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Role of 5.8S rRNA in Zebrafish and Human Blood Coagulation
Hemolytic disorders are characterized by hemolysis and are prone to thrombosis. Previously, it has been shown that the RNA released from damaged blood cells activates clotting. However, the nature of RNA released from hemolysis is still elusive. We found that after hemolysis, the red blood cells from both zebrafish and humans release 5.8S rRNA. This RNA activated coagulation in zebrafish and human plasmas. Using both natural and synthetic 5.8S rRNA and its synthetic truncated fragments, we found that the 3'-end 26 nucleotide-long RNA (3'-26 RNA) and its stem-loop secondary structure were necessary and sufficient for clotting activity. Corn trypsin inhibitor (CTI), a coagulation factor XII (FXII) inhibitor blocked 3'-26 RNA-mediated coagulation activation of both zebrafish and human plasma. CTI also inhibited zebrafish coagulation in vivo. 5.8S rRNA monoclonal antibody inhibited both 5.8S rRNA- and 3'-26 RNA-mediated zebrafish coagulation activity. Both 5.8S rRNA and 3'-26 RNA activates normal human plasma but did not activate FXII-deficient human plasma. Taken together, these results suggested that the activation of zebrafish plasma is via FXII-like protein. Since zebrafish has no FXII and hepatocyte growth factor activator (Hgfac) has sequence similarities to FXII, we knocked down the hgfac in adult zebrafish. We found that plasma from this knockdown fish does not respond to 3'-26 RNA. In conclusion, we identified 5.8S rRNA released in hemolysis activates clotting in human and zebrafish plasma. Only 3'-end 26 nucleotides of the 5.8S rRNA is needed for the clotting activity. Furthermore, we showed that fish Hgfac plays a role in 5.8S rRNA-mediated activation of coagulation.
A Genetic Approach to Identify Proteins that Interact with Eukaryotic Microtubule Severing Proteins via a Yeast Two Hybrid System
Microtubules (MT) are regulated by multiple categories of proteins, including proteins responsible for severing MTs that are therefore called MT-severing proteins. Studies of katanin, spastin, and fidgetin in animal systems have clarified that these proteins are MT-severing. However, studies in plants have been limited to katanin p60, and little is known about spastin or fidgetin and their function in plants. I looked at plant genomes to identify MT-severing protein homologues to clarify which severing proteins exist in plants. I obtained data from a variety of eukaryotic species to look for MT-severing proteins using homology to human proteins and analyzed these protein sequences to obtain information on the evolution of MT-severing proteins in different species. I focused this analysis on MT-severing proteins in the maize and Arabidopsis thaliana genomes. I created evolutionary phylogenetic trees for katanin-p60, katanin-p80, spastin, and fidgetin using sequences from animal, plant, and fungal genomes. I focused on Arabidopsis spastin and worked to understand its functionality by identifying protein interaction partners. The yeast two-hybrid technique was used to screen an Arabidopsis cDNA library to identify putative spastin interactors. I sought to confirm the putative protein interactions by using molecular tools for protein localization such as the YFP system. Finally, a Biomolecular Fluorescence Complementation (BiFC) assay was initiated as a proof of concept for confirmation of in vivo protein-protein interaction.
The Role of the Actin Cytoskeleton in Asymmetric Cell Division in Maize
Stomata are specialized plant structures required for gaseous exchange with the outer environment. During stomata formation, the cytoskeleton plays an important role in controlling the division of the individual cells leading to the generation of the stomata complex. Two mutants that affect microfilament and microtubule organization in subsidiary mother cells include brk1 and dcd1. While only 20% of the subsidiary cells in the brk1 and dcd1 single mutants are abnormally shaped, it was reported that there is a synergistic effect between the brk1 and dcd1 mutations in the brk1; dcd1 double mutant since 100% of the subsidiary cells are abnormal. The focus of this research is to try to understand this synergistic effect by investigating the actin cytoskeleton and nuclear position in the single and double mutants. The reported results include the observation that the size of actin patch was largest in the wild-type subsidiary mother cells (SMCs) and smallest in dcd1 and brk1; dcd1 SMCs and that brk1 and brk1; dcd1 double mutants had fewer actin patches than wild-type and dcd1 SMCs. Additionally, we observed that some SMCs that did not have actin patches still underwent nuclear migration suggesting that nuclear migration may not be solely dependent on actin patch formation. Finally, during SMC cytokinesis, a large percentage of double mutant (brk1; dcd1) cells showed an off-track development of the phragmoplast as compared to the single mutants and the wild-type plant explaining the large number of abnormally shaped subsidiary cells in the double mutants.
A Behavioral Model for Detection of Acute Stress in Bivalves
A behavioral model for acute responses in bivalves, was developed using time series analysis for use in a real-time biomonitoring unit. Stressed bivalves closed their shell and waited for the stressful conditions to pass. Baseline data showed that group behavior of fifteen bivalves was periodic, however, individuals behaved independently. Group behavior did not change over a period of 20 minutes more than 30 percent, however, following toxic exposures the group behavior changed by more than 30 percent within 20 minutes. Behavior was mathematically modeled using autoregression to compare current and past behavior. A logical alarm applied to the behavior model determined when organisms were stressed. The ability to disseminate data collected in real time via the Internet was demonstrated.
Development, Validation, and Evaluation of a Continuous, Real-time, Bivalve Biomonitoring System
A biological monitoring tool to assess water quality using bivalve gape behavior was developed and demonstrated. The purpose of this work was to develop methodologies for screening water quality appropriate to the goals of the watershed paradigm. A model of bivalve gape behavior based on prediction of behavior using autoregressive techniques was the foundation of the bivalve biomonitoring system. Current technology was used in developing the system to provide bivalve gape state data in a continuous real-time manner. A laboratory version of the system, including data collection and analysis hardware and software, was developed for use as a toxicological assay for determination of effective concentrations of toxicant(s) or other types of stress on bivalve gape behavior. Corbicula fluminea was monitored and challenged with copper, zinc, and chlorpyrifos using the system. Effective concentrations of 176±23µg/L copper, 768±412µg/L zinc, and 68µg/L chlorpyrifos were observed using a natural water with high dissolved organic carbon concentrations. A rugged field version of the bivalve biomonitoring system was developed and deployed in two locations. The field systems were fitted with a photovoltaic array, a single board computer, and a CDPD telemetry modem for robust remote operation. Data were telemetered at a time relevant rate of once every ten minutes. One unit was deployed in Lake Lewisville, Denton County, TX in February 2000. Data were telemetered and archived at a 92% success rate. Bivalve gape data demonstrated significant behavioral deviations on average 5 times per month. A second unit was deployed in Pecan Creek, Denton, TX in June 2001. Data from this site were telemetered and archived at a 96% success rate. Over the months of June-August 2001, 16 significant behavioral deviations were observed, 63% of which were correlated with changes in physical/chemical parameters. This work demonstrated the relative sensitivity of bivalve gape as a toxicological endpoint …
Evaluation of Diet, Water, and Culture Size for Ceriodaphnia Dubia Laboratory Culturing
Six reagent waters, eleven diets, and two culture sizes were evaluated for culturing C. dubia. Different filtration techniques were used to prepare the reagent waters. The eleven diets were comprised of two algae augmented with eight supplements. Reproduction and growth were assessed to discern differences among C. dubia raised in mass cultures and cultured in individual cups, during which, bacterial population densities, lipid, protein, and carbohydrate concentrations of the diets were measured. Results showed that a glass-distilled, carbon filtered, deionized reagent water and a Selenastrum capricornutum- Cerophyl® diet were optimum for culturing. Mass culturing supported the highest reproduction and growth, while no correlation was found between nutritional measurements and production.
