The 17^th Annual

NC State University
Undergraduate Research Symposium

Biological Sciences:

Molecular, Biochemical, Genetics, Cell Biology

Abstracts

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

Biological Sciences abstracts

Applied Sciences (Crop, Poultry, Animal, and Horticultural Sciences)

Ecology, Environmental, Conservation, Botanical
Molecular, Biochemical, Genetics, Cell Biology
Zoology, Physiology, Behavior, Neurobiology

Engineering & Technology abstracts
Humanities, Social Sciences, Psychology abstracts
Physical and Mathematical Sciences abstracts

Student Author(s):	Al-Nadaf, Sami
Department(s):	Biological Sciences
Research Mentor(s):	Edward J. Noga/Clinical Sciences
Title of Presentation:	The Evidence for the Production of Hemoglobin-beta (Hb-ß) Protein, an Innate Immune Defense, in Fish Gut Epithelium

An organism’s innate immune system is the primary defense against disease causing pathogens. An essential part of the innate immune system is antimicrobial peptides (AMPs). AMPs have a function in the non-specific immune response in organisms. In previous studies in the Aquatic Medicine Laboratory, we were able to isolate hemoglobin-beta (Hb-ß ) peptides from the gill of channel catfish (Ictalurus punctatus) (Ullal et al., Submitted). These Hb-ß peptides were found to be potent antimicrobial agents expressed in the skin and the gill epithelium against the parasite ich (Ichthyophithirius multifiliis). Using in situ hybridization procedure on paraffin embedded tissue sections of the gill and skin epithelium from ich-infected channel catfish, Ullal et al. (Submitted) was able to determine that Hb-ß peptides were being synthesized and expressed by the cells in the gill and skin epithelium in response to ich. Furthering this research project, my project was to determine whether other cells are able to synthesize and express the Hb-ß peptide sequence in other fish species. The stomach and intestine sections of channel catfish were examined for the Hb-ß transcript by using the same in situ hybridization procedure. To test for the Hb-ß expression in other fish species, a cell line from carp (Cyprinus carpio) skin epthelium, epithelioma papulosum cyprini (EPC) was examined using a modified in situ hybridization procedure for a monolayer cell culture. Potentially these findings, suggest that more than one type of cell across different species is able to synthesize and express AMPs from hemoglobin in a non-specific immune defense system.

Student Author(s):	Amend, Sarah R.
Department(s):	Biological Sciences
Research Mentor(s):	Edward Williams, MS, CGC/Genetic Services, LabCorp Kenneth J. Friedman, PhD, FACMG/Molecular Genetics, LabCorp
Title of Presentation:	Cystic Fibrosis Extended Mutation Panels Provide Comparable Detection to Standard Mutation Panels

Cystic fibrosis (CF) is a common autosomal recessive genetic disease occurring in approximately 1 in every 3700 births. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene on chromosome 7; to date, over 1500 CFTR mutations have been discovered, but most of these mutations are rare. In 1997, the National Institute of Health made a recommendation that a program for CF carrier screening be instituted. In 2001, the first panel of 25 CF mutations was formulated by the American College of Obstetricians and Gynecologists and the American College of Medical Genetics. In 2004, this recommended panel was revised to 23 CF mutations. Since that time, extended panels have been developed to target specific ethnic population while maintaining the value of a pan-ethnic screen. The extended panels had not been compared with existing panels to determine if they added useful information. Data from LabCorp’s extended panel CF 70, the standard panel of CF 32, and the recommended panel of 23 mutations were analyzed. Comparing data from the extended panel of the CF 70 to the recommended panel of 23, in a sample size of 21,377 individuals, 80 mutations detected by the extended panel would not have been detected in the recommended panel, an increased test sensitivity of 0.374%. The thin increase in test sensitivity (0.374%) suggests that expanded panels targeting rare CF mutations provide marginal benefits for the general population, but certain individuals may be served by their decision to pursue extended panel testing. These results raise concerns as to the overall utility of extended panels, both for use as universal panels and as screening for particular ethnic groups.

Student Author(s):	Aremu-Cole, Moyo B.
Department(s):	Genetics
Research Mentor(s):	Robert G. Franks/Genetics Fang Bao/Genetics
Title of Presentation:	Reverse Genetic analysis Using SEUSS-LIKE2 AINTEGUMENTA Double-mutants to Determine Functional Similarity between SLK2 and SEUSS Genes in the Flowering Plant Arabidopsis thaliana

SEUSS (SEU) and the three SEUSS-LIKE (SLK) genes SLK1, SLK2, and SLK3, comprise a family of transcriptional co-regulators in Arabidopsis thaliana that display protein sequence similarity. Mutations in the SEUSS gene result in disrupted flower development and morphology. Other members of the SLK gene family however have yet to be characterized in regards to the loss-of-function mutant phenotypes. The objective of this study is to determine the degree of functional similarity shared by SLK2 and SEUSS. The slk2 mutant plant lines are grown in autoclaved soil, under standard temperature and light conditions until the adult stage is reached. At the adult stage, genomic DNA is isolated from the mature rosette leaves to be used for genotyping and confirmation of the T-DNA insertion site. Gene specific primers are used to distinguish wild type, heterozygous and homozygous individuals from the segregating populations. Wild type has no T-DNA insertions, homozygous lines have insertions in both parental chromosomes, and finally heterozygous lines have an insertion in just one of the two homologous parental chromosomes. Amplified PCR products are then visualized by electrophoresis on ethidium-bromide stained gels. Double mutant combinations are created through the analysis of progeny from specific genetic crosses of the single mutants. Floral morphology defects are characterized in the T-DNA mutant alleles and in the seu slk2 double mutants. The mutants are characterized using light microscopy with a stereo dissecting microscope. Floral organ number and type are recorded from several flowers for each genotype. Statistical differences in the number or type of floral organ is determined with the Students T-Test.

Student Author(s):	Bathurst, Erin N.
Department(s):	Microbiology
Research Mentor(s):	Eric S. Miller/Microbiology
Title of Presentation:	mRNA Translational Coupling Signals: The Tryptophan Operon of E. coli

The trpE and trpD genes of the tryptophan operon of Escherichia coli code for the two protein subunits of anthranilate synthase-phosphoribosyl transferase (AS-PRT), a crucial enzyme catalyzing the first two steps of tryptophan synthesis. The stop codon of trpE and start codon of trpD overlap by one nucleotide, leading to a phenomenon known as translational coupling. Because the ribosome continues to translate the second gene without disengaging from the mRNA transcript, translation of trpD is dependent on the translation of the upstream trpE gene. It has long been assumed that the stoichiometry of the two AS-PRT subunits, produced from one mRNA transcript, is in part maintained by efficient and obligatory translational coupling of trpE and trpD. The cell is able to limit the build-up of unnecessary products by sustaining the constant ratio of protein subunits. In this project, the trpED gene pair was isolated from E. coli and mutations were introduced by the PCR-based method of gene splicing with overlap extension. Multiple configurations were cloned to test the role of mRNA sequences in signaling translational coupling. Subsequent fluorescence enzyme assays will be used to evaluate the effect of the various mutations on AS-PRT activity, which in turn should give insight into the degree of translational coupling occurring. Data from this study can lead to a better understanding of the ribosome-mRNA interactions occurring during translational coupling.

Student Author(s):	Bonner, Bethany D.
Department(s):	Biology, Winston-Salem State University
Research Mentor(s):	Mary F. Paine/Division of Pharrmacotherapy and Experimental Therapeutics
Title of Presentation:	Comparison of the Intestinal Permeability of Two Novel Antiparasitic Agents

Human African trypanosomiasis, or African sleeping sickness, is a parasitic disease that consists of two stages. Early stages of the infection are characterized by general symptoms, including fever and lethargy, whereas late stages of infection are characterized by severe neurological disorders. Current agents used to treat either stage must be given parenterally, which is especially challenging in remote areas of Africa. The promising antiparasitic agent, furamidine (DB75), has been shown to have efficacy towards early stage infection in animal models, but it has poor systemic exposure when given orally. DB75 has poor oral absorption because it exists as a dication at physiological pH, making the agent highly hydrophilic. Pafuramidine (DB289), a prodrug of DB75, is much more lipophilic, which greatly increases permeability across the intestinal epithelium. As such, DB289 has improved oral potency towards early stage infection. Despite its improved intestinal permeability, DB289 has little promise towards late stage infection, which is likely due to inadequate concentrations of DB75 reaching the brain. Structural analogs of DB289 have been tested as treatment for late stage infection. One analog includes DB868. DB868 is a prodrug of DB829, which differs from DB75 by two nitrogen, one added to each phenyl ring. DB868 is a compound shown to have efficacy towards late stage infection in mouse models. This improved efficacy could be due to: (1) a superior permeability of DB829 across the blood-brain barrier, (2) greater extent of conversion of DB868 to DB829 in the liver, and/or (3) a higher permeability of DB868 across intestinal epithelium. To test this third hypothesis, the human intestinal cell line, Caco-2, was used to compare the intestinal permeability of DB868 to DB289.

