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- Development of cardiovascular regulation in embryos of the domestic fowl (Gallus gallus), with a partial comparison to embryos of the desert tortoise (Gopherus agassizii)
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In adult vertebrates, cardiovascular regulation is accomplished by numerous systems with neural, hormonal and local components responsible for the majority of regulation. These regulatory components work in concert to maintain the essential function of blood perfusion to adult tissues. Given the essential nature of this function it is therefore surprising that the development of cardiovascular regulation during gestation is poorly understood. The majority of what is known is based on a single vertebrate model, the fetal lamb. The fetal lamb has been used in multiple studies due to the clear clinical applications and has been pivotal in understanding the onset of regulation in developing vertebrates. However, study on the fetal lamb is limited to the latter 40% of gestation and has the added complication of an in-utero developmental strategy. Therefore the primary focus of this dissertation was to characterize basic cardiovascular regulation in the chicken embryo to provided the needed information for it's use an alternative to the fetal lamb. Developing chicken embryos rely on both alpha and beta adrenergic tones to maintain normal heart rate and arterial blood pressure during incubation. However, on day 21, just prior to hatch, these animals lose both tones on arterial pressure suggesting the onset of adult regulation. Cholinergic tone, however, was absent throughout chicken development indicating that it must mature during the neonatal life. Adult cardiovascular reflexes become apparent late in chicken development with a clear baroreflex specifically operating initially on day. However, an adult response to changes in ambient gas tension was absent during incubation suggesting embryos possess unique regulatory systems that are absent in adult chickens. This mechanism is comprised entirely of adrenergic systems with no cholinergic action during change in ambient gas tension. Similar developmental patterns were determined in embryos of the desert tortoise suggesting fundamental differences between in-utero and ex-utero developing vertebrates. digital.library.unt.edu/ark:/67531/metadc2223/ - Structural analysis of the TOL pDK1 xylGFJQK region and partial characterization of the xylF and xylG gene products
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TOL plasmids encode enzymes responsible for utilization of toluene and related aromatic compounds by Pseudomonas putida, ultimately converting them to central metabolic intermediates. The nucleotide sequence for the 5.6 kb xylGFJQK region of the pDK1 TOL meta operon was determined. DNA sequence analysis revealed the presence of five open reading frames corresponding to xylG (1458 bp), xylF (846 bp), xylJ (783 bp), xylQ (936 bp) and xylK (1047 bp), encoding predicted protein products of 51.6, 31.3, 27.8, 32.8, and 36.6 kDa in size, respectively. The average G+C content of the xylLTEGFJQK region was 65.7%, somewhat higher than the 58.9% seen in the immediately upstream xylXYZ region and substantially more than the 50% G+C content reported for the upper TOL operon of this plasmid. Homology comparisons were made with genes and proteins of related catabolic plasmids. The dmpCDEFG and pWWO xylGFJQK regions exhibit consistently high levels of nucleotide and amino acid homology to pDK1 xylGFJQK throughout the entire region. In contrast, although the nucleotide sequence homology of the Acinetobacter atdCDE region to xylGFJ is high, the homology of atdFG to xylQK is markedly less. Such radical changes in homology between corresponding regions of different operons, combined with variable base and codon usage patterns within and between operons, provides additional support for the idea that the upper and lower operons encoding enzymes of aromatic pathways have evolved independently of one another and that these operons have continued to exchange genetic material with homologous expression units through a series of recombination events. Recombinant plasmids were constructed for individual expression of each of the xylGFJQK genes. HMSD (XylG) and HMSH (XylF) were partially purified and characterized with respect to substrate specificity and kinetic mechanism. Evidence was obtained suggesting that the HMSD reaction occurs via a steady state ordered mechanism or a random mechanism where binding of the first substrate effects the enzyme's affinity for the second substrate. digital.library.unt.edu/ark:/67531/metadc2270/ - Conformational Studies of Myosin and Actin with Calibrated Resonance Energy Transfer
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Resonance energy transfer was employed to study the conformational changes of actomyosin during ATP hydrolysis. To calibrate the technique, the parameters for resonance energy transfer were defined. With conformational searching algorithms to predict probe orientation, the distances measured by resonance energy transfer are highly consistent with the atomic models, which verified the accuracy and feasibility of resonance energy transfer for structural studies of proteins and oligonucleotides. To study intramyosin distances, resonance energy transfer probes were attached to skeletal myosin's nucleotide site, subfragment-2, and regulatory light chain to examine nucleotide analog-induced structural transitions. The distances between the three positions were measured in the presence of different nucleotide analogs. No distance change was considered to be statistically significant. The measured distance between the regulatory light chain and nucleotide site was consistent with either the atomic model of skeletal myosin subfragment-1 or an average of the three models claimed for different ATP hydrolysis states, which suggested that the neck region was flexible in solution. To examine the participation of actin in the powerstroke process, resonance energy transfer between different sites on actin and myosin was measured in the presence of nucleotide analogs. The efficiencies of energy transfer between myosin catalytic domain and actin were consistent with the actoS1 docking model. However, the neck region was much closer to the actin filament than predicted by static atomic models. The efficiency of energy transfer between Cys 374 and the regulatory light chain was much greater in the presence of ADP-AlF4, ADP-BeFx, and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached crossbridges which appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin. The resonance energy transfer data exclude a number of versions of the swinging lever arm model, and indicate that actin participation is indispensable for conformational changes leading to force generation. The conformational selection during weak binding at the actomyosin interface may precock the myosin head for the ensuing powerstroke. digital.library.unt.edu/ark:/67531/metadc2438/ - Mitochondrial DNA Sequence Variation in Populations of the Nine-Banded Armadillo, Dasypus novemcinctus
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Four populations of nine-banded armadillos, Dasypus novemcinctus, were investigated in the south-central United States in order to assess genetic variation in an isolated population (Electric Island, Lake Hamilton, Garland County, Arkansas); a semi-isolated population (Arkansas Post, Arkansas County, Arkansas), and two free ranging populations (southern Arkansas and central Texas). A 233 basepair sequence of the D-loop region of mitochondrial DNA was sequenced in individuals from each population. Individuals and populations were compared to assess relatedness among populations and individuals. Higher sequence diversity was detected in the semi-isolated population, while lower sequence diversity was observed in the isolated and free ranging populations. Overall, all populations exhibited low genetic variation when compared to genetic variation for other mammals. The results support the hypothesis that rapid range expansion combined with the organism's unique reproductive strategies have promulgated low genetic variation in the North American populations of nine-banded armadillos. digital.library.unt.edu/ark:/67531/metadc2577/ - BioInformatics, Phylogenetics, and Aspartate Transcarbamoylase
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In this research, the necessity of understanding and using bioinformatics is demonstrated using the enzyme aspartate transcarbamoylase (ATCase) as the model enzyme. The first portion of this research focuses on the use of bioinformatics. A partial sequence of the pyrB gene found in Enterococcus faecalis was submitted to GenBank and was analyzed against the contiguous sequence from its own genome project. A BLAST (Basic Local Alignment Search Tool; Atschul, et al., 1990) was performed in order to hypothesize the remaining portion of the gene from the contiguous sequence. This allowed a global comparison to other known aspartate transcarbamoylases (ATCases) and once deduced, a translation of the sequence gave the stop codon and thus the complete sequence of the open reading frame. When this was complete, upstream and downstream primers were designed in order to amplify the gene from genomic DNA. The amplified product was then sequenced and used later in phylogenetic analyses concerning the evolution of ATCase. The second portion of this research involves taking multiple ATCase nucleotide sequences and performing phenetic and phylogenetic analyses of the archaea and eubacter families. From these analyses, ancestral relationships which dictate both structure and function were extrapolated from the data and discussed. digital.library.unt.edu/ark:/67531/metadc2580/ - A Data Fusion Framework for Floodplain Analysis using GIS and Remotely Sensed Data
