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Genetic and Cellular Analysis of Anoxia-Induced Cell Cycle Arrest in Caenorhabditis elegans
The soil-nematode Caenorhabditis elegans survives oxygen deprivation (anoxia < 0.001 kPa of O2, 0% O2) by entering into a state of suspended animation during which cell cycle progression at interphase, prophase and metaphase stage of mitosis is arrested. I conducted cell biological characterization of embryos exposed to various anoxia exposure times, to demonstrate the requirement and functional role of spindle checkpoint gene san-1 during brief anoxia exposure. I conducted a synthetic lethal screen, which has identified genetic interactions between san-1, other spindle checkpoint genes, and the kinetochore gene hcp-1. Furthermore, I investigated the genetic and cellular mechanisms involved in anoxia-induced prophase arrest, a hallmark of which includes chromosomes docked at the nuclear membrane. First, I conducted in vivo analysis of embryos carried inside the uterus of an adult and exposed to anoxic conditions. These studies demonstrated that anoxia exposure prevents nuclear envelope breakdown (NEBD) in prophase blastomeres. Second, I exposed C. elegans embryos to other conditions of mitotic stress such as microtubule depolymerizing agent nocodazole and mitochondrial inhibitor sodium azide. Results demonstrate that NEBD and chromosome docking are independent of microtubule function. Additionally, unlike anoxia, exposure to sodium azide causes chromosome docking in prophase blastomeres but severely affects embryonic viability. Finally, to identify the genetic mechanism(s) of anoxia-induced prophase arrest, I conducted extensive RNA interference (RNAi) screen of a subset of kinetochore and inner nuclear membrane genes. RNAi analysis has identified the novel role of 2 nucleoporins in anoxia-induced prophase arrest.
Genetic and Environmental Factors that Mediate Survival of Prolonged Oxygen Deprivation in the Nematode Caenorhabditis Elegans
Ischemic events of even a very short duration are not tolerated Ill in humans. The human cost of ischemia, when looked at as combined cardiovascular disease, dwarfs all other causes of death in the United States. Annually, CVD kills as many people in the US as does cancer, chronic lower respiratory disease, accidents, and diabetes mellitus combined. In 2005 (the latest year for which final statistics are available), CVD was responsible for 864,480 deaths or 35.3 percent of total deaths for the year. In my study, I have used the nematode Caenorhabditis elegans to determine genetic and environmental modulators of oxygen deprivation a key component of ischemia. I have found that animals with mutations in insulin like signaling pathways, neuronal function, electron transport chain components, germline function, and animals that are preconditioned by being raised on a diet of E. coli HT115 bacteria at 25°C have an enhanced ability to survive long-term (>72 hours) anoxia (<.005 kPa O2) at 20°C. The enhanced anoxia survival phenotype partially correlates with increased levels of carbohydrate stores in the nematodes. Suppression of this enhanced anoxia survival phenotype is possible by altering expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, the FOXO transcription factor DAF-16, and 5’-AMP kinase.
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.
A Genetic Assessment of the Mating System of a Suburban Red-Shouldered Hawk Population in Southwest Ohio
Considering the high reproductive investment of the social male and the cost to the female of losing this benefit by soliciting copulations outside the social pair bond, it is expected that most raptor populations would exhibit low to no occurrence of extra-pair paternity (EPP). This holds true for the majority of raptor species studied to date with only one exception of an urban Cooper's hawk (Accipiter cooperii) study which reported an unexpectedly high extra-pair young frequency of 19.29%. In our study we examined the frequency of EPP within a red-shouldered hawk (Buteo lineatus) population residing in the suburban/urban matrix of southwest Ohio. During the breeding seasons of 2018 and 2019, 181 breeding age and nestling individuals were color-banded and sampled for genetic analysis using nine microsatellite loci. After genotyping a total of 40 broods (with at least two nestlings per brood) and both presumptive parents of each brood, no clear evidence of EPP was detected. However, at one nest site, the entire brood of four chicks was not sired by the adult male observed during the courtship period, nor another adult male observed tending the chicks later in the season. We suspect that this particular nest represented two instances of rapid mate replacement rather than extra-pair fertilization by a third unsampled male, because none of the chicks were sired by either of the two adult males observed at the nest. We also reviewed potential factors contributing to our finding of overall genetic monogamy in our study population in comparison to other raptor taxa EPP studies. Our results suggested that factors other than habitat composition alone play an important role in determining the type of breeding strategy exhibited by different raptor populations.
Genetic Characterization of Central and South American Populations of Scarlet Macaw (Ara macao)
The wild populations of the Scarlet Macaw subspecies native to southern Mexico and Central America, A. m. cyanoptera, have been drastically reduced over the last half century and are now a major concern to local governments and conservation groups. Programs to rebuild these local populations using captive bred specimens must be careful to reintroduce the native A. m. cyanoptera, as opposed to the South American nominate subspecies (A. m. macao) or hybrids of the two subspecies. Molecular markers for comparative genomic analyses are needed for definitive differentiation. Here I describe the isolation and sequence analysis of multiple loci from 7 pedigreed A. m. macao and 14 pedigreed A. m. cyanoptera specimens. The loci analyzed include the 18S rDNA genes, the complete mitogenome as well as intronic regions of selected autosomally-encoded genes. Although the multicopy18S gene sequences exhibited 10% polymorphism within all A. macao genomes, no differences were observed between any of the 21 birds whose genomes were studied. In contrast, numerous polymorphic sites were observed throughout the 16,993 bp mitochondrial genomes of both subspecies. Although much of the polymorphism was observed in the genomes of both subspecies, subspecies-specific alleles were observed at a number of mitochondrial loci, including 12S, 16S, CO2 and ND3. Evidence of possible subspecies-specific alleles were also found in three of four screened nuclear loci. Collectively, these mitochondrial and nuclear loci can be used as the basis to distinguish A. m. cyanoptera from the nominate subspecies, A. m. macao, as well as identify many hybrids, and most importantly will contribute to further reintroduction efforts.
Genetic Differentiation of the Geomys Pocket Gopher Complex of Texas
Genetic variation was analyzed for populations of seven taxa comprising four cytotypes of the Geomys bursarius chromosome complex, including G. b. major, G. b. knoxjonesi, and the Edwards Plateau taxa, G. b. llanensis and G. b. texensis. Genetic relationships of the Edwards Plateau gophers with other taxa and between themselves were examined. Genetic similarity, number of fixed allelic differences, and ectoparasite distribution indicate the Edwards Plateau gophers are a distinct gene pool. Isolation of the Edwards Plateau taxa precludes contact zone analysis. However, genetic differentiation is typical of that between other species of Geomys, and the Edwards Plateau taxa should be recognized as G. texensis. Distributions of allelic frequencies indicate little justification in retaining the subspecific status of the Edwards Plateau forms.
