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Corbicula fluminea Invasion as a Secondary Effect of Hydrilla verticillata Management via Triploid Grass Carp (Ctenopharyngodon idella) (open access)

Corbicula fluminea Invasion as a Secondary Effect of Hydrilla verticillata Management via Triploid Grass Carp (Ctenopharyngodon idella)

A study of Asian clam (Corbicula fluminea Müller) colonization in relation to changes in aquatic vegetation community as a result of management of Hydrilla verticillata (L. f.) Royle with grass carp was conducted at the Lewisville Aquatic Ecosystem Research Facility (LAERF), Lewisville, TX, from April 2015 through October 2016. Percent vegetation cover, C. fluminea abundance and water quality metrics (pH, turbidity, conductivity, DO, calcium, chlorophyll a) from 16 experimental subjects were analyzed. Treatments included four replicated grass carp stocking densities; 1-control with no fish stocked (n = 4), 2-low density of 40-43 fish per vegetated ha (n = 4), 3-medium density of 72-81 fish per vegetated ha (n = 4) and 4-high density of 110-129 fish per vegetated ha (n = 4). Data analysis showed statistical significance in the relation of C. fluminea abundance to percent vegetation cover (multiple linear regression, r2 = 0.820), grass carp stocking densities (two-way analysis of variance, p = <0.001) and chlorophyll a (multiple linear regression, r2 = 0.339). Findings of this research indicate the possibility that management of hydrilla had enabled establishment of secondary invasive species.
Date: December 2017
Creator: Holbrook, David Lee
System: The UNT Digital Library
The Influence of Disease Mapping Methods on Spatial Patterns and Neighborhood Characteristics for Health Risk (open access)

The Influence of Disease Mapping Methods on Spatial Patterns and Neighborhood Characteristics for Health Risk

This thesis addresses three interrelated challenges of disease mapping and contributes a new approach for improving visualization of disease burdens to enhance disease surveillance systems. First, it determines an appropriate threshold choice (smoothing parameter) for the adaptive kernel density estimation (KDE) in disease mapping. The results show that the appropriate threshold value depends on the characteristics of data, and bandwidth selector algorithms can be used to guide such decisions about mapping parameters. Similar approaches are recommended for map-makers who are faced with decisions about choosing threshold values for their own data. This can facilitate threshold selection. Second, the study evaluates the relative performance of the adaptive KDE and spatial empirical Bayes for disease mapping. The results reveal that while the estimated rates at the state level computed from both methods are identical, those at the zip code level are slightly different. These findings indicate that using either the adaptive KDE or spatial empirical Bayes method to map disease in urban areas may provide identical rate estimates, but caution is necessary when mapping diseases in non-urban (sparsely populated) areas. This study contributes insights on the relative performance in terms of accuracy of visual representation and associated limitations. Lastly, the study contributes …
Date: December 2017
Creator: Ruckthongsook, Warangkana
System: The UNT Digital Library
Phylogenetic and Functional Characterization of Cotton (Gossypium hirsutum) CENTRORADIALIS/TERMINAL FLOWER1/SELF-PRUNING Genes (open access)

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

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 …
Date: December 2017
Creator: Prewitt, Sarah F.
System: The UNT Digital Library
The Generation of Recombinant Zea mays Spastin and Katanin Proteins for In Vitro Analysis (open access)

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

Plant microtubules play essential roles in cell processes such as cell division, cell elongation, and organelle organization. Microtubules are arranged in highly dynamic and ordered arrays, but unlike animal cells, plant cells lack centrosomes. Therefore, microtubule nucleation and organization are governed by microtubule-associated proteins, including a microtubule-severing protein, katanin. Mutant analysis and in vitro characterization has shown that the highly conserved katanin is needed for the organization of the microtubule arrays in Arabidopsis and rice as well as in a variety of animal models. Katanin is a protein complex that is part of the AAA+ family of ATPases. Katanin is composed of two subunits, katanin-p60, a catalytic subunit and katanin-p80, a regulatory subunit. Spastin is another MT-severing protein that was identified on the basis of its homology to katanin. In animal cells, spastin is also needed for microtubule organization, but its functionality has not yet been investigated in plants. To initiate an exploration of the function of katanin-p60 and spastin in Zea mays, my research goal was to generate tools for the expression and purification of maize katanin-p60 and spastin proteins in vitro. Plasmids that express katanin-p60 and spastin with N-terminal GST tags were designed and constructed via In-Fusion® cloning …
Date: December 2017
Creator: Alodailah, Sattam Sonitan
System: The UNT Digital Library
Stability of Myosin Subfragment-2 Modulates the Force Produced by Acto-Myosin Interaction of Striated Muscle (open access)

