment fraction used for beach nourishment should not exceed that of pre-nourished beach plus 5%, and that the average percentage by weight of the coarse-grained sediment fraction used for beach nourishment should not exceed that of the pre-nourished beach plus 4%. Our results can be integrated with sea turtle nesting statistics to provide insight on whether any differences in physical properties of renourished versus natural sand affect nesting patterns.

Student Author(s):	Morgan, Tiffany D.
Department(s):	Food Science
Research Mentor(s):	Todd R. Klaenhammer/Food Science
Title of Presentation:	Environmental Tolerances of the S-layer Gene slpB of Lactobacillus acidophilus NCFM

S--layers have been identified in most species of bacteria and hundreds of species of archaea. They are reported to be important in a number of diverse functions including cell shape, stress tolerance, and adherence to human and animal intestinal cells. Previous research has identified a chromosomal region harboring two S-layer genes, slp A, and slp B in Lactobacillus acidophilus ATCC4356 (Boot et al. 1993). A related strain, Lactobacillus acidophilus NCFM, harbors a similar chromosomal region including both S-layer genes, which were found to be identical or nearly identical to ATCC4356 on the nucleotide level, respectively. The slp A gene was functionally inactivated by chromosomal insertion of an integration plasmid (pTRK826) via homologous recombination. An integrant, designated as NCK1377, was monitored for genetic stability and tolerance to environmental stress. The stability of the integration event within the S-layer region was stable for at least 20 generations. However, long-term evaluation of the NCK1377 culture revealed the occurrence of a chromosomal inversion re-establishing a functional S-layer, mediated through the expression of the previously silent slp B gene (NCK1377-CI) (unpublished). The L. acidophilus mutant expressing the slp B mediated S-layer exhibited responses to NaCl, EtOH, and Oxgall exposure that were distinct from both the parental culture expressing slp A and the S-layer deficient strain NCK1377. Extended exposure of NCK1377–CI to 15% (v/v) ethanol resulted in no change in viability in contrast to the wild–type strain and NCK1377. Compared to NCK1377, cells of NCK1377--CI and NCFM showed decreased survival at concentrations above 1% (w/v) of Oxgall. NCK1377--CI cells were more sensitive to concentrations above 2.0% (w/v) NaCl, in contrast to NCK1377 and the wild-type strain. This study indicated that expression of two different native S-layer genes mediated varying responses of L. acidophilus to environmental stress.

Student Author(s): Morton, Nolen E.

Department(s): Genetics

Research Mentor(s): Greg Gibson/Genetics

Title of Presentation: Genotyping Hypertension in African Americans

Hypertension, or chronically high blood pressure, is the most common health problem for Americans and can result in heart attack, heart failure, or stroke. The disease affects over 40% of African Americans; this is a much higher occurrence than in other Americans. Many factors are involved in the development of hypertension, including gene-by-gene, and gene-by-environment interactions. A study done by Rotimi et al found that blacks from rural Nigeria, Jamaica, and the US all carry a higher percentage than Caucasians of an allele that has been previously associated with hypertension, but the actual rate of hypertension is higher in the US African American population than in the Nigerian population, probably due to a less-active lifestyle and a higher intake of salts and fats. This suggests that while blacks may be more susceptible to hypertension due to genetic factors, they will only develop the disease in a certain environment. For this study, which is being completed in conjunction with North Carolina Central University, the main objective was to figure out protocols for extracting DNA from dried blood samples and for using this DNA to genotype polymorphisms in genes related to hypertension. To do this, I chose to examine polymorphisms in 5 genes that had previously been shown to be associated with hypertension in other ethnic groups. Protocols for extracting DNA from dried blood spots were tested and genotyping was completed using allele specific restriction digests of PCR fragments. For three of the genes, a higher incidence of alleles associated with hypertension was observed; associations between genotypes and the individuals’ phenotypes have yet to be examined. Genotyping of the remaining genes is in progress.