Genetic Mechanisms for Anoxia Survival in C. Elegans
Oxygen deprivation can be pathological for many organisms, including humans. Consequently, there are several biologically and economically relevant negative impacts associated with oxygen deprivation. Developing an understanding of which genes can influence survival of oxygen deprivation will enable the formulation of more effective policies and practices. In this dissertation, genes that influence adult anoxia survival in the model metazoan system, C. elegans, are identified and characterized. Insulin-like signaling, gonad function and gender have been shown to influence longevity and stress resistance in the soil nematode, C. elegans. Thus, either of these two processes or gender may influence anoxia survival. The hypothesis that insulin-like signaling alters anoxia survival in C. elegans is tested in Aim I. The hypotheses that gonad function or gender modulates anoxia survival are tested in Aim II. Insulin-like signaling affects anoxia survival in C. elegans. Reduction of insulin-like signaling through mutation of the insulin-like receptor, DAF-2, increases anoxia survival rates in a gpd-2/3 dependent manner. The glycolytic genes gpd-2/3 are necessary for wild-type response to anoxia, and sufficient for increasing anoxia survival through overexpression. Gonad function and gender both affect anoxia survival in C. elegans. A reduction of ovulation and oocyte maturation, as measured by oocyte flux, is associated with enhanced anoxia survival in all cases examined to date. Reduction of function of several genes involved in germline development and RTK/Ras/MAPK signaling reduce ovulation and oocyte maturation while concurrently increasing anoxia survival. The act of mating does not influence anoxia survival, but altering ovulation through breeding or chemical treatment does. The male phenotype also increases anoxia survival rates independent of genotype. These studies have identified and characterized over ten different genotypes that affect adult survival of anoxia in C. elegans. Before these studies were conducted, there were no genes known to influence adult anoxia survival in C. …
Genetic Modification of Fatty Acid Profiles in Cotton
The industrial uses of cottonseed oil are limited by its fatty acid composition. Genetic modification of cotton lipid profiles using seed-specific promoters could allow cotton growers to produce valuable new oils in the seed without adverse effects on fiber quality and yield, therefore making this crop more commercially profitable. Transgenic cotton callus harboring a diverged fatty acid desaturase gene (FADX) from Momordica charantia was characterized for production of alpha-eleostearic acid (conjugated double bonds: 18:3 D9 cis, 11 trans, 13 trans), not normally found in cotton. Gas chromatography (GC) in conjunction with mass spectrometry (MS) confirmed production of alpha-eleostearic acid in the transgenic cotton tissues. A second series of transformation experiments introduced the cotton fatty acid thioesterase B (FATB) cDNA, fused to the seed-specific oleosin promoter into cotton to promote the over-expression of FATB, to generate cotton with increased palmitate in the cottonseed. PCR amplification, as well as fatty acid analysis by gas chromatography, confirmed introduction of the FATB cDNA in transgenic tissues. Collectively, these results demonstrate the feasibility of manipulating the fatty acid composition in cotton via transgenic approaches and form the basis for continued efforts to create novel oils in cottonseed.
The Genetics of Pigmentation in Corynebacterium poinsettiae ATCC 9682
Corynebacterium poinsettiae mutant strains blocked in carotenoid biosynthesis were obtained by treatment with the mutagen N-methyl-N1-nitro-N-nitrosoguanidine. Additional carotenoid (Crt) mutant strains were obtained from a previous study conducted in our laboratory. Fifty-nine Crt mutants affected in carotenoid biosynthesis were examined by a normal phase high performance liquid chromatography (HPLC) system. Pigment extracts of Crt mutants and C. poinsettiae wild type strains were resolved by an isocratic system with hexane:acetone:dicholoromethane, 11.35:1.73:1.00 (by vol.) as the eluting solvent. In addition to the five major peaks, twelve minor peaks were observed in the wild type C. poinsettiae strain used in this study. Crt mutant and wild type strain peak heights were measured from the individual chromatograms and the peak height data set created was analyzed using the Statistical Analysis System program to perform a cluster analysis. The cluster analysis revealed five carotenoid mutant groups. Carotenoid pigments which accumulated or were absent in each of the cluster groups are reported. Cluster group 1 mutants (CrtA) are blocked in the dehydrogenase(s) which is(are) responsible for the dehydrogenations between phytoene and lycopene. Cluster group 2 mutants (CrtB) appear to be blocked at a second dehydrogenase specific for the dehydrogenation from C.p. 470 to C.p. 496. Cluster group 3 mutants (CrtC) are blocked at a cyclization step in the pathway which involves cyclization of C.p. 496 to C.p. 470 and which may cyclize C.p. 473 to C.p. 450. The genes CrtA and CrtB map only 0.5 map units from each other while CrtA and CrtC map 2.1 map units from one another. Mutants which accumulate end products but which lack certain precursors indicate a branched pathway for pigment biosynthesis exists in this organism. Media for the formation, fusion and regeneration of C. poinsettiae protoplasts are reported and a protocol for the use of these media in genetic …
Genic Differentiation and Evolution in the Ground Squirrel Subgenus Ictidomys (Spermophilus)
The genetic structure of 26 natural populations of three species (S. tridecemlineatus, S. mexicanus, and S. spilosoma) of the Ictidomys subgenus of ground squirrels was analyzed using chromosomal and electrophoretic techniques. Chromosomal variation was not observed in S. mexicanus, and only slight karyotypic variation was found in the other two species. Chromosomal evidence indicated hybridization between S. tridecemlineatus and S. mexicanus, placing these species within the classical definition of semispecies. Analysis of electrophoretic variation at 29 genetic loci indicated close genetic relationships between these species. Evolution in Ictidomys appears to be linked with Pleistocene events, and speciation appears to have occurred within the last 155,000 years.
Genic Differentiation Between Two Chromosomal Races of Pocket Gophers, Geomys bursarius
Genic data from two chromosomal races of Geomys bursarius from a contact zone in central Texas indicated that the two races possessed distinct gene pools which would define them as separate species. Data from proteins encoded from 21 loci in this study substantiated this hypothesis. A pattern of alternately fixed alleles at the ADH-l, MDH-2, LDH-l, and IDH-1 loci with no apparent gene flow in zones of contact strongly suggested that these two races should be designated as separate species. Levels of heterozygosity and high FST values indicate that genomic structuring within Geomys is most heavily influenced by high levels of inbreeding and low migration rates. Fossorial rodents were suggested to undergo speciation primarily through parapatric means.
