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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.
- 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 Human Baroreceptor Reflex Regulation of Blood Pressure Following 15 Days of Simulated Microgravity Exposure
- Prolonged exposure to microgravity is known to invoke physiological changes which predispose individuals to orthostatic intolerance upon readaptation to the earth's gravitational field. Attenuated baroreflex responsiveness has been implicated in contributing to this inability to withstand orthostatic stress. To test this hypothesis, eight individuals were exposed to 15 days of simulated microgravity exposure using the 6° head-down bed rest model. Prior to, and after the simulated microgravity exposure, the following were assessed: a) aortic baroreflex function; b) carotid baroreflex function; c) cardiopulmonary baroreflex function; and d) the degree of interaction between the cardiopulmonary and carotid baroreflexes.
- Analysis of Phytoplankton Responses to Water Chemistry Dynamics in a Moderately Eutrophic North Texas Reservoir
- Canonical correspondence analysis (CCA) was used to explore relationships between measured environmental variables and in situ phytoplankton communities in a moderately eutrophic North Texas Reservoir.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Biological and Toxicological Responses Resulting from Dechlorination of a Major Municipal Wastewater Treatment Plant Discharge to the Trinity River
- Federal regulations such as the Clean Water Act (P.L. 92-500), and its amendments, direct the Environment Protection Agency (EPA) to implement programs to control the releases of conventional pollutants and toxics into the waterways of the United States. The EPA began requiring treatment plants to conduct toxicity tests (biomonitoring) of their effluent discharges. To control toxicity caused by chlorination of wastewater discharges, the EPA also began requiring some treatment facilities to dechlorinate their wastewater before discharging. This research was funded by the EPA to document the changes that occurred in the Trinity River from the dechlorination of the effluent from Ft. Worth's Village Creek municipal wastewater treatment plant. The study occurred over a two year period beginning in August 1990. A wide variety of biological field assessments and toxicological assays were used to measure various responses. Seven river stations, covering approximately twenty river miles, and the treatment plant effluent were assessed. Two of the river stations were upstream from the treatment plant and used as reference sites. The remaining five river stations were downstream from the treatment plant, spread out over seventeen river miles. The study evaluated the impact of chlorination prior to dechlorination, which served as a baseline. Responses determined during dechlorination were compared to the baseline data. An overall improvement in species richness and diversity was seen at those river stations which had previously been adversely impacted by chlorine. Aquatic toxicity tests, such as those required to be used by dischargers, were conducted during this study. Periodic toxicity was observed with these tests in the effluent and river samples after dechlorination was initiated. Those tests, along with in situ toxicity assays, proved to be good predictors of biological community responses.
- 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.
- Blood Pressure Regulation During Simulated Orthostatism Prior to and Following Endurance Exercise Training
- Cardiovascular responses and tolerance to an orthostatic stress were examined in eight men before and after eight months of endurance exercise training. Following training, maximal oxygen consumption and blood volume were increased, and resting heart rate reduced. Orthostatic tolerance was reduced following training in all eight subjects. It was concluded that prolonged endurance training decreased orthostatic tolerance and this decrease in tolerance appeared associated with attenuated baroreflex sensitivity and alterations in autonomic balance secondary to an increased parasympathetic tone noted with training.
- Carbon Flux in Reservoir Sediments
- The central objective of the study was to fractionate sedimenting organic materials according to their source (allochthonous or autochthonous) and ultimately to determine the degree of biodegradability of contributions from either source with particular reference to activities at the mud-water interface.
- Cardio-respiratory ontogeny and the transition to bimodal respiration in an air breathing fish, the blue gourami (Trichogaster trichopterus): Morphological and physiological development in normoxia and hypoxia.