Student Author(s):	Moye, Hollis C.
Department(s):	Molecular Biomedical Sciences, NCSU College of Veterinary Medicine
Research Mentor(s):	M. Christine McGahan/Molecular Biomedical Sciences
Title of Presentation:	Vascular Endothelial Growth Factor Secretion by Human Retinal Pigment Epithelial Cells is Regulated by Iron

Vascular endothelial growth factor, (VEGF), an angiogenic factor, is responsible for the promotion of vascularization throughout various

cells in the body.  VEGF production is regulated by hypoxia-inducible factor-1 (HIF-1), a dimer of HIF-1α and HIF-1β.  HIF-1α originates

outside of the nucleus and when iron is present, causes HIF-1α degradation.  However, in the presence of an iron chelator, HIF-1α

translocates to the nucleus where it heterodimerizes with HIF-1β to form the active HIF-1.  This results in the increased production of its

downstream product, VEGF.  In this present study, we demonstrate that retinal pigment epithelial (RPE) cells synthesize and secrete VEGF

and that iron regulates these processes.  We found that ferric ammonium citrate (FAC) decreases VEGF accumulation in the cell

conditioned medium of RPE cells.  Dipyridyl (DP), an iron chelator, increased accumulation, resulting in VEGF levels twice as high as the

control.  Interestingly, the combination of FAC and DP resulted in higher VEGF accumulation than was observed with DP treatment alone.

Since RPE cells closely interact with the retina, dysregulation of VEGF secretion by these cells may affect retinal function and have several

pathological consequences. Further research of iron and iron chelators and their use within the VEGF pathway could lead to therapeutic

treatments for retinal disease.

Student Author(s):	Nelson, Audrey R.
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	Paul F. Agris/Molecular and Structural Biochemistry
Title of Presentation:	Folding Interactions of an mRNA 5’-UTR that Regulates Gene Expression: Interaction with a Regulatory tRNA

Genes for aminoacyl tRNA synthetases (aaRS) in pathogenic gram positive bacteria (Streptococcus, Staphylococcus, Bacillus) are uniquely regulated through unacylated tRNA's interaction with the 5’-untranslated region (UTR) of the mRNA. The anticodon of the tRNA interacts with a codon sequence occurring in the specifier loop of the UTR. Those genes regulated through mRNA specifier loops have evolved with codon sequences for different tRNAs in order to selectively regulate the different aaRS genes. For this UTR to positively affect gene expression through regulation of transcription, the RNA folding interaction must be significantly stable to engage the codon sequence with the tRNA anticodon. A model system was engineered in which the interaction between common and specifier could be analyzed to see if it was structurally and functionally similar to the system in vivo. Gel shift analysis and fluorescent quenching resulted in experimental Kds for complex formation that demonstrate strong binding. Titration of ASLgly into solution of complex characterized the trimolecular interaction which was shown to be specific through control titration of ASLphe. Through a lack of quenching with the titration of ASLphe, the binding of ASLgly to the Specifier Loop in vitro was shown to be both achievable and specific.

Student Author(s):	Peele, Lauren N.
Department(s):	Animal Science
Research Mentor(s):	William L. Flowers/Animal Science
Title of Presentation:	Animal Usage of a Ficus spp. in a Panamanian Rain Forest

An ecosystem is as large or small as its defining boundaries. A single tree and its inhabitants and visitors, for example, can be considered an entire ecosystem. In this study, I observed one such ecosystem and how the individual animals used a particular Ficus spp. Because my study was conducted in a rain forest, I expected to see many different animal species, with a particular abundant representation of the class Insecta. My observations did follow this general trend of great animal diversity, with many insect species. There was also a decrease in abundance of species observed as I moved up the phylogenic tree.

Student Author(s):	Ridge, Ashley M.
Department(s):	Inspire Pharmaceuticals
Research Mentor(s):	Vittitow, Jason/Inspire Pharmaceuticals
Title of Presentation:	Analysis of the Metabolism of INS37217, a P2Y2 Receptor Agonist, using HPLC

Retinal detachment disrupts the function and cellular organization of the retina, often resulting in severe and permanent visual loss in humans. INS37217 is a drug in Phase II trials that works to increase the transport of fluid from the subretinal space leading to reattachment of the retina. The purpose of this experiment is to evaluate the pharmacokinetic metabolism of INS37217 after injection into adult rabbit eyes. We specifically look to see the timing and location of metabolism as well as identifying metabolites that are formed. To achieve these goals adult rabbit eyes were given 50 ul intravitreal injections of INS37217. Aqueous humor and vitreous samples were isolated from the eye and analyzed using high-performance liquid chromatography (HPLC). To quantitate the levels of INS37217 and its metabolites, linear regression analysis was used to compare the samples with different standard concentrations of INS37217. From this it was determined that INS37217 is metabolized into deoxycytidine 5'-diphosphate and uridine 5'-diphosphate. It was also noted that there are higher concentrations of INS37217 in the vitreous than in the aqueous and these concentrations decrease over time. There are no active metabolites of INS37217 formed when injected into the vitreous cavity and no suspected toxic agents have been found. In conclusion, INS37217 may be useful in the clinic to help repair retinal detachments and decrease the overall amount of vision loss caused by them.