Genomic Island Discovery through Enrichment of Statistical Modeling with Biological Information
Horizontal gene transfer enables acquisition and dissemination of novel traits including antibiotic resistance and virulence among bacteria. Frequently such traits are gained through the acquisition of clusters of functionally related genes, often referred to as genomic islands (GIs). Quantifying horizontal flow of GIs and assessing their contributions to the emergence and evolution of novel metabolic traits in bacterial organisms are central to understanding the evolution of bacteria in general and the evolution of pathogenicity and antibiotic resistance in particular, a focus of this dissertation study. Methods for GI detection have also evolved with advances in sequencing and bioinformatics, however, comprehensive assessment of these methods has been lacking. This motivated us to assess the performance of current methods for identifying islands on broad datasets of well-characterized bacterial genomes and synthetic genomes, and leverage this information to develop a novel approach that circumvents the limitations of the current state-of-the-art in GI detection. The main findings from our assessment studies were 1) the methods have complementary strengths, 2) a gene-clustering method utilizing codon usage bias as the discriminant criterion, namely, JS-CB, is most efficient in localizing genomic islands, specifically the well-studied SCCmec resistance island in methicillin resistant Staphylococcus aureus (MRSA) genomes, and 3) in general, the bottom up, gene by gene analysis methods, are inherently limited in their ability to decipher large structures such as GIs as single entities within bacterial genomes. We adapted a top-down approach based on recursive segmentation and agglomerative clustering and developed a GI prediction tool, GEMINI, which combined compositional features with segment context information to localize GIs in the Liverpool epidemic strain of Pseudomonas aeruginosa. Application of GEMINI to the genome of P. aeruginosa LESB58 demonstrated its ability to delineate experimentally verified GIs in the LESB58 genome. GEMINI identified several novel islands including pathogenicity islands and revealed the …
Glucose and Altered Ceramide Biosynthesis Impact the Transcriptome and the Lipidome of Caenorhabditis elegans
The worldwide rise of diabetes and obesity has spurred research investigating the molecular mechanisms that mediate the deleterious effects associated with these diseases. Individuals with diabetes and/or obesity are at increased risk from a variety of health consequences, including heart attack, stroke and peripheral vascular disease; all of these complications have oxygen deprivation as the central component of their pathology. The nematode Caenorhabditis elegans has been established as a model system for understanding the genetic and molecular regulation of oxygen deprivation response, and in recent years methods have been developed to study the effects of excess glucose and altered lipid homeostasis. Using C. elegans, I investigated transcriptomic profiles of wild-type and hyl-2(tm2031) ( a ceramide biosynthesis mutant) animals fed a standard or a glucose supplemented diet. I then completed a pilot RNAi screen of differentially regulated genes and found that genes involved in the endobiotic detoxification pathway (ugt-63 and cyp-25A1) modulate anoxia response. I then used a lipidomic approach to determine whether glucose feeding or mutations in the ceramide biosynthesis pathway or the insulin-like signaling pathway impact lipid profiles. I found that gluocose alters the lipid profile of daf-2(e1370) (an insulin-like receptor mutant) animals. These studies indicate that a transcriptomic approach can be used to discover novel pathways involved in oxygen deprivation response and further validate C. elegans as a model for understanding diabetes and obesity.
Glucose-Induced Developmental Delay is Modulated by Insulin Signaling and Exacerbated in Subsequent Glucose-Fed Generations in Caenorhabditis elegans
In this study, we have used genetic, cell biological and transcriptomic methods in the nematode C. elegans as a model to examine the impact of glucose supplementation during development. We show that a glucose-supplemented diet slows the rate of developmental progression (termed "glucose-induced developmental delay" or GIDD) and induces the mitochondrial unfolded protein response (UPRmt) in wild-type animals. Mutation in the insulin receptor daf-2 confers resistance to GIDD and UPRmt in a daf-16-dependent manner. We hypothesized that daf-2(e1370) animals alter their metabolism to manage excess glucose. To test this, we used RNA-sequencing which revealed that the transcriptomic profiles of glucose-supplemented wildtype and daf-2(e1370) animals are distinct. From this, we identified a set of 27 genes which are both exclusively upregulated in daf-2(e1370) animals fed a glucose-supplemented diet and regulated by daf-16, including a fatty acid desaturase (fat-5), and two insulin-like peptides (ins-16 and ins-35). Mutation of any of these genes suppresses the resistance of daf-2(e1370) to GIDD. Additionally, double mutation of ins-16 and ins-35 in a daf-2(e1370) background results in an increase in constitutive dauer formation which is suppressed by glucose supplementation. Further investigation of the insulin-like peptides revealed that ins-16 mutation in a wild-type background results in upregulation of ins-35 and DAF-16 nuclear translocation regardless of diet; however, unlike daf-2(e1370), this translocation is not associated with resistance to GIDD. Taken together, these data suggest that glucose-supplemented daf-2(e1370) animals maintain developmental trajectory in part through upregulation of specific insulin-like peptide genes and fatty acid desaturation and contribute to a deeper understanding of the mechanisms underlying the resistance of daf-2(e1370) animals to GIDD. We also showed another fascinating aspect of GIDD: it becomes more pronounced in subsequent generations exposed to a glucose-supplemented diet, suggesting that the parental glucose diet has an impact on the developmental progression of their offspring.
Glucose Induces Sensitivity to Oxygen Deprivation and Alters Gene Expression in Caenorhabditis Elegans
An organisms’ diet represents an exogenous influence that often yields colossal effects on long-term health and disease risk. The overconsumption of dietary sugars for example, has contributed to significant increases in obesity and type-2 diabetes; health issues that are costly both economically and in terms of human life. Individuals who are obese or are type-2 diabetic often have compromised oxygen delivery and an increased vulnerability to oxygen-deprivation related complications, such as ischemic strokes, peripheral arterial disease and myocardial infarction. Thus, it is of interest to identify the molecular changes glucose supplementation or hyperglycemia can induce, which ultimately compromise oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, I determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide biosynthesis and antioxidant activity, modulates anoxia survival. Additionally, a glucose-supplemented diet induces lipid accumulation. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified are homologous to human genes that are differentially regulated in response to metabolic diseases, suggesting that there may be conserved gene expression responses between C. elegans supplemented with glucose and a diabetic and/or obese state observed in humans. These findings support the utility of C. elegans to model specific aspects of the T2D disease process (e.g., glucose-induced sensitivity to oxygen deprivation) and identify potentially novel regulators of common complications seen in hyperglycemic and T2D patients (e.g., macrovascular complications).
