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  Partner: UNT Libraries
 Department: Department of Biological Sciences
 Decade: 2010-2019
 Collection: UNT Theses and Dissertations
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.
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.
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.
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.
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.
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.
Characterization of Arthrobacter Globiformis Aspartate Transcarbamylase Concentrations of Substrates
This thesis consists of one major section with two subsections. The first subsection investigates the activity of Arthrobacter globiformis aspartate transcarbamylase's specific activity with increasing concentrations of the enzyme's substrate. Dihydroorotase (DHOase) activity was also measured with increasing concentrations of the substrate dihydroorotate. The second subsection collected data in order to classify the enzyme, resulting in a classification into the category of class A ATCases with bifunctional ATCase-DHOase complexes. The thesis provides evidence to broaden understanding of the ATCase and DHOase enzymes for members of the family that Arthrobacter belongs to.
A Characterization Of Jackson Blue Spring, Jackson County, Florida
Jackson Blue is a first magnitude spring in the karst terrane of northeast Florida. Previous studies have identified inorganic fertilizer as the source of high nitrate levels in the spring. Agricultural land use and karst vulnerability make Jackson Blue a good model for conservation concerns. This work offers an aggregation of studies relating to the springshed, providing a valuable tool for planning and conservation efforts in the region. An analysis of nitrate levels and other water quality parameters within the springshed did not reveal significantly different values between agricultural and forested land use areas. Confounding factors include: high transmissivity in the aquifer, interspersed land use parcels, and fertilizer application in forested areas due to commercial pine stand activity.
Characterization of Triclocarban, Methyl- Triclosan, and Triclosan in Water, Sediment, and Corbicula Fluminea (Müller, 1774) Using Laboratory, in Situ, and Field Assessments
In the last decade emerging contaminants research has intensified in a bid to answer questions about fate, transport, and effects as these chemicals as they get released into the environment. The chemicals of interest were the antimicrobials; triclocarban (TCC) and triclosan (TCS), and a metabolite of triclosan, methyl triclosan (MTCS). This research was designed to answer the question: what is the fate of these chemicals once they are released from the waste water treatment plant into receiving streams. Three different assessment methods; field monitoring, in-situ experiments, and laboratory studies were used to answer the overall question. TCS, TCC, and MTCS levels were measured in surface water, sediment and the Asiatic clam Corbicula fluminea. Field studies were conducted using four sites at Pecan Creek, Denton TX. Levels of all three chemicals in clams were up to fives orders of magnitude the water concentrations but an order of magnitude lower than in sediment. Highest sediment levels of chemicals were measured in samples from the mouth of Pecan Creek (highest organic matter). TCC was the most and TCS was the least accumulated chemicals. In-situ and lab studies both indicated that uptake of these chemicals into the clams was very rapid and measurable within 24hours of exposure. The after clams were transferred into clean water most of the compounds were depurated within 14 days.
Chronic Hypoxia and Hyperoxia Modifies Morphology and Vegf Expression of the Lungs of the Developing Chicken (Gallus Gallus Domesticus)
This study determines effects of oxygen levels on morphology and VEGF expression of developing chicken lungs following incubation in normoxia (21% O2), hypoxia (15% O2) or hyperoxia (30% O2), until developmental days 16 or 18. Lung morphology was assessed using light microscopy, while VEGF expression was determined with ELISA. In hypoxia, the proportion of parabronchial tissue and parabronchi including lumina increased from day 16 to 18 (61 to 68% and 74.2 to 82.2%, respectively). Non-parabronchial tissue was higher in hypoxia than in hyperoxia on day 16 (26 to 20%). However, by day 18, there were no differences between groups. VEGF expression was 33% higher in hypoxia than in hyperoxia on day 16 (736 vs. 492 pg/ml). On day 18, VEGF expression was 43% higher in hyperoxia than in normoxia (673 to 381pg/ml), and remained elevated by 40% in hypoxia over normoxia (631 pg/ml). VEGF may be a mechanism by which parabronchial tissue is stimulated from day 16 to 18 following exposure to chronic hypoxia.
Clustering Algorithms for Time Series Gene Expression in Microarray Data
Clustering techniques are important for gene expression data analysis. However, efficient computational algorithms for clustering time-series data are still lacking. This work documents two improvements on an existing profile-based greedy algorithm for short time-series data; the first one is implementation of a scaling method on the pre-processing of the raw data to handle some extreme cases; the second improvement is modifying the strategy to generate better clusters. Simulation data and real microarray data were used to evaluate these improvements; this approach could efficiently generate more accurate clusters. A new feature-based algorithm was also developed in which steady state value; overshoot, rise time, settling time and peak time are generated by the 2nd order control system for the clustering purpose. This feature-based approach is much faster and more accurate than the existing profile-based algorithm for long time-series data.
Comparative Phyto-uptake Across Distribution Coefficients of Pharmaceutical Compounds and Aquatic Macrophytes: Carbamazepine and Amiodarone Uptake in Lemna Spp
Few studies have been conducted on the effectiveness of phytoremediation of pharmaceutical compounds, although the persistent and non-acutely toxic nature of many of these compounds in today's water bodies may yield an ideal application for this practice. To quantify the potential effectiveness of plant uptake, kinetic and proportional bioconcentration factors (BCFk, and BCFp, respectively) in nanograms (ng) carbamazepine and amiodarone per gram (g) wet weight plant tissue for Lemna spp. were determined utilizing a 14-day continuous flow-through study. Samples were analyzed using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) running in positive ion mode. Kinetic BCF was estimated at 0.538, while proportional BCF was estimated at 0.485. Kinetic BCF for the amiodarone study was estimated at 23.033, whereas proportional BCF was estimated at 41.340. Possible contamination of the C18 column and peristaltic pump failure may have impacted uptake results. In light of variability and current lack of research in the field, this work should be considered exploratory rather than conclusive.
Comparison of Heteranthera Dubia (Jacq.) MacM.-associated Macroinvertebrates Between Georgraphical Regions in the United States
Macroinvertebrates associated with the aquatic plant, water stargrass (Heteranthera dubia), were sampled from 12 waterbodies in four regions of the United States from June to August 2005. Taxa richness, evenness, and diversity were lowest in the Lower Midwest (LMW) region, and higher in Northern sites, especially the Upper Midwest (UMW), and Northeast (NE). While relative abundance varied from site to site and region to region, utilization of the plant by functional groups remained fairly constant. Collector-gatherers consistently comprised the largest portion of invertebrates sampled. The shredder/ herbivore functional group comprised an average of 17 % of total groups. Through an exhaustive literature review, it was found that shredder/ herbivores of water stargrass have not been reported in the literature. Because of this, the herbivore group was analyzed separately and consisted of 2,383 specimens representing 23 species. The most common groups were Rhopalosiphum sp., Nectopsyche spp. and chironomids. No differences were found in herbivore diversity or evenness between sampling regions, but species richness was significantly different.