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Throughout history floods have been part of the human experience. They are recurring phenomena that form a necessary and enduring feature of all river basin and lowland coastal systems. In an average year, they benefit millions of people who depend on them. In the more developed countries, major floods can be the largest cause of economic losses from natural disasters, and are also a major cause of disaster-related deaths in the less developed countries. Flood disaster mitigation research was conducted to determine how remotely sensed data can effectively be used to produce accurate flood plain maps (FPMs), and to identify/quantify the sources of error associated with such data. Differences were analyzed between flood maps produced by an automated remote sensing analysis tailored to the available satellite remote sensing datasets (rFPM), the 100-year flooded areas "predicted" by the Flood Insurance Rate Maps, and FPMs based on DEM and hydrological data (aFPM). Landuse/landcover was also examined to determine its influence on rFPM errors. These errors were identified and the results were integrated in a GIS to minimize landuse / landcover effects. Two substantial flood events were analyzed. These events were selected because of their similar characteristics (i.e., the existence of FIRM or Q3 data; flood data which included flood peaks, rating curves, and flood profiles; and DEM and remote sensing imagery.) Automatic feature extraction was determined to be an important component for successful flood analysis. A process network, in conjunction with domain specific information, was used to map raw remotely sensed data onto a representation that is more compatible with a GIS data model. From a practical point of view, rFPM provides a way to automatically match existing data models to the type of remote sensing data available for each event under investigation. Overall, results showed how remote sensing could contribute to the complex problem of flood management by providing an efficient way to revise the National Flood Insurance Program maps. digital.library.unt.edu/ark:/67531/metadc2557/ - Production and characterization of a novel extracellular polysaccharide produced by Paenibacillus velaei, sp. nov
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Paenibacillus velaei, sp. nov. is a soil bacterium capable of producing an unusually large amount of exopolysaccharide (EPS). The EPS contains glucose, mannose, galactose and fucose in a molar ratio of 4:2:1:1. The molecular weight of the EPS is higher than 2x106. The viscosity of 1% EPS is 1300 cP when measured at a shear rate of 1 sec-1. Physiological parameters for optimal production of the EPS were studied and it was found that 1.4 g dry weight per 1 l of medium was produced when the bacteria were grown at 30EC and the pH adjusted at 7± 0.2 in a medium containing glucose as the carbon source. Growing the bacteria on different carbon sources did not alter the quantity or the composition of the EPS produced. No toxicity effects were observed in mice or rats when EPS was administered in amounts ranging from 20 to 200 mg per kg body weight. The data obtained from physical, chemical and biological properties suggest that the EPS may be employed in several industrial and environmental applications. It is an excellent emulsifier, it holds 100 times its own weight in water, it is not toxic, and it can be used to remove mercury, cadmium and lead from aqueous solutions. digital.library.unt.edu/ark:/67531/metadc2551/ - Development of a procedure to evaluate groundwater quality and potential sources of contamination in the East Texas Basin
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This study contributes a procedure, based on data analysis and geostatistical methods, to evaluate the distribution of chemical ratios and differentiate natural and anthropogenic contaminant sources of groundwater quality in the East Texas Basin. Four aquifers were studied, Sparta, Queen City, Carrizo and Wilcox. In this study, Carrizo- Wilcox is considered as one aquifer, and Sparta-Queen City as another. These aquifers were divided into depth categories, 0-150 feet for Sparta-Queen City and 300-600 feet and 600-900 feet for Carrizo-Wilcox in order to identify individual sources of contamination. Natural sources include aquifer mineral make up, salt domes and lignite beds. Major anthropogenic sources include lignite and salt dome mining and oil-gas production. Chemical ratios selected were Na/Cl, Ca/Cl, Mg/Cl, SO4/Cl, (Na+Cl)/TDS, SO4/Ca and (Ca+Mg)/(Na+K). Ratio distributions and their relationships were examined to evaluate physical-chemical processes occurring in the study area. Potential contaminant sources were used to divide the Basin into three areas: Area 1 to the east, Area 2 in the west and Area 3 in the center. Bivariate analysis was used to uncover differences between the areas. The waters in Area 1 are potentially impacted primarily from oil field waters. Sources present in Area 2 include lignite beds and oil field operations. Area 3 is the cap rock of salt domes that can contain gypsum and anhydrite. Based on the exploratory data analysis (Na+Cl)/TDS, (Ca+Mg)/(Na+K), and SO4/Ca ratios were chosen for geostatistical analysis. Chemical ratios that provided indications of cation exchange, salt domes and oil fields were (Na+Cl)/TDS, (Ca+Mg)/(Na+K) and SO4/Ca. In the Sparta-Queen City 150 zone the procedure did not provide a good method for differentiating between contaminant sources. However, the procedure was effective to indicate impacted ground water in the Carrizo-Wilcox 600 and 900 foot zones. digital.library.unt.edu/ark:/67531/metadc2769/ - Age-Dependent Effects Of Chronic GABAA Receptor Blockade In Barrel Cortex
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GABAA receptor binding is transiently increased in rat whisker barrels during the second postnatal week, at a time when neurons in the developing rat cortex are vulnerable to excitotoxic effects. To test whether these GABAA receptors might serve to protect neurons from excessive excitatory input, polymer implants containing the GABAA receptor antagonist bicuculline were placed over barrel cortex for a 4-day period in young (postnatal days 8 - 12) and adult rats. In the cortex of young, but not adult rats, the chronic blockade of GABAA receptors resulted in substantial tissue loss and neuron loss. The greater loss of neurons in young rats supports the hypothesis that a high density of GABAA receptors protects neurons from excessive excitatory input during a sensitive period in development. digital.library.unt.edu/ark:/67531/metadc2760/ - Riparian Forest Width and the Avian Community in a Greenbelt Corridor Setting
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The forest avian community of the Ray Roberts Greenbelt (Denton Co., Texas) was characterized for two years using point count station sampling, from fall 1998 to summer 2000. Richness data for both breeding seasons were correlated with two-spatial metrics: width of the riparian forest and distance to the nearest edge. There were significant correlations between forest interior species richness and both spatial metrics, for both breeding seasons. Based on these data, a minimum riparian forest width threshold of 400-meters is suggested to provide habitat for forest interior species, which have lost considerable habitat through forest fragmentation. Partners in Flight breeding bird priority concern scores were used to create a habitat priority index for the Trinity River bottomland hardwood forest system digital.library.unt.edu/ark:/67531/metadc2757/ - The developmental physiology of the zebrafish: Influence of environment and cardiovascular attributes
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Temperature effects on the development of the zebrafish embryos and larvae and adults were examined. It was found that the earlier in development a temperature change was performed on an embryo, the more significant the change in survival and/or subsequent development. Thus, viable temperature ranges for zebrafish widened significantly as development proceeded. Adults reared and bred at 25oC produced embryos that were significantly more successful at the lower range of rearing temperatures compared to embryos produced from adults reared at 28oC. The majority of this study focused on the physiological effects of swim training during development in the zebrafish. The earlier in development the zebrafish larvae were trained, the greater the mortality. Trained free swimming larvae had a significantly higher routine oxygen consumption after 11 days of training, and a higher mass specific routine metabolic rate after 8 and 11 days of training. Trained free swimming larvae consumed significantly less oxygen during swimming and were more efficient at locomotion, compared to control larvae. Training enhanced survival during exposure to extreme hypoxia in all age groups. Performance aspects of training were investigated in attempt to quantify training effects and in most cases, trained fish performed significantly better than controls. As blood vessels formed during development, they decreased in cross sectional area from days two to six. It was also shown that the variability in visual stroke volume measurements could be reduced significantly by using a third dimension in the analysis with a more accurate volume equation. Finally, the ontogeny of cardiac control was evaluated. The adrenergic receptors were the first to respond to pharmacological stimulation but were closely followed by cholinergic pharmacological stimulation a few days later. There was a significant cholinergic tone present in day 15 zebrafish larvae which persisted. Although an adrenergic tone was not documented in this study, this does not prove its lack of existence. digital.library.unt.edu/ark:/67531/metadc2854/ - A novel mechanism for site-directed mutagenesis of large catabolic plasmids using natural transformation
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Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total transforming DNA concentration, transforming DNA conformation, and gene dosage on transformation efficiency are presented. Addition of Acinetobacter plasmid DNA sequences to the manipulated constructs did not have an effect on transformation rates. Results suggest that a broadly applicable and efficient method to carry out site-directed genetic manipulations of large plasmids has been identified. The ability to easily reintroduce the recombinant DNA molecules back into the original host organism was maintained. digital.library.unt.edu/ark:/67531/metadc2828/ - 4-Ethoxymethylphenol: a novel phytoestrogen that acts as an agonist for human estrogen receptors.