- As selection pressures exist for not only adults, but for every life history stage, it is important to understand how environmental factors shape developing animals. Despite the significance placed on aquatic hypoxia as a driving force in the evolution of air breathing, this is the first known study to examine the effects of hypoxia on cardio-respiratory ontogeny of an air breathing fish. Blue gouramis are obligatory air breathing fish that possess a labyrinth-like structure that serves as the air breathing organ. Gouramis were reared for up to 90 d in normoxia or hypoxia, and morphological and physiological development was observed. Hypoxic larvae had increased lamellar and labyrinth organ surface areas. Bradycardia and increased gill ventilation rates were observed when larvae from either rearing group were briefly exposed to hypoxia. Hypoxic larvae also showed a reduced heart rate and gill ventilation rate in the absence of a hypoxic stimulus, possibly indicative of a more comprehensive, long-term respiratory plasticity. The similarity of routine oxygen consumption between rearing groups suggests that metabolic demand did not change for hypoxic larvae, but that they were more efficient at oxygen acquisition. This is further supported by increased resistance time of hypoxic gouramis to extreme hypoxia. The onset of air breathing was between 20 and 25 d post-fertilization, and was not affected by either rearing or exposure environment. It may be that this behavior is associated with the inability of smaller larvae to successfully overcome water surface tension, rather than with the necessity of aerial respiration at this stage. Hypoxia is commonly experienced by most air breathing fishes, and studies of hypoxia-induced developmental effects may provide critical insights into the evolution of air breathing. The studies presented here provide novel data on the plasticity of cardio-respiratory development of an air breathing fish reared in hypoxia, and can serve as a solid foundation for future studies.
- Comparative biochemistry and genetic analysis of nucleoside hydrolase in Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas fluorescens.
- The pyrimidine salvage enzyme, nucleoside hydrolase, is catalyzes the irreversible hydrolysis of nucleosides into the free nucleic acid base and D-ribose. Nucleoside hydrolases have varying degrees of specificity towards purine and pyrimidine nucleosides. In E. coli, three genes were found that encode homologues of several known nucleoside hydrolases in protozoa. All three genes (designated yaaF, yeiK, and ybeK) were amplified by PCR and cloned. Two of the gene products (yeiK and ybeK) encode pyrimidine-specific nucleoside hydrolases, while the third (yaaF) encodes a nonspecific nucleoside hydrolase. All three were expressed at low levels and had different modes of regulation. As a comparative analysis, the homologous genes of Pseudomonas aeruginosa and P. fluorescens (designated nuh) were cloned. Both were determined to encode nonspecific nucleoside hydrolases. The nucleoside hydrolases of the pseudomonads exhibited markedly different modes of regulation. Both have unique promoter structures and genetic organization. Furthermore, both pseudomonad nucleoside hydrolase were found to contain an N-terminal extension of 30-35 amino acids that is shown to act as a periplasmic-signaling sequence. These are the first two nucleoside hydrolases, to date,that have been conclusively demonstrated to be exported to the periplasmic space. The physiological relevance of this is explained.
- Comparative morphology of sensilla styloconica on the proboscis of North American Nymphalidae and other selected taxa (Lepidoptera): systematic and ecological considerations.
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Sensilla styloconica on the proboscis of 107 species of North American and tropical butterflies were comparatively studied using the scanning electron microscope. Focus was on 76 species of North American Nymphalidae representing 45 genera and 11 subfamilies. Nomenclature for generalized and specific types of nymphalid sensilla is proposed. Written descriptions and micrographs are presented for each species studied. Morphological features were generally consistent for all or most species within genera and sometimes within subfamilies, with specified exceptions. Statistical analysis revealed significant differences for six of eight variables tested between two distinct feeding guilds of North American Nymphalidae. Average number, density, extent of proboscis coverage with sensilla, their total length, and shoulder spine length were all significantly greater in the non-nectar feeding guild than in nectar feeders, and may indicate adaptation for greater efficiency in feeding on flat surfaces. The greater frequency of apical shoulder spines in non-nectar feeders may represent adaptation for protection of sensory pegs from mechanical abrasion during feeding, or for anchoring the flexible proboscis tip to the surface. Correlation analysis revealed 9 out of 28 positive correlations in nectar feeders and 5 out of 28 in non-nectar feeders. Results of preliminary cladistic analysis were not considered to be meaningfully robust due to few available characters. The stylar characters identified in this study should be more useful in future analyses when included with characters from other lines of evidence. The presence of sensilla styloconica in all subfamilies of Nymphalidae, except Danainae, largely supports Ehrlich's (1958) higher classification concept for the family. The presence of less conspicuous sensilla in the Danainae, and other characteristics are presented as further evidence that they should be reconsidered for full family status. Sensilla styloconica in nymphalid butterflies appear to function as extensions that provide greater sensory reach during feeding. The role of these sensilla in liquid uptake, pollen feeding, and host plant selection is discussed.
- A Comparative Study of Passive Transfer Mechanisms of Tuberculin and Chemical Contact Delayed Hypersensitivities in the Guiea Pig
- This study is concerned with a critical comparison of the passive transfer mechanisms of tuberculin and chemical contact hypersensitivities in the guinea pig by use of a four phase experimental approach.
- A contravention of established principles of interspecific allometric metabolic scaling in developing silkworms, Bombyx mori.