A Comparison of Mercury Localization, Speciation, and Histology in Multiple Fish Species From Caddo Lake, a Fresh Water Wetland
This work explores the metabolism of mercury in liver and spleen tissue of fish from a methylmercury contaminated wetland. Wild-caught bass, catfish, bowfin and gar were collected. Macrophage centers, which are both reactive and primary germinal centers in various fish tissues, were hypothesized to be the cause of demethylation of methylmercury in fish tissue. Macrophage centers are differentially expressed in fish tissue based on phylogenetic lineage, and are found primarily in the livers of preteleostean fish and in the spleen of teleostean fish. Histology of liver and spleen was examined in both control and wild-caught fish for pathology, size and number of macrophage centers, and for localization of mercury. Total mercury was estimated in the muscle tissue of all fish by direct mercury analysis. Selenium and mercury concentrations were examined in the livers of wild-caught fish by liquid introduction inductively coupled plasma mass spectrometry (ICP-MS). Total mercury was localized in histologic sections by laser ablation ICP-MS (LA-ICP-MS). Mercury speciation was determined for inorganic and methylmercury in liver and spleen of fish by bas chromatography-cold vapor atomic fluorescence spectroscopy (GC-CVAFS). Macrophage center tissue distribution was found to be consistent with the literature, with a predominance of centers in preteleostean liver and in spleens of teleostean fish. Little evidence histopathology was found in the livers or spleens of fish examined, but differences in morphology of macrophage centers and liver tissue across species are noted. the sole sign of liver pathology noted was increased hepatic hemosiderosis in fish with high proportions of liver inorganic mercury. Inorganic mercury was found to predominate in the livers of all fish but bass. Organic mercury was found to predominate in the spleens of all fish. Mercury was found to accumulate in macrophage centers, but concentrations of mercury in this compartment were found to vary less in relation to total mercury than hepatocyte mercury. No association was found between selenium content and inorganic mercury proportions. Overall, findings from this study to not support a primary role for macrophage centers in the demethylation of methylmercury in fish tissues.
Designing Tools to Probe the Calcium-dependent Function of Arabidopsis Tonneau2
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Plants possess unique features in many aspects of development. One of these features is seen in cell wall placement during cytokinesis, which is determined by the position of the preprophase band (PPB) and the subsequent expansion of the phragmoplast that deposits the new cell wall. During phragmoplast expansion, the phragmoplast tracks to the cortical division site, which was delineated by the PPB. Thus the position of the PPB determines the orientation of the division plane. In Arabidopsis thaliana, TONNEAU2 (TON2) is required for PPB formation and has been shown to interact with a type A subunit of the PP2A phosphatase in the yeast two-hybrid system. In Arabidopsis tonneau2 (ton2) mutants, abnormalities of the cortical microtubule cytoskeleton, such as disorganization of the interphase microtubule array and lack of PPB formation before mitosis markedly affects cell shape and arrangement as well as overall plant morphology. Loss of dcd1/add1, the maize ton2 homologues gives rise to a similar phenotype in Zea mays. The TON2 protein has two EF hand domains which are calcium-binding sites. Since calcium has been known to play key roles in several areas of plant functioning, the following question was raised: “Does calcium binding contribute to the localization and function of TONNEAU at the PPB?” To address this question, a series of constructs were generated to determine if TON2 binds calcium. Additionally, Ca2+ binding sites were mutated in constructs containing the TON2 gene fused to GFP or YPF. These constructs were then transformed into ton2 mutant plants and the localization of TON2 fusion protein and whether the construct is capable of rescuing the mutant phenotype were observed. Although, localization of TON2 to the PPB was not observed, the presence of the constructs were confirmed in the transformed plants using selection markers and by observing fluorescence under a confocal microscope.
Determination of Bioconcentration Potential of Selected Pharmaceuticals in Fathead Minnow, Pimephales Promelas, and Channel Catfish, Lctalurus Punctatus
The primary objective of this work was to determine the tissue-specific bioconcentration factors (BCFs) of the selected pharmaceuticals: norethindrone (NET), ibuprofen (IBU), verapamil (VER), clozapine (CLZ) and fenofibrate (FFB) in two freshwater fishes: fathead minnow and channel catfish. BCF tests on fathead followed the standard OECD 42-day test while a 14-day abridged test design was used in catfish exposures. Additional objectives included a) comparing the measured BCFs to the US EPA's BCFWIN model predicted values, b) comparing the BCF results from the standard and reduced tests, and c) prediction of chronic risk of the pharmaceuticals in fish using the human therapeutic plasma concentrations. Each test included uptake and depuration phases to measure tissue-specific kinetic BCFs. The results indicated that all the pharmaceuticals, except IBU, have the potential for accumulation in fish. Estimated BCFs for NET, VER and FFB may not be significant in view of the current regulatory trigger level (BCF &#8805; 2000); however, CLZ's BCF in the liver had approached the criterion level. Significant differences were noticed in the tissue-specific uptake levels of the pharmaceuticals with the following general trend: (liver/kidney) > (gill/brain) > (heart/muscle) > plasma. IBU uptake was highest in the plasma. When compared to the measured BCFs, predicted values for NET, IBU, VER and FFB were slightly overestimated but did not differ largely. However, the measured BCF of CLZ in the liver was approximately two-orders of magnitude higher than the predicted level. The tissue-BCFs for the two species were not widely different indicating the potential usefulness of the reduced BCF test. Comparison of fish and human plasma levels indicated that NET, CLZ and VER have the potential to cause chronic effects in fish.
DNA Degradation as an Indicator of Post-Mortem Interval
The question of post-mortem interval (PMI) or time since death is often the most sought after piece of information associated with a medical death investigation. Based on the observation that DNA degradation disproportionately affects the analysis of larger genetic loci, it was proposed that DNA degradation, as a result of autolysis or putrefaction, could prove suitable as a potential rate-of-change indicator of PMI. Nine randomly amplified polymorphic DNA (RAPD) analysis primers and three sets of directed amplification primers were evaluated to determine their suitability for use in assessing the degree of DNA fragmentation in tissue samples. They were assessed for amplicon specificity, total DNA target sensitivity, allele monomorphism and the observance of degradation-based profile changes. Markers meeting the requisite criteria were then used to assess a range samples degraded under controlled and uncontrolled conditions. Tissue samples collected from seven domestic pigs (Sus scrofa) were incubated under controlled laboratory or uncontrolled field conditions to produce samples simulating those potentially collected in a forensic case. DNA samples isolated from these specimens were then analyzed at those loci which had been determined to meet the requisite criteria. Collectively, data generated from these analyses indicate that genetic profiles generated by this approach can provide information useful for estimating the post-mortem interval, with the locus and amplicons used being most useful during the first 72 hours after death.