Stability of Myosin Subfragment-2 Modulates the Force Produced by Acto-Myosin Interaction of Striated Muscle

Myosin subfragment-2 (S2) is a coiled coil linker between myosin subfragment-1 and light meromyosin (LMM). This dissertation examines whether the myosin S2 coiled coil could regulate the amount of myosin S1 heads available to bind actin thin filaments by modulating the stability of its coiled coil. A stable myosin S2 coiled coil would have less active myosin S1 heads compared to a more flexible myosin S2 coiled coil, thus causing increased force production through acto-myosin interaction. The stability of the myosin S2 coiled coil was modulated by the binding of a natural myosin S2 binding protein, myosin binding protein C (MyBPC), and synthetic myosin S2 binding proteins, stabilizer and destabilizer peptide, to myosin S2. Competitive enzyme linked immunosorbent assay (cELISA) experiments revealed the cross specificity and high binding affinity of the synthetic peptides to the myosin S2 of human cardiac and rabbit skeletal origins. Gravitational force spectroscopy (GFS) was performed to test the stability of myosin S2 coiled coil in the presence of these myosin S2 binding proteins. GFS experiments demonstrated the stabilization of the myosin S2 coiled coil by the binding of MyBPC and stabilizer peptide to myosin S2, while the binding of destabilizer peptide to the same resulted …
Date: December 2017
Creator: Singh, Rohit Rajendraprasad
System: The UNT Digital Library
Reproduction and Metabolic Responses to Acute and Chronic Hypoxia in Ovoviviparous Blaberid Cockroaches, with a Focus on Blaptica dubia (open access)

Reproduction and Metabolic Responses to Acute and Chronic Hypoxia in Ovoviviparous Blaberid Cockroaches, with a Focus on Blaptica dubia

The major components of the tracheal system of insects are an extension of the exoskeleton, and the size of the exoskeleton is fixed in the adult stage, so any increase in metabolic demand that may accompany reproduction must be met by a relatively unaltered tracheal system that the female set in place at ecdysis, when entering adulthood. Acute hypoxia tends to elicit an increase in ventilation in insects, and here, I observe increased interburst VCO2 release, and a tendency towards a more continuous gas exchange pattern being preferred over discontinuous gas exchange when Blaptica dubia and Eublaberus posticus are exposed to a descending regime of hypoxia. Additionally, higher temperatures appear to increase sensitivity to hypoxia in these species, an expected result because both species, like most ectothermic animals, display a Q10 effect, increasing metabolic rates as temperature increases. The reproductive mode of B. dubia is considered to be lecithotrophic pseudoviviparity (or type A ovoviviparity), and by the time the embryos are born, they have more than doubles in volume from the time of oviposition. This gain is apparent in the wet mass of the embryo, with no change occurring in dry mass. The egg mass that can be attributed to …
Date: August 2017
Creator: Mallery, Christopher Sean
System: The UNT Digital Library
Development of a Targeted Protein Residue Analysis Approach in Archaeology (open access)

Development of a Targeted Protein Residue Analysis Approach in Archaeology

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
System: The UNT Digital Library
The Acute Toxic Effects of the Synthetic Cannabinoid, JWH-018 on the Cardiovascular and Neuroendocrine Systems in Ictalurus punctatus (Channel Catfish) (open access)

The Acute Toxic Effects of the Synthetic Cannabinoid, JWH-018 on the Cardiovascular and Neuroendocrine Systems in Ictalurus punctatus (Channel Catfish)

