UNT Theses and Dissertations - 30 Matching Results

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Presence of Wolbachia, A Potential Biocontrol Agent: Screening for Vertebrate Blood Meal Source and West Nile Virus in Mosquitoes in the North Texas Region

Description: 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 ...
Date: August 2016
Creator: Adiji, Olubu Adeoye
Partner: UNT Libraries

Genetic Analysis of Medicago truncatula Plants with a Defective MtIRE Gene

Description: Leguminous plants are able to fix nitrogen by establishing a symbiotic relationship with soil dwelling bacteria, called rhizobia. The model plant Medicago truncatula forms a partnership with Sinorhizobium meliloti whereby the plant gains bioavailable nitrogen and in exchange the bacteria gains carbohydrates. This process occurs within nodules, which are structures produced on the roots of the plants within which nitrogen is fixed. M. truncatula incomplete root elongation (MtIRE) was localized to the infection zone, which is zone II of indeterminate nodules. It was shown to encode a signaling kinase so it was anticipated to play a role in nodulation. Mutants of MtIRE in the R108 background, mutagenized with the Tnt1 retrotransposon, were obtained from reverse screen, and were assessed to determine if a disrupted MtIRE gene was the cause of nitrogen fixation defective nodules. Mutant line NF1320, having a mutant phenotype, showed typical Mendelian segregation of 3:1 when backcrossed to R108. Experimental results show that MtIRE gene is not the cause of the mutant phenotype, but was linked to the causative locus. MtIRE co-segregated with the mutant phenotype 83%. Southern blot and the first version of the M. truncatula genome (version 3.5) reported a single MtIRE gene and this was shown to be on chromosome 5 but the latest version of the M. truncatula genome (version 4.0) showed a second copy of the gene on chromosome 4. The genome sequence is based on the A17 reference genome. Both genes are 99% identical. Genetic markers that originate from flanking sequence tags (FSTs) on both chromosome 4 and 5 were tested in an attempt to find an FST that co-segregated with the mutant phenotype 100%. An FST derived from a Tnt1 insertion in Medtr4g060930 (24F) co-segregated with the mutant phenotype closely, with 76% co-segregation. Medtr4g060930 (24F) is on chromosome 4, making it ...
Date: August 2015
Creator: Alexis, Naudin
Partner: UNT Libraries

The Role of the Actin Cytoskeleton in Asymmetric Cell Division in Maize

Description: 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.
Date: August 2014
Creator: Alhassan, Hassan Hamdan
Partner: UNT Libraries

The Generation of Recombinant Zea mays Spastin and Katanin Proteins for In Vitro Analysis

Description: 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.
Date: December 2017
Creator: Alodailah, Sattam Sonitan
Partner: UNT Libraries

Studies in Trypsin as an Alarm Substance in Zebrafish

Description: 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 ...
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Date: August 2018
Creator: Alsrhani, Abdullah Falleh
Partner: UNT Libraries

Role of GPR17 in Thrombocyte Aggregation in Adult Zebrafish

Description: GPR17, a uracil nucleotide cysteinyl leukotriene receptor, belongs to the GPCR (G protein coupled receptor) family. It has been shown recently that inhibiting this protein in the nervous system in mice can lead to blockage of oligodendrocyte maturation, which supports myelin repair. Interestingly, our laboratory found GPR17 in thrombocytes. However, we do not know whether it has any function in thrombocyte aggregation or the nature of the ligand. In this paper, we studied the role of GPR17 in hemostasis, which is a fundamental defense mechanism in the event of injury. Using zebrafish as a model system, our laboratory has studied specifically thrombocytes, which play a significant role in hemostasis. The major reasons to use zebrafish as a model system are that their thrombocytes are functionally equivalent to human platelets, the adult fish are amenable to knockdown experiments, and they are readily available in the market. This study was performed by using a piggy back knockdown method where we used a chemical hybrid of control morpholino and an antisense oligonucleotide sequence leads to the degradation the mRNA for GPR17. After knockdown GPR17 in thrombocytes, the percent difference of the thrombocytes aggregation between the control and knockdown blood samples was measured by flow cytometry. We used various thrombocyte agonists to study differences in aggregation between the control and knockdown blood samples. The study showed that knockdown of GPR17 resulted in no significant differences in percent thrombocyte aggregation between control and agonist treated samples except for a slight increase in collagen-treated samples. Thus, it appears that GPR17 has no significant role in hemostasis.
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Date: December 2015
Creator: Bohassan, Maruah Hejey
Partner: UNT Libraries