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Estrogen is the natural agonist of the estrogen receptor (ER). However, certain plant-derived compounds or phytoestrogens have been identified that mimic estrogens and act as agonists and/or antagonists of ERs, depending on subtype and target tissue. Understanding how phytoestrogens interact with ERs, and therefore effect the estrogenic response, may prove beneficial in hormone replacement therapy and in the prevention and treatment of hormone-related diseases. Using Thin Layer Chromatography, gas chromatography/mass spectrometry (GC/MS), and proton nuclear nagnetic resonance (HNMR), I identified 4-ethoxymethylphenol (4EM) found in Maclura pomifera. While most phytoestrogens are heterocyclic compounds, 4EM is a simple phenol that acts as an agonist of ER-alpha and -beta in HeLa and MCF-7 cells. To study the effect of 4EM on ER-alpha and -beta activity, I performed transient transfection assays and showed that 4EM activates ER dependent gene transcription in a dose dependent manner in both ER subtypes. Further, 4EM- mediated transcription in ER-alpha, like estrogen, was enhance in the presense of co-activators, SRC-1 (steroid receptor coactivator-1), CBP (CREB binding proteins), and E6-AP (E6-associated protein) and inhibited by trans-4- hydroxytamoxifen (4HT). I found that 4EM was specific for ER and did not activate transcription of the progesterone receptor in HeLa cells. digital.library.unt.edu/ark:/67531/metadc3003/ - Comparative morphology of sensilla styloconica on the proboscis of North American Nymphalidae and other selected taxa (Lepidoptera): systematic and ecological considerations.
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Sensilla styloconica on the proboscis of 107 species of North American and tropical butterflies were comparatively studied using the scanning electron microscope. Focus was on 76 species of North American Nymphalidae representing 45 genera and 11 subfamilies. Nomenclature for generalized and specific types of nymphalid sensilla is proposed. Written descriptions and micrographs are presented for each species studied. Morphological features were generally consistent for all or most species within genera and sometimes within subfamilies, with specified exceptions. Statistical analysis revealed significant differences for six of eight variables tested between two distinct feeding guilds of North American Nymphalidae. Average number, density, extent of proboscis coverage with sensilla, their total length, and shoulder spine length were all significantly greater in the non-nectar feeding guild than in nectar feeders, and may indicate adaptation for greater efficiency in feeding on flat surfaces. The greater frequency of apical shoulder spines in non-nectar feeders may represent adaptation for protection of sensory pegs from mechanical abrasion during feeding, or for anchoring the flexible proboscis tip to the surface. Correlation analysis revealed 9 out of 28 positive correlations in nectar feeders and 5 out of 28 in non-nectar feeders. Results of preliminary cladistic analysis were not considered to be meaningfully robust due to few available characters. The stylar characters identified in this study should be more useful in future analyses when included with characters from other lines of evidence. The presence of sensilla styloconica in all subfamilies of Nymphalidae, except Danainae, largely supports Ehrlich's (1958) higher classification concept for the family. The presence of less conspicuous sensilla in the Danainae, and other characteristics are presented as further evidence that they should be reconsidered for full family status. Sensilla styloconica in nymphalid butterflies appear to function as extensions that provide greater sensory reach during feeding. The role of these sensilla in liquid uptake, pollen feeding, and host plant selection is discussed. digital.library.unt.edu/ark:/67531/metadc3002/ - Characterization of cDNA and genomic clones for a palmitoyl-acyl carrier protein thioesterase (FatB1) and an osmotin-like PR5 protein in Gossypium hirsutum.
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Putative cotton cDNA clones and cognate genomic clones for a palmitoyl-acyl carrier protein (ACP) thioesterase (PATE) and an osmotin-like pathogenesis-related 5 (PR5) protein have been isolated and characterized. PATE is a class B fatty acid thioesterase with specificity for saturated long-chain fatty acids such as palmitate, and is implicated as a key enzyme to be targeted for regulation of fatty acid synthesis in order to alter cotton seed oil profiles. A nearly full-length 1.7-kb cDNA clone was isolated using a hybridization probe derived from an Arabidopsis PATE cDNA clone designated TE 3-2. A 17-kb genomic segment encompassing the PATE gene was also isolated, which has six exons and five introns with high sequence identity with other FatB cDNA/gene sequences. The deduced PATE preprotein amino acid sequence of 413 residues has putative signal sequences for targeting to the chloroplast stroma. PR5 proteins called osmotins are made in response to fungal pathogen stress or osmotic stress (water deprivation or salt exposure). Osmotins may actually form pores in fungal membranes, leading to osmotic rupture and destruction of the fungal cells. A cotton osmotin-like PR5 cDNA insert of 1,052 base-pairs was isolated and shown to encode a preprotein of 242 amino acids and is predicted to be secreted to the extracellular matrix as a neutral isoform. The deduced amino acid sequence has 16 cysteine residues that are highly conserved in osmotin-like proteins and are important in stabilizing the three-dimensional structure seen in thaumatin, zeamatin, and PR5-d. The intronless cognate cotton genomic clone has two putative ethylene response elements (GCC boxes) found in other PR5 gene promoter regions, as well as several tentative promoter/enhancer elements possibly involved in spatial/temporal gene expression. digital.library.unt.edu/ark:/67531/metadc3072/ - Comparative biochemistry and genetic analysis of nucleoside hydrolase in Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas fluorescens.
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The pyrimidine salvage enzyme, nucleoside hydrolase, is catalyzes the irreversible hydrolysis of nucleosides into the free nucleic acid base and D-ribose. Nucleoside hydrolases have varying degrees of specificity towards purine and pyrimidine nucleosides. In E. coli, three genes were found that encode homologues of several known nucleoside hydrolases in protozoa. All three genes (designated yaaF, yeiK, and ybeK) were amplified by PCR and cloned. Two of the gene products (yeiK and ybeK) encode pyrimidine-specific nucleoside hydrolases, while the third (yaaF) encodes a nonspecific nucleoside hydrolase. All three were expressed at low levels and had different modes of regulation. As a comparative analysis, the homologous genes of Pseudomonas aeruginosa and P. fluorescens (designated nuh) were cloned. Both were determined to encode nonspecific nucleoside hydrolases. The nucleoside hydrolases of the pseudomonads exhibited markedly different modes of regulation. Both have unique promoter structures and genetic organization. Furthermore, both pseudomonad nucleoside hydrolase were found to contain an N-terminal extension of 30-35 amino acids that is shown to act as a periplasmic-signaling sequence. These are the first two nucleoside hydrolases, to date,that have been conclusively demonstrated to be exported to the periplasmic space. The physiological relevance of this is explained. digital.library.unt.edu/ark:/67531/metadc3290/ - Development, validation, and evaluation of a continuous, real-time, bivalve biomonitoring system.
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A biological monitoring tool to assess water quality using bivalve gape behavior was developed and demonstrated. The purpose of this work was to develop methodologies for screening water quality appropriate to the goals of the watershed paradigm. A model of bivalve gape behavior based on prediction of behavior using autoregressive techniques was the foundation of the bivalve biomonitoring system. Current technology was used in developing the system to provide bivalve gape state data in a continuous real-time manner. A laboratory version of the system, including data collection and analysis hardware and software, was developed for use as a toxicological assay for determination of effective concentrations of toxicant(s) or other types of stress on bivalve gape behavior. Corbicula fluminea was monitored and challenged with copper, zinc, and chlorpyrifos using the system. Effective concentrations of 176±23µg/L copper, 768±412µg/L zinc, and 68µg/L chlorpyrifos were observed using a natural water with high dissolved organic carbon concentrations. A rugged field version of the bivalve biomonitoring system was developed and deployed in two locations. The field systems were fitted with a photovoltaic array, a single board computer, and a CDPD telemetry modem for robust remote operation. Data were telemetered at a time relevant rate of once every ten minutes. One unit was deployed in Lake Lewisville, Denton County, TX in February 2000. Data were telemetered and archived at a 92% success rate. Bivalve gape data demonstrated significant behavioral deviations on average 5 times per month. A second unit was deployed in Pecan Creek, Denton, TX in June 2001. Data from this site were telemetered and archived at a 96% success rate. Over the months of June-August 2001, 16 significant behavioral deviations were observed, 63% of which were correlated with changes in physical/chemical parameters. This work demonstrated the relative sensitivity of bivalve gape as a toxicological endpoint and the feasibility of its use in a continuous, real-time, bivalve biomonitoring system. Technical aspects of collecting, telemetering, and analyzing this type of data in a time-relevant manner were developed resulting in a system appropriate for use as a means of data collection within the watershed paradigm. digital.library.unt.edu/ark:/67531/metadc3295/ - Removal of selected water disinfection byproducts, and MTBE in batch and continuous flow systems using alternative sorbents.