- Established interspecific metabolic allometric relationships do not adequately describe the complexity and variable physiological states of developing animals. Consequently, intraspecific allometric relationships of oxygen consumption and carbon dioxide production as a function of body mass; the respiratory quotient; the function of the silk cocoon; and body composition were investigated for each distinct developmental stage of the silkworm, Bombyx mori. Whole animal O2 consumption in Bombyx ranged from 0.00064 + 0.000047 ml O2 .hr-1 at larval instar I to 0.77 + 0.06 ml O2 .hr-1 in pre-pupal, falling to 0.21+ 0.01 ml O2 .hr-1 in the pupae. Those instars having a significant relationship between O2 consumption as a function of body mass, the slope of the line relating O2 consumption to body mass varied between 0.99 and 1.02, while across all instars the slope was 0.82. Developmental allometry should be presented for individual developmental stages because the individual allometric exponents of the stages can be significantly different from the overall allometric exponent throughout development and in some cases, the overall allometric exponent can be a statistical artifact. The first larval instar of Bombyx mori has the lowest cross sectional area of high metabolic tissue of the midgut (27%) and had one of the highest percentages of some metabolically inert tissues (i.e. lipid, 7.5%). Body composition of the first instar does not support the idea that smaller mass animals having the highest O2 consumption are composed of a greater percentage of metabolically active organs when compared to larger animals. However, this developmental stage has the highest percentage of the mitochondrial marker cytochrome oxidase, which correlates well with the high O2 consumption rate of the smaller mass. Therefore, established interspecific principles should not be assumed to function as valid models for intraspecific developmental relationships of metabolism as a function of body mass. Developmental allometry should include an analysis of individual stages of development as well as an analysis of development as a whole to gain a comprehensive understanding of the complexity of allometry of the developing animal such as the silkworm.
- 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 ≥ 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.
- Development of Cardiovascular Regulation in Embryos of the Domestic Fowl (Gallus Gallus), with a Partial Comparison to Embryos of the Desert Tortoise (Gopherus Agassizii)
- In adult vertebrates, cardiovascular regulation is accomplished by numerous systems with neural, hormonal and local components responsible for the majority of regulation. These regulatory components work in concert to maintain the essential function of blood perfusion to adult tissues. Given the essential nature of this function it is therefore surprising that the development of cardiovascular regulation during gestation is poorly understood. The majority of what is known is based on a single vertebrate model, the fetal lamb. The fetal lamb has been used in multiple studies due to the clear clinical applications and has been pivotal in understanding the onset of regulation in developing vertebrates. However, study on the fetal lamb is limited to the latter 40% of gestation and has the added complication of an in-utero developmental strategy. Therefore the primary focus of this dissertation was to characterize basic cardiovascular regulation in the chicken embryo to provided the needed information for it's use an alternative to the fetal lamb. Developing chicken embryos rely on both alpha and beta adrenergic tones to maintain normal heart rate and arterial blood pressure during incubation. However, on day 21, just prior to hatch, these animals lose both tones on arterial pressure suggesting the onset of adult regulation. Cholinergic tone, however, was absent throughout chicken development indicating that it must mature during the neonatal life. Adult cardiovascular reflexes become apparent late in chicken development with a clear baroreflex specifically operating initially on day. However, an adult response to changes in ambient gas tension was absent during incubation suggesting embryos possess unique regulatory systems that are absent in adult chickens. This mechanism is comprised entirely of adrenergic systems with no cholinergic action during change in ambient gas tension. Similar developmental patterns were determined in embryos of the desert tortoise suggesting fundamental differences between in-utero and ex-utero developing vertebrates.
- The developmental physiology of the zebrafish: Influence of environment and cardiovascular attributes
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Temperature effects on the development of the zebrafish embryos and larvae and adults were examined. It was found that the earlier in development a temperature change was performed on an embryo, the more significant the change in survival and/or subsequent development. Thus, viable temperature ranges for zebrafish widened significantly as development proceeded. Adults reared and bred at 25oC produced embryos that were significantly more successful at the lower range of rearing temperatures compared to embryos produced from adults reared at 28oC. The majority of this study focused on the physiological effects of swim training during development in the zebrafish. The earlier in development the zebrafish larvae were trained, the greater the mortality. Trained free swimming larvae had a significantly higher routine oxygen consumption after 11 days of training, and a higher mass specific routine metabolic rate after 8 and 11 days of training. Trained free swimming larvae consumed significantly less oxygen during swimming and were more efficient at locomotion, compared to control larvae. Training enhanced survival during exposure to extreme hypoxia in all age groups. Performance aspects of training were investigated in attempt to quantify training effects and in most cases, trained fish performed significantly better than controls. As blood vessels formed during development, they decreased in cross sectional area from days two to six. It was also shown that the variability in visual stroke volume measurements could be reduced significantly by using a third dimension in the analysis with a more accurate volume equation. Finally, the ontogeny of cardiac control was evaluated. The adrenergic receptors were the first to respond to pharmacological stimulation but were closely followed by cholinergic pharmacological stimulation a few days later. There was a significant cholinergic tone present in day 15 zebrafish larvae which persisted. Although an adrenergic tone was not documented in this study, this does not prove its lack of existence.
