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Biodiversity of Dragonflies and Damselflies (Odonata) of the South-Central Nearctic and Adjacent Neotropical Biotic Provinces
The south-central United States serves as an important biogeographical link and dispersal corridor between Nearctic and Neotropical elements of western hemisphere odonate faunas. Its species are reasonably well known because of substantial collections, but there has been no concerted effort to document the extent of biodiversity and possible geographic affinities of dragonflies and damselflies of this region. The recent discoveries of Argia leonorae Garrison, Gomphus gonzalezi Dunkle and Erpetogomphus heterodon Garrison from southern and western Texas and northern Mexico suggest that Odonata species remain to be discovered in this area, particularly from far south Texas and northern Mexico. I have documented a total of 12,515 records of Odonata found in 408 counties within the south-central U.S. A total of 73 species of damselflies and 160 species of dragonflies was revealed in the region. The 233 (197 in Texas) Odonata species are distributed among 10 families and 66 genera. Illustrated family, generic, and species-level keys are provided. Since the beginning of this work in the Fall of 1993, one species has been added each to the Louisiana and Oklahoma faunas, and 12 species have been added, previously unreported from Texas, including four new to the U.S. The area of highest Odonata biodiversity overall (161 spp.) is in the Austroriparian biotic province. The greatest degree of faunal similarity between the south-central U.S. and other intra-continental regions was observed for the eastern (64%) United States. Diversity is a function of area, and as expected, the numbers of breeding birds and Odonata, in each contiguous U.S. state are positively correlated (r=0.376, n=33, p=0.031). There is, however, no strong correlation between land area and species diversity within the region, but those natural biotic provinces (Austroriparian, Texan, Balconian) where aquatic systems and topographic heterogeneity are the greatest provide a broader spectrum of potential Odonata habitats and …
Landscape forest modeling of the Luquillo Experimental Forest, Puerto Rico.
This thesis contributes to modeling the dynamics of forest community response to environmental gradients and disturbances over a mountain landscape. A gap model (FACET) was parameterized for species of various forest types (Tabonuco, Colorado, Dwarf and Palm), for many terrain conditions and was modified and extended to include species response to excess soil moisture and hurricanes. Landscape cover types were defined by dominance of species of each forest type and canopy height. Parameters of the landscape model (MOSAIC) were calculated from multiple runs of FACET. These runs were determined by combining terrain variables (elevation and soil) and hurricane risk. MOSAIC runs were analyzed for distribution patterns. Geographic Information Systems software was used to process terrain variables, hurricane risk and MOSAIC model output.
Impact of Anti-S2 Peptides on a Variety of Muscle Myosin S2 Isoforms and Hypertrophic Cardiomyopathy Mutants Revealed by Fluorescence Resonance Energy Transfer and Gravitational Force Spectroscopy
Myosin subfragment-2 (S2) is an intrinsically unstable coiled coil. This dissertation tests if the mechanical stability of myosin S2 would influence the availability of myosin S1 heads to actin thin filaments. The elevated instability in myosin S2 coiled coil could be one of the causes for hypercontractility in Familial Hypertrophic Cardiomyopathy (FHC). As hypothesized FHC mutations, namely E924K and E930del, in myosin S2 displayed an unstable myosin S2 coiled coil compared to wild type as measured by Fluorescence Resonant Energy Transfer (FRET) and gravitational force spectroscopy (GFS). To remedy this, anti-S2 peptides; the stabilizer and the destabilizer peptides by namesake were designed in our lab to increase and decrease the stability of myosin S2 coiled coil to influence the actomyosin interaction. Firstly, the effectiveness of anti-S2 peptides were tested on muscle myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by the stabilizer and decreased by the destabilizer across the cardiac and skeletal myosin S2 isoform but not for the smooth muscle isoform. The destabilizer peptide had dissociation binding constants of 9.97 × 10-1 μM to MYH7 isoform, 1.00 μM to MYH2 isoform, and no impact on MYH11, and the stabilizer peptide had dissociation binding constants of 2.12 × 10-2 μM to MYH7 isoform, 3.41 × 10-1 μM to MYH2 isoform, and no impact on MYH11 revealed by FRET. In presence of the stabilizer, FRET assay, affinity of the E930del and E924K increased by 10.23 and 0.60 fold respectively. The force required to uncoil muscle myosin S2 peptides in the presence of the stabilizer peptide was more than in its absence in muscle myosin S2 isoforms of MYH7 (1.80 fold higher), MYH2 (1.40 fold higher), and E930del (2.60 fold higher) and no change for MYH11 …
DNA-DNA Hybridization of Methane Oxidizing Bacteria
Bacteria classified in the family Methylomonadaceae must derive their carbon from one-carbon compounds. They are characterized by the possession of internal membranes of two types. Type I membranes are layered and fill the middle of the cells while type II membranes form concentric layers around the periphery of the cells. Also, there are two metabolic pathways by which the methylobacteria assimilate one-carbon compounds. Further evidence of this dichotomy was sought by DNA-DNA saturation hybridization of DNAs from both types of methylobacteria. Very low DNA-DNA homology was seen between types I and II or within the types. It was not possible, therefore, to correlate the degree of genetic relatedness with either the nature of the internal membranes or the pathway of carbon assimilation.