Student Author(s):	Rittiner, Joseph E.
Department(s):	Chemistry/Plant Pathology
Research Mentor(s):	Stefan Franzen/Chemistry Richard Guenther/Plant Pathology Steve Lommel/Plant Pathology
Title of Presentation:	In Vitro Reassembly of Infective Red Clover Necrotic Mosaic Virus Virion Particles Containing Non-native RNA

Small RNA plant viruses have shown promise as tools for therapeutic and diagnostic nanotechnology applications. While virus particles, or virions, of soil born viruses have ideal physical properties for application to nanotechnology their integration has been hampered by the lack of an in vitro assembly method. Red clover necrotic mosaic virus (RCNMV) is a member of the dianthovirus family of soil born viruses. RCNMV’s virions are composed of 180 copies of a 37 kDa capsid protein producing T=3 icosahedra which package the two genomic ssRNAs of 4 and 1.5 kb. A method has been developed that uses purified capsid protein and transcript RNA to assemble viral particles in vitro. The capsid protein was obtained by disrupting viruses and purified by size exclusion. The virus reassembly is made by mixing purified capsid protein and viral RNA transcripts initially at pH 10 and then equilibrating the mixture to pH 5.5 in a dialysis cassette. The progression of the reassembly reaction was monitored using Dynamic Light Scattering (DLS). DLS analysis indicated the reassembly method yielded particles of diamter~35nm. Transmission Electron Microscopy (TEM) was used to further characterize the reassembled particles. A comparison of reassemble particles with wild type virion finds the two indistinguishable to the limits of particle resolution. To further validate the spectral data that indicate virions were being assembled in vitro a biological assay was developed. The biological assay tests if in vitro assembled particles made with viral RNA transcript encoding a foreign reporter gene can infect N. clevelandi, a research hostplant. The viral RNA was modified by the substitution of the Green Fluorescent Protein (GFP) gene for the

viral capsid protein gene. When reassembled particles containing the GFP transcript were rub inoculated on plants detectable fluorescence was observed on inoculated leaves one week after inoculation.

Student Author(s):	Ruffin, April R. Dean, Megan
Department(s):	Animal Science
Research Mentor(s):	Brenda Alston-Mills/Animal Science
Title of Presentation:	Affect of Different Diets on Sexual Behavior and Reproductive Organs in Mice

Studies provide evidence that a typical American diet, which is high in fat and low in fiber, decreases the age at menarche and increases the risk for breast cancer. The objective of this study was to determine how different diets affect sexual behavior, developmental characteristics, and physiological changes of reproductive organs in mice that may alter the risk for developing breast cancer. The diet groups consisted of a control, soy, lactalbumin, estrogen, soy-lactalbumin, and estrogen-lactalbumin. Sexual behavioral differences were observed by video taping interactions between male and female mice from the different experimental groups. The number of caudal and rostral behaviors initiated, the number of times females crossed the center of the cage, and the number of mountings during a 10-minute period were counted. Mammary gland fat pad invasion was scored on a scale from 1 to 5, which is based on mammary gland differentiation in relation to the lymph node. Blood serum estrogen levels were detected using the RIA method. Total DNA was extracted from mammary gland tissue and homogenized and purified to determine total DNA present. A TCA/BCA analysis was performed to determine the total amount of protein in a gland and compared with the levels of DNA. We noticed that the estrogen group showed the highest levels of interest in sexual activities and the lactalbumin group also showed interest. Fat pad invasion was greatest in soy and smallest in the estrogen group. Serum estrogen levels were highest in the soy-lactalbumin group when compared to the estrogen group. Total DNA levels in the soy group were highest when compared to the control group. There were no significant differences in total protein between groups.