Lipogenic Proteins in Plants: Functional Homologues and Applications

Description: Although cytoplasmic lipid droplets (LDs) are the major reserves for energy-dense neutral lipids in plants, the cellular mechanisms for packaging neutral lipids into LDs remain poorly understood. To gain insights into the cellular processes of neutral lipid accumulation and compartmentalization, a necessary step forward would be to characterize functional roles of lipogenic proteins that participate in the compartmentalization of neutral lipids in plant cells. In this study, the lipogenic proteins, Arabidopsis thaliana SEIPIN homologues and mouse (Mus Musculus) fat storage-inducing transmembrane protein 2 (FIT2), were characterized for their functional roles in the biogenesis of cytoplasmic LDs in various plant tissues. Both Arabidopsis SEIPINs and mouse FIT2 supported the accumulation of neutral lipids and cytoplasmic LDs in plants. The three Arabidopsis SEIPIN isoforms play distinct roles in compartmentalizing neutral lipids by enhancing the numbers and sizes of LDs in various plant tissues and developmental stages. Further, the potential applications of Arabidopsis SEIPINs and mouse FIT2 in engineering neutral lipids and terpenes in plant vegetative tissues were evaluated by co-expressing these and other lipogenic proteins in Nicotiana benthamiana leaves. Arabidopsis SEIPINs and mouse FIT2 represent effective tools that may complement ongoing strategies to enhance the accumulation of desired neutral lipids and terpenes in plant vegetative tissues. Collectively, our findings in this study expand our knowledge of the broader cellular mechanisms of LD biogenesis that are partially conserved in eukaryotes and distinct in plants and suggest novel targets that can be introduced into plants to collaborate with other factors in lipid metabolism and elevate oil content in plant tissues.
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Date: December 2018
Creator: Cai, Yingqi
Partner: UNT Libraries

Rapid Metabolic Response of Plants Exposed to Light Stress

Description: Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. So, in our study we have taken an metabolomics approach to identify the rapid response of plants to light stress. In the first part we have focused on the ultrafast (0-90 sec) metabolic response of local tissues to light stress and in the second part we analyzed the metabolic response associated with rapid systemic signaling (0-12 min). Analysis of the rapid response of Arabidopsis to light stress has revealed 111 metabolites that significantly alter in their level during the first 90 sec of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is dependent on an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for rapid metabolic responses. Taken together, our analysis could be used as an initial road map for the identification of different pathways that could be used to augment the rapid response of plants to abiotic stress. In addition, it highlights the important role of glutathione in initial stage of light stress response. Light-induced rapid systemic signaling and systemic acquired acclimation (SAA) are thought to play an important role in the response of plants to different abiotic stresses. Although molecular and metabolic responses to light stress have been extensively studied in local leaves, and to a lesser degree in systemic leaves, very ...
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Date: May 2018
Creator: Choudhury, Feroza Kaneez
Partner: UNT Libraries

