Tree cores of Fraxinus pennsylvanica were used in a dendrochronological analysis investigating the species' responses to climate and flooding. The objective was to develop a model that incorporates the effects of precipitation, temperature, and flooding on radial growth in this species in north-central Texas. The trees exhibited strong climatic signals. The study clearly shows that all three factors have significant impacts on tree ring growth both prior to and during growth; however, the nature and extent of these impacts are highly dependent on what time of year they occur. The large temporal variations in growth responses emphasize the importance of considering the timing of environmental events when studying tree growth responses.

Cryopreservation in suspension is commonplace for a variety of cell types. However, cryopreservation of adherent cells has achieved limited success. This research aimed to cryopreserve adherent nerve cell networks in vitro in a manner that preserved network morphology and physiology. Successful implementation would enable long term storage of adherent neuronal networks on microelectrode arrays and on-demand access for use in pharmacological and toxicological testing. Based upon morphological assessments, excellent post-thaw preservation was obtained and post-thaw cultures survived in a transitional medium for up to 3.5 hours. However, transitions to native culture medium post-thaw presented difficulties, ultimately resulting in necrosis. A discussion of methods to supplement the current research and increase post-thaw viability is included in the thesis.

Prostanoids are oxygenated derivatives of arachidonic acid with a wide range of physiological effects in vertebrates including modulation of inflammation and innate immune responses. Nonsteroidal anti-inflammatory drugs (NSAIDs) act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid to prostanoids. In order to better understand the potential of environmental NSAIDS for interruption of normal levels COX products in fishes, we developed an LC/MS/MS-based approach for tissue analysis of 7 prostanoids. Initial studies examining muscle, gut and gill demonstrated that prostaglandin E2 (PGE2) was the most abundant of the measured prostanoids in all tissues and that gill tissue had the highest and most consistent concentrations of PGE2. After short-term 48-h laboratory exposures to concentrations of 5, 25, 50 and 100 ppb ibuprofen, 50.0ppb and 100.0 ppb exposure concentrations resulted in significant reduction of gill tissue PGE2 concentration by approximately 30% and 80% respectively. The lower exposures did not result in significant reductions when compared to unexposed controls. Measured tissue concentrations of ibuprofen indicated that this NSAID had little potential for bioaccumulation (BCF 1.3) and the IC50 of ibuprofen for inhibition of PGE2 production in gill tissue was calculated to be 0.4 µM. Short-term laboratory exposure to ibuprofen did not result in …

Faithful segregation of chromosomes is ensured by the spindle checkpoint. If a kinetochore does not correctly attach to a microtubule the spindle checkpoint stops cell cycle progression until all chromosomes are attached to microtubules or tension is experienced while pulling the chromosomes. The C. elegans gene, san-1, is required for spindle checkpoint function and anoxia survival. To further understand the role of san-1 in the spindle checkpoint, an RNAi screen was conducted to identify genetic interactions with san-1. The kinetochore gene hcp-1 identified in this screen, was known to have a genetic interaction with hcp-2. Interestingly, san-1(ok1580);hcp-2(ok1757) had embryonic and larval lethal phenotypes, but the phenotypes observed are less severe compared to the phenotypes of san-1(ok1580);hcp-1(RNAi) animals. Both san-1(ok1580);hcp-1(RNAi) and san-1(ok1580);hcp-2(RNAi) produce eggs that may hatch; but san-1(ok1580):hcp-1(RNAi) larvae do not survive to adulthood due to defects caused by aberrant chromosome segregations during development. Y54G9A.6 encodes the C. elegans homolog of bub-3, and has spindle checkpoint function. In C.elegans, bub-3 has genetic interactions with san-1 and mdf-2. An RNAi screen for genetic interactions with bub-3 identified that F31F6.3 may potentially have a genetic interaction with bub-3. This work provided genetic evidence that hcp-1, hcp-2 and F31F6.2 interact with spindle checkpoint …