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A study was conducted to evaluate the sorption characteristics of six disinfection byproducts (DBPs) on four sorbents. To investigate sorption of volatile organic compounds (VOCs), specially designed experimental batch and continuous flow modules were developed. The investigated compounds included: chloroform, 1,2-dichloroethane (DCE), trichloroethylene (TCE), bromodichloromethane (BDCM), methyl tertiary butyl ether (MTBE), bromate and bromide ions. Sorbents used included light weight aggregate (LWA), an inorganic porous material with unique surface characteristics, Amberlite® XAD-16, a weakly basic anion exchange resin, Amberjet®, a strongly basic anion exchange resin, and granular activated carbon (GAC). Batch experiments were conducted on spiked Milli-Q® and lake water matrices. Results indicate considerable sorption of TCE (68.9%), slight sorption of bromate ions (19%) and no appreciable sorption for the other test compounds on LWA. The sorption of TCE increased to 75.3% in experiments utilizing smaller LWA particle size. LWA could be a viable medium for removal of TCE from contaminated surface or groundwater sites. Amberlite® was found unsuitable for use due to its physical characteristics, and its inability to efficiently remove any of the test compounds. Amberjet® showed an excellent ability to remove the inorganic anions (>99%), and BDCM (96.9%) from aqueous solutions but with considerable elevation of pH. Continuous flow experiments evaluated GAC and Amberjet® with spiked Milli-Q® and tap water matrices. The tested organic compounds were sorbed in the order of their hydrophobicity. Slight elevation of pH was observed during continuous flow experiments, making Amberjet® a viable option for removal of BDCM, bromate and bromide ions from water. The continuous flow experiments showed that GAC is an excellent medium for removal of the tested VOCs and bromate ion. Each of the test compounds showed different breakthrough and saturation points. The unique design of the continuous flow apparatus used in the study proved to be highly beneficial to assess removal of volatile organic compounds from aqueous solutions. digital.library.unt.edu/ark:/67531/metadc3285/ - Modeling of land use change effects on storm water quantity and quality in the City of Carrollton and the North Texas Area.
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Development and population are rapidly increasing in urbanizing areas of North Texas and so is the need to understand changes in storm water runoff flow and its contamination by nutrients, sediment, pesticides and other toxicants. This study contributes to this understanding and has two primary components: first, development of a graphical user interface for a geographic information system and storm water management database, and second, performing a two-scale hydrological modeling approach (the US Corp of Engineers HEC-HMS model and the US Environmental Protection Agency SWMM model). Both primary components are used together as a toolkit to support the storm water management program of the City of Carrollton, located in North Texas. By focusing limited city resources, the toolkit helps storm water managers in the process of compliance with federal regulations, especially the National Pollution Discharge Elimination System permit, and provides guidance for reporting, planning and investigation. A planning example was conducted by modeling potential changes in storm water quality due to projections of land use based on the City of Carrollton's Comprehensive Plan. An additional component of this study is the evaluation of future changes in surface water quantity and quality in the North Central Texas area, specifically in a rural but rapidly urbanizing subbasin area of the greater Lake Lewisville watershed. This was accomplished using the US Corp of Engineers HEC-HMS hydrological model. Precipitation scenarios were derived from years of historically high, medium, and low annual precipitation. Development scenarios were derived from current land use in the Lake Lewisville sub basin, current land use in the city of Carrollton, and from Markov projections based on recent land use change calculated from satellite images of 1988 and 1999. This information is useful for future land use planning and management of water resources in North Texas. digital.library.unt.edu/ark:/67531/metadc4368/ - Structure-Function Studies on Aspartate Transcarbamoylase and Regulation of Pyrimidine Biosynthesis by a Positive Activator Protein, PyrR in Pseudomonas putida
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The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was transferred to P. putida from E. coli and shown to function in this heterologous host. Chapter II details the enzymology of Pseudomonas aspartate transcarbamoylase (ATCase), its allosteric regulation and how it is assembled. The E. coli ATCase is a dodecamer of two different polypeptides, encoded by pyrBI. Six regulatory (PyrI) and six catalytic (PyrB) polypeptides assemble from two preformed trimers (B3) and three preformed regulatory dimers (I2) in the conserved 2B3:3I2 molecular structure. The Pseudomonas ATCase also assembles from two different polypeptides encoded by pyrBC'. However, a PyrB polypeptide combines with a PyrC. polypeptide to form a PyrB:PyrC. protomer; six of these assemble into a dodecamer of structure 2B3:3C'2. pyrC' encodes an inactive dihydroorotase with pyrB and pyrC' overlapping by 4 bp. Chapter III explores how catabolite repression affects pyrimidine metabolism. The global catabolite repression control protein, Crc, has been shown to affect pyrimidine metabolism in a number of ways. This includes orotate transport for use as pyrimidine, carbon and nitrogen sources. Orotate is important because it interacts with PyrR in repressing the pyr genes. Chapter IV describes PyrR, the positive activator of the pyrimidine pathway. As with other positive activator proteins, when pyrimidine nucleotides are depleted, PyrR binds to DNA thereby enhancing expression of pyrD, pyrE and pyrF genes. When pyrimidine nucleotides are in excess, the PyrR apoprotein binds to orotate, its co-repressor, to shut down all the pyrimidine genes. Like many positive activators, PyrR is subject to autoregulation and has catalytic activity for uracil phosphoribosyltransferase inducible by orotate. digital.library.unt.edu/ark:/67531/metadc4362/ - Analysis of a cotton gene cluster for the antifungal protein osmotin.