Greater, Lesser, Guessers: A Look into the Hybridization of Greater and Lesser Prairie-Chickens
My thesis focuses on the conservation consequences of the hybridization of Lesser Prairie-Chickens in Kansas. Specifically, examining how past land management practices altering the species ranges impact the distinctiveness of Lesser Prairie-Chickens. Each chapter is an individual publication that addresses if the Greater and Lesser Prairie-Chicken are distinct when applying the morphological and biological species concepts. Chapter 2 compares the evolutionary history and morphological construct of Lesser Prairie-Chickens and other Galliformes using morphometric analysis. Chapter 3 uses low-resolution microsatellite data to reflect recent changes at the population level. This study aims to observe the Greater and Lesser Prairie-Chicken using the morphological and biological species concepts, two of the many species concepts, to determine the distinctiveness and rate of hybridization for these closely related species.
Green Improvements: A Consumer's Guide to Environmentally and Economically Responsible Home Repairs and Improvements for the North Central Texas Region
The Consumer's Guide is designed to help consumers by providing guidelines for the purchase of specific energy-efficient household appliances- water heaters, air conditioning and heating systems, windows, dishwashers, refrigerators, clothes washers, and dryers. This serves two major purposes: to decrease the environmental impact of those products and to save consumers money over the lifetime of the products. The seven major appliances covered in this work are things that consumers tend to purchase quickly when their older models wear out and with little research into their energy and/or water efficiency. The guide begins with a general introduction and an explanation of the need for energy conservation. Explanations of how they work, purchasing tips, installation tips, maintenance tips, tips for additional energy efficiency, and case studies are given for each appliance. Printable pamphlets are included at the end.
The Growth of Azotobacter vinelandii on p-Hydroxybenzoic Acid from Soil Medium
The purpose of this study was to search for the substrates utilized by Azotobacter vinelandii in dialysed soil media. Also, we sought to determine the relationship between these substrates and the growth and morphological variations of A. vinelandii. p-Hydroxybenzoic acid was shown to be used as the carbon and energy source by A. vinelandii in dialysed soil medium. The amount of this compound in the soil dialysed soil medium ranged from 14 to 21 micrograms per gram of soil. In a dialysed soil medium, p-hydroxybenzoic acid induced A. vinelandii to form minute bodies, similar to the filtrable forms reported by Gonzalez and Vela, although no growth of minute bodies was detected.
A Habitat Evaluation and Management Plan for a Riparian Ecosystem
Ecological research involving habitat studies was conducted on the Elm Fork of the Trinity River in Denton County, Texas, from spring 1985 to spring 1986. Habitat Evaluation Procedures and Habitat Suitability Index Models developed by the United States Fish and Wildlife Service were applied to a 1419 hectares study area to determine the quality of habitat for four species: beaver, Castor canadensis, wood duck, Aix sponsa, pileated woodpecker, Dryocopus pileatus, and white crappie, Poxomis annularis. Population estimates were generated. A wildlife management plan was developed for the study area. Habitat Suitability Index Models were found to be overly conservative, underestimating the quality of habitat in areas of ecological transition.
Habitat Evaluation Procedures at Ray Roberts Lake: an Analysis of the Relationship with Ecological Indicators and a Study of Observer and Temporal Variability
Habitat Evaluation Procedure data gathered at Ray Roberts Lake in 1989 and 1990 were analysed for temporal variability, observer variability and relationships between Habitat Units (HUs) and species density/diversity. observer variability within a group was analysed by cluster analysis and bootstrapping. Five out of 36 sites showed significant differences in Habitat Suitability Index (HSI) values within the group. A nonparametric Mann-Whitney test was used to analyze temporal variability. One of 6 sites showed a significant difference in HSI values between years. Using Spearman's Rank Correlation Coefficient, a correlation was found between indicator species density and HUs. No significant correlation was indicated between species diversity and HUs.
Habitat Fragmentation by Land-Use Change: One-Horned Rhinoceros in Nepal and Red-Cockaded Woodpecker in Texas
This research focuses on the spatial analysis of the habitat of two vulnerable species, the one-horn rhinoceros in the grasslands of southern Nepal, and the red-cockaded woodpecker in the Piney woods of southeast Texas, in the USA. A study sites relevant for biodiversity conservation was selected in each country: Chitwan National Park in Nepal, and areas near the Big Thicket National Preserve in Texas. Land-use differs in the two study areas: the first is still undergoing agrarian development while the second is in a technological phase and undergoing urbanization processes. Satellite remote sensing images were used to derive land-cover maps by supervised classification. These maps were then processed by Geographic Information Systems methods to apply habitat models based on basic resources (food and cover) and obtain habitat suitability maps. Several landscape metrics were computed to quantify the habitat characteristics especially the composition and configuration of suitable habitat patches. Sensitivity analyses were performed as the nominal values of some of the model parameters were arbitrary. Development potential probability models were used to hypothesize changes in land-use of the second study site. Various scenarios were employed to examine the impact of development on the habitat of red-cockaded woodpecker. The method derived in this study would prove beneficial to guide management and conservation of wildlife habitats.
Head Trauma Release of Histamine from Dural Mast Cells Alters Blood-Brain Barrier: Attenuation with Zolantidine
This study employed a new model of mild-to-moderate head trauma to specifically identify the role of dural mast cell (MC) histamine in trauma-induced increased permeability in the blood-brain barrier (BBB). A single line was scored partially through the left dorsal parietal skull. Immediately following the trauma, degranulation was seen in 39% of the MCs on the left and in 2% on the right. After a 20 min survival period, left duras showed 55% with MC degranulation (fewer with complete degranulation) compared to 34% on the right. In the other experiments two parallel lines were scored following the injection of Evan's blue. Histamine assay showed histamine increased in the left cortex to 154% at 5 min, 174% at 10 min, and 151% at 20 min. Fluorescent quantitation of extravasated Evan's blue at 20 min following the trauma gave an increase of 1385% over the value measured for the right cortex. Zolantidine, a selective histamine H2 receptor antagonist, administered at 10- and 20- mg/kg 30 min before the trauma blocked 65% of the Evan's blue extravasation compared with the control and 2.5 mg group.
Heart rate and oxygen consumption during the critical prenatal period in chicken embryos (Gallus gallus): Influence of light cues and the onset of pulmonary ventilation.