Effects of Suspended Multi-Walled Carbon Nanotubes on Daphnid Growth and Reproduction
Multi-walled carbon nanotube aggregates can be suspended in the aqueous phase by natural organic matter. These aggregates are ingested by filter feeding zooplankton. Ingested aggregates result in decreased growth and decreased reproduction. These effects may be caused by reduction in energy input from normal feeding behavior. pH alters natural organic matter structure through changes in electrostatic repulsion. Altered natural organic matter structure changes multi-walled carbon nanotube aggregate size. This size variation with variation in pH is significant, but not large enough a change in size to alter toxicity, as the aggregate size range remains well within the particle size selection of the organisms.
Analysis of Multipartite Bacterial Genomes Using Alignment-Free and Alignment-Based Pipelines
In this work, we have performed comparative evolutionary analysis, functional genomics analysis, and machine learning analysis to identify the molecular factors that discriminate between multipartite and unipartite bacteria, with the goal to decipher taxon-specific factors and those that are prevalent across the taxa underlying the these traits. We assessed the roles of evolutionary mechanisms, namely, horizontal gene transfer and gene gain, in driving the divergence of bacteria with single and multiple chromosomes. In addition, we performed functional genomic analysis to garner support for our findings from comparative evolutionary analysis. We found genes such as those encoding conserved hypothetical protein DR_A0179 and hypothetical protein DR_A0109 in Deinococcus radiodurans R1, and Putative phage phi-C31 gp36 major capsid-like protein and hypothetical protein RSP_3729 in Rhodobacter sphaeroides 2.4.1, which are located on accessory chromosomes in both bacteria and were not found in the inferred ancestral sequences, and on the primary chromosomes, as well as were not found in their closest relatives with single chromosome within the same clade. These genes emphasize the important potential roles of the secondary chromosomes in helping multipartite bacteria to adapt to specialized environments or conditions. In addition, we applied machine learning algorithms to predict multipartite genomes based on gene content of multipartite genomes and their unipartite relatives, and leveraged this to identify genes that are deemed important by machine learning in discriminating between multipartite and unipartite genomes. This approach led to the identification of marker genes that could be used in discriminating between bacteria with multipartite genomes and. bacteria with single chromosome genomes Furthermore, we examined modules in gene co-expression networks of multipartite Rhodobacter sphaeroides 2.4.1 and its close unipartite relative Rhodobacter capsulatus SB 1003 that were enriched in genes differentially expressing under stressful conditions representing different experiments. This led to the identification of 6 modules in the Rhodobacter …
The Generation of Recombinant Zea mays Spastin and Katanin Proteins for In Vitro Analysis
Plant microtubules play essential roles in cell processes such as cell division, cell elongation, and organelle organization. Microtubules are arranged in highly dynamic and ordered arrays, but unlike animal cells, plant cells lack centrosomes. Therefore, microtubule nucleation and organization are governed by microtubule-associated proteins, including a microtubule-severing protein, katanin. Mutant analysis and in vitro characterization has shown that the highly conserved katanin is needed for the organization of the microtubule arrays in Arabidopsis and rice as well as in a variety of animal models. Katanin is a protein complex that is part of the AAA+ family of ATPases. Katanin is composed of two subunits, katanin-p60, a catalytic subunit and katanin-p80, a regulatory subunit. Spastin is another MT-severing protein that was identified on the basis of its homology to katanin. In animal cells, spastin is also needed for microtubule organization, but its functionality has not yet been investigated in plants. To initiate an exploration of the function of katanin-p60 and spastin in Zea mays, my research goal was to generate tools for the expression and purification of maize katanin-p60 and spastin proteins in vitro. Plasmids that express katanin-p60 and spastin with N-terminal GST tags were designed and constructed via In-Fusion® cloning after traditional cloning methods were not successful. The constructs were expressed in E. coli, then the recombinant proteins were purified. To determine if the GST-tagged proteins are functional, ATPase activity and tubulin polymerization assays were performed. While both GST-katanin-p60 and GST-spastin hydrolyzed ATP indicating that the ATPase domains are functional, the results of the tubulin polymerization assays were less clear and further experimentation is necessary.
Novel Role of Trypsin in Zebrafish
It has been shown previously in our laboratory that zebrafish produce trypsin from their gills when they are under stress, and this trypsin is involved in thrombocyte activation via PAR2 during gill bleeding. In this study, I investigated another role of the trypsin that is secreted from zebrafish. This investigation has demonstrated a novel role of trypsin in zebrafish. Not only did this investigation demonstrate the role of trypsin in zebrafish behavior, but also it showed that PAR2 might be the receptor that is involved in trypsin-mediated behavioral response. In addition, we have shown that Gq and ERK inhibitors are able to block the trypsin pathway and prevent the escaping behavior. Finally, the results of this investigation suggest that the cells that respond to trypsin are surface cells, which have an appearance similar to that of neuromast cells.
Studies in Trypsin as an Alarm Substance in Zebrafish
Previous studies have shown that fish release alarming substances into the water to alert their kin to escape from danger. In our laboratory, we found that zebrafish produce trypsin and release it from their gills into the environment when they are under stress. By placing the zebrafish larvae in the middle of a small tank and then placing trypsin at one end of the tank, we observed that the larvae moved away from the trypsin zone and almost to the opposite end of the tank. This escape response was significant and did not occur in response to the control substances, bovine serum albumin (BSA), Russell's viper venom (RVV), and collagen. Also, previously, we had shown that the trypsin could act via a protease-activated receptor-2 (PAR2) on the surface of the cells. Therefore, we hypothesized that trypsin would induce a change in neuronal activity in the brain via PAR2-mediated signaling in cells on the surface of the fish body. To investigate whether the trypsin-responsive cells were surface cells, we generated a primary cell culture of zebrafish keratinocytes, confirmed these cells' identity by specific marker expression, and then incubated these cells with the calcium indicator Fluo-4 and exposed them to trypsin. By using calcium flux assay in a flow-cytometer, we found that trypsin-treated keratinocytes showed an increase in intracellular calcium release. To test whether PAR2 mediates the escape response to trypsin, we treated larvae with a PAR2 antagonist and showed that the trypsin-initiated escape response was abrogated. Furthermore, par2a mutants with knockdown of par2a by the piggyback knockdown method failed to respond to trypsin. Trypsin treatment of adult fish led to an approximately 2-fold increase in brain c-fos mRNA levels 45 mins after trypsin treatment, suggesting that trypsin signals may have reached the brain, probably via a spinothalamic pathway. Taken together, our …
Hooking Mortality of Largemouth Bass Caught on Controversial Artificial Lures and Live Bait : Lake Ray Roberts, Texas
A total of 192 largemouth bass were caught at Lake Ray Roberts, Texas (1995) to investigate five controversial bass angling techniques relative to hooking mortality. The bait types were Texas-rigged scented and non-scented plastic worms, Carolina-rigged scented and non-scented plastic worms, and live golden shiners. Overall hooking mortality was 21.87% and mortality was dependent upon bait type. Highest mortality resulted from the Texas-rigged scented lures, while the lowest mortality was generated by live golden shiners. A creel survey indicated that few anglers were having success with the investigated baits. Factors that had a confirmed effect on hooking mortality were hooking location and water temperature. Hooking mortality was not excessive compared to other similar studies.