Ecological Significance and Underlying Mechanisms of Body Size Differentiation in White-tailed Deer
Body size varies according to nutritional availability, which is of ecological and evolutionary relevance. The purpose of this study is to test the hypothesis that differences in adult body size are realized by increasing juvenile growth rate for white-tailed deer (Odocoileus virginianus). Harvest records are used to construct growth rate estimates by empirical nonlinear curve fitting. Results are compared to those of previous models that include additional parameters. The rate of growth increases during the study period. Models that estimate multiple parameters may not work with harvest data in which estimates of these parameters are prone to error, which renders estimates from complex models too variable to detect inter-annual changes in growth rate that this simpler model captures
The Ecology and Paleobiogeography of Freshwater Mussels (Family: Unionidae) from Selected River Basins in Texas
This dissertation has two overall objectives: first, to demonstrate the utility of paleozoological data for ongoing and future mussel-conservation efforts in Texas and second, to evaluate whether simple measures of habitat (e.g., water depth, velocity and particle size) are important for demonstrating the within-habitat spatial separation of mussels. Although these topics may seem disparate, both are important for increasing our understanding of unionid ecology and biogeography. Chapters 1 through 3 examine the use of paleozoological data for mussel conservation. Although these types of data are not new they have rarely been used in mussel conservation efforts within Texas. This is unfortunate because paleozoological data can provide an excellent record of the mussel fauna prior to wide-scale modern impacts and in areas where historical survey data are lacking. Chapter 4 examines whether assessments of microhabitat for mussels using simple measures of habitat (e.g., water velocity, depth and particle size) are useful. Recent studies have suggested that these measures do not explain the mussel distribution in flowing streams. If this is correct, instream flow studies using this approach need to be revised. Results of Chapter 4 indicate that mussels in the lower Brazos River basin are constrained in distribution by the availability of heterogenous substrate. Appendix A, details the first account of a living population of Truncilla macrodon, which is a candidate species for the Endangered Species Act (ESA). The population was found while conducting mussel instream flow studies in the lower Brazos River basin.
Ecology and Recolonization of Benthic Macroinvertebrates in a Groundwater-dependent Stream in North Central Texas During a Supra-seasonal Drought
Extreme climatic events such as droughts are known to eliminate aquatic biota and alter community structure and function. Perennial headwater springs provide important drought refugia to benthic macroinvertebrates and an important source of colonists via drift or aerial adults to intermittent streams post-drought. During a supra-seasonal drought in North-central Texas summer and fall 2006, benthic macroinvertebrates from persistent groundwater-dependent macrohabitats of varying hydrological connectivity and riparian shading were studied: perennial riffles, connected pools, shaded disconnected pools, and full sun disconnected pools. Riffles were a distinct habitat with significantly higher taxa richness, proportion of lotic taxa, diversity and evenness than other macrohabitats. Macrohabitats were found to be important refugia for 106 benthic macroinvertebrates and 4 microcrustacean taxa. Throughout the extreme drought, perennially flowing habitats were refugia to 19 taxa (17.9% total taxa) not collected in disconnected pools. Shaded disconnected pools contained lotic taxa not previously known to be able to complete their lifecycles in lentic habitats, emphasizing the importance of groundwater effluent and shading. With the resumption of flow at a downstream intermittent site of Ash Creek in mid-October 2006, an annual recolonization study was conducted comparing the perennial headwaters’ benthic macroinvertebrate taxa richness, densities and community ecology with the downstream intermittent site. The headwaters supported higher mean taxa richness than the intermittent site over the duration of the study (ANOVA P < 0.001). However, the unexpected result of overall decreasing taxa richness at the perennial headwater site from August 2006 to April 2008 appears to reflect lag effects of the supra-seasonal drought combined with effects of multiple spates of 2007, which are factors confounding the point of recovery for taxa richness. Recovery of taxa richness at the intermittent site took 9 months compared to 1 to 2 months reported in other arid and semi-arid streams in the United States recovering from seasonal drying and floods. Sustainable use of groundwater resources and conservation of riparian corridors is vital to protecting groundwater-dependent ecosystems that play a vital role in maintaining regional biodiversity by serving as biotic refugia during catastrophic disturbance.
The Effect of Menthol on Nicotine Metabolism: a Cross Species Evaluation
The effect of menthol on nicotine metabolism was examined in liver S9 fractions of four different species and in the in vivo mouse model. The purpose of this study was to investigate three parameters: (1) biotransformation of nicotine to cotinine in various species (human, mouse, rat and trout) using in vitro methods; (2) to determine if the addition of menthol with nicotine altered biotransformation of nicotine to cotinine; (3) and to assess similar parameters in an in vivo mouse model. The major findings of this study include: (1) mice appear to metabolize nicotine, over time, in a manner similar to humans; (2) menthol decreased cotinine production, over time, after a single dose in mice; and (3) menthol increased cotinine production, over time, after repeated doses, in mice.
Effect of Rancher’s Management Philosophy, Grazing Practices, and Personal Characteristics on Sustainability Indices for North Central Texas Rangeland
To assess sustainability of privately owned rangeland, a questionnaire was used to gathered data from ranches in Cooke, Montague, Clay, Wise, Parker, and Jack counties in North Central Texas. Information evaluated included: management philosophy, economics, grazing practices, environmental condition, quality of life, and demographics. Sustainability indices were created based on economic and land health indicator variables meeting a minimum Cronbach‘s alpha coefficient (α = 0.7). Hierarchical regression analysis was used to create models explaining variance in respondents’ indices scores. Five predictors explained 36% of the variance in rangeland economic sustainability index when respondents: 1) recognized management inaction has opportunity costs affecting economic viability; 2) considered forbs a valuable source of forage for wildlife or livestock; 3) believed governmental assistance with brush control was beneficial; 4) were not absentee landowners and did not live in an urban area in Texas, and; 5) valued profit, productivity, tax issues, family issues, neighbor issues or weather issues above that of land health. Additionally, a model identified 5 predictors which explained 30% of the variance for respondents with index scores aligning with greater land health sustainability. Predictors indicated: 1) fencing cost was not an obstacle for increasing livestock distribution; 2) land rest was a component of grazing plans; 3) the Natural Resource Conservation Service was used for management information; 4) fewer acres were covered by dense brush or woodlands, and; 5) management decisions were not influenced by friends. Finally, attempts to create an index and regression analysis explaining social sustainability was abandoned, due to the likely-hood of type one errors. These findings provide a new line of evidence in assessing rangeland sustainability, supporting scientific literature concerning rangeland sustainability based on ranch level indicators. Compared to measuring parameters on small plots, the use of indices allows for studying replicated whole- ranch units using rancher insight. Use of sustainability indices may prove useful in future rangeland research activities.
Effects of Airway Pressure, Hypercapnia, and Hypoxia on Pulmonary Vagal Afferents in the Alligator (Alligator Misssissippiensis)
The American alligator (Alligator mississippiensis) is an aquatic diving reptile with a periodic breathing pattern. Previous work has identified pulmonary stretch receptors (PSR), both rapidly- and slowly-adapting, and intrapulmonary chemoreceptors (IPCs) that modulate breathing patterns in alligators. The purpose of the present study was to identify the effects of prolonged lung inflation and deflation (simulated dives) on PSR and/or IPC firing characteristics in the alligator. The effects of airway pressure, hypercapnia, and hypoxia on dynamic and static responses of pulmonary stretch receptors (PSR) were studied in juvenile alligators (mean mass = 246 g) at 24°C. Receptor activity appeared to be a mixture of slowly-adapting PSRs (SARs) and rapidly-adapting PSRs (RARs) with varying thresholds and degrees of adaptation, but no CO2 sensitivity. Dives were simulated in order to character receptor activity before, during, and after prolonged periods of lung inflation and deflation. Some stretch receptors showed a change in dynamic response, exhibiting inhibition for several breaths after 1 min of lung inflation, but were unaffected by prolonged deflation. For SAR, the post-dive inhibition was inhibited by CO2 and hypoxia alone. These airway stretch receptors may be involved in recovery of breathing patterns and lung volume during pre- and post-diving behavior and apneic periods in diving reptiles. These results suggest that inhibition of PSR firing following prolonged inflation may promote post-dive ventilation in alligators.