Integrating Selective Herbicide and Native Plant Restoration to Control Alternanthera philoxeroides (Alligator Weed)
Exotic invasive aquatic weeds such as alligator weed (Alternanthera philoxeroides) threaten native ecosystems by interfering with native plant communities, disrupting hydrology, and diminishing water quality. Development of new tools to combat invaders is important for the well being of these sensitive areas. Integrated pest management offers managers an approach that combines multiple control methods for better control than any one method used exclusively. In a greenhouse and field study, we tested the effects of selective herbicide application frequency, native competitor plant introduction, and their integration on alligator weed. In the greenhouse study, alligator weed shoot, root, and total biomass were reduced with one herbicide application, and further reduced with two. Alligator weed cumulative stem length and shoot/root ratio was only reduced after two applications. In the greenhouse, introduction of competitors did not affect alligator weed biomass, but did affect shoot/root ratio. The interaction of competitor introduction and herbicide did not significantly influence alligator weed growth in the greenhouse study. In the field, alligator weed cover was reduced after one herbicide application, but not significantly more after a second. Introduction of competitor species had no effect on alligator weed cover, nor did the interaction of competitor species and herbicide application. This study demonstrates that triclopyr amine herbicide can reduce alligator weed biomass and cover, and that two applications are more effective than one. To integrate selective herbicides and native plant introduction successfully for alligator weed control, more research is needed on the influence competition can potentially have on alligator weed growth, and the timing of herbicide application and subsequent introduction of plants.
The Eosinophil and Lysophospholipase Responses in Mice Infected with Trichinella spiralis: A Role for the Lymphocyte and Macrophage
The relationship among eosinophils, lysophospholipase activity and the immune response in animals infected with Trichinella spiralis was studied using in vivo and in vitro techniques. In an in vivo experiment, anti-thymocyte serum (ATS) was administered to mice infected with T. spiralis and its effects on intestinal lysophospholipase (EC 3.1.1.5.) activity, peripheral blood, bone marrow and intestinal eosinophilia were measured in the same experimental animal. The ATS caused a significant temporally related suppression of both the tissue lysophospholipase response and eosinophilia, in all three compartments. These findings support the hypothesis that parasite-induced eosinophilia is the cause of the increased lysophospholipase activity of parasitized tissue and that the responses are thymus cell-dependent. In vitro experiments demonstrated that the eosinophil was the primary inflammatory cell source of lysophospholipase among eosinophils, neutrophils macrophages and lymphocytes. The role of other cells and antigen in the production of the enzyme by the eosinophil was also investigated in vitro• Results demonstrated that eosinophils cultured with both T. spiralis antigen and other leukocytes yielded enzyme activities significantly greater than eosinophils cultured alone or with only antigen. More specific experiments showed that T-lymphocytes were the cells responsible for influencing the eosinophils' lysophospholipase activity in the presence of antigen, and that their influence was enhanced by the presence of macrophages. These results suggested that increased lysophospholipase activity present in parasitized tissue was not only due to increased numbers of eosinophils infiltrating parasitized tissue but was also due to each eosinophil synthesizing more of the enzyme. The necessity for antigen and other cells suggests a role for cell cooperation in the production of the enzyme, specifically T-lymphocytes and macrophage interaction with the eosinophil. A lymphocyte soluble factor collected from sensitized lymphocytes stimulated with specific antigen or concanavalin A was found to enhance the eosinophil lysophospholipase activity when added to cultures of …
Identification and Characterization of an Arabidopsis thaliana Mutant with Tolerance to N-lauroylethanolamine
N-Acylethanolamines (NAEs) are fatty acid derivatives in plants that negatively influence seedling growth. N-Lauroylethanolamine (NAE 12:0), one type of NAE, inhibits root length, increases radial swelling of root tips and reduces root hair numbers in a dose dependent manner in Arabidopis thaliana L. (ecotype Columbia). A forward genetics approach was employed by screening a population of T-DNA “activation-tagged” developed by the Salk Institute lines for NAE resistance to identify potential genes involved in NAE signaling events in Arabidopsis thaliana L. (ecotype Columbia). Seeds of the activation tagged lines were grown at 0, 25, 30, 50, 75 and 100 µM N-lauroylethanolamime (NAE 12:0). Ten plants which displayed NAE tolerance (NRA) seedling phenotypes, compared with wildtype (Columbia, Col-0) seedlings were identified. I focused on one mutant line, identified as NRA 25, where the tolerance to NAE 12:0 appears to be mediated