All populations of two-lined salamanders outside of the Appalachian Mountains in North Carolina are currently assigned to the southern two-lined salamander (Eurycea cirrigera). However, recent work suggests that more than one species of two-lined salamander occurs along the North Carolina and Virginia border. One of the species, the northern two-lined salamander (Eurycea bislineata), has not previously been recorded in North Carolina. I have sampled five populations from localities further west than previously collected populations of putative northern two-lined salamanders. From each population, I have amplified, purified, and sequenced 1,500 base pairs of the mitochondrial gene ND2. Here, I present the results of a Bayesian phylogenetic reconstruction.WNT5A, a secreted protein of the WNT signaling pathway, is well known for its role in the cellular differentiation and organ development, as well as proliferation and cellular migration. Studies have shown that it plays a role in many human cancers, acting as both a tumor suppressor, and oncogene. In this study, we analyzed WNT5A regulation from its two major transcription start sites, termed promoter A and promoter B. These promoters give rise to two distinct protein isoforms, identified as isoforms A and B. These proteins differ at the N-terminus; isoform A has an additional 15 amino acids in comparison to isoform B. In osteosarcoma cell line, SaOS-2, isoform A is overexpressed, whereas isoform B is not expressed at all. Moreover, both isoforms A and B are expressed in normal osteoblasts. In addition, the colorectal cell line, HCT-116, both isoforms are absent. This leads to the question whether isoform A has a distinct function that from isoform B. It is possible the distinct functions of the WNT5A isoforms are responsible for the oncogenic or tumor suppressor activity of WNT5A in different cancers. To determine this, we analyzed and characterized the functional distinctions between WNT5A isoforms A and B, specifically cellular proliferation, and migration. Together, our data suggest that both isoforms A and B are functional distinct.Throughout vertebrate evolution, the cardiac outflow vasculature has changed from a branchial arch system to a systemic and pulmonary circulatory system. However, all vertebrate hearts and outflow tracts develop from a single heart tube. In the chick and mouse, cardiac neural crest cells divide the single outflow tract into the aorta and pulmonary arteries. Additionally, cardiac neural crest cells provide the smooth muscle of the aortic arch arteries, help to remodel the aortic arch arteries into asymmetrical structures, and contribute cardiac ganglia. Yet, the role of cardiac neural crest cells in vertebrates with an undivided outflow is not well understood. I re-evaluate the role of cardiac neural crest cells in zebrafish, Danio rerio, and hypothesize that neural crest cells in fish contribute to the smooth muscle of gill arch arteries, the ventral aorta and cardiac ganglia, but they do not contribute to myocardium as previously described. I also study the outflow tract development of the turtle Trachemys scripta to understand the process of outflow septation in a vertebrate with a divided outflow tract but incomplete ventricle division. I hypothesize that cardiac neural crest cells are also responsible for the outflow tract septation of reptiles. I perform a comparison study of turtle outflow tract formation to that of the chick outflow tract, as much is known about the chick. These results demonstrate that the pattern of cardiac neural crest cell contribution to vertebrate vasculature remains predictable and consistent, enabling future studies to focus on changes in vascular patterning caused by cardiac neural crest cells among different vertebrate lineages.Autophagy is a survival mechanism utilized by all eukaryotic cells during nutrient starvation in which the cell recycles proteins and other cytoplasmic components to the vacuole to degrade and release. Understanding this mechanism can potentially improve cell survival after stroke and heart attack, thus minimizing tissue damage. Macroautophagy, the better known autophagy pathway, involves recycling cellular components via a double-membrane bound vesicle, whereas microautophagy is poorly understood and involves the vacuole directly engulfing the cytoplasmic components. Previous internal studies showed glucose starvation inhibited macroautophagy in S. cerevisiae (budding yeast) cells; however, some autophagic activity was still detected by an enzymatic assay. To determine whether this activity was due to microautophagy, we investigated the consequences of deleting a critical gene for macroautophagy, Atg5, and genes that potentially influence microautophagy, Vtc1 and Vtc2, in a Pho8Δ60 strain, allowing us to measure autophagy quantitatively through the Pho8Δ60 enzymatic assay. We starved these strains for nitrogen, glucose, or both nitrogen and glucose for ∼30 hr and