To examine if a rhythm can be entrained in either heart rate or oxygen consumption in late stage embryos (days 17-19.5) with light as a zeitgeber, chicken embryos were incubated in complete darkness (D:D) and 12:12 light:dark cycle (L:D). Light had no impact on oxygen consumption (390 µL O2∙min-1∙egg-1) but increased heart rate for non-internally pipped embryos (260 to 270 beats∙min-1 during light cycle). Oxygen consumption increased independent of pipping while heart rate increased (255 to 265 beats∙min-1) in D:D embryos due to pipping. A light-induced rhythm or effect occurred in heart rate but not oxygen consumption, suggesting heart rate and oxygen consumption may be uncoupled.
Heat Shock Proteins in Ascaris suum
Ascaris suum were exposed to a number of stressors, including heavy metals and both high (40°C) and low (18°C) temperatures. The 70kD and 90kD heat shock proteins (HSPs) in the different A. suum tissues were analyzed by Western blot and quantitated by Macintosh Image Program.
Hematocrit, hematocrit Regulation and its effect on oxygen consumption in the late stage chicken embryo (Gallus domesticus).
Hematocrit and hematocrit regulation have the potential to affect developing embryos. To examine the ability of chicken embryos at day 15 to regulate hematocrit, they were subjected to either repeated saline injections (5% of total blood volume) or repeated blood removal (5% of total blood volume). Embryos showed an ability to maintain hematocrit (~20%) despite blood volume increases up to 115% of initial blood volume. Embryos were not able to maintain hematocrit in the face of dramatic blood volume loss. Oxygen consumption of embryos could be affected by their level of hematocrit. To examine this, chicken embryos at day 15, 16, and 17 of incubation were given a high hematocrit (~50-60%) sample of blood (400 μl) to artificially increase the hematocrit of the embryos (~10-12%). Despite the increase in oxygen availability, when monitored over a period of six hours, embryos showed no difference (0.36 ± 0.01 (ml O2 - min-1- egg-1) in metabolism from baseline measurements at day 15, 16 and 17.
Hematological Parameters of the Bluegill, Lepomis machrochirus (Rafinesque), Including Effects of Turbidity, Chloramines, and Flexibacter columnaris
Normal ranges of values for hematological parameters of bluegill gathered seasonally from three lakes were determined. Sexual, seasonal, and inter-lake variations were found. Effects of 2-wk exposure to turbidity on blood parameters included an increase in rbc size and a decrease in small lymphocytes. Effects of 3-hr exposure were increases in rbc count, hemoglobin, and pH and decreases in PG2 and large lymphocytes. The effects of 0.44 and 0.88 ppm chloramines were an increase in blood pH, a decrease in MEV, and severe spastic reactions resulting in loss of equilibrium or death in 90% of the fish. Effects of Flexibacter columnaris included an increase in transformed lymphocytes and a decrease in small lymphocytes.
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.
A High-fat Meal Alters Post-prandial mRNA Expression of SIRT1, SIRT4, and SIRT6
Sirtuins (SIRT) regulate the transcription of various genes involved in the development of diet-induced obesity and chronic disease; however, it is unknown how they change acutely following a high-fat meal. The purpose of this study was to determine the effect of a high-fat meal (65% kcals/d; 85% fat recommendation), on SIRT1-7 mRNA expression in blood leukocytes at 1, 3, and 5-h post-prandial. Men and women (N=24) reported to the lab following an overnight fast (>12H). Total RNA was isolated and reverse transcribed prior to using a Taqman qPCR technique with 18S rRNA as a normalizer to determine SIRT1-7 mRNA expression. An additional aliquot of serum was used to measure triglycerides. Data was analyzed using a RM ANOVA with P<0.05. Triglycerides (P<0.001; 124%) peaked at 3-h. SIRT 1 (P=0.004; 70%), and SIRT 6 (P=0.017; 53%) decreased expression at 3-h. SIRT4 (P=0.024) peaked at 5H relative to baseline (70%) and 3-h (68%). To our knowledge, this is the first study to report that consumption of a high-fat meal transiently alters SIRT mRNA expression consistent in a pattern that mirrors changes in serum triglycerides. Decrease in expression of SIRT1 and SIRT6 combined with an increased SIRT4 would be consistent with an increase in metabolic disease risk if maintained on a chronic basis.
Hindrance of the Myosin Power Stroke Posed by the Proximity to the Troponin Complex Identified Using a Novel LRET Fluorescent Nanocircuit
A novel luminescence resonance energy transfer (LRET) nanocircuit assay involving a donor and two acceptors in tandem was developed to study the dynamic interaction of skeletal muscle contraction proteins. The donor transmits energy relayed to the acceptors distinguishing myosin subfragment-1 (S1) lever arm orientations. The last acceptor allows the detection of S1's bound near or in between troponin complexes on the thin filament. Additionally, calcium related changes between troponin T and myosin were detected. Based on this data, the troponin complex situated every 7 actin monomers, hinders adjacently bound myosins to complete their power stroke; whereas myosins bound in between troponin complexes undergo complete power strokes.
Histochemical Characterization of Lymphocytes in Preleukemic and Leukemic AKR Mice
The AKR strain of mice have a genetic trait for spontaneous development of lymphocytic leukemia. In this study, leukemic mice were found to have significantly larger (p<0.01) thymuses and spleens than preleukemic mice. The enlarged leukemic tissues were densely packed with a light staining cell, with a hollow-appearing nucleus. Tissues from preleukemic mice were observed to be infiltrated with a smaller, darker-staining lymphocyte. Fluorescent antibody staining was done on preleukemic and leukemic tissues, using three antisera against murine lymphocyte theta antigen, and an antiserum against murine IgG. Significantly brighter fluorescence, (p <0.05) with theta-specific antisera, was found in leukemic thymuses,spleens, and kidneys than in the same preleukemic tissues. Leukemic tissues had significantly brighter fluorescence (p <0.05) than preleukemic tissues with IgG antiserum.
Histological age estimation of the midshaft clavicle using a new digital technique.
Histological methods to estimate skeletal age at death, in forensic cases, are an alternative to the more traditional gross morphological methods. Most histological methods utilize counts of bone type within a given field for their estimation. The method presented in this paper uses the percentage area occupied by unremodeled bone to estimate age. The percentage area occupied by unremodeled bone is used in a linear regression model to predict skeletal age at death. Additionally, this method uses digital software to measure area rather than the traditional technique in which a gridded microscope is used to estimate area. The clavicle was chosen as a sample site since it is not a weight bearing bone and has little muscular insertion. These factors reduce the variation seen as a result of differences in lifestyle or activity pattern.
Home range analysis of rehabilitated and released great horned owls (Bubo virginianus) in Denton County, Texas, through radio telemetry.