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Three overlapping genomic clones covering 29.0 kilobases of cotton DNA were found to encompass a cluster of two presumptive osmotin genes (OSMI and OSMII) and two osmotin pseudogenes (OSMIII and OSMIV). A segment of 16,007 basepairs of genomic DNA was sequenced from the overlapping genomic clones (GenBank Accessions AY303690 and AF304007). The two cotton osmotin genes were found to have open reading frames of 729 basepairs without any introns, and would encode presumptive osmotin preproteins of 242 amino acids. The open reading frames of the genes are identical in sequence to two corresponding cDNA clones (GenBank Accessions AF192271 and AY301283). The two cDNA inserts are almost full-length, since one lacks codons for the four N-terminal amino acids, and the other cDNA insert lacks the coding region for the 34 N-terminal amino acids. The cotton osmotin preproteins can be identified as PR5 proteins from their similarities to the deduced amino acid sequences of other plant osmotin PR5 preproteins. The preproteins would have N-terminal signal sequences of 24 amino acids, and the mature 24 kilodalton isoforms would likely be targeted for extracellular secretion. Prospective promoter elements, including two ethylene response elements, implicated as being positive regulatory elements in the expression of a number of PR-proteins, occur in the 5'-flanking regions. The mature osmotin proteins accumulate in cotton plants treated with the inducers ethephon and hydrogen peroxide. Thus, the two cotton osmotin genes encode osmotin proteins. The coding regions of the two genes have been expressed and isolated as fusion polypeptides in a bacterial expression system. Binary constructs containing the open reading frames of the two osmotin genes under the control of the 35S CaMV promoter have been generated for eventual production of transgenic Arabidopsis and cotton plants for potential constitutive expression of the osmotin proteins for increased resistance against fungal pathogens. digital.library.unt.edu/ark:/67531/metadc4350/ - Biogeography of Montane Mammals on the Colorado Plateau and Adjacent Regions
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This study identifies the biogeographic factors that structure small mammal communities on mountains of the Colorado Plateau and adjacent regions. Forty six isolated ranges were characterized across a 5-state study area encompassing the Colorado Plateau, including the central high plateaus of Utah and the Basin and Range Province (i.e. the Great Basin and mountains of Arizona and New Mexico). Presence/absence data of 25 montane mammal species were used to explore the interactions between historical and ecological processes affecting local and regional diversity patterns. Multivariate analyses, such as non-metric dimensional scaling, were used to explore factors which influence community composition. Results of these analyses revealed the Colorado River as a significant biogeographic barrier that affects montane mammal community structure. MtDNA cytochrome b sequence variation was analyzed among populations of the long-tailed vole, Microtus longicaudus, sampled from five interior ranges of the Colorado Plateau- Abajo, LaSal, Henry, and Chuska Mts., and Boulder Mountain of the Aquarius Plateau-and analyzed using traditional phylogenetic approaches (parsimony and likelihood) as well as nested clade analysis. Results support previous documentation of a major east-west phylogeographic break occurring between populations southeast of the Colorado River (eastern Arizona, Colorado, Wyoming and New Mexico) and all other western populations, which include a central clade, a northwest clade, and an Alaskan island clade. Evidence also supports differentiation of a 'southern Rockies' clade and a distinct 'southwest island' clade. Populations of M. longicaudus north and west of the Colorado River (Boulder and Henry Mts.) share two haplotypes, form a well-supported subclade with populations from the Kaibab plateau, and are closely related to the Northwest clade. Past approaches to studying montane mammal communities utilizing theory based on island biogeography have overemphasized area and isolation as the only forces structuring insular communities. As a result, there has been a lack of recognition of the influences of environmental factors, species turnover, and barriers that create and maintain regional diversity on the Colorado Plateau and adjacent areas. digital.library.unt.edu/ark:/67531/metadc4467/ - Callus Development and Organogenesis in Cultured Explants of Cowpea (Vigna unguiculata (L.) Walp
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Cowpea, Vigna unguiculata (L.) Walp is an excellent source of protein, vitamins and minerals and a major food crop many parts of Africa. Optimal production levels are hampered by insect pests and diseases. Biotechnological techniques such as tissue culture and genetic engineering can aid in the development of varieties with resistance to insect pests and diseases. The objective of this study was to investigate conditions necessary for the development of a reproducible tissue culture system that can be applied to regenerate transformed cells from culture. The in vitro manipulation of cowpea using Murashige and Skoog (MS) medium, auxins and cytokinins resulted in the formation of callus and rhizogenesis. Calli that were formed were separated into six classes based on color and texture. Yellowish friable callus, yellowish compact, soft yellowish callus and green and white were composed of largely vacuolated cells and were non-regenerative. Friable green callus was the most prevalent callus type and could form of roots in some hormone combinations. Green spots were formed on hard compact green callus. The green spots became nodular, forming root primordia and ultimately giving rise to roots. None of the six calli types gave rise to the formation of shoots. Embryogenic callus was induced from cowpea explants cultured on MS medium supplemented with dicamba and picloram. Embryogenic suspension cultures were initiated from callus induced on MS supplemented with 3.0 mg/L dicamba or picloram and conditions for maintenance of embryogenic suspension cultures were evaluated. Somatic embryos were formed in suspension cultures. Attempts to convert and germinate the somatic embryos resulted in the formation of callus or formation of appendages on the somatic embryos or in the death of the embryos. The appendages formed roots on prolonged culture. Further research is needed to determine appropriate optimal conditions for embryo conversion and germination and ultimately plant recovery from culture. digital.library.unt.edu/ark:/67531/metadc4655/ - Pyrimidine Enzyme Specific Activity at Four Different Phases of Growth in Minimal and Rich Media, and Concomitant Virulence Factors Evaluation in Pseudomonas aeruginosa
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Pseudomonas aeruginosa is a Gram-negative rod, aerobic, non-fermenting, oxidase positive, pigment producing, and nutritionally versatile bacterium. Infections by P. aeruginosa are the most important cause of morbidity and mortality in immunocompromised patients, given virulence factor production that suppresses antibiotic therapy and promotes persistent infection. This research is the first comprehensive report of the pyrimidine biosynthetic pathway for all phases of growth in minimal and rich media coupled with the evaluation of virulence factor production of P. aeruginosa in comparison to four other bacterial species (Pseudomonas putida, Pseudomonas fluorescens, Burkholderia cepacia, and Escherichia coli wild-type strains). Cellular growth and passing genetic information to the next generation depend on the synthesis of purines and pyrimidines, the precursors of DNA and RNA. The pyrimidine biosynthetic pathway is essential and found in most organisms, with the exception of a few parasites that depend upon the pyrimidine salvage pathway for growth. Both the pyrimidine biosynthetic and salvage enzymes are targets for chemotherapeutic agents. In our laboratory, research on pyrimidine auxotrophic mutants showed the role of the pyrimidine biosynthetic pathway and its intermediates on P. aeruginosa metabolism and impaired virulence factors production. The present research shows that pyrimidine enzymes are active in all phases of growth, including the production of two forms of ATCase in the late log phase in P. aeruginosa. This finding may be explained by the displacement of the inactive PyrC' by the active PyrC or PyrC2 to form a new and larger pyrBC encoded ATCase. Pseudomonas aeruginosa wild-type appears to produce by far the most virulence factors, haemolysin, iron chelation, rhamnolipid, adherence, and three types of motility (swimming, swarming, and twitching) investigated in this study, when compared to the other four wild-type strains. Growth analysis was carried out as typically done in minimal medium but also in rich medium to simulate conditions in the blood and lung tissues of humans as P. aeruginosa infections develop. digital.library.unt.edu/ark:/67531/metadc4918/ - Isolation and Characterization of Polymorphic Loci from the Caribbean Flamingo (Phoenicopterus ruber ruber): New Tools for Wildlife Management
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Methods to determine genetic diversity and relatedness within populations are essential tools for proper wildlife management. Today the approach of choice is polymerase chain reaction-based microsatellite analysis. Seven new polymorphic loci were isolated from a microsatellite-enriched Caribbean flamingo genomic library and used to characterize survey populations of Caribbean and African greater flamingos. In addition, four of these loci were used to verify parentage relationships within a captive-breeding population of African greater flamingos. Parentage predictions based upon gamekeeper observations of breeding and nesting did not always agree with genetic-based parentage analyses of the nine suggested family groups. Four family groups were supported (groups I, II, III and VI) by there results. However, an analysis of the remaining five suggested groups, with a total of eight offspring/dam and eight offspring/sire suggested relationships, yielded seven exclusions of the suggested dam and six exclusions of the suggested sire. This put the overall suggested dam exclusion rate at 35% and exclusion rate for suggested sires at 29%. Although the keeper observation data for our family groups must be considered a variable of concern at this time, these findings are certainly suggestive that more carefully controlled studies may reveal that flamingos are not monogamous as long accepted, but rather socially monogamous or even promiscuous. Thus we have now been able to both characterize and demonstrate the utility of our polymorphic microsatellite loci. We hope these results will interest additional wildlife facilities in further parentage and behavioral studies that will collectively aid to improve monitoring and maintenance of genetic diversity, and as provide better insight into breeding habits of both wild and captive populations. digital.library.unt.edu/ark:/67531/metadc4908/ - Identification and characterization of an incomplete root hair elongation (IRE)-like gene in Medicago truncatula (L.) root nodules.