Analysis of Young and Mature Thrombocytes in Zebrafish

Description: Eukaryotic platelets are small cell fragments that are released into the bloodstream from megakaryocytes, and their production is initiated in the bone marrow. They are mainly involved in blood hemostasis and thrombus formation. The newly synthesized platelets are called reticulated platelets or young platelets. Zebrafish thrombocytes are equivalent to mammalian platelets and have similar characteristics and functions. Likewise, zebrafish has both young and mature thrombocytes. Only young thrombocytes as reticulated platelets are labeled with thiazole orange. Similarly, labeling zebrafish thrombocytes with a specific concentration of DiI-C18 showed two populations of thrombocytes (DiI+ and DiI-). Again, only young thrombocytes showed DiI+ labeling. The mechanism of selective labeling of young thrombocytes by is unknown. Furthermore, there is no zebrafish line where young and mature thrombocytes are differentially labeled with fluorescence proteins. Therefore, in this study, we identified and confirmed that the RFP labeled cells of Glofish were young thrombocytes. In addition, we found that myosin light chain 2 (MLC2) promoter is expressed in young thrombocytes. We also generated a transgenic zebrafish line, GloFli fish, where the young and mature thrombocytes are labeled with red and green fluorescence proteins respectively. Furthermore, this study showed a two-fold increase in glycerol-phospholipids (GP) in mature thrombocytes compared to young thrombocytes suggesting the lipid composition may be important for differential labeling. Therefore, we tested the liposomes prepared with different ratios of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and observed that the lower amounts of PE favor the DiI-C18 labeling whereas higher concentrations of PC are less efficient. Also, in both PE and PC, increased concentrations of both resulted in decreased binding. These results are consistent with our observation that mature thrombocytes have higher concentrations GP and thus DiI-C18 may not bind to them efficiently compared to young thrombocytes.
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Date: August 2018
Creator: Fallatah, Weam
Partner: UNT Libraries

Alterations in Fatty Acid Amide Hydrolase (Faah) Transcript Levels and Activity Lead to Changes in the Abiotic Stress Susceptibility of Arabidopsis Thaliana

Description: N-Acylethanolamines (NAEs) are a class of bioactive lipids, and FAAH is one of the enzymes responsible for degrading NAEs in both plants and animals. in plants, FAAH appears to be closely associated with ABA, a phytohormone which has long been associated with plant stress responses, since the overexpression of FAAH in Arabidopsis results in ABA hypersensitivity. Therefore, it is reasonable to speculate that alterations in FAAH transcript levels will result in altered stress responses in plants. to investigate this hypothesis experiments were carried out in which wild type (WT), FAAH-overexpressing (OE), and T-DNA insertional FAAH knockouts of Arabidopsis (faah) were grown in MS media under stress conditions. the stress conditions tested included chilling stress, heavy metal stress induced by cadmium or copper, nutrient limitations induced by low phosphorus or low nitrogen, salt stress induced with NaCl, and osmotic stress induced with mannitol. the OE plants were consistently hypersensitive to all stress conditions in relation to wild type plants. Inactive FAAH overexpressors did not have the hypersensitivity to the salt and osmotic stress of the active OE plants and were instead tolerant to these stresses. FAAH2 (faah2) knockouts and FAAH 1 and 2 double knockouts (faah 1+2) were based on some root development parameters somewhat more tolerant than WT plants, but more sensitive in terms of shoot growth. Collectively the data suggests that FAAH activity may interact with stress-responsive pathways in plants, perhaps including pathways involving ABA.
Date: May 2012
Creator: Gonzalez, Gabriel
Partner: UNT Libraries

Isolation and Genomic Characterization of 45 Novel Bacteriophages Infecting the Soil Bacterium Streptomyces griseus

Description: Bacteriophages, or simply "phages," are the most abundant biological entities on the planet and are thought to be the largest untapped reservoir of available genetic information. They are also important contributors to both soil health and nutrient recycling and have significantly influenced our current understanding of molecular biology. Bacteria in the genus Streptomyces are also known to be important contributors to soil health, as well as producing a number of useful antibiotics. The genetic diversity of large (> 30) groups of other actinobacteriophages, i.e. phages infecting a few close relatives of the Streptomycetes, has been explored, but this is the first formal effort for Streptomyces-infecting phages. Described here are a group of 45 phages, isolated from soil using a single Streptomycete host, Streptomyces griseus ATCC 10137. All 45 phages are tailed phages with double-stranded DNA. Siphoviruses predominate, six of the phages are podoviruses, and no myoviruses were observed. Notably present are seven phages with prolate icosahedral capsids. Genome lengths and genome termini vary considerably, and the distributions of each are in line with findings among other groups of studied actinobacteriophages. Interestingly, the average G+C among the 45 phages is around 11% lower than that of the isolation host, a larger disparity than reported for other groups of actinobacteriophages. Eighteen of the phages carry between 17 and 45 tRNAs and 12 of those carry a single tmRNA. Forty-three phages were grouped into seven clusters and two subclusters based on dot plot analysis, average nucleotide identities, and gene content similarities. Two phages were not clustered with other phages in this dataset. A total of 5250 predicted genes were sorted into 1300 gene "phamilies," with about 8% of the total phamilies having only a single member. Analysis of gene content among the 45 phages indicates first that most clusters presented here appear to ...
Date: December 2018
Creator: Hale, Richard
Partner: UNT Libraries