Effects of CFT Legumine™ Rotenone on Macroinvertebrates in Four Drainages of Montana and New Mexico
Rotenone is considered essential in the restoration of native fish populations; however, the technique is contentious and criticized, specifically concerning impacts to invertebrates. Knowledge of effects to non-target organisms is important for the management and conservation of fish populations. This thesis has two general objectives: (1) demonstrate the influence CFT Legumine™ rotenone has on benthic macroinvertebrates for restoration projects in Montana and New Mexico and (2) evaluate the immediate response by means of invertebrate drift. Chapters 2 and 4 incorporate results from four different restoration projects that examine benthic macroinvertebrate response. Results indicate treatment effects are minimal for Specimen and Cherry Creek projects in Montana. New Mexico projects, Comanche and Costilla Creek suggest a greater influence. Potassium permanganate used to neutralize rotenone, influenced communities in three of the four projects. Regardless, invertebrates in all four projects recovered one-year after treatment. Chapter 3 examines macroinvertebrate drift during rotenone treatment. Results suggest a delayed response compared to previous literature. Rotenone appears to have the greatest immediate influence on the early life stages of Ephemeroptera and Plecoptera. To reduce impacts of rotenone to invertebrates, managers should apply CFT Legumine and use the minimal dosage and duration to complete the projects goal of removing non-indigenous fish species.
Effects of Layer Double Hydroxide Nanoclays on the Toxicity of Copper to Daphnia Magna
Nanoparticles may affect secondary pollutants such as copper. Layer Double Hydroxides (LDH) are synthetically produced nanoparticles that adsorb copper via cation exchange. Pretreatment of copper test solutions with LDH nanoparticles followed by filtration removal of LDH nanoparticles demonstrated the smallest LDH aggregates removed the most copper toxicity. This was due to increased surface area for cation exchange relative to larger particle aggregates. Co-exposure tests of copper chloride and clay were run to determine if smaller clay particles increased copper uptake by D. magna. Coexposure treatments had lower LC50 values compared to the filtration tests, likely as a result of additive toxicity. LDH nanoclays do reduce copper toxicity in Daphnia magna and may serve as a remediation tool.
Effects of Suspended Multi-Walled Carbon Nanotubes on Daphnid Growth and Reproduction
Multi-walled carbon nanotube aggregates can be suspended in the aqueous phase by natural organic matter. These aggregates are ingested by filter feeding zooplankton. Ingested aggregates result in decreased growth and decreased reproduction. These effects may be caused by reduction in energy input from normal feeding behavior. pH alters natural organic matter structure through changes in electrostatic repulsion. Altered natural organic matter structure changes multi-walled carbon nanotube aggregate size. This size variation with variation in pH is significant, but not large enough a change in size to alter toxicity, as the aggregate size range remains well within the particle size selection of the organisms.
Endocannabinoid System in a Planarian Model
In this study, the presence and possible function of endocannabinoid ligands in the planarian is investigated. The endocannabinoids ananadamide (AEA) and 2-arachidonoylglycerol (2-AG) and entourage NAE compounds palmitoylethanolamide (PEA), stearoylethanolamide (SEA) and oleoylethanolamide (OEA) were found in Dugesia dorotocephala. Changes in SEA, PEA, and AEA levels were observed over the initial twelve hours of active regeneration. Exogenously applied AEA, 2-AG and their catabolic inhibition effected biphasic changes in locomotor velocity, analogous to those observed in murines. The genome of a close relative, Schmidtea mediterranea, courtesy of the University of Utah S. med genome database, was explored for cannabinoid receptors, none were found. A putative fatty acid amide hydrolase (FAAH) homolog was found in Schmidtea mediterranea.
Endocrine Disruption of Levonorgestrel in Early-life Stages of Fathead Minnows, Pimephales Promelas
Pharmaceuticals have routinely been detected in the environment resulting in a growing concern about whether these drugs could elicit effects on aquatic organisms. The concerns are centered on the highly conserved nature of mammalian therapeutic targets in fish. These pharmaceuticals are found at very low levels in the environment, which can result in sub-lethal effects in aquatic organisms. Therefore, 28 d early-life stage studies were conducted on six pharmaceuticals to assess their impacts on survival and growth fathead minnow larvae. Two pharmaceuticals tested, carbamazepine and fenofibrate, resulted in no alterations to survival and growth. However, amiodarone, clozapine, dexamethasone, and levonorgestrel (LNG) reduced survival at concentrations tested with LNG being the most potent at 462 ng/L. Survival was increased with amiodarone and clozapine; however LNG significantly decreased growth at 86 ng/L. Therefore, the most potent pharmaceutical tested was the synthetic progestin LNG with survival and growth impacts at concentrations less than 1 μg/L. Further analysis was conducted by measuring specific endocrine related mRNA transcript profiles in FHM larvae following the 28 d ELS exposure to LNG. Transcripts of 3β-HSD, 20β-HSD, and FSH were significantly down-regulated following 28 d exposure to both 16.3 and 86.9 ng/L LNG. Also, CYP19a expression was significantly down-regulated at 86.9 and 2392 ng/L LNG. Subsequently, a second study examined time periods that may be most sensitive (e.g., windows of sensitivity) for FHM larvae exposed to LNG. Larvae were exposed to a single concentration of LNG (i.e. LOECgrowth of 86.2 ng/L as determined in the 28 d ELS study) for different time periods starting with fertilized egg through 28 dph. Growth and mRNA expression of the four differentially expressed transcripts from the first study were measured. Regardless of the duration of exposure, LNG significantly decreased growth in fathead minnow larvae at day 28. For both 20β-HSD and CYP19a, mRNA expression was decreased following exposure to LNG; however, these transcripts returned to baseline levels after removal of LNG. 3β-HSD and FSH showed similar trends after exposure to LNG with 7-14 d and 14-28 d exposures exhibiting a decrease in expression; however, FSH expression returned to baseline once removed for LNG exposure. Based on these data, 3β-HSD was the only transcript to remain down regulated after LNG exposure. Together these data suggest LNG can negatively impact FHM larval survival and growth, with significant alterations in endocrine related responses. However, these changes in endocrine related responses may not directly correlate to the changes in growth demonstrated with LNG exposure to fathead minnows. Therefore, additional research is warranted to ascertain additional mechanisms, either endocrine related or non-endocrine functions, related to changes in growth of larval fathead minnows.
Evaluating the Habitat Requirements of the Golden Orb Mussel (Quadrula Aurea) for Conservation Purposes
Many freshwater mussels are imperiled, due to a number of interrelated factors such as habitat alteration, degradation of water quality, and impoundments. The Golden Orb mussel (Quadrula aurea, I. Lea, 1859) is endemic to the state of Texas and is currently a candidate for the endangered species list, as the number of known populations has been declining in recent years. Little is currently known about Q. aurea aside from basic distribution data. This study is focused on evaluating a combination of macro-habitat and micro-habitat variables to determine their influence on the distribution and density of this species. Macro-habitat variables, including dominant land cover, surface geology, and soil erodibility factor, did not have a significant relationship with mussel distributions. The best model of micro-habitat variables that impacts the Q. aurea distributions is comprised of relative substrate stability (RSS) at moderate flows and current velocity at low flows. For all mussel species in this study, current velocity at low flows is the primary variable that influences distribution. Q. aurea are associated with habitats where larger sediment particles (large gravel and cobble) help to stabilize the substrate in areas with higher current velocities. An understanding of the preferred habitats for Q. aurea can be used to help focus conservation efforts and practices.