Retinoic acid Treatment Affects Kidney Development and Osmoregulatory System in the Developing Chicken (Gallus Gallus)
Development is a dynamic process characterized by critical periods in which organ systems are sensitive to changes in the surrounding environment. In the current study, critical windows of embryonic growth and kidney development were assessed in the embryonic chicken. All‐trans retinoic acid (tRA) influences not only organogenesis and cell proliferation, but also targets metanephric kidney nephrogenesis. Embryonic chickens were given a single injection of tRA on embryonic day 8. tRA decreased embryo, kidney, and heart mass from day 16 to day 18. However, mass specific kidney and heart masses showed no differences. Whole blood, plasma, and allantoic fluid osmolality were altered in tRA treated embryos from day 16 to day 18. In addition, hematocrit, red blood cell count, and hemoglobin concentration were altered in tRA treated embryos. The results suggest that although nephrogenesis was not affected by tRA, the developing osmoregulatory system was altered in tRA treated embryos.
Isolation and Bioinformatic Characterization of Four Novel Bacteriophages from Streptomyces toxytricini
Six initial phage isolates with high titer lysates were obtained using Streptomyces toxytricini B-5426 as the host bacterium. These isolates were named Goby, Toma, Yosif, Yara, Deema, and Hsoos. However, upon completion of the sequencing, it was found that the Yara and Hsoos isolates were identical, as were Goby and Deema. As a result, final analysis was completed on only the four unique isolates. All of the phages mentioned above were isolated from soil samples from different locations. Also, they had different sizes of plaques, ranging from 0.3 – 0.9mm. Yosif had the largest plaque size. Yara's head diameter was 79nm with tail diameter of 94nm.
Quantifying the Effects of Single Nucleotide Changes in the TATA Box of the Cauliflower Mosaic Virus 35S Promoter on Gene Expression in Arabidopsis thaliana
Synthetic biology is a rapidly growing field that aims to treat cellular biological networks in an analogous way to electrical circuits. However, the field of plant synthetic biology has not grown at the same pace as bacterial and yeast synthetic biology, leaving a dearth of characterized tools for the community. Due to the need for tools for the synthetic plant biologist, I have endeavored to create a library of well-characterized TATA box variants in the cauliflower mosaic virus (CaMV) 35S promoter using the standardized assembly method Golden Braid 2.0. I introduced single nucleotide changes in the TATA box of the CaMV 35S promoter, a genetic part widely used in plant gene expression studies and agricultural biotechnology. Using a dual-luciferase reporter system, I quantified the transcriptional strength of the altered TATA box sequences and compared to the wild-type sequence, both in transient protoplast assays and stable transgenic Arabidopsis thaliana plants. The library of TATA-box modified CaMV 35S promoters with varying transcriptional strengths created here can provide the plant synthetic biology community with a series of modular Golden Braid-adapted genetic parts that can be used dependably and reproducibly by researchers to fine-tune gene expression levels in complex, yet predictable, synthetic genetic circuits.
Investigation of Strategies for Improving STR Typing of Degraded and Low Copy DNA from Human Skeletal Remains and Bloodstains
Forensic STR analysis is limited by the quality and quantity of DNA. Significant damage or alteration to the molecular structure of DNA by depurination, crosslinking, base modification, and strand breakage can impact typing success. Two methods that could potentially improve STR typing of challenged samples were explored: an in vitro DNA repair assay (PreCR™ Repair Mix) and whole genome amplification. Results with the repair assay showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally-damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR™ assay. The data suggest that the use of PreCR™ in casework should be considered with caution due to the assay’s varied results. As an alternative to repair, whole genome amplification (WGA) was pursued. The DOP-PCR method was selected for WGA because of initial primer design and greater efficacy for amplifying degraded samples. Several modifications of the original DOP-PCR primer were evaluated. These modifications allowed for an overall more robust amplification of damaged DNA from both contemporary and historical skeletal remains compared with that obtained by standard DNA typing and a previously described DOP-PCR method. These new DOP-PCR primers show promise for WGA of degraded DNA.
Integrating life cycle analysis and the ecological footprint calculator to foster sustainable behaviors
Many tools have been developed to assess global, national or regional sustainable development policies. However, as governments develop sustainable policies, individuals must also feel empowered to affect their personal impact on the planet. This thesis integrates three sustainability concepts that lend themselves to individual sustainability: The natural step, life cycle assessment, and the ecological footprint. TNS serves to provide the meaning and substance toward sustainable development. LCA helps provide the framework for assessing sustainability. The EF calculator determines the driving components and measures the qualitative decisions made through TNS and LCA. From the analysis of the household footprint calculator a simplified footprint calculator was developed to assist individuals and communities in setting benchmarks and goals as they move away from over-consumption and towards a sustainable lifestyle.
Developing a Generalizable Two-Input Genetic AND Logic Gate in Arabidopsis thaliana for Multi-Signal Processing
With effective engineering using synthetic biology approaches, plant-based platforms could conceivably be designed to minimize the production costs and wastes of high-value products such as medicines, biofuels, and chemical feedstocks that would otherwise be uneconomical. Additionally, modern agricultural crops could be engineered to be more productive, resilient, or restorative in different or rapidly changing environments and climates. To achieve these complex goals, information-processing genetic devices and circuits containing multiple interacting parts that behave predictably must be developed. A genetic Boolean AND logic gate is a device that computes the presence or absence of two inputs (signals, stimuli) and produces an output (response) only if both inputs are present. Here, we optimized individual genetic components and used synthetic protein heterodimerizing domains to rationally assemble genetic AND logic gates that integrate two hormonal inputs in whole plants. These AND gates produce an output only in the presence of both abscisic acid and auxin, but not when either or neither hormone is present. Furthermore, we demonstrate the AND gate can also integrate two plant stresses, cold temperature and bacterial infection, to produce a specific response. The design principles used here are generalizable, and therefore multiple orthogonal AND gates could be assembled and rationally layered to process complex genetic information in plants. In addition to bioproduction, these layered logic gates may also be used in circuits to probe fundamental questions in plant biology such as hormonal crosstalk.
The Effects of Probiotics on Growth, and Metabolism in Juvenile Oreochromis mossambicus (Mozambique Tilapia)
Improving growth, lowering mortality rates, and having a faster turnaround to harvest is essential for the future of commercial aquaculture. The primary goal of this study was to determine if introducing a single strain probiotic Lactobacillus rhamnosus IMC 501 into the feed regimen of a commercially important aquaculture freshwater fish, Mozambique tilapia (Oreochromis mossambicus), would decrease mortality; change metabolic rates; and increase tissue wet mass (MW), standard length, growth rate and feed conversion rate (FCRs). IMC501 was added to the fishmeal in four increasing concentrations and compared to a control without probiotics. Results from two-way ANOVAs showed that both treatment levels and elapsed time had a significant effect on both mean standard length and wet mass; in the latter case, time points and treatments interacted with one another, showing that tilapia grew best with a moderate level of probiotics present. The growth benefits of probiotics continued for months after the initial treatments. Oxygen consumption (metabolic rate) was measured using closed respirometry and resulted in recording the first values for juvenile tilapia treated with probiotics. For oxygen consumption, there were significant treatment and time effects with significant interactions, indicating that metabolism increased with probiotics once the dosage exceeded three times the industry level. These results are consistent with the observed increases in mass, length and growth rates. These results demonstrate the importance of conducting dose-response experiments in order to determine the most effective concentration of probiotics in juvenile freshwater fish. Importantly, probiotics at the right concentration increase metabolic rates and can positively influence tilapia growth, which is of interest for the optimization of Mozambique tilapia production in aquaculture.