by a single dominant, nuclear gene. Thermal asymmetric interlaced (TAIL) PCR identified the location of the T-DNA insert as 3.86 kbp upstream of the locus At1g68510. Quantitative PCR indicated that the transcript level corresponding to At1g68510 is upregulated approximately 20 fold in the mutant relative to wildtype. To determine whether the NAE tolerance in NRA 25 is associated with overexpression of At1g68510 I created overexpressing lines of At1g68510 with and without GFP fusions behind the 2X35S CaMV promoter. As predicted, results with overexpressing lines of At1g68510 also exhibited enhanced resistance to NAE when compared with the wildtype. Confocal images of the fusion proteins suggest that GFP-At1g68510 is concentrated in the nucleus and this was confirmed by counterstaining with 4', 6-Diamidino-2-phenylindol (DAPI). Futhermore, At1g68510 overexpressing lines and NRA 25 line also exhibited tolerance to abscisic acid (ABA) during seedling germination. The findings suggests that At1g68510 overexpression mediates seedling tolerance to both ABA and NAE, a mechanism independent of fatty acid amide hydrolase …
Proteomic Responses in the Gill of Zebrafish Following Exposure to Ibuprofen and Naproxen
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most abundant environmental pharmaceutical contaminants. In this study, a proteomic analysis was conducted to identify proteins differentially expressed in gill tissue of zebrafish (Danio rerio) after a 14-day exposure to the NSAIDs ibuprofen or naproxen. A total of 104 proteins with altered expression as indicated by 2-dimensional electrophoresis were analyzed by liquid chromatography with ion trap mass spectrometry (MS/MS). A total of 14 proteins fulfilled our requirements for identification which included consistency among replicate gels as well as successful MS/MS ion searches with the MASCOT database. The most prominent feature of the differential protein expression observed after NSAID exposure was an up-regulation of proteins belonging to the globin family which are involved in the transport of oxygen from gills and availability of heme molecules required for synthesis of cyclooxygenase. Differential expression was observed at exposure concentrations as low as 1-10 µg/L indicating that altered gene expression may occur in fish subjected to environmentally realistic levels of NSAID exposure.
Solvent Effects and Bioconcentration Patterns of Antimicrobial Compounds in Wetland Plants
This study looked at effects of organic solvents dimethylsulfoxide, dimethylformamide and acetone at 0.01%, 0.05% and 0.1% concentration on germination and seedling development wetland plants. Even at 0.01% level, all solvents affected some aspect of seed germination or seedling growth. Acetone at 0.01% was least toxic. Root morphological characteristics were most sensitive compared to shoot morphological characteristics. This study also looked at bioconcentration patterns of antimicrobial compounds triclosan, triclocarban and methyl-triclosan in wetland plants exposed to Denton Municipal Waste Water Treatment Plant effluent. Bioconcentration patterns of antimicrobial compounds varied among species within groups as well as within organs of species. The highest triclocarban, triclosan and methyltriclosan concentration were in shoot of N. guadalupensis, root of N. lutea and in shoots of P. nodous respectively.
Identification, Characterization and Engineering of UDP-Glucuronosyltransferases for Synthesis of Flavonoid Glucuronides
Flavonoids are polyphenolics compounds that constitute a major group of plant specialized metabolites, biosynthesized via the phenylpropanoid/polymalonate pathways. The resulting specialized metabolites can be due to decoration of flavonoid compounds with sugars, usually glucose, by the action of regiospecific UDP-glycosyltransferase (UGT) enzymes. In some cases, glycosylation can involve enzymatic attachment of other sugar moieties, such as glucuronic acid, galactose, rhamnose or arabinose. These modifications facilitate or impact the bioactivity, stability, solubility, bioavailability and taste of the resulting flavonoid metabolites. The present work shows the limitations of utilizing mammalian UDP-glucuronosyltransferases (UGATs) for flavonoid glucuronidation, and then proceeds to investigate plant UG(A)T candidates from the model legume Medicago truncatula for glucuronidating brain-targeted flavonoid metabolites that have shown potential in neurological protection. We identified and characterized several UG(A)T candidates from M. truncatula which efficiently glycosylate various flavonoids compounds with different/multiple regiospecificities. Biochemical characterization identified one enzyme, UGT84F9, that efficiently glucuronidates a range of flavonoid compounds in vitro. In addition, examination of the ugt84f9 gene knock-out mutation in M. truncatula indicates that UGT84F9 is the major UG(A)T enzyme that is necessary and sufficient for attaching glucuronic acid to flavonoid aglycones, particularly flavones, in this species. Finally, the identified UG(A)T candidates were analyzed via homology modeling and site-directed mutagenesis towards increasing the repertoire of UG(A)Ts applicable for synthesis of flavonoid glucuronides with potential human health benefits in neurological protection.