Pseudomonas fluorescens NCIMB 11764 (Pf11764) is uniquely able to grow on the poison cyanide as its sole nitrogen source. It does so by converting cyanide oxidatively to carbon dioxide and ammonia, the latter being assimilated into cellular molecules. This requires a complex enzymatic machinery that includes nitrilase and oxygenase enzymes the nature of which are not well understood. In the course of a proteomics analysis aimed at achieving a better understanding of the proteins that may be required for cyanide degradation by Pf11764, an unknown protein of 17.8 kDa was detected in cells exposed to cyanide. Analysis of this protein by ESI-coupled mass spectrometry and bioinformatics searches gave evidence of strong homology with a protein (Hyp1) of unknown function (hypothetical) present in the bacterium Photorhabdus luminescens subsp. laumondii TTO1 (locus plu_1232). A search of available microbial genomes revealed a number of Hyp1 orthologs the genes of which are found in a conserved gene cluster known as Nit1C. Independent studies revealed that in addition to Hyp1, Pf11764 possesses a gene (nit) specifying a nitrilase enzyme whose closest homologue is a nitrilase found in Nit1C gene clusters (77% amino acid identity). DNA sequence analysis has further revealed that indeed, hyp1Pf11764 and nitPf11764 …

Weekly collections were conducted from May to December, 2007 (153 trap nights, total) in Denton, Texas, in and around large storm drains and overpass drainage facilities in residential and non-residential areas, using Centers for Disease Control (CDC) light traps and gravid traps. A total of 1964 mosquitoes were collected, representing 24 species within 6 genera: Aedes, Anopheles, Culiseta, Culex, Psorophora, and Uranotaenia. Culex was the most abundant genus, representing 75% of all mosquitoes collected; Aedes was the second most abundant, representing 12 % of all mosquitoes collected. Cx. quinquefasciatus was the dominant species collected via gravid traps; Cx. (Melanoconion) species were the dominant species collected via CDC light traps. Data of gravid traps and light traps were analyzed separately using nonparametric correlation analysis, comparing environmental data and physical characteristics to total abundance of mosquitoes. There was no significant correlation found when comparing the three dominant species collected in light traps (unidentified Cx. (Melanoconion) sp, Cx. quinquefasciatus, and Ae. vexans) to environmental characteristics and physical characteristics. Analysis of Cx. quinquefasciatus collected in gravid traps indicated no significant correlation between abundance, environmental data, and physical characteristics. Linear regression models were analyzed to determine if either environmental variables or physical characteristics of the …

This research investigated the impact of invertebrates to four species of native aquatic macrophytes: V. americana, P. nodosus, P. illinoensis, and N. mexicana. Two treatments were utilized on each plant species, an insecticide treatment to remove most invertebrates and a non-treated control. Ten herbivore taxa were collected during the duration of the study including; Synclita, Paraponyx, Donacia, Rhopalosiphum, and Hydrellia. Macrophyte biomass differences between treatments were not measured for V. americana or N. mexicana. The biomasses of P. nodosus and P. illinoensis in non-treated areas were reduced by 40% and 63% respectively. This indicated that herbivory, once thought to be insignificant to aquatic macrophytes, can cause substantial reductions in biomass.

The focus of this study was to design and evaluate a trapping system that would reduce populations of common carp within water bodies in conjunction with establishment of native aquatic macrophytes founder colonies. A pond study and field study were conducted. A pond study was performed at the Lewisville Aquatic Ecosystem Research Facility, located in Lewisville, Texas, followed by a field study within a constructed wetland located in southern Dallas, Texas. For the pond study, twelve funnel traps were constructed (four reps of each type: control, dual-walled and ring cage). Two anti-escape devices were tested with funnels including steel fingers and hinged flaps. Ring cage and dual-walled treatments were planted using native pondweeds, while controls were left unplanted (additional bait and a drift fence scenarios were also tested). Common carp were introduced into the study pond. Chi-square statistical analyses were utilized and showed ring cage treatments using fingers as well as the use of a drift fence to be most effective. Following completion of the pond study, the two most effective treatments (controls and ring cages) were tested within the Dallas, Texas wetland; no carp were caught during the field test.

Surfactants are used in a wide assortment of "down-the-drain" consumer products, yet they are often discharged in wastewater treatment plant effluent into receiving water, potentially causing environmental harm. The objective of this project was to predict surfactant toxic units and in-stream nutrients in the upper Trinity River watershed. Surface and pore water samples were collected in late summer 2005. General chemistries and surfactant toxic units were calculated. GIS models of anthropogenic and natural factors were collected and analyzed according to subwatersheds. Multiple regression analyses using the Maximum R2 improvement method were performed to predict surfactant toxic units and in-stream nutrients using GIS and in-stream values. Both geospatial and in-stream parameters generated multiple regression models for surfactant surface and pore water toxic units, as well as in-stream nutrients, with high R2 values. Thus, GIS and in-stream parameter modeling have the potential to be reliable and inexpensive method of predicting surfactant toxic units and nutrient loading in the upper Trinity River watershed.

