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Glucose and Altered Ceramide Biosynthesis Impact the Transcriptome and the Lipidome of Caenorhabditis elegans

Description: The worldwide rise of diabetes and obesity has spurred research investigating the molecular mechanisms that mediate the deleterious effects associated with these diseases. Individuals with diabetes and/or obesity are at increased risk from a variety of health consequences, including heart attack, stroke and peripheral vascular disease; all of these complications have oxygen deprivation as the central component of their pathology. The nematode Caenorhabditis elegans has been established as a model system for understanding the genetic and molecular regulation of oxygen deprivation response, and in recent years methods have been developed to study the effects of excess glucose and altered lipid homeostasis. Using C. elegans, I investigated transcriptomic profiles of wild-type and hyl-2(tm2031) ( a ceramide biosynthesis mutant) animals fed a standard or a glucose supplemented diet. I then completed a pilot RNAi screen of differentially regulated genes and found that genes involved in the endobiotic detoxification pathway (ugt-63 and cyp-25A1) modulate anoxia response. I then used a lipidomic approach to determine whether glucose feeding or mutations in the ceramide biosynthesis pathway or the insulin-like signaling pathway impact lipid profiles. I found that gluocose alters the lipid profile of daf-2(e1370) (an insulin-like receptor mutant) animals. These studies indicate that a transcriptomic approach can be used to discover novel pathways involved in oxygen deprivation response and further validate C. elegans as a model for understanding diabetes and obesity.
Date: August 2016
Creator: Ladage, Mary Lee

Maturation of Endothermic Capacity within the Avian Developmental Spectrum: A Characterization of Thermoregulatory Metamorphosis

Description: An avian embryo is ectothermic, with body temperature determined by environmental temperature. Upon hatching, the neonate begins a conversion so that endothermic capacity becomes feasible and body temperature becomes independent of environment. Whole animal metabolic rate and ventilation response, cardiovascular development, and maturation of muscle mitochondrial flux were the focus of this dissertation because of the direct role in shivering thermogenesis. Precocial ducks and altricial Double-crested Cormorants exhibit increasing hematocrit and disproportionate increases in fractional heart mass resulting in greater oxygen delivery capacity and increased capacity of muscles to utilize oxygen compared with ectothermic American Alligator and Common Snapping turtles. By selecting for faster growth and higher meat yield in the domestic chicken, differences in whole-animal, tissue, cellular, and regulatory responses are evident between broiler and layer type birds. In the altricial red-winged blackbird, despite appearance of a whole animal endothermic response sometime after 7 dph, capacity of skeletal muscles involved in shivering thermogenesis peaks prior to that time. Thus, full development of endothermy is delayed in this species, allowing the altricial nestling to allocate energy towards growth rather than metabolic maintenance. Hypothyroidism in neonate red-winged blackbirds results in delayed maturation of the cardiovascular system and mitochondrial oxidative capacity of skeletal muscle. Such deficiencies were quickly recovered once the animals returned to a normothyroid state, apparently at the cost of increasing body mass. Insights into onset of thermoregulation provide a more thorough understanding of metabolic and physical transitions a hatchling bird must undergo to reach the adult endothermic phenotype. Endothermic capacity will continue to be at the forefront of physiological research because of the significance of changes between the energetic relations of an animal that must occur with its environment.
Date: August 2016
Creator: Sirsat, Sarah Goy

Respiratory Responses in the Freshwater Snail (Pomacea Bridgesii) are Differently Affected by Temperature , Body Mass,and Oxygen Avalability