Student Author(s):	Burroughs, James L.
Department(s):	Molecular and Structural Biochemistry Plant Pathology
Research Mentor(s):	Gary A. Payne/Plant Pathology Ryan Georgianna/Functional Genomics
Title of Presentation:	Disruption of afldmat, a Gene in Aspergillus flavus with a Putative Role in the Synthesis of the Mammalian Toxin Cyclopiazonic Acid

The potent carcinogen aflatoxin is one of many toxic secondary metabolites produced by the fungus Aspergillus flavus. Over 50 biosynthetic gene clusters for secondary metabolites are predicted to occur in A. flavus based on the genome sequence. The goal of this study was to delete a gene (afldmat) in cluster 42 that encodes for a dimethyl allyl tryptophan synthase (DMAT). We hypothesize that this gene is involved in the biosynthesis of cyclopiazonic acid, a highly selective inhibitor of a Ca2+ ATPase in mammals. Gene deletion experiments in A. flavus require an efficient genetic transformation system based on the complementation of nutritional auxotrophs. Chemical mutagenesis, targeted gene disruption, and a directed gene loss strategy were used to create strain AFC-1 that contains specific gene mutations for arginine biosynthesis (argD) and uracil biosynthesis (pyrG). This strain was shown to be phenotypically similar to the wild type strain when grown on media supplemented with arginine and uracil, and to produce wild type concentrations of aflatoxin. A DNA construct for targeted gene deletion of afldmat was constructed with argD as the selectable marker for transformation. Transformation of AFC-1 with this construct resulted in 33 putative transformants. Characterization of these mutants by PCR showed that two transformants lacked a copy of afldmat. Current research is focused on further genetic characterization of these deletion strains and analyses for their ability to produce cyclopiazonic acid.

Student Author(s):	Carr, Benjamin D.
Department(s):	Microbiology
Research Mentor(s):	Jonathan W. Olson/Microbiology
Title of Presentation:	Directed Evolution of C. jejuni and Identification of Putative Signal Transducers in Biofilm Formation

Campylobacter jejuni is the second most common cause of food poisoning in the U.S., with Campylobacteriosis resulting in 2 to 10 days of gastrointestinal distress and, rarely, Guillain-Barre syndrome. The avian gut is an optimal environment for C. jejuni, so commercial poultry flocks serve as its main reservoir and contaminated meat is a common infection source. Transmission within and between flocks, however, depends on the survival of C. jejuni outside of the host. One environmental survival mechanism is biofilm formation, a process in which bacteria adhere to a physical interface and alter their physiology and morphology to coordinate multicellular activity. Bacteria use local concentrations of small signaling molecules to detect the population threshold at which biofilm formation is initiated, a process called quorum sensing. Previous studies have shown that C. jejuni employs the Auto-Inducer II quorum sensing system and possesses a LuxS gene, which encodes its signaling molecule. However, the genes that mediate signal transduction remain unknown. Possible candidates include cj1226c and dccS, which have some homology to luxQ and luxO of the Vibrio spp., respectively. Mutants in these genes exhibit attenuated motility and biofilm formation phenotypes. In addition to site-directed mutagenesis, another project currently in progress applies evolutionary pressure to C. jejuni cultures to increase or decrease biofilm formation. Strains have been isolated with half the biofilm formation capacity of the wild-type, and twice that of the wild-type. The genomes of mutant strains will be sequenced to identify biofilm-related mutations.

Student Author(s):	Cheek, Hannah D.
Department(s):	Crop Science
Research Mentor(s):	Candace H. Haigler/Crop Science and Plant Biology Bir Singh/Crop Science
Title of Presentation:	Effects of Natural and Synthetic Auxin on Cotton Fiber and Seed Development in Culture

Cotton fibers are essential to our textile industry and also one of the purest forms of cellulose (>90%). The quality of cotton fibers can be altered by the amount and quality of cellulose in the fiber secondary wall. Natural auxin, indole-3-acetic acid (IAA), and synthetic auxin, naphthalene acetic acid (NAA), are both commonly used in plant tissue cultures. We tested how IAA and NAA affect the transition to secondary wall deposition in cultured cotton fibers (Gossypium hirsutum). We also determined how the two hormones affected fiber length and secondary wall thickness. Ovules were dissected from cotton flowers and cultured in media containing either IAA or NAA, along with GA3. Developing ovules/fibers were collected at specified days after flowering, and fiber length and weight, as well as seed weight, were measured. Additionally, fibers were observed under the microscope to determine the timing of the primary-to-secondary wall transition. Based on these observations, cultured ovules/fibers have been harvested, frozen, and stored for later analysis of expression patterns of marker genes for primary and secondary wall deposition. We found that fibers were longer when grown in NAA vs. IAA, and the ovules weighed more. However, the NAA-grown fibers weighed less, and microscopy indicated clearly that IAA-grown fibers deposited secondary wall cellulose substantially earlier than NAA-grown fibers. For ovule/fiber growth data, t-test will be used to assess significance of differences between means. Preliminarily, the data show that NAA delays the onset and reduces the extent of secondary wall deposition in fibers when compared to IAA. At the same time, NAA appears to enable prolonged fiber elongation. These data set the stage for experiments to try to manipulate the timing of secondary wall deposition in cultured cotton fibers as a means of understanding the control of this developmental transition.

Student Author(s):	Chong, Jessica
Department(s):	Biological Sciences
Research Mentor(s):	Allen Foegeding/Food Science
Title of Presentation:	Effect of Agar, Glycerol and Gelatin Concentrations on Textural Properties of Gels

The purpose of this study was to develop a series of model foods for use in mastication investigations, filling four sections of a texture map. In the first phase of the project, we determined if a standard flavoring-coloring agent could be used for agar-glycerol gels without modifying the texture of agar-based model foods. Results showed that the gels were within quadrants I (soft) and IV (brittle) of the texture map. In the second phase, the goal was to make model foods with a high level of strength (fracture stress) and deformability (fracture strain), which are within quadrants II (rubbery) and III (tough). We focused on the effects of gelatin and glycerol concentration on the rheological properties of the gel; results showed the gels reached II but not III. These data showed that model foods with soft or short (I), brittle (IV), or rubbery (II) textures can be made with agar or gelatin gels modified by glycerol. The model foods will be used in a collaborative study with scientists in the Dental School at the University of North Carolina at Chapel Hill.

Student Author(s):	Connolly-Brown, Arwen M.
Department(s):	Plant Biology
Research Mentor(s):	Jianli Lu/Plant Biology DeYu Xie/Plant Biology
Title of Presentation:	Suspension Culture of PAP1 Transgenic Tobacco cells and Anthocyanin Biosynthesis

Anthocyanin, which is universally produced by land flora, is a group of the commonest red pigments giving various red or pink or blue colors to flower and bright red colors to leaves in autumn. Its biosynthesis is controlled by multiple enzymes and regulated by several families of transcription factors. PAP1 gene encodes a R2R3-MYB transcription factor essentially involved in the biosynthesis of anthocyanin. In this presentation, we report the establishment of PAP1 transgenic tobacco cell suspension culture and the properties of anthocyanin biosynthesis. Plant tissue culture of PAP1 transgenic tobacco plants was developed by using leaf tissues as explants cultured on solid medium. Two cell lines, 6R producing high production of anthocyanin and 6W producing less anthocyanin level, were obtained. In addition, plant tissue culture line P3 of wild-type tobacco plant was established as control experiments. Plant cell suspension culture from each of the three lines was developed in liquid medium. In order to comparatively study the growth of cells and dynamic biosynthesis of anthocyanin in suspension cells, we designed experiments by growing cells in 25 ml liquid medium contained in 125 ml E-flask for 25 days. Flasks were shaken at the speed of 120 rpm/min on a rotation shaker under lighting/dark (16/8 hrs) and 25oC condition. Suspension cells and liquid medium were harvested at 0, 5, 10, 15, 20 and 25 days. The medium and cells were separated through filtering. The cells were weighed to establish a growth curve and were extracted by using ethyl acetate to measure the level of anthocyanin. Liquid medium was also extracted by ethyl acetate. Our results showed that the growth of the three cell lines had an obvious property of “S” shape from 0 day to 25 days; the wild-type cells, P3, grew faster than the transgenetic lines, 6R, and 6W. The suspension cells of 6R line produced the highest level of anthocyanin production. P3 line did not produce anthocyanin as we expected. The dynamic properties of anthocyanin biosynthesis corresponding to cell growth will be discussed in our presentation.

Student Author(s):	Cox, Christina M.
Department(s):	Biological Sciences
Research Mentor(s):	Susanne M. Gollin/Human Genetics, Graduate School of Public Health, University of Pittsburgh
Title of Presentation:	ATM Gene Deletion and CCND1 Gene Amplification in Human Breast Cancer Cells

Human cancers have been commonly found to exhibit gene amplification. Cyclin D1, also known as CCND1, has already been shown to experience significant amplification in sqamous cell carcinomas of the head and neck (SCCHN). We propose that like SCCHN, amplification of CCND1 occurs by the breakage-fusion-bridge (BFB) cycle model in human breast cancer cells and that in step one of the BFB cycle model, one of the genes that is deleted is the ataxia telangiectasia mutated (ATM) gene. ATM works to help repair mutations in the genome, while CCND1 assists in passage through the cell cycle. Using fluorescence in situ hybridization, we detected the presence or lack thereof, of the two genes of interest, ATM and CCND1, in a sample of ten different cell lines. We found that where there is CCND1 amplification, there is consistently also, ATM loss. This may be useful information when researched on a larger scale as a diagnostic tool for cancer patients to determine the aggressiveness of breast tumors and which therapies might be most effective in treating them.