Student Author(s):	Schaffer, Hannah C.
Department(s):	Animal Science
Research Mentor(s):	Scott Whisnant/Animal Science
Title of Presentation:	Effects of Caffeine on Sperm Function and Embryo Development

For reproduction to occur in mammals, sperm must be capacitated and undergo the acrosome reaction before they are able to penetrate the oocyte. This is normally done in vivo; secretions in the female’s reproductive tract stimulate these events. In vitro, however, other methods are needed to capacitate the sperm before fertilization. Traditionally, heparin is added to the semen to stimulate these results. The addition of caffeine, a phosphodiesterase inhibitor, will also lead to capacitation, and is sometimes used to gain higher capacitation, acrosome reaction and fertilization rates. The objective to this study was to analyze the effect that caffeine treated sperm will have on capacitation, acrosome reaction, fertilization and subsequent embryo development in cattle. Ovaries were obtained from a local abattoir. Follicular fluid was aspirated from follicles to obtain cumulus oocyte complexes (COCs). The COCs were isolated from the aspirate and placed in maturation media, and matured to the Metaphase II stage. Frozen thawed bull semen was prepared and analyzed to determine sperm concentration and insemination dosage. Caffeine in the amount of 3 μg/ml was added to the sperm preparation media. The sperm were added to the wells containing the matured COCs. After 24 hours, the presumptive zygotes were washed to remove sperm and semen remnants, and allowed to mature to the blastocyst stage. The embryos were counted on days 5, 7, and 9 to monitor development. Results showed that being fertilized with caffeine treated sperm had little effect on embryo development.

Student Author(s):	Seate, Jessica L.
Department(s):	Animal Science
Research Mentor(s):	Kimberly Ange-van Heugten/Animal Science
Title of Presentation:	Wildlife: Diseases and Care

In Companion Animal Management, ANS 400, I learned to care for not only companion animals, but also wild and exotic ones. I learned about their behavioral characteristics and the diseases that affect them. Since communities are expanding and destroying animal environments, we are seeing huge amounts of stranded wild animals. Consequently, people are beginning to take in these wild animals and are ignorant of possible issues. I gained an understanding of these potential concerns via ANS 400 and I wanted to educate the public as to how to preserve wildlife.

The Piedmont Wildlife Rehabilitation Center (PWC) receives numerous calls asking what to do if a stranded or injured wild animal is encountered. I created a web page to educate the public as to how to react in this situation and how to become aware of the diseases animals carry and how important it is to not keep them as pets for human and animal safety. Disease education is extremely important especially when you come in contact with a wild or unfamiliar animal. For instance, two of the main diseases PWC treats are the zoonotic and potentially life threatening Aspergillosis and Salmonellosis.

This website development project compliments ANS 400 because I have provided a knowledgeable source of information for the public to learn about wild animal care and management that can be incorporated into future ANS 400 semesters. Using the Internet to present this information was a natural decision due to the wide audience it reaches. Anyone in the world can simply search for wildlife information and via our technological society I can help teach them responsible animal care via my website. Hopefully this web page will help the animals, educate the public, and help those, like myself, whose future endeavors are veterinary animal care.

Student Author(s):	Shah, Syed A
Department(s):	Biological Sciences
Research Mentor(s):	Jonathan C. Allen/Food Science
Title of Presentation:	A Comparison of Spray-drying and Freeze-drying Techniques on the Preservation of Immunoglobulin A and Vitamin A in Human Milk

Human milk is the most complete food for newborns and infants because it contains the right amount of nutrition and protection. Established human milk banks are interested in drying human milk to ease storage and shipping and for fortification with extra nutrition in a low volume. Cow milk is dried in the commercial industry primarily by spray-drying, but freeze-drying techniques may improve nutrient retention. An experiment was conducted to find out which one of these two methods preserves the most vitamin A, and IgA (acting as nutritional and defensive markers, respectively) content in the milk. Milk from a single donor was randomly selected or pooled from samples over the first two weeks of lactation, and the two samples were both spray dried and freeze dried. Total solids of the original milk samples were measured by evaporation to constant weight. Vitamin A content in the samples was measured by High Performance Liquid Chromatography (HPLC). Finally, the two samples were analyzed with an Enzyme Linked Immunosorbent Assay (ELISA) to compare the amount of IgA activity against an E. coli antigen that the two samples contained after the drying processes. The HPLC showed that freeze-dried sample was able to preserve more vitamin A per micro liter than the spray-dried sample. However, the ELISA did not yield reliable results, probably because one or more of its components (the antigen, the enzyme, or the substrate) was not working as expected. Only a small amount of IgA activity that was similar for the spray-dried, freeze-dried, and control samples was indicated by the ELISA. Additional experiments can be performed to systematically eliminate the sources of error in ELISA to improve the results.