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Cloning and molecular characterization of new genes constitutes a useful approach in studying the symbiotic interactions between the model plant Medicago truncatula and Synorhizobium meliloti. Large numbers of expressed sequence tags (ESTs) available for Medicago truncatula, along with numerous cDNA, oligonucleotides, and Affimetrix DNA microarray chips, represent useful tools for gene discovery. In an attempt to identify a new gene that might be involved in the process of nodulation in Medicago truncatula, preliminary data reported by Fedorova et al. (2002), who identified 340 putative gene products or tentative consensus sequences (TCs) expressed only in nodules, was used. This research was focused on TC33166 (TC103185), which has 3 ESTs in the TC, and whose strongest BLASTX hit of TC103185 is the incomplete root hair elongation (IRE) protein kinase-like protein (NP_192429) from Arabidopsis thaliana. The Arabidopsis IRE gene is required for normal root hair growth, and a role in apical growth was suggested (Oyama et al., 2002). Infection thread growth can be looked at as an inward growth of the root hair. Thus, TC103185 was a good candidate for identifying a gene that may be involved in early events of nodulation. MtIRE (GenBank accession AC122727) is organized in 17 exons and 16 introns, similarly to the Arabidopsis IRE gene. MtIRE is a new member of the IRE family and it is a putative Ser/Thr protein kinase. MtIRE is a nodule- and flower-specific gene, suggesting that nodulation may have recruited it from other developmental processes. MtIRE is likely to be involved in the invasion process, or in the maturation of the symbiosome, or of the cells that contain rhizobia, rather than infection thread initiation and elongation or in nitrogen fixation. Nodule invasion precedes the onset of MtIRE expression and the expression pattern changes in time within the nodule. RNA interference results support MtIRE expression data and suggest a possible role in preventing extensive defense responses. Our study demonstrates the existence of an Arabidopsis IRE homolog in Medicago truncatula root nodules with an entirely new function and regulation. digital.library.unt.edu/ark:/67531/metadc5215/ - Evaluation of City of Denton Sub-Watershed by Benthic Macroinvertebrate Field Experimental Approach
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In this study, two different field experiments were designed to assess the relative influence of urbanization on benthic communities. During spring and summer, four urban and one reference sites from Denton County, Texas were selected for benthic macroinvertebrate evaluation. Statistically significant differences in colonized benthic macroinvertebrate taxa on artificial substrates were observed among the four urban sites and the reference site. Oligochaetes and chironomids were the dominant taxa at all sites. Identification of chironomid larvae at the subfamily and genus level to detect differences between sites had higher statistical power than the evaluation based on total chironomids. At the reference site, Caenis, Cladotanytarsus, Orthocladius, and Ceratopogonidae were the dominant taxa, while the urban sites were dominated by Dero, Physella, Ancylidae, Chironomus, Dicrotendipes, Glyptotendipes, Polypedilum, Pseudochironomus, Stenochironomus, and Tanytarsus. These differences may have been dependent upon differences in hydrologic regime and water quality between sites. Significant differences (ANOVA, p < 0.01) in water quality parameters (alkalinity, hardness, nitrates, phosphates, chlorides, sulfates, calcium, magnesium, potassium, and triazine) were found among water samples collected from the reference and urban sites. During the transfer period, most of the Ephemeroptera and Trichoptera taxa and a few other taxa disappeared from artificial substrates that were colonized at the reference site and then moved to the urban sites. Also, local abundant taxa from the urban site significantly (t test, p < 0.05) increased in number on the transferred artificial substrates. Seasonal differences in colonization patterns were also observed between the spring and summer experimental periods, which indicate that temporal variation is equally important, as is the anthropogenic effect in benthic community evaluation. Field survival and growth experiments using Erpetogomphus designatus larvae were designed to detect differences between evaluated sites. Larvae were collected from the reference site, measured in the laboratory, and exposed at the urban sites for six weeks in using specially designed cages. The exposed larvae demonstrated a higher mortality rate at the urban sites compared to the reference site. digital.library.unt.edu/ark:/67531/metadc5314/ - Influence of Cholesterol Import on Aspergillus fumigatus Growth and Antifungal Suscepibility
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Invasive pulmonary aspergillosis is a life-threatening fungal infection commonly observed in immunocompromised patients and has a mortality rate approaching 100% once the disease is disseminated. Aspergillus fumigatus is the most common pathogen. Early diagnosis improves the prognosis but is very difficult since most signs and symptoms are nonspecific. Antifungal therapy, usually based on sterol biosynthesis inhibitors, is also of limited efficacy. In my attempts to discover a diagnostic sterol marker for aspergillosis, I observed that A. fumigatus incorporates large amounts of cholesterol from serum-containing medium. This observation suggested the hypothesis that exogenous cholesterol from the host can be imported by A. fumigatus and used as a substitute for ergosterol in the cell membrane. This proposed mechanism would reduce the efficacy of antifungal drugs that act as sterol biosynthesis inhibitors. Experiments to test this hypothesis were designed to determine the effects of serum-free and serum-containing medium on growth of A. fumigatus in the presence and absence of azole antifungal agents. The results showed a marked increase in growth in the presence of human serum. Cultures in media containing cholesterol but no serum also showed enhanced growth, a result indicating that a non-cholesterol component of serum is not primarily responsible for the increased growth. However, sterol analysis of A. fumigatus cultured in the absence of inhibitors showed little or no change in ergosterol levels. This result suggested that the imported cholesterol was not being used as membrane sterol. However, in parallel experiments using Itraconazole, an antifungal agent that attenuates sterol biosynthesis by inhibiting the sterol 14a-demethylase (ERG11), ergosterol levels decreased with increasing doses of inhibitor. Moreover, serum-containing medium partially rescued A. fumigatus from the effects of Itraconazole, and a similar rescue effect was observed with serum-free media containing cholesterol. From the preceding results, it can be concluded that human serum enhances A. fumigatus growth, that cholesterol import rescues Aspergillus from the effects of antifungal agents, that the potency of some azole antifungals is decreased by cholesterol, and that imported cholesterol may substitute for membrane ergosterol in the presence of sterol biosynthesis inhibitors. digital.library.unt.edu/ark:/67531/metadc5539/ - Bioavailability and toxicity of 2,4,6-trinitrotoluene in sediment.
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TNT (2,4,6-trinitrotoluene) is a persistent contaminant at many military installations and poses a threat to aquatic ecosystems. Data from environmental fate and toxicity studies with TNT revealed that sediment toxicity test procedures required modification to accurately assess sediment TNT toxicity. Key modifications included aging TNT-spiked sediments 8-14 d, basing lethal dose on measured sediment concentrations of the molar sum of TNT and its main nitroaromatic (NA) transformation products (SNA), basing sublethal dose on average sediment SNA concentrations obtained from integration of sediment SNA transformation models, avoiding overlying water exchanges, and minimizing toxicity test durations. Solid phase microextraction fibers (SPMEs) were investigated as a biomimetic chemical measure of toxicity and bioavailability. Both organism and SPME concentrations provided measures of lethal dose independent of exposure scenario (TNT-spiked sediment or TNT-spiked water) for Tubifex tubifex. Among all benthic organisms tested (Chironomus tentans, Ceriodaphnia dubia, T. tubifex) and matrixes, median lethal dose (LC50) estimates based on SPME and organism concentrations ranged from 12.6 to 55.3 mmol SNA/ml polyacrylate and 83.4 to 172.3 nmol SNA/g tissue, ww, respectively. For Tubifex, LC50s (95% CI) based on SNA concentrations in sediment and SPMEs were 223 (209-238) nmol SNA/g, dw and 27.8 (26.0-29.8) mmol SNA/ml, respectively. Reproductive effects occurred at slightly lower exposures. Median effective dose (EC50) estimates (95% CI) for Tubifex cocoon production, based on sediment and SPME concentrations, were 118 (114-122) nmol SNA/g, dw and 21.8 (21.2-22.4) mmol SNA/ml, respectively. Bioconcentration experiments with Tubifex revealed that compound hydrophobicity predicted the toxicokinetics and bioconcentration of these compounds from water, however, there was a large discrepancy between the toxicokinetics of absorbed versus metabolically-generated aminodinitrotoluenes. A large portion of bioconcentrated, radiolabeled TNT transformation products could not be identified. In addition to their ability to provide matrix-independent measures of dose, SPME concentrations were more accurate indicators of bioavailable NAs than were sediment concentrations. digital.library.unt.edu/ark:/67531/metadc5549/ - Cyanide Assimilation in Pseudomonas fluorescens NCIMB 11764: Characterization of Cyanide Oxygenase as a Pterin-Dependent Multicomponent Enzyme Complex
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Cyanide utilization in Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated by an enzyme referred to as cyanide oxygenase (CNO), previously shown to require components in both high (H) (>30 kDa) and low (L) (<10 kDa) molecular weight cell fractions. In this study, tetrahydrobiopterin (H4biopterin) was identified as a cofactor in fraction L, thus making CNO appear as a pterin- dependent hydroxylase. CNO was purified 150-fold (specific activity 0.9 U/mg) and quantitatively converted cyanide to formate and ammonia as reaction products. When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted. CNO was found to be an aggregate of known enzymes that included NADH oxidase (Nox), NADH peroxidase (Npx), cyanide dihydratase (CynD) and carbonic anhydrase (CA). A complex multi-step reaction mechanism is proposed in which Nox generates hydrogen peroxide which in turn is utilized by Npx to catalyze the oxygenation of cyanide to formamide accompanied by the consumption of one and two molar equivalents of oxygen and NADH, respectively. The further hydrolysis of formamide to ammonia and formate is thought to be mediated by CynD. The role of H4biopterin and of the enzyme CA in the proposed process remains unclear, but the involvement of each in reactive oxygen and radical chemistry is consistent with the proposed formation of such species in the catalytic process. H4biopterin may additionally serve as a protein stabilizing agent along with a protein co-purifying with CynD identified as elongation factor Tu, a known chaperone. At least two of the CNO components (Nox and CynD) are complex oligomeric proteins whose apparent association with Npx and CA appears to be favored in bacterial cells induced with cyanide allowing their purification in toto as a multiprotein enzyme complex. digital.library.unt.edu/ark:/67531/metadc5548/ - Application of cultured neuronal networks for use as biological sensors in water toxicology and lipid signaling.