found that Vtc2, not Vtc1 or Atg5, was required for autophagic activity in glucose-starved cells. We concluded that microautophagy occurs in glucose-starved cells, and some of the genes in the vacuolar transporter chaperone (VTC) complex are more important for microautophagy in glucose-starved cells than others. In the future, we plan to test the impact of deleting all four of the genes in the VTC complex on microautophagy and to test whether selective microautophagy of cell organelles occurs during glucose starvation.Desmognathus are medium sized lungless salamanders distributed across the Appalachian Mountains. Historically, there has been debate about how many species of Desmognathus there are. Currently there are six recognized species of Desmognathus: ochrophaeus, orestes, carolinensis, apalachicolae, ocoee, and abditus. These six species were recognized in part, based on molecular data. To date, there has not been a comprehensive range wide molecular phylogeny for Desmognathus. Here, we present a range wide molecular phylogeny that reveals the relationships of the six recognized Desmognathus, as well as several, apparently unnamed, lineages. To understand the morphological variation within these lineages we have photographed and measured specimens from twenty one localities. For each of these localities we sequenced a 600 base pair fragment of COX1 mitochondrial DNA. In the 1960s Martof and Rose collected over 4,000 Desmognathus from twenty one localities and made twelve different measurements for the specimens. To leverage their large morphological date set, we collected a series of thirty salamanders from the same localities. We made the same measurements and used our data to supplement their existing data. Here we demonstrate considerable levels of morphological homoplasy in Mountain Duskies. There is both extensive homoplasy and homology with respect to dorsal pigmentation. By supplementing Martof and Rose's large morphological dataset and sequencing each of their populations, I should be able to distinguish between homoplasy and homology.The objective of this study was to access the acid and bile salt tolerance of microencapsulated probiotic bacteria. Strains of B. bifidum NRC and L. paracasei 441 with 2% active cells were incubated at 37° C for 18 h in MRS broth. Alginate with NaCl was used for microencapsulation. Survival and viability of microencapsulated strains under different conditions were established. Selected strains in MRS broth cultures were challenged to pH (2 and 1.5) and bile salts (0.5 and 1.0%) for three hours. Our results showed that the total viable counts of the two strains were increased in the different pH levels. Microencapsulated B. bifidum NRC and L. paracasei 441cells had higher viability (6.90, 6.70 CFU/ml) than free cells (5.69, 5.70 CFU/ml) during exposure to low pH at 1.5. Similarly, the bacterial count of microencapsulated B. bifidum NRC cells when exposed to the bile salt for three hours was slightly higher (7.91 CFU/ml) than that of non-microencapsulated (7.80 CFU/ml) in the presence of bile salts (1.0%). Bacterial count of microencapsulated and non-microencapsulated for L. paracasei 441strain was 7.88 CFU/ml and 7.85 CFU/ml respectively. Our results suggested that the microencapsulated strains have higher viability at 1.5 pH and 1.0% bile salts. These encapsulated strains could be used to improve probiotics viability in food products.Acetaminophen (APAP) is an analgesic commonly administered to both sick children and adults. Studies of APAP toxicity, during embryonic development in the zebrafish (Danio rario) as a model organism, have focused on physical changes in development (David, et al. 2009), but an active area of research has begun to focus on its effects at the molecular level. Studies suggest that biomarkers for oxidative stress are elevated in APAP-treated rats (Jin, et al. 2012). To extend these studies, zebrafish embryos were exposed to two concentrations of APAP, 0.37 g/L and 0.076 g/L, based on previous research that observed anatomical changes in the heart and liver in zebrafish (Xu, et al. 2012 & He, et al. 2012). RNA was isolated at 24, 48, and 72 hr post fertilization (hpf) and mRNA levels of the oxidative stress markers, heme oxygenase 1a (hmox1a) and glutathione S-transferase pi 2 (gstp2), were measured using RT-PCR. TATA box binding protein (tbp) was used as an internal reference to normalize the data. At the times and APAP concentrations evaluated, the mRNA levels of hmox1a and gstp2 do not appear to be affected. Since higher doses of APAP were required to cause changes in gene expression in the rat and a longer exposure time to APAP may be required to see changes in the zebrafish at the molecular level, these stress indicators will next be evaluated using a higher dose of APAP, 0.75 g/L and 1 g/L, and for longer-term effects (96 hpf and 120 hpf).Planaria, Dugesia tigrina (freshwater flatworms), are some of the simplest organisms with