Presence of Wolbachia, A Potential Biocontrol Agent: Screening for Vertebrate Blood Meal Source and West Nile Virus in Mosquitoes in the North Texas Region
West Nile virus (WNV) is a geographically endemic mosquito-borne flavivirus that has spread across the United States infecting birds, mosquitos, humans, horses and other mammals. The wide spread nature of this virus is due to the ability of the mosquito vector to persist in broad, ecological diverse environments across the United States. In this study, mosquito populations in North Texas region were sampled for detection of Wolbachia, blood meal source, and WNV. The ultimate goal of this study was to examine the potential of a biocontrol agent, Wolbachia sp. that colonizes the hindgut of various insects, including mosquitos, as a natural means to interrupt virus transmission from mosquitos to other hosts, including humans. In Australia, Wolbachia sp. from fruit flies (Drosophila melanogaster) have been successfully used to block transmission of a similar pathogenic virus from mosquitos responsible for transmission of Dengue fever. Here, mosquitoes were collected using CDC style Gravid Traps in Denton, Texas, from October 2012 through September 2014. Collected mosquitoes were identified, sexed, and categorized as to the amount of host blood in their alimentary system using a Zeiss Axio Zoom microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY). Culex quinquefaciatus was the dominant blood engorged species collected. Smaller populations of Culex tarsalis and Aedes albopictus, another known vector for WNV were also collected. Mosquito larva were also collected from the UNT water research field station and reared to adults. Cx. tarsalis was the dominant mosquito taken from this habitat. Samples of Cx. quinquefasciatus, Cx. tarsalis and A. albopictus were analyzed for Wolbachia sp. and to identify host blood in the mosquito alimentary system. Total DNA extraction from the pool of mosquito samples was by both commercially available DNA extraction kits (Qiagen, Valencia, CA) and salt extraction technique. Polymerase chain reaction (PCR) was used to amplify and identify Wolbachia …
Traffic-Generated Air Pollution-Exposure Mediated Expression of Factors Associated with Progression of Multiple Sclerosis in a Female Polipoprotein E Knockout Mouse Model
Environmental air pollution is one risk factor associated with the onset and progression of multiple sclerosis (MS). In this project, we investigated the effects of ubiquitous traffic-generated pollutants, namely a mixture of gasoline and diesel vehicle exhaust (MVE), on signaling pathways associated with the pathophysiology of MS in the central nervous system (CNS) of either ovary intact (ov+) or ovariectomized (ov-) female Apolipoprotein (Apo) E-/-. Specifically, we investigated whether a subchronic inhalation exposure to MVE (200 PM μg/m3; 6 hr/d, 7d/wk, 30d) vs. filtered air (FA) controls altered myelination, T cell infiltration, blood-brain barrier (BBB) integrity, or production of reactive oxygen species (ROS) and expression of neuroinflammation markers in the CNS ov+ and ov- Apo E-/- mice. Our results revealed that inhalation exposure to MVE resulted in increased demyelination and CD4+ and CD8+ T cell infiltration, associated with alterations in BBB integrity. Disruption of the BBB was evidenced by decreased tight junction (TJ) protein expression, increased matrix metalloproteinase (MMPs) activity, and increased permeability of immunoglobin (Ig) G, which were more pronounced in the MVE ov- group. Moreover, MVE-exposure also promoted ROS and neuroinflammatory signaling in the CNS of ov+ and ov- mice, compared to FA groups. To analyze mechanisms that may contribute to MVE-exposure mediated inflammatory signaling in the CNS, we examined the NF-κB signaling pathway components, namely IKK subunits, IKKα, and IKKβ, as well as RelA. MVE -exposure did not alter the expression of either IKKα and IKKβ or RelA. However, increased expression of IKKα and IKKβ mRNA was observed in both FA ov- and MVE ov- groups, indicating female sex steroid hormone signaling involvement. Investigation of hormone receptors expression revealed a reduction in cerebral ERα mRNA expression, compared to ov+ mice; however, MVE-exposure resulted in an even further decrease in expression of ERα mRNA, while ERβ and PRO …
Chronic Ventricular Sympathectomy : Effects on Myocardial Metabolism
Chronic ventricular sympathectomy elicits changes in the coronary circulation, myocardial oxygen consumption and size of infarction resulting fromcoronary occlusion. These changes indicate a change occurring in the basic metabolism of the heart in response to the removal of its sympathetic nervous input. This hypothesis was tested using two groups of dogs, a shamoperated control and a ventricular sympathectomized group. The sympathectomy procedure was an intrapericardial surgical technique which selectively removes ventricular sympathetic input. Four weeks after surgery, left ventricular tissue samples were obtained and rapidly frozen to -80°C. Selected metabolic variables were then compared between the two groups.