Student Author(s):	Gass, C. Elizabeth
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	Antonella Longo/Molecular and Structural Biochemistry Robert Rose/Molecular and Structural Biochemistry
Title of Presentation:	Optimization of the Pitx1 Purification Protocol for Crystallization Studies

Pitx1 (Paired-like homeodomain transcription factor 1) is a homeobox protein that functions as both a transcriptional regulator on the pituitary POMC (pro-opiomelanocortin) promoter and as a bicoid-related factor in embryonic organogenesis. Our research is focused on determining the crystal structure of the Pitx1 protein bound to the POMC promoter sequence. In order to obtain sufficient concentrations of protein to allow for crystallization studies, the protocol for Pitx1 purification must be optimized. The current protocol, in which the protein is tagged with six histidine residues and bound to Ni-Sepharose beads prior to elution, has produced yields too low to be useful for crystallization studies, with a maximum yield of 0.476 mg from 1L of cell culture in our trials. Moreover, we have been unable to concentrate the protein solution to more than 0.475 mg/mL. Tracking the purification process via Bradford assays and SDS-Page gels has indicated that the low yield is not due to under-expression but rather to partial insolubility of the protein or a tendency of the protein to precipitate on the Ni-Sepharose beads. Accordingly, modifications to the protein preparation protocol may involve washing the beads with a buffer containing a higher salt concentration or increasing the solubility of the lysed cell pellet. Once a higher yield of Pitx1 has been achieved, the protein will be bound to oligonucleotides that mimic the POMC promoter, which may allow us to increase the concentration of the resulting complex. This complex will then be set on trays containing polyethylene glycol (PEG) ion screens. Initial trials with PEG ion screening indicate that current protein yields are not sufficiently concentrated to allow for crystal formation.

Student Author(s):	Gould, Jeremy E.
Department(s):	Plant Biology
Research Mentor(s):	Wendy Boss/Plant Biology Yang Ju Im/Plant Biology Amy Grunden/ Microbiology
Title of Presentation:	Agrobacterium tumefaciens Mediated Transformation of Lycopersicon esculentum with the SOR Gene Identified in the Hyperthermophile Pyrococcus furiosus

When exposed to stressful environments such as heat and drought, plants produce intracellular reactive oxygen species (ROS) which are toxic to the plant. The archaeal hyperthermophile Pyrococcus furiosus lives in deep sea volcanic vents on the ocean floor in anaerobic conditions. However, when it is thrown out of the vents and exposed to cooler oxygen-rich water, it must be able to detoxify the reactive oxygen species. P. furiosus makes a protein, superoxide reductase, which is very effective in detoxifying ROS. The "superoxide reductase" gene (SOR) has been cloned and sequenced (Jenney et al., Science 286: 306-9, 1999). Our hypothesis was that if we express P. furiosus in plants, the plants would show enhanced stress tolerance by being more capable of detoxifying the ROS that is formed during stressful periods. We have tested this concept in a model system Arabidopsis, but it has not been tested in a crop plant. The goal of this research was to transform the model crop plant Lycopersicon esculentum cv. Micro-Tom with SOR and test the stress tolerance of the transformants. Our approach was to grow sterile tomato seedlings on agar, excise hypocotyls and cotyledons from 7-9 day old seedlings, incubate with Agrobacterium expressing the SOR gene and select for transformants on kanamycin medium. We have our first putative transformed shoots and have transplanted them to rooting medium. Once roots are formed, plants will be transferred to soil, analyzed for production of the SOR protein and then transformed plants will be grown to seed. The transformed seedlings will be screened and selected for future studies of stress tolerance.

Student Author(s):	Hamilton, Peter J.
Department(s):	Textile Engineering, Chemistry, and Science
Research Mentor(s):	Sam M. Hudson/Textile Engineering, Chemistry, and Science
Title of Presentation:	Contact Activation of the Plasma Coagulation Cascade Using Chitosan Films

Chitosan is a cationic poly-beta(1-4)-2-amino-2-deoxy-D-glucose, obtained through the deacetylation of chitin, a polysaccharide found naturally as the structural element in the exoskeleton of crustaceans. Chitosan and its derivatives are known to possess hemostatic potential; however the detailed coagulation mechanisms that influence hemostatic actions are still being investigated. This study attempts to simplify the blood coagulation mechanism by removing all cellular components and examining only the protein components of the coagulation cascade, seen as the plasma portion of whole blood. The interaction between chitosan films and the plasma coagulation cascade is explored by linearly scaling the amount of 1 cm x 1 cm chitosan film squares suspended in a fixed amount of platelet poor porcine plasma. Increasing numbers of film squares correspond to increasing “surface activation” sites that serve to initiate the plasma coagulation cascade. A positive result of activating the plasma coagulation cascade would be seen as a polymerization of fibrin proteins resulting in a gel network. This test method was repeated for chitosan films with varying molecular weights and degrees of acetylation with no positive result. The same test method was performed with whole porcine blood, with a positive result, suggesting that a cellular component of whole blood is influential in the interaction between blood and chitosan films.

Student Author(s):	Hammond, Catherine E. Rueschhoff, Elizabeth
Department(s):	Plant Biology Plant Pathology
Research Mentor(s):	Heike Sederoff/Plant Biology Margaret Daub/Plant Pathology; Plant Biology
Title of Presentation:	The Role of Vitamin B6 in Arabidopsis thaliana in Carbohydrate Metabolism and Sensing

Plants synthesize vitamins essential for human nutrition. One of these vitamins is pyridoxal 5’-phosphate, which is the active form (or vitamer) of vitamin B6. Vitamin B6 is well known as a coenzyme in many reactions, including amino acid metabolism and carbohydrate metabolism. It has also been shown to be an antioxidant in oxidative stress responses in plants. We used Arabidopsis thaliana as a model system to study the metabolism of vitamin B6 synthesis in plants. Two pathways of vitamin B6 metabolism have been identified: the de novo pathway, which is present in bacteria, fungi and plants, and the salvage pathway, which is present in all organisms. The de novo pathway is responsible for synthesis of pyridoxal 5’-phosphate. The salvage pathway interconverts pyridoxal 5’-phosphate with its other vitamer forms (see Figure 1). Using Arabidopsis mutants defective in genes from both the de novo pathway (pdx1.3) and the salvage pathway (sos4), we have shown that vitamin B6 metabolism is involved in carbohydrate metabolism and sugar sensing.

Student Author(s):	Henderson, Katelyn J.
Department(s):	Plant Biology
Research Mentor(s):	Imara Perera/Plant Biology
Title of Presentation:	Expression of a Ca²⁺ Biosensor in Transgenic Arabidopsis to Measure Cytoplasmic Free Calcium in vivo

The phosphoinositide (PI) pathway is a critical signaling pathway in plants and animals. To study stress responses in plants, our lab has altered the PI pathway in Arabidopsis by expressing a mammalian type I inositol polyphosphate 5-phosphatase (InsP 5-ptase). This enzyme specifically hydrolyzes the second messenger, 1,4,5-inositol trisphosphate (InsP₃). InsP₃, has been shown to increase in response to many different stimuli and can release Ca²⁺ from intracellular stores. The Ca²⁺ acts as a second messenger activating certain protein kinases, turning on gene expression, and ultimately leading to a cellular response. To further characterize the PI pathway, we will compare Ca²⁺ signaling in the InsP 5-ptase and wild type plants in response to stimuli. Many current techniques to monitor Ca²⁺ are invasive and not well suited for studying plant cells. The aim of this study was to express a synthetic Ca²⁺ binding protein called yellow cameleon (YC 3.6) in both wild type and the transgenic InsP 5-ptase plants for in vivo Ca²⁺ measurements. This biosensor consists of two modified fluorescent proteins linked by camodulin (CaM) and a CaM-binding peptide (M13). Upon binding Ca²⁺, fluorescence resonance energy transfer will occur and the ratio of emission by the two fluorescent proteins will describe the concentration of Ca²⁺ in vivo. Wild type and a transgenic InsP 5-ptase line of Arabidopsis were transformed by the floral-dip protocol using Agrobacterium tumefaciens carrying the YC 3.6 gene. Transformants were screened by selecting with kanamycin for the wild type and Basta for the InsP 5-ptase plants. Because the InsP 5-ptase plants were previously transformed, they already carried the resistance to kanamycin. Seedlings were examined for fluorescence and the presence of YC 3.6 was confirmed by PCR of genomic DNA using gene specific primers. Future work will include selecting homozygous transformants and confirming transgene expression.

Student Author(s):	Hwang, Hye M.
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	Debra A. Clare/Food, Bioprocessing and Nutrition Sciences Prachuab Kwanyuen/USDA-ARS Chris R. Daubert/ Food, Bioprocessing and Nutrition Sciences
Title of Presentation:	Effect of Tranglutaminase Polymerization on Biochemical and Functional Properties of Heated and Non-heated Soy Proteins

Several characteristics of soy protein were investigated after treatment with microbial transglutaminase (TGase). Also, these differences were compared between heated (mSPI) and non-heated (SPI) protein substrates. Upon incubation with the enzyme, SPI and mSPI formed high molecular weight polymers, visualized after SDS-PAGE. Based on equivalent treatment times, mSPI dispersions showed less crosslinking, likely caused by the formation of aggregates during heating which limited their accessibility to the enzymatic catalytic site. To quantify polymerization reactions, o -phthaldehyde (OPA) fluorescent protein assays were used. When SPI dispersions were incubated at 60°C for 1.5 h, no change was observed in the concentration of soluble reactive amino groups. However, upon incubation with TGase (60°C, 1.5 h), a SPI gel was rapidly formed and OPA analysis was not possible. The concentration of reactive amino groups was lower in mSPI dispersions suggesting decreased solubility due to thermal processing and OPA analysis revealed 14% covalent linkage after a 1.5 h reaction time. The apparent viscosity of TGase treated SPI and mSPI dispersions, and control mSPI protein solutions (heated, non-heated), decreased with increasing shear rate, termed shear thinning. By contrast, the apparent viscosity of control SPI samples (heated, non-heated) remained fairly constant, exhibiting Newtonian flow behavior. Also, a yield stress was observed at lower shear rates. The apparent viscosity of enzyme treated SPI and mSPI dispersions was enhanced although the rates of catalysis differed. In general, the effect of chemical and enzymatic modification reactions on functional properties, such the apparent viscosity, is of great interest to the industry. With these approaches, we anticipate the development of novel soy-based protein ingredients for utilization in the food industry.