the ability to learn, and have been used in numerous learning studies. In order to determine the effect of different agents on learning using this model organism, there must first be a reliable method of training. In this study, 54 Planaria were trained for directional preference in a y-maze with negative stimuli: light and electrical shock. They were trained in a specified direction in the maze over a three-day period consisting of ten trials per day. If they attempted to travel in the opposite direction to which they were being trained, they were negatively stimulated. It appeared that Planaria could be successfully trained with the administration of electrical shock; however, light administration was not an effective training method. Since the tactile sensory nervous system is more developed than the visual system in Planaria, this may explain the differences in the success of these training methods.This study serves to find a drug that can be used by the body to stimulate fatty acid metabolism to a useable energy source for cellular respiration while overriding the normal mechanism. This conversion process mostly occurs in a starvation state. L-glutamate dehydrogenase (GDH) catalyzes glutamate to alphaketoglutarate (AKG) that feeds into the citric acid cycle. When excess food intake occurs, GDH is inhibited by GTP, ATP and palmitoyl-CoA, slowing the synthesis of AKG. Overcoming endogenous GDH inhibition can have therapeutic benefits including stimulating fatty acid oxidation and basal insulin secretion. This experiment began with designing a kinetic assay that could measure the reversal of inhibition of the GDH enzyme as done by the body when converting fatty acids to glucose. Once this was accomplished, the assay was miniaturized and automated for high-throughput small molecule screening in 384-well microtiter dishes to discover potential GDH activators. To start, plates are loaded with a buffer solution containing l-Glutamate, NADH and +/−GTP (no GTP for control wells), then test compounds or dimethyl sulfoxide (DMSO) as the vehicle control. Next, a buffer solution containing GDH is applied to all wells and it is immediately read on a BMG Pherastar fluorospectrometer at 30-second intervals for five minutes to detect NADH. The points are plotted, the slope determined, and wells with slopes 50% greater than negative control (relative to the uninhibited positive control) are selected. After confirmation, these hits will be used in a cell-based assay to determine effects on cellular respiration. Any molecules showing desired results will be considered for future animal studies. Should this prove successful, this will provide a novel approach to helping people with Diabetes lose excess weight in a controlled manner.Hester-Dendy samplers were placed in riffles and pools to map colonization and drift patterns along a reach of Crabtree Creek that historically has dramatic fluctuations in flow as a result of rainfall. Data showed the species that colonized the samplers, primarily mayflies, caddisflies and dipterans, represent a small proportion of biota that typically inhabit the area. Riffle samplers were consistently colonized by more individuals than pool samplers on any given sample day with a higher proportion of Baetis and Hydropsyche relative to pools, which showed greater numbers of Maccaffertium. In riffle habitats there was a clear positive relationship between the increase in flow and the increase in colonization. However, when flow rates reached scouring levels little to no colonization occurred. At high flow rates a sampler, which was previously occupied, was stripped of colonizers. There was also a strong negative relationship between species colonization and turbidity; however temperature did not appear to be a factor.The success of any phylogenetic survey depends on a sampling regime of appropriate geographic scope and scale. Unfortunately how to design an appropriate sampling regime is often obscure. In the case of morphological conservative species it can be even more difficult to design an adequate sampling regimen because all populations look similar and there is nothing to suggest where the most informative collections should be made. Here I report the results of a robust sampling regimen that I believe are of broad utility, especially for low vagility organisms. As a case study we sampled ∼700 populations across the distribution of all described dusky salamander (Desmognathus) species. Because most species of Desmognathus are semiaquatic, upland dispersal is probably minimal; instead, most inter-population movement likely occurs via streamside (and/or other wetland) conduits that are eventually circumscribed within a given river drainage system. Thus, river drainages provide a logical starting point for investigating distribution patterns and evolutionary relationships of Desmognathus. To enhance resolution and repeatability, I have identified a second-level sampling component to