Cannabinoid (CB) receptors have been found in most vertebrates that have been studied. The location of various CB receptors in the body and brain are known, but their physiological functions are not fully understood. The effects CBs have on the cardiovascular system have been of growing interest in recent years. Increasing reports from emergency departments and law enforcement agencies detail acute cardiovascular and psychological effects from synthetic CB intoxication, such as JWH-018. This major health concern is substantiated by governmental agencies like the CDC and NIDA. This pilot study investigates the acute toxic effects of the synthetic CB, JWH-018, on the cardiovascular and neuroendocrine systems in Ictalurus punctatus (channel catfish). Research in organisms besides the traditional mammal models can provide new insights into CB function and physiology. Ictalurus punctatus lend multiple benefits as a model organism that permits researchers to investigate in vivo effects of both cardiovascular and neuroendocrine systems without much influence from traditional sampling methods, and further more provide ample size and tissue to perform specific cardiovascular experiments. Multiple methods were used to assess cardiovascular function and sympathetic nervous system activation. Two different doses, low (500 µg/kg) and high 1,500 µg/kg, of JWH-018 were evaluated in the study. …
Date: August 2017
Creator: Taylor, Dedric Esmond
System: The UNT Digital Library
Evaluating the Role of UV Exposure and Recovery Regimes in PAH Photo-Induced Toxicity to Daphina Magna (open access)

Evaluating the Role of UV Exposure and Recovery Regimes in PAH Photo-Induced Toxicity to Daphina Magna

Polyaromatic hydrocarbons (PAHs) are contaminants synthesized through incomplete combustion of carbon based substances. PAHs are known to be photodynamic and toxicity increases exponentially when in contact with ultraviolet radiation (UV). The effect of UV absent recovery periods and potential for latent toxicity during photo-induced toxicity are previously unknown and are not included within the toxicity model. Results of equal interval tests further support the current reciprocity model as a good indicator of PAH photo-induced toxicity. Interval test results also indicate a possible presence of time-dependent toxicity and recovery thresholds and should be included into toxicity risk assessments. Moreover, results of latent effects assays show that latent mortality is a significant response to PAH photo-induced toxicity and should be included into toxicity risk assessments. The present research demonstrates that UV exposure time rate is a significant driving force of PAH photo-induced toxicity.
Date: August 2017
Creator: Gnau, Jennifer Leigh
System: The UNT Digital Library
Neurological Responses to a Glucose Diet in Caenorhabditis elegans (open access)

Neurological Responses to a Glucose Diet in Caenorhabditis elegans

TRPV channels play a role in both mammalian insulin signaling, with TRPV1 expression in pancreatic beta-cells, and in C. elegans insulin-like signaling through expression of OSM-9, OCR-1, and OCR-2 in stress response pathways. In response to a glucose-supplemented diet, C. elegans are know to have sensitivity to anoxic stress, exhibit chemotaxis attraction, and display reduced egg-laying rate. Transcriptome analysis reveals that glucose stimulates nervous system activity with increased transcript levels of genes regulating neurotransmitters. Ciliated sensory neurons are needed for a reduced egg-laying phenotype on a glucose-supplemented diet. Egg-laying rate is not affected when worms graze on glucose-supplemented Delta-PTS OP50 E. coli, which is defective in glucose uptake. This suggests a possible sensory neuron obstruction by exopolysaccharides produced by standard OP50 E. coli on glucose, eliciting a starvation response from the worm and causing reduced egg-laying rate. Glucose chemotaxis is affected in specific TRPV subunit allele mutants: ocr-2(vs29) and osm-9(yz6), serotonin receptor mutants: ser-1(ok345) and mod-1(ok103), and G-alpha protein mutant: gpa-10(pk362). TRPV deletion mutants had no effect on glucose chemotaxis, alluding to the modality role pf TRPV alleles in specific sensory neurons. The role of serotonin in a reduced egg-laying rate with glucose remains unclear.
Date: August 2017
Creator: Dumesnil, Dennis
System: The UNT Digital Library
Generating Molecular Biology Tools to Investigate the Ca2+ Binding Ability of Arabidopsis TON2 (open access)

