Browsing by Subject "Oxidative Stress"
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Item Differential Regulation of Phosphatidylinositol 4-Phosphate 5 Kinase Beta during Hypertonic and Oxidative Stress(2009-01-08) Chen, Mark; Yin, Helen L.Of the total amount of phospholipids in the cell, phosphoinositides account for only a small fraction, yet they play an indispensable role in regulating cellular homeostasis. The phosphatidylinositol derivative, PI(4,5)P2 (PIP2)is an essential mediator of cellular processes such as signal transduction, membrane traffic, actin cytoskeleton, ion homeostasis, growth and apoptosis. Despite the importance of this signaling molecule in controlling a diverse array of functions, little is known about the regulation of the kinases that produce PIP2 as well as its precursor, phosphatidylinositol 4 phosphate (PI4P). Our laboratory found that hypertonic and oxidative stress increases and decreases PIP2 levels, respectively. In this thesis, I sought to understand how these two different types of stresses alter PIP2 by examining the effects on the phosphatidylinositol 5-phosphate 4 kinases that generate PIP2. Using RNAi and in vitro kinase activity assays, I found that PIP5Kbeta is regulated by both stimuli in opposite directions and is responsible for the net change in PIP2 levels. Hypertonic stress activates PIP5Kbeta through PP1 phosphatase dependent ser/thr dephosphorylation which leads to increased kinase activity and plasma membrane localization. The PIP2 produced by PIP5Kbeta during this event leads to an increase in actin polymerization and stress fiber formation. Conversely, oxidative stress leads to a decrease in PIP5Kbeta activity, membrane detachment and stress fiber dissolution. Oxidative stress induces simultaneous tyrosine phosphorylation and ser/thr dephosphorylation, establishing that tyrosine phosphorylation decreases PIP5Kbeta activity and is the dominant modification of PIP5Kbeta that overrides the activating effects of ser/thr dephosphorylation. I further identified the tyrosine kinase Syk as the kinase responsible for PIP5Kbeta phosphorylation during oxidative stress. The phosphorylation site was mapped to position Y105 which was shown to control both activity and localization of PIP5Kbeta. Oxidative stress also increased PI4P which is likely to be at least partly due to activation of a PI4K. Using RNAi and wortmannin to selectively inhibit the activity of different PI4K isoforms, I sought to identify the PI4K isoform that is responsible for this increase.Item Preventing hypertension: the role of oxidative stress(2003-08-14) Nesbitt, Shawna D.Item Reactive oxygen biology & the cardiovascular system: a conspiracy of extremes(2003-01-09) Garcia, Joseph A.Item Role of Complement Factor H Polymorphism and Diet in Neuroinflammation(2014-02-04) Parnell, Samuel; Kasumu, Ade; Aredo, Bogale; Chen, Xiao; Ufret-Vincenty, RafaelBACKGROUND: Multiple lines of evidence point towards an important role for complement factor H (Cfh) in neuroinflammation. Evidence of activated microglia and activated astrocytes has been found in the brains of both Parkinson's disease and Alzheimer's disease patients.1 In addition, Cfh has been shown to be present in amyloid beta plaques in Alzheimer's disease.2 Our laboratory has developed a mouse model of early AMD based on expressing variant Cfh molecules in mice.3 The Cfh transgenic mice develop deposits under the retinal pigment epithelium, which resemble early changes seen on AMD. We believe these findings indicate that the variant Cfh molecules are less able to control chronic low grade inflammation at the tissue level. METHODS: Young (6m old) and aging (18m old) Cfh transgenic and control B6 mice were divided into groups and fed either a control diet or a high-fat diet for 5 months. Brains were collected after perfusion with 2% PFA/PBS and were post-fixed overnight in 4% PFA/PBS. Next, the brains were transferred to a 30% sucrose solution, weighed, and sectioned. Immunohistochemistry was then performed on 30 μm brain sections with antibodies specific for inflammatory, oxidative stress, and microglial markers. Stained images were visualized using a Leica fluorescence microscope with an objective lens of 10x. The microglia over the photographed field were counted and averaged in order to obtain a cells/field value for each mouse. Statistical analysis was then performed on the data. RESULTS: CfhTg brains weighed less than the corresponding B6 brains (0.447 vs. 0.482g; p=0.00024). Many of the tested antibodies, particularly those associated with oxidative stress, did not stain the sections, perhaps due to the fixation method. However, the anti-TREM2 and anti-Iba 1 antibodies stained well. There was no difference in the number of Iba-1+ microglia in the dentate gyrus of CfhTg vs. B6 mice (p=0.607). Younger mice seemed to have higher numbers of these cells compared to older mice (73.3 vs. 52.6 microglia/field; p=0.0352). In addition, the mice fed a high fat diet appeared to have less microglia per field compared to the mice fed a normal control diet (53.1 vs. 72.8 microglia/field; p=0.0469). TREM2 is considered to be a marker for microglial activation. Neither age (p=0.65), nor a high fat diet (p=0.435) appeared to affect the level of TREM2 expression. However, there was a trend towards higher numbers of TREM2+ cells in CfhTg mice compared to B6 mice (p=0.17), particularly in the old group of mice (p=0.12). CONCLUSIONS: Reduced brain mass in CfhTg mice suggests increased CNS oxidative stress and tissue injury. There was no difference in the number of Iba-1+ microglial cells in CfhTg vs B6 mice. However, there was a trend towards increased microglial activation in Tg mice. More brains will be analyzed with alternative methods of tissue collection and additional antibodies to corroborate these findings.Item Spatiotemporal Regulation of the NADP(H) Phosphatase Nocturnin(August 2021) Laothamatas, Isara; Mishra, Prashant; Takahashi, Joseph; Conrad, Nicholas; Green, Carla B.Periodic changes in the environment are ubiquitous in the natural world. Among these, the most biologically relevant rhythm is the 24-hour geophysical day/night cycle. As an adaptive strategy, many organisms have evolved an endogenous biological clock to temporally organize their physiology and anticipate daily changes in the environment. At its core, the mammalian "circadian clock" is a molecular oscillator driven by a genetic transcription/translation feedback loop, which orchestrates the rhythmic expression of thousands of genes. An intimate link between circadian clocks and metabolism is established by the rhythmic transcription of output genes involved in almost every metabolic pathway. Among these oscillating genes, Nocturnin (also Noct; protein name: NOC) has one of the highest amplitude rhythms at the mRNA level. Mice with a loss-of-function in Noct possess metabolic phenotypes, where they are protected from high-fat diet-induced obesity and LPS-induced septic shock. However, the mechanism by which this occurs is not well-understood. Here, in collaboration with Green lab members and the Liou lab, I used both in vitro biochemical and in vivo cellular and mouse models to elucidate the molecular and physiological function of NOC. Even though NOC is highly-conserved with the endonuclease/exonuclease/phosphatase (EEP) domain-containing CCR4 family of deadenylases, we show that highly-purified recombinant NOC lacks ribonuclease activity. Instead, NOC catalyzes the dephosphorylation of NADP(H), and its activity level is associated with the cellular response to oxidative stress. Furthermore, we describe two isoforms of NOC and their spatiotemporal regulation in the mouse liver. Cytoplasmic NOC is constitutively-expressed throughout the day and associates externally with the endoplasmic reticulum and other membranes via N-terminal glycine myristoylation. In contrast, mitochondrial NOC levels are highly circadian with peak expression during the early dark phase. Overall, our work suggests that NOC links circadian clocks to metabolism by regulating local intracellular concentrations of NADP(H) in a manner that changes throughout the day.