Description: Pomacea bridgesii is a snail species native to tropical and sub-tropical regions, where it usually faces variability in water, temperature and oxygen level. This study of the effect of temperature on mass-specific oxygen consumption (ṀO2) and its relation to body weight shows that the ṀO2 of juvenile snails in normoxia (18-21 kPa) acclimated at temperature of 25°C ranged from 5 to 58 µMol O2/g/h, with a mean of 41.4 ± 18.3 µMol O2/g/h (n=7). Adult snails in normoxia at 25°C show less variation, ranging from 13 to 23 µMol O2/g/h , with a mean of 24.4± 6.1 µMol O2/g/h (n=12). The Q10 value for juvenile snails was higher in the interval 25-30°C (Q 10=5.74) than in the interval 20-25°C (Q10= 0.286). In adult snails, Q10 was higher in the interval 20-25°C (Q10=3.19). ṀO2 of P. bridgesii in relation to body weight showed a negative linear correlation between metabolic rate and body weight with b values between 0.23 and 0.76. Also, both juvenile and adult snails exhibited weak O2 regulation. In general, the different respiratory characteristics between juvenile and adult snails might be related to the differences of individual life history, which caused them to perform differently in face of temperatures change. Additionally, Pomacean snails species originated in tropical habitats where there is a lack of thermal fluctuation. For this reason, Pomacean snails may be less likely to have evolved effective thermal acclimation capabilities.
Date: August 2016
Creator: Frifer, Wenasa Salem

Characterizing the Molecular Changes of Austrofundulus Limnaeus As It Develops Towards and Enters Diapause Ii

Description: Austrofundulus limnaeus is a species of annual killifish which inhabits ephemeral ponds in South America. The species is able to survive seasonally desiccating ponds due to their ability to produce robust embryos. The embryos of this species are capable of entering a developmental arrest, termed diapause II, which precedes the onset of drought. While in this arrested state embryos exhibit the greatest tolerance to anoxia of any characterized vertebrate at 25ºC. Furthermore, when raised at 30ºC, embryos escape the entrance to diapause II and go on to develop directly. Currently, little is known about the molecular mechanisms which induce and maintain this developmentally arrested state. In this study I have developed methods to analyze changes in histone modifications in the context of diapause II. Histone modifications were chosen due to their extreme conservation and well characterized role as modulators of gene expression in other systems. Results utilizing adapted immunobased assays show significant changes in the global amount of H3S10P, H3K27me and H3K4me, as the embryos progress from early embryogenesis through the exit of diapause. Additionally, it is revealed that there exists a degree of phenotypic plasticity with regards to the entrance into diapause II which is modulated by the environment (severe hypoxia 0.1% O2). This work builds a foundation for future histone modification studies and contributes the development of several tools to the field. This study contributes to a greater molecular understanding of the cue(s) which influence the remarkable phenomenon of obligate developmental arrest in a vertebrate embryo.
Date: December 2015
Creator: Toni, Lee S.

The Effect of Post-exercise Ethanol Consumption on the Acute Hormonal Response to Heavy Resistance Exercise in Women

Description: The purpose of this study was to examine the hormonal response to acute ethanol ingestion following a bout of heavy resistance exercise in women. Eight resistance trained women completed two identical acute heavy resistance exercise tasks (AHRET). From 10-20 minutes post-AHRET, participants consumed either a grain ethanol or a placebo beverage. Blood was collected before (PRE) and immediately after the AHRET (IP) and then every 20 minutes for five hours. Blood collected after beverage ingestion was pooled into 3 batches (phases: 20-40 minutes, 60-120 minutes, and 140-300 minutes post-exercise) and analyzed for serum total testosterone (TT), free testosterone (FT), insulin-like growth factor-I (IGF-I), human growth hormone (GH), cortisol (COR), and estradiol (E2) concentrations. Circulating concentrations of TT were significantly greater at P20-40 than at PRE, P60-120, and P140-300. Circulating concentrations of FT were significantly greater at P20-40 than at all other times. Circulating concentrations of GH were significantly greater at IP than at PRE, P60-120, and P140-300. Circulating concentrations of COR were significantly greater at P20-40 than at all other times. Additionally, COR concentrations at P140-300 were significantly lower than at all other times. Circulating concentrations of IGF-1 were significantly greater at P20-40 than at P60-120 and P140-300. Circulating concentrations of E2 were significantly greater at P20-40 than at all other times. In summary, the present study demonstrated an acute modulation of the neuroendocrine milieu following a heavy resistance exercise bout in women. Ethanol ingestion appeared to have no significant effect on the characteristics of acute hormonal augmentation in TT, FT, GH, COR, IGF-1, or E2.
Date: December 2015
Creator: Budnar, Jr., Ronald G.

