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Modulation of the Coelomic Fluid Protein Profile in the Earthworm, Lumbricus Terrestris, After Exposure to Copper as Copper Sulfate

Description: Proteomic techniques were used to analyze the protein profile of earthworm, Lumbricus terrestris, coelomic fluid collected by either whole body dissection method or the coelomic cavity puncture method. Data demonstrated that collection of coelomic fluid using the coelomic cavity puncture method protocol resulted in a 32% reduction, 377 +/- 4.5 vs 253+/- 19.9 (p=0.0007), in the number of individual proteins. It was determined that the coelomic cavity puncture method yielded a "cleaner" preparation, one less contaminated with extraneous proteins from intestinal tissue, gut contents, and body wall materials. This protocol was used in all later studies. The same proteomic techniques were used to evaluate the effects that exposure to Cu (1.0 μg/cm2) as CuSO4 had on the earthworm coelomic fluid profile. Comparison of protein profile from exposed earthworms demonstrated a significant reduction in the number of proteins expressed (184 ± 2.64 vs 253 ±19.9 p=0.0192) when compared to control organisms. Cu exposure also resulted in a modulation of the protein profile with treated earthworms expressing 47 new proteins that were not identified in unexposed worm coelomic fluid. Additionally, 116 proteins found in coelomic fluid collected from normal worms were absent in Cu exposed organisms. Finally, 137 proteins were conserved or found in both control and exposed organisms; however of these proteins, 24 were up-regulated, 105 were down-regulated, and 8 were unchanged as a result of Cu exposure.
Date: May 2010
Creator: Herring, Reese

Virulence Factor Production in PyrE Mutants of Pseudomonas Aeruginosa

Description: It has been shown previously in our lab that mutations in the pyrimidine pathway reduced the ability of Pseudomonas aeruginosa to produce virulence factors. Knockout mutations in pyrB, pyrC and pyrD genes of the pyrimidine pathway showed that virulence factor production was decreased. Pyoverdin, pyocyanin, hemolysin, iron chelation, motility, and adherence are all considered virulence factors. Here I further investigate the effects of mutations in the pyrimidine pathway by studying a pyrE mutant. I studied the effect of the pyrE mutation on the production of the above virulence factors. Just like the effect of pyrB, pyrC and pyrD mutations,the pyrE mutation also showed that the bacteria were deficient in producing virulence factors when compared to the wild type. The broader impact of this research would be the possibility of finding drugs that could treat patients infected with P. aeruginosa and possibly extend the lives of chronically infected patients with cystic fibrosis.
Date: May 2010
Creator: Niazy, Abdurahman

Adaptive Advantages of Carotenoid Pigments in Alpine and Subalpine Copepod Responses to Polycyclic Aromatic Hydrocarbon Induced Phototoxicity

Description: Alpine zooplankton are exposed to a variety of stressors in their natural environment including ultraviolet radiation. Physiological coping mechanisms such as the accumulation of photoprotective compounds provide these zooplankton protection from many of these stressors. Elevated levels of carotenoid compounds such as astaxanthin have been shown to help zooplankton survive longer when exposed to ultraviolet radiation presumably due to the strong antioxidant properties of carotenoid compounds. This antioxidant capacity is important because it may ameliorate natural and anthropogenic stressor-induced oxidative stress. While previous researchers have shown carotenoid compounds impart increased resistance to ultraviolet radiation in populations of zooplankton, little work has focused on the toxicological implications of PAH induced phototoxicity on zooplankton containing high levels of carotenoid compounds. This thesis discusses research studying the role that carotenoid compounds play in reducing PAH induced phototoxicity. By sampling different lakes at elevations ranging from 9,500' to 12,700' in the front range of the Colorado Rocky Mountains, copepod populations containing different levels of carotenoid compounds were obtained. These populations were then challenged with fluoranthene and ultraviolet radiation. Results discussed include differences in survival and levels of lipid peroxidation among populations exhibiting different levels of carotenoid compounds, and the toxicological and ecological implications of these results.
Date: May 2010
Creator: Kovach, Matthew James

Stretching the Flexible Myosin II Subfragment Using the Novel Gravitational Force Spectroscope, and the Uncoiling of S2

