2006 NCSU Summer UGRS Independent Researcher

The 5th Annual

NC State University

Undergraduate Summer Research Symposium

Independent Researcher

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

Student Author(s):	Bisbee, Diana Y.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Biomolecular Chemical Engineering
Research Mentor(s):	Melissa Cox/Biotechnology Sue Carson/Biotechnology
Title of Presentation:	Cloning and Mutagenesis of a Xanthomonas campestris pathovar vesicatoria Putative Siderophore Biosynthesis Gene

The plant pathogen Xanthomonas campestris pv. vesicatoria (Xcv) strain 85-10 infects pepper plants through the stomata, causing bacterial spot disease. This bacterial infection has a significant effect on crop production. Iron is an essential component for life of most bacteria. However, iron in the environment is mostly found in the form of insoluble ferric (Fe3+) iron. Furthermore, pathogenic bacteria have a difficult time obtaining iron because their hosts sequester iron in iron-binding proteins not obtainable to most bacteria. Many successful pathogenic bacteria overcome these obstacles through the production and secretion of low molecular weight iron chelators called siderophores. Siderophores allow ferric iron to be reduced and transported into the cell via specific active transport through a TonB-dependent, receptor-mediated event. While much is known about iron-utilization in human pathogens, iron-utilization in plant pathogens including Xcv has not been widely studied. The genome of Xcv strain 85-10 has recently been sequenced and published in GenBank. Using bioinformatics tools, we identified a putative operon with predicted siderophore biosynthesis gene homologs. The goal of this research was to clone and mutate a specific gene within the putative siderophore biosynthesis complex to determine whether the gene encodes a functional siderophore biosysnthesis protein.

Student Author(s):	Burleyson, Casey D.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Marine, Earth, and Atmospheric Sciences
Research Mentor(s):	Sandra Yuter/Marine, Earth, and Atmospheric Sciences
Title of Presentation:	The Probability of Rainfall Evaporating Before Reaching the Surface

Weather radars are often used to provide quantitative precipitation estimates from passing storms. Scanning radars, such as the National Weather Service WSR-88D, observe the atmosphere from a few hundred meters to several kilometers in altitude. The lowest level observed by the NWS radar increases with increasing range from the radar. The NWS radars are unable to identify conditions in which precipitation at the lowest level scanned evaporates before reaching the surface. When this evaporation occurs radar derived precipitation estimates yield an overestimate of cumulative precipitation. This project uses a statistical analysis of radar returns at varying height levels and the frequency of virga events (precipitation falling from clouds that does not reach the surface) to determine the probability of measurable precipitation at the surface for given thresholds of reflectivity (dBZ) aloft. Data were collected from a vertically-pointing radar (METEK MicroRainRadar) and an accompanying Joss-Waldvogel disdrometer located in Scholls, Oregon. Probabilities were calculated for 2 dBZ intervals in three bins of rain rate [R>0 mm/hr, R>0.5 mm/hr, and R>1 mm/hr] with a minimum of 50 events used to calculate the probability. Probabilities of rainfall at the surface generally increase with increasing observed reflectivity at a given height. Increasing the rain rate threshold lowered the probabilities considerably. For example, at 1650m altitude probability of rainfall at the surface when a 20dBZ echo is observed is 100% for R>0 mm/hr, 73% for R>0.5 mm/hr, and 47% for R>1 mm/hr. Variability in the height of the freezing level within precipitation from mixed phase clouds impacts observed reflectivities. Future work will condition the probabilities on the freezing level height. Results of this study will be applied to refining NWS radar derived quantitative precipitation estimates and in the nowcasting of storms observed by scanning radars.

Student Author(s):	Burroughs, James L. Georgianna, D. Ryan Brown, Matt P.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Plant Pathology and Center for Integrated Fungal Research Functional Genomics Graduate Program
Research Mentor(s):	Gary Payne/Plant Pathology
Title of Presentation:	The afl-4 Mutation in Aspergillus flavus Strain 241 Is Not the Result of a Genetic Mutation in the Global Regulator laeA