- Discovery and Characterization of Two Tn5 Generated pyrA Mutants in Pseudomonas putida and the Generation of Hfr Strains
- A pyrA mutation in Pseudomonas putida was isolated using transposon mutagenesis for the first time. Transposon Tn5 was used to inactivate the pyrA gene for carbamoylphosphate synthetase in these mutants. Accordingly, these mutants were defective in pyrimidine and arginine biosynthesis. The suicide vector, pM075, from Pseudomonas aeruginosa, was used to introduce the transposon into the cells. Tn5 was subsequently used to supply homology so that the plasmid pM075 could be introduced in its entirety into the Pseudomonas putida chromosome at the locus of the Tn5 insertion in the pyrA gene. Consequently, these strains exhibited high frequency of recombination and were capable of chromosome mobilization.
- 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.
- Effects of carbaryl (1-naphthyl-n-methylcarbamate) on Trichocorixa reticulata (Hemiptera: corixidae) and Glyptotendipes barbipes (Diptera: chironomidae)
- My study of the effects of carbaryl in aquatic systems under controlled laboratory conditions emphasized four major objectives: (1) to determine the acute toxicity of carbaryl to the herbivorous adult and immature Trichocorixa reticulata (Guerin)(Hemiptera: Corixidae), and to the omnivorous larvae of Glyptotendipes barbipes (Staeger) (Diptera: Chironomidae) under static bioassay; (2) to adapt a quantitative method of analysis for carbaryl in water and whole insect tissue extract; (3) to measure the accumulation of the insecticide by G. barbipes under static exposure; and (4) to quantify the uptake and loss of carbaryl by G. barbipes under daily-renewed sublethal dosages.
- Effects of methanol, atrazine, and copper on the ultrastructure of Pseudokirchneriella subcapitata (Selenastrum capricornutum).
- The toxicity of methanol, atrazine, and copper to Pseudokirchneriella subcapitata (Korshikov) Hindák historically referred to as Selenastrum capricornutum Printz were determined following 96 hrs growth in a modified Goram's growth media. Methanol and atrazine inhibited fluorescence readings in the cultures by 50% (IC50) at concentrations of 2% and 82 µg/l respectively. These toxicity values compared favorably to other published reports. The IC50 for copper was 160 µg/l which is substantially higher than reported values. This is understandable because of the high chelating capacity of Goram's media. The use of stereologically derived relative volume in the chloroplasts, mitochondria, lipid bodies, phosphate bodies, and nucleus was investigated to determine if it could be used as a sensitive endpoint in toxicity tests. The volume fractions for the chloroplasts and mitochondria were normally distributed in control cells while the nuclei, phosphate bodies, and lipid bodies were not. The chloroplasts were the most dominate organelle occupying a mean relative volume of 46% and mitochondria occupied a mean relative volume of 3%. The nucleus and phosphate bodies occupied a median relative volume of 7% and 2% respectively. The lipid bodies were rare in section profile and no meaningful median relative volume could be calculated. Up to the 82nd percentile of sectioned profiles contained no recognizable lipid bodies. The use of relative volume was not a sensitive endpoint for use in toxicity tests. No significant differences in relative volume could be detected in the nucleus or phosphate bodies following any treatment. Limited differences were detected in the mitochondria, chloroplasts, and lipid bodies. The only significant differences that appear to be biologically significant occurred in methanol treated cells where an increase in the lipid bodies' relative volume was apparently concentration dependent. Significant differences in the relative volume of mitochondria and chloroplasts do not appear to be biologically significant.