Evaluation of the Developmental Effects and Bioaccumulation Potential of Triclosan and Triclocarban Using the South African Clawed Frog, Xenopus Laevis
Triclosan (TCS) and triclocarban (TCC) are antimicrobials found in U.S. surface waters. This dissertation assessed the effects of TCS and TCC on early development and investigated their potential to bioaccumulate using Xenopus laevis as a model. The effects of TCS on metamorphosis were also investigated. For 0-week tadpoles, LC50 values for TCS and TCC were 0.87 mg/L and 4.22 mg/L, respectively, and both compounds caused a significant stunting of growth. For 4-week tadpoles, the LC50 values for TCS and TCC were 0.22 mg/L and 0.066 mg/L; and for 8-week tadpoles, the LC50 values were 0.46 mg/L and 0.13 mg/L. Both compounds accumulated in Xenopus. For TCS, wet weight bioaccumulation factors (BAFs) for 0-, 4- and 8-week old tadpoles were 23.6x, 1350x and 143x, respectively. Lipid weight BAFs were 83.5x, 19792x and 8548x. For TCC, wet weight BAFs for 0-, 4- and 8-week old tadpoles were 23.4x, 1156x and 1310x. Lipid weight BAFs were 101x, 8639x and 20942x. For the time-to-metamorphosis study, TCS showed an increase in weight and snout-vent length in all treatments. Exposed tadpoles metamorphosed approximately 10 days sooner than control tadpoles. For the hind limb study, although there was no difference in weight, snout-vent length, or hind limb length, the highest treatment was more developed compared to the control. There were no differences in tail resorption rates between the treatments and controls. At relevant concentrations, neither TCS nor TCC were lethal to Xenopus prior to metamorphosis. Exposure to relatively high doses of both compounds resulted in stunted growth, which would most likely not be evident at lower concentrations. TCS and TCC accumulated in Xenopus, indicating that the compound has the potential to bioaccumulate through trophic levels. Although TCS may increase the rate of metamorphosis in terms of developmental stage, it did not disrupt thyroid function and metamorphosis in regards to limb development and tail resorption.
Examination of the Relationship Between Glucuronic Acid and Vascular Damage in Rats
The goal of this experiment was to examine the role of glucuronic acid in the development of vascular damage in the kidneys and retinas of diabetic individuals. Glucuronic acid was provided to rats in their water at various concentrations in order to increase plasma levels of the compound. Kidneys and retinas were excised and compared to control specimens using microscopy to determine the effect of elevated blood glucuronic acid levels on the occurrence of microaneurysms in renal capillary networks. No differences were seen between the treatment and control groups. Further study needs to be conducted to determine a more suitable time frame for this experiment.
Exploring the Evolutionary History of North American Prairie Grouse (Genus: Tympanuchus) Using Multi-locus Coalescent Analyses
Conservation biologists are increasingly using phylogenetics as a tool to understand evolutionary relationships and taxonomic classification. The taxonomy of North American prairie grouse (sharp-tailed grouse, T. phasianellus; lesser prairie-chicken, T. pallidicinctus; greater prairie-chicken, T. cupido; including multiple subspecies) has been designated based on physical characteristics, geography, and behavior. However, previous studies have been inconclusive in determining the evolutionary history of prairie grouse based on genetic data. Therefore, additional research investigating the evolutionary history of prairie grouse is warranted. In this study, ten loci (including mitochondrial, autosomal, and Z-linked markers) were sequenced across multiple populations of prairie grouse, and both traditional and coalescent-based phylogenetic analyses were used to address the evolutionary history of this genus. Results from this study indicate that North American prairie grouse diverged in the last 200,000 years, with species-level taxa forming well-supported monophyletic clades in species tree analyses. With these results, managers of the critically endangered Attwater's prairie-chicken (T. c. attwateri) can better evaluate whether outcrossing Attwater's with greater prairie-chickens would be a viable management tool for Attwater's conservation.
Expression of G-protein Coupled Receptors in Young and Mature Thrombocytes and Knockdown of Gpr18 in Zebrafish
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In this study, a novel method based on biotinylated antibodies and streptavidin coated magnetic beads was used to separate the thrombocyte subpopulations from zebrafish whole blood. DiI-C18, a lipophilic dye, labels only young thrombocytes when used at low concentrations. Commercially available biotinylated anti-Cy3 antibody was used to label the chromophore of DiI-C18 on the young thrombocytes and streptavidin coated magnetic beads were added subsequently, to separate young thrombocytes. The remaining blood cells were probed with custom-made biotinylated anti-GPIIb antibody and streptavidin magnetic beads to separate them from other cells. Further, thrombocytes are equivalents of mammalian platelets. Platelets play a crucial role in thrombus formation. The G-protein coupled receptors (GPCRs) present on the platelet surface are involved during platelet activation and aggregation processes. So, thrombocytes were studied for the presence of GPCRs. The GPCR mRNA transcripts expressed in the young and mature thrombocytes were subjected to densitometry analysis and pixel intensities of the bands were compared using one way ANOVA. This analysis did not show significant differences between the young and mature GPCR mRNA transcripts but identified a novel GPCR, GPR18 that was not reported in platelets earlier. To study the function of this GPCR, it was knocked down using GPR18 specific antisense morpholino and vivo morpholino. The immunofluorescence experiment indicated the presence of GPR18 on thrombocytes. The results of the assays, such as, time to occlusion (TTO) and time to aggregation (TTA) in response to N-arachidonyl glycine (NAG) as an agonist, showed prolongation of time in GPR18 larval and adult morphants respectively, suggesting that GPR18 plays a role in thrombus formation in zebrafish. In conclusion, our results indicate that GPR18 may be present in zebrafish thrombocytes, it may be involved in thrombus formation and that NAG may be an agonist at GPR18 on thrombocytes.
Field and Laboratory Fish Tissue Accumulation of Carbamazepine and Amiodarone
The goals of this dissertation work were to assess the bioaccumulation potential of carbamazepine and amiodarone, two widely used ionizable pharmaceutical compounds that possess mid-range and high LogD values, respectively, and to evaluate alternative methods to assess chemical accumulation in bluntnose minnows, catfish, and tilapia. Results indicated that carbamazepine does not appreciably bioaccumulate in fish tissue with BCFk and BAF carbamazepine values < 10. Amiodarone, however, with a log D of 5.87 at pH 7.4, accumulated in fish tissues with kinetic BCF values <2,400. Collectively, the data suggest that full and abbreviated laboratory-derived BCFs, BCFMs derived from S9 loss-of-parent assays, as well as field BAF values are similar for each of the two drugs. In summary, the results from this dissertation indicated: 1) The reduced design BCF test is a good estimate for the traditional OECD 305 test. 2) In vitro S9 metabolism assays provide comparable BCF estimates to the OECD 305 test. 3) Metabolism may play a large role in the accumulation of drugs in fish. 4) Reduced BCF tests and in vitro assays are cost effective and can reduce vertebrate testing.
