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Browsing by Author "Frye, Melinda A., committee member"

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    Avian influenza A virus transmission and the emergence of drug resistance
    (Colorado State University. Libraries, 2011) Achenbach, Jenna Elizabeth, author; Bowen, Richard A., advisor; Landolt, Gabriele A., committee member; Avery, Anne C., committee member; Frye, Melinda A., committee member
    As avian influenza A viruses (AIV) continue to circulate worldwide both naturally, within the reservoir host of wild waterfowl, and cross species barriers, eventually establishing itself in new host species, it is imperative to study the natural reservoir in respect to virus change and transmissibility. This dissertation will focus on the transmissibility of a mallard virus from mallards to other wild and domestic species as well as elucidate the possible outcomes of oseltamivir contamination in the environment and its effect on influenza A virus infected mallards. Low pathogenicity (LP) AIVs of the H5N2 and H7N3 subtypes were utilized to evaluate the ability of transmission of a mallard derived virus to other species present in a co-habitation (barnyard) scenario. Other species in contact with the mallards were chickens, blackbirds, rats, and pigeons. Viral replication was assessed directly from ducks in the barnyard with assessment of the other animals in the barnyard through sero-conversion. Additional animals of each species were directly inoculated with these two viruses and assessed for viral replication. The H5N2 virus was transmitted to other ducks and chickens in the barnyard through either direct or environmental contamination, but not to rats or blackbirds. The H7N3 virus was transmitted to other ducks, chickens, pigeons, and rats. Chickens and blackbirds directly inoculated with both virus strains shed significant amount of virus and seroconverted, but rats and pigeons (except for one pigeon) failed to shed virus but did develop antiviral antibodies. Knowing that both mallard viruses can directly transmit without adaptation, show the mallard to be a good model to further evaluate the outcome of oseltamivir contamination in the environment and its effect on AIV infected mallards. The environment has been shown to be contaminated with significant amounts of oseltamivir carboxylate (OC) in an area of high drug prescription use. We analyzed the outcomes of AIV in infected mallards when they have access to OC in their drinking water. Two separate LPAIV H5N2 viruses were tested for their ability to mutate under drug pressure. One H5N2 virus did not demonstrate any altered sequence after 7-10 days of drug access and infection. The other H5N2 virus did show mutations in the neuraminidase gene that led to an increase in resistance to oseltamivir caused by a specific mutation at E119V. This resistant virus was further evaluated for its ability to transmit between infected and naïve mallards. While the resistant virus did transmit duck to duck, the mutation at position 119 was not detected after challenge or transmission showing instability of this mutation. This could either be a reversion to wild-type or possibly the low level presence of wild-type present in the resistant strain stock that outcompeted with the mutant strain to succeed in the host. This shows, that in these duck experiments, the E119V mutation is not stable in the absence of drug pressure and unlikely to succeed in the host.