Influence of a Human Lipodystrophy Gene Homologue on Neutral Lipid Accumulation in Arabidopsis Leaves

Description: CGI-58 is the defective gene in the human neutral lipid storage disease called Chanarin-Dorfman syndrome. This disorder causes intracellular lipid droplets to accumulate in nonadipose tissues, such as skin and blood cells. Here, disruption of the homologous CGI-58 gene in Arabidopsis thaliana resulted in the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy of isolated lipid droplets from cgi-58 loss-of-function mutants showed they contain triacylglycerols with common leaf specific fatty acids. Leaves of mature cgi-58 plants exhibited a marked increase in absolute triacylglycerol levels, more than 10-fold higher than in wild-type plants. Lipid levels in the oil-storing seeds of cgi-58 loss-of-function plants were unchanged, and unlike mutations in beta-oxidation, the cgi-58 seeds germinated and grew normally, requiring no rescue with sucrose. We conclude that the participation of CGI-58 in neutral lipid homeostasis of nonfat-storing tissues is similar, although not identical, between plant and animal species. This unique insight may have implications for designing a new generation of technologies that enhance the neutral lipid content and composition of corp plants.
Date: August 2016
Creator: James, Christopher Neal
Partner: UNT Libraries

Forward Genetic Characterization of Medicago truncatula Tnt1 Insertion Mutants Defective in Nodule Development and Symbiotic Nitrogen Fixation

Description: Legumes are unique plants because they form special structures “nodules”, via symbiotic relationships with rhizobial bacteria present in the soil. Once rhizobia mature inside nodules, they fix atmospheric nitrogen providing a source of bioavailable nitrogen to the plant. To discover novel genetic components involved in the legume-rhizobia symbiosis by using forward genetic screening, we have isolated Medicago truncatula Tnt1 insertion mutants in the R108 ecotype, which are defective in nodule development and symbiotic nitrogen fixation in response to Sinorhizobium meliloti. Out of three mutants NF11044, NF11217 and NF8324, one of the mutants showed brown nodules and Fix- phenotype that is defective in symbiotic nitrogen fixation. The other two mutants showed white nodules and Fix- phenotype, also indicator of defects in symbiotic nitrogen fixation. To identify the underlying mutation causing the phenotype, we have developed molecular genetic markers by obtaining genomic sequences flanking the Tnt1 insertions by TAIL-PCR and Illumina sequencing. To carry out co-segregation analysis, back-crossed BC1F2 segregating populations were obtained. These are being phenotyped, genotyped and analyzed for co-segregation of the phenotype with the Tnt1 genetic markers. Back-crossing also has the effect of reducing the Tnt1 insertions, which are not linked to the nodulation defective phenotypes. Out of the three mutants, NF8324 harbors exactly the same insertion as in the rsd-1 Tnt1 mutant NF11265. The defect in NF11217 is caused by a Tnt1 insertion in the previously described PLC gene; the site of this insertion is close to that found in a different mutant, NF0217. For mutant NF11044, we developed linkage markers that place the defective locus on chromosome 7. To further characterize co-segregation in NF11044, a mapping population has been created by crossing the mutant with other ecotypes: A17 and A20. We tested mutants and wild type plants with linkage marker A20 X NF11044 BC1F2 that segregates 3:1(wild ...
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Date: May 2015
Creator: Kadel, Khem L.
Partner: UNT Libraries

Metabolism and Action of Polyunsaturated N-acylethanolamines in Arabidopsis thaliana Seedlings