Environmental Modulation of the Onset of Air-breathing of the Siamese Fighting Fish and the Blue Gourami

Description: This study determined the effect of hypoxia on air-breathing onset and physiological and morphological characters in larvae of the air breathing fishes Trichopodus trichopterus and Betta splendens. Larvae were exposed intermittently (12/12 h daily) to 20, 17, and 14 kPa of PO2 from 1 to 40 days post-fertilization. Survival, onset of air breathing, wet body mass, O2, Pcrit were measured every 5 dpf. Hypoxia advanced by 4 days, and delayed by 9 days, the onset of air breathing in Betta and Trichopodus, respectively. Hypoxia increased larval body length, wet mass, and labyrinth organ respiratory surface of Betta, but did not affect these factors in Trichopodus. Hypoxic exposure increased O2 by 50-100% at each day throughout larval development in Betta, but had no effect on larval Trichopodus. Hypoxia decreased Pcrit in Betta by 37%, but increased Pcrit in Trichopodus by 70%. Larval Betta reared in hypoxia showed a modified heart rate:opercular rate ratio (3:1 to 2:1), but these changes did not occur in Trichopodus. Compared to Betta, the blood of Trichopodus had a higher P50 and much smaller Bohr and Root effects. These interspecific differences are likely due to ecophysiological differences: Betta is a non- obligatory air-breather after 36 dpf with a slow lifestyle reflected in its low metabolism, while Trichopodus is an obligatory air-breather past 32 dpf with an athletic fast lifestyle and accompanying high metabolism.
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Date: December 2015
Creator: Mendez Sanchez, Jose Fernando

A High-fat Meal Alters Post-prandial Mrna Expression of Sirt1, Sirt4, and Sirt6

Description: Sirtuins (SIRT) regulate the transcription of various genes involved in the development of diet-induced obesity and chronic disease; however, it is unknown how they change acutely following a high-fat meal. The purpose of this study was to determine the effect of a high-fat meal (65% kcals/d; 85% fat recommendation), on SIRT1-7 mRNA expression in blood leukocytes at 1, 3, and 5-h post-prandial. Men and women (N=24) reported to the lab following an overnight fast (>12H). Total RNA was isolated and reverse transcribed prior to using a Taqman qPCR technique with 18S rRNA as a normalizer to determine SIRT1-7 mRNA expression. An additional aliquot of serum was used to measure triglycerides. Data was analyzed using a RM ANOVA with P<0.05. Triglycerides (P<0.001; 124%) peaked at 3-h. SIRT 1 (P=0.004; 70%), and SIRT 6 (P=0.017; 53%) decreased expression at 3-h. SIRT4 (P=0.024) peaked at 5H relative to baseline (70%) and 3-h (68%). To our knowledge, this is the first study to report that consumption of a high-fat meal transiently alters SIRT mRNA expression consistent in a pattern that mirrors changes in serum triglycerides. Decrease in expression of SIRT1 and SIRT6 combined with an increased SIRT4 would be consistent with an increase in metabolic disease risk if maintained on a chronic basis.
Date: December 2015
Creator: Best Sampson, Jill Nicole

Impact of a Genetically Engineered Probiotic Therapy and Igf-1 Genomics in the Pahenu2 Mouse Model of Pku

Description: Absence of functional phenylalanine hydroxylase results in phenylketonuria (PKU). Viable treatments remain few, expensive and secondary conditions such as osteopenia occur in most PKU patients. Objective 1: Given the recently described roles of gut microbes to aid host digestion, an orally administered genetically engineered probiotic as the delivery vehicle for enzyme replacement therapy was created. The engineered probiotic, pHENOMMenal, produced phenylalanine ammonia lyase with significant production of trans-cinnamate (phenylalanine cleavage product) in vitro and resulted in a reduction of 515 μM in blood phenylalanine when fed to PKU animals for 14 days (from 2307µM ± 264µM to 1792µM ± 261µM, n = 6, P < 0.05). The control probiotic produced no change in blood phenylalanine. Thus, pHENOMMenal treatment in PKU mice demonstrated engineered microbes could compensate for a metabolic deficiency of the host. Objective 2: Evaluate the PAHenu2 mouse model of PKU for a genetic discrepancy causing ocular enlargement and delayed development observed only after the PAHenu2 mutation was crossed to the C57BL/6J mouse. When compared to healthy littermates, ELISA indicated a consistent but insignificant decrease in plasma IGF-1 and an increase in ocular IGF-1 in PKU animals. SNP screening demonstrated a differential inheritance of IGF-1 alleles in healthy and PKU animals based on PAH allele inheritance. Ocular and developmental phenotypes in the PAHenu2 colony match those described in previous IGF-1 studies. Understanding the IGF-1 inheritance discrepancy will enable better osteopenia research using PAHenu2 mice and allow breeding of a healthier mouse colony for continued research. Collectively the results from this work describe a new therapeutic approach for treatment of PKU as well as a better understanding of the PAHenu2 mouse model to study this disease.
Date: December 2015
Creator: Durrer, Katherine Elaine

