Browsing by Subject "fluorescence microscopy"
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Item Comparative Neurotoxicity of Methylmercury and Mercuric Chloride In Vivo and In Vitro(2010-10-12) Thuett, Kerry A.It is impossible to remove methylmercury (MeHg) from biological systems because MeHg is found throughout our environment in many fresh and salt water fish. The consumption of fish is important to human nutrition and health. The mechanism of MeHg neurotoxicity must be understood to minimize adverse exposure consequences. The dissertation objective was to: 1) compare mechanisms of MeHg neurotoxicity between animals exposed as adults and those exposed during gestation, and 2) develop an in vitro test model of in vivo MeHg exposure. Total mercury (Hg) levels in tissue / cells were determined by combustion / trapping / atomic absorption. Cell death was determined by Fluoro-Jade histochemical staining and activated caspase 3 immunohistochemistry for in vivo studies, and Trypan blue exclusion, lactate dehydrogenase activity, and cytotoxicity assays for in vitro studies. Mitochondrial membrane potential (MMP), intracellular calcium ion concentration ([Ca2+]i), and production of reactive oxygen species (ROS) were determined using fluorescence microscopy or microplate reader assays. Young adult C57Bl/6 mice were exposed to a total dose of 0, 1.0, or 5.0 mg/kg body weight MeHg divided over postnatal days (P)35 to 39. Pregnant female mice were exposed to a total does of 0, 0.1, or 1.0 mg/kg body weight MeHg divided over gestational days (G)8 to 18. SY5Y cells were exposed to 0, 0.01, 0.1, or 1.0 ?M MeHg or HgCl2 for 24, 48, or 72 hours. Total Hg in brains of young adult mice, mouse pups, and SY5Y cells accumulated in a dose-dependent manner. Cell death increased in SY5Y cells exposed to the highest concentrations of MeHg and HgCl2 used in this study. Cell death increased in the molecular and granule cerebellar cell layers of young adult mice exposed to the highest doses of MeHg used in this study. P0 mouse pups showed no increase in cell death within the cerebellum following MeHg exposure. Cerebella of mice at P10 exhibited decreased dying cells only in the external germinal layer. Low concentrations of MeHg affected MMP in both in vivo and in vitro studies, but did not result in decreased MMP typically associated with higher MeHg concentrations. [Ca2+]i was increased throughout the in vivo experiments in an age- , sexand brain region-dependent manner. Generation of ROS was decreased in both in vivo and in vitro studies with both the MeHg and HgCl2 (in vitro) treatments. In summary, low and moderate MeHg exposure, both in vivo and in vitro, altered mitochondrial function, Ca2+ homeostasis, and ROS differently than what is reported in the literature for higher MeHg exposure concentrations. SY5Y cells were sensitive to low-levels of MeHg and HgCl2 and responded similarly to cells in the whole animal studies, thus making SY5Y cells realistic candidates for mechanistic MeHg studies. Cell culture and whole animal neuronal functional studies at chronic low-level MeHg exposure are limited. These data suggest that low-levels of MeHg may affect neuronal function. Therefore, further chronic low-level MeHg neuronal functional studies are warranted.Item The morphology of polymer modified asphalt and its relationship to rheology and durability(Texas A&M University, 2008-10-10) Kraus, Zachary RothmanPolymers are added to asphalt binders primarily to stiffen the binder at higher temperatures and thus to protect the pavement against rutting at summertime temperatures early in the pavement's life. Also, it has been noted that polymers typically increase the ductility of a binder and that some polymer-asphalt combinations are especially effective. Furthermore, it is hypothesized that enhancing a binder's ductility, and maintaining this enhancement with binder oxidative aging, contributes to enhanced binder durability in pavements. However, polymer-asphalt interactions and how they might contribute to improved binder performance is not well understood. The goal of this work was to probe the relationship of polymer morphology on asphalt binder rheology and mixture durability. Experiments were conducted on asphalt mixtures and binders, and as a function of oxidative aging. PFC mixtures, which are an open mixture designed to allow enhanced water drainage, were of specific interest. These mixtures were tested for Cantabro Loss, an indicator of a mixture's likelihood of failure by raveling. Asphalt binders were tested using dynamic shear rheometry (DSR), which provided the DSR function, (G' /?'/G'), a measure of binder stiffness that includes both the elastic modulus and the flow viscosity), ductility (used to measure the elongation a binder could withstand before failure), gel permeation chromatography (GPC), used to estimate the relative amount of polymer) and fluorescence microscopy (used to image the polymer morphology in the asphalt binder). From these data, relationships were assessed between binder morphology and binder rheology and between binder rheology and mixture durability, all as a function of binder oxidative aging. Polymer morphology related to ductility enhancement. Polymer morphology related to a change in the DSR function, relative to the amount of polymer, as measured by the polymer GPC peak height. Cantabro loss correlated to the DSR function (R2=0.963). The overall conclusion is that polymer morphology, as indicated by fluorescence microscopy, relates to both the rheological properties of the binder and the Cantabro loss of the mixture. These relationships should yield a better understanding of polymer modification, increased mixture durability (decreased raveling) and improved rheological properties (DSR function and ductility).