Engineered Microbial Consortium for the Efficient Conversion of Biomass to Biofuels
Current energy and environmental challenges are driving the use of cellulosic materials for biofuel production. A major obstacle in this pursuit is poor ethanol tolerance among cellulolytic Clostridium species. The first objective of this work was to establish a potential upper boundary of ethanol tolerance for the cellulosome itself. The hydrolytic function of crude cellulosome extracts from C. cellulolyticum on carboxymethyl cellulose (CMC) with 0, 5, 10, 15, 20 and 25% (v/v) ethanol was determined. Results indicated that the endoglucanase activity of the cellulosome incubated in 5% and 10% ethanol was significantly different from a control without ethanol addition. Furthermore a significant difference was observed in endoglucanase activity for cellulosome incubated in 5%, 10%, 15%, 20% and 25% ethanol in a standalone experiment. Endoglucanase activity continued to be observed for up to 25% ethanol, indicating that cellulosome function in ethanol will not be an impediment to future efforts towards engineering increasing production titers to levels at least as high as the current physiological limits of the most tolerant ethanologenic microbes. The second objective of this work was to study bioethanol production by a microbial co-culture involving Clostridium cellulolyticum and a recombinant Zymomonas mobilis engineered for the utilization of oligodextrans. The recombinant Z. mobilis ZM4 pAA1 and wild type ZM4 were first tested on RM medium (ATCC 1341) containing 2% cellobiose as the carbon source. Ethanol production from the recombinant Z. mobilis was three times that observed from the wild type Z. mobilis. Concomitant with ethanol production was the reduction in OD from 2.00 to 1.580, indicating the consumption of cellobiose. No such change in OD was observed from the wild type. The recombinant ZM4 was then co-cultured with C. cellulolyticum using cellobiose and microcrystalline cellulose respectively as carbon sources. Results indicate that the recombinant ZM4 acted synergistically with C. cellulolyticum …
Evaluation of virulence in wild type and pyrimidine auxotrophs of Pseudomonas aeruginosa using the eukaryotic model system Caenorhabditis elegans.
The human opportunistic pathogen, Pseudomonas aeruginosa PAO1, has been shown to kill the nematode Caenorhabditis elegans. C. elegans has been a valuable model for the study of bacterial pathogenesis, and has reinforced the notion that common virulence and host defense mechanisms exist. Recently, the pyrimidine pathway was shown to regulate virulence levels. Therefore, mutations in the pyrimidine pathway of PAO1 showed decrease virulence in the nematode. When starving the nematode, bacterial resistance was also shown to increase. It was hypothesized that starvation induced the DAF pathway, which regulates the transcription of genes involved with the antibacterial defense mechanism. Further research will be conducted to test this theory by performing RNAi experiments for the genes functioning in the antibacterial defense mechanism.
Inquiry-based science for high school students: a forensic unit
This project constitutes an instructional unit for honors biology that involves the use of science in the field of criminal investigation and forensics. Before beginning the unit, the learners should have mastered basic laboratory skills, including use of the microscope. They should also have an understanding of the basic structure and function of DNA and its role in heredity and protein synthesis. The standard time frame is 24 days with 70-minute periods, but can be easily adjusted to meet classroom needs. Several instructional strategies enhance student learning and make science fun. The unit is inquiry-driven and activity-based. Students are surprised by the crime, gather and analyze evidence, and work towards proposing an explanation. This real world problem involves the use of cooperative learning and a variety of assessment techniques.
Fatty Acid Amide Hydrolases in Upland Cotton (Gossypium hirsutum L.) and the Legume Model Medicago truncatula
Fatty acid amide hydrolase (FAAH) is a widely conserved amidase in eukaryotes, best known for inactivating the signal of N-acylethanolamine (NAE) lipid mediators. In the plant Arabidopsis thaliana, FAAH-mediated hydrolysis of NAEs has been associated with numerous biological processes. Recently, the phylogenetic distribution of FAAH into two major branches (group I and II FAAHs) across angiosperms outside of Arabidopsis (and in other Brassicaceae), suggests a previously unrecognized complexity of this enzyme. Although A. thaliana has long been used to assess biological questions for plants, in this case it will fall short in understanding the significance of multiple FAAHs in other plant systems. Thus, in this study, I examined the role (s) of six FAAH isoforms in upland cotton (Gossypium hirsutum L.) and two FAAHs in the legume Medicago truncatula.
Effects of Water Quality, Instream Toxicity, and Habitat Variability on Fish Assemblages in the Trinity River, Texas
The Trinity River flows through the Dallas-Ft. Worth Metroplex in north central Texas where it receives effluents from numerous point sources including seven large regional wastewater treatment facilities. Historically, the Trinity River has been impacted by massive wastewater loadings which often constitute > 80% of the total river discharge during low flow periods. Normally, high mass loadings correspond to the summer months, compounding the effects of a naturally stressful period, characterized by high temperatures and low dissolved oxygen concentrations. Samples from 12 stations were collected quarterly over an 18 month period from the Trinity River and two tributaries. Water samples were analyzed for a variety of water quality variables, including metals, priority pollutants, pesticides, and general water quality parameters. Water samples were also tested for acute and subchronic effects with several test species. Fish were collected at each station and assemblages were characterized using traditional classification techniques and the Index of Biotic Integrity. In addition, sediment samples were assessed for toxic effects which could have adversely impacted fish recruitment and in situ biomonitoring experiments were performed. Quantitative habitat characterization analyses were performed to gain additional information that could possibly explains differences in fish assemblage structure related to habitat variability. Data were analyzed using regression, univariate, multivariate, and descriptive statistical techniques and new approaches for analyzing impact assessment data were discussed. Results indicated that the most substantial impacts on fish assemblages were confined to a segment of the river where a sequence of point sources, in close proximity to each other, were overloading the river's capacity to sufficiently dilute and/or detoxify the effluent. Data also indicated the presence of episodic toxicity from nonpoint sources. In addition, toxic effects in sediment samples and differences in habitat were detected and may have contributed to measured differences among fish assemblages in the Trinity River.
Effector Response of the Aspartate Transcarbamoylase From Wild Type Pseudomonas Putida and a Mutant with 11 Amino Acids Deleted at the N-terminus of PyrB.
Like its enteric counterpart, aspartate transcarbamoylase (ATCase) from Pseudomonas putida is a dodecamer of two different polypeptides. Unlike the enterics, the Pseudomonas ATCase lacks regulatory polypeptides but employs instead inactive dihydroorotases for an active dodecamer. Previous work showed that PyrB contains not only the active site but also the effector binding sites for ATP, UTP and CTP at its N-terminus. In this work, 11 amino acids were deleted from the N-terminus of PyrB and the ATCase with the truncated protein was expressed in E. coli pyrB- and purified. The wild type enzyme was similarly treated. Velocity-substrate plots without effectors gave Michaelis-Menten kinetics in all cases. Deleting 11 amino acids did not affect dodecameric assembly but altered effector responses. When carbamoylphosphate was varied, the mutant enzyme was inhibited by UTP while the wild type enzyme was activated 2-fold. When the aspartate was varied, CTP had no effect on the mutant enzyme but strongly inhibited the wild type enzyme.