Description: Familial Hypertrophic cardiomyopathy (HCM) causes ventricle walls to thicken and often leads to sudden death especially in adults. Mutations in the subfragment 2 (S2) of β-cardiac myosin are implicated in the genetic disorder. This S2 region is a coiled-coil rod region resulting from the dimeric form of myosin II. It has been proposed that an elastic quality allows normal S2 to absorb force during the powerstroke according to the sliding filament model. To test the flexibility of single molecules of S2 against levels of physiological force, the Gravitational Force Spectrometer (GFS) is being developed. This novel system employs a standard microscope on an equatorial mount that allows the spectrometer to be rotated freely in space. Stationary glass beads are attached to a microscope slide where the molecule is tethered between the stationary bead and a smaller mobile bead. The GFS is oriented so that the force of gravity can act on the mobile bead and so impart a small force to the tethered subfragment. Additionally, a video system in conjunction with ImageJ software makes a distance measurement of the molecule possible with a resolution of around 11 nm. The S2 can be stretched parallel or perpendicular to the coiled coil to elucidate different structural properties of the rod. This study is the first to show structural evidence that S2 in vertebrate skeletal myosin uncoils proportionally to physiological force loads. Because of this, the usefulness and promise of the novel GFS is highlighted, and the biological role of S2's flexibility can be directly commented on. If the dimer undergoes uncoiling at physiological force loads as shown, then it is reasonable to think that this might occur in nature in response to the stress of the powerstroke on a single molecule. This unwinding could be to absorb force as a mechanism to ...
Date: May 2010
Creator: Dunn, James W.

Effects of Suspended Multi-Walled Carbon Nanotubes on Daphnid Growth and Reproduction

Description: Multi-walled carbon nanotube aggregates can be suspended in the aqueous phase by natural organic matter. These aggregates are ingested by filter feeding zooplankton. Ingested aggregates result in decreased growth and decreased reproduction. These effects may be caused by reduction in energy input from normal feeding behavior. pH alters natural organic matter structure through changes in electrostatic repulsion. Altered natural organic matter structure changes multi-walled carbon nanotube aggregate size. This size variation with variation in pH is significant, but not large enough a change in size to alter toxicity, as the aggregate size range remains well within the particle size selection of the organisms.
Date: May 2010
Creator: Alloy, Matthew Michael

Comparison of Heteranthera Dubia (Jacq.) MacM.-associated Macroinvertebrates Between Georgraphical Regions in the United States

Description: Macroinvertebrates associated with the aquatic plant, water stargrass (Heteranthera dubia), were sampled from 12 waterbodies in four regions of the United States from June to August 2005. Taxa richness, evenness, and diversity were lowest in the Lower Midwest (LMW) region, and higher in Northern sites, especially the Upper Midwest (UMW), and Northeast (NE). While relative abundance varied from site to site and region to region, utilization of the plant by functional groups remained fairly constant. Collector-gatherers consistently comprised the largest portion of invertebrates sampled. The shredder/ herbivore functional group comprised an average of 17 % of total groups. Through an exhaustive literature review, it was found that shredder/ herbivores of water stargrass have not been reported in the literature. Because of this, the herbivore group was analyzed separately and consisted of 2,383 specimens representing 23 species. The most common groups were Rhopalosiphum sp., Nectopsyche spp. and chironomids. No differences were found in herbivore diversity or evenness between sampling regions, but species richness was significantly different.
Date: May 2010
Creator: Harms, Nathan Earl

Thermal Stress During Pre-Incubation Induces Subsequent Developmental Plasticity in Northern Bobwhites

Description: Northern bobwhite populations have declined concurrent with global warming. The focal period of this study was the 12-d pre-incubation period, when bobwhite eggs remain in the nest without the thermal protection of the incubating parent. This study first established the storage and thermal limits of bobwhite eggs, then investigated how global warming may impact oviparous embryos and how bobwhite embryos react to acute and chronic doses of simulated drought temperatures during pre-incubation. First, the maximum storage limit of bobwhite eggs was determined by storing eggs ≤21 d and measuring hatching success and pH of egg albumen and yolk. Hatching success of stored eggs declined after 14 d, when yolk and albumen pH reached levels detrimental to embryonic development. Secondly, thermal limits were determined by exposing bobwhite eggs to hyperthermic temperatures (38-52 °C). Bobwhite embryos survived 50 °C for 1 h, 49 °C for 3 h and 46 °C for 6 h. Results indicate an adaptation to the naturally occurring temperature extremes that can occur in the bobwhite's southern range during pre-incubation. Subsequently, bobwhite eggs were exposed to either low constant (LC), low fluctuating (LF), high constant (HC), or high fluctuating (HF) temperatures during pre-incubation to determine if the nature of temperatures differentially affected development. Although eggs exposed to high heat loads (HC and HF), and low heat loads (LF and LC) had equal heating degree-hours within groups, they exhibited differential growth during pre-incubation. Oxygen consumption, hatch timing, and hatching success were also affected by the thermal regimes. Eggs in simulated drought (HF) had a 47% lower hatch rate than eggs in simulated non-drought (LF) indicating that thermal stress during pre-incubation may contribute to population declines during drought. Finally, northern bobwhite eggs were exposed to acute or chronic doses of simulated drought temperatures, which tested for critical periods of development during ...
Date: August 2010
Creator: Reyna, Kelly Shane