Generating Molecular Biology Tools to Investigate the Ca2+ Binding Ability of Arabidopsis TON2

The position of the cell division plane in plants is determined by the position of the preprophase band. The pre prophase band (PPB) is a ring of microtubules centered around the nucleus on the inner side of plasma membrane that establishes the cortical division site. The PPB forms at the end of G2 and breaks down at the end of prophase leaving behind protein markers of its position that are collectively called the cortical division site. During cytokinesis the phragmoplast expands towards the cortical division site and mediates the fusion of the new cell plate with the mother cell at that position. Several proteins necessary for PPB formation in plants have been identified, including maize DCD1 and ADD1 and Arabidopsis TON2, which are all type 2A protein phosphatase (PP2A)B" regulatory subunits. DCD1, ADD1, and TON2 localize to the PPB and the cortical division site through metaphase. The PP2A subunits each have two EF-hand domains, which are predicted to bind calcium ions. Since calcium ions are important for some aspects of cell division, we designed a series of constructs to test if TON2 binds calcium. TON2 protein was cloned into expression vectors, pET42a, and expression of TON2 protein was confirmed via …
Date: August 2017
Creator: Shao, Danyang
System: The UNT Digital Library
The Effects of Air Pollution on the Intestinal Microbiota: A Novel Approach to Assess How Gut Microbe Interactions with the Environment Affect Human Health (open access)

The Effects of Air Pollution on the Intestinal Microbiota: A Novel Approach to Assess How Gut Microbe Interactions with the Environment Affect Human Health

This thesis investigates how air pollution, both natural and anthropogenic, affects changes in the proximal small intestine and ileum microbiota profile, as well as intestinal barrier integrity, histological changes, and inflammation. APO-E KO mice on a high fat diet were randomly selected to be exposed by whole body inhalation to either wood smoke (WS) or mixed vehicular exhaust (MVE), with filtered air (FA) acting as the control. Intestinal integrity and histology were assessed by observing expression of well- known structural components tight junction proteins (TJPs), matrix metallopeptidase-9 (MMP-9), and gel-forming mucin (MUC2), as well known inflammatory related factors: TNF-α, IL-1β, and toll-like receptor (TLR)-4. Bacterial profiling was done using DNA analysis of microbiota within the ileum, utilizing 16S metagenomics sequencing (Illumina miSeq) technique. Overall results of this experiment suggest that air pollution, both anthropogenic and natural, cause a breach in the intestinal barrier with an increase in inflammatory factors and a decrease in beneficial bacteria. This evidence suggests the possibility of air pollution being a potential causative agent of intestinal disease as well as a possible contributing mechanism for induction of systemic inflammation.
Date: May 2017
Creator: Fitch, Megan N.
System: The UNT Digital Library
Delivery of CRISPR/Cas9 RNAs into Blood Cells of Zebrafish: Potential for Genome Editing in Somatic Cells (open access)

Delivery of CRISPR/Cas9 RNAs into Blood Cells of Zebrafish: Potential for Genome Editing in Somatic Cells

Factor VIII is a clotting factor found on the intrinsic side of the coagulation cascade. A mutation in the factor VIII gene causes the disease Hemophilia A, for which there is no cure. The most common treatment is administration of recombinant factor VIII. However, this can cause an immune response that renders the treatment ineffective in certain hemophilia patients. For this reason a new treatment, or cure, needs to be developed. Gene editing is one solution to correcting the factor VIII mutation. CRISPR/Cas9 mediated gene editing introduces a double stranded break in the genomic DNA. Where this break occurs repair mechanisms cause insertions and deletions, or if a template oligonucleotide can be provided point mutations could be introduced or corrected. However, to accomplish this goal for editing factor VIII mutations, a way to deliver the components of CRISPR/Cas9 into somatic cells is needed. In this study, I confirmed that the CRISPR/Cas9 system was able to create a mutation in the factor VIII gene in zebrafish. I also showed that the components of CRISPR/Cas9 could be piggybacked by vivo morpholino into a variety of blood cells. This study also confirmed that the vivo morpholino did not interfere with the gRNA binding …
Date: August 2017
Creator: Schneider, Sara Jane
System: The UNT Digital Library
The Role of Thyroid Hormone across Avian Development Spectrum: Investigations on Systemic Development, Metabolism and Ontogeny of Endothermy (open access)