The afl-4 mutation in Aspergillus flavus strain 241 is the only genetically mapped aflatoxin biosynthesis mutation not localized to linkage group VII (LG VII). LG VII contains the aflatoxin biosynthetic cluster and has been karyotypically mapped to a 4.9 Mb chromosome. Strain 241 has a unique phenotype characterized by a lack of aflatoxin production, a tan pigmentation, and suboptimal growth at 37C. AflR, which is a transcriptional regulator of aflatoxin biosynthesis genes, is not expressed in strain 241. Transformation of 241 with additional copies of aflR only partially restores its ability to produce aflatoxin, whereas overexpression of aflR in this strain with a constitutive promoter fully complements aflatoxin production. These observations suggest that aflR is not being transcriptionally activated in 241, and that the afl-4 mutation is upstream of aflR. One known transcriptional regulator of aflR is laeA, which is thought to be a global regulator of secondary metabolism. Deletion of laeA inhibits transcription of alfR and consequently prevents production of aflatoxin. Because strain 241 exhibits similar phenotypic characteristics to fungal strains where laeA is deleted and relatively few upstream regulators of aflR are known, we hypothesized that the afl-4 mutation may have resulted from a mutated copy of laeA. To test our hypothesis we PCR amplified laeA from strain 241 and cloned it into a plasmid vector. Purified plasmid containing the laeA gene was sequenced and compared with the A. flavus whole genome sequence. Alignment of our sequence with the genome revealed no detected DNA mutations indicating that afl-4 is not the result of a mutation in laeA.

Student Author(s):	Caudle, David
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Biochemistry and Chemical Engineering
Research Mentor(s):	Sue Carson/Biotechnology
Title of Presentation:	Cloning and Mutagenesis of an Iron-citrate Receptor (fecA) Gene Homolog in Xanthomonas campestris pathovar vesicatoria

The plant pathogen Xanthomonas campestris pathovar vesicatoria is the cause of bacterial spot disease, a common infection of pepper and tomato plants resulting in significant leaf damage and crop loss. The genome of Xanthomonas campestris pathovar vesicatoria strain 85-10 has recently been sequenced and made publicly available. This gram negative bacterium, like most living organisms, requires iron as an essential element for its survival. Many gram-negative organisms use similar mechanisms for iron uptake. This includes secretion and reuptake of ferric-specific chelators, or siderophores, through the use of several inner and outer membrane proteins. Through the use of bioinformatics tools, we assessed that Xanthomonas campestris pathovar vesicatoria strain 85-10 contains sequences homologous to siderophore biosynthesis and siderophore uptake genes from other gram-negative bacteria. This research focused on a putative gene with homology to a family of TonB-dependent outer-membrane iron receptors in other Gram-negative organisms. It is adjacent to a putative siderophore biosynthesis operon, but is not predicted to be in the same operon. We have cloned a large fragment of this gene and are in the process of creating a “knock-out” mutant in order to observe if a functional deletion of the gene impairs the organism’s ability to acquire iron and to infect its natural host.

Student Author(s):	Chestnut, Michael II
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Electrical and Computer Engineering
Research Mentor(s):	Dr. Osburn/Electrical and Computer Engineering
Title of Presentation:	Trimming of 193 nm Photoresist Lines for Nanopatterning

The smallest feature that can be resolved and patterned by optical lithography is limited by the diffraction of light. Even using the shortest wavelength exposures, i.e.,193 nm, feature sizes are limited to about 100 nm in size. In this work we have develop an isotropic etching process to “trim” photoresist lines from the sides to reduce the linewidth and allow us to produce even smaller nanopatterns. Polymeric layers of 193 nm photoresist ( 300 nm of V-41) on top of a bottom antireflective coating (80 nm of ____) material were spin coated and then reactive ion etched (RIE) in O2 in a Plasma Therm tool. );sThe uniformity of the as-spun resist wafers was 0.15% (1 however, the resist RIE etch rate showed a systematic variation from center to edge of the wafer, i.e., was about 10% higher at the edge than at the center. The vertical etch rate of resist was found to be 53% of that of the BARC. The vertical etch rate of resist, as measured on an ellipsometer, was found to vary linearly with etch time and had a strong dependence on the plasma power. Pressure and O2 flow rate had only a minor effect on etch rate. For the trimming process, 110 nm features were exposed in resist in the 193 nm ASML scanner and then developed. The resist trimming process was chosen to provide a 50% overetch of the 80 nm BARC layer, corresponding to the vertical removal of about 62 nm of resist. An Hitachi scanning electron microscope, in top down mode, was used to measure linewidths before and after resist trimming to quantify the lateral etch rate of resist; cross sectional pictures of the resist/BARC were obtained in the JOEL SEM.