Student Author(s):	Kemp, Jayme L.
Department(s):	Pediatric Dentistry- UNC Chapel Hill School of Dentistry
Research Mentor(s):	Eric T. Everett/Pediatric Dentistry
Title of Presentation:	Analysis of KUSA-A1 Osteoblast Cell Lines to be Used for Co-culture of Osteoclastogenesis Following Treatment with Mitomycin-C

Osteoclasts are multinucleated cells which originate from hematopoietic cells of the monocyte-macrophage lineage and are responsible for bone resorption. Osteoclast formation (osteoclastogenesis) requires selected factors produced by bone forming cells called osteoblasts. A co-culture system of osteoblasts/stromal cells regulating osteoclast differentiation was established in order to examine the regulatory mechanism of osteoclast generation. The purpose of this experiment was to determine whether KUSA-A1 cells (an immortalized osteoblast cell line derived from the bone marrow stroma of a mouse) can be used in co-culture with osteoclast progenitors to induce osteoclastogenesis. KUSA-A1 cells are known to proliferate quickly, which presents a problem in co-culture design. In order to keep these cells from over populating the tissue culture system we needed to determine the optimal concentration of Mitomycin-C (an inhibitor of cell division). This drug slows down the growth rate of the KUSA-A1 cells while maintaining the natural features of the osteoblasts. In order for osteoclastogenesis to be successful, the osteoblasts/stromal cells must continue to proliferate at very low rates to leave enough room for osteoclast differentiation, and the osteoblasts/stromal cells must maintain all of their natural features. The hypothesis stated that Mitomycin-C would be successful, and the KUSA-A1 cells treated with Mitomycin-C can be used for co-culture of osteoclastogenesis. Through repeated experiments, we found that Mitomycin-C did indeed slow down the growth of the KUSA-A1 cells and these cells remained viable. We also observed that Mitomycin-C treatment slightly affected the gene expression pattern of the cells which may indicate a change in cell function. Further studies are needed to determine if KUSA-A1 cells treated with Mitomycin-C are capable of supporting osteoclastogenesis in a co-culture system.

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

Campylobacter jejuni is a gram-negative spiral bacterium that is a major cause of bacterial food poisoning in the U.S. 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. From preliminary data, the optical density and the number of colony forming units from a growth curve for 27 days indicated the formation and disintegration of a biofilm in liquid culture. This study focuses on C. jejuni and its ability to form biofilm and proteomic changes influencing biofilm formation. Proteomic analysis was used to identify proteins that are differentially expressed between wildtype (strain 11168) and a luxS mutant. The LuxS mutant is a mutant involved with decreased quorum sensing and delayed biofilm formation. Using two-dimensional gel analysis, differentially expressed proteins were excised from the gel and characterized by mass spectrometry. After characterization, knock-out mutants of target proteins, fumarate reductase, isocitrate dehydrogenase, and cj 0427 were created for further analysis. Biofilm formation in C. jejuni may play a role in transmission.

Student Author(s):	Klocke, Sarah K.
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	John Cavanagh/Molecular and Structural Biochemistry
Title of Presentation:	AbrB: Cloning and Sequencing of the C-terminal Domain

Bacillus subtilus is a bacterium commonly found in soil. In its natural habitat it is frequently exposed to harsh and inhospitable conditions. B. subtilis, has the unique ability to save itself by forming spores. AbrB is a B. subtilus transtition state regulator protein, transition-state regulators play an central and essential role in spore formation and survival of the cell. They are responsible for the transition from vegetative growth and cellular response to non-ideal environmental conditions. [2]. The N-terminal domain of AbrB has been isolated and characterized extensively while the structure and biological role of the C-terminus remain unknown [4, 5, 6]. To facilitate the study of AbrB's C-terminus, the polymerase chain reaction (PCR) will be used to truncate the full length AbrB protein to its C-terminal domain (residues 54-94). Upon isolation of the truncated DNA a new plasmid will be constructed using pET-21b from Novagen. The new plasmid will be sequenced to verify that the resulting construct contains the correct sequence for the C-terminus of AbrB, residues 54-94. Once verified, the C- terminal AbrB plasmid will be tested for protein expression. After the expression protocol is developed and optimized the resulting protein will be used with NMR and mass spectrometry to obtain the C- terminal structure. Elucidating the structure of the C-terminus will be critical in determining the biological role of the protein.

Student Author(s):	Klompstra, Diana M.
Department(s):	Biological Sciences
Research Mentor(s):	Laura D. Mathies/Genetics
Title of Presentation:	The Role of a Novel Forkhead Transcription Factor in Caenorhabditis elegans Gonadogenesis

Little is known about the complex process leading to the development of mature organs from multipotent precursor cells. The genes regulating this process and how they interact (i.e. gene regulatory networks) remain to be discovered. We use the gonad of the nematode Caenorhabditis elegans as a model for organogenesis. In C. elegans, gonadogenesis begins with the formation of a four-celled gonadal primordium. Two of these cells, the somatic gonadal precursors (SGPs), will give rise to all of the somatic tissues of the adult reproductive system. The Hand bHLH gene hnd-1 is known to act earliest in the gene regulatory network controlling gonadogenesis and is important for the maintenance and survival of the SGPs. It is not known what gene(s) regulates hnd-1 or how the SGPs are specified. Analysis of the promoter region of hnd-1 shows possible FoxF transcription factor binding sites. The only FoxF transcription factor in C. elegans is encoded by the gene let-381. It is known that a FoxF transcription factor in Drosophila melanogaster directly regulates the fly Hand bHLH gene. Based on this evidence, we predict that this regulation is conserved and that LET-381 regulates hnd-1 in C. elegans. We applied classical and molecular genetics approaches to ask if LET-381 regulates hnd-1. By finding out how let-381 acts in gonadogenesis, we can add to the gene regulatory network that governs gonad development so that we can better understand organ formation and move closer to understanding how SGP fate is specified.

Student Author(s):	Lorick, Stephanie R.
Department(s):	Entomology
Research Mentor(s):	Fred L. Gould/Entomology Gissella Vásquez/Entomology Marie Estock/Entomology
Title of Presentation:	Quantity of Receptor Genes HR13 and HR15 in Male Moth Species Heliothis subflexa and Heliothis virescens Are Unaffected by Moth Age and Mating Status

The moth species Heliothis subflexa and Heliothis virescens are closely related and overlap in geographic range. H. virescens is a severe agriculture pest on cotton, tobacco, and soybean; while H. subflexa is a pest on physalis plants (e.g. tomatillo). Both species are difficult to control, and pheromones have been used for monitoring moth flight and to disrupt moth mating. The male moth antennae have pheromone receptor proteins that bind to components of the female pheromone blend. This allows the male to detect the presence of a female moth of the same species. HR13 and HR15 are two receptors implicated in the male's response. HR13 binds to a pheromone component common to females of both species. HR15 is less well characterized and could be important to one or both species. Two hypotheses were tested: 1) HR13 and HR15 mRNA quantities are equal in the two species, 2) The mRNA quantities are unaffected by age and mating status. mRNA from both species was extracted from the male antennae of individuals that varied in age and mating status. The mRNA was used to make cDNA using reverse transcriptase. Quantitative Real Time Polymerase Chain Reaction (QRT-PCR) was utilized to estimate quantities of HR13 and HR15 cDNA. The quantity of HR13 mRNA was greater than that of HR15 in both species. Finally, in both species there was no significant difference in the relative quantity of HR13 and HR15 mRNA due to age or mating status.

Student Author(s):	Lowder, Casey D.
Department(s):	Biological Sciences
Research Mentor(s):	Wendy F. Boss/Plant Biology
Title of Presentation:	Characterization of the Impact of Plant-specific PIPKs on Membrane-associated Actin Filament Formation in vivo

Phosphatidylinositol phosphate kinases (PIPKs) are essential enzymes in the phosphoinositide pathway, an important pathway in sensing and responding to environmental stimuli. Unlike human PIPKs, at least one Arabidopsis thaliana PIPK, AtPIPK1, has been previously shown to bind directly to filamentous actin (F-actin). Our lab has identified a critical peptide region within AtPIPK1, the linker region, which is essential for actin binding in vitro. In order to characterize direct interaction between the AtPIPK1 linker region and actin in vivo, we first had to have a method for visualizing actin in vivo. To this end, we expressed cyan and yellow fluorescent-tagged actin binding proteins in Nicotiana tabacum (NT-1) cells. These cells will eventually be retransformed with green fluorescent tagged AtPIPK1 linker region peptides. The localization of actin binding protein and linker peptide will be determined by visualizing the proteins using confocal microscopy. Thus far, we have produced tobacco cells expressing cyan or yellow fluorescent protein linked to actin binding protein2 (35S:ABD2-CFP and 35S:ABD2-YFP). The constructs were obtained from Dr. Elison Blancaflor and were transformed into Agrobacterium tumefaciens using the freeze-thaw method. Agro-transformants were co-cultivated with wild-type NT-1 cells and transgenic NT-1 cells were selected on media containing hygromycin. We generated an AtPIPK1 linker region construct in a pENTR vector using PCR based cloning. The resulting construct was verified by restriction enzyme digestion and PCR for correct orientation. Verified constructs were recombined into a GFP-containing plant binary vector (pK7WGF2). We are currently in the process of selecting tobacco cells expressing AtPIPK1 linker region peptide tagged with green fluorescent protein (GFP-linker). This construct will be used in the future to transform 35S:ABD2-CFP and 35S:ABD2-YFP expressing tobacco cells. We anticipate that the linker region of AtPIPK1 will be critical for actin binding in vivo and when imaged, the GFP-linker will co-localize with 35S:ABD2-CFP and 35S:ABD2-YFP.

