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- 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.
- 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.
- N-Acylethanolamine metabolism during seed germination: Molecular identification of a functional N-acylethanolamine amidohydrolase.
- N-Acylethanolamines (NAEs) are endogenous lipid metabolites that occur in a variety of dry seeds, and their levels decline rapidly during the first few hours of imbibition (Chapman et al., 1999, Plant Physiol., 120:1157-1164). Biochemical studies supported the existence of an NAE amidohydrolase activity in seeds and seedlings, and efforts were directed toward identification of DNA sequences encoding this enzyme. Mammalian tissues metabolize NAEs via an amidase enzyme designated fatty acid amide hydrolase (FAAH). Based on the characteristic amidase signature sequence in mammalian FAAH, a candidate Arabidopsis cDNA was identified and isolated by reverse transcriptase-PCR. The Arabidopsis cDNA was expressed in E. coli and the recombinant protein indeed hydrolyzed a range of NAEs to free fatty acids and ethanolamine. Kinetic parameters for the recombinant protein were consistent with those properties of the rat FAAH, supporting identification of this Arabidopsis cDNA as a FAAH homologue. Two T-DNA insertional mutant lines with disruptions in the Arabidopsis NAE amidohydrolase gene (At5g64440) were identified. The homozygous mutant seedlings were more sensitive than the wild type to exogenously applied NAE 12:0. Transgenic seedlings overexpressing the NAE amidohydrolase enzyme showed noticeably greater tolerance to NAE 12:0 than wild type seedlings. These results together provide evidence in vitro and in vivo for the molecular identification of Arabidopsis NAE amidohydrolase. Moreover, the plants with altered NAE amidohydrolase expression may provide new tools for improved understanding of the role of NAEs in germination and seedling growth.
- N-Acylethanolamine (NAE) profiles change during Arabidopsis thaliana seed germination and seedling growth.
- An understanding of the potential roles as lipid mediators of a family of bioactive metabolites called N-acylethanolamines (NAEs) depends on their accurate identification and quantification. The levels of 18C unsaturated NAEs (e.g. NAE18:2, NAE 18:3, etc.) in wild-type seeds (about 2000 ng/g fw) generally decreased by about 80% during germination and post-germinative growth. In addition, results suggest NAE-degradative fatty acid amide hydrolase (FAAH) expression does not play a major role in normal NAE metabolism as previously thought. Seedlings germinated and grown in the presence of abscisic acid (ABA), an endogenous plant hormone, exhibited growth arrest and secondary dormancy, similar to the treatment of seedlings with exogenous Nlauroylethanolamine (NAE12:0). ABA-mediated growth arrest was associated with higher levels of unsaturated NAEs. Overall, these results are consistent with the concept that NAE metabolism is activated during seed germination and suggest that the reduction in unsaturated NAE levels is under strict temporal control and may be a requirement for normal seed germination and post-germinative growth.
- N-Acylethanolamines and Plant Phospholipase D
- Recently, three distinct isoforms of phospholipase D (PLD) were identified in Arabidopsis thaliana. PLD α represents the well-known form found in plants, while PLD β and γ have been only recently discovered (Pappan et al., 1997b; Qin et al., 1997). These isoforms differ in substrate selectivity and cofactors required for activity. Here, I report that PLD β and γ isoforms were active toward N-acylphosphatidylethanolamine (NAPE), but PLD α was not. The ability of PLD β and γ to hydrolyze NAPE marks a key difference from PLD α. N-acylethanolamines (NAE), the hydrolytic products of NAPE by PLD β and γ, inhibited PLD α from castor bean and cabbage. Inhibition of PLD α by NAE was dose-dependent and inversely proportional to acyl chain length and degree of unsaturation. Enzyme kinetic analysis suggested non-competitive inhibition of PLD α by NAE 14:0. In addition, a 1.2-kb tobacco (Nicotiana tabacum L.) cDNA fragment was isolated that possessed a 74% amino acid identity to Arabidopsis PLD β indicating that this isoform is expressed in tobacco cells. Collectively, these results provide evidence for NAE producing PLD activities and suggest a possible regulatory role for NAE with respect to PLD α.
- Adaptive Advantages of Carotenoid Pigments in Alpine and Subalpine Copepod Responses to Polycyclic Aromatic Hydrocarbon Induced Phototoxicity
- Alpine zooplankton are exposed to a variety of stressors in their natural environment including ultraviolet radiation. Physiological coping mechanisms such as the accumulation of photoprotective compounds provide these zooplankton protection from many of these stressors. Elevated levels of carotenoid compounds such as astaxanthin have been shown to help zooplankton survive longer when exposed to ultraviolet radiation presumably due to the strong antioxidant properties of carotenoid compounds. This antioxidant capacity is important because it may ameliorate natural and anthropogenic stressor-induced oxidative stress. While previous researchers have shown carotenoid compounds impart increased resistance to ultraviolet radiation in populations of zooplankton, little work has focused on the toxicological implications of PAH induced phototoxicity on zooplankton containing high levels of carotenoid compounds. This thesis discusses research studying the role that carotenoid compounds play in reducing PAH induced phototoxicity. By sampling different lakes at elevations ranging from 9,500' to 12,700' in the front range of the Colorado Rocky Mountains, copepod populations containing different levels of carotenoid compounds were obtained. These populations were then challenged with fluoranthene and ultraviolet radiation. Results discussed include differences in survival and levels of lipid peroxidation among populations exhibiting different levels of carotenoid compounds, and the toxicological and ecological implications of these results.