Student Author(s):	De la Rosa, Indhira H.
Home Institution:	Universidad Metropolitana
Program:	Independent Researcher
Department(s):	Biochemistry
Research Mentor(s):	Carla Mattos/Biochemistry
Title of Presentation:	Barrier to Autointegration Factor (BAF): Purification, Crystallization and Multiple Solvent Crystal Structure

Barrier to autointegration factor (BAF) is a conserved protein in metazoan. BAF is necessary for nuclear assembly, chromatin organization, gene expression and is exploited by retroviruses. BAF is localized in the nucleus and facilitates retroviral DNA integration through unknown mechanisms. BAF form stable dimers that bind to nonspecific dsDNA and to the LEM domain proteins (LAP2, Emerin and MAN1). BAF have been well study for it’s multiple functions in the cells, but the protein structures and the binding sites are not well known. In this project we are interested in characterizing new binding sites on this novel protein. It is important to study this region of interaction because it will give us a better idea about BAF protein – protein interaction mechanisms and regulatory functions in the cells. Previous experiments have shown that BAF has DNA binding sites; we are also looking to map these DNA binding sites in order to understand the ability of BAF to promote viral DNA integration into the host cell. For our experiments we expressed BAF in e. coli BL21 (DE3) and purified the protein using methods like the Fast Protein Liquid Chromatography ( Anion exchange and gel filtration). In order to find new protein binding sites we have to generate BAF crystals and collocate them in to organic solvents, this method is calls the Multiple Solvent Crystal structures (MSCS). The MSCS is a novel method in which we have different organic solvents cluster on the crystal protein surfaces. The MSCS is a complementary strategy for computational techniques and X –ray that will give us a precise way to determined binding sites. Our results suggest that we purified BAF successfully. We are growing BAF crystals to be ready to expose them to the MSCS and finally map the binding sites.

Student Author(s):	Desai, Divya M.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	Carla Mattos/Molecular & Structural Biochemistry
Title of Presentation:	Solving Protein:protein Interaction in Cdc25B Using the Multiple Solvent Crystal Structure (MSCS) Method

Cdc25B phosphatase is an important regulator of the cell cycle. Cdc25B helps regulate the cell cycle by binding to the Cdk2-CyclinA complex which controls the transition from the S to the G2 phase of the cell cycle. Cdc25B activates the Cdk2/CyclinA complex by removing two inhibitory phosphates from Thr-14 and Tyr-15 located on Cdk2. Site-directed mutagenesis and molecular dynamics simulation of the ternary complex show protein: protein interactions between Cdk2 and Cdc25B. However, a crystal structure of the ternary complex is still unavailable. The goal of this project is to solve the crystal structure of Cdc25B in a variety of organic solvents, following the Multiple Solvent Crystal Structures (MSCS) method. Cdc25B was transformed in BL21 E coli cells and purified by cation exchange and gel filtration chromatography. Protein purity was assessed by SDS-PAGE and phosphatase activity was checked by the pNpp assay. Crystal trials of Cdc25 B were set up according to previously published conditions. Crystals will be soaked in different organic solvents to obtain a detailed map of solvent binding sites on the protein surface of Cdc25B. This solvent binding site map should further validate the experimentally determined model of the Cdc25B/Cdk2/CycA complex and provide additional structural insights into the protein interaction surface of Cdc25B.

Student Author(s):	Green, Mindy R.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Biotechnology
Research Mentor(s):	Joanna Miller/ Biotechnology
Title of Presentation:	Identification and Functional Testing of a Possible Dicer, a Key RNA Interference Protein, from Danio rerio (zebrafish)

RNA interference uses double stranded RNA (dsRNA) to silence gene _expression by degradation or by inhibiting translation of targeted messenger RNA (mRNA). DsRNA is recognized by the Dicer protein which cleaves long dsRNA into smaller fragments called small interfering RNAs (siRNAs). The siRNAs are then recognized by a complex called the RNA Induced Silencing Complex (RISC). RISC binds to the siRNAs, unwinds the double strands and releases the sense strand. The antisense strand remains to silence complementary mRNA. RNAi occurs in a wide variety of organisms, from fission yeast to humans.