Student Author(s):	Shepherd, Mark N.
Department(s):	Genetics
Research Mentor(s):	James W. Mahaffey/Genetics
Title of Presentation:	The Drosophila Genes disconnected and disconnected-related May Have Different Roles in Drosophila Limb Development

The disconnected (disco) and disco-related (disco-r) genes of Drosophila melanogaster encode partially redundant zinc finger transcription factors that are expressed in and control developmental fates of many cells from mid-embryogenesis to adult stage. These include cells found within the imaginal discs. The imaginal discs are blocks of cells set aside in the embryo that will develop into the adult body during the pupil stage. Previously, disco expression within the leg imaginal discs had been inferred through the use of a Lac Z reporter carried in an enhancer-trap insertion element. The expression of Lac Z indicated that disco expression in the leg discs formed concentric rings where the presumptive leg joints would form. However, based on detection of mRNA from disco and disco-r using in situ hybridizations of the imaginal discs of OreR pupae, we determined that the Lac Z reporter is not indicative of disco expression in the leg discs; rather it corresponds to disco-r expression. mRNA detection demonstrated that disco is expressed in the distal tip of the leg discs, while disco-r is expressed in the concentric circles that will form the joints of the leg. Based on these observations, it appears disco and disco-r may have different roles in limb development, though they are redundant in other developmental processes, for example in embryonic development. We are using ectopic expression studies to examine the different roles of disco and disco-r during leg development.

Student Author(s):	Shin, Peter G.
Department(s):	Zoology
Research Mentor(s):	Marianne Niedzlek-Feaver/Zoology
Title of Presentation:	The Histological Study on the Patterns of Sperm Transfer and Organization inside the Spermatheca during Different Mating Time Intervals of Grasshoppers, Schistocerca americana and Dissostiera carolina

This research experiment involves the study on the patterns of sperm transfer and organization inside the spermatheca of the grasshoppers, Schistocerca americana and Dissostiera carolina. A female is taken at intervals of 5 minutes, 15 minutes, 30 minutes, 45 minutes, one hour, and several hours during the interrupted mating process and is frozen to stop the progress of injected sperm from the male. Each female grasshopper is dissected isolating the spermatheca which is composed of several chambers. The spermatheca is fixed in Carnoy's fixature, dehydrated with an ethanol series, and embedded in paraffin. Transverse sections are then made by thin cuts of the structure. Feulgin's stain which is specific for DNA and therefore, helping in detecting the sperm inside the spermatheca is used for analysis. In addition, the three dimensional construction of the spermatheca will be done. Ultimately, the path of the sperm into the various chambers of the spermatheca may give insight into whether it is used for fertilization versus nutrient.

Student Author(s):	Stanton, Laura M.
Department(s):	Zoology
Research Mentor(s):	James W. Moyer/ Plant Pathology
Title of Presentation:	The Effect of Pepper Host on the Haplotype Population of Resistance-breaking Tomato Spotted Wilt Virus (TSWV) Isolates

Tomato Spotted Wilt Virus (TSWV) is a virus that causes severe symptoms in plants of high economic importance. Technological advancements have led to the development of crops with varying levels of resistance to TSWV. However, some TSWV isolates have the ability to overcome this resistance. In this study, to elucidate the differences between TSWV-susceptible and TSWV-resistant peppers, biological and molecular characteristics of a TSWV isolate from California known to break resistance in TSWV-resistant peppers was determined. A host range consisting of TSWV-resistant and TSWV-susceptible peppers was conducted to verify the resistance breaking property. The TSWV resistance-breaking isolate was inoculated once into Nicotiana benthamiana to propagate the virus particles. The resistance breaking isolate was then mechanically inoculated by a series of passages into TSWV-susceptible pepper and TSWV-resistant pepper. First passage into TSWV-resistant pepper resulted in systemic infection of the plant, but second passage was void. This was an abnormal finding based on the hypothesis that the isolate should become more concentrated with each passage. The haplotype population structure was determined prior to treatment and following the multiple passages in TSWV- susceptible peppers by cloning genes located on the S segment of the TSWV genome, the segment involved in resistance breaking capabilities in pepper. Twenty clones of three segments of the non-structural protein (NSs) were sequenced and analyzed.