DNA Degradation as an Indicator of Post-Mortem Interval

Description: The question of post-mortem interval (PMI) or time since death is often the most sought after piece of information associated with a medical death investigation. Based on the observation that DNA degradation disproportionately affects the analysis of larger genetic loci, it was proposed that DNA degradation, as a result of autolysis or putrefaction, could prove suitable as a potential rate-of-change indicator of PMI. Nine randomly amplified polymorphic DNA (RAPD) analysis primers and three sets of directed amplification primers were evaluated to determine their suitability for use in assessing the degree of DNA fragmentation in tissue samples. They were assessed for amplicon specificity, total DNA target sensitivity, allele monomorphism and the observance of degradation-based profile changes. Markers meeting the requisite criteria were then used to assess a range samples degraded under controlled and uncontrolled conditions. Tissue samples collected from seven domestic pigs (Sus scrofa) were incubated under controlled laboratory or uncontrolled field conditions to produce samples simulating those potentially collected in a forensic case. DNA samples isolated from these specimens were then analyzed at those loci which had been determined to meet the requisite criteria. Collectively, data generated from these analyses indicate that genetic profiles generated by this approach can provide information useful for estimating the post-mortem interval, with the locus and amplicons used being most useful during the first 72 hours after death.
Date: August 2010
Creator: Watson, William H.

Metabolic Engineering of Raffinose-Family Oligosaccharides in the Phloem Reveals Alterations in Patterns of Carbon Partitioning and Enhances Resistance to Green Peach Aphid

Description: Phloem transport is along hydrostatic pressure gradients generated by differences in solute concentration between source and sink tissues. Numerous species accumulate raffinose-family oligosaccharides (RFOs) in the phloem of mature leaves to accentuate the pressure gradient between source and sinks. In this study, metabolic engineering was used to generate RFOs at the inception of the translocation stream of Arabidopsis thaliana, which transports predominantly sucrose. To do this, three genes, GALACTINOL SYNTHASE, RAFFINOSE SYNTHASE and STACHYOSE SYNTHASE, were expressed from promoters specific to the companion cells of minor veins. Two transgenic lines homozygous for all three genes (GRS63 and GRS47) were selected for further analysis. Sugars were extracted and quantified by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and 21-day old plants of both lines had levels of galactinol, raffinose, and stachyose approaching 50% of total soluble sugar. All three exotic sugars were also identified in phloem exudates from excised leaves of transgenic plants whereas levels were negligible in exudates from wild type leaves. Differences in starch accumulation or degradation between wild type and GRS63 and GRS47 lines were not observed. Similarly, there were no differences in vegetative growth between wild type and engineered plants, but engineered plants flowered earlier. Finally, since the sugar composition of the phloem translocation stream is altered in these plants, we tested for aphid feeding. When green peach aphids were given a choice between WT and transgenic plants, WT plants were preferred. When aphids were reared on only WT or only transgenic plants, aphid fecundity was reduced on the transgenic plants. When aphids were fed on artificial media with and without RFOs, aphid reproduction did not show differences, suggesting the aphid resistance is not a direct effect of the exotic sugars.
Date: August 2010
Creator: Cao, Te

Molecular systematics of Baird's pocket gopher (Geomys breviceps)