Using a known Dicer sequence, we performed a BLAST search in the Danio rerio (zebrafish) genome and identified a known functional Dicer (accession number AY386319) as well as two other genes with domains consistent with Dicer proteins: accession numbers XM_677923and XM_678382. Our goal was to clone one of these and test it to see if it was a novel functional Dicer, as it is not uncommon for organisms to have more than one Dicer. We designed primers to PCR-amplify the gene from the first accession number above. We extracted RNA from a zebrafish and performed Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) using multiple enzymes and conditions to amplify our gene of interest. Agarose gel electrophoresis was used to analyze the PCR products; we expect a 5.4 kilobase band for a full-length PCR product. A band was excised and TA cloned. Possible clones were analyzed by restriction digests. Once the sequence is confirmed, it will be subcloned into a Schizosaccharomyces pombe (S.pombe) _expression plasmid. The plasmid will be introduced into a S. pombe strain which had its Dicer deleted. Our possible Dicer will be expressed and we can determine if this zebrafish Dicer can substitute for S.pombe Dicer in a thiabendazole sensitivity assay.

Student Author(s):	Holsinger, Hannah L.
Home Institution:	Virginia Tech
Program:	Independent Researcher
Department(s):	Food Science USDA-ARS Food Science Research Unit
Research Mentor(s):	Ilenys Diaz-Muniz/Food Science
Title of Presentation:	Evaluation of the Leuconostoc mesenteroides Metabolic Potential for Glucose Utilization and Acetic Acid Production Using Bioinformatics

Leuconostoc mesenteroides is a lactic acid bacterium, which predominates in some vegetable fermentation. The L. mesenteroides genome was sequenced and is currently available to the scientific community. This research takes advantage of the completed genome sequence to initiate an evaluation of the global metabolic potential of L. mesenteroides. This evaluation was initiated by constructing a putative pyruvic acid cycle and glucose utilization maps for L. mesenteroides . The putative maps were created using known pathways, the genome sequence, and metabolic flux analyses. Although, most of the standard tricarboxilic acid cycle-related enzymes were not identified in the L. mesenteroides genome sequence, a putative pyruvic acid cycle and a putative route for acetic acid production were identified. Sections of the putative maps have been validated and documented in the current literature. Efforts to evaluate the functionality of sections on the maps not previously characterized included enzyme activity assays (i.e., alcohol dehydrogenase), detection of end products such as formic acid, and a small scale gene expression study. The functionality of previously uncharacterized sections on the map was studied under conditions similar to those present in fermented vegetable products. The knowledge derived from this study will help in the design of optimized conditions for vegetable fermentations.

Student Author(s):	Houseworth, Paul J.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Physics
Research Mentor(s):	Karen E. Daniels/Physics
Title of Presentation:	Instabilities of Droplets Spreading on Gels

The spreading of a droplet of one simple fluid over another is well understood. For example, a drop of oil on water will spread in a circular pattern. Alternatively, when the substrate is a complex fluid, like a gel, a droplet of another fluid spreading along the gel’s surface will instead form arms, provided that the surface tension of the drop is significantly larger than that of the gel. We show this pattern to be a result of fracturing of the gel's macromolecular network at the surface. By utilizing light scattering through gels containing one micron polystyrene beads, we can quantitatively determine the cross sectional shape of the arms. We investigate how the depth and width of these arms change, both over time and for different strength gels. This data will be used to form mathematical models of these interactions.

Student Author(s):	Lithgo, Abigail K..
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Chemical and Biomedical Engineering
Research Mentor(s):	Ruben Carbonell/Chemical and Biomedical Engineering
Title of Presentation:	Purification of Porcine Parvovirus by Gel Filtration Chromatography

The pharmaceutical industry is required to determine and remove pathogens, such as viruses and bacteria, from human therapeutic products. Porcine parvovirus (PPV) is a viral disease found in the majority of unvaccinated pigs around the world causing reproductive problems such as miscarriages, and can be found in products such as porcine insulin used to treat diabetes. It is also a good model virus for small, nonenveloped viruses that can be difficult to remove from process streams by chemical inactivation, heat or pH changes, or filtration. The goal of this work is to purify PPV from cell culture supernatant using gel filtration chromatography on a large scale, and this purified PPV would be used in further research of virus removal and detection techniques. To perform the experiment, PPV supernatant is loaded onto a gel filtration column and the exiting effluent is tested with gel electrophoresis for purity, BCA assays for total protein content, and MTT assays to determine the amount of infectious virus present. So far, experiments have shown that infectious virus can be recovered from the gel filtration column. More trials on the column, optimizing virus loading conditions and flow rate, are planned to further determine the purification abilities of the column.