- Adherence and haemagglutination of Moraxella catarrhalis.
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M. catarrhalis is a gram-negative diplococci frequently associated with infections of the upper respiratory tract. During the past decade, some preliminary studies have attempted to elucidate mechanisms of adherence and haemagglutination of M. catarrhalis. These studies have reported, in many cases, inconsistent results. There are two purposes of this research. First, identify mechanisms that may potentially be associated with the adherence and haemagglutination of M. catarrhalis. Second, suggest research directions that may be fruitful in clarifying these mechanisms.
- The adolescent stress response to a naturalistic driving stressor
- The proposed study examined the role of anxiety and risk-taking in driving performance in adolescents. In addition to examining the sample as a whole, gender differences were assessed given earlier reports from our laboratory and others indicating that males and females differ with respect to risky behaviors to driving performance and anxiety. Adolescents' subjective and physiological responses to a driving simulator task were assessed. Anxiety was measured via self report and salivary cortisol. Participants provided a baseline saliva sample and 3 post-task samples for cortisol analysis. Subjective anxiety scores were obtained at both baseline and following the driving stressor. Information concerning impulsivity, as well as other psychological constructs was also collected at baseline. Unlike the pilot study, there were no relationships (with or without respect to gender) between salivary cortisol and both self-reported anxiety (state and trait) or impulsively measures for this sample. These results suggest that this group of adolescents may not have been anxious about the driving task. This discrepancy may stem from error introduced by the smaller sample size obtained from the initial findings or to other factors remaining outside the parameters of the current study. The task did, however, induce a slight hypothalamic pituitary adrenal (HPA) axis response indicating some physiological arousal. Males had significantly higher cortisol levels at baseline than females and at time point 3 while approaching significance at time points 2 and 4. Females possessed significantly higher trait anxiety than males and all post task cortisol levels were positively correlated to age while time points 2 and 4 (with time point 3 approaching significance, p=0.09) were inversely correlated with Self Depreciation scores. Additionally, females had Persecutory Ideas scores that were also negatively correlated with cortisol at time points 3 and 4. For both the entire sample and males only, the correlation between post-task cortisol and driving performance was positive and approached significance (p=0.07 and p=0.08, respectively), suggesting that some HPA activation may be facilitative for successful driving task performance. Correlations between driving performance and psychological constructs were explored and discussed with and without respect to gender.
- Age-Dependent Effects Of Chronic GABAA Receptor Blockade In Barrel Cortex
- 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.
- Aging Is a Determinant in Anoxia Stress Tolerance in Caenorhabditis Elegans
- Oxygen availability is critical for survival for most organisms. The nematode, C. elegans, has been useful for studying genetic regulation of anoxia tolerance due to the oxygen deprivation response mechanisms shared with other metazoans. Studies examining long-term anoxia (72h, LTA) tolerance have only been conducted at adult day 1. To investigate the effect of aging on anoxia tolerance wild-type and mutant strains were exposed to LTA between adult day 1 and day 9. Wild-type isolates and daf-16(mu86) (FOXO transcription factor regulated by insulin-signaling) and aak-2(gt33) (catalytic subunit of AMP-activated protein kinase) strains were anoxia sensitive at day 1 and displayed increased LTA tolerance with aging correlated with reproductive senescence followed by a decline in survivorhsip through day 9. The daf-2(e1370) (insulin receptor homologue of C. elegans), glp-1(e2141) (a lin-12/Notch receptor) and fog-2(q71) (required for spermatogenesis) strains were LTA-tolerant through day 5. I conclude that aging influences LTA-tolerance in a strain- and age-dependent manner. In addition to being LTA-tolerant the daf-2(e1370) and glp-1(e2141) strains have a longevity phenotype that is suppressed by loss of kri-1 or daf-12. While loss of kri-1 did not suppress the LTA-tolerant phenotype of glp-1(e2141) at day 1 the portion of impaired survivors increased at day 3 and by day 5 tolerance was suppressed. Similarly, when exposed to 4 days of anoxia the glp-1(e2141);daf-12(rh41rh611) double mutant had a reduced survivor rate at all ages analyzed compared to glp-1(e2141) controls. To better understand formation of an anoxia-tolerant physiology I exposed adults to one or more 24h bouts. Recurrent bouts increased LTA tolerance in wild-type hermaphrodites in a dose-dependent manner. Bout-treated daf-16(mu86) animals had increased survival rate compared to controls yet maximum survival remained below age-matched wild-type. Anoxia bouts decreased LTA-tolerance in aak-2(gt33) mutants, indicating the requirement for ATP regulation in establishing an LTA-tolerant phenotype. These data support the idea that anoxia tolerance is multi-factorial and influenced by environment, metabolism, food, reproduction, sex phenotype and likely additional factors.
- Alterations in Fatty Acid Amide Hydrolase (Faah) Transcript Levels and Activity Lead to Changes in the Abiotic Stress Susceptibility of Arabidopsis Thaliana
- N-Acylethanolamines (NAEs) are a class of bioactive lipids, and FAAH is one of the enzymes responsible for degrading NAEs in both plants and animals. in plants, FAAH appears to be closely associated with ABA, a phytohormone which has long been associated with plant stress responses, since the overexpression of FAAH in Arabidopsis results in ABA hypersensitivity. Therefore, it is reasonable to speculate that alterations in FAAH transcript levels will result in altered stress responses in plants. to investigate this hypothesis experiments were carried out in which wild type (WT), FAAH-overexpressing (OE), and T-DNA insertional FAAH knockouts of Arabidopsis (faah) were grown in MS media under stress conditions. the stress conditions tested included chilling stress, heavy metal stress induced by cadmium or copper, nutrient limitations induced by low phosphorus or low nitrogen, salt stress induced with NaCl, and osmotic stress induced with mannitol. the OE plants were consistently hypersensitive to all stress conditions in relation to wild type plants. Inactive FAAH overexpressors did not have the hypersensitivity to the salt and osmotic stress of the active OE plants and were instead tolerant to these stresses. FAAH2 (faah2) knockouts and FAAH 1 and 2 double knockouts (faah 1+2) were based on some root development parameters somewhat more tolerant than WT plants, but more sensitive in terms of shoot growth. Collectively the data suggests that FAAH activity may interact with stress-responsive pathways in plants, perhaps including pathways involving ABA.