Description: Baird's pocket gopher (Geomys breviceps) is found in eastern Texas, eastern Oklahoma, central and western Arkansas, and western Louisiana. The cytochrome-b gene was sequenced and analyzed for 16 pocket gophers from throughout the range of the species. Similar phylogenetic trees were obtained using maximum-parsimony, maximum-likelihood, neighbor-joining, and Bayesian analyses. Two major clades were formed with northern individuals belonging to clade I and southern individuals belonging to clade II. G. b. sagittalis was paraphyletic in relation to G. b. breviceps in all analyses. Based on inconsistencies between the taxonomic classification and systematic relationships within Baird's pocket gopher, a taxonomic restructuring appears warranted.
Date: August 2010
Creator: Bodine, Deanna Martinez

Wind Energy-related Wildlife Impacts: Analysis and Potential Implications for Rare, Threatened and Endangered Species of Birds and Bats in Texas

Description: Texas currently maintains the highest installed nameplate capacity and does not require publicly available post-construction monitoring studies that examine the impacts of wind energy production on surrounding fauna. This thesis examines potential wind energy impacts on avian and bat species in Texas through a three-part objective. The first two objectives synthesize literature on variables attractive to species within wind development areas and estimate impacted ranges outside of Texas, based on studies examining wind energy's environmental impacts. The third objective focuses on Texas wind development potential for interaction with rare, threatened and endangered species of birds and bats using GIS analysis with a potential hazard index (PHI) model, which addresses broad-spectrum, high risk variables examined within the first two objectives. Assuming areas with higher wind speeds have potential for wind development, PHI values were calculated for 31 avian and ten bat species, based on an analysis of species range data obtained from the Texas Parks and Wildlife Department and wind data obtained from the National Renewable Energy Laboratory. Results indicate one avian species, Tympanuchus pallidicinctus, is at high risk for wind development interaction on an annual basis, with 20 species of birds and nine species of bats at higher risk during the spring season. This macro-scale approach for identifying high risk species in Texas could be used as a model to apply to other conterminous states' preliminary evaluation of wind energy impacts.
Date: August 2010
Creator: Graham, Tara L.

The Influence of in Vitro Gill and Liver Metabolism of Xenobiotics on Fish Bioconcentration

Description: This dissertation examines the ability of in vitro biotransformation assays to provide an indication of metabolic potential. The potential for xenobiotic compounds to bioconcentrate in aquatic organisms is expressed through the bioconcentration factor (BCF). The metabolic loss of ibuprofen, norethindrone and propranolol was measured using rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) gill and liver S9 fractions, microsomes and cell suspensions. Metabolic transformation rates (kM) were extrapolated from in vitro intrinsic clearance of parent compound (CLm) and integrated into a refined BCF model. In general, CLm of test compounds was greater in liver S9 fractions and hepatocytes. However, the influence of hepatic metabolism on kM and BCF was limited by hepatic blood flow (20-25%) compared to gill blood flow (~100%). A significant difference was noted between BCF solely based on KOW and BCF including kM. These studies indicate that the inclusion of kM in BCF models can bring predicted bioconcentration estimates closer to in vivo values. Primary cell suspensions are preferred over subcellular fractions as cell suspensions possess both phase I and phase II enzyme activity. Further study was conducted on ibuprofen biotransformation pathways. As fish do not contain the same cytochrome P450 (CYP) 2C homologs known to metabolize ibuprofen in mammals, it cannot be assumed that piscine biotransformation is similar. Metabolite analysis found 2-hydroxy-ibuprofen as the major metabolite in S9 and microsomal fractions. Additional assays involving the induction and inhibition of specific CYP isozymes support CYP1A2 as an alternative metabolic pathway.
Date: August 2010
Creator: Gomez, Cristi Frasier

Role of N-Acylethanolamines in Plant Defense Responses: Modulation by Pathogens and Commercial Antimicrobial Stressors

Description: N-acyl ethanolamines (NAEs) are a class of lipids recently recognized as signaling molecules which are controlled, in part, by their degradation by fatty acid amide hydrolase (FAAH). On the basis of previous studies indicating increased NAE levels in a tobacco cell suspension-xylanase elicitor exposure system and the availability of FAAH mutants, overexpressor and knockout (OE and KO) genotypes in Arabidopsis thaliana, further roles of NAEs in A. thaliana plant defense was investigated. The commonly occurring urban antimicrobial contaminant triclosan (TCS) has been shown to suppress lipid signaling associated with plant defense responses. Thus, a second objective of this study was to determine if TCS exposure specifically interferes with NAE levels. No changes in steady state NAE profiles in A. thaliana-Pseudomonas syringae pv. syringae and A. thaliana-flagellin (bacterial peptide, flg22) challenge systems were seen despite evidence that defense responses were activated in these systems. There was a significant drop in enoyl-ACP reductase (ENR) enzyme activity, which catalyzes the last step in the fatty acid biosynthesis pathway in plants, on exposure of the seedlings to TCS at 10 ppm for 24 h and decreased reactive oxygen species (ROS) production due to flg22 in long term exposure of 0.1 ppm and short term exposure of 5 ppm. However, these responses were not accompanied by significant changes in steady state NAE profiles.
Date: August 2010
Creator: Vadapalli, Vatsala