The Role of Thyroid Hormone across Avian Development Spectrum: Investigations on Systemic Development, Metabolism and Ontogeny of Endothermy

Achievement of endothernic capacity is vital for independence from ambient temperature changes, sustained activity, optimal biochemical reactions and optimization of parental care. During early avian development, the core tenets of transition from ectothermy to endothermy are development of metabolic capacity (oxygen consumption, mitochondrial bioenergetics), enhanced cardiovascular function (heart rate and cardiac output), pulmonary ventilation and thermogenic capacity. Thyroid hormones, particularly T3, are key metabolic regulators of basal metabolism, thermogenesis, pulmonary ventilation and mitochondrial respiration. Thyroid hormone fluctuation patterns during both precocial and altricial avian endothermic transition suggest a prominent role in maturation of endothermy, cardiovascular, respiratory and skeletal muscle physiology. This body of work explores effects of T3 manipulations in two avian species: the precocial Pekin duck and the altricial Red-winged Blackbird. Increased plasma T3 during late incubation resulted in increased cardiac mass, elevated resting and intrinsic heart rate, intrinsic mean arterial pressure, increased cholinergic tone and blunted alpha-adrenergic tone in the precocial Pekin duck. In both Pekin duck and Red-winged blackbird, plasma T3 levels correlated with changes in the trajectory of endothermic ontogeny, systemic oxygen consumption, thermogenesis, maturation of pulmonary ventilatory function, altered growth and effects on skeletal and cardiac mitochondrial bioenergetics. These observations support the role of thyroid …
Date: August 2017
Creator: Sirsat, Tushar S
System: The UNT Digital Library
Phenotypic Analysis of Medicago truncatula NPF1.7 Over-Expressing Plants Grown under Different Nitrate Conditions (open access)

Phenotypic Analysis of Medicago truncatula NPF1.7 Over-Expressing Plants Grown under Different Nitrate Conditions

Plants have many nitrate transporters; in the model legume Medicago truncatula, MtNPF1.7 is among them. MtNPF1.7 is important for M. truncatula growth and it has been established that MtNPF1.7 is a high affinity nitrate transporter. M. truncatula plants with mutations in MtNPF1.7 gene show defects during plants growth, with striking abnormalities in nodule development and root architecture. Nitrogen fixation is an energy expensive process; when legumes have sufficient bioavailable nitrogen like nitrate available, it suppresses nodulation and nitrogen fixation. Previous preliminary results in our lab showed that plants constitutively expressing MtNPF1.7 have a growth phenotype in the absence of nitrate, but no data was available on how M. truncatula plants constitutively expressing MtNPF1.7 are affected by the presence of nitrate. For my research, I confirmed the preliminary results on the growth of M. truncatula plants overexpressing NPF1.7 and examined these plants' phenotypes when nitrate was not provided in the growth media and when it was provided at two different concentrations. Compared with wild type A17, plants constitutively expressing MtNPF1.7 gene grow larger, have more lateral roots and more nodules when grown in the absence of nitrate and when 0.2 mM KNO3 was provided. At 1 mM KNO3, there are fewer …
Date: December 2017
Creator: Cai, Jingya
System: The UNT Digital Library
Homologs of Mammalian Lysosomal Lipase in Arabidopsis and Their Roles in Lipid Droplet Dynamics (open access)

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 …
Date: December 2017
Creator: McClinchie, Elizabeth A
System: The UNT Digital Library
Niche Expansion of an Invasive Predator (Neovison vison), Prey Response, and Facilitative Interactions with Other Invasive Mammals at the Southern End of the Americas: Conservation Challenges and Potential Solutions (open access)

Niche Expansion of an Invasive Predator (Neovison vison), Prey Response, and Facilitative Interactions with Other Invasive Mammals at the Southern End of the Americas: Conservation Challenges and Potential Solutions