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This dissertation research explored the capabilities of neuronal networks grown on substrate integrated microelectrode arrays in vitro to be applied to toxicological research and lipid signaling. Chapter 1 details the effects of chlorine on neuronal network spontaneous electrical activity and pharmacological sensitivity. This study demonstrates that neuronal networks can maintain baseline spontaneous activity, and respond normally to pharmacological manipulations in the present of three times the chlorine present in drinking water. The findings suggest that neuronal networks may be used as biological sensors to monitor the quality of water and the presence of novel toxicants that cannot be detected by conventional sensors. Chapter 2 details the neuromodulatory effects of N-acylethanolamides (NAEs) on the spontaneous electrical activity of neuronal networks. NAEs are a group of lipids that can mimic the effects of marijuana and can be derived from a variety of plant sources including soy lecithin. The most prominent NAEs in soy lecithin, palmitoylethanolamide (PEA) and linoleoylethanolamide (LEA), were tested individually and were found to significantly inhibit neuronal spiking and bursting activity. These effects were potentiated by a mixture of NAEs as found in a HPLC enriched fraction from soy lecithin. Cannabinoid receptor-1 (CB1-R) antagonists and other cannabinoid pathway modulators indicated that the CB1-R was not directly involved in the effects of NAEs, but that enzymatic degradation and cellular uptake were more likely targets. The results demonstrate that neuronal networks may also be a viable platform for the elucidation of biochemical pathways and drug mechanisms of action. digital.library.unt.edu/ark:/67531/metadc5557/ - Characterization of infection arrest mutants of Medicago truncatula and genetic mapping of their respective genes.
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In response to compatible rhizobia, leguminous plants develop unique plant organs, root nodules, in which rhizobia fix nitrogen into ammonia. During nodule invasion, the rhizobia gain access to newly divided cells, the nodule primordia, in the root inner cortex through plant-derived cellulose tubes called infection threads. Infection threads begin in curled root hairs and bring rhizobia into the root crossing several cell layers in the process. Ultimately the rhizobia are deposited within nodule primordium cells through a process resembling endocytosis. Plant host mechanisms underlying the formation and regulation of the invasion process are not understood. To identify and clone plant genes required for nodule invasion, recent efforts have focused on Medicago truncatula. In a collaborative effort the nodulation defect in the lin (lumpy infections) mutant was characterized. From an EMS-mutagenized population of M. truncatula, two non-allelic mutants nip (numerous infections with polyphenolics) and sli (sluggish infections) were identified with defects in nodule invasion. Infection threads were found to proliferate abnormally in the nip mutant nodules with only very rare deposition of rhizobia within plant host cells. nip nodules were found to accumulate polyphenolic compounds, indicative of a host defense response. Interestingly, nip was also found to have defective lateral root elongation suggesting that NIP has a role in both nodule and lateral root development. NIP was found to map at the upper arm of chromosome 1. In sli, infection threads were observed to bring rhizobia from infection threads to newly divided nodule primordium cells in the roots inner cortex. Polyphenolic accumulation in sli nodule/bumps was found. Lateral roots in sli were found to be clustered at the top of the root, indicating that sli like nip may be defective in lateral root development. digital.library.unt.edu/ark:/67531/metadc5567/ - The structure and function of troponin T upon metal ion binding and the detection of nucleic acid sequence variations.
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Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild hypertrophy, but a high incidence of sudden death. Detection of those genetic mutations would facilitate the clinical diagnosis and initiation of treatment at an early stage. This dissertation also investigated a novel hybridization proximity assay (HYPA) combining molecular beacon and luminescence resonance energy transfer (LRET) technologies. Experimental results suggest that a shared stem probe design produces a more consistent response upon hybridization, whereas the internally labeled probe was less consistent, but can yield the highest responses. Using the optimally designed molecular probes, the HYPA provides a detection of alterations in nucleic acid structure of as little as a single nucleotide. This novel HYPA is expected to expand its applications in the analysis and screening of genetic diseases. digital.library.unt.edu/ark:/67531/metadc5565/ - Influence of Sediment Exposure and Water Depth on Torpedograss Invasion of Lake Okeechobee, Florida
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Torpedograss (Panicum repens) was first observed in Lake Okeechobee in the 1970s and appears to have displaced an estimated 6,400 ha of native plants, such as spikerush (Eleocharis cellulosa), where inundation depths are often less than 50 cm. Two series of studies evaluated substrate exposure and water depth influences on torpedograss establishment and competitiveness. Results revealed that fragments remain buoyant for extended periods and so facilitate dispersal. Once anchored to exposed substrate fragments can readily root and establish. Subsequently, torpedograss thrives when subjected to inundations to 75 cm and survives prolonged exposure to depths greater than 1 m. These findings suggest that fluctuating water levels contribute to torpedograss dispersal and colonization patterns and that low water levels increase marsh area susceptible to invasion. The competition study found that spikerush grown in monoculture produces significantly more biomass when continually inundated to shallow depths (10 to 20 cm) than when subjected to drier conditions (-25 cm) or greater inundations (80 cm). In contrast, torpedograss establishes more readily on exposed substrate (-25 to 0 cm) compared to inundate substrates. During the first growing season biomass production increases as substrate exposure interval increases. However, during the second year, established torpedograss produces more biomass when grown on intermittently wet (0 cm) compared to permanently dry (-25 cm) or intermittently inundated (10 cm) substrates. No difference in production was observed between substrates permanently inundated (10 cm) and any other regime tested. During the first two years of torpedograss invasion, regardless of treatment, spikerush suppresses invasion and torpedograss had little effect on established spikerush, indicating that spikerush-dominated areas are capable of resisting torpedograss invasion. Even so, disturbances that might cause mortality of long hydroperiod species, such as spikerush, may create open gaps in the native vegetation and thus facilitate torpedograss establishment and expansion. digital.library.unt.edu/ark:/67531/metadc5607/ - Automated Low-cost Instrument for Measuring Total Column Ozone
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Networks of ground-based and satellite borne instruments to measure ultraviolet (UV) sunlight and total column ozone have greatly contributed to an understanding of increased amounts of UV reaching the surface of the Earth caused by stratospheric ozone depletion. Increased UV radiation has important potential effects on human health, and agricultural and ecological systems. Observations from these networks make it possible to monitor total ozone decreases and to predict ozone recovery trends due to global efforts to curb the use of products releasing chemicals harmful to the ozone layer. Thus, continued and expanded global monitoring of ozone and UV is needed. However, existing automatic stratospheric ozone monitors are complex and expensive instruments. The main objective of this research was the development of a low-cost fully automated total column ozone monitoring instrument which, because of its affordability, will increase the number of instruments available for ground-based observations. The new instrument is based on a high-resolution fiber optic spectrometer, coupled with fiber optics that are precisely aimed by a pan and tilt positioning mechanism and with controlling programs written in commonly available software platforms which run on a personal computer. This project makes use of novel low-cost fiber optic spectrometer technology. A cost advantage is gained over available units by placing one end of the fiber outdoors to collect sunlight and convey it indoors, thereby allowing the spectrometer and computer to be placed in a controlled environment. This reduces the cost of weatherproofing and thermal compensation. Cost savings also result from a simplified sun targeting system, because only a small pan and tilt device is required to aim the lightweight fiber optic ends. Precision sun-targeting algorithms, optical filter selection, and software to derive ozone from spectral measurements by the spectrometer are a major contribution of this project. This system is a flexible platform which may be adapted to study other atmospheric constituents such as sulfur dioxide, nitrous oxides, and haze. digital.library.unt.edu/ark:/67531/metadc5792/ - Tests of a New Model of Paclitaxel-Induced Neuropathy and the Effects of Paclitaxel on the Dorsal Root Ganglia