- The Effects of Organic Surface Amendments on Soil Nutrients and Initial Tree Establishment
- This study examined the effects of replicating woodland soil surface horizonation on the nutrient status of underlying soils and the initial establishment and growth of trees. A total of 283 container grown trees were planted in a bufferzone around a future landfill site. Control amendments consisted of an 8 cm layer (0.5 m3) of wood chips applied in a circular area of 4.6 m2 around the trees' planting pit. For the treatment, a 2.5 cm layer of composted biosolids (0.15 m3 or 80 Mg/ha) was applied in a circular area of 4.6 m2 around the trees' planting pit followed by an 8 cm layer (0.5 m3) of wood chips. The results indicate that the replication of woodland soil surface attributes using composted biosolids can significantly improve the nutrient status of underlying soil. Some significant effects were seen under control conditions, too. However, the effects on tree establishment and growth parameters were, for the most part, not statistically significant.
- Effects of Peripheral Nerve Injury on the Cells of the Dorsal Root Ganglion: a Role for Primary Cilia
- Primary cilia are ubiquitous sensory organelles found on most cell types including cells of the dorsal root ganglia (DRG). The DRG are groups of peripheral neurons that relay sensory information from the periphery to the CNS. Other cell types in the DRG include a type of glial cell, the satellite glial cells (SGCs). The SGCs surround the DRG neurons and, with the neurons, form functional sensory units. Currently are no reports describing the numbers of DRG cells that have cilia. We found that 26% of the SGCs had primary cilia. The incidence of cilia on neurons varied with neuron size, a property that roughly correlates with physiological characteristics. We found that 29% of the small, 16% of the medium and 5% of the large neurons had primary cilia. Primary cilia have been shown to have a role in cell proliferation in a variety of cell types. In some of the cells the cilia mediate the proliferative effects of Sonic hedgehog (Shh). In the CNS, Shh signaling through primary cilia affects proliferation during development as well as following injury, but no studies have looked at this function in the PNS. The SGCs and neurons of the DRG undergo complex changes following peripheral nerve injury such as axotomy. One marked change seen after axotomy is SGC proliferation and at later stages, neuronal death. We found that following axotomy there is a significant increase in the percentage of SGCs with primary cilia. We also found a significant increase in the percentage of medium-sized neurons with primary cilia. In other experiments we tested the idea that Shh plays a role in SGC proliferation. When Shh signaling was blocked following axotomy we found decreased proliferation of SGCs. This is the first report of a change in the percentage of cells with cilia following injury in the PNS, and the first report of a role for Shh in SGC proliferation following axotomy.
- Effects of triclosan, triclocarban, and caffeine exposure on the development of amphibian larvae.
- Triclosan and triclocarban are antimicrobials found in numerous consumer products, while caffeine is the most commonly consumed stimulant by humans. This study was undertaken to determine the effects of triclosan, triclocarban, and caffeine on the development and physiology of amphibian larvae. LC50 values of triclosan and triclocarban were determined after 96 hours for three North American larval species: Acris crepitans blanchardii, Bufo woodhousii woodhousii, Rana sphenocephala, and for a common amphibian developmental model: Xenopus laevis. Amphibian larvae were most sensitive to triclosan and triclocarban exposure during early development based upon 96-hour LC50 values. Heart rates for X. laevis and North American larvae exposed to triclosan were variable throughout development. However, significantly lower heart rates were observed in all larvae exposed to triclocarban. Metabolic rates of X. laevis and R. sphenocephala larvae exposed to triclosan were significantly affected in larvae exposed to ½ LC50 and the LC50 concentration. Metabolic rates of X. laevis larvae exposed to triclocarban were significantly affected by exposure to ½ LC50 concentrations in three of four stages investigated. No significant differences were observed in North American larvae exposed to triclocarban. Tissue uptake, lipid uptake, tissue bioconcentration factor (BCF) and lipid BCF of triclosan and triclocarban were investigated in three developmental stages of X. laevis, and in one developmental stage of B. woodhousii woodhousii, and R. sphenocephala. For most tissue and lipid uptake values, a significant increase was observed as exposure concentration increased. Tissue and lipid BCF values were dependent upon both stage and species. Chronic and acute effects of caffeine were determined in X. laevis larvae. Acute 96-hour LC50 values in four developmental stages were > 75,000 ug L-1 caffeine and heart rates were significantly different at the two earliest developmental stages. Larvae chronically exposed to caffeine reached metamorphosis at the same time as controls. Changes in chronic heart rate were dependent upon stage of development and exposure concentration. This research indicates that the toxicity of amphibian larvae exposed to triclosan, triclocarban, and caffeine appears to be dependent upon species and developmental stage, with early developmental stages being most sensitive to contaminant exposure.