Regulation of pyrimidine biosynthesis and virulence factor production in wild type, Pyr- and Crc- mutants in Pseudomonas aeruginosa.
Previous research in our laboratory established that pyrB, pyrC or pyrD knock-out mutants in Pseudomonas aeruginosa required pyrimidines for growth. Each mutant was also discovered to be defective in the production of virulence factors. Moreover, the addition of exogenous uracil did not restore the mutant to wild type virulence levels. In an earlier study using non-pathogenic P. putida, mutants blocked in one of the first three enzymes of the pyrimidine pathway produced no pyoverdine pigment while mutants blocked in the fourth, fifth or sixth steps produced copious quantities of pigment, just like wild type P. putida. The present study explored the correlation between pyrimidine auxotrophy and pigment production in P. aeruginosa. Since the pigment pyoverdine is a siderophore it may also be considered a virulence factor. Other virulence factors tested included casein protease, elastase, hemolysin, swimming, swarming and twitching motilities, and iron binding capacity. In all cases, these virulence factors were significantly decreased in the pyrB, pyrC or pyrD mutants and even in the presence of uracil did not attain wild type levels. In order to complete this comprehensive study, pyrimidine mutants blocked in the fifth (pyrE) and sixth (pyrF) steps of the biosynthetic pathway were examined in P. aeruginosa. A third mutant, crc, was also studied because of its location within 80 base pairs of the pyrE gene on the P. aeruginosa chromosome and because of its importance for carbon source utilization. Production of the virulence factors listed above showed a significant decrease in the three mutant strains used in this study when compared with the wild type. This finding may be exploited for novel chemotherapy strategies for ameliorating P. aeruginosa infections in cystic fibrosis patients.
Spatial Variations and Cultural Explanations to Obesity in Ghana
While obesity is now recognized as a major health concern in Ghana, the major drivers, causal factors, and their spatial variation remain unclear. Nutritional changes and lack of physical activity are frequently blamed but the underlying factors, particularly cultural values and practices, remain understudied. Using hot spot analysis and spatial autocorrelation, this research investigates the spatial patterns of obesity in Ghana and the explanatory factors. We also use focus group discussions to examine the primary cultural factors underlying these patterns. The results show that wealth, high education, and urban residence are the best positive predictors of obesity, while poverty, low education, and rural residence are the best (negative) predictors of obesity. Consequently, improving the socioeconomic status, for example, through higher levels of education and urbanization may increase obesity rates. Furthermore, the cultural preference for fat body as the ideal body size drives individual aspiration for weight gain which can lead to obesity. Thus, reducing obesity rates in Ghana is impossible without addressing the underlying cultural values.
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.
Hypoxia-Induced Cardiac Arrest Alters Central Nervous System Concentrations of the GLYT2 Glycine Transporter in Zebrafish (Danio rerio)
Hypoxia as a stressor has physiological implications that have been a focal point for many physiological studies in recent years. In some studies, hypoxia had large effects on the organ tissue degeneration, which ultimately effects multiple ecological processes. These organ tissue studies played a part in the development of new fields like neurocardiology, a specialty that studied the relationship between the brain and the heart. This thesis focuses on how hypoxia-induced cardiac arrest alters the amounts of GLYT2, a glycine reuptake transporter, in the central nervous system of zebrafish, Danio rerio. At 7 days post-fertilization (dpf), zebrafish were exposed to acute, severe hypoxia until they lost equilibrium, and minutes later, subsequent cardiac arrest occurred. Zebrafish were then placed into recovery groups to measure the GLYT2 levels at multiple points in zebrafish recovery. Fish were then sacrificed, and their brains dissected. Using immunofluorescence, the outer left optic tectum of the zebrafish was imaged, and mean image pixel fluorescent intensity was taken. There were significant changes (one-way ANOVA) in the levels of GLYT2 compared to that of the control groups during the course of recovery. GLYT2 levels continued to rise through the 24-hour recovery mark but did not show significant difference after 3 hours of recovery. This suggest that GLYT2 levels increased rapidly in the first 3 hours of recovery and continued to increase through 24 hours at a slower rate. Changes in GLYT2 levels may affect motor and sensory information, movement, visualization, and audition in these zebrafish. Further research should be conducted to determine how long it takes for GLYT2 levels to return to baseline, as well as behavioral measurements through each recovery period as it relates to glycine function.
Pyrimidine Enzyme Specific Activity at Four Different Phases of Growth in Minimal and Rich Media, and Concomitant Virulence Factors Evaluation in Pseudomonas aeruginosa
Pseudomonas aeruginosa is a Gram-negative rod, aerobic, non-fermenting, oxidase positive, pigment producing, and nutritionally versatile bacterium. Infections by P. aeruginosa are the most important cause of morbidity and mortality in immunocompromised patients, given virulence factor production that suppresses antibiotic therapy and promotes persistent infection. This research is the first comprehensive report of the pyrimidine biosynthetic pathway for all phases of growth in minimal and rich media coupled with the evaluation of virulence factor production of P. aeruginosa in comparison to four other bacterial species (Pseudomonas putida, Pseudomonas fluorescens, Burkholderia cepacia, and Escherichia coli wild-type strains). Cellular growth and passing genetic information to the next generation depend on the synthesis of purines and pyrimidines, the precursors of DNA and RNA. The pyrimidine biosynthetic pathway is essential and found in most organisms, with the exception of a few parasites that depend upon the pyrimidine salvage pathway for growth. Both the pyrimidine biosynthetic and salvage enzymes are targets for chemotherapeutic agents. In our laboratory, research on pyrimidine auxotrophic mutants showed the role of the pyrimidine biosynthetic pathway and its intermediates on P. aeruginosa metabolism and impaired virulence factors production. The present research shows that pyrimidine enzymes are active in all phases of growth, including the production of two forms of ATCase in the late log phase in P. aeruginosa. This finding may be explained by the displacement of the inactive PyrC' by the active PyrC or PyrC2 to form a new and larger pyrBC encoded ATCase. Pseudomonas aeruginosa wild-type appears to produce by far the most virulence factors, haemolysin, iron chelation, rhamnolipid, adherence, and three types of motility (swimming, swarming, and twitching) investigated in this study, when compared to the other four wild-type strains. Growth analysis was carried out as typically done in minimal medium but also in rich medium to simulate conditions …
The Influence of a Return of Native Grasslands upon the Ecology and Distribution of Small Rodents in Big Bend National Park
In the southwestern United States there is a delicate balance between the existing grasslands and the rodent fauna. The purpose of this investigation was to determine the influence of secondary succession of native grasslands upon the ecology and distribution of small rodents. Two methods of determining the rodent species were plot quadrates and trap lines using Sherman live traps.