Raptor rehabilitation has become commonplace globally, yet studies on the survival and adaptation of great horned owls (Bubo virginianus) after release has been neglected to an appreciable extent. The primary objective of this study is to provide quantitative data on the success of rehabilitated and released great horned owls in the North Texas region. Owls (N=12) were rehabilitated and released onto the Ray Roberts Greenbelt Corridor in Denton County, Texas, and monitored using radio telemetry to evaluate home range (November 2002 - February 2005). With approximately 75% of the birds released for this study surviving until transmitter battery failure, it is believed that the rehabilitation process was successful for these birds.
Homologs of Mammalian Lysosomal Lipase in Arabidopsis and Their Roles in Lipid Droplet Dynamics
Lipid droplets (LDs) are organelles with many functions in cells and numerous protein interactors facilitate their biogenesis, maintenance, and turnover. The mammalian lipase responsible for LD turnover during lipophagy, LipA, has two candidate homologs in Arabidopsis: MPL1 and LIP1. One or both of these plant homologs may function in a similar manner to mammalian LipA, providing an LD breakdown pathway. To test this hypothesis, wild type (WT) Arabidopsis plants, MPL1 over-expressing (OE) mutants, and T-DNA insertion mutants of MPL1 (mpl1) and LIP1 (lip1) were examined for LD phenotypes in normal conditions and in environments where LD numbers are known to fluctuate. Plants to be imaged by confocal microscopy were exposed to heat stress and wounding to increase LD accumulation, senescence was induced in leaves to deplete lipids, and LDs were imaged throughout the day/night period to observe their diurnal regulation. The mutation of both MPL1 and LIP1 lead to an increase in LDs within the leaf mesophyll cells, although the spatial distribution of the LDs differed between the two mutants. mpl1 mutants had disrupted diurnal regulation of their LDs, but lip1 mutants did not. Alternately, lip1 mutants retained LDs during dark-induced senescence, and mpl1 mutants did not. Together these results suggest that MPL1 and LIP1 are likely both important for LD dynamics; however they appear have roles in different aspects of LD accumulation and turnover.
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.
Hypoxia and the Development of Endothermic Capacity in Chickens (Gallus Gallus)
Adult chickens employ endothermy – internal generation of heat that maintains a constant body temperature (Tb). Prior to hatching, chicken embryos are ectothermic - controlling Tb by external heat sources. Upon hatching, the hatchling transitions from an ectotherm to an endotherm that has been shown to be delayed by hypoxia. In this study, whole animal oxygen consumption () and liver, heart, and skeletal muscle citrate synthase activity (CSA) and were measured during this transition to endothermy in chickens incubated in normoxia and hypoxia (15% O2). The only significant differences in occurred in 48 hour old hatchlings where was lower in normoxic hatchlings. There were no differences in CS activity between age and incubation oxygen levels. Additionally, preliminary 2-D protein gels of embryo and hatchling liver show changes in the proteome upon hatching. Results suggest that hypoxia had no significant effect on CSA and a minimal effect on .
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.
Hypoxic and hyperoxic incubation affects the ductus arteriosus in the developing chicken embryo (Gallus gallus).
Developing chicken embryos have two ductus arteriosus (DA) that shunt blood away from the lungs and to the chorioallantoic membrane, the embryonic gas exchanger. In mammals, DA closure is stimulated by an increase in blood gas O2 that occurs as the animal begins to breathe with its lungs. The goal of this study was to determine the influence of O2 levels during incubation on the vascular reactivity and morphology of the O2-sensitive DA and to examine the effects of changing O2 levels during late incubation on the morphology of the DA from chicken embryos. In comparison to normoxia, hypoxia (15%) reduced venous O2 levels in day 16 and day 18 embryos and reduced aircell O2 values in day 16, day 18, and internally pipped (IP) embryos, whereas hyperoxia (30%) increased venous O2 levels and aircell O2 level in day 16, day 18, and IP embryos. In comparison to normoxia, hypoxia delayed closure of the DA, whereas hyperoxia accelerated DA closure. In comparison to the left DA from externally pipped (EP) normoxic embryos, the left DA from EP hypoxic embryos exhibited a significantly weaker contractile response to O2. The DA from day 18 hypoxic embryos exhibited a significantly weaker contractile response to norepinephrine and phenylephrine when compared with the DA from day 18 normoxic and hyperoxic embryos. The effect of incubation in hypoxia / hyperoxia during different developmental windows on the DA O2-induced contractile response was observed only in IP embryos that were incubated in normoxia for 16 days and were then moved to hyperoxia. Incubation in hypoxia / hyperoxia resulted in differences in embryo mass, yolk mass, and heart mass. There is an association between the decreased contractile response to O2 and delayed closure in the proximal portion of the DA from hypoxic embryos; as well as an increased contractile …
Identification and Characterization of a Mutation Causing Stunted Growth in Arabidopsis that is Linked to Phosphate Perception
Plant yield is an agronomic trait dependent on the transport of photosynthate from mature source leaves to sink tissues. Manipulating phloem transport may lead to increased yield, however in a previous study, Arabidopsis thaliana overexpressing sucrose transporter AtSUC2 in the phloem resulted in stunted growth and an apparent P-deficiency. In the course of further characterizing the phenotype and identifying the causative mutation, this research included 1) reverse genetics to test genes hypothesized to modulate carbon-phosphate interactions; 2) whole genome sequencing to identify all T-DNA insertions in plants displaying the phenotype; 3) genetic crosses and segregation analysis to isolate the causative mutation; and 4) transcriptomics to capture gene-expression profiles in plants displaying the phenotype. These phenotypes were traced to a T-DNA insertion located on chromosome 4. Transcriptomics by RNA-Seq and data analysis through bioinformatics pipelines suggest disruptions in metabolic and transport pathways that include phosphate, but do not support a direct role of well-established phosphate acquisition mechanisms. Gene At1G78690 is immediately downstream of the T-DNA insertion site and shows modestly increased expression relative to wild type plants. At1G78690 encodes O-acyl transferase, which is involved in processing N-acylphosphotidyl ethanolamine (NAPE) to N-acyl ethanolamine (NAE). Exogenous NAE application causes stunted growth in specific conditions. From the experiments described herein, At1G78690 emerges as the strongest candidate for causing the observed phenotypes.
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 …
Identification and characterization of an incomplete root hair elongation (IRE)-like gene in Medicago truncatula (L.) root nodules.