Student Author(s):	Lucking, Jonathan R.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Microbiology
Research Mentor(s):	HC Liu/ Animal Science
Title of Presentation:	Anti-Meq: Isolation and Characterization of a Novel Marek's Disease Virus (MDV) Protein

Marek's disease (MD) is a ubiquitous lymphoproliferative disease of chickens induced by a herpesvirus, the Marek's disease virus (MDV). MDV causes a significant economic loss to the poultry industry. Although effective vaccines are currently in use for preventing the disease, the increasing frequency of highly virulent strains of MDV may severely erode the efficacy of those vaccines. Therefore a comprehensive understanding of MD pathogenesis is crucial for developing a better control strategy to combat MD. A key goal of research on MD pathogenesis is to elucidate the biological function of MDV gene(s) that promote oncogenesis. One unique gene (MDV077) which is antisense to the oncogene Meq is of special interest. In this project, we performed a comprehensive yeast two-hybrid system interaction screening to identify host protein(s) that specifically interact with MDV077. Putative interactions detected from the yeast two-hybrid system will be confirmed using a direct biochemical binding assay, i.e., protein pull-down assay.

Student Author(s):	Makwana, Satya T.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Microbiology
Research Mentor(s):	Michael R. Hyman/Microbiology Denise M. Aslett/Microbiology
Title of Presentation:	Heavy Stable Nitrogen Effect on Escherichia coli GC 4468 Growth

Stable isotope probing (SIP) has been an important tool in determining the functions of microorganisms in their natural environments. While much work has been done with SIP, not much is known about the effect the heavy stable isotope has on the microorganism of interest. The purpose of this research is to measure the effect that the stable isotope has on Escherichia coli GC 4468 growth. The bacteria were inoculated in a vial containing 30 mls of minimal media including ammonium chloride of a different isotope of nitrogen (N15). These vials were incubated for a period of 48 hours and then a serial dilution was done followed by plate counting. Optical density measurements were also taken at an absorbency of 600 nm. The results show quite a bit of variation every time the experiment was run. On average, the growth of N15 cells was less than that of the N14 cells. The inconsistency from the plate counting and OD 600 methods make these results inconclusive about the growth rate. Further analysis should be done with a growth curve in M9 N15 minimal media.

Student Author(s):	Morris, Eric B. Evans, Ted W.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Forestry and Environmental Resources
Research Mentor(s):	Glenn P. Catts/Forestry and Environmental Resources Ted H. Shear/Forestry and Environmental Resources
Title of Presentation:	Comparison of National Weather Service Model Predictions of Spatial Precipitation Patterns with a Network of Field Rain Gauges on Hofmann Forest

Hofmann Forest is a contiguous 80,000 acre forested wetland on the coastal plain of North Carolina. Precipitation inputs can vary considerably over such a large contiguous coastal tract less than 20 miles from the Atlantic Ocean. Knowledge of the spatial distribution of precipitation input is valuable for better understanding the ecology of the pocosin system, as well as operational management efforts.

Since 2001, precipitation levels at Hofmann Forest have been monitored using periodic manual readings of a network of nine graduated cylinder rain gauges distributed across the property. Recently the National Weather Service (NWS) has made spatial model estimates of precipitation using a combination of radar remote sensing integrated with field observations. NWS estimates are available via the internet and can be downloaded as geographic information system (GIS) files containing a lattice set of point estimates of daily rainfall.

To examine correlation between NWS radar-based, spatial estimates and Hofmann Forest rain gauge measurements, GIS data files for 15 large rain events in 2004 and 2005 (60mm total precipitation or more) were downloaded and spatially interpolated to produce estimates for the nine Hofmann Forest measurement sites for each event. Some rainfall events spanned multiple days and 24-hr precipitation estimates from multiple GIS files were summed to provide an appropriate comparison. After the precipitation estimates from field measurements and the NWS radar-based models were assembled into a single dataset, least squares analysis was applied and resulting correlations indicated that a high degree of agreement (R-squared = 0.67) existed between the variables.

Student Author(s):	Patel, Dhrumil
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Mathematics
Research Mentor(s):	Michael Shearer/Mathematics Karen Daniels/Physics
Title of Presentation:	Shearing a Granular Material Leads to Particle Size Segregation: Experiments and Theory

Experiments were conducted on a bidisperse mixture of glass spheres of different sizes. The granular mixture occupies the annular region between two fixed concentric cylinders; it is sheared by rotating the supporting plate on which the material rests. The upper surface is a free boundary. Quantitative data on the rate of mixing and the rate of segregation between the two particle sizes are obtained using a camera and image processing with MATLAB. These data are used to extract significant parameters in order to test agreement with a simple mathematical model of particle size segregation. The model is thought to capture mixing and segregation found in large-scale natural events such as granular avalanches and in small scale industrial use such as materials processing.