Exploring Flavonoid Glycosylation in Kudzu (Pueraria lobata)
The isoflavones in kudzu roots, especially the C-glycosylated isoflavone puerarin, have been linked to many health benefits. Puerarin contains a carbon-carbon glycosidic bond that can withstand hydrolysis. The C-glycosylation reaction in the biosynthesis of puerarin has not been thoroughly investigated, with conflicting reports suggesting that it could take place on daidzein, isoliquiritigenin, or 2,7,4ʹ-trihydroxyisoflavanone. Kudzu species were identified for use in comparative transcriptomics. A non-puerarin producing kudzu was identified as Pueraria phaseoloides and a puerarin producing kudzu was identified as Pueraria montana lobata. Through the use of the plant secondary product glycosyltransferase (PSPG) motif, glycosyltransferases (UGTs) were identified from the transcriptomes. The UGTs that had higher digital expression in P. m. lobata were examined further using additional tools to home in on the UGT that could be responsible for puerarin biosynthesis. One of the UGTs identified, UGT71T5, had previously been characterized from kudzu as a C-glycosyltransferase involved in puerarin biosynthesis through in vitro enzyme activity (with daidzein) and a gain of function approach in soybean hairy roots. Previous studies have not supported the end-product of a pathway such as daidzein as the target for C-glycosylation, and no genetic analysis of UGT function had been conducted in kudzu. The activity of recombinant UGT71T5 with daidzein was confirmed in the present work. Following the development of a kudzu hairy root system, UGT71T5 expression was then knocked down by RNA interference (RNAi). When compared to control hairy roots there was a large reduction in puerarin content in the UGT71T5-RNAi roots, confirming the role of this enzyme in puerarin biosynthesis. Isotopic labeling of kudzu plants revealed that labeled daidzein could be directly incorporated into puerarin; however, the percent incorporation of daidzein was substantially lower than that of L-phenylalanine, a compound at the start of the pathway to isoflavone synthesis. The knockdown of 2-hydroxisoflavanone synthase …
Analysis of the Cytochrome P450 and UDP-Glucuronosyltransferase Families and Vitamin D3- Supplementation in Anoxia Survival in Caenorhabditis elegans
Alteration in diet and knockdown of detoxification genes impacts the response of C. elegans to oxygen deprivation stress. I hypothesized that feeding worms a vitamin D3-supplementation diet would result in differential oxygen deprivation stress response. We used a combination of wet lab and transcriptomics approach to investigate the effect of a vitamin-D3 supplemented diet on the global gene expression changes and the anoxia response phenotype of C. elegans (Chapter 2). C. elegans genome consists of 143 detoxification genes (cyp and ugt). The presence of a significant number of genes in these detoxification families was a challenge with identifying and selecting specific cyp and ugt genes for detailed analysis. Our goal was to understand the evolution, phylogenetic, and expression of the detoxification enzymes CYPs and UGTs in C. elegans (Chapter 3). We undertook a phylogenetic and bioinformatics approach to analyze the C. elegans, detoxification family. Phylogenetic analysis provided insight into the association of the human and C. elegans xenobiotic/endobiotic detoxification system. Protein coding genes in C. elegans have been predicted to be human orthologs. The results of this work demonstrate the role of C. elegans in the identification and characterization of vitamin D3 induced alterations in gene expression profile and anoxia response phenotypes and the identification of human orthologs for the detoxification enzymes and provides insight into the gene expression pattern.
Nesting Ecology and Reproductive Correlates in the Desert-nesting Gray Gull Larus Modestus
General objectives of my study were to describe the reproductive ecology of gray gulls in the large Lealtad colony, with emphasis on demographic parameters and physiological adaptations of eggs and chicks, which would complete some original objectives established in the early 1980's by Guerra and Fitzpatrick. Specifically, my study focused on describing, then comparing with other desert and non-desert nesting larids, interactive effects of ambient physical conditions and nest-site predation on eggs and chicks.
Immune Response of the Rat to Outer Membrane Proteins of Legionella Pneumophila
Outer membrane proteins (OMPs) were recovered from eleven strains (eight serogroups) of Legionella pneumophila by sequential treatment with Tris buffer (pH 8), citrate buffer(pH 2.75) and Tris buffer (pH 8). Transmission electron microscopy revealed clearly the separation of the outer membrane from the bacteria. The development of delayed hypersensitivity was also noted by measuring the area of arythema and induration produced by intradermal injections of the MPSs from Chicago 8 strain. The adjuvants enhanced greatly both active and cell-meditated immunity (CMI). Transient lymphocytopenia with a slight rise in neutrophils was noted in each of the immunized groups. Intraperitoneal challenge, seven days after the OMP booster, of one LD (1.5 x10^6) of legionellae resulted in lymphocytopenia with elevated neutrophils. All immunized rats survived the challenge, although those in the saline-OMP group were clearly the sickest. Post-challenge, legionella antibody titers rose greatly and CMI was heightened. Passive immunization (homologous and heterologous) was found to protect the rats from a challenge of on LD. Actively-immunized rats retained their immunity for at least six months as determined by their resistance to a second challenge.
Determination of Habitat Preferences of Pronghorn (Antilocapra americana) on the Rolling Plains of Texas Using GIS and Remote Sensing
The Rocker b Ranch on the southern Rolling Plains has one of the last sizeable populations of pronghorn (Antilocapra americana) in Texas. To investigate habitat utilization on the ranch, pronghorn were fitted with GPS/VHF collars and were released into pastures surrounded by a variety of fences to determine how fence types affected habitat selection. Habitat parameters chosen for analysis were vegetation, elevation, slope, aspect, and distances to water, roads, and oil wells. Results showed that pronghorn on the ranch crossed modified fencing significantly less than other types of fencing. Pronghorn selected for all habitat parameters to various degrees, with the most important being vegetation type. Habitat selection could be attributed to correspondence of vegetation type with other parameters or spatial arrangements of physical features of the landscape. Seasonal differences in habitat utilization were evident, and animals tended to move shorter distances at night than they did during daylight hours.