Description: The lipoxygenase (LOX) pathway plays an important role in the oxidative metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). The LOX pathway functions in conjugation with hydrolysis by fatty acid amide hydrolase (FAAH) and to produce oxidized NAEs during seed germination and early seedling development. When Arabidopsis seedlings were grown in low micromolar concentrations of lauroylethanolamide (NAE 12:0), growth retardation and elevated endogenous PU-NAE levels were observed due to the competitive inhibition of LOX by NAE 12:0. The elevated levels of endogenous PU-NAEs were more pronounced in genotypes with reduced NAE hydrolase capacity (faah knockouts), and less evident with overexpression of FAAH. Alterations in PU-NAE metabolism were studied in seedlings of various lox and FAAH mutants. The partitioning of PU-NAEs into oxylipin metabolites was exaggerated in the presence of exogenous linolenoylethanolamide (NAE18:3) and resulted in bleaching of cotyledons. The bleaching phenotype was restricted to a narrow developmental window (3-to-5 days after sowing), and was attributed to a reversible disruption of thylakoid membranes in chloroplasts. Biochemical and genetic evidence suggested that 9-hydro(pero)xy and 13-hydro(pero)xy octadecatrienoylethanolamides (9- and 13-NAE-H(P)OT), but not their corresponding hydro(pero)xy free fatty acids, induced cotyledon bleaching. The LOX-mediated metabolites of NAE18:3 shared some overlapping effects on seedling development with those of linoleoylethanolamide (NAE18:2) such as a reduction in seedling root growth. On the other hand, NAE18:3 oxylipin metabolites also exhibited distinct effects during seedling development such as the inhibition of photomorphogenesis. Biochemical and genetic evidence indicated that a LOX-mediated metabolite of NAE18:2, 9-hydro(pero)xy octadecadienoylethanolamide (9-NAE-H(P)OD), acted as a potent negative regulator of seedling root development, and this depended on an intact abscisic acid (ABA) signaling pathway. Synergistic inhibition of root elongation between 9-NAE-H(P)OD and ABA was restricted to a narrow developmental window (3-to-5 d after sowing) of seedling development. Genetic evidence with Arabidopsis mutants in ABA synthesis (aba1, aba2), perception (pyr1, ...
Date: August 2015
Creator: Keereetaweep, Jantana
Partner: UNT Libraries

Metabolic Engineering in Plants to Control Source/sink Relationship and Biomass Distribution

Description: Traditional methods like pruning and breeding have historically been used in crop production to divert photoassimilates to harvested organs, but molecular biotechnology is now poised to significantly increase yield by manipulating resource partitioning. It was hypothesized that metabolic engineering in targeted sink tissues can favor resource partitioning to increase harvest. Raffinose Family Oligosaccharides (RFOs) are naturally occurring oligosaccharides that are widespread in plants and are responsible for carbon transport, storage and protection against cold and drought stress. Transgenic plants (GRS47, GRS63) were engineered to generate and transport more RFOs through the phloem than the wild type plants. The transgenic lines produced more RFOs and the RFOs were also detected in their phloem exudates. But the 14CO2 labeling and subsequent thin layer chromatography analysis showed that the RFOs were most likely sequestered in an inactive pool and accumulate over time. Crossing GRS47 and GRS63 lines with MIPS1 plants (that produces more myo-inositol, a substrate in the RFO biosynthetic pathway) did not significantly increase the RFOs in the crossed lines. For future manipulation of RFO degradation in sink organs, the roles of the endogenous α-galactosidases were analyzed. The alkaline α-galactosidases (AtSIP1 and AtSIP2 in Arabidopsis) are most likely responsible for digesting RFOs in the cytoplasm and may influence the ability to manipulate RFO levels in engineered plants. Atsip1/2 (AtSIP1/AtSIP2 double-knockout plants) were generated and phenotypically characterized based on seed germination patterns, flowering time, and sugar content to observe the impact on RFO sugar levels. The observations and analysis from these lines provide a basis for further insight in the manipulation of resource allocation between source and sink tissues in plants for future research.
Date: August 2013
Creator: Lahiri, Ipsita
Partner: UNT Libraries