Functional Characterization of Mtnip/latd’s Biochemical and Biological Function
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Symbiotic nitrogen fixation occurs in plants harboring nitrogen-fixing bacteria within the plant tissue. The most widely studied association is between the legumes and rhizobia. In this relationship the plant (legumes) provides the bacteria (rhizobia) with reduced carbon derived from photosynthesis in exchange for reduced atmospheric nitrogen. This allows the plant to survive in soil, which is low in available of nitrogen. Rhizobia infect and enter plant root and reside in organs known as nodules. In the nodules the bacteria fix atmospheric nitrogen. The association between the legume, Medicago truncatula and the bacteria Sinorhizobium meliloti, has been studied in detail. Medicago mutants that have defects in nodulation help us understand the process of nitrogen fixation better. One such mutant is the Mtnip-1. Mtnip-1 plants respond to S. meliloti by producing abnormal nodules in which numerous aberrant infection threads are produced, with very rare rhizobial release into host plant cells. The mutant plant Mtnip-1 has an abnormal defense-like response in root nodules as well as defects in lateral root development. Three alleles of the Mtnip/latd mutants, Mtnip-1, Mtlatd and Mtnip-3 show different degrees of severity in their phenotype. Phylogenetic analysis showed that MtNIP/LATD encodes a protein belonging to the NRT1(PTR) family of nitrate, peptide, dicarboxylate and phytohprmone transporters. Experiments with Mtnip/latd mutants demonstrats a defective nitrate response associated with low (250 μM) external nitrate concentration rather than high (5 mM) nitrate concentration. This suggests that the mutants have defective nitrate transport. To test if MtNIP/LATD was a nitrate transporter, Xenopus laevis oocytes and Arabidopsis thaliana mutant plants Atchl1-5, defective in a major nitrate transporter AtNRT1.1(CHL1), were used as surrogate expression systems. Heterologous expression of MtNIP/LATD in X. laevis oocytes and Atchl1-5 mutant plants conferred on them the ability to take up nitrate from external media with high affinity, thus demonstrating that MtNIP/LATD was a high affinity nitrate transporter. Km for MtNIP/LATD was determined to be approximately160 μM in the X. laevis system and 113 μM in the Arabidopsis Atchl1-5 mutant lines thus supporting the previous observation of MtNIP/LATD being a high affinity nitrate transporter. X. laevis expressing the mutant Mtnip-1 and Mtlatd, were unable to transport nitrate. However X. laevis oocytes, expressing the less severe mutant allele Mtnip-3 were able to transport nitrate suggesting another role of the Mtnip/latd besides high affinity nitrate transport. Experimental evidence suggested that MtNIP/LATD might transport another substrate beside nitrate. MtNIP/LATD levels are regulated by phytohormones. Experiments performed with ABA (abscisic acid), IAA (indole acetic acid) and histidine as substrates in X. laevis system show that the MtNIP/LATD mRNA injected oocytes efflux IAA but do not transport histidine or ABA. When wild type A17 and mutant Mtnip-1 and Mtnip-3 plants, grown in the presence of different sources of nitrogen were screened in herbicide chlorate, a structural analog of nitrate, the A17 and Mtnip-3 mutant showed levels of susceptibility that was different from mutant Mtnip-1 lines. Evidence suggested that the amount of chlorate transported into the plants were regulated by the C:N status of the A17 and Mtnip-3 plants. This regulation was missing in the Mtnip-1 lines thus suggesting a sensor function of MtNIP/LATD gene.
Functional Characterization of Plant Fatty Acid Amide Hydrolases
Fatty acid amide hydrolase (FAAH) terminates the endocannabinoid signaling pathway that regulates numerous neurobehavioral processes in animals by hydrolyzing a class of lipid mediators, N-acylethanolamines (NAEs). Recent identification of an Arabidopsis FAAH homologue (AtFAAH) and several studies, especially those using AtFAAH overexpressing and knock-out lines suggest that a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, I provide evidence to support this concept by identifying candidate FAAH cDNA sequences in diverse plant species. NAE amidohydrolase assays confirmed that several of the proteins encoded by these cDNAs indeed catalyzed the hydrolysis of NAEs in vitro. Kinetic parameters, inhibition properties, and substrate specificities of the plant FAAH enzymes were very similar to those of mammalian FAAH. Five amino acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the plant FAAH sequences. Site-directed mutation of each of the five putative catalytic residues in AtFAAH abolished its hydrolytic activity when expressed in Escherichia coli. Contrary to overexpression of native AtFAAH in Arabidopsis that results in enhanced seedling growth, and in seedlings that were insensitive to exogenous NAE, overexpression of the inactive AtFAAH mutants showed no growth enhancement and no NAE tolerance. However, both active and inactive AtFAAH overexpressors displayed hypersensitivity to ABA, suggesting a function of the enzyme independent of its catalytic activity toward NAE substrates. Yeast two-hybrid screening identified Arg/Ser-rich zinc knuckle-containing protein as a candidate protein that physically and domain-specifically interacts with AtFAAH and its T-DNA knock-out Arabidopsis was hypersensitive to ABA to a degree similar to AtFAAH overexpressors. Taken together, AtFAAH appears to have a bifurcating function, via NAE hydrolysis and protein-protein interaction, to control Arabidopsis growth and interaction with phytohormone signaling pathways. These studies help to functionally define the group of enzymes that metabolize NAEs in plants, and further will expand the knowledge-base of lipid metabolism and signaling for manipulation of various physiological processes important to plant growth and responses to environmental stress.
Functional Neural Toxicity and Endocrine Responses in Mice Following Naphthalene Exposure
Polycyclic aromatic hydrocarbons (PAHs) are a well studied and diverse class of environmental toxicants. PAHs act via the aryl hydrocarbon receptor (AhR), and studies have suggested that PAHs may elicit neurological and estrogenic effects. Doses of PAHs between 50 to 150 ppm may elicit neurotoxicity in rodent models. The present study investigated the effects of naphthalene on in vivo steroidogenesis in Swiss Webster male mice, and in vitro neural function of Balb-C/ICR mice frontal cortex neurons. These data suggest that naphthalene may not elicit steroidogenic effects at concentrations ranging from 0.2 to 25 mg/kg/day, following a 7 day subcutaneous dosing regime. In addition, naphthalene may cause functional toxicity of frontal cortex neurons at concentrations of 32 to 160 ppm naphthalene.