Hepatotoxicity of Mercury to Fish

Description: Tissue samples from spotted gar (Lepisosteus oculatus) and largemouth bass (Micropterus salmoides) were collected from Caddo Lake. Gar and bass livers were subjected to histological investigation and color analysis. Liver color (as abs at 400 nm) was significantly correlated with total mercury in the liver (r2 = 0.57, p = 0.02) and muscle (r2 = 0.58, p = 0.01) of gar. Evidence of liver damage as lipofuscin and discoloration was found in both species but only correlated with liver mercury concentration in spotted gar. Inorganic mercury was the predominant form in gar livers. In order to determine the role of mercury speciation in fish liver damage, a laboratory feeding study was employed. Zebrafish (Danio rerio) were fed either a control (0.12 ± 0.002 µg Hg.g-1 dry wt), inorganic mercury (5.03 ± 0.309 µg Hg.g-1 dry wt), or methylmercury (4.11 ± 0.146 µg Hg.g-1 dry wt) diet. After 78 days of feeding, total mercury was highest in the carcass of zebrafish fed methylmercury (12.49 ± 0.369 µg Hg.g-1 dry wt), intermediate in those fed inorganic mercury (1.09 ± 0.117 µg Hg.g-1 dry wt), and lowest in fish fed the control diet (0.48 ± 0.038 µg Hg.g-1 dry wt). Total mercury was highest in the viscera of methylmercury fed zebrafish (11.6 ± 1.86 µg Hg.g-1 dry wt), intermediate in those fed inorganic diets (4.3 ± 1.08 µg Hg.g-1 dry wt), and lowest in the control fish (below limit of detection). Total mercury was negatively associated with fish length and weight in methylmercury fed fish. Condition factor was not associated with total mercury and might not be the best measure of fitness for these fish. No liver pathologies were observed in zebrafish from any treatment.
Date: August 2010
Creator: Barst, Benjamin Daniel

Functional Neural Toxicity and Endocrine Responses in Mice Following Naphthalene Exposure

Description: Polycyclic aromatic hydrocarbons (PAHs) are a well studied and diverse class of environmental toxicants. PAHs act via the aryl hydrocarbon receptor (AhR), and studies have suggested that PAHs may elicit neurological and estrogenic effects. Doses of PAHs between 50 to 150 ppm may elicit neurotoxicity in rodent models. The present study investigated the effects of naphthalene on in vivo steroidogenesis in Swiss Webster male mice, and in vitro neural function of Balb-C/ICR mice frontal cortex neurons. These data suggest that naphthalene may not elicit steroidogenic effects at concentrations ranging from 0.2 to 25 mg/kg/day, following a 7 day subcutaneous dosing regime. In addition, naphthalene may cause functional toxicity of frontal cortex neurons at concentrations of 32 to 160 ppm naphthalene.
Date: August 2010
Creator: Colbert, Crystal

Interactions of N-Acylethanolamine Metabolism and Abscisic Acid Signaling in Arabidopsis Thaliana Seedlings

Description: N-Acylethanolamines (NAEs) are endogenous plant lipids hydrolyzed by fatty acid amide hydrolase (FAAH). When wildtype Arabidopsis thaliana seeds were germinated and grown in exogenous NAE 12:0 (35 µM and above), growth was severely reduced in a concentration dependent manner. Wildtype A. thaliana seeds sown on exogenous abscisic acid (ABA) exhibited similar growth reduction to that seen with NAE treatment. AtFAAH knockouts grew and developed similarly to WT, but AtFAAH overexpressor lines show markedly enhanced sensitivity to ABA. When low levels of NAE and ABA, which have very little effect on growth alone, were combined, there was a dramatic reduction in seedling growth in all three genotypes, indicating a synergistic interaction between ABA and NAE. Notably, this synergistic arrest of seedling growth was partially reversed in the ABA insensitive (abi) mutant abi3-1, indicating that a functional ABA signaling pathway is required for the full synergistic effect. This synergistic growth arrest results in an increased accumulation of NAEs, but no concomitant increase in ABA levels. The combined NAE and ABA treatment induced a dose-dependent increase in ABI3 transcript levels, which was inversely related to growth. The ABA responsive genes AtHVA22B and RD29B also had increased expression in both NAE and ABA treatment. The abi3-1 mutant showed no expression of ABI3 and AtHVA22B, but RD29B expression remained similar to wildtype seedlings, suggesting an alternate mechanism for NAE and ABA interaction. Taken together, these data suggest that NAE metabolism acts through ABI3-dependent and independent pathways in the negative regulation of seedling development.
Date: August 2010
Creator: Cotter, Matthew Q.