- Analysis and Development of Post Secondary Curriculum on Sustainability
- This thesis examines existing curricula at colleges and universities about sustainability and uses results to develop an introductory post secondary course curriculum. The proposed course is organized around three major elements - - science, philosophy, and economics - - all integral to understanding sustainability. Materials needed to teach the proposed 3-semester hour course including syllabus, teaching modules, transparencies, handouts, and exams were developed. Suggestions on how to teach a one-semester hour course on sustainability and a workshop on sustainability are also presented. The following research and curriculum development was a project established and funded by the Texas Energy Office, Renewable Resources and Sustainability Program.
- Analysis and expression of the cotton gene for the D-12 fatty acid desaturases 2-4 (FAD2-4)
- A genomic clone containing a 16.9-kb segment of cotton DNA was found to encompass a D-12 fatty acid desaturases (FAD2-4) gene. The FAD2-4 gene has a single, large intron of 2,780 bp in its 5'-untranslated region, just 12 bp upstream from the ATG initiation codon of the FAD2-4 opening reading frame. A number of prospective promoter elements, including several light-responsive sequences, occur in the 5'-flanking region. The coding region of the gene is 1155 bp with no introns, and would encode a FAD2-4 polypeptide of 384 amino acids. The putative protein had four membrane-spanning helices, hallmarks of an integral membrane protein, and would probably be located in the endoplasmic reticulum. The FAD2-4 gene is indeed a functional gene, since yeast cells transformed with a plasmid containing the coding region of the gene synthesize an appreciable amount of linoleic acid (18:2), not normally made in wild-type yeast cells. The FAD2-4 gene has many structural similarities to the cotton FAD2-3 gene that was also analyzed in this laboratory.
- Analysis of a Cotton Gene Cluster for the Antifungal Protein Osmotin
- 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.
- Analysis of the Expression Profiles of Two Isoforms of the Antifungal Protein Osmotin from Gossypium hirsutum
- The expression of two cotton osmotin genes was evaluated in terms of the mRNA and protein expression patterns in response to chemical inducers such as ethylene, hydrogen peroxide, and sodium chloride. Reverse transcriptase-polymerase chain reactions (RT-PCR) indicated that osmotin mRNAs are expressed constitutively in root tissues of cotton plants, and that they are rapidly induced in leaf and stem tissues upon ethylene treatment. Real time RT-PCR indicated that osmotin transcript levels were induced 2 to 4 h after treatment with ethephon. The osmotin mRNA levels appear to increase 12 h after treatment, decrease, and then increase again. The osmotin protein expression patterns were analyzed in Western blot analyses using an anti-osmotin antibody preparation. A 24-KDa protein band was detected from cotton plants treated with the inducers. The 24-KDa osmotin proteins were induced 4 h after treatment with ethephon, while down-regulated 96 h after treatment. Multiple osmotin isoforms were observed to be induced in cotton plants upon treatment with ethephon by two-dimensional gel electrophoresis. One goal of this dissertation research was to genetically engineer two cotton osmotin genes to routinely overproduce their antifungal proteins in transgenic Arabidopsis and cotton plants as a natural defense against fungal infections, using co-cultivation with Agrobacterium tumefaciens cells harboring pCAMBIA 2301 vector constructs containing the osmotin genes. Many transgenic Arabidopsis and cotton plants were generated. However, genomic blotting analyses indicated the absence of the osmotin transgenes, but the presence of GUS genes from the vector cassette. Alkaline blot analyses of the vector DNAs from transformed Agrobacterium cells confirmed that an anomalous DNA structural rearrangement or aberrant recombination event probably occurred in the Agrobacterium cells, interdicting the integration of osmotin transgenes into the Arabidopsis and cotton plants. This research provides crucial baseline information on expression of cotton osmotin mRNAs and proteins.
- Analysis of the one-horned rhinoceros (Rhinoceros unicornis) habitat in the Royal Chitwan National Park, Nepal.
- This study analyzes the remaining suitable habitat of the one-horned rhinoceros, Rhinoceros unicornis, in Royal Chitwan National Park of Nepal. An April 2003 Landsat image was classified into eight land cover types: wetland, sand, water, mixed forest, sal forest, agriculture, settlement, and grassland. This image was converted into habitat suitability maps using cover, food, and water. The rhinoceros prefers grassland habitat with oxbow lakes and closed canopy during the monsoon season. Nominal values of five parameters were used to create a map of habitat suitability index. The map was categorized into four habitat classes: highly unsuitable, unsuitable, moderately suitable habitat, and suitable. Landscape metrics, patch metrics and class metrics associated with habitat were determined through the use of FRAGSTATS.
