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 Department: Department of Biological Sciences
Characteristics of Primary Cilia and Centrosomes in Neuronal and Glial Lineages of the Adult Brain

Characteristics of Primary Cilia and Centrosomes in Neuronal and Glial Lineages of the Adult Brain

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Date: May 2015
Creator: Bhattarai, Samip Ram
Description: Primary cilia are sensory organelles that are important for initiating cell division in the brain, especially through sonic hedgehog (Shh) signaling. Several lines of evidence suggest that the mitogenic effect of Shh requires primary cilia. Proliferation initiated by Shh signaling plays key roles in brain development, in neurogenesis in the adult hippocampus, and in the generation of glial cells in response to cortical injury. In spite of the likely involvement of cilia in these events, little is known about their characteristics. Centrosomes, which are associated with primary cilia, also have multiple influences on the cell cycle, and they are important in assembling microtubules for the maintenance of the cell’s cytoskeleton and cilia. The cilia of terminally differentiated neurons have been previously examined with respect to length, incidence, and receptors present. However, almost nothing is known about primary cilia in stem cells, progenitors, or differentiated glial cells. Moreover, it is not known how the properties of cilia and centrosomes may vary with cell cycle or proliferative potential, in brain or other tissues. This dissertation focuses first on neurogenesis in the hippocampal subgranular zone (SGZ). The SGZ is one of the few brain regions in mammals that gives rise to a substantial ...
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Cytotoxicity and Functional Toxicity of Mefloquine and Search for Protective Compounds

Cytotoxicity and Functional Toxicity of Mefloquine and Search for Protective Compounds

Date: May 2015
Creator: Holmes, Katelyn
Description: Mefloquine hydrochloride is an antimalarial agent that has been used for the past 40 years. Numerous reports of neurological side effects have recently led the FDA to issue a strong warning regarding long-term neurological effects. This warning lead to the U.S. Army’s Special Forces and other components to discontinue its use in July of 2013. Despite reported adverse side effects, mefloquine remains in circulation and is recommended to travelers going to specific Asian countries. Mefloquine has been used as a treatment for those already infected with the malaria parasite (blood concentrations ranging from 2.1 to 23 µM), and as prophylaxis (blood concentrations averaging 3.8 µM) (Dow 2003). The purpose of this study was to quantify Mefloquine’s toxicity using spontaneously active nerve cell networks growing on microelectrode arrays in vitro and to identify compounds that alleviate or reduce toxic effects. The current literature on mefloquine toxicity is lacking electrophysiological data. These data will contribute to research on the mechanism of adverse side effects associated with mefloquine use. Sequential titration experiments were performed by adding increasing concentrations of mefloquine solution to cultured neurons. Network responses were quantified and reversibility was examined. In each network, activity decreases were normalized as a percent of ...
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The Effect of Curcumin Supplementation on Physical and Biological Indices of Delayed Onset Muscle Soreness and Inflammation Following Muscle Injury

The Effect of Curcumin Supplementation on Physical and Biological Indices of Delayed Onset Muscle Soreness and Inflammation Following Muscle Injury

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Date: May 2015
Creator: Venable, Adam Steven
Description: In this project, the effects of dietary polyphenols on exercise-induced muscle damage and vascular health are examined. Dietary polyphenols exert well-known anti-inflammatory effects; however, how these effects are realized with respect to vascular health and EIMD is relatively unknown. I begin by reviewing the available literature surrounding the impact of three dietary polyphenols (curcumin, catechins, and quercetin) on inflammation associated with EIMD. It is well established that their primary means of anti-inflammation is through alterations of NF-κB and AP-1 transcription activities. Given this, their inclusion into training strategies seems reasonable. Consistent evidence is presented making a case for the anti-inflammatory effects of dietary polyphenols following EIMD. I follow this review up by completing an in-depth study on the consumption of curcumin prior to EIMD. I found curcumin (1000 mg/day) can reduce subjective soreness and decrease inflammation compared to placebo controls. To further understand the effects of dietary polyphenols on health, I investigate the effects of a four-week supplementation period of cocoa (catechins) on vascular. I concluded that atherogenic risk in obese women is reduced after consumption of cocoa. In addition to these experimental projects, I developed two novel methods that can be used to investigate vascular health (EMP concentration) and ...
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Improving Processing Efficiency for Forensic DNA Samples

