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- 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/ - Inherent Problems Associated with the Identification of Genes Responsible for Allowing B. cepacia to Adhere to Human Lung Carcinoma A549 Cells
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In this project a bacteria's ability to bind to human lung tissue was investigated. To carry out this study Pseudomonas aeruginosa, Eschericia coli and Burkholderia cepacia were used. B. cepacia served as the bacterium of interest. Isolating the gene which confers upon this bacterium the ability to bind to lung tissue was the main objective of this study. P. aeruginosa has been identified as being the bacteria most responsible for causing serious lung infections that can result in cystic fibrosis. This bacterium therefore served as the positive control in this study. On the contrary, E. coli does not possess this binding ability and served as the negative control. This paper gives a detailed outline of the different procedures necessary for the successful completion of this project. Firstly, a broad guideline of the important steps involved are explored. This is followed by a discussion on potential problems and possible solutions. Throughout the document, illustrations of expected results are indicated so as to further guide the researcher. digital.library.unt.edu/ark:/67531/metadc5819/ - Inquiry-based science for high school students: a forensic unit
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This project constitutes an instructional unit for honors biology that involves the use of science in the field of criminal investigation and forensics. Before beginning the unit, the learners should have mastered basic laboratory skills, including use of the microscope. They should also have an understanding of the basic structure and function of DNA and its role in heredity and protein synthesis. The standard time frame is 24 days with 70-minute periods, but can be easily adjusted to meet classroom needs. Several instructional strategies enhance student learning and make science fun. The unit is inquiry-driven and activity-based. Students are surprised by the crime, gather and analyze evidence, and work towards proposing an explanation. This real world problem involves the use of cooperative learning and a variety of assessment techniques. digital.library.unt.edu/ark:/67531/metadc2585/ - Integrating life cycle analysis and the ecological footprint calculator to foster sustainable behaviors
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Many tools have been developed to assess global, national or regional sustainable development policies. However, as governments develop sustainable policies, individuals must also feel empowered to affect their personal impact on the planet. This thesis integrates three sustainability concepts that lend themselves to individual sustainability: The natural step, life cycle assessment, and the ecological footprint. TNS serves to provide the meaning and substance toward sustainable development. LCA helps provide the framework for assessing sustainability. The EF calculator determines the driving components and measures the qualitative decisions made through TNS and LCA. From the analysis of the household footprint calculator a simplified footprint calculator was developed to assist individuals and communities in setting benchmarks and goals as they move away from over-consumption and towards a sustainable lifestyle. digital.library.unt.edu/ark:/67531/metadc3294/ - 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/ - A laboratory course in experimental genetics for the biology major.
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This manual has been designed for a class of twenty- four students concurrently enrolled in the lecture course. The laboratory aids in the learning process and fosters an interest in the science of genetics. This manual and the experiments contained within are both informative and fun. The manual correlates with and expands upon the genetics course. Each investigation, with the exception of the Drosophila melanogaster project, can be completed in a 3-4 hour timeframe. This manual provides a “hands on” experience of theories simply discussed in the lecture course. This manual is intended to be a one-source manual where each investigation is designed to include an adequate introduction. Special attention has been given for each investigation with both the student and instructor in mind. digital.library.unt.edu/ark:/67531/metadc3007/ - Life history and case building behaviors of Phylloicus ornatus (Banks)(Trichoptera: Calamoceratidae) In two spring fed tributaries in the central Edwards Plateau bioregion of Texas
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The life history and case-making behaviors of Phylloicus ornatus from two springfed first order streams in the Edwards Plateau Bioregion of Texas were studied from January 1998 to November 1999. Field larval, pupal and adult samples and laboratory rearings indicated a multivoltine cycle. First instars differ from late instars in number of labral setae and in having a unique spur-like claw on each lateral hump. Larval development was asynchronous with second through fifth instars and pupae present most months. First instars were present April through July, October and November. Case making of first instar and case reconstruction of later instars extracted from their cases was documented by videophotography. digital.library.unt.edu/ark:/67531/metadc2555/ - Linkage of a nitrilase-containing Nit1C gene cluster to cyanide utilization in Pseudomonas fluorescens NCIMB 11764.