- Analysis of the Trypanosoma brucei Genome and Identification and Characterization of a Gene Family Encoding Putative EF-Hand Calcium-Binding Proteins
- The flagellum of Trypanosoma brucei contains a family of antigenically related EF-hand calcium-binding proteins which are called the calflagins. Genomic Southern blots indicated that multiple copies of calflagin genes occur in T brucei. All of the copies were contained in a single 23 kb Xhol-Xhol fragment. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. Two new members of the calflagin family were found from genomic clone sequences. The deduced amino acid sequences of the genomic clones showed the calflagin genes were arranged tandemly along the genomic fragments and were similar to previously described calflagins. The calflagin genes were related by two unrelated 3' flanking sequences. An open reading frame that was unrelated to any calflagin was found at the 5' end of the 2.5 kb genomic fragment. Each encoded protein (~24,000u) contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. In general, variability among the T. brucei calflagins is greater than related proteins in T. lewisii and T. cruzi. This variability results from amino acid substitutions at the amino and carboxy termini, and duplication of internal segments.
- Anatomical and Morphological Responses of Cardiospermum Halicacabum L. (Balloon Vine), to Four Levels of Water Availability
- C. halicacabum (Sapindaceae) is an invasive plant that is considered a nuisance species in Texas riparian environments. Little is known of the tolerance of C. halicacabum to flooding and drought; however, this information may provide insight into the characteristics that contribute to C. halicacabum purported invasiveness. C. halicacabum seedlings (n = 92) were exposed to one of four levels of water availability (flooded, saturated, intermediate and dry) over six weeks under greenhouse conditions. Plant performance was affected by water availability; however, there was no effect on survivorship. Flooded and saturated plants exhibited morphological adaptations; producing adventitious roots, hypertrophy, and aerenchyma tissue. Morphological measures, anatomical responses, and patterns of biomass allocation all indicate that C. halicacabum is able to survive periodic inundation, perform in saturation, and establish and thrive on the drier end of a moisture gradient.
- Animal contribution to human medicine
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The use of animal models in research has led to a fierce debate between animal rights activists and scientists. The former claim that little useful information is gained from animal studies and the suffering of animals does not preclude any treatments which may be used to treat human illnesses. Yet, research scientists claim that in vivo animal models are of absolute necessity to developing treatments and cures to disease. To determine the necessity of animal use, one must examine the models currently in research. Have the animal models for disorders such as cystic fibrosis and muscular dystrophy given scientists enough information to develop effective treatments? This paper will examine the role of animal subjects in several disease research protocols, as well as the applicability of the research.
- 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.
- Applications of Molecular Genetics to Human Identity.
- The primary objectives of this project were: 1. to develop improved methods for extraction of DNA from human skeletal remains, 2. to improve STR profiling success of low-copy DNA samples by employing whole genome amplification to amplify the total pool of DNA prior to STR analysis, and 3. to improve STR profiling success of damaged DNA templates by using DNA repair enzymes to reduce the number/severity of lesions that interfere with STR profiling. The data from this study support the following conclusions. Inhibitory compounds must be removed prior to enzymatic amplification; either during bone section pretreatment or by the DNA extraction method. Overall, bleach outperformed UV as a pretreatment and DNA extraction using silica outperformed microconcentration and organic extraction. DNA repair with PreCR A outperformed both whole genome amplification and repair with PreCR T6. Superior DNA extraction results were achieved using the A6 PMB columns (20 ml capacity column with 6 layers of type A glass fiber filter), and DNA repair with PreCR A led to an overall improvement in profile quality in most cases, although whole genome amplification was unsuccessful. Rapid, robust DNA isolation, successful amplification of loci from the sample-derived DNA pool, and an elimination of DNA damage and inhibitors may assist in providing sufficient genetic information from cases that might otherwise lie on the fringe of what is possible to obtain today.
- Applications of remote sensing and GIS to modeling fire for vegetative restoration in Northern Arizona
- An accurate fire model is a useful tool in predicting the behavior of a prescribed fire. Simulation of fire requires an extensive amount of data and can be accomplished best using GIS applications. This paper demonstrates integrative procedures of using of ArcGIS™, ERDAS Imagine™, GPS, and FARSITE© to predict prescribed fire behavior on the Kaibab-Paiute Reservation. ArcGIS was used to create a database incorporating all variables into a common spatial reference system and format for the FARSITE model. ArcGIS Spatial Analyst was then used to select optimal burn sites for simulation. Our predictions will be implemented in future interagency efforts towards vegetative restoration on the reservation.
- The aquatic insect communities of Holbrook Creek and Cochetopa Creek in Colorado.
- The first objective for this problem in lieu of thesis project was to gather, identify to the lowest practical taxonomic level and organize all available aquatic insects collected from high altitude Colorado aquatic systems during the summers of 1994, 1996, 1998, and 2002 for the University of North Texas Environmental Science Field Course (BIOL 5650). The curated collection will be housed in the Elm Fork Natural History Museum, located at the University of North Texas. The second objective was to provide a summary and discussion of the occurrence and distribution of the aquatic insects collected from Mt. Blanca in 1994, 1996, and 1998 and to create a taxa list of aquatic insects collected from Cochetopa Creek during the summer of 2002.
- Aspartate transcarbamoylase of Aeromonas hydrophila
- This study focused on the enzyme, aspartate transcarbamoylase (ATCase) from A. hydrophila, a Gram-negative bacterium found in fresh water. The molecular mass of the ATCase holoenzyme from A. hydrophila is 310 kDa. The enzyme is likely composed of 6 catalytic polypeptides of 34 kDa each and 6 regulatory polypeptides of 17 kDa each. The velocity-substrate curve for A. hydrophila ATCase is sigmoidal for both aspartate and carbamoylphosphate. The Km for aspartate was the highest to date for an enteric bacterium at 97.18 mM. The Km for carbamoylphosphate was 1.18 mM. When heated to 60 ºC, the specific activity of the enzyme dropped by more than 50 %. When heated to 100 ºC, the enzyme showed no activity. The enzyme's activity was inhibited by ATP, CTP or UTP.