The Developmental Physiology of the Zebrafish: Influence of Environment and Cardiovascular Attributes
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, …
Functional Characterization of Mtnip/latd’s Biochemical and Biological Function
Symbiotic nitrogen fixation occurs in plants harboring nitrogen-fixing bacteria within the plant tissue. The most widely studied association is between the legumes and rhizobia. In this relationship the plant (legumes) provides the bacteria (rhizobia) with reduced carbon derived from photosynthesis in exchange for reduced atmospheric nitrogen. This allows the plant to survive in soil, which is low in available of nitrogen. Rhizobia infect and enter plant root and reside in organs known as nodules. In the nodules the bacteria fix atmospheric nitrogen. The association between the legume, Medicago truncatula and the bacteria Sinorhizobium meliloti, has been studied in detail. Medicago mutants that have defects in nodulation help us understand the process of nitrogen fixation better. One such mutant is the Mtnip-1. Mtnip-1 plants respond to S. meliloti by producing abnormal nodules in which numerous aberrant infection threads are produced, with very rare rhizobial release into host plant cells. The mutant plant Mtnip-1 has an abnormal defense-like response in root nodules as well as defects in lateral root development. Three alleles of the Mtnip/latd mutants, Mtnip-1, Mtlatd and Mtnip-3 show different degrees of severity in their phenotype. Phylogenetic analysis showed that MtNIP/LATD encodes a protein belonging to the NRT1(PTR) family of nitrate, peptide, dicarboxylate and phytohprmone transporters. Experiments with Mtnip/latd mutants demonstrats a defective nitrate response associated with low (250 μM) external nitrate concentration rather than high (5 mM) nitrate concentration. This suggests that the mutants have defective nitrate transport. To test if MtNIP/LATD was a nitrate transporter, Xenopus laevis oocytes and Arabidopsis thaliana mutant plants Atchl1-5, defective in a major nitrate transporter AtNRT1.1(CHL1), were used as surrogate expression systems. Heterologous expression of MtNIP/LATD in X. laevis oocytes and Atchl1-5 mutant plants conferred on them the ability to take up nitrate from external media with high affinity, thus demonstrating that MtNIP/LATD …
Measurement of Feedback Inhibition In Vivo and Selection of ATCase Feedback Altered Mutants in Salmonella typhimurium
Aspartate transcarbamoylase (ATCase; encoded by pyrBI genes) is one of the most studied regulatory enzymes in bacteria. It is feedback inhibited by cytidine triphosphate (CTP) and activated by adenosine triphosphate (ATP). Much is known about the catalytic site of the enzyme, not nearly as much about the regulatory site, to which CTP binds. Until now a positive selection for feedback-modified mutants was not available. The selection we have developed involves the use of a pyrA deletion in S. typhimurium. This strain lacks carbamoylphosphate and requires both a pyrimidine and arginine for growth. In this strain citrulline is used to satisfy the pyrimidine and arginine requirements. The minimal flow through the pyrimidine pathway from the citrulline-produced carbamoylphosphate is exquisitely sensitive to feedback control of ATCase by CTP. By elevating the CTP pool, via exogenous cytidine, in a strain that also contains a cytidine deaminase mutant (cdd) growth can be stopped completely, indicating 100% inhibition. It was therefore possible to measure in vivo feedback inhibition of ATCase among the citrulline users and to isolate a family of ATCase regulatory mutants with either modified or no response to effectors.
Nucleotide Sequence Determination, Subcloning, Expression and Characterization of the xy1LT Region of the Pseudomonas putida TOL Plasmid pDK1
The complete nucleotide sequence of the region encoding the DHCDH function of the pDK1 lower operon was determined. DNA analysis has shown the presence of two open reading frames, one gene consisting of 777 nucleotides encoding a polypeptide of 27.85 kDa and another gene of 303 nucleotides encoding a polypeptide of 11.13 kDa. The results of enzymatic expression studies suggest that DHCDH activity is associated only with xy1L. However although the addition of xy1T cell-free extracts to xy1L cell-free extracts does not produce an increase in DHCDH activity, subclones carrying both xy1L and xy1T exhibit 300- 400% more DHCDH activity than subclones carrying only xy1L.
In vitro Cultures of Morus alba for Enhancing Production of Phytoestrogens
Plant estrogens have long been associated with health benefits. The potential of tissue culture techniques for the production of several secondary metabolites has been known for many years. Tissue cultures stimulate the production or induce the biosynthesis of novel compounds not found in the mature plant. Tissue culture of Morus alba, family Moraceae, is known to contain phytoestrogens, was established on plant-hormone supplemented Murashige and Skoog (MS) medium. Petiole and the stem tissue from mature trees were the best explants for initiation and proliferation of calli. The best callus proliferation was obtained on MS medium containing 1-napthalene acetic acid (1mg/ml) and benzylaminopurine (0.5mg/ml) for M. alba. Comparison of phytoestrogens of Moraceae species from in vivo and in vitro tissue isolation were carried out. The estrogenic activities of callus extracts were assayed in an estrogen-responsive yeast system expressing the human estrogen receptor alpha. Male callus extracts had higher estrogenic activity than male and female extracts from in vivo and in vitro tissues. Isolation and characterization of phytoestrogens from above tissues were carried out using solid phase extraction, high perfomance liquid chromatography and mass spectrometry techniques. Biochanin A, an isoflavonoid, was isolated as one of the compounds in male callus extracts. Biochanin A has been known to have an antiestrogenic acitivity in mammals. Isoflavonoid compounds have been characterized as strong protein tyrosine kinase inhibitors in variety of animal cells. Isoflavones are structurally similar to estradiol, and display agonistic and antagonistic interactions with the estrogen receptor. Isoflavones possess therapeutic and preventive properties such as being used for postmenopausal osteoporosis, breast cancer, and inhibition of tumors.
Ecology of Chironomids Associated with Myriophyllum Spicatum L. and Heteranthera Dubia Macm
Macroinvertebrate communities inhabiting an exotic, Myriophyllum spicatum, and a native, Heteranthera dubia macrophyte were studied from March 1999 to June 2000 in experimental ponds. Although macrophyte architecture explained some variation in macroinvertebrate abundance between the two macrophytes, most variation was explained by the sampling months. Total number of macroinvertebrates was found to be positively correlated with epiphyton biomass which differed significantly between the two plant types and among sampling months. Taxa richness did not vary between the two plant types. Chironomid larvae were the most abundant organisms and dominated by Apedilum elachistus on both plant communities. Annual production of five chironomid species was estimated by the size-frequency method. Production estimates (P) in g dry wt m-2 yr-1 of plant surface area for the predator Tanypodinae larvae were: Larsia decolarata, P= 0.77 and 0.67, Labrundinia virescens, P= 0.59 and 0.35 on M. spicatum and H. dubia, respectively. Larvae of Cricotopus sylvestris and Psectrocladius vernalis were collected from M. spicatum from March to mid-June. Production of C. sylvestris was found to be 0.46 g dry wt m-2, whereas it was 0.07 g dry wt m-2 for P. vernalis for this period. Apedilum elachistus exhibited the highest productivity: 9.9 g dry wt m-2 yr-1 of plant surface area on M. spicatum, and 8.5 g dry wt m-2 yr-1 on H. dubia. These production estimates are among the highest production values reported for a single species. Additionally, post-ovipositing development times for five chironomid species collected from Myriophyllum and Heteranthera were determined. Three different temperatures (15°, 20° and 25°C) were chosen to rear eggs under 12L: 12D photoperiod. Egg development times ranged between 1-4 days. Larval development times ranged from 44 days at 20°C for Tanypus neopunctipennis to as few as 9 days at 20°C for Larsia decolorata.