Commonly, when it is desirable to replace an essential gene with an allelic series of mutated genes, or genes with altered expression patterns, the complementing constructs are introduced into heterozygous plants, followed by the selection of homozygous null segregants. To overcome this laborious and time-consuming step, the newly developed two-component system utilizes a site-specific recombinase to excise a wild-type copy of the gene of interest from transformed tissues. In the first component (the first vector), a wild-type version of the gene is placed between target sequences recognized by FLP recombinase from the yeast 2 μm plasmid. This construct is transformed into a plant heterozygous for a null mutation at the endogenous locus, and progeny plants carrying the excisable complementing gene and segregating homozygous knockout at the endogenous locus are selected. The second component (the second vector) carries the experimental gene along with the FLP gene. When this construct is introduced, FLP recombinase excises the complementing gene, leaving the experimental gene as the only functional copy. The FLP gene is driven by an egg apparatus specific enhancer (EASE) to ensure excision of the complementing cDNA in the egg cell and zygote following floral-dip transformation. The utility of this system is being …

Raptor rehabilitation has become commonplace globally, yet studies on the survival and adaptation of great horned owls (Bubo virginianus) after release has been neglected to an appreciable extent. The primary objective of this study is to provide quantitative data on the success of rehabilitated and released great horned owls in the North Texas region. Owls (N=12) were rehabilitated and released onto the Ray Roberts Greenbelt Corridor in Denton County, Texas, and monitored using radio telemetry to evaluate home range (November 2002 - February 2005). With approximately 75% of the birds released for this study surviving until transmitter battery failure, it is believed that the rehabilitation process was successful for these birds.

This thesis describes the development of an optimized method for culturing dissociated, monolayer neuronal networks from murine frontal cortex and midbrain. It is presented as a guidebook for use by cell culture specialists and laboratory personnel who require updated and complete procedures for use with microelectrode array (MEA) recording technology. Specific cell culture protocols, contamination prevention and control, as well common problems encountered within the cell culture facility, are discussed. This volume offers value and utility to the rapidly expanding fields of MEA recording and neuronal cell culture. Due to increasing interest in determining the mechanisms underlying Parkinson's disease, the newly developed procedures for mesencephalon isolation and culture on MEAs are an important research contribution.

The Medicago truncatula NIP gene is located on M. truncatula Linkage Group 1. Informative recombinants showed crossovers that localize the NIP gene between markers 146O17 and 23C16D. Marker 164N9 co-segregates with the NIP gene, and the location of marker 164N9 is between markers 146O17 and 23C16D. Based upon data from the Medicago genome sequencing project, a subset of the model legume Medicago truncatula bacterial artificial chromosomes (BACs) were used to create a physical map on the DNA in this genetic internal. BACs near the potential NIP gene location near marker 164N9 were identified, and used in experiments to predict the physical map by a BAC-by-BAC strategy. Using marker 164N9 as a center point, and chromosome walking outward, the physical map toward markers 146O17 and 23C16D was built. The chromosome walk consisted of a virtual walk, made with existing sequence of BACs from the Medicago genome project, hybridizations to filters containing BAC DNA, and PCR reactions to confirm that predicted overlapping BACs contained DNA that yielded similar PCR products. In addition, the primers which are made for physical mapping via PCR could be good genetic markers helpful in discovering the location of the NIP gene. As a result of efforts repotted …

The chicken ductus arteriosi (DA) are two embryonic blood vessels that shunt blood away from the non-ventilated lungs and towards the body and chorioallantoic membrane. I show that prostaglandins have a diminished role in maintaining chicken DA patency and nitric oxide inhibits oxygen induced contraction of the day 19 proximal DA in a time dependent manner. The pathways governing oxygen induced contraction in the chicken DA are similar to those found in mammals and include contributions from ROS, Kv channels, L-type Ca2+ channels, and the Rho kinase pathway. Longer exposure to high oxygen generates increased oxygen induced constriction of the day 19 DA that may be mediated through the Rho kinase pathway.