- Assembly of Pseudomonas putida Aspartate Transcarbamoylase and Possible Roles of the PyrC' Polypeptide in the Folding of the Dodecameric Enzyme
- Aspartate transcarbamoylase (ATCase) of Pseudomonas putida consists of two different polypeptides, PyrB and PyrC' (Schurr et al, 1995). The role of the PyrC' and the assembly of PyrB and PyrC' have been studied. The ATCase made in vitro of P.putida PyrB with P.putida PyrC', and of E.coli PyrB with P.putida PyrC ' were generated under two different conditions, denaturation and renaturation, and untreated. It was found that PyrC' plays a role in the enzymatic regulation by ATP, CTP and UTP. In addition to playing a role in substrate binding, the PyrB polypeptide is also involved in effector binding (Kumar et al., manuscript in preparation). The most energetically preferred form of the P.putida WT is a dodecamer with a molecular mass of 480 kDa. The ratio between the PyrB and the PyrC' is 1:1. In studies of nucleotide binding, it was discovered that the P.putida PyrB was phosphorylated by a protein kinase in the cell extract. In the presence of 20 mM EDTA, this phosphorylation was inhibited and the inhibition could be overcome by the addition of divalent cations such as Zn2+ and Mg2+. This result suggested that the phosphorylation reaction required divalent cations. In the CAD complex of eukaryotes, phosphorylations of the CPSase and the linker region between ATCase and DHOase did not occur in the presence of UTP and it was hypothesized (Carrey, 1993) that UTP and phosphorylation(s) regulated the conformational change in the enzyme complex. Therefore, the same idea was approached with P.putida ATCase, where it was found that 1.0 mM UTP inhibited the phosphorylation of PyrB by more than 50%. These results suggested that the regulation of the conformational change of the P.putida ATCase might be similar to that of CAD. Furthermore, peptide mapping for phosphorylation sites was performed on P.putida ATCase WT, WT --11 amino acids and WT --34 amino acids from the N-terminus of the PyrB polypeptide. The results showed that the phosphorylation sites were located on the fragment that contained amino acid number-35 to amino acid number-112 from the N-terminus of the PyrB polypeptide.
- Assessing Effects of an Environmental Education Field Science Program Fostering Responsibility at an Urban Middle School
- The study investigated the ability of an extracurricular program to influence environmental responsibility of sixth and seventh graders. The Children's Environmental Attitude and Knowledge Survey (CHEAKS) was evaluated for appropriateness in assessing the worth of this particular environmental education strategy emphasizing water quality fieldwork and technology. CHEAKS is designed with psychometric reliability and validity that may be used in comparing disparate programs. Wilcoxon two sample tests were used to analyze data gathered from two student groups; one participated in an "Enviro-Mentals Club"; the other received no treatment. Analysis showed no significant change in environmental attitudes between groups, but did show significance (p <= 0.05) in environmental knowledge growth. Therefore, the investigated program had marginal success in influencing environmental responsibility.
- Assessing Outcomes of a Recycling Education and Service Program within an Elementary School
- During the spring 2004 a pilot school recycling program was implemented within Robert E. Lee Elementary. The primary goal of the program was to determine how recycling education in the school would affect curbside recycling rates within the surrounding community. The program was a cooperative effort between the University of North Texas, City of Denton Solid Waste Department and Keep Denton Beautiful. Throughout the first months of the study during the spring 2004, an increase in curbside recycling within the Robert E. Lee Elementary attendance zone was observed, with a dramatic decrease in participation over the summer and a rapid increase once again during the second full semester of the study. In a survey conducted with 3rd and 5th grade students at the pilot project school, most students expressed positive attitudes about recycling. Students whose survey responses indicated a high level of knowledge about what could be recycled were 37% more likely to claim to recycle regularly, than those students that scored low on the knowledge portion of the survey. Although the total amount of waste generation (recyclable and non-recyclable) at Robert E. Lee Elementary did not decrease during the study, the campus was able to divert recyclable material from their trash at a much higher rate than two other local elementary campuses with paper-only recycling and no associated recycling education program. Based upon the success of the recycling program at Robert E. Lee Elementary, the City of Denton Recycling Division has agreed to move forward with offering recycling to more schools within the Denton Independent School District during the 2005-2006 school year.
- Assessing the Effects of a Municipal Wastewater Treatment Plant Effluent on Zooplankton, Phytoplankton and Corbicula Flumina in a Constructed Wetland
- Wetland wastewater treatment offers low-cost, energy efficient alternatives to conventional wastewater technologies. In this study, an artificial wetland was constructed at the City of Denton, Texas Pecan Creek Water Reclamation Plant to facilitate diazinon removal from treated effluent.