standardize finescale geographic coverage: level IV ecoregions, which denote areas of general similarity in ecosystems as well as in the type and quantity of environmental resource. I compare and contrast the results of our survey with previous work in the genus to highlight some of the unique findings uncovered with this sampling regime.Rapamycin is a drug used to suppress the growth of tumors in many different types of cancer. Rapamycin inhibits the signaling of the mammalian target of Rapamycin (mTOR) and causes a decreased proliferation rate of eukaryotic cells. In this study the growth effects of Rapamycin (40 µM, 20 µM, 10 µM, 5 µM, 0.5 µM, 50 nM, 5 nM) on Lactobacillus acidophilus and Escherichia coli were tested to see if the drug might inhibit the gut microbiota when administered to cancer patients. Lactobacillus acidophilus and Escherichia coli are essential components of the gut microbiome; they increase the functioning of the immune system and help to prevent invasion of pathogens. While there have been many studies conducted on the tumor suppressive effects of Rapamycin, this study tested the effects this chemotherapeutic agent had on two components of normal microbiota in the human gastrointestinal tract. The hypothesis that Rapamycin inhibits the growth of Lactobacillus acidophilus and Escherichia coli in a dose-dependent manner was rejected. Other chemotherapy drugs are now being investigated.Azotobacter sp. ATCC 49359 (known informally as “Azotobacter zettuovi”), was isolated in Holland in 1960 by S. Rowinski and deposited in the American Type Culture Collection (ATCC) by P. Jurtshuk of the University of Texas Houston. Azotobacter sp. ATCC 49359 is a gramnegative, nitrogen-fixing, free-living, soil bacterium that remains officially unrecognized and uncharacterized. Phylogenetic analysis of the 16S and RNase P genes, from this lab, placed Azotobacter sp. ATCC 49359 in the genus Azomonas, most closely related to Azomonas insignis. Azomonas, Azotobacter, Azorhizophilus and Pseudomonas are all genera of the family Pseudomonadacea. The taxonomy of this family is based on superficial morphological and biochemical traits, and as a result is a poor match to the phylogenetic relationships between member species. This study will use multi-locus sequence typing (MLST) of nine genes, consisting of house-keeping and DNA repair, as well as genes for traits specific to this family, to further support the placement of Azotobacter sp. ATCC 49359 in the genus Azomonas. Additional methods of characterization will be used according to the specifications of the International Journal of Systematic and Evolutionary Microbiology (IJSEM) to support the placement of Azotobacter sp. ATCC 49359 in the genus Azomonas and to formally recognize it as “Azomonas zettuovi”.Antibiotics are usually the strongest and last line of defense against bacterial pathogens and the vast majority of antibiotics today originate from soil microbes. In particular, actinomycetes have been an exceptional source of novel antibiotics and other bioactive compounds. Actinomycetes are Gram-positive, saprophytic bacteria found in soil, water and colonizing plants. Of all the bioactive compounds that have been obtained from microbes, slightly less than half originated from actinomycetes. Actinomycetes have been intensively studied in a variety of places, including previously unexplored habitats, extreme environments, yet there is currently no publication characterizing the antibacterial activity of actinomycetes and other microbes collected from soils affected by serpentinization. Serpentinization occurs on a global scale and has been observed in marine and terrestrial environment; this metamorphic process occurs when ultramafic rocks, characteristic of the Earth's lower crust and upper mantle, are uplifted by tectonic activity and interact with water. This interaction creates an environment rich in electron donors such as hydrogen and methane; short-chain hydrocarbons and small organic acids are also produced to a lesser extent. Serpentinization leads to alkaline conditions in excess of pH 10, limited access to dissolved inorganic carbon and terminal electron acceptors, and low biological diversity. These factors make serpentinizing environments unique and challenging for inhabiting microbes. Microbes, especially actinomycetes, which can withstand the unique niche caused by serpentinization and warrant further study because of the potentially undiscovered bioactive compounds they produce. Recent surveys of serpentinized soils indicate a significant portion of microbes found there belong to the group actinomycetes. This further advocates the notion that serpentinized soils may contain unidentified bioactive compounds.Camelids contain heavy chain antibodies (hcAbs) from which single domain antibodies (sdAbs) are derived. Sharks contain hcAbs from which VNAR are derived. For this reason, Shark sdAbs have not been specifically focused on greatly in