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    Lipids and oxidative stress as mediators of endothelial pathophysiology in obesity
    (Colorado State University. Libraries, 2011) Donovan, Elise Laura, author; Miller, Benjamin F., advisor; Hamilton, Karyn L., advisor; Hickey, Matthew S., committee member; Frye, Melinda A., committee member
    Because obesity is a well established independent risk factor for diabetes and coronary artery disease (CAD), it is important to identify factors associated with obesity that are responsible for disease progression and interventions to decrease risk of developing obesity associated co-morbidities. Two of the many mediators of obesity associated risk for diabetes and CAD are oxidative stress and oxidized phospholipids, which have been implicated in vascular disease initiation and progression through endothelial cell activation, macrophage recruitment and advanced plaque rupture. Plasma platelet activating factor acetylhydrolase (Lp-PLA2) is an enzyme that circulates bound to LDL cholesterol and degrades platelet activating factor (PAF), a potent inducer of the platelet coagulation cascade and thrombosis. In addition, Lp-PLA2 degrades oxidized phospholipids to lysophospholipid products and fatty acids that may also induce inflammatory changes in multiple cell types, including vascular endothelial cells. Exogenous antioxidant supplementation has been examined as a means of decreasing vascular oxidative stress. However, data show that exogenous antioxidant supplementation has little or no effect on CVD outcomes, and in some cases it may increase mortality. A novel approach to protecting cells from oxidative stress is to increase cellular endogenous antioxidant defenses. NF-E2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) promoter region of many genes including phase II antioxidant enzymes. Protandim is a combination of phytochemicals that is thought to induce Nrf2 stabilization and translocation to the nucleus, with subsequent increases in phase II antioxidant enzymes and protection against oxidative stress. The overall objectives of the three studies we performed were to 1) globally analyze obesity associated lipid and oxidative stress using lipidomics techniques 2) determine the effects of identified obesity associated oxidative and lipid stress on the vascular endothelium 3) determine whether Protandim treatment could protect vascular endothelial cells from an oxidative challenge, and 4) characterize Lp-PLA2 in human adipose and skeletal muscle. Experiment 1 tested the hypothesis that oxidized phospholipids would be greater in morbidly obese gastric bypass patients compared to lean surgical controls, and that global lipid profiles would differ between groups. To test this hypothesis we performed a combined targeted and global lipidomic analysis of plasma lipids from morbidly obese gastric bypass patients and lean controls. We identified a group of ether-linked lipids that were greater in obese subjects compared to lean, and further examined whether a representative lipid from this group induced pathophysiological phenotypic changes in vascular endothelial cells. Experiment 2 tested the hypothesis that Protandim would protect human coronary artery endothelial cells (HCAEC) against an oxidative challenge by increasing phase II antioxidant enzymes in a Nrf2 dependent manner. To do this we performed a series of in vitro experiments treating HCAEC with Protandim and determined that Protandim induced Nrf2 nuclear localization, increased phase II antioxidant enzyme expression, and protected cells from undergoing apoptosis in response to an oxidative challenge. Silencing Nrf2 prior to the oxidative challenge inhibited the Protandim induced protection. Experiment 3 tested the hypothesis that Lp-PLA2 would be detectable in human adipose tissue and that Lp-PLA2 would be greater in adipose from morbidly obese gastric bypass patients compared to lean. In addition, we examined whether adipose Lp-PLA2 may be related to circulating Lp-PLA2 activity, inflammation, and glucose intolerance. We have identified ether-linked lipids that are elevated in obese subjects compared to lean. We found that Lp-PLA2 is expressed in human adipose for the first time, adipose Lp-PLA2 is co-localized with macrophages, and report relationships between Lp-PLA2 and indices of glucose homeostasis and inflammation. Lastly, we found that Protandim protects endothelial cells from an oxidative challenge in a Nrf2 dependent manner. Collectively, these data provide insight into the oxidative and lipid stress milieu that occurs in obese subjects.
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    Simplified membrane-like systems describing the physical behaviors of cholesterol and anti-diabetic drugs
    (Colorado State University. Libraries, 2013) Trujillo, Alejandro M., author; Crans, Debbie C., advisor; Roess, Deborah A., committee member; Frye, Melinda A., committee member; Van Orden, Alan K., committee member
    This work evaluates the properties contributing to natural membrane permeability by using simplified systems. Absorption mechanisms are a critical step in evaluating the action of orally active drugs. Reverse micelles (RMs) were used as a membrane-like model to analyze the permeation through spectroscopy. The properties exerted by the ligand and ligand substituents were evaluated in the context of membrane permeation. The polydentate ligand of anti diabetic dipicolinatooxvanadium(V) [VO2dipic])-, 2,6-pyridinedicarboxylate (dipic2-) was observed for permeability in sodium bis(2-ethylhexyl)sulfosuccinate (AOT) RMs. Measurements using proton nuclear magnetic resonance (1H NMR) spectroscopy revealed the permeation and hydrophobic stability at physiological pH for dipic2-. Substituents, NH2, OH, H, Cl, NO2 were evaluated forinfluencing the stability and permeability of [VO2(dipic)]-; in AOT RMs. Using infrared spectroscopy (IR), substituent changes significantly influenced the permeation of the vanadium complex series. Properties contributing to the membrane permeation of drugs may also be altered by membrane composition. Cholesterol is present in intestinal membranes and is known to possess properties reducing permeability. A system composed of cetyltrimethylammonium bromide (CTAB), 1-pentanol, cholesterol, and an aqueous phase formed RMs characterized by NMR and dynamic light scattering (DLS). Cholesterol altered the RM structure and proton transfer rates between the water and 1-pentanol of the system. Combined, this work illustrates that ligands, substituents, and membrane components influence the uptake of orally administered drugs.