Photoactivatable Quantum Dots in Super-Resolution Microscopy of Muscle
Super-resolution 3D imaging was achieved using newly synthesized photoactivatable quantum dot (PAQ dot) probes. Quantum dots were modified with a novel quencher system to make them photoactivatable. The unique properties of these PAQ dots enable single-fluorophore localization in three dimensions using a confocal microscopy optical sectioning method. Myosin and tropomyosin of rabbit myofibrilar bundles were specifically labeled with the newly synthesized PAQ dot. A sufficient number of single quantum dots were photoactivated, localized and reduced to their centroid and then reconstructed to a super-resolution image. The acquired super-resolution image shows a lateral and an axial sub-diffraction resolution and demonstrates ultrafine striations with widths less than 70 nm that are not evident by conventional confocal microscopy. The striations appear to be related to nebulin thin filament binding protein. This newly developed imaging system is cutting edge for its high resolution and localization as well its simplicity and convenience.
Role of MicroRNAs and Their Downstream Targets in Zebrafish Thrombopoiesis
Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, there is limited information on microRNAs' role in zebrafish thrombopoiesis. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, I identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. Knockdown of three microRNAs, mir-7148, let-7b, and mir-223, by the piggyback method in zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. I then verified these findings in zebrafish larvae after the knockdown of the above microRNAs followed by an arterial laser thrombosis assay. I concluded mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. Furthermore, I explored let-7b downstream genes in thrombocytes detected by RNA-seq analysis and chose 14 targets based on their role in cell differentiation (rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b) that are transcriptional regulators. The qRT-PCR analysis of expression levels the above genes following let-7b knockdown showed significant changes in the expression of 13 targets. I then studied the effect of the 14 targets on thrombocytes production and identified 5 genes (irf5, tgif1, irf8, cebpa, and rorca) that showed thrombocytosis and one gene ikzf1 that showed thrombocytopenia. Furthermore, I tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223. I also identified that tgif1, cebpa, …
Degradation of Phenolic Acids by Azotobacter Species Isolated from Sorghum Fields
Sorghum plants excrete phenolic acids which reduce subsequent crop yields. These acids accumulate in field soil by combining with soil and clay particles to form stable complexes which remain until degraded by bacterial metabolism. The amount of phenolic acids in soil samples were obtained by gas chromatography measurements, while Azotobacter populations were obtained by plate counts in 40 sorghum field samples from Denton County, Texas. One can conclude that increasing the Azotobacter population in the soil increased the degradation rate of phenolic acids proportionally. It is proposed that seed inoculation will introduce selected strains of Azotobacter into the soil. The presence of Azotobacter should increase crop size in subsequent plantings.
Site Directed Mutagenesis of Dienelactone Hydrolase
The clcD gene encoding dienelactone hydrolase (DLH) is part of the clc gene cluster for the utilization of the B-ketoadipate pathway intermediate chlorocatechol. The roles that individual amino acids residues play in catalysis and binding of the enzyme were investigated. Using PCR a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHi to ZscoRI subclone in the plasmid pUC19.
The Physiology of Azotobacter Vinelandii Cysts
The value of the adenylate energy charge [(ATP)+1/2(ADP)/(ATP)+(ADP)+(AMP)] in Azotobacter vinelandii cells was monitored during growth and germination in flask cultures. The miximal value of 0.88 was attained during mid-log phase; this declined gradually to 0.50 by late stationary phase. When these cultures were transferred to encystment media, the adenylate energy charge decreased to an average value of 0.40 as the vegetative cells encysted and remained unchanged during the next 20 days. Encystment cultures wre composed of vegetative cells, encysting cells and mature cysts but the proportionate value of the energy charge could be assigned. Viability of the total population remained 95% or higher during the entire period studied. Azotobacter vinelandii cysts cultivated on phosphate-sufficient media. Although cell protein and nucleic acids were unaffected by phosphate deficiency, cell wall structures, oxygen uptake and sncystment were significantly affected. Phosphate-limited cysts contained much larger amounts of poly-beta-hydroxybutyric acid but had a lower adenylate energy charge than did control cysts. The ATP/ADP ratio was much lower in phosophate-deficient cysts than in the control cysts. The data indicate a "substrate saving" choice of three metabolic pathways available to cells of Azotobacter under different growth conditions.
Effects of Phytohormones on Scenedesmus quadricauda
The literature on the effects of phytohormone on algae is clouded with contradictory reports. Reports have been published which substantiate and deny the effects of phytohormones in enhancing the growth and developmental processes in algae. The overall aim of this study was to investigate the response, if any, of the phytohormones indole-3-acetic acid (IAA), gibberellic acid A3 (GA) and kinetin on the physiology of the green alga, Scenedesmus quadricauda. Results obtained for the uptake of 14^C-IAA an(j l4C-kinetin by Scenedesmus strongly support the presumption that the alga does not absorb the hormones. The retention of the phytohormones by the alga is due to adsorption, and is independent of hormone concentration. Most of the label was adsorbed by the outer pectic layers of the cell wall.