Student Author(s):	Steed, Laurie E. Adeeyo, Brittany O. Mccarroll, D. Shane Valanejad, Leila
Department(s):	Food Science
Research Mentor(s):	Jonathan Allen/Food Science
Title of Presentation:	Biochemical Characterization of Sweet Potato Protein Fractions

White skinned sweet potatoes have been consumed raw in Japan for hundreds of years as a remedy for anemia, hypertension, and diabetes. In 2000, a team of Japanese researchers isolated an acidic glycoprotein fraction believed to contribute to this nutraceutical activity, and developed a commercial product, caiapo, that is presently available on the market. Since then, human testing has proven caiapo effective at lowering blood glucose levels in Type II diabetes patients. To date, this component has only been isolated from Japanese white skinned sweet potatoes and its potential for use in treating diabetic patients within the United States is virtually unexplored. Our research focused on characterizing protein fractions from North Carolina sweet potatoes to determine if they are similar to those in caiapo. A laboratory procedure was developed that involved preparation of a crude sweet potato homogenate, ammonium sulfate fractionation, dialysis, BCA protein assay, and SDS-PAGE electrophoresis. The primary objective centered on comparing the SDS-PAGE protein banding patterns obtained from the Nancy Hall cultivar, a white skinned sweet potato presently grown in North Carolina, with the pattern observed using a Japanese sweet potato caiapo fraction. Electrophoresis showed that the caiapo fraction and Nancy Hall sweet potato proteins produced very similar banding patterns, a result suggesting a protein homology between the two sweet potato cultivars. Also, glycoprotein staining patterns revealed similarity. These findings represent a significant opportunity for the North Carolina farming industry to consider developing new markets for important sweet potato products that will offer anti-diabetic health benefits. With this development, the economic impact could provide a substantial boost to the North Carolina sweet potato industry.

Student Author(s):	Strother, Krista M.
Department(s):	American Kennel Club, Canine Health Foundation
Research Mentor(s):	Kimberly Ange-van Heugten/Animal Science
Title of Presentation:	2005 Parent Club Survey

Americans are spending more money than ever on their pets, with a large majority of this money being spent on medical bills. In 2003 alone almost $7.9 billion was invested by pet owners in veterinary medical expenses. One concern in the pet and veterinary industries involves the crossing of pure animals to create a purebred breed. Many dog owners have a strong desire to own a purebred dog and practice “line breeding” which is technically the “interbreeding of individuals within a particular line of descent usually to perpetuate desirable characters”. The (CHF) Canine Health Foundation, which is an affiliate of the (AKC) American Kennel Club works to promote research by providing grants to researchers to learn more so they can help improve the lives of purebred dogs.

Working in conjunction with the Canine Health Foundation, I created, distributed and compiled the results for the 2005 Parent Club Survey. This survey was created on an electronic database and sent to approximately 100 AKC breed organizations. Questions regarding the structure of their organization were presented as well as the major health problems that their dog breed possesses. The information from this survey will allow the Canine Health Foundation to identify major diseases and conditions that need the most grant money to fund their research.

94 parent clubs responded to the survey, representing more than half of the AKC certified breeds. 59% feel that they have a strong relationship with the CHF, 86 % have conducted surveys of their own to find out more amongst their members and 85 % of those communicated those results to their members. The distribution of the breed categories (ex. herding, hound, sporting, toy, etc.) remained fairly consistent with each group representing about 14% of the overall results. In general, the findings showed that the most commonly researched condition in purebred dogs is hip dysplasia, followed by epilepsy and autoimmune disorders.

Student Author(s):	Su, Yizhen
Department(s):	Microbiology
Research Mentor(s):	Jonathan Olson/Microbiology
Title of Presentation:	PCR Identification of Metabolic Gene Variance in Select Campylobacter Species by Gel Electrophoresis

The goal of this project is to test and assess the metabolic versatility of various members of the Campylobacter genus. The specific species that will be assessed in this project are: C. coli, C. doylei, C. fetus, C. lari, and C. upsaliensis. C. jejuni is a very common cause of gasterenteritis, infections occurs more frequently than Salmonella, Shigella, or Escherichia coli O157:H7. Most infections are resulted from contact and consumption of contaminated poultry (1). The C. jejuni metabolic mechanism has certain unique respiratory components that allows it to survive in aerobic and anaerobic conditions with diverse pathways. The genome sequence of C. jejuni contain the genes for hydrogenase and formate dehydrogenase, which are required for the utilization of the respirators substrates H2 and formate, respectively. Also present are the genes coding two different terminal oxidases and a nitrate reductase. Activities for these donors and acceptors have been determined for C. jejuni. This project will use PCR to determine the presence of those genes in the listed Campylobacter species.

Student Author(s): Wentz, Kelly N.