Phenotypic Morphological Plasticity Induced by Environmental Salt Stress in the Brine Shrimp, Artemia Franciscana

Description: Phenotypic plasticity is the ability of an organism to express different phenotypes in response to biotic or abiotic environmental cues. The ability of an organism to make changes during development to adjust to changes in its environment is a key to survival. Sexually reproducing organisms that have short life cycles and that are easy to raise in the laboratory are more conducive for developmental phenotypic plasticity. Considerable research has already been carried out on the brine shrimp, Artemia franciscana, regarding its morphology due to changing salinities. There is, however, little research considering subsequent generations and how there morphology might be affected by parental experiences. This study has examined: 1) the morphological effects of different rearing regimes of different salinity levels, and 2) the epigenetic transgenerational transfer of these morphological traits in A. franciscana. Measurements included rate of growth (as measured by instar), body size, body length, and other morphological traits. A gradual increase to more hyperosmotic conditions during development produced brine shrimp that were larger in size and also more developmentally advanced. Salinity stress experienced by adults had increased the growth rate in the F1 offspring of A. franciscana. Collectively, these data indicate that Artemia franciscana is a tractable model for investigating phenotypic plasticity. These findings have added to the ever-growing field of developmental phenotypic plasticity while also providing more information on the natural history and adaptive abilities of A. franciscana.
Date: December 2015
Creator: Jones, Shaun Gray

The Role of Cysteinyl Leukotriene Receptor 2 in Thrombocyte Aggregation

Description: Cysteinyl leukotriene receptor 2, a G-protein coupled receptor known to be expressed and functional on human platelets. However, it seems that upon ligand activation the cysteinyl leukotriene receptor 2 activates a variety of signaling pathways in multiple cell types among different species. Previously, a former laboratory member Vrinda Kulkarni found cysteinyl leukotriene receptor 2 to be expressed on the surface of adult zebrafish thrombocytes. In this work I studied the characteristics of aggregation in adult zebrafish thrombocytes with the knockdown of cysteinyl leukotriene receptor 2. I used a newly developed knockdown method to study the function of cysteinyl leukotriene receptor 2. Knockdown of the cysteinyl leukotriene was confirmed using RT-PCR results showed p=.001, reduced sell surface level of expression of the cysteinyl leukotriene receptor 2 results showed that p=.002. I found that the knockdown of cysteinyl leukotriene receptor 2 results in prothrombotic thrombocytes by using flow cytometry p=.0001.
Date: December 2015
Creator: Reyna, Julianna

Thresholds and Legacy Effects of Tropical Floodplain Fish Assemblages in Response to Flood Attributes

Description: Natural flow regimes are critical for sustaining biodiversity and river integrity. Floods and droughts form an important component of river systems and control population sizes and species diversity across space and time. Modification of flow regimes, including disruption of the timing, magnitude and duration of flooding, is a global problem, and many new impoundments are planned for large river-floodplain ecosystems in the tropics. Flow modifications may cause dramatic non-linear responses in population sizes and have lasting effects through time, but such topics are poorly investigated over multi-year scales, especially in highly diverse tropical ecosystems. Using a long-term dataset from the Upper Paraná River floodplain, Brazil, I tested for threshold and legacy effects of fish assemblages to flood attributes, such as timing, magnitude, duration, rate of change and variation. Specifically, I hypothesized that long duration, high magnitude floods would elicit threshold responses in long-distance migratory fish species and these responses result in significant legacy effects detectable over multiple years. Consistent positive threshold responses to increasing flood duration and magnitude were detected for many species and not significantly correlated with reproductive guilds. Legacy effects were prevalent (i.e. identified for more than 90% of species) and including flood attributes from previous years increased variance explained in species abundances by 15-20% compared to contemporary flood attributes alone. Contrary to my hypotheses, flood duration did not elicit strong legacy effects and species from the same reproductive guild did not have similar legacy effects models. The prevalence of legacy effects across almost all species in this diverse study system highlights the need to consider such dynamics in other systems. My results provide targets for management and conservation actions, such as environmental flow releases from upstream reservoirs. Environmental flows releases may play a significant role in sustainability of the floodplain and other tropical floodplain ecosystems affected by ...
Date: December 2015
Creator: Hoeinghaus, Ana Paula Ferrari