Cloning and molecular characterization of new genes constitutes a useful approach in studying the symbiotic interactions between the model plant Medicago truncatula and Synorhizobium meliloti. Large numbers of expressed sequence tags (ESTs) available for Medicago truncatula, along with numerous cDNA, oligonucleotides, and Affimetrix DNA microarray chips, represent useful tools for gene discovery. In an attempt to identify a new gene that might be involved in the process of nodulation in Medicago truncatula, preliminary data reported by Fedorova et al. (2002), who identified 340 putative gene products or tentative consensus sequences (TCs) expressed only in nodules, was used. This research was focused on TC33166 (TC103185), which has 3 ESTs in the TC, and whose strongest BLASTX hit of TC103185 is the incomplete root hair elongation (IRE) protein kinase-like protein (NP_192429) from Arabidopsis thaliana. The Arabidopsis IRE gene is required for normal root hair growth, and a role in apical growth was suggested (Oyama et al., 2002). Infection thread growth can be looked at as an inward growth of the root hair. Thus, TC103185 was a good candidate for identifying a gene that may be involved in early events of nodulation. MtIRE (GenBank accession AC122727) is organized in 17 exons and 16 introns, similarly to the Arabidopsis IRE gene. MtIRE is a new member of the IRE family and it is a putative Ser/Thr protein kinase. MtIRE is a nodule- and flower-specific gene, suggesting that nodulation may have recruited it from other developmental processes. MtIRE is likely to be involved in the invasion process, or in the maturation of the symbiosome, or of the cells that contain rhizobia, rather than infection thread initiation and elongation or in nitrogen fixation. Nodule invasion precedes the onset of MtIRE expression and the expression pattern changes in time within the nodule. RNA interference results support MtIRE …
Identification and Characterization of Genes Required for Symbiotic Nitrogen Fixation in Medicago truncatula Tnt1 Insertion Mutants
In this dissertation I am using M. truncatula as a model legume that forms indeterminate nodules with rhizobia under limited nitrogen conditions. I take advantage of an M. truncatula Tnt1 mutant population that provides a useful resource to uncover and characterize novel genes. Here, I focused on several objectives. First, I carried out forward and reverse genetic screening of M. truncatula Tnt1 mutant populations to uncover novel genes involved in symbiotic nitrogen fixation. Second, I focused on reverse genetic screening of two genes, identified as encoding blue copper proteins, and characterization of their mutants' potential phenotypes. Third, I further characterized a nodule essential gene, M. truncatula vacuolar iron transporter like 8 (MtVTL8), which encodes a nodule specific iron transporter. I characterized the expression pattern, expression localization and function of MtVTL8. Additionally, I characterized several residues predicted to be essential to function using a model based on the known crystal structure of Eucalyptus grandis vacuolar iron transporter 1 (EgVIT1), a homologous protein to MtVTL8. I identified several potential essential residues of the MtVTL8 protein, mutagenized them, and through complementation experiments in planta and in yeast assessed functionality of the resulting protein. This helped us to better understand the potential mechanism by which MtVTL8 functions.
Identification and Characterization of the Pyrimidine Biosynthetic Operon in Streptomyces griseus
To further understand the ATCase/DHOase bifunctional complex formed in Streptomyces, the genes encoding these and other pyrimidine enzymes were identified and characterized. Polymerase chain reaction (PCR) was utilized in this effort. Primers were constructed by selecting conserved regions of pyrimidine genes from known gene and protein sequences of a wide variety of organisms. These sequences were then optimized to Streptomyces codon usage. PCR products were obtained from internal sites within pyrimidine genes and also from primer combinations of different genes. The size, orientation, and partial sequence of the resulting products shows that Streptomyces has a gene organization of pyrR followed by pyrB, pyrC, carA, carB, and pyrF in an operon similar to that found in other Gram-positive bacteria.
Identification and Characterization of Two Putative Sulfate Transporters Essential for Symbiotic Nitrogen Fixation in Medicago truncatula
The process of symbiotic nitrogen fixation (SNF) in legume root nodules requires the channeling and exchange of nutrients within and between the host plant cells and between the plant cells and their resident rhizobia. Using a forward genetics approach in the Medicago truncatula Tnt1 mutant population followed by whole genome sequencing, two putative sulfate transporter genes, MtSULTR3;5 and MtSULTR3;4b, were identified. To support the hypothesis that the defective putative sulfate transporter genes were the causative mutation for the mutants' phenotypes, the M. truncatula Tnt1 population was successfully reverse screened to find other mutant alleles of the genes. The F2 progeny of mutants backcrossed with wildtype R108 demonstrated co-segregation of mutant phenotypes with the mutant alleles confirming that the mutated mtsultr3;5 and mtsultr3;4b genes were the cause of defective SNF in the mutant lines mutated in the respective genes. This finding was further established for mtsultr3;4b by successful functional complementation of a mutant line defective in the gene with the wildtype copy of MtSULTR3;4b. A MtSULTR3;4b promoter-GUS expression experiment indicated MtSULTR3;4b expression in the vasculature and infected and uninfected plant cells of root nodules. MtSULTR3;4b was found to localize to the autophagosome membrane when expressed in Nicotiana benthamiana. A transcriptomics study on the mutant nodules revealed the probable impact of mutated mtsultr3;5 and mtsultr3;4b on expression of genes involved in N fixation and on other biological processes, including possible effects of the mutated genes on the transcriptional regulation of sulfate assimilation pathway in the respective mutants' nodules. The RNAseq study also demonstrated the mis-regulation of nodule zone-specific genes in mtsultr3;5 and mtsultr3;4b mutants. A PCR-based approach was used to study the transcription of MtSULTR3;5 and MtSULTR3;4b in the respective mutant lines. The study demonstrated formation of readthrough chimeric gene-Tnt1 transcripts in mtsultr3;5 mutant alleles and truncated chimeric gene-Tnt1 transcripts and aberrantly …
Identification and quantification of lipid metabolites in cotton fibers: Reconciliation with metabolic pathway predictions from DNA databases.
The lipid composition of cotton (Gossypium hirsutum, L) fibers was determined. Fatty acid profiles revealed that linolenate and palmitate were the most abundant fatty acids present in fiber cells. Phosphatidylcholine was the predominant lipid class in fiber cells, while phosphatidylethanolamine, phosphatidylinositol and digalactosyldiacylglycerol were also prevalent. An unusually high amount of phosphatidic acid was observed in frozen cotton fibers. Phospholipase D activity assays revealed that this enzyme readily hydrolyzed radioactive phosphatidylcholine into phosphatidic acid. A profile of expressed sequence tags (ESTs) for genes involved in lipid metabolism in cotton fibers was also obtained. This EST profile along with our lipid metabolite data was used to predict lipid metabolic pathways in cotton fiber cells.
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.