Genetic Analysis of Development and Behavior in Hypoxia and Cellular Characterization of Anoxia Induced Meiotic Prophase Arrest in Caenorhabditis Elegans
It was hypothesized that chronic hypoxia will affect various biological processes including developmental trajectory and behavior. To test this hypothesis, embryos were raised to adulthood in severe hypoxic environments (0.5% O2 or 1% O2, 22°C) and analyzed for survival rate, developmental progression, and altered behaviors. Wildtype hermaphrodites survive chronic hypoxia yet developmental trajectory is slowed. The hermaphrodites raised in chronic hypoxia had different phenotypes in comparison to the normoxic controls. First, hermaphrodites exposed to chronic hypoxia produced a significantly lower number of embryos and had a slight increase in male progeny. This suggests that chronic hypoxia exposure during development affects the germline. Second, animals raised in chronic hypoxia from embryos to young adults have a slight increase in lifespan when re-exposed to a normoxic environment, indicating that chronic hypoxia does not negatively decrease lifespan. Finally, hermaphrodites that were raised in hypoxia will lay the majority of their eggs on the area of the agar plate where the bacterial lawn is not present. This is in contrast to animals in normoxia, which lay the majority of their eggs on the bacterial lawn. One hypothesis for this hypoxia-induced egg-laying behavior is that the animal can sense microenvironments in hypoxia. To examine if various pathways are involved with chronic-hypoxia responses RNAi and assayed genetic mutants were used. Specifically, genetic mutations affecting oxygen sensing (egl-9), aerotaxis (npr-1), TFG-ß signaling (dbl-1, daf-7) and predicted oxygen-binding proteins (globin-like genes) were phenotypically analyzed. Results indicate that mutations in several of these genes (npr-1, dbl-1) resulted in a decrease in hypoxia survival rate. A mutation in egl-9 also had a detrimental affect on the viability of an animal raised in chronic hypoxia. However, a similar phenotype was not observed in the vhl-1 mutation indicating that the phenotype may not be due to a mere increase in HIF-1 levels, per se. A mutation in the globin-like gene (glb-13(tm2825)) suppressed the hypoxia-induced egg-laying phenotype. That is, the glb-13(tm2825) animal raised in chronic hypoxia laid eggs on the bacterial lawn at a significantly higher rate in comparison to wildtype controls, thus suggesting that globin-like molecules may be involved with the sensing of microenvironments. Together, this research lays the foundation for understanding the implications of chronic hypoxia in developing organisms.
Genetic and Environmental Factors that Mediate Survival of Prolonged Oxygen Deprivation in the Nematode Caenorhabditis Elegans
Ischemic events of even a very short duration are not tolerated Ill in humans. The human cost of ischemia, when looked at as combined cardiovascular disease, dwarfs all other causes of death in the United States. Annually, CVD kills as many people in the US as does cancer, chronic lower respiratory disease, accidents, and diabetes mellitus combined. In 2005 (the latest year for which final statistics are available), CVD was responsible for 864,480 deaths or 35.3 percent of total deaths for the year. In my study, I have used the nematode Caenorhabditis elegans to determine genetic and environmental modulators of oxygen deprivation a key component of ischemia. I have found that animals with mutations in insulin like signaling pathways, neuronal function, electron transport chain components, germline function, and animals that are preconditioned by being raised on a diet of E. coli HT115 bacteria at 25°C have an enhanced ability to survive long-term (>72 hours) anoxia (<.005 kPa O2) at 20°C. The enhanced anoxia survival phenotype partially correlates with increased levels of carbohydrate stores in the nematodes. Suppression of this enhanced anoxia survival phenotype is possible by altering expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, the FOXO transcription factor DAF-16, and 5’-AMP kinase.
Habitat Fragmentation by Land-Use Change: One-Horned Rhinoceros in Nepal and Red-Cockaded Woodpecker in Texas
This research focuses on the spatial analysis of the habitat of two vulnerable species, the one-horn rhinoceros in the grasslands of southern Nepal, and the red-cockaded woodpecker in the Piney woods of southeast Texas, in the USA. A study sites relevant for biodiversity conservation was selected in each country: Chitwan National Park in Nepal, and areas near the Big Thicket National Preserve in Texas. Land-use differs in the two study areas: the first is still undergoing agrarian development while the second is in a technological phase and undergoing urbanization processes. Satellite remote sensing images were used to derive land-cover maps by supervised classification. These maps were then processed by Geographic Information Systems methods to apply habitat models based on basic resources (food and cover) and obtain habitat suitability maps. Several landscape metrics were computed to quantify the habitat characteristics especially the composition and configuration of suitable habitat patches. Sensitivity analyses were performed as the nominal values of some of the model parameters were arbitrary. Development potential probability models were used to hypothesize changes in land-use of the second study site. Various scenarios were employed to examine the impact of development on the habitat of red-cockaded woodpecker. The method derived in this study would prove beneficial to guide management and conservation of wildlife habitats.
Hepatotoxicity of Mercury to Fish
Tissue samples from spotted gar (Lepisosteus oculatus) and largemouth bass (Micropterus salmoides) were collected from Caddo Lake. Gar and bass livers were subjected to histological investigation and color analysis. Liver color (as abs at 400 nm) was significantly correlated with total mercury in the liver (r2 = 0.57, p = 0.02) and muscle (r2 = 0.58, p = 0.01) of gar. Evidence of liver damage as lipofuscin and discoloration was found in both species but only correlated with liver mercury concentration in spotted gar. Inorganic mercury was the predominant form in gar livers. In order to determine the role of mercury speciation in fish liver damage, a laboratory feeding study was employed. Zebrafish (Danio rerio) were fed either a control (0.12 ± 0.002 µg Hg.g-1 dry wt), inorganic mercury (5.03 ± 0.309 µg Hg.g-1 dry wt), or methylmercury (4.11 ± 0.146 µg Hg.g-1 dry wt) diet. After 78 days of feeding, total mercury was highest in the carcass of zebrafish fed methylmercury (12.49 ± 0.369 µg Hg.g-1 dry wt), intermediate in those fed inorganic mercury (1.09 ± 0.117 µg Hg.g-1 dry wt), and lowest in fish fed the control diet (0.48 ± 0.038 µg Hg.g-1 dry wt). Total mercury was highest in the viscera of methylmercury fed zebrafish (11.6 ± 1.86 µg Hg.g-1 dry wt), intermediate in those fed inorganic diets (4.3 ± 1.08 µg Hg.g-1 dry wt), and lowest in the control fish (below limit of detection). Total mercury was negatively associated with fish length and weight in methylmercury fed fish. Condition factor was not associated with total mercury and might not be the best measure of fitness for these fish. No liver pathologies were observed in zebrafish from any treatment.
Hypoxia and the Development of Endothermic Capacity in Chickens (Gallus Gallus)
Adult chickens employ endothermy – internal generation of heat that maintains a constant body temperature (Tb). Prior to hatching, chicken embryos are ectothermic - controlling Tb by external heat sources. Upon hatching, the hatchling transitions from an ectotherm to an endotherm that has been shown to be delayed by hypoxia. In this study, whole animal oxygen consumption () and liver, heart, and skeletal muscle citrate synthase activity (CSA) and were measured during this transition to endothermy in chickens incubated in normoxia and hypoxia (15% O2). The only significant differences in occurred in 48 hour old hatchlings where was lower in normoxic hatchlings. There were no differences in CS activity between age and incubation oxygen levels. Additionally, preliminary 2-D protein gels of embryo and hatchling liver show changes in the proteome upon hatching. Results suggest that hypoxia had no significant effect on CSA and a minimal effect on .
