Browsing by Subject "Oxidative Stress"

Now showing 1 - 4 of 4
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    Investigating The Role Of Cystic Fibrosis Transmembrane Conductance Regulator On Oxidative Stress In Colorectal Cancer
    (2020-05) Singhania, Mekhla
    Colorectal cancer (CRC) is the third leading cause of cancer-related deaths among both men and women in the United States, with an estimated 53,200 deaths in 2020 (Siegel, Miller, & Jemal, 2020). Our lab identified Cystic Fibrosis Transmembrane Regulator (CFTR) as a CRC tumor suppressor gene in mice and humans. However, the mechanism by which CFTR acts as a tumor suppressor gene in CRC is unknown. To identify potential pathways, we compared gene expression in CFTR-low expressing CRC vs. CFTR high expressing tumors in several publicly available databases using gene set enrichment analysis. These analyses revealed that a common subset of genes is responsive in CFTR-low expressing tumors and in HIF1α-high tumors. HIF1α protein is induced by oxidative stress and is known to play a protective role in the oxidative stress pathway. To investigate the significance of this correlation, I hypothesized that downregulation of CFTR protects against oxidative stress. To test this hypothesis, I compared cell viability in human Caco-2 CRC cell lines: one in which CFTR has been knocked out by CRISPR-Cas9 modification (CFTR-KO) vs. matched parental controls (CFTR-WT). Each cell line was treated with menadione to induce oxidative stress. When cell viability was measured using an MTT assay, a trend was observed, where CFTR-KO cells were less affected by menadione than CFTR-WT. This suggests that CFTR deficiency may help colon cancer cells to survive better in an oxidative stress environment. To determine if CFTR deficiency promotes this survival of CRC cells by change in ROS levels, ROS detection assays were carried out to compare ROS levels between CFTR WT and CFTR KO cell lines. We observed a trend where CFTR-WT cells had higher ROS levels than CFTR-KO in this oxidative stress environment. This suggests that CFTR deficiency maybe promoting survival of CRC cells in an oxidative stress environment by reducing ROS levels. This work will help to better understand the role of CFTR as a tumor suppressor gene in CRC.
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    Lanthanide-Based Probes for Oxidative Stress
    (2014-06) Peterson, Katie
    Oxidative stress, or the imbalance of reactive oxidative species and antioxidants, is implicated in a wide variety of physiological functions and diseases. Currently, little is known about the biological concentrations and the exact roles of individual species. In particular, the cellular concentration of hydroxyl radical and the etiology of this reactive oxygen species in disease states are unclear. The photophysical properties of luminescent lanthanide-based imaging agents and the magnetic properties of fluorinated contrast agents make them favorable candidates to monitor oxidative species in biological environments. Luminescent lanthanide-based probes for hydroxyl radical are presented. These probes utilize aromatic acid pre-antennas that sensitize terbium emission upon hydroxylation. The ability of hydroxylated and non-hydroxylated aromatic acids including benzoate, benzamide, isophthalate, isophthalamide, trimesate, and trimesamide to sensitize Tb DO3A was evaluated by time-delayed luminescence spectroscopy. The formation of a weak ternary complex between hydroxytrimeasamide and Tb-DO3A was confirmed by temperature-dependent titrations. The luminescence response of the bimolecular Tb DO3A and trimesamide probe to hydroxyl radical generated by the photolysis of hydrogen peroxide was investigated. The system exhibits excellent selectivity for hydroxyl radical over other biologically relevant reactive oxygen and nitrogen species. Next, fluorinated magnetic resonance imaging contrast agents responsive to hydroxyl radical are described. The 3,5-difluorobenzoic acid probe is water soluble and ratiometrically responds to hydroxyl radical. Upon hydroxylation, a fluoride ion is released. The relative signal intensity of the product and that of the unreacted contrast agent can then be used to monitor the analyte in a ratiometric manner by 19F NMR and 19F MRI. The selectivity of the system towards hydroxyl radical compared to other reactive oxygen and nitrogen species is also measured. Paramagnetic, lanthanide-based contrast agents incorporating the sensing moiety are also evaluated for increased sensitivity of detection compared to the diamagnetic analogs. Additionally, a family of lanthanide-based luminescent complexes based on a macrocyclic core featuring different sensitizing antennas and variable pendant arms are investigated in terms of their biological compatibility. The cellular uptake of Tb-DOTA complexes containing hydroxyisophthalamide (IAM), methoxyisophthalamide (IAM(OMe)), or phenathridine (Phen) antenna were comparable despite their differences in hydrophobicity. The luminescence quenching of Tb-DOTA-IAM(OMe) was also investigated in cell lysate by time-delayed spectroscopy. Pendant arms varying in hydrophobicity and charge were used to evaluate the effect of structural and electronic properties on cellular viability and cell association as measured by a MTT assay and ICP-MS, respectively. Regardless of the amide substituents, complexes based on Tb-DOTAm-IAM(OMe) core exhibited low cytotoxicity and low cellular association. Thus, complexes based on this platform are well-suited for the detection of extracellular analytes.