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Pseudomonas fluorescens NCIMB 11764 (Pf11764) is uniquely able to grow on the poison cyanide as its sole nitrogen source. It does so by converting cyanide oxidatively to carbon dioxide and ammonia, the latter being assimilated into cellular molecules. This requires a complex enzymatic machinery that includes nitrilase and oxygenase enzymes the nature of which are not well understood. In the course of a proteomics analysis aimed at achieving a better understanding of the proteins that may be required for cyanide degradation by Pf11764, an unknown protein of 17.8 kDa was detected in cells exposed to cyanide. Analysis of this protein by ESI-coupled mass spectrometry and bioinformatics searches gave evidence of strong homology with a protein (Hyp1) of unknown function (hypothetical) present in the bacterium Photorhabdus luminescens subsp. laumondii TTO1 (locus plu_1232). A search of available microbial genomes revealed a number of Hyp1 orthologs the genes of which are found in a conserved gene cluster known as Nit1C. Independent studies revealed that in addition to Hyp1, Pf11764 possesses a gene (nit) specifying a nitrilase enzyme whose closest homologue is a nitrilase found in Nit1C gene clusters (77% amino acid identity). DNA sequence analysis has further revealed that indeed, hyp1Pf11764 and nitPf11764 are contained in a cluster that includes also a gene specifying an oxygenase. Given the possible connection of Nit1C-endoded nitrilase and oxygenase enzymes to enzymatic cyanide degradation, there is strong reason for thinking that the genes specifying these enzymes contribute to bacterial growth on cyanide in those bacteria containing the Nit1C cluster. Because the biological function of the Hyp1 protein is currently unknown, it was cloned and the protein expressed in E. coli so that its properties could further be explored. Unfortunately, the expression of the protein in an insoluble form complicated these analyses. However, at least two lines of evidence suggest a possible role as a regulator of gene expression. First, over-expression of the protein was accompanied by the parallel elevation of the putative vector-encoded b-lactamase, implying that Hyp1Pf11764 can affect the expression of other genes. Second, a comparison of the amino acid sequence of select peptide fragments of Hyp1Pf11764, by conducting searches for homology with proteins in the existing nonredundant protein database, consistently revealed motifs in common with those present in bacterial response regulators that are part of two-component signal transduction systems widely distributed in bacteria. digital.library.unt.edu/ark:/67531/metadc10993/ - Macroinvertebrate Community Structure as an Indicator of Watershed Health in the Upper Trinity River Basin, North Central Texas
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This study describes macroinvertebrate community structure and assesses its potential in detecting point and non-point sources of disturbance associated with rural and urban areas in the Upper Trinity River Basin. Geospatial techniques were used to quantify landuse within the watershed in a GIS. At rural sites near the headwaters of the Trinity River, collector-gathering burrowers that are adapted to minimal flow comprised the majority of taxa. Destinies of taxa compositions at downstream sites increased and shifted toward psammophilic and rheophilic invertebrates, including primarily collector-filtering clingers, that are characteristic of shifting sand habitats in large prairie rivers. Benthic community structure generally benefited from point source impacts including wastewater treatment plant effluents that maintained higher flow. Community indices were negatively associated with forest landuse and positively associated with urban landuse. Partial CCA determined that flow and landuse contributed equally to species dispersions. Comparisons with historical biomonitoring studies in upper Trinity River Basin indicate improved watershed health. digital.library.unt.edu/ark:/67531/metadc2445/ - 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/ - 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/ - 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/ - Molecular and biochemical characterization of phospholipase D in cotton (Gossypium hirsutum L) seedlings.