the past and we wished to determine if they would also prove to be thermally stable. Twenty clones were transformed and cultured and the melting temperatures of 19 out of the 20 clones were determined. We transformed the cells using pHelp, Turner DE3 strain of E. coli, and plasmid DNA encoding one of the 20 VNAR from one of the 20 different cell lines of each type of shark. We inoculated the cells and grew them in terrific broth (TB) media. From these cells we extracted the protein and determined the melting point using a dye melt technique. Using about half of the samples, we performed circular dichroism (CD) as another, more accurate way to test the melting temperatures and to observe whether or not the shark proteins refolded. Using the dye melts, we found melting temperatures ranging from 42°C to 77°C. When we performed CD on the samples, we found that the melting temperatures usually fell within a 5°C range of those from the dye melt. We concluded that shark VNAR do not refold in the same manner as camelid sdAbs. Some of the proteins were shown to have above a 70% refolding ability, but it was not conclusive for a majority of the samples that were tested.Evolving software systems have instructions that are typically encoded by symbols or commands that do not change their meaning in terms of their corresponding machine functions. Here we explore the before-and-after scenarios for how instruction symbols may be reassigned during digital evolution experiments. We made adjustments to the digital evolution software AVIDA codebase to implement two basic steps. We configured AVIDA to support multiple forms of an instruction that have different symbolic encodings but the same machine function. We then substituted all copies of one of the redundant symbolic encodings in an evolved organism to correspond to a different machine function and examined the consequences. Our analysis workflow enabled us to identify different scenarios where substitutions of redundant symbolic encodings reduced fitness. There are broader implications for how this digital evolution experiment may relate to codon degeneracy in biological systems.The salamander genus Pseudotriton (family Plethodontidae) has several races, yet red salamanders (Pseudotriton ruber) and mud salamanders (Pseudotriton montanus) remain the only identified species of the genus. Pseudotriton is widely distributed across the eastern United States, ranging from New York in the north to Florida in the south westward to the Mississippi River. While the distinctiveness of the two species of Pseudotriton has long been recognized, the genetic variation and structure of populations has never been analyzed. In general plethodontid salamanders are characterized by a pattern of extreme geographic partitioning and cryptic speciation but to date no studies have addressed these issues in Pseudotriton. We have sampled 110 populations spanning the extent of this genera's distribution. For each population sampled, we extracted DNA, amplified and sequenced a 1686 base pair fragment of the mtDNA genome. Here we present the results of a Bayesian phylogenetic reconstruction for this genus.Riboswitches are an important aspect of the regulation of some genes in bacteria. The binding of effector molecules to mRNA leader regions specifies their secondary structures, which modulates transcription termination and therefore the expression of downstream genes. This study examines the FMN riboswitch located upstream of the rib operon in Photorhabdus luminescens, and seeks to determine if this region of regulatory RNA is a potential target for novel antimicrobials. The pathogenicity of this bacterium on its host, Caenorhabditis elegans, will be examined via mutagenesis to lock the riboswitch in the “on” or “off” conformations using the lambda-Red recombineering system. If the bacterium loses virulence when the FMN riboswitch is locked in the “off” confirmation, it may mean that the bacteria cannot bypass the rib pathway and can therefore not sequester FMN from its host. This finding would focus attention on the FMN riboswitch as a potential target for novel antimicrobials for use against multi-drug resistant bacteria.Chapel Hill landfill closed in July 2013 resulting in transportation of city trash to a remote site, thus adding cost and creating pollution. Since ∼50% of school cafeteria trash and ∼35% of city trash are compostable, composting in schools and communities will significantly reduce greenhouse gases and transportation cost. Four objectives were developed by the Trash Terminators team: to practice and educate composting at our school, to duplicate the program in other district schools, to conduct community outreach, and to propose municipal composting to local governments. We first set up composting bins in the school cafeteria, and contracted with a composting company for collecting compostable waste. In this school year, 10.24 tons (>80%) of trash will be either recycled or