Student Author(s):	Maradia, Dhara K.
Department(s):	Biochemistry
Research Mentor(s):	Cynthia Hemenway/Biochemistry
Title of Presentation:	Making p850PVX Clone by Isolating 850nt from pMon8453, Containing Potato Virus X cDNA

Potato Virus X (PVX) is a rod-shaped virus containing a capped and polyadenylated 6438 nucleotide RNA genome. PVX is an excellent model system for studying RNA replication because of its small, single genomic RNA that is functionally monocistronic and is available as an infectious clone. In addition, this virus replicates to high levels in plants and protoplasts. These features of PVX make it very efficient for biochemical and genetic studies of protein and RNA components required for infection, isolation and biochemical characterization of viral proteins and replication complexes, passaging of mutants for revertant analyses, and the expression of foreign genes in plants. The Hemenway lab found that replication of PVX is dependent on long-distance interactions between terminal and internal conserved elements in the genome. For my project I made a clone, p850PVX, which will be used to study this process. To make the p850PVX clone, I cut out 850nt from the 3’ end of pMon8453 that includes the hexanucleotide motif in the 3’ non-translating region and upstream complementary elements. Subsequently, this 850nt insert was ligated in the transcription vector pGEM to obtain the final clone. The clone p850PVX will specifically be used to analyze the interaction between the 3’ hexanucleotide motif and a conserved complementary element upstream of capsid protein gene.

Student Author(s):	Medearis, Sarah A.
Department(s):	Microbiology
Research Mentor(s):	Matthew D. Koci/Poultry Science
Title of Presentation:	Development of an Expression Construct to Examine the Effects of Polymorphisms within Mx on Antiviral Activity

The Mx protein is part of the interferon mediated host response to viral infection. Genes encoding Mx and their ability to inhibit viral replication have been described in various vertebrate animal species. Polymorphisms within Mx among breeds of mice have been described to affect the host’s ability to resist viral infection. In chickens, an amino acid change at position 631 (Asp to Ser) has been described as a major determinant of antiviral activity. Our laboratory has recently identified numerous polymorphisms associated with distinct genetic lines of chickens. The purpose of the current study was to develop an expression system to allow for the systematic analysis of each of the alleles of chicken Mx. To achieve this, Mx expression was induced by interferon and influenza in primary chicken embryo fibroblast cells and confirmed with RT-PCR. The full length cDNA was amplified, cloned into a PCR cloning vector and sequenced. The cloned Mx gene was found to have a serine at amino acid position 631, suggesting it would not have antiviral activity. Mx was then subcloned into an expression vector. Using site-directed mutagenesis, this construct will allow us to test the effect of various polymorphisms have on the antiviral activity of Mx. The result of the studies based on this expression system will enhance our understanding of antiviral mechanisms of Mx. Ultimately, these studies will allow for the development of transgenic animals with the effective Mx to aid in the host’s resistance to viral activity.

Student Author(s):	Medalla, Nicole C.
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	John Cavanagh/Molecular and Structural Biochemistry
Title of Presentation:	Sub-cloning, Expression, and Purification of Red Clover Necrotic Mosaic Virus-Movement Protein

Plant viruses are parasites, are intracellular and can not replicate without a host do to the absence of a molecular machinery. The Red Clover Necrotic Mosaic Virus (RCNMV) is a plant virus in the Dianthovirus genus and the family Tombusviridae. Systemic infection of a plant by a virus requires two forms of spread from the cells initially infected: short-distance movement from cell to cell probably via plasmodesmata and long-distance movement via the vascular system. Virus cell-to-cell movement is not a passive process but requires virus-encoded movement proteins. RCNMV contains one such protein, a 35kD protein called RNA-2 swiss prot ID P10838. To date no structural characterization has been done on any plant viral movement protein. In order to begin the structural work, the gene of interest, has been truncated to include residues 1-168 only. The n- terminus was inserted into the pET 28a Novagen system. By using various techniques in cloning, expression, and purification of both the DNA and Protein components the new plasmid has been constructed and work on the expression protocol has begun. The sequence of the RCNMV 1-168 pET 28a construct will be verified and the protein expression is examined. The protein expression protocol will be optimized and structural studies using NMR will begin. This will be the first plant viral movement protein to be structurally characterized.

Student Author(s):	Moye, Virginia A.
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	Paul F. Agris/Molecular and Structural Biochemistry
Title of Presentation:	Characterization of the 5-formylcytidine (f⁵C) Modification Involved in C-A Wobble Binding of Mitochondrial tRNA Methionine (mtRNA^Met) with the AUA Codon

Mitochondrial tRNA methionine is uniquely post-transcriptionally modified at the first position of the anticodon. This 5-formylcytidine (f⁵C) modification allows mtRNA^Met to read both the AUG codon for methionine and the AUA codon for isoleucine in both the A- and P-sites of the ribosome during protein synthesis and is also responsible for significantly increased ribosomal binding. The f⁵C modification of mtRNA^Met is highly conserved in a range of species from squid to humans, and the mechanism is of particular interest because in order for the tRNA to read the AUA codon, a bond must be formed between the f⁵C and the final A of the isoleucine codon. To investigate this modification and its influence on the structure of mtRNA^Met, thermal characterization experiments were performed to compare the tRNA possessing the f⁵C modification and the unmodified tRNA. The modification appears to destabilize the structure of tRNA^Met, making it more flexible and giving it the symmetry necessary for binding. This destabilization is evident by the reduction of the T_m of the modified as compared to the unmodified mtRNA^Met. To further understand this modification, a bioinformatics search is currently being performed to identify the enzyme responsible for this modification. In the future, information gained by characterization of the modification may be used in techniques such as RNA silencing to develop highly specialized short interfering RNA sequences for treatment of disease. Identifying the enzyme responsible for the modification may enable the development of therapeutics for mitochondrial genetic disorders caused by misreading of base pairs.

Student Author(s):	Oluronbi, Ruby A.
Department(s):	Biochemistry
Research Mentor(s):	Rupangi C. Vasavada/Department of Medicine, Division of Endocrinology, University of Pittsburgh
Title of Presentation:	Determination of the effects of Parathyroid Hormone Related Protein on Cell Cycle Regulators to enhance β-Cell Proliferation

Diabetes is a disease that results from the body’s inability to produce insulin due to insufficient functional pancreatic β-cell mass, or the inability to use insulin properly, or both. Parathyroid hormone related protein (PTHrP) is known to enhance functional β-cell mass in mice by increasing β-cell proliferation. It is also known that PTHrP mediates its proliferative effect on the β-cell through activation of the atypical Protein Kinase C (PKC) signaling pathway. However, nothing is known regarding the regulation of cell cycle molecules by PTHrP in the β-cell. Based on the effects of PTHrP on cell cycle regulation in other cell types, we hypothesize that PTHrP may increase β-cell proliferation through the regulation of cell cycle inhibitors. Therefore, this study examines the effects of PTHrP on the expression of members of the Cip/Kip and INK families of cyclin-dependent kinase inhibitors.

Student Author(s):	Overton, Matthew H.
Department(s):	Biochemistry Microbiology
Research Mentor(s):	Stefan Franzen/Chemistry Richard H. Guenther/Plant Pathology Steven A. Lommel/Plant Pathology
Title of Presentation:	Characterization of Novantrone Infusion in Red Clover Necrotic Mosaic Virus

Selective and specific delivery of cytotoxic compounds to cancerous cells is the goal for next generation cancer treatment. One way to selectively deliver cytotoxic compounds is to load them in a nanoparticle that has been engineered to target only cancerous cells. This research seeks to develop a method that allows for the characterization of the infusibility of a novel plant virus nanoparticle with regards to a chemical of therapeutic interest, Novantrone. It also seeks to develop a simple method to isolate infused virus particles from unincorporated drug. In this study, Red clover necrotic mosaic virus (RCNMV) is modified with buffers which cause it to swell, enhancing the ability of the added drugs to become encapsulated. After twenty-four hours of incubation with the drug, the infused virus sample is purified and the amount of drug encapsulated is determined by absorbance and fluorescence profiles of the infused sample. Experiments to determine the limit of infusibility of the virus were conducted by altering the starting concentration of RCNMV in each experiment to determine the ideal concentration for maximum infusibility of 100 uL of a 3.18 mM Novantrone solution. The results demonstrate that size exclusion chromatography is effective in separating viral particles from free drug. The infusibility experiments show that a limit of approximately 175 molecules of Novantrone per virion can be encapsulated in a standard 10 mg/mL virus solution. These studies also show that viral concentrations between 1.25 mg/mL and 0.625 mg/mL exhibit drug infusibility saturation with approximately 500 infused particles per virion whereas increasing viral concentrations show a decreasing pattern of infused molecules per virus. These trends suggest that the load of drug per virus can be controlled by adjusting the ratio of drug to virus during formulation.