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    T cell mediated satellite cell function: implications for age-associated changes in skeletal muscle regeneration
    (Colorado State University. Libraries, 2010) Dumke, Breanna R., author; Lees, Simon J., advisor; Gotshall, Robert W., advisor; Frye, Melinda A., committee member
    Sarcopenia is an age-associated loss of skeletal muscle mass and strength. Recent evidence suggests that an age-associated loss of muscle precursor cell (MPC) functionality contributes to sarcopenia. Current research also suggests that T cells of the immune system may influence skeletal muscle repair via signaling with MPCs. The objective of the present study was to examine the influence of activated T cells on MPCs. MPCs were collected from the gastrocnemius and plantaris from 3-mo-old (young) and 32-mo-old (old) animals. Splenic T cells were also harvested using anti-CD3 Dynabead isolation. T cells were activated for 48 hours with co-stimulation of 100 IU/ml Interleukin-2 (IL-2) and 5 ug/ml of anti-CD28. Co-stimulation increased 5-bromo-2'-deoxyuridine (BrdU) incorporation (proliferation) of T cells from 13.382% (SEM=4.55, n=5) in control to 64.77% (SEM= 6.02, n=5). Additionally, T cell cytokines increased MPC proliferation by 23.98% (SEM=5.69, n=4) in young MPCs but decreased by 1.58% (SEM=4.09, n=4) in old MPCs. T cell cytokines were also found to be chemoattractant. Young MPCs migrated at a rate of 1.36 (SEM=0.56, n=4) with T cell cytokines. Old MPCs, however, did not migrate with T cell cytokines -0.05 (SEM= 0.214, n=4). These data suggest that T cells may play a critical role in mediating MPC function. Furthermore, aging may alter T cell-induced MPC function. These findings have implications for developing strategies aimed at increasing MPC proliferation and the regenerative capacity of aged skeletal muscle.
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    Upregulation of heme oxygenase-1 and activation of Nrf2 by the phytochemicals in protandim
    (Colorado State University. Libraries, 2010) Reuland, Danielle Judith, author; Hamilton, Karyn L., advisor; Miller, Benjamin F., committee member; Frye, Melinda A., committee member
    Increased production of reactive oxygen species has been implicated in the pathogenesis of cardiovascular disease (CVD), with enhanced endogenous antioxidants proposed as a potential mechanism for promoting redox balance. Protandim is a well-defined combination of five widely studied medicinal plants derived from botanical sources [Bacopa monniera, Silybum marianum (milk thistle), Withania somnifera (Ashwagandha), Camellia sinensis (green tea), and Curcuma longa (turmeric)]. The purpose of this study was to determine if treatment of cardiomyocytes with Protandim induces phase II detoxification enzymes, including the endogenous antioxidant heme oxygenase-1 (HO-1), with activation of nuclear factor E2 p45-related factor 2 (Nrf2), and protection from oxidative stress induced apoptosis. In cultured cardiomyocytes, treatment with Protandim was associated with activation of Nrf2 and a significant increase in HO-1. Protandim supplemented cells were protected against hydrogen peroxide-induced apoptosis as assessed by TUNEL (35% apoptotic in untreated vs. 5% apoptotic in Protandim treated). These findings support the use of Protandim as a potential method for upregulation of antioxidant defenses and protection of heart cells against an oxidative challenge. Future studies will focus on optimizing phytochemical induction of Nrf2-mediated antioxidant defenses in relevant in vivo models of CVD.