Effect of Acute Alcohol Ingestion on Resistance Exercise Induced Mtorc1 Signaling in Human Muscle

Description: The purpose of this project was to further elucidate the effects post-exercise alcohol ingestion. This project had many novel aspects including using a resistance exercise (RE) only exercise design and the inclusion of women. To our knowledge, we are the first to investigate the effect of post-RE alcohol ingestion in women. In the first chapter of this project, information on the prevalence of alcohol use and the importance of skeletal muscle as a dynamic and metabolic tissue was provided. In chapter two, the effects of post-RE alcohol ingestion in men and women are detailed. The major findings of this study was that although RE elicited similar mTORC1 signaling both in men and in women, alcohol ingestion appeared to only attenuate RE-induced phosphorylation of the mTORC1 signaling pathway in men. The third chapter focused on examining the effects of post-RE alcohol ingestion on acute testosterone bioavailability. The primary findings of this study was that alcohol substantially elevated serum total and free testosterone concentrations during recovery from a bout of resistance exercise. The fourth chapter detailed factors that contribute to bone density in men. The major findings of this study was that young adult male long-distance runners who participated in resistance training at least once per week had greater bone mineral density than their non-resistance trained and non-exercise trained peers.
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Date: August 2015
Creator: Anthony A. Duplanty

Glucose Induces Sensitivity to Oxygen Deprivation and Alters Gene Expression in Caenorhabditis Elegans

Description: An organisms’ diet represents an exogenous influence that often yields colossal effects on long-term health and disease risk. The overconsumption of dietary sugars for example, has contributed to significant increases in obesity and type-2 diabetes; health issues that are costly both economically and in terms of human life. Individuals who are obese or are type-2 diabetic often have compromised oxygen delivery and an increased vulnerability to oxygen-deprivation related complications, such as ischemic strokes, peripheral arterial disease and myocardial infarction. Thus, it is of interest to identify the molecular changes glucose supplementation or hyperglycemia can induce, which ultimately compromise oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, I determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide biosynthesis and antioxidant activity, modulates anoxia survival. Additionally, a glucose-supplemented diet induces lipid accumulation. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified are homologous to human genes that are differentially regulated in response to metabolic diseases, suggesting that there may be conserved gene expression responses between C. elegans supplemented with glucose and a diabetic and/or obese state observed in humans. These findings support the utility of C. elegans to model specific aspects of the T2D disease process (e.g., glucose-induced sensitivity to oxygen deprivation) and identify potentially novel regulators of common complications seen in hyperglycemic and T2D patients (e.g., macrovascular complications).
Date: August 2015
Creator: Garcia, Anastacia M.

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

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 number of new neurons throughout adulthood. The neuron lineage contains a progression of identifiable precursor cell types with different proliferation rates. This present study found that primary cilia were present in every cell type in the neuronal lineage in SGZ. Cilium length and incidence were positively correlated among these cell types. Ciliary levels of adenylyl cyclase type III (ACIII) levels relative to ADP-ribosylation factor-like protein 13b (Arl13b) was higher in neurons than in precursor cells and glia, and also changed with the cell cycle. G-protein coupled receptors, SstR3, MCHR1, and Gpr161 receptors were only found in neuronal cilia. The levels ...
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Date: May 2015
Creator: Bhattarai, Samip Ram

Cytotoxicity and Functional Toxicity of Mefloquine and the Search for Protective Compounds