Novel Approaches for Enhancing Resistance to Fusarium graminearum in Arabidopsis and Wheat by Targeting Defense and Pathogenicity Factors
Fusarium head blight (FHB) is an important disease of small grain cereals including wheat that affects grain quality and yield. The fungus Fusarium graminearum (Fg) is the major agent of this disease. Lack of natural resistance has limited ability to control wheat losses to this disease. Developing new approaches is critical for increasing host plant resistance to this fungus. This work has identified four processes that can be targeted for enhancing host plant resistance to FHB. The first involves targeting the pattern-triggered immunity mechanism to promote host plant resistance. Two other approaches involved reducing activity of susceptibility factors in the host to enhance plant resistance. The susceptibility factors targeted include accumulation of the phytohormone jasmonic acid and the 9-lipoxygenase pathway that oxidizes fatty acids. Besides suppressing host defenses against Fg, jasmonic acid also directly acts on the fungus to promote fungal growth. 9- lipoxygenases similarly suppress host defenses to promote fungal pathogenicity. Another approach that was developed involved having the plant express double stranded RNA to target fungal virulence genes for silencing. This host-induced gene silencing approach was employed to target two fungal virulence genes, the lipase encoding FGL1 and salicylate hydroxylase encoding FgNahG, which the fungus secretes into the host to promote turnover of the plant defense signaling metabolite salicylic acid. FGL1 in contrast acts on host lipids to release fatty acids, which suppress the deposition of callose that provides a physical barrier to limit fungal spread.
Assessing Student Perceptions in Short Research Experiences and Course Research Experiences in Undergraduate Biology Laboratories
This study examined students' perception between short research experiences (SRE) courses and full-semester course research experiences (CRE) using the Persistence in the Sciences (PITS) survey and the interview questionnaire. The study also aimed to correlate the influence of student's demographic as a predictive indicator for Project Ownership Scores (POS) and Quantitative Literacy (QL) score means. The three courses studied at the University of North Texas were Biology for Science Majors Laboratory (BIOL 1760 SRE), Microbiology with Tiny Earth (BIOL 2042 Tiny Earth SRE), and Introductory Biology Research Laboratory I (BIOL 1750 SEA-PHAGES CRE). The mean scores for the PITS categories leaned favorably towards the research component of each laboratory course assessed in this study. The interview questionnaire showed 66% of the students in the SRE courses and 90% of the students in the CRE course preferred the research component of the lab. Paired survey demographic analysis for BIOL 1760 SRE showed significance for the Science Community Values with associate/bachelor's degree. BIOL 1750 SEA-PHAGES CRE showed significance in three of the six categories when comparing means for Project Ownership Emotion, Self-Efficacy, and Science Identity with Gender. Binary logistics was used to build a regression model to predict demographics with approximately 65% to 75% accuracy for each course. When analyzing students' QL score, the demographic category "Ethnicity" showed significance for BIOL 2042 Tiny Earth SRE. Categorizing the correct response into two categories for the QL test scores, the SRE and CRE courses, and analyzing the PITS scores for paired data sets showed that there was significance in the Networking category for the question "I have discussed my research in this course with professors other than my course instructor." The validated PITS, POS, and interview questionnaire could be a tool for use to analyze laboratories at UNT that offer a SRE or CRE …
Long-Term Citizen Science Water Monitoring Data: An Exploration of Accuracy over Space and Time
The Texas Stream Team (TST) is one of an increasing number of citizen science water monitoring programs throughout the US which have been continuously collecting surface water quality data under quality assurance protocols for decades. Volunteer monitoring efforts have generated monitoring datasets that are long-term, continuous, and cover a large geographic area - characteristics shown to be valuable for scientists and professional agencies. However, citizen science data has been of limited use to researchers due to concerns about the accuracy of data collected by volunteers, and the decades of water quality monitoring data collected by TST volunteers is not widely used, if at all. A growing body of studies have attempted to address accuracy concerns by comparing volunteer data to professional data, but this has rarely been done with large-scale, existing datasets like those collected by TST. This study assesses the accuracy of the volunteer water quality data collected across the state of Texas by the TST citizen science program between 1992-2017 by comparing it to professional data from corresponding stations during the same time period, as well as comparing existing and experimental data from a local TST partner agency. The results indicate that even large-scale, existing volunteer and professional data with unpaired samples that may have been taken months apart can show statewide agreement of 80% for all parameters (DO = 77%, pH = 79%, conductivity = 85%) over the 38 years of sampling included in the analyses, across all locations. The local case study using paired datasets for which a greater number of factors were controlled for show an even higher agreement between volunteers and professionals (DO = 91%, pH = 87%, conductivity = 100%) and show no significant difference between experimental and existing sampling data. The results from this study indicate that TST has been collecting water …