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N-Acylethanolamines (NAEs) are enriched in seed-derived tissues and are believed to be formed from the membrane phospholipid, N-acylphosphatidylethanolamine (NAPE) via the action of phospholipase D (PLD). In an effort to identify a functional NAPE-PLD in cotton seeds and seedlings, we have screened a cotton seedling cDNA (cotyledon mRNA from 48 h dark grown seedlings) library with a 1.2 kb tobacco partial cDNA fragment encoding the middle third of a putative PLDβ/γ (genbank accession, AF195614) isoform. Six plaques were isolated from the Uni-ZAP lambda library, excised as pBluescript SK(-) phagemids and subjected to nucleotide sequence analysis. Alignment of derived sequences with Arabidopsis PLD family members indicated that the cDNAs represent six different PLD gene products -three putative PLD β isoforms and three putative PLD δ isoforms. The PLD β isoforms, designated Ghpldβ1a, GHpldβ1b and a truncated Ghpldβ1b isoform. Both the full-length PLD β proteins contained characteristic HKxxxxD catalytic domains, a PC-binding domain, a PIP2-binding domain and a C2 domain. In addition both cotton PLD β isoforms had a N-terminal "SPQY" rich domain which appeared to be unique to these PLDs. The three PLD δ isoforms, designated Ghpldδ1a, Ghpldδ1b and Ghpldδ1b-2 encode full-length PLDδ proteins, and like the above PLDs, contained the characteristic catalytic and regulatory domains. The expression of Ghpldδ1b showed hydrolytic and transphosphatidylation activity toward radiolabelled phosphatidylcholine (PC) but it appears Ghpldδ1b does not utilize NAPE as a substrate to produce NAEs nor does it seem to be suppressed by NAEs. digital.library.unt.edu/ark:/67531/metadc4732/ - A morphological study of the avian (Gallus domesticus) ductus arteriosi during hatching.
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The ductus arteriosi (DA) are two blood vessels connecting the pulmonary arteries to the descending aorta in the avian embryo. Following hatching, the DA closes, separation of the systemic and pulmonary circulation. I present the morphological changes that occur in the chicken DA during prepipping, internal pipping, external pipping, and hatching. The avian DA consists of two distinct tissue types, a proximal and a distal portion. Histological examination shows developmental differences between the proximal and distal portions of the DA with regard to lumen occlusion, endothelial cells, smooth muscle and elastin. Endothelial cell proliferation begins to occur as early as external pipping, with the lumen almost completely occluded by the 3rd day of post-hatching life. Expression of vascular endothelial growth factor (VEGF) increases in avian endothelial cells during hatching. I provide a morphological timeline of changes in the DA as the chicken develops from embryo to hatchling. digital.library.unt.edu/ark:/67531/metadc5233/ - A Multimedia Atlas of Dissection for Comparative Anatomy of the Vertebrates
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Traditional methods of teaching the laboratory course for Comparative Anatomy of the Vertebrates could be improved by applying current computer technology to construct an interactive, multimedial atlas of dissection. Five specimens used in comparative anatomy courses at most institutions were chosen as representative members of the Phylum Chordata: amphioxus, lamprey, dogfish shark, mud puppy, and cat. Specimens were dissected according to the modified method of Wischnitzer, 1993, and each stage was photographed with a Kodak DC120 digital zoom camera. These images were processed on a Power Macintosh 7600 computer with Adobe Photoshop v. 5.0. The atlas was constructed from these images using Macromedia Authorware v. 4.0.3. Each image contains a series of interactive objects that display a highlight and descriptive text as the cursor passes over each object. digital.library.unt.edu/ark:/67531/metadc2224/ - 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/ - The potential of coelomocyte chemotaxis as an immune biomarker in the earthworm, Lumbricus terrestris
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Coelomocyte migration responses, both random and chemotatic, were examined in the earthworm Lumbricus terrestris. Coelomocyte random migration patterns towards non-stimulatory, non-chemotatic solutions were described. Migration responses to immunostimulatory agents lipopolysaccharides (LPS), N-formly-methionyl-leucyl-phenylalanine (FMLP), sheep erythrocytes, Saccharomyces cerevisiae, Aeromonas hydrophila, Eisenia fetida and Rhabditis pellio were characterized. Chemotaxis was reported to LPS, FMLP, sheep erythrocytes, S. cerivesae and E. fetida. Bio-indicator potential of chemotaxis is discussed relative to variability in migration responses. digital.library.unt.edu/ark:/67531/metadc2272/ - 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/ - Purification of Cyanide-Degrading Nitrilase from Pseudomonas Fluorescens NCIMB 11764.
