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  Access Rights: Public
  Partner: UNT Libraries
 Degree Discipline: Molecular Biology
 Collection: UNT Theses and Dissertations
Genetic Analysis of Development and Behavior in Hypoxia and Cellular Characterization of Anoxia Induced Meiotic Prophase Arrest in Caenorhabditis Elegans

Genetic Analysis of Development and Behavior in Hypoxia and Cellular Characterization of Anoxia Induced Meiotic Prophase Arrest in Caenorhabditis Elegans

Date: August 2011
Creator: Little, Brent Ashley
Description: It was hypothesized that chronic hypoxia will affect various biological processes including developmental trajectory and behavior. To test this hypothesis, embryos were raised to adulthood in severe hypoxic environments (0.5% O2 or 1% O2, 22°C) and analyzed for survival rate, developmental progression, and altered behaviors. Wildtype hermaphrodites survive chronic hypoxia yet developmental trajectory is slowed. The hermaphrodites raised in chronic hypoxia had different phenotypes in comparison to the normoxic controls. First, hermaphrodites exposed to chronic hypoxia produced a significantly lower number of embryos and had a slight increase in male progeny. This suggests that chronic hypoxia exposure during development affects the germline. Second, animals raised in chronic hypoxia from embryos to young adults have a slight increase in lifespan when re-exposed to a normoxic environment, indicating that chronic hypoxia does not negatively decrease lifespan. Finally, hermaphrodites that were raised in hypoxia will lay the majority of their eggs on the area of the agar plate where the bacterial lawn is not present. This is in contrast to animals in normoxia, which lay the majority of their eggs on the bacterial lawn. One hypothesis for this hypoxia-induced egg-laying behavior is that the animal can sense microenvironments in hypoxia. To examine if ...
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Genetic and Cellular Analysis of Anoxia-Induced Cell Cycle Arrest in Caenorhabditis elegans

Genetic and Cellular Analysis of Anoxia-Induced Cell Cycle Arrest in Caenorhabditis elegans

Date: December 2008
Creator: Hajeri, Vinita A.
Description: 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. ...
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Genetic and Environmental Factors that Mediate Survival of Prolonged Oxygen Deprivation in the Nematode Caenorhabditis Elegans

Genetic and Environmental Factors that Mediate Survival of Prolonged Oxygen Deprivation in the Nematode Caenorhabditis Elegans

Date: August 2010
Creator: LaRue, Bobby Lee, Jr.
Description: 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 ...
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Impaired virulence factor production in a dihydroorotate dehydrogenase mutant (pyrD) of  Pseudomonas aeruginosa.

Impaired virulence factor production in a dihydroorotate dehydrogenase mutant (pyrD) of Pseudomonas aeruginosa.

Date: December 2005
Creator: Ralli, Pooja
Description: Previous research in our laboratory showed that when knockout mutations were created in the pyrB and pyrC genes of the pyrimidine pathway in Pseudomonas aeruginosa, not only were the resultant mutants auxotrophic for pyrimidines but they were also impaired in virulence factor production. Such a correlation had not been previously reported for P. aeruginosa, a ubiquitous opportunistic pathogen in humans. In an earlier study it was reported that mutants blocked in one of the first three enzymes of the pyrimidine pathway in the non-pathogenic strain P. putida M produced no pyoverdin pigment while mutants blocked in the later steps produced copious amounts of pigment, just like the wild type. This study probed for the same connection between pyrimidine auxotrophy and pigment production applied in P. aeruginosa. To that end a knockout mutation was created in pyrD, the fourth step in the pyrimidine pathway which encodes dihydroorotate dehydrogenase. The resulting mutant required pyrimidines for growth but produced wild type pigment levels. Since the pigment pyoverdin is a siderophore it may also be considered a virulence factor, other virulence factors were quantified in the mutant. These included casein protease, hemolysin, elastase, swimming, swarming and twitching motility, and iron binding capacity. In all ...
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Investigating the Ability of Pseudomonas Aeruginosa Pyre Mutants to Grow and Produce Virulence Factors

Investigating the Ability of Pseudomonas Aeruginosa Pyre Mutants to Grow and Produce Virulence Factors

Date: December 2014
Creator: Niazy, Abdurahman A
Description: Pseudomonas aeruginosa are medically important bacteria that are notorious for causing nosocomial infections. To gain more knowledge into understanding how this organism operates, it was decided to explore the pyrimidine biosynthetic pathway. Pyrimidine synthesis, being one half of the DNA structure, makes it a very important pathway to the organism’s survivability. With previous studies being done on various genes in the pathway, pyrE has not been studied to the fullest extent. To study the function of pyrE, a site directed mutagenesis was done to completely knock out pyrE, which encodes the protein orotate phosphoribosyl transferase that is responsible for converting orotate into orotate monophosphate (OMP). A mutation in this step leads to accumulation and secretion of orotate into the medium. Analyzing virulence factors produced by the mutant and comparing to the wild type, some intriguing features of the mutant were discovered. One of the findings was the over expression of virulence factors pyoverdin and pyocyanin. Pyocyanin over expression, based on the results of this study, is due to the accumulation of orotate while over production of pyoverdin is due to the accumulation of dihydroorotate. The other virulence factors studied were motility assays, exoproducts, and growth analysis. All virulence factor production ...
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Investigation of Strategies for Improving STR Typing of Degraded and Low Copy DNA from Human Skeletal Remains and Bloodstains