- Assessing the Spatial and Temporal Distribution of MTBE and BTEX Compounds in Lake Lewisville, Texas February 1999 - February 2000
- The spatial and temporal distribution of Methyl Tertiary-Butyl Ether (MTBE) and BTEX (Benzene, Toluene, Ethylbenzene, Xylenes) compounds were assessed in a multipurpose reservoir, Lake Lewisville, Texas between February 1999 and February 2000. Concentrations of MTBE ranged from 0.0 - 16.7 mg/L. Levels of MTBE in the lake were related to watercraft. BTEX concentrations were never detected above 2.0 mg/L during the sampling period. Finished drinking water from Denton and the Upper Trinity Regional Water District (UTRWD) Treatment Plants were also tested for MTBE and BTEX. MTBE and BTEX were not detected in UTRWD water samples. Denton's finished water samples never exceeded 2.2 mg/L for MTBE and BTEX was not detected except for one replicate of 1.1 mg/L toluene.
- An Assessment of Storm Water Toxicity from the Dallas/Fort Worth Metroplex and Denton, Texas
- With the advent of national storm water regulations, municipalities with populations greater than 100,000 are required to obtain National Pollutant Discharge Elimination System Permits (NPDES) for storm water discharges. In addition to the sampling required for the permit process, the City of Fort Worth contracted with the University of North Texas' Institute of Applied Sciences to conduct acute toxicity testing using Pimephales prcmelas and Ceriodaphnia dubia on storm water samples received from the Dallas/Fort Worth Metroplex. A Toxicity Identification Evaluation (TIE) was performed on four samples that exhibited acute toxicity to C. dubia. High levels of metals as well as diazinon were some of the probable toxicants found.
- An Assessment of the Use of Seeding, Mowing, and Burning in the Restoration of an Oldfield to Tallgrass Prairie in Lewisville, Texas
- An examination of the effectiveness of seeding, burning, and mowing in the reestablishment of tallgrass prairie species on overgrazed and abandoned pastureland. The study site is a 20 acre tract on U.S. Corps of Engineers land below Lake Lewisville in Denton County, Texas. The site was partitioned into thirty-nine 40 by 40 meter plots with seeding (carried out in 1996) and management treatment (burning, mowing, and no maintenance carried out in 1998) randomly applied following a two level design. For each plot, nine stratified-random 0.1 m2 subplots were examined and shoot counts for each species recorded. The effects of the treatments on individual species and species richness were analyzed with a two-way ANOVA followed by a SNK multiple range test, both on ranked data. Community level analysis was conducted with both a MANOVA on ranked data and a Canonical Correspondence Analysis on raw data. Results indicate that seeding positively affected species richness, particularly when combined with either burning or mowing in the early spring. Mowing also significantly increased species richness in areas that were not seeded, while burning negatively affected species richness on unseeded plots. Treatments significantly affected community composition with treatments having the most clear effect on spring and summer forbs.
- Attenuation of Escherichia Coli Aspartate Transcarbamoylase Expressed in Pseudomonas Aeruginosa Mutant and Wild Type Strains
- No apparent repression of pyr gene expression in Pseudomonas aeruginosa is observed upon addition of exogenous pyrimidines to the growth medium. Upon introduction of the subcloned Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) into a P. aeruginosa pyrB mutant strain, repression was observed in response to exogenously fed pyrimidine compounds. The results proved that it is possible to bring about changes in pyrimidine nucleotide pool levels and changes in transcriptional regulation of gene expression as a result. Thus, the lack of regulatory control in P. aeruginosa pyr gene expression is not due to an inability to take up and incorporate pyrimidine compounds into metabolic pools, or to an inability of the RNA polymerase to respond to regulatory sequences in the DNA but is probably due to a lack of specific regulatory signals in the promoter of the genes themselves.
- 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.
- Autonomic reflexes of the heart during acute myocardial ischemia
- This study investigated whether acute myocardial ischemia of the anterior left ventricular wall induced an increase in cardiac sympathetic efferent nerve activity and thereby affected regional myocardial blood flow and contractile function.
- Autophosphorylation and Autoactivation of an S6/H4 Kinase Isolated From Human Placenta
- A number of protein kinases have been shown to undergo autophosphorylation, but few have demonstrated a coordinate increase or decrease in enzymatic activity as a result. Described here is a novel S6 kinase isolated from human placenta which autoactivates through autophosphorylation in vitro. This S6/H4 kinase, purified in an inactive state, was shown to be a protein of Mr of 60,000 as estimated by SDS-PAGE and could catalyze the phosphorylation of the synthetic peptide S6-21, the histone H4, and myelin basic protein. Mild digestion of the inactive S6/H4 kinase with trypsin was necessary, but not sufficient, to activate the kinase fully
- Autoradiographic localization of carbachol-induced second messenger response in the rat spinal cord following inflammation.
- This study examined central mechanisms of persistent pain using an autoradiographic technique to localize phosphoinositide hydrolysis (PI) in the rat spinal cord dorsal horn. The lateral half of laminae I-II showed the highest levels of baseline PI turnover and carbachol-stimulated PI turnover in normal animals as well as after inflammation. Inflammation resulted in increased baseline PI turnover in this region of the ipsilateral (76%) and contralateral (65%) dorsal horns. Carbachol increased PI turnover in this region in normal rats (55%) and following inflammation (ipsilateral: 46%, contralateral: 45%). The absolute magnitudes of these increases were 1.85, 2.71, and 2.51 nCi/mg, respectively. The results of this study demonstrate the involvement of PI turnover in neural mechanisms of persistent pain, and provide evidence for the involvement of cholinergic systems in this process. Because spinal cholinergic systems have been reported to be anti-nociceptive, the present results appear to reflect an upregulation of anti-nociceptive activity in response to inflammation. Thus, the spinal cholinergic system may be a regulatory site within the anti-nociceptive pathway, and may provide an attractive target for the development of new therapeutic agents.
- Bacterial challenge in Lumbricus terrestris: A terrestrial invertebrate immunotoxicity model.