Student Author(s):	Saldarriaga, Ivandario Vendra, Lakshmi
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Mechanical Engineering
Research Mentor(s):	Afsaneh Rabiei/Mechanical Engineering
Title of Presentation:	The Probability of Rainfall Evaporating Before Reaching the Surface

Metal foams display interesting properties, such as energy absorption, thermal insulation, and sound dampening, which make them worth investigating and developing. The research at NC State focused on creating closed cell composite metal foam that is homogeneous in cell structure and with increased plateau stress compared to metal foams being produced currently. The research used gravity casting techniques to create the foam, which was comprised of hollow metal spheres with an aluminum matrix. Two different types of hollow spheres were used; one type was made of low-carbon steel, while the other was made of stainless steel. The foam had a density of 2.4 g/cmSUP3/SUP. The properties of the foam were then observed using static compression and compression-compression fatigue testing. The stress strain curve obtained from the static compression displayed three distinct regions, an initial elastic region, followed by plateau plastic region, and ending with a densification region. For two low-carbon steel sphere samples, plateau stresses were calculated to be 58 and 60 MPa. For a stainless steel sphere sample, the plateau stress was calculated to be 73 MPa. At 50% strain, the metal foam with low-carbon steel sphere samples absorbed 24 MJ/mSUP3/SUP. The foam was modeled as a crash box for automotive bumpers. The models demonstrated good results, with the foam crash boxes absorbing impacts of up to 20 miles per hour. The foam's energy absorption properties show promising applications for various other transportation industries.

Student Author(s):	Sapp, Tracy L.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Biomolecular and Chemical Engineering
Research Mentor(s):	Susan Carson/Biotechnology
Title of Presentation:	Cloning and mutagenesis of a putative siderophore receptor gene of Xanthamonas campestris pathovar vesicatoria

Xanthamonas campestris pathovar vesicatoria (Xcv) is responsible for black rot disease in pepper plants. Iron is essential for bacterial metabolic functions including respiration and electron transfer, but it is difficult for bacteria to obtain because iron exists primarily in an insoluble form in the environment. Pathogens have the additional obstacle that host iron is generally sequestered by binding proteins, rendering it unavailable. In response, many bacteria evolved mechanisms to overcome these problems. One of these mechanisms is the production of low molecular weight iron-binding molecules called siderophores. Bacteria that utilize this system secrete siderophores into their environment which then bind iron and are brought back into the cell. The outer-membrane of Gram-negative bacteria is impermeable to large hydrophilic molecules that are unable to pass through porin channels. Therefore, bacteria must acquire ferric-siderophores by an energy-dependent process, in which a specific outer-membrane receptor binds and transports the ferric-siderophore. While iron-utilization systems have been extensively studied in E. coli and other human pathogens, little has been studied in plant pathogens. The genome of cv strain 85-10 was recently sequenced. Using bioinformatics tools, we identified a putative operon encoding homologs of a siderophore receptor gene, periplasmic binding protein gene, cytoplasmic membrane permeases, and ATPases. The goal of this project was to clone and create a functional knockout of the outer membrane receptor gene homolog in this putative operon and to determine its function in iron-utilization and plant pathogenesis. We have cloned the gene and inserted an antibiotic resistance cassette into the recombinant gene. We are currently attempting to cross the mutated copy back into Xanthomonas. Once accomplished, we will test the mutant strain for growth on various iron-sources and for its ability to infect its host pepper plant.

Student Author(s):	Sargent, Luke C.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Physics
Research Mentor(s):	Karen E. Daniels/Physics
Title of Presentation:	Characterizing the Growth of Wisps in Droplets Spreading on Gel Substrates

In contrast to the well-studied uniform spreading of droplets on solid and liquid substrates, a droplet spreading on a viscoelastic material (such as a gel) spreads by way of 'wisps' of the liquid which propagate outward from the center of droplet impact. We characterize the length of the wisps as a function of time to gain a better understanding of the mechanisms involved in their growth. We present data on how the growth rate varies with such parameters as gel strength, droplet surface tension, and number of wisps.