Mass Spectrometry-Based Identification of Ceramic-Bound Archaeological Protein Residues: Method Validation, Residue Taphonomy, and Prospects
Despite the variety of successful reports of the preservation, recovery, and identification of archaeological proteins in general, there are few positive reports regarding mass spectrometry-based identification of ceramic-bound proteins. In large part, this shortage is due to the lack of consideration for the unique taphonomic histories of such residues and, in general, methods development. Further, because negative results are rarely published, there is no baseline to which results can be compared. This paper attempts to address these challenges via a multi-pronged approach that uses mass spectrometry and complementary approaches to evaluate ceramic-bound protein preservation in both controlled, actualistic experiments, and in archaeological artifacts. By comparing the results obtained from protein-spiked, experimentally-aged ceramic to those obtained from both faunal and ceramic archaeological materials, an enhanced perspective on protein preservation and subsequent recovery and identification is revealed. This perspective, focusing on taphonomy, reveals why negative results may be the norm for ceramic artifacts when non-targeted methods are employed, and provides insight into how further method development may improve the likelihood of obtaining positive results.
Producing a Film on Oil Spill Research for the Public
The Deepwater Horizon oil drilling rig exploded on April 20, 2010, off the coast of Louisiana in the Gulf of Mexico. Following the spill, British Petroleum, leaser of the rig, set up a funding institution known as the Gulf of Mexico Research Initiative (GoMRI) to support research and understanding of the spill on the environments and peoples of the gulf. This outreach project was created alongside research of the RECOVER consortium, funded by GoMRI, to communicate what is happening within research labs around the country to understand the effect that the spill had on fish in pelagic and coastal regions of the gulf. The outreach project is composed of a short film (Deepwaters: The Science of a Spill, 18 min) and related outreach materials posted to Instagram (@FishandOilSpills).
Ecological Significance and Underlying Mechanisms of Body Size Differentiation in White-tailed Deer
Body size varies according to nutritional availability, which is of ecological and evolutionary relevance. The purpose of this study is to test the hypothesis that differences in adult body size are realized by increasing juvenile growth rate for white-tailed deer (Odocoileus virginianus). Harvest records are used to construct growth rate estimates by empirical nonlinear curve fitting. Results are compared to those of previous models that include additional parameters. The rate of growth increases during the study period. Models that estimate multiple parameters may not work with harvest data in which estimates of these parameters are prone to error, which renders estimates from complex models too variable to detect inter-annual changes in growth rate that this simpler model captures
Comparison of Aspartate Transcarbamoylase and Pyrimidine Salvage in Sporosarcina urea, Sprolactobacillus inulinus, Lactobacillus fermentum, and Micrococcus luteus
The enzyme that catalyzes the committed step in pyrimidine biosynthesis, aspartate transcarbamoylase, has been compared in selected endospore-forming organisms and in morphologically similar control organisms. The ATCases and pyrimidine salvage from Sporosarcina ureae, Sporolactobacillus inulinus, Lactobacillus fermentum, and Micrococcus luteus were compared to those of Bacillus subtilis. While the ATCases from Sporosarcina ureae, Sporolactobacillus inulinus, and L. fermentum were found to exhibit characteristics to that of Bacillus with respect to molecular weight and kinetics, M. luteus ATCase was larger at approximately 480 kDa. Furthermore, pyrimidine salvage in Sporosarcina ureae and M. luteus was identical to those of B. subtilis, while pyrimidine salvage of Sporolactobacillus inulinus and L. fermentum resembled that of the pseudomonads.
Evaluation of the Economic, Social, and Biological Feasibility of Bioconverting Food Wastes with the Black Soldier Fly (Hermetia illucens)
Food waste in the waste stream is becoming an important aspect of integrated waste management systems. Current efforts are composting and animal feeding. However, these food waste disposal practices rely on slow thermodynamic processes of composting or finding farmers with domestic animals capable of consuming the food wastes. Bioconversion, a potential alternative, is a waste management practice that converts food waste to insect larval biomass and organic residue. This project uses a native and common non-pest insect in Texas, the black soldier fly, which processes large quantities of food wastes, as well as animal wastes and sewage in its larval stage. The goal of this research is to facilitate the identification and development of the practical parameters of bioconversion methods at a large cafeteria. Three major factors were selected to evaluate the practicality of a bioconversion system: (1) the biological constraints on the species; (2) the economic costs and benefits for the local community; (3) the perception of and interaction between the public and management agencies with respect to the bioconversion process. Results indicate that bioconversion is feasible on all levels. Larvae tolerate and consume food waste as well as used cooking grease, reducing the overall waste volume by 30-70% in a series of experiments, with an average reduction of 50%. The economical benefits are reduced collection costs and profit from the sale of pupae as a feedstuff, which could amount to as much as $1,200 per month under optimal conditions. Social acceptance is possible, but requires education of the public, specifically targeting school children. Potential impediments to social acceptance include historical attitudes and ignorance, which could be overcome through effective educational efforts.
Hepatotoxicity of Mercury to Fish
Tissue samples from spotted gar (Lepisosteus oculatus) and largemouth bass (Micropterus salmoides) were collected from Caddo Lake. Gar and bass livers were subjected to histological investigation and color analysis. Liver color (as abs at 400 nm) was significantly correlated with total mercury in the liver (r2 = 0.57, p = 0.02) and muscle (r2 = 0.58, p = 0.01) of gar. Evidence of liver damage as lipofuscin and discoloration was found in both species but only correlated with liver mercury concentration in spotted gar. Inorganic mercury was the predominant form in gar livers. In order to determine the role of mercury speciation in fish liver damage, a laboratory feeding study was employed. Zebrafish (Danio rerio) were fed either a control (0.12 ± 0.002 µg Hg.g-1 dry wt), inorganic mercury (5.03 ± 0.309 µg Hg.g-1 dry wt), or methylmercury (4.11 ± 0.146 µg Hg.g-1 dry wt) diet. After 78 days of feeding, total mercury was highest in the carcass of zebrafish fed methylmercury (12.49 ± 0.369 µg Hg.g-1 dry wt), intermediate in those fed inorganic mercury (1.09 ± 0.117 µg Hg.g-1 dry wt), and lowest in fish fed the control diet (0.48 ± 0.038 µg Hg.g-1 dry wt). Total mercury was highest in the viscera of methylmercury fed zebrafish (11.6 ± 1.86 µg Hg.g-1 dry wt), intermediate in those fed inorganic diets (4.3 ± 1.08 µg Hg.g-1 dry wt), and lowest in the control fish (below limit of detection). Total mercury was negatively associated with fish length and weight in methylmercury fed fish. Condition factor was not associated with total mercury and might not be the best measure of fitness for these fish. No liver pathologies were observed in zebrafish from any treatment.