In order to assess the enhancement of seedling survival and growth during drought conditions, five-hundred bare-root seedlings each of Shumard oak (Quercus shumardii Buckl.) and green ash (Fraxinus pennsylvanica Marsh.) were planted each with four soil amendments at a Wildlife Management Area in Lewisville, Texas. The treatments were a mycorrhizal inoculant, mulch fabric, and two superabsorbent gels (TerraSorb® and DRiWATER®). Survival and growth measurements were assessed periodically for two years. Research was conducted on vegetation, soil, and site history for baseline data. Both superabsorbent gels gave significant results for Shumard oak survival, and one increased green ash diameter. For overall growth, significant results were found among DRiWATER®, mycorrhizae, and mulch treatments.

The study of root nodule symbiosis between nitrogen-fixing bacteria and leguminous plant species is important because of the ability to supplement fixed nitrogen fertilizers and increase plant growth in poor soils. Our group has isolated a mutant called nip in the model legume Medicago truncatula that is defective in nodule symbiosis. The nip mutant (numerous infections with polyphenolics) becomes infected by Sinorhizobium meliloti but then accumulates polyphenolic defense compounds in the nodule and fails to progress to a stage where nitrogen fixation can occur. Analysis of the transcriptome of nip roots prior to inoculation with rhizobia was undertaken using Affymetric Medicago Genome Array microarrays. The total RNA of 5-day old uninoculated seedlings was analyzed in triplicate to screen for the NIP gene based on downregulated transcript levels in the mutant as compared to wild type. Further microarray data was generated from 10 days post inoculation (dpi) nip and wild type plants. Analysis of the most highly downregulated transcripts revealed that the NIP gene was not identifiable based on transcript level. Putative gene function was assigned to transcripts with altered expression patterns in order to characterize the nip mutation phenotypically as inferred from the transcriptome. Functional analysis revealed a large number …

The rapid development of once-rural landscapes often produces detrimental effects on surface water quality entering local reservoirs through vulnerable stream channels. This study presents a methodology that incorporates geographic information systems (GIS) and remote sensing techniques for the creation of a stream corridor evaluation mechanism, coined the water quality corridor management (WQCM) model. Specifically, the study focuses on determining the viability of the WQCM model in assessing the stream corridor conditions within a northern Denton County pilot study region. These results will aid in the prediction and evaluation of the quality of stream water entering reservoirs that serve as the primary drinking water source for local municipalities.

A bacterial challenge assay was developed utilizing the earthworm, Lumbricus terrestris, in order to assess potential immunotoxic effects from exposure to specific polychlorinated biphenyl congeners. Earthworms were inoculated with Aeromonous hydrophila, establishing a 10-day LD50. In vitro assays for effects of PCBs on phagocytosis agreed with mammalian studies, demonstrating potent suppression of phagocytosis by the non-coplanar PCB congener 138 and no suppression by the coplanar congener 126. However, when the effects of the two PCB congeners were evaluated for suppression of resistance to a whole animal infection challenge assay, coplanar PCB 126 decreased the ability of L. terrestris to withstand infection while non-coplanar PCB 138 did not.

A novel luminescence resonance energy transfer (LRET) nanocircuit assay involving a donor and two acceptors in tandem was developed to study the dynamic interaction of skeletal muscle contraction proteins. The donor transmits energy relayed to the acceptors distinguishing myosin subfragment-1 (S1) lever arm orientations. The last acceptor allows the detection of S1's bound near or in between troponin complexes on the thin filament. Additionally, calcium related changes between troponin T and myosin were detected. Based on this data, the troponin complex situated every 7 actin monomers, hinders adjacently bound myosins to complete their power stroke; whereas myosins bound in between troponin complexes undergo complete power strokes.