Student Author(s):	Oyegunwa, Olusegun A.
Department(s):	Plant Biology
Research Mentor(s):	Wendy F. Boss/Plant Biology Yang Ju Im/ Plant Biology
Title of Presentation:	In vitro Analysis of an Arabidopsis thaliana PIPK1 Binding Partner

Interacting partners of Arabidopsis thaliana phosphatidylinositol phosphate kinase 1 (AtPIPK1AtPIPK1), profilin 1 and profilin 5, have been identified using isolated Arabidopsis proteins (Davis et al., 2007). Our goal for this project was to determine whether one or both of the profilin isoforms interact directly with AtPIPK1. In addition, because actin binds both AtPIPK1 and profilin, we asked whether profilin affected actin-AtPIPK1binding. We hypothesized that profilin would compete with AtPIPK1 for actin binding. For these experiments, we expressed full-length AtPIPK1 along with its domains, MORN and ∆MORN in Escherichia coli, and attempted to observe how actin interacts with AtPIPK1, profilin, MORN and ∆MORN in in vitro experiments using Gluthathione S-transferase and Bovine Serum Albumin as controls. Our biggest challenge thus far has been trying to optimize actin polymerization conditions and conditions for protein-protein interaction. We have approached this problem by varying concentrations of actin and binding proteins to improve polymerization and to decrease non-specific binding. In addition, we tested three different test tubes and found the non siliconized, 1.5ml tubes were optimal for polymerizing the actin and recovering the pellet (actin filaments). Once we optimized acting binding conditions, we discovered that production of full length recombinant proteins decreased with time, therefore, we are planning to retransform Escherichia coli with AtPIPK1 constructs and will test for acting binding once we produce sufficient recombinant protein. Reference: Davis JBC 282: 14121-14131, 2007

Student Author(s):	Parr, Meredith A.
Department(s):	Biological Sciences
Research Mentor(s):	Nanette M Nascone-Yoder/Molecular Biomedical Sciences
Title of Presentation:	Heterotaxin: A Novel Pyridine Compound that Perturbs Left-Right Asymmetric Organ Morphogenesis

Proper orientation of internal organ situs is dependent on correct interpretation of left-right asymmetric cues by developing organs. To investigate the molecular mechanisms of asymmetric organ morphogenesis, we employed a phenotype-based chemical genetic screen in embryos of the frog Xenopus laevis, which develop organ asymmetries analogous to higher vertebrates. In a screen of 44 natural product-like compounds, one compound mixture termed “Heterotaxin” specifically reversed or isomerized the asymmetry of the heart and gut without affecting other aspects of development. To examine the interpretation of left-right cues in this context, we used in situ hybridization to define the expression of three genes that are normally expressed in left-side specific patterns in vertebrate embryos, the nodal-related gene Xnr-1, the nodal antagonist and lefty homologue Antivin and the homeobox gene Pitx2. Heterotaxin-treated embryos have either unilateral left, unilateral right, bilateral or absent Pitx2 and Xnr expression in the lateral plate mesoderm, suggesting that global left-right asymmetry is randomized by Heterotaxin. Interestingly, Antivin expression appears completely absent on both sides of Heterotaxin-treated embryos. These anomalous gene expression patterns give important clues to the normal functions of these asymmetrical genes in asymmetric organ development. The discovery of Heterotaxin provides a novel tool to uncover the etiology of heterotaxia, and underscores the utility of a chemical genetic approach to organ morphogenesis.

Student Author(s):	Puetzer, Jennifer L.
Department(s):	Biomedical Engineering
Research Mentor(s):	Susan H. Bernacki/Biomedical Engineering Elizabeth G. Loboa/Biomedical Engineering
Title of Presentation:	Effects of Cyclic Hydrostatic Pressure on Chondrogenesis of Human Adipose-Derived Adult Stem Cells

The purpose of this study was to determine if, in the absence of chondrogenic media, human adipose-derived adult stem cells (hASCs) would initiate chondrogenic differentiation in response to cyclic hydrostatic pressure (CHP). We hypothesized that CHP alone would be enough to induce chondrogenesis of hASCs as evidenced by upregulation of mRNA expression of Sox9, aggrecan, collagen II, and/or cartilage oligomeric matrix protein (COMP). Human ASCs were isolated from two donors and separately seeded in 2% type VII agarose constructs at a cell seeding density of 9 x 106 cells/ml. Control constructs were placed in static culture in an oil-filled container while experimental constructs were loaded under CHP of 7.5 MPa for 4 hours per day at 1 Hz for up to 21 days in an oil filled 1L pressure vessel connected to a hydraulic cylinder powered by an MTS 858 Mini Bionix II load frame. Real time RT-PCR analysis for mRNA expression of type II collagen, aggrecan, Sox9, and COMP was performed on samples taken at 0, 7, 14, and 21 days. Real Time RT-PCR revealed an upregulation in the average donor mRNA expression of aggrecan, Sox9, and COMP of the loaded samples compared to the unloaded samples on day 7. The GAPDH values for the loaded samples for both donors were extremely low on day 21. There was no mRNA expression of type II collagen in either donor at any time point. The upregulation of aggrecan, Sox9, and COMP mRNA expression in the loaded samples on day 7 suggests early chondrogenesis. The low GAPDH expression levels in the CHP-loaded hASCs of both donors on day 21 potentially suggest that hASCs cell viability might have decreased as a result of continued CHP. The findings of this study exemplify the importance of considering mechanical load for cartilage tissue engineering using hASCs.

Student Author(s):	Ricks, Jennifer J.
Department(s):	Biochemistry
Research Mentor(s):	Hai Jiang/Biology, Massachusetts Institute of Technology Michael T. Hemann/Biology, Massachusetts Institute of Technology
Title of Presentation:	Tumor Environment Plays a Critical Role in Determining Response to Doxorubicin in ATR-Deficient Cells

Following DNA damage caused by radiation or chemotherapy, signaling pathways are activated in the cell that lead to cell cycle arrest, DNA repair, and/or cell death. Suppression of genes involved in these pathways can be used to examine the role of these processes in chemotherapeutic sensitivity or resistance. We have shown that small hairpin RNA molecules targeting the gene ATM and Rad3-related (ATR) confer resistance to the chemotherapeutic doxorubicin (Dox) in cell culture, but sensitize tumors to Dox in mice. We hypothesized that this response difference is due to 1) a difference in the time frames of the two experiments, 2) a significant difference in the doses of Dox administered in the two settings, or 3) microenvironmental factors that are present in mice but absent in cell culture. Our results support the hypothesis that the tumor microenvironment is responsible for this difference, as altering the time frame or dosage of the culture experiments failed to sensitize ATR-deficient cells to Dox. Importantly, cell cycle analysis of tumors after Dox treatment shows a profound difference in the two settings. These data suggest that specific genetic mutations can produce distinct, and even opposing, effects on therapeutic response when examined in relevant physiological contexts.

Student Author(s):	Rouf, Cynthia
Department(s):	Zoology
Research Mentor(s):	Simon Gregory/Duke University, Department of Medicine, Center for Human Genetics Svati Shah/Duke University, Department of Medicine, Center for Human Genetics Beth S. Sutton/Duke University, Department of Medicine, Center for Human Genetics
Title of Presentation:	Association of Gene Biliverdin Reductase with Coronary Artery Disease Risk

In the United States coronary artery disease (CAD) kills one person every minute on average, and last year alone CAD was responsible for an estimated $151.6 billion in economic losses. While many environmental elements can contribute to CAD development, numerous independent studies have used pedigree analyses and twin studies to show that family history is among the strongest risk factors, and that many CAD risk factors such as lipid levels, hypertension, body mass index, and diabetes also exhibit significant heritabilities, providing cogent evidence that multiple genes play a significant role in CAD susceptibility. The identification of those genes underlying CAD risk will open doors to more efficient and personalized prevention, diagnosis, and treatment for this heavy socioeconomic burden. Accordingly, our lab group is searching across the human genome to identify, prioritize, and validate candidate genes based on the convergence of multiple avenues of evidence, including the following: location of the genes in linkage peaks, evidence from mouse models, pathophysiological role of the protein product, and statistical association of the gene with CAD phenotypes. Specifically, my work within the group focuses on selecting and validating one of those candidate genes, biliverdin reductase(BLVRA). Gene BLVRA's enzyme product converts a heme degredation product (biliverdin) into a useful antioxidant (bilirubin) strongly correlated with decreased CAD. My results from genotyping 10 haplotype tagging SNPs to thoroughly test all the polymorphisms in gene region suggest that the downregulated genotypes at the BLVRA locus increase risk for CAD. The results also narrow in on one strongly and consistently associated haplotype block of the gene within which the risk conferring polyorphism(s) likely lie. Continuing research on gene BLVRA involves a deeper look into the functional polymorphisms within that haplotype block, and the identification/validation of other CAD risk genes in the BLVRA pathway.

Student Author(s):	Saylor, Katherine D.
Department(s):	Biological Sciences
Research Mentor(s):	Rachael Thomas/Molecular Biomedical Sciences
Title of Presentation:	A 15Mb-Resolution, Genome-Anchored, Cytogenetically-Validated BAC Map for the Domestic Cat

Feline injection-site associated sarcomas (ISAS) can develop after a cat has been vaccinated. ISAS are typically more aggressive, larger, and have a higher recurrence rate than sarcomas that are not associated with vaccination (non-ISAS). Consequently ISAS must usually be surgically excised with considerably larger margins than are typically required for non-ISAS cases. It can, however, be extremely difficult to distinguish conclusively between these tumor subtypes on the basis of their clinical presentation and/or histopathology. This study aims to verify the theory that the number and distribution of chromosome copy number aberrations (CNAs) is associated with tumor subtype, and thus can be used to distinguish between them. We have developed a genomic microarray for detection of tumor-associated CNAs in feline sarcomas. As part of this process, genomic DNA was isolated from around 200 domestic cat bacterial artificial chromosome (BAC) clones and physically assigned using multicolor fluorescence in situ hybridization (FISH) analysis, to map each clone to its unique chromosomal location. Their location was also compared to its predicted assignment within the cat genome sequence assembly. The majority of BAC clones mapped to the expected chromosome, and in the expected relative order, whilst a small proportion of clones did not show the expected FISH results. We present the optimization of this clone resource into a ~15Mb resolution cytogenetic map of the cat genome, that is now being used for cytogenetic analysis of cat tumors.