Endocannabinoid System in a Planarian Model

Description: In this study, the presence and possible function of endocannabinoid ligands in the planarian is investigated. The endocannabinoids ananadamide (AEA) and 2-arachidonoylglycerol (2-AG) and entourage NAE compounds palmitoylethanolamide (PEA), stearoylethanolamide (SEA) and oleoylethanolamide (OEA) were found in Dugesia dorotocephala. Changes in SEA, PEA, and AEA levels were observed over the initial twelve hours of active regeneration. Exogenously applied AEA, 2-AG and their catabolic inhibition effected biphasic changes in locomotor velocity, analogous to those observed in murines. The genome of a close relative, Schmidtea mediterranea, courtesy of the University of Utah S. med genome database, was explored for cannabinoid receptors, none were found. A putative fatty acid amide hydrolase (FAAH) homolog was found in Schmidtea mediterranea.
Date: December 2010
Creator: Mustonen, Katie Lynn

Induced Bradycardia Effects on Angiogenesis, Growth and Development in Early Development in Chicken Embryos, Gallus Domesticus

Description: Cardiac performance, angiogenesis and growth was investigated during early chicken development. Heart rate, and thus arterial pulse pressure and cardiac output, were altered with the bradycardic drug ZD7288. Heart rates at 72 h of development of control embryos and those dosed with chicken Ringer were not different at 171 bpm. Acute and chronic application of ZD7288 caused significant bradycardia. Chronic dosing of Ringer and ZD7288 changed neither eye diameter nor development rate. Chronic dosing of ZD7288 did not significantly alter CAM vessel density close to the embryo (2, 3 and 4 mm) but at farther distances (5 and 6 mm) chronic dosing with both Ringer and ZD7288 decreased vessel density by 13 - 16%. Chronic dosing with ZD7288 also reduced body mass by 20%. Thus, lowered heart rate and cardiac output had little effect on vessel density or developmental stage, but did reduce embryo growth.
Date: December 2010
Creator: Ruck, Sylvia A.

Photoactivatable Quantum Dots in Super-Resolution Microscopy of Muscle

Description: Super-resolution 3D imaging was achieved using newly synthesized photoactivatable quantum dot (PAQ dot) probes. Quantum dots were modified with a novel quencher system to make them photoactivatable. The unique properties of these PAQ dots enable single-fluorophore localization in three dimensions using a confocal microscopy optical sectioning method. Myosin and tropomyosin of rabbit myofibrilar bundles were specifically labeled with the newly synthesized PAQ dot. A sufficient number of single quantum dots were photoactivated, localized and reduced to their centroid and then reconstructed to a super-resolution image. The acquired super-resolution image shows a lateral and an axial sub-diffraction resolution and demonstrates ultrafine striations with widths less than 70 nm that are not evident by conventional confocal microscopy. The striations appear to be related to nebulin thin filament binding protein. This newly developed imaging system is cutting edge for its high resolution and localization as well its simplicity and convenience.
Date: December 2010
Creator: Akel, Amal

Evaluation of the Developmental Effects and Bioaccumulation Potential of Triclosan and Triclocarban Using the South African Clawed Frog, Xenopus Laevis