Superior swimming stamina in adult fish is presumably passed on to their offspring, but the ontogeny of the appearance of superior stamina and the requisite enhanced cardio-respiratory support for locomotion in larval fishes has not been determined. Is the expression of the suite of parental traits enabling superior swimming stamina in their offspring dependent upon their achieving juvenile/adult morphology, or does it appear earlier in their larvae? To answer this, adults were classified into three groups based on swimming stamina, followed by measurement of length, mass, and width. Larval offspring from the two parental groups -high stamina larvae (HSL) and low stamina larvae (LSL)- were reared at 27°C in aerated water (21% O2). Routine and active heart rate, routine and active mass specific oxygen consumption were recorded through 21dpf, and cost of transport (COT) and factorial aerobic scope were derived from oxygen consumption measurements. Routine heart rate at 2dpf of LSL was 164 ± 1 b·min-1, compared to only 125 ± 2 b·min-1 for HSL. Routine heart rate subsequently peaked at 203 ± 1 b·min-1 at 5dpf in the HSL group, compared to 207 ± 1 b·min-1, at 4dpf in the LSP larvae. Active heart rate at 5 dpf of …

Large amounts of industrial fertilizers are used to maximize crop yields. Unfortunately, they are not completely consumed by plants; consequently, this leads to soil pollution and negative effects on aquatic systems. An alternative to industrial fertilizers can be found in legume plants that provide a nitrogen source that is not harmful for the environment. Legume plants, through their symbiosis with soil bacteria called rhizobia, are able to reduce atmospheric nitrogen into ammonia, a biological nitrogen source. Establishment of the symbiosis requires communication on the molecular level between the two symbionts, which leads to changes on the cellular level and ultimately results in nitrogen-fixing nodule development. Inside the nodules hypoxic environment, the bacterial enzyme nitrogenase reduces atmospheric nitrogen to ammonia. Medicago truncatula is the model legume plant that is used to study symbiosis with mycorrhiza and with the bacteria Sinorhizobium meliloti. The focus of this work is the M. truncatula nodulation mutant nip (numerous infections and polyphenolics). The NIP gene plays a role in the formation and differentiation of nodules, and development of lateral roots. Studying this mutant will contribute knowledge to understanding the plant response to infection and how the invasion by rhizobia is regulated. Previous genetic mapping placed NIP …

Proteomic techniques were used to evaluate the protein profile of the earthworm, (Lumbricus terrestris), following a bacterial challenge. One control group received no injection; a second control group received injections of phosphate buffer solution (PBS). The experimental group received injections of PBS containing (Aeromonas hydrophila). After incubation for 12 hours at 20°C, coelomic fluid was collected from each group for analysis by 2-D electrophoresis. There were significant differences in spot appearance and density between control and experimental groups. Sixteen spots showed a two-fold increase in density and 63 showed at least a two-fold decrease in density between samples from control and bacteria-challenged earthworms, respectively, suggesting up- and down-modulation of proteins potentially involved in the earthworm's response to bacterial challenge.

An understanding of the potential roles as lipid mediators of a family of bioactive metabolites called N-acylethanolamines (NAEs) depends on their accurate identification and quantification. The levels of 18C unsaturated NAEs (e.g. NAE18:2, NAE 18:3, etc.) in wild-type seeds (about 2000 ng/g fw) generally decreased by about 80% during germination and post-germinative growth. In addition, results suggest NAE-degradative fatty acid amide hydrolase (FAAH) expression does not play a major role in normal NAE metabolism as previously thought. Seedlings germinated and grown in the presence of abscisic acid (ABA), an endogenous plant hormone, exhibited growth arrest and secondary dormancy, similar to the treatment of seedlings with exogenous N­lauroylethanolamine (NAE12:0). ABA-mediated growth arrest was associated with higher levels of unsaturated NAEs. Overall, these results are consistent with the concept that NAE metabolism is activated during seed germination and suggest that the reduction in unsaturated NAE levels is under strict temporal control and may be a requirement for normal seed germination and post-germinative growth.

Dispersal of aquatic insects is difficult to measure with traditional direct trapping methodologies. However, genetic markers are an ideal surrogate to indirectly infer dispersal and gene flow. For this research, a portion of the cytochrome oxidase I gene was used to evaluate gene flow and dispersal of Epeorus pleuralis located in the northern Appalachian headwater streams of the Allegheny, Genesee, and Susquehanna watersheds. A total of 536 basepairs from 16 individual insects were used for analysis. Thirteen haplotypes were discovered, two of which were shared between the Allegheny and Genesee streams. Although no shared haplotypes were found in the Susquehanna, analysis of molecular variance results suggest that there is not a significant genetic difference between the three populations and attributes the majority of variation to within population differences.