Identification of Three Symbiosome Targeting Domains in the MtENOD8 Protein and Cell-to-cell MtENOD8 mRNA Movement in Nodules
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The model legume, Medicago truncatula, is able to enter into a symbiotic relationship with soil bacteria, known as rhizobia. This relationship involves a carbon for nitrogen exchange in which the plant provides reduced carbon from photosynthesis in exchange for reduced, or “fixed” atmospheric nitrogen, which allows the plant to thrive in nitrogen depleted soils. Rhizobia infect and enter plant root organs, known as nodules, where they reside inside the plant cell in a novel organelle, known as the symbiosome where nitrogen fixation occurs. the symbiosome is enriched in plant proteins, however, little is known about the mechanisms that direct plant proteins to the symbiosome. Using the M. truncatula ENOD8 (MtENOD8) protein as a model to explore symbiosome protein targeting, 3-cis domains were identified within MtENOD8 capable of directing green fluorescent protein (GFP) to the symbiosome, including its N-terminal signal peptide (SP). the SP delivered GFP to the vacuole in the absence of nodules suggesting that symbiosome proteins share a common targeting pathway with vacuolar proteins. a time course analysis during nodulation indicated that there is a nodule specific redirection of MtENOD8-SP from the vacuole to the symbiosome in a MtNIP/LATD dependent manner. GFP expression by the MtENOD8 promoter revealed spatial discrepancy between promoter activity and protein localization. in situ localization of MtENOD8 mRNA showed localization to infected cells, where the protein is found, suggesting mRNA cell-to-cell movement. Expression of MtENOD8 in Arabidopsis showed that the SP did not direct GFP to the vacuole indicating that vacuolar targeting of MtENOD8’s SP may be legume specific. Taken together, the research presented here indicates that the MtENOD8 symbiosome protein has evolved redundant domains for targeting, which has part of a common pathway with vacuolar proteins. Observed spatial discrepancy between the MtENOD8 promoter and protein shows additional mechanisms of gene regulation through cell-to-cell mRNA movement, previously unknown in nodules.
Immunohistochemistry of the Gills of the Channel Catfish Ictalurus Punctatus: Cells and Neurochemicals That May Be Involved in the Control of Cardioventilatory Reflexes
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In teleost fishes the neurochemicals involved in sensing and responding to hypoxia are unresolved. Serotonergic branchial neuroepithelial cells (NECs) are putative O2 chemoreceptors believed to be homologous to the neural crest (NC) derived APUD (amine-precursor uptake and decarboxylation) pulmonary NECs and carotid body type-1 glomus cells. Branchial NECs contain serotonin (5-HT), thought to be central to the induction of the hypoxic cardioventilatory reflexes. However, application of 5-HT in vivo does not elicit cardioventilatory reflexes similar to those elicited by hypoxia. But previous in vitro neural recordings from glossopharyngeal (IX) afferents innervating O2 chemoreceptors in the trout gill show the same discharge response to hypoxic conditions as does that of acetylcholine (ACh) application. This evidence strongly supports the cholinergic hypothesis of chemoreceptor impulse origin rather than a serotonergic-induced impulse origin model. We therefore hypothesized that NECs contain ACh among other neurochemicals in cells belonging to the APUD series. Although serotonergic branchial NECs did not colocalize with ACh using immunohistochemical methods, several populations of ACh and/or tyrosine hydroxylase (TH) (catecholaminergic) positive, dopamine (DA) negative, cells were found throughout the second gill arch of the channel catfish Ictalurus punctatus. In addition, the NC derivation marker zn-12 labelled the HNK-1-like epitope (Human natural killer) expressed by lamellar pillar cells’ collagen column-associated pillar cell adhesion molecules (CC-PCAMs), evidence confirming their hypothesized NC origin.
Induced Bradycardia Effects on Angiogenesis, Growth and Development in Early Development in Chicken Embryos, Gallus Domesticus
Cardiac performance, angiogenesis and growth was investigated during early chicken development. Heart rate, and thus arterial pulse pressure and cardiac output, were altered with the bradycardic drug ZD7288. Heart rates at 72 h of development of control embryos and those dosed with chicken Ringer were not different at 171 bpm. Acute and chronic application of ZD7288 caused significant bradycardia. Chronic dosing of Ringer and ZD7288 changed neither eye diameter nor development rate. Chronic dosing of ZD7288 did not significantly alter CAM vessel density close to the embryo (2, 3 and 4 mm) but at farther distances (5 and 6 mm) chronic dosing with both Ringer and ZD7288 decreased vessel density by 13 - 16%. Chronic dosing with ZD7288 also reduced body mass by 20%. Thus, lowered heart rate and cardiac output had little effect on vessel density or developmental stage, but did reduce embryo growth.
The Influence of in Vitro Gill and Liver Metabolism of Xenobiotics on Fish Bioconcentration
This dissertation examines the ability of in vitro biotransformation assays to provide an indication of metabolic potential. The potential for xenobiotic compounds to bioconcentrate in aquatic organisms is expressed through the bioconcentration factor (BCF). The metabolic loss of ibuprofen, norethindrone and propranolol was measured using rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) gill and liver S9 fractions, microsomes and cell suspensions. Metabolic transformation rates (kM) were extrapolated from in vitro intrinsic clearance of parent compound (CLm) and integrated into a refined BCF model. In general, CLm of test compounds was greater in liver S9 fractions and hepatocytes. However, the influence of hepatic metabolism on kM and BCF was limited by hepatic blood flow (20-25%) compared to gill blood flow (~100%). A significant difference was noted between BCF solely based on KOW and BCF including kM. These studies indicate that the inclusion of kM in BCF models can bring predicted bioconcentration estimates closer to in vivo values. Primary cell suspensions are preferred over subcellular fractions as cell suspensions possess both phase I and phase II enzyme activity. Further study was conducted on ibuprofen biotransformation pathways. As fish do not contain the same cytochrome P450 (CYP) 2C homologs known to metabolize ibuprofen in mammals, it cannot be assumed that piscine biotransformation is similar. Metabolite analysis found 2-hydroxy-ibuprofen as the major metabolite in S9 and microsomal fractions. Additional assays involving the induction and inhibition of specific CYP isozymes support CYP1A2 as an alternative metabolic pathway.
Integrating Selective Herbicide and Native Plant Restoration to Control Alternanthera philoxeroides (Alligator Weed)
Exotic invasive aquatic weeds such as alligator weed (Alternanthera philoxeroides) threaten native ecosystems by interfering with native plant communities, disrupting hydrology, and diminishing water quality. Development of new tools to combat invaders is important for the well being of these sensitive areas. Integrated pest management offers managers an approach that combines multiple control methods for better control than any one method used exclusively. In a greenhouse and field study, we tested the effects of selective herbicide application frequency, native competitor plant introduction, and their integration on alligator weed. In the greenhouse study, alligator weed shoot, root, and total biomass were reduced with one herbicide application, and further reduced with two. Alligator weed cumulative stem length and shoot/root ratio was only reduced after two applications. In the greenhouse, introduction of competitors did not affect alligator weed biomass, but did affect shoot/root ratio. The interaction of competitor introduction and herbicide did not significantly influence alligator weed growth in the greenhouse study. In the field, alligator weed cover was reduced after one herbicide application, but not significantly more after a second. Introduction of competitor species had no effect on alligator weed cover, nor did the interaction of competitor species and herbicide application. This study demonstrates that triclopyr amine herbicide can reduce alligator weed biomass and cover, and that two applications are more effective than one. To integrate selective herbicides and native plant introduction successfully for alligator weed control, more research is needed on the influence competition can potentially have on alligator weed growth, and the timing of herbicide application and subsequent introduction of plants.