The Cape Horn Biosphere Reserve is located at the southern tip of South America. This large archipelago is considered one of the last pristine areas left on the world. Despite it being an unpopulated area with most of the native forest cover intact, it has not been exempt from biological invasions, one of the major drivers of biodiversity loss. Three species that naturally interact in their native range in North America – American beavers (Castor canadensis), muskrats (Ondatra zibethicus), and American mink (Neovison vison) – were independently introduced in this remote region. In my dissertation, I investigated (i) the hypothesis of niche expansion in the invasive mink population on Navarino Island towards terrestrial habitats; (ii) potential mink impact on breeding success of forest-bird populations; (iii) habitat selection of small-rodent species and their perception on the mink's novel predation risk; and (iv) the dynamics of multiple-species invasions under the hypothesis of an invasional meltdown. Additionally, I worked within the framework of environmental philosophy. I provide an example of combining ecological and cultural dimensions within the International Long-Term Ecological Research network to disentangle the ethical dilemmas that surround the management of invasive species. I finally proposed a management plan based on the …
Date: December 2017
Creator: Crego, Ramiro Daniel
System: The UNT Digital Library
Development of von Willebrand Factor Zebrafish Mutant Using CRISPR/Cas9 Mediated Genome Editing (open access)

Development of von Willebrand Factor Zebrafish Mutant Using CRISPR/Cas9 Mediated Genome Editing

von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.
Date: May 2017
Creator: Toffessi Tcheuyap, Vanina
System: The UNT Digital Library
Adrenergic and Cholinergic Regulation of Cardiovascular Function in Embryonic Neotropic Cormorants (Phalacrocorax basilianus) (open access)

Adrenergic and Cholinergic Regulation of Cardiovascular Function in Embryonic Neotropic Cormorants (Phalacrocorax basilianus)

Investigations of cholinergic and adrenergic tone on heart rate (fH) and mean arterial pressure (Pm) during embryonic development have been conducted on numerous avian species. While these investigations have documented that adrenergic tone, a continuous stimulation, on fH and Pm is vital to embryonic development in the birds studied to date, development of cholinergic tone on fH has been shown to vary even within species. Further, past studies have been bias to focus primarily on precocial species while altricial species remain poorly understood in this context. The goal of this investigation was to investigate the role of cholinergic and adrenergic tone on fH and Pm of an altricial species, the neotropic cormorant (P. brasilianus) to address this bias. The embryonic neotropic cormorant possesses B-and-a adrenergic tone on fH and Pm at 70% and 90% incubation while cholinergic tone on fH occurs at 90% incubation. This pattern of control is similar to that previously reported for several species of precocial birds suggesting the development of tonic cardiovascular regulation may be conserved across avian taxa.
Date: May 2017
Creator: Cummins, James B.
System: The UNT Digital Library
Studies on Zebrafish Thrombocyte Function (open access)

Studies on Zebrafish Thrombocyte Function

Thrombocytes are important players in hemostasis. There is still much to be explored regarding the molecular basis of the thrombocyte function. In our previous microarray analysis data, we found IFT122 (an intraflagellar transport protein known to be involved in cilia formation) transcripts in zebrafish thrombocytes. Given recent discoveries of non-ciliary roles for IFTs, we examined the possibility that IFT122 affects thrombocyte function. We studied the role of IFT122 in thrombocyte function. We also found that IFT122 plays a central role in thrombocyte activation initiated by the agonists ADP, collagen, PAR-1 peptide and epinephrine. Although the receptors for ADP, PAR-1 peptide and epinephrine are present in the zebrafish genome, the collagen receptor GPVI was missing. In this study, we identified G6fL as a collagen receptor in zebrafish thrombocytes. Furthermore, IFT knockdown results in reduction in Wnt signaling. The Wnt signaling has been shown to be involved in megakaryocyte proliferation and proplatelets production. Therefore, defects in IFT could lead to thrombocytopenia. Splenectomy is performed in humans to treat such conditions. Therefore, in this study we developed a survival surgery protocol for splenectomy. We have shown that number of thrombocytes and their microparticles increase following splenectomy in zebrafish. Thus overall the studies on …
Date: May 2017
Creator: Pulipakkam Radhakrishnan, Uvaraj
System: The UNT Digital Library
Markov Model of Segmentation and Clustering: Applications in Deciphering Genomes and Metagenomes (open access)