brk1 and dcd1 Act Synergistically in Subsidiary Cell Formation in Zea mays

Description: Subsidiary mother cell (SMC) divisions during stomatal complex formation in Zea mays are asymmetric generating a small subsidiary cell (SC) and a larger epidermal cell. Mutants with a high number of abnormally shaped subsidiary cells include the brick1 (brk1) and discordia1 (dcd1) mutants. BRK1 is homologous to HSPC300, an ARP2/3 complex activator, and is involved in actin nucleation while DCD1 is a regulatory subunit of the PP2A phosphatase needed for microtubule generation (Frank and Smith, 2002; Wright et al. 2009). Possible causes of the abnormal SCs in brk1 mutants include a failure of the SMC nucleus to polarize in advance of mitosis, no actin patch, and transverse and/or no PPBs (Gallagher and Smith, 2000; Panteris et al 2006). The abnormal subsidiary mother cell division in dcd1 is due to correctly localized, but disorganized preprophase bands (PPBs; Wright et al. 2009). The observation that brk1 has defects in PPB formation and that the dcd1 phenotype is enhanced by the application of actin inhibitors led us to examine the dcd1; brk1 double mutant (Gallagher and Smith, 1999). We found that dcd1; brk1 double mutants demonstrate a higher percentage of aberrant SCs than the single mutants combined suggesting that these two mutations have a synergistic and additive effect on SC formation. Our observations and results are intriguing and the future step will be to quantitate the abnormal PPBs and phragmoplasts in the double and single mutants using immunolocalization of tubulin and actin as well as observations of live cells expressing tubulin-YFP.
Date: August 2014
Creator: Malhotra, Divya
Partner: UNT Libraries

Homologs of Mammalian Lysosomal Lipase in Arabidopsis and Their Roles in Lipid Droplet Dynamics

Description: 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.
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Date: December 2017
Creator: McClinchie, Elizabeth A
Partner: UNT Libraries

Regulation of Alternative Sigma Factors During Oxidative and Ph Stresses in the Phototroph Rhodopseudomonas Palustris

Description: Rhodopseudomonas palustris is a metabolically versatile phototrophic α-proteobacterium. The organism experiences a wide range of stresses in its environment and during metabolism. The oxidative an pH stresses of four ECF (extracytoplasmic function) σ-factors are investigated. Three of these, σ0550, σ1813, and σ1819 show responses to light-generated singlet oxygen and respiration-generated superoxide reactive oxygen species (ROS). The EcfG homolog, σ4225, shows a high response to superoxide and acid stress. Two proteins, one containing the EcfG regulatory sequence, and an alternative exported catalase, KatE, are presented to be regulated by σ4225. Transcripts of both genes show similar responses to oxidative stress compared to σ4225, indicating it is the EcfG-like σ-factor homolog and controls the global stress response in R. palustris.
Date: August 2014
Creator: Perry, Leslie M.
Partner: UNT Libraries

Expression of G-protein Coupled Receptors in Young and Mature Thrombocytes and Knockdown of Gpr18 in Zebrafish

Description: In this study, a novel method based on biotinylated antibodies and streptavidin coated magnetic beads was used to separate the thrombocyte subpopulations from zebrafish whole blood. DiI-C18, a lipophilic dye, labels only young thrombocytes when used at low concentrations. Commercially available biotinylated anti-Cy3 antibody was used to label the chromophore of DiI-C18 on the young thrombocytes and streptavidin coated magnetic beads were added subsequently, to separate young thrombocytes. The remaining blood cells were probed with custom-made biotinylated anti-GPIIb antibody and streptavidin magnetic beads to separate them from other cells. Further, thrombocytes are equivalents of mammalian platelets. Platelets play a crucial role in thrombus formation. The G-protein coupled receptors (GPCRs) present on the platelet surface are involved during platelet activation and aggregation processes. So, thrombocytes were studied for the presence of GPCRs. The GPCR mRNA transcripts expressed in the young and mature thrombocytes were subjected to densitometry analysis and pixel intensities of the bands were compared using one way ANOVA. This analysis did not show significant differences between the young and mature GPCR mRNA transcripts but identified a novel GPCR, GPR18 that was not reported in platelets earlier. To study the function of this GPCR, it was knocked down using GPR18 specific antisense morpholino and vivo morpholino. The immunofluorescence experiment indicated the presence of GPR18 on thrombocytes. The results of the assays, such as, time to occlusion (TTO) and time to aggregation (TTA) in response to N-arachidonyl glycine (NAG) as an agonist, showed prolongation of time in GPR18 larval and adult morphants respectively, suggesting that GPR18 plays a role in thrombus formation in zebrafish. In conclusion, our results indicate that GPR18 may be present in zebrafish thrombocytes, it may be involved in thrombus formation and that NAG may be an agonist at GPR18 on thrombocytes.
Date: May 2013
Creator: Potbhare, Vrinda Nikhil
Partner: UNT Libraries