Improving Processing Efficiency for Forensic DNA Samples

Date: May 2015
Creator: Connon, Catherine Cupples
Description: The goal of this project was to reduce processing time for forensic DNA testing without incurring significant added costs and/or the need for new instrumentation, while still generating high quality profiles. This was accomplished by: 1) extraction normalization using the ChargeSwitch® Forensic DNA Purification Kit such that a small range of DNA concentrations was consistently obtained, eliminating the need for sample quantification and dilution; 2) developing fast PCR protocols for STR primer sets using shorter amplification methods, low volume reactions and non-fast thermal cyclers; and 3) developing a quicker 3130xl Genetic Analyzer detection method using an alternative polymer/array length combination. Extraction normalization was achieved through a reduction in bead quantity, thereby forcing an increase in bead binding efficiency. Four products (AmpliTaq Gold® Fast PCR Master Mix, KAPA2G™ Fast Multiplex PCR Kit, SpeedSTAR™ HS DNA Polymerase and Type-it Microsatellite PCR Kit) were evaluated for low volume (3μl) fast PCR on a 384-well Veriti® thermal cycler with the Identifiler primer set. KAPA2G™ was selected for 3μl fast PCR protocols using PowerPlex 16 HS and Identifiler Plus primer sets (42-51min), as well as 5μl and 6μl Identifiler fast reactions on a 9700 thermal cycler (51-60min). Alternative detection (POP-6™/22cm) achieved 24-28min run times, but ...
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Manipulations of Sucrose/proton Symporters and Proton-pumping Pyrophosphatase Lead to Enhanced Phloem Transport But Have Contrasting Effects on Plant Biomass

Manipulations of Sucrose/proton Symporters and Proton-pumping Pyrophosphatase Lead to Enhanced Phloem Transport But Have Contrasting Effects on Plant Biomass

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Date: May 2015
Creator: Khadilkar, Aswad S
Description: Delivery of photoassimilate, mainly sucrose (Suc) from photoautotrophic source leaves provides the substrate for the growth and maintenance of sink tissues such as roots, storage tissues, flowers and fruits, juvenile organs, and seeds. Phloem loading is the energized process of accumulating solute in the sieve element/companion cell complex of source leaf phloem to generate the hydrostatic pressure that drives long-distance transport. In many plants this is catalyzed by Suc/Proton (H+) symporters (SUTs) which are energized by the proton motive force (PMF). Overexpression of SUTs was tested as means to enhance phloem transport and plant productivity. Phloem specific overexpression of AtSUC2 in wild type (WT) tobacco resulted in enhanced Suc loading and transport, but against the hypothesis, plants were stunted and accumulated carbohydrates in the leaves, possibly due to lack of sufficient energy to support enhanced phloem transport. The energy for SUT mediated phloem loading is provided from the PMF, which is ultimately supplied by the oxidation of a small proportion of the loaded photoassimilates. It was previously shown that inorganic pyrophosphate (PPi) is necessary for this oxidation and overexpressing a proton-pumping pyrophosphatase (AVP1) enhanced both shoot and root growth, and augmented several energized processes like nutrient acquisition and stress responses. ...
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The Microbial Retting Environment of Hibiscus Cannabinus and Its Implications in Broader Applications

The Microbial Retting Environment of Hibiscus Cannabinus and Its Implications in Broader Applications

Date: May 2015
Creator: Visi, David K.
Description: Fiber-yielding plants is an area of increased interest due to the potential use in a variety of green-based materials. These biocomposites can be incorporated into multiple uses; for example, to replace building materials and interior vehicular paneling. The research here aims to focus in on the crop Hibiscus cannabinus for utilization into these functions. H. cannabinus is economically attractive due to the entire process being able to be accomplished here in the United States. The plant can be grown in a relatively short growth period (120-180 days), and then processed and incorporated in a biocomposite. The plant fiber must first be broken down into a useable medium. This is accomplished by the retting process, which occurs when microbial constituents breakdown the heteropolysaccharides releasing the fiber. The research aims to bridge the gap between the primitive process of retting and current techniques in molecular and microbiology. Utilizing a classical microbiological approach, which entailed enrichment and isolation of pectinase-producing bacteria for downstream use in augmented microbial retting experiments. The tracking of the bacteria was accomplished by using the 16S rRNA which acts as “barcodes” for bacteria. Next-generation sequencing can then provide data from each environment telling the composition and microbial diversity of ...
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Neurotoxicity of the Industrial Solvent 4-Methylcyclohexanemethanol: Involvement of the GABA Receptor

Neurotoxicity of the Industrial Solvent 4-Methylcyclohexanemethanol: Involvement of the GABA Receptor

Date: May 2015
Creator: Gibson, Jason
Description: A recent chemical spill of 4-Methylcyclohexanemethanol (4-MCHM) in West Virginia left 300,000 people without water. Officials claimed that this compound is not lethally toxic, but potentially harmful if swallowed or inhaled, and can cause eye and skin irritation. Sittig's Handbook of Toxic and Hazardous Chemical Carcinogens reports high exposures from skin contact or inhalation may cause damage to the heart, liver, kidneys, and lungs, and may result in death. However, no quantitative data seem to exist and no references can be found on neurotoxicity. We have investigated the neurotoxicity of 4-MCHM using mammalian nerve cell networks grown on microelectrode arrays. Network spontaneous activity from multiple units (range 48 – 120 per network) were used as the primary readout. Individual units were followed based on spike waveforms digitized at 40 kHz (Plexon MNAP system). Dose response curves show the effective inhibitory concentration at 50 percent decrease (EC50) to average 27.4 microM SD±6.17. However, in the presence of 40 microM bicuculline, a competitive GABAA antagonist, the EC50 shifts to 70.63uM SD ±4.3; implying that early, low concentration exposures to 4-MCHM involve GABA activation. Initial activity loss occurs without active unit loss (defined as 10 or more template threshold crossing per min), indicating ...
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Restoration Techniques for Northern Bobwhites