- Effects of Whisker-Trimming on GABAA Receptors in S1 Cortex
- A number of studies have shown that sensory deprivation is associated with selective decreases in GABA, GAD, and GABA receptors, in deprived areas of visual and somatosensory cortex. Those studies focused on layer 4, a recipient of direct thalamocortical sensory input. However, supragranular layers 2/3 have been recently identified as a major locus of functional plasticity in sensory deprivation and long-term potentiation. To examine whether GABAA receptors in layers 2/3 are affected by sensory deprivation, rats had mystacial vibrissae in middle row C or rows ABDE trimmed for 6 weeks beginning in early adulthood. Layers 2/3 above the deprived and adjacent whisker barrels were located in tangential sections, using patterns of radial blood vessels as fiducial marks. In deprived whisker barrel columns, [3H]muscimol binding to GABAA receptors decreased by 12.8% ± 1.2 (P < 0.001) in layers 2/3 and 11.4% ± 1.2 (P<0.001) in layer 4. Altered levels of GABAA α1 subunit (Fritschy et al., 1994) were indicated by reduced optical density of immunostaining, both in deprived layers 2/3 (6.4% ± 0.7; P< 0.001) and in layer 4 (3.4% ± 1.0; P < 0.005). Interestingly, Nissl staining density also decreased in deprived layers 2/3 (12.7% ± 1.8 P < 0.001) and in 4 (6.0 ± 0.7 (P < 0.001). The percent decreases were greater in layers 2/3 than in 4 for both GABAA α1 (P < 0.05) and Nissl substance (P < 0.005). The present results suggest that down-regulation in GABAA receptors may underlie the physiological signs of disinhibition observed in neurons of layer 2/3 and 4 in deprived whisker barrel columns.
- 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.
- Enzyme Assays Using Earthworms for Assessing Innate and Nonspecific Immunotoxicity of Xenobiotics
- Principal objectives of my research were to: (1) report for the first time that coelomocytes are able to reduce NBT dye and confirm the presence of lysozyme-like activity in earthworm; (2) develop a standard methodology for determination of NBT reduction and lysozyme-like activity in earthworms; (3) compare NBT reduction and lysozyme-like activity in earthworms with those of murine and human cells and fluids; and (4) demonstrate the sensitivity of earthworm NBT reduction and lysozyme-like activity as the assays using matrics in refuse-derived fuel fly ash (RDFF) and CuSO4.
- The Eosinophil Response in Mice Infected with Trichinella spiralis or Trichinella pseudospiralis as Indicated by Phospholipase B Activity
- The host eosinophil response was compared in mice infected with either T. spiralis or T. pseudospiralis by determination of levels of splenic and intestinal phospholipase B, a marker enzyme for eosinophils. Primary infection of naive mice and challenge infection of homologously sensitized mice with T. pseudospiralis resulted in significantly lower tissue phospholipase B activities than infection with T. spiralis. Mice homologously challenged with T. pseudospiralis did exhibit an anamnestic eosinophil response compared to mice given a primary T. pseudospiralis infection. This anamnestic response, however, was significantly lower than the eosinophil response seen in sensitized mice given a homologous T. spiralis challenge. Mice sensitized to T. spiralis or T. pseudospiralis and heterologous challenge demonstrated an elevated eosinophil response compared to mice given a primary infection with either parasite. The heterologous challenge response, however, was not as intense as found for sensitized mice given a homologous challenge.
- Establishing genetic and physiological baselines for the black-tailed prairie dog (Cynomys ludovicianus).
- The black-tailed prairie dog (Cynomys ludovicianus) has experienced dramatic declines over much of its historical range due to habitat loss, plague, poisonings, and shootings. Many populations now occur as isolated genetic relicts. A multiple locus genetic profile was obtained using microsatellite analyses of six polymorphic nucleotide repeats from 319 black-tailed prairie dogs collected from 16 colonies throughout the state of Texas. This assessment revealed that existing populations have sufficient variation at all six loci to verify the usefulness of this approach as a primary genetic tool in conservation and preservation. The data reveals regional-dependent frequency patterns as well as support for founder/bottleneck effects for several of the 16 sites. Although the prairie dog population in Texas as a whole may appear genetically diverse, considerable genetic divergence has already occurred among the subpopulations (FST = 0.164). Isolation by distance is supported by genic differentiation analysis (P < 0.001) and pairwise correlation analysis between genetic distance and geographic distance (P < 0.001). Prairie dogs from six (COC, LUBA, LUBC, LUBD, LUBE, and TAR) of the original 16 sites have been relocated or exterminated, or were in the process of being relocated. Results indicated the following colonies (COT, DAL, HOW, and HUD) are of sufficient size and possess ample genetic diversity to be characterized as candidate foundation populations for future preservation efforts. The proximity of small colonies (< 20 hectares) such as HEMB, LUBB, and PEC, to other colonies should be examined to determine if they are isolated or part of a metapopulation. Colonies (HAR, HEMA, and SCH) with low genetic diversity would be ideal candidates for supplementation. Alternatively, these colonies could be relocated or blended with other similar but genetically distinct colonies. Baselines for healthy, pet prairie dog hematology and blood chemistries were also established. Results signify that data gathered from pet prairie dog blood analyses should be referenced against hematology and blood chemistry baselines established using pet prairie dog subjects.