- A bacterial challenge assay was developed utilizing the earthworm, Lumbricus terrestris, in order to assess potential immunotoxic effects from exposure to specific polychlorinated biphenyl congeners. Earthworms were inoculated with Aeromonous hydrophila, establishing a 10-day LD50. In vitro assays for effects of PCBs on phagocytosis agreed with mammalian studies, demonstrating potent suppression of phagocytosis by the non-coplanar PCB congener 138 and no suppression by the coplanar congener 126. However, when the effects of the two PCB congeners were evaluated for suppression of resistance to a whole animal infection challenge assay, coplanar PCB 126 decreased the ability of L. terrestris to withstand infection while non-coplanar PCB 138 did not.
- Bacterial Cyanide Assimilation: Pterin Cofactor and Enzymatic Requirements for Substrate Oxidation
- Utilization of cyanide as the sole nitrogen source by Pseudomonas fluorescens NCIMB 11764 (Pf11764) occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated oxygenolytically by an enzyme referred to as cyanide oxygenase (CNO), which exhibits properties of a pterin-dependent hydroxylase. The pterin requirement for Pf11764 CNO was satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 µM). These compounds included, for example, biopterin, monapterin and neopterin, all of which were also identified in cell extracts. A related CNO-mediated mechanism of cyanide utilization was identified in cyanide-degrading P. putida BCN3. This conclusion was based on (i) the recovery of CO2 and NH3 as enzymatic reaction products, (ii) the dependency of substrate conversion on both O2 and NADH, and (iiii) utilization of cyanide, O2 and NADH in a 1:1:1 reaction stoichiometry. In contrast to findings reported for Pf11764, it was not possible to demonstrate a need for exogenously added pterin as a cofactor for the PpBCN3 enzyme system. However, results which showed that cells of PpBCN3 contained approximately seven times the amount of pterin as Pf11764 (of which a significant portion was protein-bound) were interpreted as indicating that sufficient bound CNO-cofactor exists, thus eliminating any need for a supplemental source.
- A Behavioral Model for Detection of Acute Stress in Bivalves
- A behavioral model for acute responses in bivalves, was developed using time series analysis for use in a real-time biomonitoring unit. Stressed bivalves closed their shell and waited for the stressful conditions to pass. Baseline data showed that group behavior of fifteen bivalves was periodic, however, individuals behaved independently. Group behavior did not change over a period of 20 minutes more than 30 percent, however, following toxic exposures the group behavior changed by more than 30 percent within 20 minutes. Behavior was mathematically modeled using autoregression to compare current and past behavior. A logical alarm applied to the behavior model determined when organisms were stressed. The ability to disseminate data collected in real time via the Internet was demonstrated.
- Benthic Macroinvertebrates of Temperate, Sub-Antarctic Streams: The Effects of Altitudinal Zoning and Temperature on the Phenology of Aquatic Insects Associated to the Robalo River, Navarino Island (55°S), Chile
- The Cape Horn Biosphere Reserve, within the remote Sub-Antarctic ecoregion is a reservoir of expressions of biological and cultural diversity. Although it is considered one of 24 wilderness areas remaining in the world, it is not free from local and global threats, such as invasive species, and climate change. Field biologists and philosophers associated to the Sub-Antarctic Biocultural Conservation Program and the Omora Ethnobotanical Park, have worked to describe the region’s biocultural diversity, linking ecological and philosophical research into education, ecotourism, and conservation, through a methodology called field environmental philosophy (FEP), which integrates ecological sciences and environmental ethics through a 4-step cycle consisting of: 1) interdisciplinary research; 2) composition of metaphors; 3) design of field activities with an ecological and ethical orientation; and 4) implementation of in situ conservation areas. In this context, the purposes of this dissertation were to: 1) provide a comprehensive review of publications regarding the conservation status of aquatic and terrestrial insects at a global scale and with an emphasis in southern South America; 2) study the distribution of benthic macroinvertebrates through the sharp altitudinal gradient of the Róbalo River watershed; 3) describe the life histories of Gigantodax sp (Simuliidae: Diptera) and Meridialaris chiloeense (Leptophlebiidae: Ephemeroptera) in the Róbalo River and to assess the potential effects of climate change on their phenology; and 4) to apply FEP methodology in order to better understand and communicate the intrinsic and instrumental values of freshwater invertebrates in the Cape Horn Biosphere Reserve.
- Beta-adrenergic Blockade Via Atenolol and Its Effects on Blood Pressure, Heart Rate, and Renal Morphology in the Developing Chicken Gallus Gallus Domesticus
- Chicken embryos were chronically exposed to the ?1- blocker atenolol during one of three stages: mesonephros (E7-E9), mesonephros-metanephros (E11-E13), or metanephros (E15-E17). Mesonephros group hearts were larger than all other groups (P < 0.01). Mesonephros and metanephros group kidneys were larger than all remaining groups (P < 0.0001). The mesonephros group nephron number was ~40% lower than control values (P = 0.002). Glomerular areas were 26% and 18% larger than the control group in the mesonephros and metanephros groups, respectively (P < 0.001). These data suggest an E7-E9 critical window of cardiovascular and renal development for atenolol. Acute atenolol exposure in E15 embryos showed an increase in mean arterial pressure with all but the highest dose. All doses significantly decreased heart rate.
- Bioaccumulation of triclocarban, triclosan, and methyl-triclosan in a North Texas wastewater treatment plant receiving stream and effects of triclosan on algal lipid synthesis.