Description: Triclosan (TCS) and triclocarban (TCC) are antimicrobials found in U.S. surface waters. This dissertation assessed the effects of TCS and TCC on early development and investigated their potential to bioaccumulate using Xenopus laevis as a model. The effects of TCS on metamorphosis were also investigated. For 0-week tadpoles, LC50 values for TCS and TCC were 0.87 mg/L and 4.22 mg/L, respectively, and both compounds caused a significant stunting of growth. For 4-week tadpoles, the LC50 values for TCS and TCC were 0.22 mg/L and 0.066 mg/L; and for 8-week tadpoles, the LC50 values were 0.46 mg/L and 0.13 mg/L. Both compounds accumulated in Xenopus. For TCS, wet weight bioaccumulation factors (BAFs) for 0-, 4- and 8-week old tadpoles were 23.6x, 1350x and 143x, respectively. Lipid weight BAFs were 83.5x, 19792x and 8548x. For TCC, wet weight BAFs for 0-, 4- and 8-week old tadpoles were 23.4x, 1156x and 1310x. Lipid weight BAFs were 101x, 8639x and 20942x. For the time-to-metamorphosis study, TCS showed an increase in weight and snout-vent length in all treatments. Exposed tadpoles metamorphosed approximately 10 days sooner than control tadpoles. For the hind limb study, although there was no difference in weight, snout-vent length, or hind limb length, the highest treatment was more developed compared to the control. There were no differences in tail resorption rates between the treatments and controls. At relevant concentrations, neither TCS nor TCC were lethal to Xenopus prior to metamorphosis. Exposure to relatively high doses of both compounds resulted in stunted growth, which would most likely not be evident at lower concentrations. TCS and TCC accumulated in Xenopus, indicating that the compound has the potential to bioaccumulate through trophic levels. Although TCS may increase the rate of metamorphosis in terms of developmental stage, it did not disrupt thyroid function and metamorphosis in ...
Date: December 2010
Creator: King, Marie Kumsher

Multiple Activities of Aspartate Transcarbamoylase in Burkholderia cepacia: Requirement for an Active Dihydroorotase for Assembly into the Dodecameric Holoenzyme

Description: The aspartate transcarbamoylase (ATCase) was purified from Burkholderia cepacia 25416. In the course of purification, three different ATCase activities appeared namely dodecameric 550 kDa holoenzyme, and two trimeric ATCases of 140 kDa (consists of 47 kDa PyrB subunits) and 120 kDa (consists of 40 kDa PyrB subunits) each. The 120 kDa PyrB polypeptide arose by specific cleavage of the PyrB polypeptide between Ser74 and Val75 creating an active polypeptide short by 74 amino acids. Both the 40 and 47 kDa polypeptides produced active trimers. To compare the enzyme activity of these trimers, an effector assay using nucleotides was performed. The 140 kDa trimer showed inhibition while the 120 kDa polypeptide showed less inhibition. To verify the composition of the pyrBC holoenzyme complex, B. cepacia dihydroorotase (DHOase, subunit size of 45 kDa) was purified by the pMAL protein fusion and purification system and holoenzyme reconstruction was performed using purified ATCase and DHOase. Both the 140 kDa and the 120 kDa trimers could produce holoenzymes of 550 kDa and 510 kDa, respectively. The reconstructed ATCase holoenzyme from cleaved ATCase showed better reconstruction compared to that from uncleaved ATCase in the conventional ATCase activity gel assay. To characterize the relationship between pyrimidine pathway and virulence factor production, motility tests and biofilm assays were conducted using pyrC- mutant. Even though no significant difference in growth rates was observed, there were significant differences between the wild type and mutant in the production of biofilm and virulence factors. This study will help us to understand the structure and regulation of ATCase holoenzyme with DHOase, and facilitate the use of B. cepacia as an applicable bio-tool. Additionally, we can potentially pursue more efficient drug targets for B. cepacia.
Date: December 2010
Creator: Kim, Hyunju