Development of a Procedure to Evaluate Groundwater Quality and Potential Sources of Contamination in the East Texas Basin
This study contributes a procedure, based on data analysis and geostatistical methods, to evaluate the distribution of chemical ratios and differentiate natural and anthropogenic contaminant sources of groundwater quality in the East Texas Basin. Four aquifers were studied, Sparta, Queen City, Carrizo and Wilcox. In this study, Carrizo- Wilcox is considered as one aquifer, and Sparta-Queen City as another. These aquifers were divided into depth categories, 0-150 feet for Sparta-Queen City and 300-600 feet and 600-900 feet for Carrizo-Wilcox in order to identify individual sources of contamination. Natural sources include aquifer mineral make up, salt domes and lignite beds. Major anthropogenic sources include lignite and salt dome mining and oil-gas production. Chemical ratios selected were Na/Cl, Ca/Cl, Mg/Cl, SO4/Cl, (Na+Cl)/TDS, SO4/Ca and (Ca+Mg)/(Na+K). Ratio distributions and their relationships were examined to evaluate physical-chemical processes occurring in the study area. Potential contaminant sources were used to divide the Basin into three areas: Area 1 to the east, Area 2 in the west and Area 3 in the center. Bivariate analysis was used to uncover differences between the areas. The waters in Area 1 are potentially impacted primarily from oil field waters. Sources present in Area 2 include lignite beds and oil field operations. Area 3 is the cap rock of salt domes that can contain gypsum and anhydrite. Based on the exploratory data analysis (Na+Cl)/TDS, (Ca+Mg)/(Na+K), and SO4/Ca ratios were chosen for geostatistical analysis. Chemical ratios that provided indications of cation exchange, salt domes and oil fields were (Na+Cl)/TDS, (Ca+Mg)/(Na+K) and SO4/Ca. In the Sparta-Queen City 150 zone the procedure did not provide a good method for differentiating between contaminant sources. However, the procedure was effective to indicate impacted ground water in the Carrizo-Wilcox 600 and 900 foot zones.
Transient Expression of BABY BOOM, WUSCHEL, and SHOOT MERISTEMLESS from Virus-Based Vectors in Cotton Explants: Can We Accelerate Somatic Embryogenesis to Improve Transformation Efficiency?
Upland cotton (Gossypium hirsutum L.) is the world's most prominent fiber crop. Cotton transformation is labor intensive and time consuming, taking 12 to 18 months for rooted T0 plants. One rate limiting step is the necessary production of somatic embryos. In other recalcitrant species, ectopic expression of three genes were shown to promote somatic embryogenesis: WUSCHEL (WUS), SHOOT MERISTEMLESS (STM), and BABY BOOM (BBM). WUS is responsible for maintaining stem-cell fate in shoot and floral meristems. STM is needed to establish and maintain shoot meristems. STM and WUS have similar functions but work in different pathways; overexpression of both together converts somatic cells to meristematic and embryogenic fate. BBM encodes an AP2/ERF transcription factor that is expressed during embryogenesis and ectopic expression of BBM reprograms vegetative tissues to embryonic growth. In prior studies, these genes were constitutively expressed, and cultures did not progress beyond embryogenesis because the embryogenic signal was not turned off. In our study, we set out to use these genes to increase the efficiency of cotton transformation and decrease the time it takes to regenerate a plant. A disarmed cotton leaf crumple virus (dCLCrV) vector delivers WUS, STM, or BBM into cotton tissue cultures through Agrobacterium tumefaciens infection. We propose that virus delivery of embryo-inducing genes is a better approach for transformation because A) inserts more than 800 nucleotides are unstable, and will spontaneously inactivate, B) virus DNA can migrate through plasmodesmata to cells around the infected cell, creating a gradient of embryonic potential, C) the virus DNA does not pass through the germ line and the seed will not contain virus. We propose this method of inducing embryogenesis will facilitate the stable transformation of cotton and will be beneficial to the cotton industry. Ectopic expression of AtBBM, AtSTM, and AtWUS GrWUS:meGFP from a constitutive CaMV 35S …
Exploration of Explanatory Variables in the Creation of Linear Regression Models and Logistic Regression Models to Predict the Performance of Preservice Teachers on the Science Portion of the EC-6 TExES Certification Examination
The purpose of this study was to analyze the current and pre-service conditions that can affect student teachers' preparedness to pass the science portion of the EC-6 Texas Examinations for Educator Standards (TExES), one of the mandatory certification exam to become a teacher in Texas. Two types of prediction models were employed in this study: binomial logistic regression and multiple linear regression. The independent variables used in this study were: final grade in BIOL 1082, classification of students, transfer status, taken college biology, taken college chemistry, taken college physics, taken college environmental science, taken college earth science, attending college part-time, number of credits taken during the semester, first-generation college student, relatives with degree in education, and current GPA. The dependent variable of this study was the posttest score on science portion of the EC-6 TExES practice exam. A total of 170 preservice teachers participated this study. This study used students enrolled in BIOL 1082, who volunteered to take a Biology for Educators QualtricsTM survey and the EC-6 TExES practice exam in a pretest (start of semester) and posttest (end of semester) form. The findings of this study revealed that the single best predictor of preservice teachers' performance on the science portion of EC-6 TExES practice certification examination was the Grade in BIOL 1082.
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 …
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