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    Oxidative Stress: aging and disuse.
    (2009-05) Chen, Chiao-nan
    Sarcopenia, the age-related decline of muscle mass and strength, is one major risk factor for frailty and mobility disability of the elderly. Muscle disuse due to bed rest or surgery (such as joint replacements) exacerbates the ongoing decline of muscle function in the elderly. The decline of muscle function with disuse is greater in aging muscles. However, the cellular mechanism responsible for the greater functional decline of aging muscles with disuse is unknown. Oxidative stress, a condition where the balance between oxidant production and removal is disrupted, is a shared mechanism of age and disuse related muscle dysfunction. Thus, the overall aim of my dissertation is to understand the role of oxidative stress in the age-related muscle dysfunction with disuse.Using an animal model of muscle disuse (hindlimb unloading), I tested the hypothesis that the ability of aging muscles to cope with the increased oxidative stress associated with muscle disuse is compromised. There are three major findings: (1) the regulation of glutathione (GSH), an essential endogenous antioxidant, is impaired in aging muscles with disuse; (2) the decline of GSH levels in aging muscles with disuse is associated with the decrease of glutamate cysteine ligase (GCL) activity and the reduction of the catalytic subunit of GCL content; (3) using proteomic techniques, I identified two proteins (carbonic anhydrase III and four-and-a-half LIM protein1, FHL1), which show changes in the oxidation levels with disuse and aging. The changes in the oxidation levels of these two proteins with disuse occur in adult rats but not old rats. However, old rats have greater baseline levels of oxidized FHL1.In summary, the series of studies demonstrate that the response of muscles with disuse is age-dependent. The ability to maintain GSH levels with disuse is compromised in aging muscles. In addition, the changes of protein oxidation with muscle disuse occur in specific proteins and that the changes are age-related.
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    The Role of Oxidative Stress in Remodeling the Cardiac Microtubule Cytoskeleton
    (2021-05) Goldblum, Rebecca
    Microtubules are cylindrical cytoskeletal polymers composed of α/β-tubulin heterodimers that make up an ordered tubulin lattice. In cells, microtubules form a network that is a key component of the cellular cytoskeleton. Under pathological conditions of oxidative stress, we and others have found that cardiomyocytes, the contractile cells in the heart, display a denser microtubule cytoskeleton, which may lead to the progressive structural and functional cellular changes associated with myocardial ischemia and systolic dysfunction. This reorganization of the microtubule network occurs despite only small increases in tubulin expression, suggesting that alterations to microtubule length regulation and stability are involved. Using biophysical reconstitution experiments and live-cell imaging, we found that oxidative stress may synergistically increase the density of microtubules inside of cells by simultaneously increasing the length of dynamic, short-lived microtubules, while fostering the longevity of stable, long-lived microtubules. We found that microtubules subjected to oxidative stress undergo cysteine oxidation, and our electron and fluorescence microscopy experiments revealed that the locations of oxidized tubulin subunits within the microtubule had structural damage within the cylindrical tubulin lattice, consisting of holes and lattice openings. For dynamic microtubules, incorporation of stabilizing GTP-tubulin into these damaged lattice regions led to an increased frequency of rescue events (the transition from shortening to growth), and thus longer microtubules. For long-lived microtubules, these same structural defects facilitate entry of the enzyme αTAT1 into the microtubule lumen, where it catalyzes the acetylation of α-tubulin. This intraluminal acetylation has been shown to increase the lifetime of stable microtubules by conferring mechanical stability to the microtubule lattice. In this way, oxidative stress triggers a dramatic, pathogenic shift from a sparse microtubule network into a dense, longitudinally aligned microtubule network inside of cardiac myocytes, likely contributing to increased cellular stiffness and contractile dysfunction. Our results provide insight into myocardial changes in ischemic heart disease by describing a mechanism for the dramatic remodeling of the microtubule cytoskeletal network within cardiac myocytes subjected to oxidative stress.