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 reference activity yielding a mean IC50 value of 5.97 ± 0.44 (SD) µM (n=6). After total activity loss, no activity was recovered with two successive medium changes. To test for network response desensitization resulting from sequential applications over 5-6 hr periods, one-point titrations at varying concentrations were conducted with fresh networks. These experiments yielded a single concentration response curve with an IC50 value of 2.97 µM. This represents a statistically significant shift (p < 0.0001) to lower concentrations of mefloquine, demonstrating that sequential applications result in network desensitization. After mefloquine exposures, cells were evaluated for irreversible cytotoxic damage. Over a ...
Date: May 2015
Creator: Holmes, Katelyn

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

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 intracellular protein and mRNA production using flow cytometry.
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Date: May 2015
Creator: Venable, Adam Steven

Improving Processing Efficiency for Forensic DNA Samples

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 with decreased resolution as compared to traditional POP-4®/36cm detection for alleles >200bp; however, 1bp resolution was still obtainable for alleles <300bp. These modifications resulted in robust databasing processes with up to a 37% reduction in processing time for buccal swabs and Buccal DNA Collectors™ using the three primer sets evaluated (3μl fast PCR reactions) and generated high quality STR profiles with ≥90% pass rates.
Date: May 2015
Creator: Connon, Catherine Cupples

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

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 functional interference with spike production. Full recovery has not been seen at concentrations above 130 microM, unless the culture was given bicuculline. Direct exposure to 400uM results in immediate, irreversible loss of spike production, followed by necrosis of glia and neurons.
Date: May 2015
Creator: Gibson, Jason

Understanding Microbial Biodegradation of Environmental Contaminants

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 research give us insights on how bacteria communities, not just individually, can adapt and become resistant to the contaminants. The present dissertation work showed specific genes and mechanisms for degradation and resistance of contaminants that could contribute to develop new bioremediation strategies.
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Date: May 2015
Creator: Vilo Muñoz, Claudia Andrea

Acute Effects of the Antibiotic Streptomycin on Neural Network Activity and Pharmacological Responses

Description: The purpose of this study is to find out that if antibiotic streptomycin decreases neuronal network activity or affects the pharmacological responses. The experiments in this study were conducted via MEA (multi-electrode array) technology which records neuronal activity from devices that have multiple small electrodes, serve as neural interfaces connecting neurons to electronic circuitry. The result of this study shows that streptomycin lowered the spike production of neuronal network, and also, sensitization was seen when neuronal network pre-exposed to streptomycin.
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Date: December 2014
Creator: Zeng, Wei Rong

Effects of Sertraline Exposure on Fathead Minnow (Pimephales promelas) Steroidogenesis

Description: Sertraline is a selective serotonin reuptake inhibitor (SSRI) that is widely used for the treatment of depression and anxiety. Due to the abundant therapeutic use of sertraline, low levels have been detected in municipal wastewater effluents suggesting that aquatic organisms may be exposed. The purpose of this study was to evaluate the steroidogenic effects of sertraline on larval (FHM) and adult female fathead minnows (FFHM), Pimephales promelas. Larval FHM were exposed to 0.1, 1, and 10 µg/L sertraline for 28 days and analyzed via RT-qPCR for differential expression of 11β-Hydroxysteroid dehydrogenase (11β-HSD), 20β-Hydroxysteroid dehydrogenase (20β-HSD), aromatase (CYP19), and nuclear thyroid receptor alpha (TRα). FFHM were exposed to 3 or 10 µg/L sertraline for 7 days with the brain and ovary excised at exposure termination. Juvenile FHM exposed to 0.1 μg/L sertraline had a significant upregulation of both 20β-HSD and TRα. FFHM exposed to 10 µg/L sertraline had a significant upregulation of 11β-HSD expression in brain tissue, while no steroidogenic changes were observed in the FFHM ovary. Similarly, in FFHM brain tissue, CYP19 and 20β-HSD expression levels were significantly higher in fish exposed to 10µg/L sertraline compared to control. The significance of these findings with respect to survival, growth and reproduction are currently unknown, but represent future research needs.
Date: December 2014
Creator: Carty, Dennis R.