Restoration Techniques for Northern Bobwhites

Date: May 2015
Creator: Newman, William L
Description: Isolated populations of northern bobwhites (Colinus virginianus) have declined causing many quail managers to attempt population restoration by releasing captive-reared bobwhites or translocating wild bobwhites. I evaluated three restoration techniques: (1) release of captive-reared bobwhites, (2) translocation of bobwhites from high densities to low densities, and (3) release of captive-reared and translocated bobwhites acclimated on site prior to release. These results show that captive-reared birds have reduced survival and fewer nesting attempts when compared to translocated birds and that acclimation time was not a factor. I hypothesized that high mortality rates were caused by captive-reared birds exhibiting different predator avoidance behavior than wild birds. Captive-reared and wild-trapped bobwhites were subjected to independent predator simulations and their responses were recorded on high definition video. Threat recognition time, reaction type, and reaction time was recorded for comparative analysis. Pen-reared birds recognized the simulated raptorial and terrestrial predator threats quicker than wild-trapped birds, but reaction times were not different among groups. However, the type of reaction was different among groups where pen-reared birds typically flushed immediately upon recognizing either simulated predator as compared to wild-trapped birds which typically ran or held when subjected to the raptorial threat and showed little to no observable ...
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Stress Response by Alternative Σ-factor, Rpoh, and Analysis of Posttranslational Modification of the Heat Shock Protein, Dnak, in Escherichia Coli

Stress Response by Alternative Σ-factor, Rpoh, and Analysis of Posttranslational Modification of the Heat Shock Protein, Dnak, in Escherichia Coli

Date: May 2015
Creator: Martinez, Sarah N
Description: Bacteria have developed specialized responses that involve the expression of particular genes present in a given regulon. Sigma factors provide regulatory mechanisms to respond to stress by acting as transcriptional initiation factors. This work focuses on σ32 during oxidative stress in Escherichia coli. The differential response of key heat shock (HS) genes was investigated during HS and oxidative stress using qPCR techniques. While groEL and dnaJ experienced increases in transcriptional response to H2O2 (10 mM), HS (42°C), and paraquat (50 mM) exposure, the abundance of dnaK over the co-chaperones was apparent. It was hypothesized that DnaK undergoes oxidative modification by reactive carbonyls at its Lys-rich C-terminus, accounting for the differential response during oxidative stress. A σ32-mediated β-galactosidase reporter was devised to detect the activity of wild-type DnaK and DnaKV634X modified to lack the Lys-rich C-terminus. Under unstressed conditions and HS, σ32 was bound at the same rate in both strains. When subjected to H2O2, the WT DnaK strain produced significantly higher β-galactosidase than DnaKV634X (one-tailed Student’s t test p=0.000002, α=0.05) and approached the same level of output as the lacZ positive control. The β-galactosidase assay indicates that DnaK undergoes Lys modification in the WT strain, preventing the protein from binding ...
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Understanding Microbial Biodegradation of Environmental Contaminants

Understanding Microbial Biodegradation of Environmental Contaminants

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Date: May 2015
Creator: Vilo Muñoz, Claudia Andrea
Description: The accumulation of industrial contaminants in the natural environments have rapidly become a serious threat for human and animal life. Fortunately, there are microorganisms capable of degrading or transforming environmental contaminants. The present dissertation work aimed to understand the genomic basis of microbial degradation and resistance. The focus was the genomic study of the following bacteria: a) Pseudomonas fluorescens NCIMB 11764, a unique bacterium with specific enzymes that allow cyanide adaptation features. Potential cyanide degradation mechanisms found in this strain included nit1C cluster, and CNO complex. Potential cyanide tolerance genes found included cyanide insensitive oxidases, nitric oxide producing gene, and iron metabolism genes. b) Cupriavidus sp. strain SK-3 and strain SK-4. The genome of both bacteria presented the bph operon for polychlorinated biphenyl (PCB) degradation, but we found differences in the sequences of the genes. Those differences might indicate their preferences for different PCB substrates. c) Arsenic resistant bacterial communities observed in the Atacama Desert. Specific bacteria were found to thrive depending on the arsenic concentration. Examples were Bacteroidetes and Spirochaetes phyla whose proportions increased in the river with high arsenic concentrations. Also, DNA repair and replication metabolic functions seem to be necessary for resistance to arsenic contaminated environments. Our ...
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