Markov Model of Segmentation and Clustering: Applications in Deciphering Genomes and Metagenomes

Rapidly accumulating genomic data as a result of high-throughput sequencing has necessitated development of efficient computational methods to decode the biological information underlying these data. DNA composition varies across structurally or functionally different regions of a genome as well as those of distinct evolutionary origins. We adapted an integrative framework that combines a top-down, recursive segmentation algorithm with a bottom-up, agglomerative clustering algorithm to decipher compositionally distinct regions in genomes. The recursive segmentation procedure entails fragmenting a genome into compositionally distinct segments within a statistical hypothesis testing framework. This is followed by an agglomerative clustering procedure to group compositionally similar segments within the same framework. One of our main objectives was to decipher distinctive evolutionary patterns in sex chromosomes via unraveling the underlying compositional heterogeneity. Application of this approach to the human X-chromosome provided novel insights into the stratification of the X chromosome as a consequence of punctuated recombination suppressions between the X and Y from the distal long arm to the distal short arm. Novel "evolutionary strata" were identified particularly in the X conserved region (XCR) that is not amenable to the X-Y comparative analysis due to massive loss of the Y gametologs following recombination cessation. Our compositional based …
Date: August 2017
Creator: Pandey, Ravi Shanker
System: The UNT Digital Library
Developing a Phylogeny Based Machine Learning Algorithm for Metagenomics (open access)

Developing a Phylogeny Based Machine Learning Algorithm for Metagenomics

Metagenomics is the study of the totality of the complete genetic elements discovered from a defined environment. Different from traditional microbiology study, which only analyzes a small percent of microbes that could survive in laboratory, metagenomics allows researchers to get entire genetic information from all the samples in the communities. So metagenomics enables understanding of the target environments and the hidden relationships between bacteria and diseases. In order to efficiently analyze the metagenomics data, cutting-edge technologies for analyzing the relationships among microbes and communities are required. To overcome the challenges brought by rapid growth in metagenomics datasets, advances in novel methodologies for interpreting metagenomics data are clearly needed. The first two chapters of this dissertation summarize and compare the widely-used methods in metagenomics and integrate these methods into pipelines. Properly analyzing metagenomics data requires a variety of bioinformatcis and statistical approaches to deal with different situations. The raw reads from sequencing centers need to be processed and denoised by several steps and then be further interpreted by ecological and statistical analysis. So understanding these algorithms and combining different approaches could potentially reduce the influence of noises and biases at different steps. And an efficient and accurate pipeline is important to …
Date: August 2017
Creator: Rong, Ruichen
System: The UNT Digital Library
Investigating Human Gut Microbiome in Obesity with Machine Learning Methods (open access)

Investigating Human Gut Microbiome in Obesity with Machine Learning Methods

Obesity is a common disease among all ages that has threatened human health and has become a global concern. Gut microbiota can affect human metabolism and thus may modulate obesity. Certain mixes of gut microbiota can protect the host to be healthy or predispose the host to obesity. Modern next-generation sequencing technique allows accessing huge amount of genetic information underlying microbiota and thus provides new insights into the functionality of these micro-organisms and their interactions with the host. Multiple previous studies have demonstrated that the microbiome might contribute to obesity by increasing dietary energy harvest, promoting fat deposition and triggering systemic inflammation. However, these researches are either based on lab cultivation studies or basic statistical analysis. In order to further explore how gut microbiota affect obesity, this thesis utilize a series of machine learning methods to analyze large amount of metagenomics data from human gut microbiome. The publicly available HMP (Human Microbiome Project) metagenomic sequencing data, contain microbiome data for healthy adults, including overweight and obese individuals, were used for this study. HMP gut data were organized based on two different feature definitions: taxonomic information and metabolic reconstruction information. Several widely used classification algorithms: namely Naive Bayes, Random Forest, SVM …
Date: August 2017
Creator: Zhong, Yuqing
System: The UNT Digital Library