The Impact of Developmental Stress on Cardiovascular Physiology of Two Archosaur Species: American Alligator (Alligator mississippiensis) and Domestic Chicken (Gallus gallus)

Description: Crocodilians and birds comprise sister taxa of archosaurs, the development of these vertebrates occurs within an egg case that leaves developing embryos susceptible to fluctuations in the nesting environment. Studies suggest that sub-optimal conditions alter morphological growth and cardiovascular physiology. Regulation of the cardiovascular system is immature in the subjects studied, and embryos may rely on humoral rather than neural control of the cardiovascular system. The primary focus of this dissertation was to assess regulatory mechanisms responsible for maintenance of arterial pressure and heart rate. Dehydration stress had marked effects on embryo growth, and altered baseline cardiovascular parameters, while leaving the response to humoral regulator, angiotensin II (Ang II), unaffected. However, dehydrated alligator embryos developed cholinergic tone on heart rate. Hypoxic incubated chicken embryos were reduced in embryo mass, and altered response to humoral regulatory components Ang I and adenosine in addition identifying a novel regulatory component of the cardiovascular response to acute hypoxia. Collectively, these studies add to the existing knowledge of cardiovascular physiology in embryonic archosaurs and suggest that some components of cardiovascular regulation are plastic following developmental stress.
Date: December 2014
Creator: Tate, Kevin B.

In Vitro Investigations of Antibiotic Influences on Nerve Cell Network Responses to Pharmacological Agents

Description: Neuronal networks, derived from mouse embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate effects of gentamicin pretreatment on pharmacological response to the L-type calcium channel blocker, verapamil. Gentamicin is a broad spectrum antibiotic used to control bacterial contamination in cell culture. The addition of gentamicin directly to medium affects the pharmacological and morphological properties of the cells in culture. A reproducible dose response curve to verapamil from untreated cultures was established and the mean EC50 was calculated to be 1.5 ± 0.5 μM (n=10). 40 μM bicuculline was added to some cell cultures to stabilize activity and verapamil dose response curves were performed in presence of bicuculline, EC50 1.4 ± 0.1 μM (n=9). Statistical analysis showed no significant difference in verapamil EC50s values obtained in presence of bicuculline and hence the data was combined and a standard verapamil EC50 was calculated as 1.4 ± 0.13 μM (n=19). This EC50 was then used to compare verapamil EC50s obtained from neuronal cell cultures with chronic and acute exposures to gentamicin. FC cultures (21- 38 days old) were found to be stable in presence of 2300 μM gentamicin. The recommended concentration of gentamicin for contamination control is 5uL /1 ml medium (108 μM). At this concentration, the verapamil EC50 shifted from 1.4 ± 0.13 μM to 0.9 ± 0.2 μM. Given the limited data points and only two complete CRCs, statistical comparison was not feasible. However, there is a definite trend that shows sensitization of cells to verapamil in presence of gentamicin. The cultures exposed to 108 μM gentamicin for 5 days after seeding showed loss of adhesion and no data could be collected for pharmacological analysis. To conclude, acute gentamicin exposure of neuronal cell cultures causes increased sensitivity to verapamil and chronic or long term exposure to ...
Date: December 2014
Creator: Sawant, Meera

Measuring Biomarkers From Dried Blood Spots Utilizing Bead-based Multiplex Technology

Description: Dried blood spots is an alternative method to collect blood samples from research subjects. However, little is known about how hemoglobin and hematocrit affect bead-based multiplex assay performance. The purpose of this study was to determine how bead-based multiplex assays perform when analyzing dried blood spot samples. A series of four experiments outline the study each with a specific purpose. A total of 167 subject samples were collected and 92 different biomarkers were measured. Median fluorescence intensity results show a positive correlation between filtered and non-filtered samples. Utilizing a smaller quantity of sample results in a positive correlation to a larger sample. Removal of hemoglobin from the dried blood spot sample does not increase detection or concentration of biomarkers. Of the 92 different biomarkers measured 56 were detectable in 100-75% of the attempted samples. We conclude that blood biomarkers can be detected using bead-based multiplex assays. In addition, it is possible to utilize a smaller quantity of sample while avoiding the use of the entire sample, and maintaining a correlation to the total sample. While our method of hemoglobin was efficient it also removed the biomarkers we wished to analyze. Thus, an alternative method is necessary to determine if removing hemoglobin increases concentration of biomarkers. More research is necessary to determine if the biomarkers measured in this study can be measured over time or within an experimental model.
Date: December 2014
Creator: Prado, Eric A.