Investigation of Strategies for Improving STR Typing of Degraded and Low Copy DNA from Human Skeletal Remains and Bloodstains

Date: August 2014
Creator: Ambers, Angie D.
Description: 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 ...
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Isolation and analysis of cotton genomic clones encompassing a fatty acid desaturase (FAD2) gene

Isolation and analysis of cotton genomic clones encompassing a fatty acid desaturase (FAD2) gene

Date: May 2001
Creator: Kongcharoensuntorn, Wisatre
Description: Polyunsaturated fatty acids are major structural components of plant chloroplast and endoplasmic reticulum membranes. Two fatty acid desaturases (designated FAD2 and FAD3) desaturate 75% of the fatty acids in the endoplasmic reticulum. The w -6 fatty acid desaturase (FAD2) may be responsible for cold acclimation response, since polyunsaturated phospholipids are important in helping maintain plant viability at lowered temperatures. To study regulation of FAD2 gene expression in cotton, a FAD2 gene was isolated from two genomic libraries using an Arabidopsis FAD2 hybridization probe and a cotton FAD2 5¢ -flanking region gene-specific probe, respectively. A cotton FAD2 gene was found to be in two overlapping genomic clones by physical mapping and DNA sequencing. The cloned DNA fragments are identical in size to cotton FAD2 genomic DNA fragments shown by genomic blot hybridization. The cotton FAD2 coding region has 1,155 bp with no introns and would encode a putative polypeptide of 384 amino acids. The cotton FAD2 enzyme has a high identity of 75% with other plant FAD2 enzymes. The enzyme has three histidine-rich motifs that are conserved in all plant membrane desaturases. These histidine boxes may be the iron-binding domains for reduction of oxygen during desaturation. To confirm that this FAD2 ...
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Isolation and Characterization of the Operon Containing Aspartate Transcarbamoylase and Dihydroorotase from Pseudomonas aeruginosa

Isolation and Characterization of the Operon Containing Aspartate Transcarbamoylase and Dihydroorotase from Pseudomonas aeruginosa

Date: May 1993
Creator: Vickrey, John F. (John Fredrick), 1959-
Description: The Pseudomonas aeruginosa ATCase was cloned and sequenced to determine the correct size, subunit composition and architecture of this pivotal enzyme in pyrimidine biosynthesis. During the course of this work, it was determined that the ATCase of Pseudomonas was not 360,000 Da but rather present in a complex of 484,000 Da consisting of two different polypeptides (36,000 Da and 44,000 Da) with an architecture similar to that of E. coli ATCase, 2(C3):3(r2). However, there was no regulatory polypeptide found in the Pseudomonas ATCase.
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Isolation of a  Pseudomonas aeruginosa Aspartate Transcarbamoylase Mutant and the Investigation of Its Growth Characteristics, Pyrimidine Biosynthetic Enzyme Activities, and Virulence Factor Production

Isolation of a Pseudomonas aeruginosa Aspartate Transcarbamoylase Mutant and the Investigation of Its Growth Characteristics, Pyrimidine Biosynthetic Enzyme Activities, and Virulence Factor Production

Date: December 2004
Creator: Hammerstein, Heidi Carol
Description: The pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.
Contributing Partner: UNT Libraries
Map-based cloning of the NIP gene in model legume Medicago truncatula.

Map-based cloning of the NIP gene in model legume Medicago truncatula.

Date: May 2007
Creator: Morris, Viktoriya
Description: Large amounts of industrial fertilizers are used to maximize crop yields. Unfortunately, they are not completely consumed by plants; consequently, this leads to soil pollution and negative effects on aquatic systems. An alternative to industrial fertilizers can be found in legume plants that provide a nitrogen source that is not harmful for the environment. Legume plants, through their symbiosis with soil bacteria called rhizobia, are able to reduce atmospheric nitrogen into ammonia, a biological nitrogen source. Establishment of the symbiosis requires communication on the molecular level between the two symbionts, which leads to changes on the cellular level and ultimately results in nitrogen-fixing nodule development. Inside the nodules hypoxic environment, the bacterial enzyme nitrogenase reduces atmospheric nitrogen to ammonia. Medicago truncatula is the model legume plant that is used to study symbiosis with mycorrhiza and with the bacteria Sinorhizobium meliloti. The focus of this work is the M. truncatula nodulation mutant nip (numerous infections and polyphenolics). The NIP gene plays a role in the formation and differentiation of nodules, and development of lateral roots. Studying this mutant will contribute knowledge to understanding the plant response to infection and how the invasion by rhizobia is regulated. Previous genetic mapping placed NIP ...
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