Phylogenetic and Functional Characterization of Cotton (Gossypium hirsutum) CENTRORADIALIS/TERMINAL FLOWER1/SELF-PRUNING Genes

Description: Plant architecture is an important agronomic trait driven by meristematic activities. Indeterminate meristems set repeating phytomers while determinate meristems produce terminal structures. The centroradialis/terminal flower1/self pruning (CETS) gene family modulates architecture by controlling determinate and indeterminate growth. Cotton (G. hirsutum) is naturally a photoperiodic perennial cultivated as a day-neutral annual. Management of this fiber crop is complicated by continued vegetative growth and asynchronous fruit set. Here, cotton CETS genes are phylogenetically and functionally characterized. We identified eight CETS genes in diploid cotton (G. raimondii and G. arboreum) and sixteen in tetraploid G. hirsutum that grouped within the three generally accepted CETS clades: flowering locus T (FT)-like, terminal flower1/self pruning (TFL1/SP)-like, and mother of FT and TFL1 (MFT)-like. Over-expression of single flower truss (GhSFT), the ortholog to Arabidopsis FT, accelerates the onset of flowering in Arabidopsis Col-0. In mutant rescue analysis, this gene driven by its native promoter rescues the ft-10 late flowering phenotype. GhSFT upstream sequence was used to drive expression of the uidA reporter gene. As anticipated, GUS accumulated in the vasculature of Arabidopsis leaves. Cotton has five TFL1-like genes, all of which delay flowering when ectopically expressed in Arabidopsis; the strongest phenotypes fail to produce functional flowers. Three of these genes, GhSP, GhTFL1-L2, and GhBFT-L2, rescue the early flowering tfl1-14 mutant phenotype. GhSPpro:uidA promoted GUS activity specifically in plant meristems; whereas, other GhTFL1-like promoters predominately drove GUS activities in plant vascular tissues. Finally, analysis of Gossypium CETS promoter sequences predicted that GhSFT, GhSP, GhTFL1-L1, GhTFL1-L2 and GhBFT-L2 are regulated by transcription factors involved in shoot and flowering development. Analysis of cotton's two MFT homologs indicated that neither gene functions to control shoot architecture. Our results emphasize the functional conservation of members of this gene family in flowering plants and also suggest this family as targets during artificial selection ...
Date: December 2017
Creator: Prewitt, Sarah F.
Partner: UNT Libraries

Identification of Genes Involved in Flocculation by Whole Genome Sequencing of Thauera aminoaromatica Strain MZ1T Floc-defective Mutants

Description: 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.
Date: December 2015
Creator: Prombutara, Pinidphon
Partner: UNT Libraries

Cytochrome P450 Gene Expression Modulates Anoxia Sensitivity in Caenorhabditis Elegans