Student Author(s):	Shah, Nishant P.
Department(s):	Biochemistry
Research Mentor(s):	Christopher Halweg/Laboratory of Molecular Genetics, Chromosome Stability Group, National Institute of Environmental Health Sciences
Title of Presentation:	Development of a Friedreichs’s Ataxia Model Cell System Using Regulated MicroRNA to Knockdown Frataxin Expression

Friedreich’s ataxia (FRDA), the most common hereditary form of ataxia, is an autosomal recessive disease that causes neural degradation. The pathogenesis is progressive ataxia, absence of vibration sense, high incidence of diabetes, cardiomyopathy, scoliosis, optic atrophy, premature death, and sensorineural hearing loss. Disease onset typically appears between five and twenty years of age with a median life span of approximately three decades. The cause of FRDA is due to a deficiency of a highly conserved mitochondrial protein called frataxin, which regulates iron homeostasis. Interestingly, FRDA is a quantitative disease in that the level of reduction of frataxin appears to correlate with severity. Since FRDA is a quantitative disease, a model system with the capacity to modulate the levels for frataxin expression would be beneficial. In order to study the effects of reduced levels of frataxin we have designed a FRDA model system which uses a stably integrated tetracycline-regulated expression system to modulate the levels of frataxin within an osteocarcoma (U2OS) cell line. In this system, the addition of tetracycline allows the Tet repressor (TetR) to disassociate with the operator sequence and allows transcription of a microRNA (miRNA) that targets degradation of frataxin RNA. We are currently in the process of creating stable cell lines and evaluating the efficiency of the miRNA mediated reduction in frataxin by real-time PCR. Once this model system has been fully evaluated, we plan to investigate the biological consequences of reduced levels of frataxin.

Student Author(s):	Springston, Mastafa M.
Department(s):	Genetics
Research Mentor(s):	Laura Reed/Genetics
Title of Presentation:	Natural Variation of Lipid Content in Wild-type Drosophila melanogaster and its Implications as a Model for Human Metabolic Syndrome

Metabolic syndrome is a complex phenotype recognized by a combination of related symptoms: central obesity, insulin resistance, elevated blood pressure, high circulating triglycerides, and low HDL cholesterol. The presence of metabolic syndrome is a high risk factor for other health problems such as coronary heart disease and type II diabetes. The intrinsic mechanisms linking the syndrome symptoms are not known since genetic risk factors for metabolic syndrome are the result of a complex genotype by environment interaction. Using Drosophila melanogaster as a model organism we are working to characterize the complex genetic architecture underlying the metabolic syndrome. Our first step in the project was to screen 156 different wild-type Drosophila lines for natural genetic variation in hemolymph glucose, lipids, and pupal weight across four dietary stresses. I measured lipid content on all 156 lines using a total triglyceride kit in a 96-well absorbance assay format. Variation in triglyceride levels among the different lines followed a normal distribution with a mean mg/ml standardized to pupal weight of .231961mg/ml per larvae. Triglyceride levels showed a strong variation among genetics lines (p-value <.0001) and showed a significant genotype by environment interaction (p-value <.0001). These findings demonstrate that significant variation in triglyceride levels does occur naturally in wild type Drosophila populations. The significant genotype by environment interaction suggests that a metabolic syndrome phenotype can be induced in certain genetic lines. This leads us to conclude that Drosophila can be a viable model for human metabolic syndrome.

Student Author(s):	Thompson, Peter M. Cooper, Beth W.
Department(s):	Molecular and Structural Biochemistry Toxicology
Research Mentor(s):	Andrew D. Wallace/Toxicology
Title of Presentation:	The Biological Activity of DEHP and its Metabolites

Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer used in many plastic products and leaches off plastic materials over time and use. DEHP has been shown to increase the protein levels of cytochrome P450 3A4 (CYP3A4), a protein responsible for metabolism of foreign compounds, including about 50% of pharmaceuticals as well as endogenous steroids. DEHP has also been shown to act as a developmental reproductive toxicant and increase liver and testicular CYP3A levels in rodents. There is growing concern that DEHP and other phthalates may also affect human reproduction. While we have some idea of what DEHP does, we know very little about the activity of DEHP metabolites. These compounds appear in the body as DEHP is metabolized by liver enzymes to mono-2-ethylhexyl phthalate (MEHP) and MEHP oxidative metabolites. There is some evidence that these metabolites could cause their own deleterious effects. Our research focused on understanding if DEHP and its metabolites induce CYP3A4 promoter activity and CYP3A4 protein levels, the mechanism of this induction, and if these compounds also affect the activity of the androgen receptor in certain cells. We found that DEHP and MEHP increase CYP3A4 promoter activity via the pregnane X receptor (PXR), that the amount of PXR is dependent on glucocorticoid receptor (GR) activation, and that the metabolites do not significantly activate PXR. However, our latest findings indicate that the metabolites may affect androgen receptor signaling.

Student Author(s):	Vinal, Kellie
Department(s):	Reproductive and Developmental Toxicology Molecular and Structural Biochemistry
Research Mentor(s):	Tatsuya Sueyoshi/Lab of Reproductive and Developmental Toxicology at NIEHS
Title of Presentation:	Crystallization of Cytoplasmic CAR Retention Protein (CCRP)

Cellular and molecular mechanisms of constitutive androstane receptor (CAR) activation are not yet well understood. The nuclear receptor CAR, when activated by xenobiotics, is responsible for regulating genes for drug transporters and metabolizing enzymes. The cytoplasmic CAR retention protein (CCRP) interacts with CAR and developing a full knowledge base about CCRP is essential for learning more about CAR. In this experiment, current biotechnological cloning techniques were used to create a GST::CCRP fusion protein that could be expressed in E. coli. This fusion protein was then purified from ten liter cultures of E. coli containing the plasmid designed for expressing the fusion protein. Finally, the CCRP was cleaved from the fusion tag protein, GST, bound to GSH agarose. The ultimate goal was to purify the CCRP in such a way that it could be crystallized within NIEHS. So far, the protein purification techniques optimal for this protein have been perfected, but the crystallization has not yet been successful. The protein CCRP exhibits poor solubility in buffer solutions of neutral pH, which are unfortunately essential for crystallization. To solve this problem, several new constructs of CCRP were created with strategic sequences of amino acids cleaved from each end of the protein. These new constructs were created in the hopes of achieving improved solubility in neutral pH buffer solutions so that crystallization would be possible. The new constructs showed increased solubility, although crystallization has not yet been achieved.

Student Author(s):	Walker, Patrick T. Zachary, Christopher L.
Department(s):	Plant Biology
Research Mentor(s):	DeYu Xie/Plant Biology
Title of Presentation:	Molecular Characterization of Antioxidant Proanthocyanidins in PAP1-ANR Transgenic Tobacco Plants

Proanthocyanidins (PAs), also called condensed tannins (CTs), are potent antioxidants with multiple medicinal and nutritive benefits to human health, such as anti-cancer and anti-arthrosclerosis. The goal of our research is to molecularly characterize proanthocyanidins in PAP1-ANR double transgenic tobacco. Our previous work established a complete biosynthetic pathway of proanthocyanidins in tobacco plants by over expressing PAP1and ANR transgenes. We grew F2 transgenic plants in greenhouse to study the molecular properties of engineered proanthocyanidins. Regular PCR analysis showed the genetic segregation of the PAP1 and ANR transgenes in F2 offspring plants. Proanthocyanidins were extracted from leaves. Hydrolysis of crude PAs extract produced two main components: pelargonidin and cyanidin. In addition, delphinidin was detected as a minor component. This result indicated that engineered PAs include epicatechin, epiafzelechin and epigallocatechin units. Different oligomeric molecular fractions of PAs were isolated from PAP1-ANR double transgenic tobacco leaf tissues by using column chromatography separation and characterized by using a HPLC-MS based profiling. The main monomeric molecules included epicatechin and epiafzelechin. Ent-gallocatechin was also detected. The detected dimeric PAs candidates included epicatechin-epicatechin, epiafzelechin-epicatechin and epicatechin-epigallocatechin. Epicatechin-4 beta-8 epicatechin (Procyanidin B2) was definitively identified as one of the main dimeric PA molecules made by transgenic plants. One candidate of identified trimeric PA molecules was predicted to be epicatechin-epicatechin-epicatechin. In addition, five yellowish compounds were extracted from transgenic plants. The characterization of these new compounds is under way.

Student Author(s):	Waugh, Lindsey N.
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	William L. Miller/Molecular and Structural Biochemistry
Title of Presentation:	Stable Expression of Smad Proteins for Purification of Factors That Drive Transcription of the FSH-beta Subunit

Follicle stimulating hormone (FSH), responsible for egg maturation in mammals, is solely produced in the gonadotropes of pituitary tissue. The beta subunit controls overall FSH production, and its regulation is therefore crucial in furthering research in the area of female hormonal control. LbetaT2 cells are transformed mouse gonadotropes, which have been used successfully to study the transcriptional elements and proteins involved in the regulation of the beta-subunit of FSH. Smad proteins are known to bind with additional unknown proteins on a part of the FSH beta promoter critical for transcription (-167 to -158 bp). To identify these unknown proteins, LbetaT2 cells were engineered to produce Smad 3/4 that contain sequences that bind streptavidin and calmodulin. Later, these binding sequences will be used to isolate the Smads and their partner proteins using streptavidin and calmodulin affinity chromatography. The plasmids that encode Smad 3/4 also contained a sequence for resistance to the antibiotic geniticin (G418); thus stable transformants were identified because of their resistance to G418. The amount of protein (Smad 3/4) being produced was determined by western blotting using commercial antibodies against Smad 3/4. Those LbetaT2 cells which were successfully transformed and produced the greatest amount of Smad 3/4 will be used in future research to isolate the unknown upstream binding proteins.