composted, and thus 574 pounds of CO2 and $550 of cost will be reduced for our school. We reached out to the leadership in our school district, and successfully convinced all district schools to compost their cafeteria trash by the end of this year. In the community, our outreach activities included composting demonstrations in public places and events, local newspaper interviews, infomercial aired on TV, and information and surveys posted through social media. We have presented to governing bodies of Chapel Hill Township, Carrboro Township and Orange County to promote municipal composting. Two State Senators have highlighted our initiatives in their newsletters. In summary, we have successfully launched a composting program in our middle school and expanded it into our communities with the goal of promoting an eco-friendly society.Cardiovascular diseases (CVD) account for a large number of mortality all over the world. Although the pathogenesis of CVD includes numerous factors, environmental toxin exposure from air pollution could elevate the risk of CVD. Acrolein, a highly reactive aldehyde species, is a major air pollutant. It is generated in high quantities from automobile exhaust and tobacco smoke. However, the action of acrolein in endothelial cells remains to be investigated. The present study examined acrolein-induced cellular injury, oxidative modifications of cellular constituents, altered intracellular glutathione (GSH), and adhesion of monocytes to endothelial cells on EAHY926 cells, a widely used endothelial cell line for study of endothelial cell dysfunction. Incubation of cells with acrolein at pathophysiological concentrations for 24 hr caused a significant decrease in cell viability, as measured by the MTT assay and an increase in the release of LDH, which further confirms a significant change in the cell morphology. Acrolein also increased the amount of thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, and protein carbonyl levels, a marker of protein damage in the cells. Incubation of cells with acrolein also resulted in a significant depletion of cellular GSH and augmented monocyte adhesion to human endothelial cells, an important step in the development of atherosclerosis. The results of this study may contribute to our ability to assess the cardiovascular risk of human exposure to acrolein.This research measured and compared the levels of physiological reactions to stress of freshman and senior student-athletes. The hypothesis was that senior student-athletes would have more physiological reactions to stress compared to freshman student-athletes. To measure the level of stress experienced by the student-athletes, a survey that measured the physiological responses to stress was distributed to 20 freshmen and 20 seniors student-athletes of the University of Mount Olive. These student-athletes were currently engaging in athletic activities such as practicing and competing. The results supported the hypothesis and showed that seniors experience more physiological reactions to stress than freshmen. There was a significant correlation between class status and physiological responses to stress. To our knowledge, this was the first study that compared physiological responses to stress between freshman and senior student-athletes. Further research should examine other factors that influence physiological responses to stress in student-athletes.Protein phosphorylation, mediated by protein kinases, underlies nearly all cell signaling processes. Recently, it has become apparent that, in addition to direct kinase-substrate interactions, cells achieve signaling specificity by differentially activating distinct pools of a given kinase within the cell. To monitor the spatiotemporal regulation of kinases within the endogenous cellular environment, we are developing a series of genetically-encodable kinase activity reporters that can be targeted to specific subcellular regions. These reporters, which rely on the phenomenon of fluorescence resonance energy transfer (FRET), are based on a modular design in which a switching domain composed of a “substrate region” (e.g., a consensus phosphorylation motif) and a “phosphoamino acid binding domain” is sandwiched etween complementary fluorescent protein (FP) color variants, such as CFP and YFP. Phosphorylation dependent conformational changes alter the relative orientation of the FPs, causing a change in FRET. We are currently developing a generalizable in vitro assay platform to characterize these reporters. Here we report the expression, purification and initial characterization of one such reporter, the A-kinase activity reporter 3 (AKAR3). AKAR3 was expressed in JM109(DE3) E. coli and purified by Ni-NTA affinity chromatography. The purified product was analyzed by gel electrophoresis and visualized by silver staining. We are currently using active human PKA purified from yeast to measure its specificity and dynamic range using a multimode plate