- Eutrophic Levels of Different Areas of a Reservoir: A Comparative Study
- It was the purpose of this investigation to attempt to demonstrate if differences in eutrophic levels existed among selected areas of Garza-Little Elm, and to demonstrate the role that sediments play in affecting eutrophication.
- Eutrophication Monitoring and Prediction
- Changes in trophic status are often related to increases or decreases in the allocthonous inputs of nutrients from changes in land use and management practices. Lake and reservoir managers are continually faced with the questions of what to monitor, how to monitor it, and how much change is necessary to be considered significant. This study is a compilation of four manuscripts, addressing one of these questions, using data from six reservoirs in Texas.
- Evaluation of City of Denton Sub-Watershed by Benthic Macroinvertebrate Field Experimental Approach
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In this study, two different field experiments were designed to assess the relative influence of urbanization on benthic communities. During spring and summer, four urban and one reference sites from Denton County, Texas were selected for benthic macroinvertebrate evaluation. Statistically significant differences in colonized benthic macroinvertebrate taxa on artificial substrates were observed among the four urban sites and the reference site. Oligochaetes and chironomids were the dominant taxa at all sites. Identification of chironomid larvae at the subfamily and genus level to detect differences between sites had higher statistical power than the evaluation based on total chironomids. At the reference site, Caenis, Cladotanytarsus, Orthocladius, and Ceratopogonidae were the dominant taxa, while the urban sites were dominated by Dero, Physella, Ancylidae, Chironomus, Dicrotendipes, Glyptotendipes, Polypedilum, Pseudochironomus, Stenochironomus, and Tanytarsus. These differences may have been dependent upon differences in hydrologic regime and water quality between sites. Significant differences (ANOVA, p < 0.01) in water quality parameters (alkalinity, hardness, nitrates, phosphates, chlorides, sulfates, calcium, magnesium, potassium, and triazine) were found among water samples collected from the reference and urban sites. During the transfer period, most of the Ephemeroptera and Trichoptera taxa and a few other taxa disappeared from artificial substrates that were colonized at the reference site and then moved to the urban sites. Also, local abundant taxa from the urban site significantly (t test, p < 0.05) increased in number on the transferred artificial substrates. Seasonal differences in colonization patterns were also observed between the spring and summer experimental periods, which indicate that temporal variation is equally important, as is the anthropogenic effect in benthic community evaluation. Field survival and growth experiments using Erpetogomphus designatus larvae were designed to detect differences between evaluated sites. Larvae were collected from the reference site, measured in the laboratory, and exposed at the urban sites for six weeks in using specially designed cages. The exposed larvae demonstrated a higher mortality rate at the urban sites compared to the reference site.
- 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.
- Food, Feeding Selectivity, and Ecological Efficiencies of Fundulus notatus (Rafinesque) (Osteichthyes; Cyprinodontidae)
- This study was made to further define the trophic dynamics of Fundulus notatus by determining its ration composition under natural conditions, measuring feeding selectivity under various laboratory conditions of prey-species composition and availability, and determining the efficiencies with which F. notatus utilizes ingested chironomid larvae.