Functional Characterization of Plant Fatty Acid Amide Hydrolases

Description: Fatty acid amide hydrolase (FAAH) terminates the endocannabinoid signaling pathway that regulates numerous neurobehavioral processes in animals by hydrolyzing a class of lipid mediators, N-acylethanolamines (NAEs). Recent identification of an Arabidopsis FAAH homologue (AtFAAH) and several studies, especially those using AtFAAH overexpressing and knock-out lines suggest that a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, I provide evidence to support this concept by identifying candidate FAAH cDNA sequences in diverse plant species. NAE amidohydrolase assays confirmed that several of the proteins encoded by these cDNAs indeed catalyzed the hydrolysis of NAEs in vitro. Kinetic parameters, inhibition properties, and substrate specificities of the plant FAAH enzymes were very similar to those of mammalian FAAH. Five amino acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the plant FAAH sequences. Site-directed mutation of each of the five putative catalytic residues in AtFAAH abolished its hydrolytic activity when expressed in Escherichia coli. Contrary to overexpression of native AtFAAH in Arabidopsis that results in enhanced seedling growth, and in seedlings that were insensitive to exogenous NAE, overexpression of the inactive AtFAAH mutants showed no growth enhancement and no NAE tolerance. However, both active and inactive AtFAAH overexpressors displayed hypersensitivity to ABA, suggesting a function of the enzyme independent of its catalytic activity toward NAE substrates. Yeast two-hybrid screening identified Arg/Ser-rich zinc knuckle-containing protein as a candidate protein that physically and domain-specifically interacts with AtFAAH and its T-DNA knock-out Arabidopsis was hypersensitive to ABA to a degree similar to AtFAAH overexpressors. Taken together, AtFAAH appears to have a bifurcating function, via NAE hydrolysis and protein-protein interaction, to control Arabidopsis growth and interaction with phytohormone signaling pathways. These studies help to functionally define the group of enzymes that metabolize NAEs in plants, and further will ...
Date: December 2010
Creator: Kim, Sang-Chul

Purification of Cyanide-Degrading Nitrilase from Pseudomonas Fluorescens NCIMB 11764.

Description: Cyanide is a well known toxicant that arises in the environment from both biological and industrial sources. Bacteria have evolved novel coping mechanisms for cyanide and function as principal agents in the biosphere for cyanide recycling. Some bacteria exhibit the unusual ability of growing on cyanide as the sole nitrogen source. One such organism is Pseudomonas fluorescens NCIMB 11764 (Pf11764) which employs a novel oxidative mechanism for detoxifying and assimilating cyanide. A unique complex of enzymes referred to as cyanide oxygenase (CNO) is responsible for this ability converting cyanide to ammonia which is then assimilated. Because one component of the four member CNO complex was previously shown to act on cyanide independent of the other members, its characterization was sought as a means of gaining a better understanding of the overall catalytic mechanism of the complex. Preliminary studies suggested that the enzyme belonged to a subset of nitrilase enzymes known as cyanide dihydratases (CynD), however, a cynD-like gene in Pf11764 could not be detected by PCR. Instead, a separate nitrilase (Nit) linked to cyanide metabolism was detected. The corresponding nit gene was shown to be one of a conserved set of nit genes traced to a unique cluster in bacteria known as Nit1C. To determine whether the previously described CynD enzyme was instead Nit, efforts were undertaken to isolate the enzyme. This was pursued by cloning and expressing the recombinant enzyme and by attempting to isolate the native enzyme. This thesis is concerned with the latter activity and describes the purification of a Nit-like cyanide-degrading nitrilase (NitCC) from Pf11764 to ~95% homogeneity. Purification was greatly facilitated by the discovery that fumaronitrile, as opposed to cyanide, was the preferred substrate for the enzyme (20 versus 1 U/mg protein, respectively). While cyanide was less effective as a substrate, the specificity for cyanide ...
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Date: December 2010
Creator: Chou, Chia-Ni

Habitat Fragmentation by Land-Use Change: One-Horned Rhinoceros in Nepal and Red-Cockaded Woodpecker in Texas

Description: This research focuses on the spatial analysis of the habitat of two vulnerable species, the one-horn rhinoceros in the grasslands of southern Nepal, and the red-cockaded woodpecker in the Piney woods of southeast Texas, in the USA. A study sites relevant for biodiversity conservation was selected in each country: Chitwan National Park in Nepal, and areas near the Big Thicket National Preserve in Texas. Land-use differs in the two study areas: the first is still undergoing agrarian development while the second is in a technological phase and undergoing urbanization processes. Satellite remote sensing images were used to derive land-cover maps by supervised classification. These maps were then processed by Geographic Information Systems methods to apply habitat models based on basic resources (food and cover) and obtain habitat suitability maps. Several landscape metrics were computed to quantify the habitat characteristics especially the composition and configuration of suitable habitat patches. Sensitivity analyses were performed as the nominal values of some of the model parameters were arbitrary. Development potential probability models were used to hypothesize changes in land-use of the second study site. Various scenarios were employed to examine the impact of development on the habitat of red-cockaded woodpecker. The method derived in this study would prove beneficial to guide management and conservation of wildlife habitats.
Date: December 2010
Creator: Thapa, Vivek