Browsing by Subject "FLIM"

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    Macromolecular Crowding Effects on Cellular NADH-enzyme Binding Kinetics
    (2017-08) Wilson, Shane
    Reduced nicotinamide adenine dinucleotide (NADH) is a major cofactor for a large number of biological enzymes that are essential in a myriad of metabolic pathways such as glycolytic and oxidative phosphorylation pathways. In addition, NADH is intrinsically fluorescent and therefore has the potential of serving as a biomarker to monitor mitochondrial dysfunctions associated with aging, cancer, and apoptosis. In this thesis, we investigate how macromolecular crowding may affect the biochemical reaction kinetics of NADH interaction with lactate dehydrogenase (LDH) as a model system in biomimetic crowding (e.g., Ficoll-enriched buffer at 0 ̶ 400 g/L). Using noninvasive, quantitative two-photon fluorescence lifetime and associated anisotropy, we exploit the sensitivities of NADH fluorescence lifetime and rotational diffusion to protein binding. To differentiate between viscosity and crowding effects on the reaction kinetics, we also conducted complementary measurements in glycerol-enriched buffer. Additionally, we are investigating the sensitivity of cellular NADH interaction with dehydrogenases to metabolic manipulations. Our quantitative and non-invasive methodology complements the traditional biochemical and thermodynamics techniques without the destruction of live cells. Intracellular NADH also exists as a mixture of free and enzyme-bound populations at dynamic equilibrium throughout living cells, which can be imaged using fluorescence lifetime imaging for both quantitative and noninvasive assessment of cellular metabolism. 2P-fluorescence lifetime imaging microscopy (FLIM) and 2P- fluorescence anisotropy of intrinsic NADH was measured in cultured mouse embryonic cells under both resting conditions and metabolic-manipulation.
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    Native NADH As A Biomarker For Oxidative Stress In Living Cells
    (2014-08) Alfveby, John
    Oxidative stress underlies the mechanisms of many diseases such as Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease, schizophrenia, diabetes, Multiple Sclerosis, bipolar disorder, and chronic fatigue syndrome. Oxidative stress is caused by excessive reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals, which are byproducts of oxygen metabolism in aerobic organisms. Environmental factors such as exposure to ultraviolet light, chemicals ingested by the diet, ionizing radiation, and cigarette smoke can also lead to production of ROS and therefore oxidative stress. As a result, there is a growing need for quantitative and noninvasive methodologies to probe oxidative stress at the single-cell level and ultimately in vivo. In this thesis, we examined the potential of natural coenzyme nicotinamide adenine dinucleotide (NADH) as a natural biomarker for oxidative stress in C3H 10T1/2 living cells in culture. NADH (fluorescent) is a coenzyme that is essential for energy metabolism via oxidative phosphorylation pathway in the inner membrane of mitochondria, which is also a major source for intrinsic ROS species generated through the electron transport chain. Our experimental multiparametric approach combined cell culture with fluorescence microscopy (confocal and two-photon) and spectroscopy (fluorescence lifetime imaging and time-resolved anisotropy) methods. Cultured cells were treated with hydrogen peroxide and rotenone in order to trigger oxidative stress. As a point of reference, conventional oxidative stress assays such as MitoSOX Red, JC-1, and H2DCFDA were used to optimize the chemical dosage and incubation time needed for observable oxidative stress. Our results help in the collective effort to establish cellular NADH autofluorescence as a natural biomarker for cellular metabolic health and oxidative stress.