Department(s): Animal Science

Research Mentor(s): Jack Odle/Animal Science

Title of Presentation: The Effect of Ractopamine Feeding Level on Fatty Acid Profiles

in Belly Fat and Backfat of Finishing Pigs

The effect of ractopamine feeding level on fatty acid profiles in belly fat and backfat of finishing pigs. One hundred fifty pigs (75 borrows and 75 gilts, initial weight 77 kg) were used to investigate the effect of ractopamine (RAC) on fatty acid profile in belly fat and backfat. Pigs, within genders, were randomly assigned to three treatments, which consisted of 0 (control diet), 5 or 10 ppm of RAC. Each treatment contained 10 pens and each pen had 5 pigs. Pigs were fed experimental diets for five weeks and were sent to a commercial slaughter facility. Belly fat and backfat were sampled from the clear plate of carcasses. Lipids were isolated from the fat samples in duplicate and fatty acid composition was analyzed by gas-liquid chromatography. The iodine value (IV, the grams of iodine bound per 100 g of fat) was then calculated from the fatty acids composition data. Feeding RAC had no effect on linoleic acid in backfat. The percentage of linoleic acid in backfat from pigs fed diet with 10 ppm RADC (18.6) was on average 7% higher than that (17.4) from pigs fed control diet and control diet with 5 ppm RAC. This resulted in 3% of increase in IV, 8% of increase in polyunsaturated fatty acid and 8.5% of decrease in the ratio of monounsaturated and polyunsaturated fatty acids from pigs consuming 10 ppm RAC compared to that from pigs consuming control and 5 ppm RAC diets. No significant difference was detected between pigs fed control diet and pigs fed control diet with 5 ppm RAC. The results indicated that feeding high level of RAC (>5ppm) will increase the enrichment of linoleic acid in backfat, but there is no overall effect (or negligible effect) of RAC (less or equal to 10 ppm) on the fatty acid profile in belly fat and backfat of finishing pigs.

Student Author(s):	West, Steven A.
Department(s):	Genetics
Research Mentor(s):	Trudy F.C. Mackay/Genetics Theodore J. Morgan/Genetics
Title of Presentation:	The Genetics of Heat Stress Resistance in Drosophila

Fruit flies (Drosophila melanogaster) are an isothermal organism with an historical range consisting primarily of habitats within the tropical climatic zones of central Africa. Approximately 10,000 - 15,000 years ago, D. melanogaster successfully colonized temperate climatic zones on the Asian and European continents. This movement out of the tropics and into temperate habitats exposed populations to thermal ‘challenges’ outside the range experienced in their ancestral tropical habitat. Following this initial colonization, D. melanogaster have successfully colonized nearly every niche on the planet with human movement around the globe. The extension in this species habitat has introduced populations to novel thermal environmental changes. In regions with diverse seasonal, clinal, and daily temperatures, it is very likely that Drosophila populations have undergone adaptive evolution which has resulted in their current cosmopolitan distribution. To understand this potentially adaptive response to novel environments we need to know what genes influence traits that are important for survival in novel thermal environments. In this current study, we designed and performed a screen to identify the genes that are involved in resistance to heat stress. We scored percent survival after exposure to heat stress in 660 single P-element insertion lines and their contemporaneous control lines. Since each of the mutant lines contained a single P-element insertion that randomly disrupts a single locus in the genome, differences in survival of mutant lines from the control allowed us to identify several candidate loci that are involved in a diverse array of biological and molecular processes that are implicated in resistance to heat stress in D. melanogaster.

Student Author(s):	Whitney, Brandon C.
Department(s):	Biological Sciences
Research Mentor(s):	Thomas Wentworth/Botany Alexa McKerrow/Botany
Title of Presentation:	Mapping the Cedar Glades of the Tennessee Central Basin

The cedar glades of the Inner Central Basin of Tennessee represent one of the globally rare rock-outcrop communities in the southeastern United States. These glades, which are mosaics of bare rock and cedar woodlands, support three federally endangered and over 25 state listed plant species. To determine the conservation status of the cedar glades, the Southeast Gap Analysis Project needs an accurate distribution map. The combination of small patch size, scattered distribution, and changes in land use around these communities make them difficult to map using satellite imagery alone. Often the bare rock looks similar to urban or other barren cover types, such as fallow fields. In this project, we overlaid a data layer of location records for diagnostic plant species onto high resolution digital aerial photographs in order to recognize the pattern of a cedar glade. We then used our photograph interpretation to construct a set of training sites to identify cedar glades from satellite imagery, utilizing feature extraction. After accuracy assessment of the distribution map, we intersected a data layer of conservation lands in the Inner Central Basin to conduct a Gap Analysis for the cedar glades. This analysis enabled us to determine the percentage of this community that is currently protected.