Student Author(s):	Westfall, Kathryn J.
Department(s):	Microbiology
Research Mentor(s):	Amy M. Grunden/Microbiology
Title of Presentation:	Development of a Protocol for Functional Terminal Restriction Fragment Length Polymorphism Analysis

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a 16S rDNA PCR-based technique for determining the composition of complex microbial systems, such as swine waste lagoons, in a rapid, culture-independent manner. In this technique, fluorescently labeled universal DNA primers are used to obtain PCR products, which are then digested with restriction enzymes, yielding fluorescently labeled Terminal Restriction Fragments (T-RFs). These T-RFs are analyzed via the Phylogenetic Assignment Tool (P.A.T.) online and by utilizing a custom designed database program, which matches specific fragment patterns to specific microorganisms, enabling their identification. While identification of microorganisms in complex systems is important, the ability to accurately determine metabolic processes occurring at a specific time in a similar rapid, culture-independent, high-throughput manner is a significant innovation. A custom T-RFLP database program called InSilico© has made it possible to design custom T-RFLP experiments, and has facilitated the development of a functional T-RFLP (fn-T-RFLP) protocol. The fn-T-RFLP protocol utilizes real-time RT-PCR of mRNA to quantitatively indicate to what degree a particular metabolic process is occurring within the organism of interest based on the expression of target gene(s). Proof-of-concept experiments for the fn-T-RFLP protocol are being completed using Microsource ® S, a microbial feed additive composed of a spore suspension of three Bacillus species. Specifically, the gene sequence for a flagellin protein, hag, which is only expressed in the beginning of spore germination, was targeted for amplification. Amplification of the hag gene will allow both identification and quantification of spore germination of the Microsource® S organisms. Once completed, the fn-T-RFLP protocol will enable similar identification and quantification of processes, including methanogenesis and nitrification, in other organisms. Fn-T-RFLP will provide a cost-efficient, rapid, high-throughput alternative to current methods of analysis of these processes.

Student Author(s):	Whitaker, Kaylan M.
Department(s):	Microbiology
Research Mentor(s):	Amy M. Grunden/Microbiology Anthony A. Devine/Microbiology
Title of Presentation:	Investigation of Whether the PF1280 Protein Regulates Expression of the Pyrococcus furiosus Superoxide Reductase (SOR) System

The hyperthermophilic archaeon Pyrococcus furiosus has been shown to have the superoxide reduction pathway for converting the toxic oxygen radical superoxide to water without the generation of more oxygen molecules that could continue oxygen radical production under oxidative stress conditions. The genes encoding all three of the superoxide reduction pathway enzymes, superoxide reductase (SOR), rubreythrin reductase (Rr), and rebredoxin (Rd) are co-localized in an operon, and based on previous gene expression data, appear to be coordinately expressed in response to oxidative stress. An analysis of the SOR operon and surrounding DNA revealed the presence of an open reading frame, designated as PF1280 that is located just upstream of the SOR pathway and appears to have discrete DNA-binding motifs. Therefore, given that the expression of the SOR pathway genes appear to be regulated and that a potential DNA-binding protein (PF1280) gene is located close to the SOR operon, the hypothesis for this study is to determine whether the PF1280 serves as a possible regulator for the superoxide reductase system. In order to determine this, the PF1280 gene was PCR amplified and cloned into an expression vector for recombinant expression in the bacterium Escherichia coli. Small-scale expression experiments were conducted to optimize the PF1280 protein expression conditions that were used for large-scale expressions. Large-scale expressions of PF1208 were done to produce sufficient protein for DNA-binding studies. Gel mobility shift assays were used to establish whether PF1280 binds to the SOR promoter DNA, which would indicate whether PF1280 functions as a DNA-binding protein, and therefore, as a potential transcriptional regulator.

Student Author(s):	Wissink, Erin M.
Department(s):	Biological Sciences Genetics
Research Mentor(s):	Serena M. Dudek/Laboratory of Neurobiology, National Institutes of Environmental Health Sciences Ramendra Saha/Laboratory of Neurobiology, National Institutes of Environmental Health Sciences
Title of Presentation:	Rapid Regulation of arc Transcription: Contribution of Chromatin Structure

Immediate early genes (IEGs) are genes that can be activated within mere minutes of a stimulus. Their activation relies on accessible chromatin structure so that transcription factors can easily bind to promoter for the gene of interest. In the brain, neuronal responses to synaptic activity and other stimuli include upregulating a set of IEGs that then mediate specific physiological responses of the neuron. One such IEG, Arc/Arg 3.1, is used in neurons to maintain long-term changes in synapse strength and memory storage. The expression of arc can be initiated within two minutes of in vivo synaptic activity in mice, and several protocols are able to mimic this response in vitro. We investigated qualities of the arc chromatin structure that could aid this rapid induction. Using chromatin immunoprecipitation (ChIP) assays, we found that the nucleosomes near the transcription start site for the arc promoter are characterized by the presence of histone 2A variant z (H2Az), which has been implicated in regulating gene expression. ChIP assays also revealed the presence of a stalled RNA Polymerase-II near the arc transcription start site, where it is poised to begin transcribing arc in response to stimulus. These data suggest that the chromatin in the arc promoter is suitably structured to rapidly initiate transcription. We hypothesize that the presence of H2Az facilitates the stalling of RNA Polymerase-II. Future experiments will feature knocking down H2Az and seeing the effect on the polymerase.

Student Author(s):	Womble, Mandy A.
Department(s):	Biological Sciences
Research Mentor(s):	Mike Dush/College of Veterinary Medicine: Molecular Biomedical Sciences Nanette Nascone-Yoder/ College of Veterinary Medicine: Molecular Biomedical Sciences
Title of Presentation:	Let's Stick Together: E-Cadherin and the Role it Plays in Xenopus laevis Gut Morphogenesis and Elongation

During development the vertebrate gut tube undergoes dramatic elongation and rotation, but the mechanisms underlying these transformations are poorly understood. The embryonic gut tube is temporarily occluded by endoderm cells before it is recanalized and the endoderm cells are reorganized to form a mature single-layered gut epithelium. If this process is perturbed (1 in 500 births), abnormal epithelialization can lead to intestinal stenosis or atresia or intestinal malrotation, predisposing the affected individual to life-threatening complications necessitating surgical correction. Previously, apoptosis was thought to drive the recanalization process but recent experiments have failed to support this theory. We hypothesized that the reorganization of E-cadherin, a cell to cell adhesion molecule, by endocytosis, provides a mechanism for gut elongation by allowing intercellular rearrangements to occur, similar to the cell movements that occur during gastrulation. Using immunohistochemical staining of cross sections of Xenopus laevis frog embryos (stages 35-46), we defined the normal subcellular localization of E-cadherin and laminin in the forming gut tube. Our results suggest that E-cadherin is localized on the entire surface of rearranging cells but is gradually remodeled to the apical domains of cells that form stable cell to cell interactions during the formation of the single layered digestive epithelium. We then treated embryos with compounds PP1 and PP2 that are known to perturb endocytosis of molecules like E-cadherin through the inhibition of SRC family kinases. These endocytosis inhibitors dramatically perturbed gut tube elongation. Immunohistochemical analyses indicated that they inhibited the normal apical localization of E-cadherin and prevented the endoderm cells from intercalating to form a single layered epithelium. This suggests that E-cadherin endocytosis may provide a mechanism for the elongation and epithelization of the gut tube, and abnormal E-cadherin endocytosis may underlie common digestive system birth defects.

Student Author(s):	Wright, R. Clay
Department(s):	Chemical Engineering
Research Mentor(s):	Bala M. Rao/Chemical Engineering
Title of Presentation:	Spatial Model for in vitro Patterned Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) can be maintained in culture in an undifferentiated state or can be caused to differentiate to a specific cell lineage. The differentiation event is controlled in-part by the competition between BMP-2 and GDF-3 signaling. These ligands are both secreted and captured by hESCs, thus acting through both autocrine and paracrine signaling. Extracellular BMP-2 and GDF-3 signals both operate via the Smad signaling pathway, albeit BMP-2 acts through Smad1, and GDF-3 acts through Smad2. Competition between Smad1 and Smad2 signaling is used to maintain the undifferentiated hESC state. In culture, hESCs may differentiate in a variety of spatial patterns. From this it can be hypothesized that these differentiation patterns are caused by the spatial gradients of diffusing signal molecules. Direct characterization of these gradients can be analyzed through the spatial-kinetic model derived here.

Student Author(s):	York, Abby M.
Department(s):	Molecular Biomedical Sciences
Research Mentor(s):	Jorge A. Piedrahita/Molecular Biomedical Sciences
Title of Presentation:	D-loop of Cloned Yucatan Miniature Pig Mitochondrial DNA Sequenced: Using SNPs for Quantification of Occidental European Mitochondrial DNA and Yucatan Miniature pig Mitochondrial DNA within Cloned Yucatan Miniature Pigs

The D-loop within mitochondrial DNA of different swine breeds is highly variable with multiple breed specific single nucleotide polymorphisms (SNP) (Kim et al., 2002). Two SNPs previously sequenced at base position 15511 and 15544 are breed specific for both Occidental European breeds and Yucatan miniature pigs (Kim et al., 2002). Yucatan miniature pigs were cloned using somatic cell nuclear transfer (SCNT) techniques with the recipient egg being of Occidental European descent. Due to maternal inheritance of mitochondrial DNA, the goal was to sequence the mitochondrial DNA of the cloned Yucatan miniature pigs using pyrosequencing techniques from PCR amplifications. The position 15511 was further sequenced using a specific SNP program within the pyrosequencing software in order to quantify how much of each breed specific SNP was present in the cloned Yucatan miniature pigs. The cloned Yucatan miniature pigs were found to have both Occidental European breed mitochondrial DNA from the recipient egg and Yucatan miniature pig mitochondrial DNA from the donor fibroblast cell, with the later only making up 10-15% of the entire amount of mitochondrial DNA present in the cloned Yucatan miniature pigs.

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Last modified April 2008 by Sharon E. Hunt, WordHunting

The 17th Annual

NC State University Undergraduate Research Symposium

The 17^th Annual

NC State University
Undergraduate Research Symposium