Spatial and Temporal Patterns of Areal and Volumetric Phytoplankton Productivity of Lake Texoma
Phytoplankton productivity of Lake Texoma was measured for one year from August 1999 to August 2000 for four stations, using the oxygen change method and laboratory incubation. Mean values of the photosynthetic parameters, PBmax and alphaB ranged from 4.86 to 46.39 mg O2.mg Chl-1.hr-1 for PBmax and 20.06 to 98.96 mg O2.mg Chl-1.E-1.m2 for alphaB. These values were in the range to be expected for a highly turbid, temperate reservoir. Estimated gross annual areal productivity ranged from 594 g C.m2.yr-1 (P.Q. = 1.2), at a station in the Washita River Zone to 753 g C.m2.yr-1 at a station in the Red River Zone, of the reservoir. Gross annual areal productivity at Station 17, in the Main Lake Zone, was 708 g C.m2.yr-1. Gross areal and volumetric productivity showed distinct seasonal variation with Photosynthetically Available Radiation (PAR) and temperature. Trophic status estimated on a station-by-station basis, using net productivity values derived from gross productivity and respiration estimates, was mesotrophic for all the stations, though one station approached eutrophy. Net productivity values ranged from 0.74 to 0.91 g C. m-2.d-1. An algal bioassay conducted at two stations in August 2000, revealed that phosphorus was most likely the nutrient limiting photosynthesis at both these stations, although the more turbid riverine station was primarily light-limited.
Evaluating Fish Impingement and Entrainment at the Comanche Peak Steam Electric Station
This study was designed to determine if impingement and entrainment by cooling water intake at the Comanche Peak Steam Electric Station have an adverse impact upon the Squaw Creek Reservoir fish population. The yearly impingement of fish was estimated to be 262,994 of 14 species. The threadfin shad (Dorosoma petenense) accounted for 96% of this total. Entrainment of eggs and larvae for a five month period was estimated to be 15,989,987 and 42,448,794 respectively. Two fish population studies were performed on Squaw Creek Reservoir to help assess impact. It was determined that the losses due to impingement and entrainment have no adverse impact upon the fish population of Squaw Creek Reservoir.
Pyrimidine Salvage Enzymes in Microorganisms: Labyrinths of Enzymatic Diversity
Pyrimidine salvage pathways are essential to all cells. They provide a balance of RNA synthesis with the biosynthetic pathway in pyrimidine prototrophs and supply all the pyrimidine requirements in auxotrophs. While the pyrimidine biosynthetic pathway is found in almost all organisms and is nearly identical throughout nature, the salvage pathway often differs from species to species, with aspects of salvage seen in every organism. Thus significant taxonomic value may be ascribed to the salvage pathway. The pyrimidine salvage pathways were studied in 55 microorganisms. Nine different salvage motifs, grouped I-IX, were identified in this study based on the presence of different combinations of the following enzymes: cytidine deaminase (Cdd), cytosine deaminase (Cod), uridine phosphorylase (Udp), uracil phosphoribosyltransferase (Upp), uridine hydrolase (Udh), nucleoside hydrolase (Nuh), uridine/cytidine kinase (Udk), 5'-nucleotidase and CMP kinase (Cmk).
Effect of Rancher’s Management Philosophy, Grazing Practices, and Personal Characteristics on Sustainability Indices for North Central Texas Rangeland
To assess sustainability of privately owned rangeland, a questionnaire was used to gathered data from ranches in Cooke, Montague, Clay, Wise, Parker, and Jack counties in North Central Texas. Information evaluated included: management philosophy, economics, grazing practices, environmental condition, quality of life, and demographics. Sustainability indices were created based on economic and land health indicator variables meeting a minimum Cronbach‘s alpha coefficient (α = 0.7). Hierarchical regression analysis was used to create models explaining variance in respondents’ indices scores. Five predictors explained 36% of the variance in rangeland economic sustainability index when respondents: 1) recognized management inaction has opportunity costs affecting economic viability; 2) considered forbs a valuable source of forage for wildlife or livestock; 3) believed governmental assistance with brush control was beneficial; 4) were not absentee landowners and did not live in an urban area in Texas, and; 5) valued profit, productivity, tax issues, family issues, neighbor issues or weather issues above that of land health. Additionally, a model identified 5 predictors which explained 30% of the variance for respondents with index scores aligning with greater land health sustainability. Predictors indicated: 1) fencing cost was not an obstacle for increasing livestock distribution; 2) land rest was a component of grazing plans; 3) the Natural Resource Conservation Service was used for management information; 4) fewer acres were covered by dense brush or woodlands, and; 5) management decisions were not influenced by friends. Finally, attempts to create an index and regression analysis explaining social sustainability was abandoned, due to the likely-hood of type one errors. These findings provide a new line of evidence in assessing rangeland sustainability, supporting scientific literature concerning rangeland sustainability based on ranch level indicators. Compared to measuring parameters on small plots, the use of indices allows for studying replicated whole- ranch units using rancher insight. Use …
The Influence of Urban Green Spaces on Declining Bumble Bees (Hymenoptera: Apidae)
Bumble bees (Bombus spp.) are adept pollinators of countless cultivated and wild flowering plants, but many species have experienced declines in recent decades. Though urban sprawl has been implicated as a driving force of such losses, urban green spaces hold the potential to serve as habitat islands for bumble bees. As human populations continue to grow and metropolitan areas become larger, the survival of many bumble bee species will hinge on the identification and implementation of appropriate conservation measures at regional and finer scales. North Texas is home to some the fastest-growing urban areas in the country, including Denton County, as well as at least two declining bumble bee species (B. pensylvanicus and B. fraternus). Using a combination of field , molevular DNA and GIS methods I evaluated the persistence of historic bumble bee species in Denton County, and investigated the genetic structure and connectivity of the populations in these spaces. Field sampling resulted in the discovery of both B. pensylvanicus and B. fraternus in Denton County's urban green spaces. While the relative abundance of B. fraternus in these spaces was significantly lower than historic levels gleaned from museum recors, that of B. pensylvanicus was significantly higher. Statistical analyses found that both bare ground and tree cover surrounding sample sites were negatively associated with numbers of bumble bee individuals and hives detected in these green spaces. Additionally, limited genetic structuring of bumble bee populations was detected, leading to the conclusion that extensive gene flow is occurring across populations in Denton County.
A morphological study of the avian (Gallus domesticus) ductus arteriosi during hatching.
The ductus arteriosi (DA) are two blood vessels connecting the pulmonary arteries to the descending aorta in the avian embryo. Following hatching, the DA closes, separation of the systemic and pulmonary circulation. I present the morphological changes that occur in the chicken DA during prepipping, internal pipping, external pipping, and hatching. The avian DA consists of two distinct tissue types, a proximal and a distal portion. Histological examination shows developmental differences between the proximal and distal portions of the DA with regard to lumen occlusion, endothelial cells, smooth muscle and elastin. Endothelial cell proliferation begins to occur as early as external pipping, with the lumen almost completely occluded by the 3rd day of post-hatching life. Expression of vascular endothelial growth factor (VEGF) increases in avian endothelial cells during hatching. I provide a morphological timeline of changes in the DA as the chicken develops from embryo to hatchling.
Evidentiary Value of Condoms: Comparison of Durable Physical and Chemical Characteristics of Condoms
Condom trace evidence must not be overlooked in sexual assault cases; understanding the chemical and physical characteristics of condoms is imperative if condoms are to be useful evidence. Previous research shows that condom identification is possible, but it is equally important to evaluate durability of condom residues versus time. Using FT-IR, this study examined vaginal swabs from subjects who self-sampled at intervals for up to 72 hours after having intercourse with a condom. This study investigated whether age and the stage of the menstrual cycle affected the durability of residues in the vagina over time. This study revealed that condoms containing nonoxynol-9, silicone-based lubricants, and particulates provide valuable information for identification, and that nonoxynol-9 specifically withstands the vaginal environment for up to 72 hours. Additionally, age and menstrual cycle both appeared to have an effect on the durability of residues although larger sample size is desirable.
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