- Genetic Mechanisms for Anoxia Survival in C. Elegans
- Oxygen deprivation can be pathological for many organisms, including humans. Consequently, there are several biologically and economically relevant negative impacts associated with oxygen deprivation. Developing an understanding of which genes can influence survival of oxygen deprivation will enable the formulation of more effective policies and practices. In this dissertation, genes that influence adult anoxia survival in the model metazoan system, C. elegans, are identified and characterized. Insulin-like signaling, gonad function and gender have been shown to influence longevity and stress resistance in the soil nematode, C. elegans. Thus, either of these two processes or gender may influence anoxia survival. The hypothesis that insulin-like signaling alters anoxia survival in C. elegans is tested in Aim I. The hypotheses that gonad function or gender modulates anoxia survival are tested in Aim II. Insulin-like signaling affects anoxia survival in C. elegans. Reduction of insulin-like signaling through mutation of the insulin-like receptor, DAF-2, increases anoxia survival rates in a gpd-2/3 dependent manner. The glycolytic genes gpd-2/3 are necessary for wild-type response to anoxia, and sufficient for increasing anoxia survival through overexpression. Gonad function and gender both affect anoxia survival in C. elegans. A reduction of ovulation and oocyte maturation, as measured by oocyte flux, is associated with enhanced anoxia survival in all cases examined to date. Reduction of function of several genes involved in germline development and RTK/Ras/MAPK signaling reduce ovulation and oocyte maturation while concurrently increasing anoxia survival. The act of mating does not influence anoxia survival, but altering ovulation through breeding or chemical treatment does. The male phenotype also increases anoxia survival rates independent of genotype. These studies have identified and characterized over ten different genotypes that affect adult survival of anoxia in C. elegans. Before these studies were conducted, there were no genes known to influence adult anoxia survival in C. elegans. Furthermore, these studies have begun to uncouple mechanisms of longevity and stress resistance.
- Head trauma release of histamine from dural mast cells alters blood-brain barrier: attenuation with Zolantidine
- This study employed a new model of mild-to-moderate head trauma to specifically identify the role of dural mast cell (MC) histamine in trauma-induced increased permeability in the blood-brain barrier (BBB). A single line was scored partially through the left dorsal parietal skull. Immediately following the trauma, degranulation was seen in 39% of the MCs on the left and in 2% on the right. After a 20 min survival period, left duras showed 55% with MC degranulation (fewer with complete degranulation) compared to 34% on the right. In the other experiments two parallel lines were scored following the injection of Evan's blue. Histamine assay showed histamine increased in the left cortex to 154% at 5 min, 174% at 10 min, and 151% at 20 min. Fluorescent quantitation of extravasated Evan's blue at 20 min following the trauma gave an increase of 1385% over the value measured for the right cortex. Zolantidine, a selective histamine H2 receptor antagonist, administered at 10- and 20- mg/kg 30 min before the trauma blocked 65% of the Evan's blue extravasation compared with the control and 2.5 mg group.
- High-Resolution Autoradiography with 33p
- The purpose of this study is to provide groundwork for future autoradiographic investigations using 33P as a tracer.
- Hypoxic and hyperoxic incubation affects the ductus arteriosus in the developing chicken embryo (Gallus gallus).
- Developing chicken embryos have two ductus arteriosus (DA) that shunt blood away from the lungs and to the chorioallantoic membrane, the embryonic gas exchanger. In mammals, DA closure is stimulated by an increase in blood gas O2 that occurs as the animal begins to breathe with its lungs. The goal of this study was to determine the influence of O2 levels during incubation on the vascular reactivity and morphology of the O2-sensitive DA and to examine the effects of changing O2 levels during late incubation on the morphology of the DA from chicken embryos. In comparison to normoxia, hypoxia (15%) reduced venous O2 levels in day 16 and day 18 embryos and reduced aircell O2 values in day 16, day 18, and internally pipped (IP) embryos, whereas hyperoxia (30%) increased venous O2 levels and aircell O2 level in day 16, day 18, and IP embryos. In comparison to normoxia, hypoxia delayed closure of the DA, whereas hyperoxia accelerated DA closure. In comparison to the left DA from externally pipped (EP) normoxic embryos, the left DA from EP hypoxic embryos exhibited a significantly weaker contractile response to O2. The DA from day 18 hypoxic embryos exhibited a significantly weaker contractile response to norepinephrine and phenylephrine when compared with the DA from day 18 normoxic and hyperoxic embryos. The effect of incubation in hypoxia / hyperoxia during different developmental windows on the DA O2-induced contractile response was observed only in IP embryos that were incubated in normoxia for 16 days and were then moved to hyperoxia. Incubation in hypoxia / hyperoxia resulted in differences in embryo mass, yolk mass, and heart mass. There is an association between the decreased contractile response to O2 and delayed closure in the proximal portion of the DA from hypoxic embryos; as well as an increased contractile response to O2 and accelerated closure in the proximal portion of the DA from hyperoxic embryos.