- Triclosan (TCS) and triclocarban (TCC), widely used antimicrobial agents found in numerous consumer products, are incompletely removed by wastewater treatment plant (WWTP) processing. Methyl-triclosan (M-TCS) is a more lipophilic metabolite of its parent compound, TCS. The focus of this study was to quantify bioaccumulation factors (BAFs) for TCS, M-TCS, and TCC in Pecan creek, the receiving stream for the City of Denton, Texas WWTP by using field samples mostly composed of the alga Cladophora sp. and the caged snail Helisoma trivolvis as test species. Additionally, TCS effects on E. coli and Arabidopsis have been shown to reduce fatty acid biosynthesis and total lipid content by inhibiting the trans-2 enoyl- ACP reductase. The lipid synthesis pathway effects of TCS on field samples of Cladophora spp. were also investigated in this study by using [2-14C]acetate radiolabeling procedures. Preliminary results indicate high TCS concentrations are toxic to lipid biosynthesis and reduce [2-14C]acetate incorporation into total lipids. These results have led to the concern that chronic exposure of algae in receiving streams to environmentally relevant TCS concentrations might affect their nutrient value. If consumer growth is limited, trophic cascade strength may be affected and serve to limit population growth and reproduction of herbivores in these riparian systems.
- 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.
- Biochemical Identification of Molecular Components Required for Cyanide Assimilation in Pseudomonas fluorescens NCIMB 11764
- Utilization of cyanide as a nutritional nitrogen source in P. fluorescens NCIMB 11764 was shown to involve a novel metabolic mechanism involving nonenzymatic neutralization outside of cells prior to further enzymatic oxidation within. Several cyanide degrading enzymes were produced by NCIMB 11764 in response to growth or exposure to cyanide, but only one of these cyanide, oxygenase (CNO), was shown to be physiologically required for assimilation of cyanide as a growth substrate.
- Bioconcentration and Morphological Effects of Triclosan on Three Species of Wetland Plants
- Triclosan (TCS) is an antimicrobial compound found in several types of common household products. After being washed down the drain, TCS will then end up in the local watershed. Although numerous studies have been conducted to evaluate the fate and effects of TCS in aquatic environments, there have been no studies evaluating the role arbuscular mycorrhizal fungi (AM) play in a plants response to TCS exposure. Three species of wetland plants native North Texas were inoculated with AM spores and exposed to 0, 0.4 g/L and 4.0 g/L TCS concentrations. Root morphology of E. prostrata and S. herbacea showed AM and exposure responses. S. herbacea produced the greatest amounts biomass and TCS bioaccumulation, in all but one treatment. It also displayed opposing results to E. prostrata in measures of root length, root surface area, relative root mass, relative shoot mass and shoot:root ratio. TCS root tissue concentrations increased with increased exposures for both E. prostrata and S. herbacea. Even though E. prostrata had the lowest levels in each measure of biomass production, it had the highest amount of root TCS bioaccumulation in the AM inoculated 4.0 g/L treatment. H. laevis was between the other two species in terms of biomass production, and did not demonstrate any exposure or inoculation effects in morphology or TCS accumulation. Overall, no clear patterns were detected, which highlights the fact that further study is required to completely understand the effects compounds like TCS can have on plant community structure, and ultimately ecosystem function.
- Bioconcentration of Triclosan, Methyl-Triclosan, and Triclocarban in the Plants and Sediments of a Constructed Wetland
- Triclosan and triclocarban are antimicrobial compounds added to a variety of consumer products that are commonly detected in waste water effluent. The focus of this study was to determine whether the bioconcentration of these compounds in wetland plants and sediments exhibited species specific and site specific differences by collecting field samples from a constructed wetland in Denton, Texas. The study showed that species-specific differences in bioconcentration exist for triclosan and triclocarban. Site-specific differences in bioconcentration were observed for triclosan and triclocarban in roots tissues and sediments. These results suggest that species selection is important for optimizing the removal of triclosan and triclocarban in constructed wetlands and raises concerns about the long term exposure of wetland ecosystems to these compounds.
- Biodiversity of Caddisflies (Trichoptera) of the Interior Highlands of North America
- Caddisflies (Trichoptera) were collected from over 500 different locations throughout the Interior Highlands (Ozark, Ouachita, Arbuckle, and Wichita Mountains) between March 1990 and March 1994. I systematically sampled representative lotic and lentic habitats in 131 natural watersheds that comprise the 17 different physiographic subregions of this area. From my examination of approximately 60,000 specimens, surveys of regional museum collections, and review of literature records, I document 229 species distributed in 16 families and 58 genera. Included in this total are 27 endemic species and 15 new regional records. Descriptions are provided for a species new to science (Cheumatopsyche robisoni), four larvae (Helicopsyche limnella, H. piroa, Marilia species A, Polycentropus crassicornis) and a female (Helicopsyche piroa). Hydropsyche reiseni Denning, previously known only from the Arbuckle Mountains, is reduced in synonymy with H. arinale Ross. Further, I provide illustrated family, generic, and selected species-level keys that reflect this regional biodiversity.
- 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.
- 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.
- Biological Nitrogen Fixation in Two Southwestern Reservoirs
- This investigation has determined the presence of biological nitrogen fixation in two reservoirs in the southwestern United States: Lake Arlington and Lake Ray Hubbard. Subsequent tests have gathered baseline data on the effects of various biological, chemical, and physical parameters on in situ nitrogen fixation in these reservoirs. Of specific importance is the relationship between nitrogen fixation arid occasional blooms of blue-green algae which produce such problems as testes and odors in these water-supply impoundments.