Identification of a Potential Factor Affecting Graduation Rates in STEM for Hispanic Students at the University of North Texas, via Analysis of Nonfiction Science Books in Spanish Language for ELLs in the Dallas ISD Schools
Latinos are the largest minority group in the U.S.; however despite the continuous growth of the Hispanic population, Latinos are severely underrepresented in STEM fields. One of the reasons that might explain why Latinos do not major in STEM is the way they encounter science curriculum in primary school. Students' limited proficiency in English may constrain their science achievement when instruction is delivered exclusively in English. A quantitative analysis with graduation rates in STEM from 2009 to 2014 at the University of North Texas was conducted, finding that there is a significant difference (p<0.05) in the number of bachelor's degrees in STEM between Hispanic, White, African American and other student populations. Interviews with teachers, librarians and publishing companies were performed to describe the limited science literature in Spanish at the Dallas ISD schools. Improving science literacy by teaching according to ELLs' linguistic skills and culture may lead to a better understanding of science curriculum throughout their education, which may translate into higher college graduation rates by Hispanic recipients in STEM.
Identification of Genes Involved in Flocculation by Whole Genome Sequencing of Thauera aminoaromatica Strain MZ1T Floc-defective Mutants
Thauera aminoaromatica MZ1T, a floc-forming bacterium isolated from an industrial activated sludge wastewater treatment plant, overproduces exopolysaccharide (EPS) leading to viscous bulking. This phenomenon results in poor sludge settling and dewatering during the clarification process. To identify genes responsible for bacterial flocculation, a whole genome phenotypic sequencing technique was applied. Genomic DNA of MZ1T flocculation-deficient mutants were subjected to massively parallel sequencing. The resultant high-quality reads were assembled and compared to the reference genome of the wild type genome. We identified nine nonsynonymous mutations and one nonsense mutation putatively involved in EPS biosynthesis. Complementation of the nonsense mutation located in an EPS deacetylase gene restored the flocculating phenotype. The FTIR spectra of EPS isolated from the wild-type showed reduced C=O peak of the N-acetyl group at 1665 cm-1 as compared to the spectra of MZ1T floc-deficient mutant EPS, suggesting that the WT EPS was partially deacetylated. Gene expression analysis also demonstrated the deacetylase gene transcript increased before flocculation occurred. The results suggest that the deacetylation of MZ1T EPS is crucial for flocculation. The information obtained from this study will be useful for preventing viscous bulking and wastewater treatment system failure, and may have potential applications in the biotechnology sector for the controlled removal of cells.
Identification of Glycine as the Factor in Peptone Which Induces Pleomorphism in Azotobacter Vinelandii
The rigid peptidoglycan layer of the cell wall is responsible for maintaining the structural integrity of bacteria. Antibiotics such as penicillin exert their anti-bacterial effect by inhibiting synthesis of peptodoglycan, and enzymes such as lysozyme destroy cell integrity by hydrolyzing specific bonds in the interior of this macromolecule. Defective cells can no longer withstand the high turgor pressure within the cell because they are no longer protected by a rigid wall and tend to become fragile and spherical or irregular in shape. While all bacteria are pleomorphic under certain conditions which do not normally affect other bacteria. This is exemplified by the pleomorphic growth of Azotobacter in nutrient agar or peptone-containing medium. The purpose of this investigation was to study the nature of peptone-induced pleomorphism of Azotobacter. The first phase of study dealt with the effects of poptone on the growth and morphology of A. vinelandii. Many diverse froms were observed in peptone-containing media, but it was shown that all cell types were related to the "fungoid" family of pleomorphic cells. Although Azotobacter failed to accumulate detectable levels of cell-wall precursors in response to glycine treatment, it was shown that glycine acted only on metabolically active cells. In addition, incorporation of glycine into cell wall of Azotobacter was not required for induction of pleomorphism. Methionine and aspartic acid, and to a lesser degree alanine and isoleucine, were found to competitively inhibit glycine toxicity.
Identification of Hox Genes Controlling Thrombopoiesis in Zebrafish
Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were both cumbersome or required breeding and production of fish where thrombocytes are GFP labeled. Therefore, I established a flow cytometry based method of counting the number of thrombocytes. I used mepacrine to fluorescently label the blood cells and used the white cell fraction. Standard antisense oligonucleotide designed to the central portion of each of the target hox mRNAs, was piggybacked by a control morpholino and intravenously injected into the adult zebrafish. The thrombocyte count was measured 48 hours post injection. In this study, I found that the knockdown of hoxc11b resulted in increased number of thrombocytes and knockdown of hoxa10b, …
Identification of the Neurobiological Basis of Hemodynamic Responses Correlated with Cognitive Stroop Task Performance After an Acute Bout of Aerobic Exercise
Cardiovascular activities may increase the brain blood flow improving neuronal activities leading to improved cognition. Consequently, the effects of an acute bout of moderate intensity aerobic exercise on brain hemodynamics and its correlation with cognitive color-word Stroop task performance were tested. The Stroop tasks were congruent (color matches word) and incongruent (color does not match word). Prefrontal (PFC) and motor cortex (MC) blood flow was recorded by fNIRS (functional near-infrared spectroscopy) while the subject was performing the Stroop tasks before and after the 30 minutes of exercise or equivalent time of rest controls (checking for practice effects). Ninety human subjects of age 24± 6, 20 ADHD (attention-deficit hyper-activity disorder), 27 High-BMI (>25), 29 males were recruited. Reaction time ‘RT' decreased (p<0.05) after exercise for both the congruent (12%) and incongruent (10%) Stroop tasks, compared to 8% with practice alone. Accuracy did not change after practice or exercise. HR changes after exercise correlated (p<0.05) with better accuracy and faster RT for the incongruent Stroop task. In general, a metabolic lag occurred in the neuronal deoxy- hemoglobin (Hb) signals behind the systemic oxy-Hb signals. PFC showed the highest effect sizes of Stroop task-responsive systemic hemodynamic changes compared to baseline irrespective of rest or exercise. Yet, PFC showed most significant (p<0.001) neuronal hemodynamic changes between the before and after exercise sessions, and these changes were opposite for right and left PFC, and opposite for congruent and incongruent Stroop tasks. Correlating the RT and mistakes with hemodynamics for both the Stroop tasks revealed that, after exercise, neuronal hemodynamic changes occurred at both PFC and MC associated with faster RT (p<0.05), and systemic hemodynamic responses occurred at PFC correlated (p<0.05) with mistakes. Overall, it was concluded that exercise changed the neuronal hemodynamic changes affecting speed; however, neuronal metabolic changes did not occur sufficiently to help …
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