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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 far outweighed that (10,000 fold) of the recombinant enzyme (NitPG) implying that the native NitCC protein purified in this work is different from that of the cloned recombinant. Further evidence of this was provided by molecular studies indicating that the two proteins differ in mass (34.5 and 38 kDa, respectively) and amino acid sequence. In summary, two different Nit enzymes are encoded by Pf11764. While the two share greater than 50% amino acid sequence identity, the results suggest that the native NitCC enzyme purified in this work functions better as a cyanide-degrading nitrilase and is one of four enzyme components comprising CNO required for Pf11764 cyanide assimilation. digital.library.unt.edu/ark:/67531/metadc33224/ - 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/ - 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/ - 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/ - Solid phase microextraction of amino-dinitrotoluenes in tissue.
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TNT (2,4,6-trinitrotoluene) readily and predominantly transforms to 2ADNT (2-amino-4,6-dinitrotoluene) and 4ADNT (4-amino-2,6-dinitrotoluene) in environmental matrixes and tissues. Solid phase microextraction (SPME) was used to extract ADNTs (amino-dinitrotoluenes) from tissue as a potential method to investigate the recalcitrance of metabolically-generated ADNTs versus absorbed ADNTs. Tubifex tubifex was allowed to metabolize TNT into ADNTs in 24-hr static non-renewal exposure test followed by 24-hr depuration in clean reconstituted hard water. Polyacrylate-coated (PA) SPME fibers were then deployed and agitated in tissue homogenates containing metabolically-generated ADNTs for 48 hr to provide a measure of available ADNTs. Extractability of ADNTs from T. tubifex tissue containing metabolically-generated ADNTs was significantly less than extractability of ADNTs from T. tubifex tissue containing absorbed ADNTs: 50-60% and 81-90% of expected extractability based on fiber-water partition ratio. The lower SPME extractability of metabolically-generated ADNTs may stem from the unavailability of metabolically-generated ADNTs sequestered in tissue or bound to tissue macromolecules during metabolism of TNT to ADNT. Tissue extractions using SPMEs may be able to estimate such bound organic residues in tissue and serve as potential indicators of toxicological bioavailability and biomagnification potential of tissue-associated organic compounds. digital.library.unt.edu/ark:/67531/metadc4649/ - 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/ - 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/ - 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/ - Temperature tolerances and predation susceptibilities of transgenic and wildtype zebra danios, Danio rerio.