Description: With an increasing population suffering from obesity or Diabetes Mellitus (DM), it is more pertinent than ever to understand how physiological changes impact cellular processes. Patients with DM often suffer from obesity, hyperglycemia, altered fatty acids that contribute to vascular dysfunction, and increased risk to ischemia. Caenorhabditis elegans is a model system used to study the conserved insulin signaling pathway, cellular responses in whole organisms and the impact a glucose diet has on oxygen deprivation (anoxia) responses. RNA-sequencing (RNA-Seq) was used to analyze the expression of genes in the anoxia sensitive populations of N2 (wild-type) fed glucose and hyl-2(tm2031), a mutant with altered ceramide metabolism. Comparison of the altered transcripts in the anoxia sensitive populations revealed 199 common transcripts- 192 upregulated and 7 downregulated. One of the gene families that have altered expression in the anoxia sensitive populations encode for Cytochrome P450 (CYP). CYPs are located both in the mitochondria and endoplasmic reticulum (ER), but the CYPs of interest are all predicted to be mainly subcellularly localized to the ER. Here, I determined that knock-down of specific cyp genes, using RNA interference (RNAi), increased anoxia survival in N2 animals fed a standard diet. Anoxia sensitivity of the hyl-2(tm2031) animals was supressed by RNAi of cyp-25A1 or cyp-33C8 genes. These studies provide evidence that the CYP detoxification system impacts oxygen deprivation responses. using hsp-4::GFP animals, a transcriptional reporter for ER unfolded protein response (UPR), I further investigated the impact of cyp knock-down, glucose, and anoxia on ER UPR due to the prediction of CYP-33C8 localization to the ER. Glucose significantly increased ER UPR and cyp knock-down non-significantly increased ER UPR. Measurements of ER UPR due to anoxia were made difficult, but representative images show an increase in ER stress post 9-hour anoxia exposure. This study provides evidence that glucose affects ER ...
Date: August 2016
Creator: Quan, Daniel L
Partner: UNT Libraries

Development of a Targeted Protein Residue Analysis Approach in Archaeology

Description: Liquid chromatography-mass spectrometry (LC-MS) based proteomic methods have provided archaeologists with a powerful tool for the discovery and identification of proteins within artifacts. Traditionally, discovery-based methods have utilized a non-targeted full mass scan method in an attempt to identify all proteins present within a given sample. However, increased sensitivity is often needed to target specific proteins in order to test hypotheses. Proteins present within archaeological materials present a unique challenge, as they are often subjected to a variety of chemical transformations both before and after burial. Any preserved proteins will be present within a complex mixture of compounds, and full mass scans often fail to detect less abundant proteins of interest. Consistent and reliable targeted methods are needed to detect protein biomarkers. Taphonomic experimentation was employed as a means to identify the effect of particular processes and conditions on the preservation of mare's milk proteins. In addition, three LC-MS methods were evaluated for their efficiency in identifying mare's milk-specific peptide biomarkers from experimental pottery samples. The ability to reliably detect the presence of these species-specific peptides can help provide evidence about past cultural groups, including the origins of dairying and animal domestication.
Date: August 2017
Creator: Scott, Ashley
Partner: UNT Libraries

Cloacal Microbiota of Captive-bred and Wild Attwater’s Prairie-chicken, Tympanuchus Cupido Attwateri

Description: The Attwater’s prairie-chicken (Tympanuchus cupido attwateri; APC) is a species of grouse native to Texas coastal prairies and is on the critically endangered species list as a result of habitat destruction and overhunting. All of the current populations were captively bred and released into the wild. Survivorship for released APCs is very low, and individuals seldom survive to reproduce in the wild. One factor contributing to this may be an alteration in the gut microbiota as a result of captivity. Factors potentially influencing the gut microbial composition in captivity include antibiotic therapy, stress, and a predominantly commercially formulated diet. Recent studies have begun to shed light on the importance of the host microbial endosymbionts. Antibiotic administration, stress, diet, age, genotype and other factors have been shown to influence microbial populations in the gastrointestinal tracts of many different vertebrates. Sequencing of 16S rRNA gene amplicons on the Ion Torrent™ platform was used in this study to identify groups of bacteria in the cloacas as a surrogate for the gut microbiota in the APC. Antibiotic-treated and untreated birds, wild-hatched and captive-bred birds, and individuals sampled before and after release to the wild were examined. Significant differences were found between wild-hatched and captive raised birds both pre- and post release. In addition, there was extensive variation among the populations at the lower taxonomic ranks between individuals for each group of APCs. Principal coordinate analysis based on the weighted UniFrac distance metric further exhibited some clustering of individuals by treatment. These data suggest that captive breeding may have long-term effects on the cloacal microbiota of APCs with unknown consequences to their long-term health and survivorship.
Date: August 2014
Creator: Simon, Stephanie E.
Partner: UNT Libraries