Student Author(s):	Seyam, Mohamed A.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Chemical and Biomolecular Engineering
Research Mentor(s):	Richard Spontak/Chemical and Biomolecular Engineering
Title of Presentation:	Cross-Linked Polymer Nanocomposites for Gas Separation

Gas separation is an important process in the chemical industry, particularly in the separation of the gaseous products resulting from the water-gas shift reaction during steam reformation of hydrocarbons. In this process, hydrogen (H₂) must be separated from a mixed gas stream containing carbon dioxide (CO₂) primarily, but also potential trace amounts of water (H₂O) and carbon monoxide (CO). The hydrogen gas is utilized in many different ways, however in the growing demand for alternative energy sources, hydrogen fuel cells have emerged as one option. It has become industrially important to reduce energy and economic demands of the current separation processes and polymer membranes provide an opportunity to achieve this goal. In this study, we have examined the gas separation properties of a cross-linked polyimide containing palladium (Pd) nanoparticles. Implementation of Pd nanoparticles into these polymer membranes decreases the free volume of the membrane as well as improves the affinity for H<sub>2</sub> over the other gas species. Additionally, chemical cross-linking of the membranes further reduces the free volume and increases chain rigidity, aiding in the prevention of the phenomenon of CO₂ plasticization and disallowing relatively larger gas molecules to penetrate.

Student Author(s):	Shah, Aditi D.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Mechanical and Aerospace Engineering
Research Mentor(s):	Afsaneh Rabiei/Mechanical and Aerospace Engineering
Title of Presentation:	Processing and Characterization of Nano-scale Hydroxyapatite Coating for Biomedical Implants

Increasing demand of the new and improved implants calls for a new generation of calcium phosphate coating. The purpose of this research is to process and characterize functionally graded hydroxyapatite coating, which is a new type of calcium phosphate coating. Functionally graded hydroxyapatite coating improves acceptance in the body as it has the similar chemical composition as the mineral component of the bone and thus helps the implant to form stronger bond with the bone. This provides mechanical stability that will allow the patient to quickly return to a normal and active lifestyle. The new coating allows a functionally graded crystallinity through the thickness of the hydroxyapatite coating, which allows the tissue to better integrate with the implant. It also minimizes motion-induced damage to the surrounding tissue thus increasing the life span of the implant. Ion Beam Assisted Deposition technique was used to process and grow a thin layer of hydroxyapatite coating, with a graded crystalline structure across the thickness of the coating. Scanning Transmission Electron Microscopy, Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy tested the microstructure of the film. The overall goal of the research is to develop a better coating to improve the implant’s acceptance in the body and to increase the service-life of orthopedic and dental implants by creating materials that form a strong, long-lasting bond with the bone. With the growing number of young patients needing orthopedic implants, this will decrease the need for implant replacement. The results from this project can be used for both dental and orthopedic implants. I as an undergraduate student have been teamed up with a graduate student working on this project to experimentally process and test the functionally graded hydroxyapatite coating.

Student Author(s):	Tucker, Latisha
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Biotechnology
Research Mentor(s):	Joanna Miller/Biotechnology
Title of Presentation:	Identification and functional testing of a novel Dicer, a key RNA interference protein, from Apis mellifera (honeybee)

RNA interference (RNAi) is a mechanism whereby double stranded RNA (dsRNA) is used to silence the expression of target genes within a cell. Although first discovered in petunias, RNAi has been shown to occur in a wide range of organisms as well. RNAi is a highly specific process that involves special mechanisms within the cell. Once double stranded RNAs have been introduced into the cell, an enzyme known as Dicer recognizes and cleaves the long RNAs into small interfering RNAs (siRNAs), approximately 21-25 nucleotides in length. The siRNAs then continue through a process that allows a RNA Induced Silencing Complex (RISC) to silence the expression of any genes with sequences homologous to the dsRNA.

The Apis mellifera (honeybee) genome sequence has recently become available. Using the sequence of a known Dicer, we performed a BLAST search and identified a possible Dicer gene (accession number XM_624507). The purpose of this experiment was to isolate the suspected Dicer gene, the first one identified from honeybees, and to test if it was functional. First RNA was prepared from honeybee thoraces (midsections), followed by a ReverseTranscription reaction to create a cDNA. Next, sequence specific primers designed to amplify the desired sequence were used in a PCR reaction. Gel electrophoresis was used to determine if a sequence of the correct length was amplified in this step. The PCR product of interest was gel-purified and TA cloned. The gene was sequenced to determine its actual coding sequence. Once confirmed, the insert was to be subcloned into a Schizosaccharomyces pombe (S.pombe) expression plasmid so that Dicer (complete or a fragment) could be made in a S. pombe strain which had its Dicer deleted. We could then determine if honeybee Dicer could substitute for S.pombe Dicer in a thiabendazole sensitivity assay.