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Both the upper and lower temperature tolerances of red fluorescent protein transgenic zebra danios and wildtype zebra danios, Danio rerio, were significantly different via two different methods; however, all differences are small (< 1°C) and probably not ecologically important. The U.S. geographic distributions of both transgenic and wildtype zebra danios will not be restricted by their upper thermal tolerances, but will be limited to the southern and western portions of the U.S. by their lower thermal tolerances. Largemouth bass did not preferentially prey upon transgenic zebra danios compared to wildtype danios or wildtypes relative to a native fish. If transgenic or wildtype zebra danios are released into southern or western U.S. waters, it is possible they could be eliminated by predation. digital.library.unt.edu/ark:/67531/metadc5581/ - 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/ - Use of Geographic Information System and Remote Sensing Technologies to Describe Mosquito Population Dynamics in the Ray Roberts Greenbelt, Denton County, Texas
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A population survey was conducted from April through September 2002 on mosquito species occurring on the Ray Roberts Greenbelt, a riparian corridor used for public recreation on the Elm Fork of the Trinity River, in Denton County, Texas. ArcGIS software was used to set up a stratified random sampling design based on habitat parameters. Multivariate analyses of sampling data and climatic variables were used to describe spatial and temporal patterns of mosquito species. A total of 33 species were collected during this study belonging to the following genera: Aedes, Anopheles, Coquillettidia, Culex, Mansonia, Ochlerotatus, Orthopodomyia, Psorophora, Toxorhynchites, and Uranotaenia. Seasonal distributions of the dominant species revealed population fluctuations. Aedes vexans was the primary species collected in April and May, occurring in low numbers throughout the rest of the sampling period. Psorophora columbiae reached its highest population density in June, with a smaller peak occurring in late July. Present from May through the end of September, Culex erraticus was the most abundant species collected with major peaks in mid-June and the end of July. Abundance of Culex salinarius followed the same general trend as that for Cx. erraticus, but with smaller numbers. The specimens were tested for a variety of arboviruses by the Texas Department of Health. One pool of Cx. erraticus and Cx. salinarius, collected in August 2002, tested positive for West Nile virus. Variables that were important factors for determining dominant species abundance were temperature, wind speed, rain accumulation occurring one-week and two-weeks prior to sampling, number of day since last rain event, dew point, and average canopy coverage. digital.library.unt.edu/ark:/67531/metadc4160/ - Use of Satellite Imagery and GIS to Model Brood-Rearing Habitat for Rio Grande Wild Turkey Populations Occurring in the Western Cross Timbers Region of Texas
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Remote sensing and GIS have become standard tools for evaluating spatial components of wildlife habitats. These techniques were implemented to evaluate Rio Grande wild turkey (Meleagris gallopavo) poult-rearing habitat in the Western Cross Timbers region of Texas. Texas Parks and Wildlife (TPWD) random roving turkey counts for 1987-1989 and 1998-2000 were selected, indicating locations where hens with poults were observed. Satellite imagery from 1988 and 1999 was classified and then processed with Patch Analyst. To add robustness, stream, road and census population densities were also evaluated for each turkey location. Analysis of the 1988 canopy cover image, comparing observed locations with randomly-selected habitat cells (N = 20) indicated significant differences (p <.05) for patch edge variables. Mean patch edge was significantly greater for habitat locations where hens with poults were observed than for those selected at random. Spatial data for 1999 did not indicate a significant difference (p < .05) between sampling groups (observed vs. random, N = 30). Significant differences (p <.05) did occur for turkey locations observed in both 1988 and 1999 (N = 7). This demonstrates the adaptability of wild turkey hens, as habitats change over time, hens continued to visit the same locations even though the habitat had significantly changed for select spatial variables. digital.library.unt.edu/ark:/67531/metadc3174/ - Utilization of Corridor Habitat by White-tailed Deer (Odocoileus virginianus) in Denton County, Texas
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White-tailed deer (Odocoileus virginianus) (N=15) movements were determined with use of radio telemetry techniques to determine the utilization of corridor habitat on the Lake Ray Roberts Greenbelt Corridor (RRGC) in north central Texas. Home ranges were calculated using three estimation types. Male white-tailed deer tend to have home ranges twice that of female home ranges. Seasonal home ranges were largest during spring (Feb. - April) and fall (Aug. - Oct.) seasons. Males had greater seasonal variation in utilization than females. No statistically significant difference (p=0.24) between white-tailed deer locations when the RRGC experiences heavy human traffic compared to days when there is light human traffic. Linearity indices indicated home ranges less linear than expected (LI = 3.02). The RRGC should be maintained at its current status to provide a variety of vegetational types and protective cover for white-tailed deer and other wildlife of Denton County. digital.library.unt.edu/ark:/67531/metadc4468/