Browsing by Subject "oxidative stress"

Now showing 1 - 3 of 3
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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.
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    Protein carbonylation in the adipocyte nucleus
    (2017-11) Hauck, Amy
    As the incidence of obesity rises globally, it has become increasingly imperative to identify the mechanisms that cause obesity-related metabolic disease. In particular, oxidative stress in the adipose tissue is known to cause metabolic dysfunction, but the mechanisms that contribute to this process remain unclear. Protein carbonylation refers to the post-translational modification of lysine, cysteine, and histidine residues by diffusible electrophilic lipids. Specifically, 4- hydroxy-2-nonenal (4-HNE) and 4-hydroxy-2-hexenal (4-HHE) are produced at high levels in obese adipose tissue as a direct result of increased oxidative stress. The studies herein focus on the hypothesis that protein carbonylation is a mechanistic link between elevated oxidative stress and metabolic dysfunction in obese adipose tissue. We found that protein carbonylation is elevated specifically in the nucleus of adipocytes as a consequence of obesity and of aging. Proteomic evaluation of these modifications revealed that the core histones and zinc finger proteins are major targets of carbonylation. Since these proteins are critical regulators of transcriptional mechanisms, these data describe a potential link between oxidative stress and altered expression of metabolic pathways in adipose tissue.
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    The role of mitochondrially derived oxidative stress in the development of insulin resistance in the adipocyte
    (2015-09) Olson, Dalay
    The number of people worldwide living with type-2-diabetes (T2D) is increasing at an alarming rate. In 2014, approximately 9% of the adult population worldwide was suffering from diabetes, with greater than 90% suffering from the type-2 form of the disease. One prominent risk factor for the development of T2D and insulin resistance is obesity. Ninety percent of type-2-diabetics are overweight or obese, highlighting the striking link between the two diseased states. Although T2D has been studied for years, the underlying mechanisms outlining the development of obesity induced insulin resistance remain poorly understood. Currently, it is well established that adipose tissue inflammation plays a major role in the induction of insulin resistance. Preliminary experiments using inflammatory cytokines to induce insulin resistance in adipocytes suggested that mitochondria maybe a site of oxidant accumulation resulting from transcriptional down-regulation of key mitochondrial antioxidants. Because oxidative stress is a hallmark feature of obese, insulin resistant adipose tissue, I was interested in identifying important metabolic pathways that were regulated by oxidative stress, independent of inflammation, within the adipocyte itself. Knockdown of Peroxiredoxin-3 (Prdx3) in adipocytes provided a tool that not only mimicked what was seen in diseased states, but also allowed for mechanistic study of the direct effects of mitochondrially generated oxidative stress on adipocyte insulin sensitivity. Importantly, these data revealed that silencing Prdx3 in adipocytes resulted in increased oxidative stress and insulin resistance mediated by rictor oxidation, decreased mTORC2 activity and subsequent decreases in S473-AKT phosphorylation.