Student Author(s):	Vadlamudi, Charita
Home Institution:	Salem College
Program:	Independent Researcher
Department(s):	Textile Engineering
Research Mentor(s):	Bhupender Gupta/Textile Engineering
Title of Presentation:	Uniformity of Fiber Accumulation During Electrospinning Process

Electrospinning is a much sought after technique for developing nano fiber structures for a range of applications, including tissue engineering. While much has been published on the spinnability of different materials, literally no information exists on the uniformity of fiber lay down on the collector plate during spinning. This study is designed to shed some light on this aspect of the electrospinning process. We determined the uniformity of fiber accumulation on aluminum foil for different processing conditions. A 15% (wt/vol) PCL in 0.5% (vol/vol) pyridine and 2 mL acetic acid solution was used in this experiment. PCL nanofibers were deposed onto aluminum foil at a flow rate of 300 ìl/hr using different voltages to the needle (10 kV, 12.5 kV, 15 kV, 17.5 kV) and cage (1/2 kV of needle, 0 kV/grounded, not connected). The weights of 17 equal sized pieces, cut using a 7/16” die, from the fiber-covered region on the foil from the center to the edges of the accumulation were determined. The weight of the fibers was determined from the known weight of the aluminum foil.

Student Author(s):	Wink, Glenna
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Molecular & Structural Biochemistry
Research Mentor(s):	Carla Mattos/Biochemistry Greg Buhrman/Biochemistry
Title of Presentation:	Q61V, Q61I, Q61K Mutants of H-Ras

Mutations of codon 61 in the GTPase H-Ras reduce GTP hydrolysis, promoting unregulated, cancerous cell growth. However, while all mutations at codon 61 reduce GTP hydrolysis equally, these mutants vary over 1000-fold in their ability to promote unregulated cell growth. This research focused on determining the structure of the valine, lysine, and isoleucine mutants of Gln61, in an attempt to illuminate reasons for the wide range of transforming efficiency exhibited by these mutants. The mutant plasmids were made by site directed mutagenesis of C-terminally truncated wt Ras and transformed into BL21 (DE3) E. coli cells for expression. Protein was purified by anion exchange and gel filtration chromatography. A nucleotide exchange reaction replaced GDP with the nonhydrolyzable analog of GTP, GMPPNP. Crystals of the Q61I mutant were obtained by the hanging drop method using a well solution of PEG Ion Screen Reagent #7, PEG 8000, and a ! protein concentration of 12mg/mL. Crystals were flash frozen in reservoir solution containing 20% glycerol as a cryoprotectant. Crystals diffracted to 1.8 Angstroms using an in-house X-ray source. The methods developed have been successful and will be used to obtain structures of the Q61V, and Q61K mutants.

Student Author(s):	Withers, James D.
Home Institution:	North Carolina State University
Program:	Independent Researcher
Department(s):	Biotechnology
Research Mentor(s):	Susan Carson/Biotechnology
Title of Presentation:	Analysis of a tonB homolog of Xanthamonas campestris pathovar vesicatoria

Xanthamonas campestris pathovar vesicatoria (Xcv) is a Gram-negative plant pathogen that causes black rot disease on pepper and tomato plants. Iron is crucial to metabolic processes of virtually all organisms, as it plays such a large role in reduction and oxidation reactions. Ionic iron is relatively insoluble, therefore bacteria have difficulty obtaining atmospheric iron. Pathogens have an additional difficulty because plant and animal hosts are able to sequester iron, rendering it unusable by bacteria unless they have a means to steal it. Many bacteria utilize siderophores, which are molecules that attract iron. The bacteria synthesize and secrete these iron-scavenging molecules, and then once the molecules bind iron, they are taken back up by the cell through specialized outer-membrane iron receptors. These outer-membrane iron-receptors generally function in an energy-dependent fashion; however, they are not able to generate this energy on their own. In gram-negative bacteria, a protein called TonB is responsible for inducing changes in the cell membrane that result in the energizing of the outer membrane receptors used for ferric uptake. The genome of Xanthamonas campestris pathovar vesicatoria strain 85-10 has recently been published. Through the use of bioinformatics tools, we assessed that Xcv has homologs of outer-membrane receptors used for iron-uptake, as well as a tonB homolog that is predicted to be located in an operon with exbB and exbD homologs. The goal of this research was to clone the Xcv tonB gene and obtain a tonB "knockout" in Xanthamonas campestris pathovar vesicatoria strain 85-10 in order to observe the effects of a functional deletion of this gene on iron-utilization and infection in the natural plant host, the pepper.

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