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This study examined a new model of paclitaxel-induced neuropathic pain and the effects of systemic paclitaxel on the gap junction protein subunit Cx43 and potassium inwardly-rectifying channel Kir4.1 within the dorsal root ganglia. In the new neuropathic pain model, subplantar injections of paclitaxel resulted in decreased conduction velocities of A-beta fiber compound action potentials in the sciatic (5.9%) and tibial nerves (6.8%) as well as in M (10.6%) and H (10.2%) waves. By using repeated recordings it was found that following paclitaxel injection, conduction velocities in the contralateral plantar nerve increased (9.2%). Systemic injections of paclitaxel resulted in reduced Kir4.1 immunolabeling in the dorsal root ganglia compared to vehicle injections. This reduction was observed in total labeling (32.4%) as well as in areas of intense labeling (28.7%). Reductions in overall Cx43 immunolabeling (25%) and area (25%) following systemic paclitaxel injections were not statistically significant. The results of these studies suggest that subplantar injections of paclitaxel can result in reduced peripheral nerve conduction velocities. The results also show that a unilateral neuropathy can result in contralateral changes in conduction velocities. The effects of paclitaxel on reducing Kir4.1 levels suggest that neuropathic pain caused by paclitaxel may share mechanisms in common with other types of neuropathies which show similar changes in Kir4.1 levels. digital.library.unt.edu/ark:/67531/metadc84251/ - Measuring Atmospheric Ozone and Nitrogen Dioxide Concentration by Differential Optical Absorption Spectroscopy
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The main objective was to develop a procedure based on differential optical absorption spectroscopy (DOAS) to measure atmospheric total column of ozone, using the automated instrument developed at the University of North Texas (UNT) by Nebgen in 2006. This project also explored the ability of this instrument to provide measurements of atmospheric total column nitrogen dioxide. The instrument is located on top of UNT’s Environmental Education, Science and Technology Building. It employs a low cost spectrometer coupled with fiber optics, which are aimed at the sun to collect solar radiation. Measurements taken throughout the day with this instrument exhibited a large variability. The DOAS procedure derives total column ozone from the analysis of daily DOAS Langley plots. This plot relates the measured differential column to the airmass factor. The use of such plots is conditioned by the time the concentration of ozone remains constant. Observations of ozone are typically conducted throughout the day. Observations of total column ozone were conducted for 5 months. Values were derived from both DOAS and Nebgen’s procedure and compared to satellite data. Although differences observed from both procedures to satellite data were similar, the variability found in measurements was reduced from 70 Dobson units, with Nebgen’s procedure, to 4 Dobson units, with the DOAS procedure.A methodology to measure atmospheric nitrogen dioxide using DOAS was also investigated. Although a similar approach to ozone measurements could be applied, it was found that such measurements were limited by the amount of solar radiation collected by the instrument. Observations of nitrogen dioxide are typically conducted near sunrise or sunset, when solar radiation experiences most of the atmospheric absorption. digital.library.unt.edu/ark:/67531/metadc103336/ - 9-Lipoxygenase Oxylipin Pathway in Plant Response to Biotic Stress
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The activity of plant 9-lipoxygenases (LOXs) influences the outcome of Arabidopsis thaliana interaction with pathogen and insects. Evidence provided here indicates that in Arabidopsis, 9-LOXs facilitate infestation by Myzus persicae, commonly known as the green peach aphid (GPA), a sap-sucking insect, and infection by the fungal pathogen Fusarium graminearum. in comparison to the wild-type plant, lox5 mutants, which are deficient in a 9-lipoxygenase, GPA population was smaller and the insect spent less time feeding from sieve elements and xylem, thus resulting in reduced water content and fecundity of GPA. LOX5 expression is induced rapidly in roots of GPA-infested plants. This increase in LOX5 expression is paralleled by an increase in LOX5-synthesized oxylipins in the root and petiole exudates of GPA-infested plants. Micrografting experiments demonstrated that GPA population size was smaller on plants in which the roots were of the lox5 mutant genotype. Exogenous treatment of lox5 mutant roots with 9-hydroxyoctadecanoic acid restored water content and population size of GPA on lox5 mutants. Together, these results suggest that LOX5 genotype in roots is critical for facilitating insect infestation of Arabidopsis. in Arabidopsis, 9-LOX function is also required for facilitating infection by F. graminearum, which is a leading cause of Fusarium head blight (FHB) disease in wheat and other small grain crops. Loss of LOX1 and LOX5 function resulted in enhanced resistance to F. graminearum infection. Similarly in wheat, RNA interference mediated silencing of the 9-LOX homolog TaLpx1, resulted in enhanced resistance to F. graminearum. Experiments in Arabidopsis indicate that 9-LOXs promote susceptibility to this fungus by suppressing the activation of salicylic acid-mediated defense responses that are important for basal resistance to this fungus. the lox1 and lox5 mutants were also compromised for systemic acquired resistance (SAR), an inducible defense mechanism that is systemically activated throughout a plant in response to a localized infection. the lox1 and lox5 mutants exhibited reduced cell death and delayed hypersensitive response when challenged with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv tomato. LOX1 and LOX5 functions were further required for the synthesis as well as perception of a SAR-inducing activity present in petiole exudates collected from wild-type avirulent pathogen-challenged leaves. Taken together, results presented here demonstrate that 9-LOX contribute to host susceptibility as well as defense against different biotic stressors. digital.library.unt.edu/ark:/67531/metadc115127/ - Identification of Three Symbiosome Targeting Domains in the MtENOD8 Protein and Cell-to-cell MtENOD8 mRNA Movement in Nodules
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The model legume, Medicago truncatula, is able to enter into a symbiotic relationship with soil bacteria, known as rhizobia. This relationship involves a carbon for nitrogen exchange in which the plant provides reduced carbon from photosynthesis in exchange for reduced, or “fixed” atmospheric nitrogen, which allows the plant to thrive in nitrogen depleted soils. Rhizobia infect and enter plant root organs, known as nodules, where they reside inside the plant cell in a novel organelle, known as the symbiosome where nitrogen fixation occurs. the symbiosome is enriched in plant proteins, however, little is known about the mechanisms that direct plant proteins to the symbiosome. Using the M. truncatula ENOD8 (MtENOD8) protein as a model to explore symbiosome protein targeting, 3-cis domains were identified within MtENOD8 capable of directing green fluorescent protein (GFP) to the symbiosome, including its N-terminal signal peptide (SP). the SP delivered GFP to the vacuole in the absence of nodules suggesting that symbiosome proteins share a common targeting pathway with vacuolar proteins. a time course analysis during nodulation indicated that there is a nodule specific redirection of MtENOD8-SP from the vacuole to the symbiosome in a MtNIP/LATD dependent manner. GFP expression by the MtENOD8 promoter revealed spatial discrepancy between promoter activity and protein localization. in situ localization of MtENOD8 mRNA showed localization to infected cells, where the protein is found, suggesting mRNA cell-to-cell movement. Expression of MtENOD8 in Arabidopsis showed that the SP did not direct GFP to the vacuole indicating that vacuolar targeting of MtENOD8’s SP may be legume specific. Taken together, the research presented here indicates that the MtENOD8 symbiosome protein has evolved redundant domains for targeting, which has part of a common pathway with vacuolar proteins. Observed spatial discrepancy between the MtENOD8 promoter and protein shows additional mechanisms of gene regulation through cell-to-cell mRNA movement, previously unknown in nodules. digital.library.unt.edu/ark:/67531/metadc115118/