Student Author(s): Williams, Nicole D.

Department(s): Food Science

Research Mentor(s): Sophia Kathariou/Food Science

Tatiana A. Vishnivetskaya/Food Science

Title of Presentation: Genomic Study of Exiguobacterium Strains Isolated from

Diverse Environments and Search for New Isolates.

Nineteen strains of the low-GC gram-positive bacterium Exiguobacterium were isolated from markedly diverse environments including ancient Siberian permafrost and Greenland glacial ice, contemporary polar and temperate soils and lakes, hot springs, and food processing plants. The genome of Exiguobacterium sp. 255-15, isolated from 2-3 million years old Siberian permafrost was fully sequenced (http://genome.ornl.gov/microbial/exig/). The 2.9 Mb genome of this isolate has 47.8 % GC content and includes 2977 candidate protein-coding sequences. Sequences for conserved genes from different functional groups have been utilized as probes in Southern hybridizations. The hybridization results identified ten genes, encoding DNA gyrase, fatty acid desaturase, chaperonin GroEL, chaperonin DnaK, translation initiation factor, and ribosome-associated protein Y that had detectable homologues in the genome of all 19 isolates. Thus, we employed these finding for a search of Exiguobacterium strains in other environments: floor drains and other environmental samples from a turkey processing plant (North Carolina); pond water (North Carolina); and subsurface pristine water collected from 880-1130 m in Lupin Mine (Canada). Low temperature and room temperature enrichments in full-strength or diluted trypticase soy broth as well as direct platings were used to isolate the bacteria. Gram-positive, catalase-positive bacteria were selected for further screening. Preliminary results suggest that Exiguobacterium-specific gene probes may be useful for identifying new isolates and evaluating genomic diversity of Exiguobacterium spp.

Student Author(s):	Wilson, Jennifer B.
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	William L. Miller/Molecular and Structural Biochemistry
Title of Presentation:	Inhibition of Follicle-Stimulating Hormone by Gonadotropin Releasing Hormone

Follicle stimulating hormone (FSH) from pituitary gonadotropes causes ovarian follicles to produce mature eggs. It also stimulates sperm production. The synthesis of this important reproductive hormone is regulated, in part, by gonadotropin releasing hormone (GnRH) which alters synthesis of its beta subunit, FSHβ, the rate limiting step in FSH synthesis. Production of FSH is stimulated or inhibited by GnRH depending on how the pituitary experiences GnRH. Acute exposure to GnRH induces FSHβ expression by activating Gq signaling pathways. This pathway has been characterized in primary pituitary gonadotropes and transformed LβT2 gonadotropes. By contrast, GnRH inhibits FSHβ expression by chronic exposure which is used medically to completely turn off the reproductive system. Recent work with primary gonadotropes clearly demonstrates GnRH-mediated inhibition of FSHβ and suggests it uses a Gs signaling pathway. The Gs pathway produces cyclic AMP to activate protein kinase A (PKA) which controls expression of many genes through phosphorylation. In this study, FSHβ expression was studied in LβT2 cells to determine if these laboratory friendly transformed cells can be used to study GnRH inhibition of FSHβ. Chemicals known to induce cAMP [Pituitary Adenylate Cyclase-Activating Peptide (PACAP), forskolin (FSK), 3-Isobutyl-1-methylxanthine (IBMX), and Cholera toxin (CTX)] were used to block FSHβ expression. Messenger RNA (mRNA) for mouse FSHβ was measured using real-time reverse transcriptase polymerase chain reaction (RT-rtPCR); RT-rtPCR of 18s ribosomal RNA served as an internal RNA marker not altered by any of the above treatments. The results show that FSHβ expression was not inhibited in LβT2 cells by GnRH or any of the reagents indicating that βT2 cells do not provide a useful model to study GnRH-mediated inhibition of FSHβ. Thus, primary pituitary gonadotropes will have to be used to investigate the inhibitory action(s) of GnRH on FSHβ expression.

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Last modified February 2005 by Sharon E. Hunt, WordHunting

The 14th Annual

NC State University Undergraduate Research Symposium

Species

The 14^th Annual

NC State University
Undergraduate Research Symposium