Dissertations / Theses on the topic 'Mitochondrial reactive oxygen species'
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Logan, Angela. "Production of reactive oxygen species in mitochondria and mitochondrial DNA damage." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609201.
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Hurd, T. R. "Interactions between mitochondrial protein thiols and reactive oxygen species." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604824.
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This work investigates the reactions of proteins with ROS when mitochondria are exposed to H2O2 or when they generate ROS endogenously. Using isolated mitochondria, those proteins that are particularly sensitive to low concentrations of H2O2 and to ROS generated by the mitochondrial electron transport chain were first identified using a method called Redox-Difference Gel Electrophoresis (Redox-DIGE). Most redox sensitive thiol proteins identified by Redox-DIGE were involved either in fatty acid oxidation or in the regulation of the pyruvate dehydrogenase complex. Next the mechanisms by which ROS selectively oxidise mitochondrial thiol proteins were investigated; it was determined that H2O2 generated by the electron transport chain may either oxidise mitochondrial thiol proteins directly or indirectly, through oxidation of the peroxiredoxin and thioredoxin redox couples. To determine if ROS generated by mitochondria might act as a redox signal by altering the functions of mitochondrial proteins, the effect of protein thiol oxidation was tested on the activity of two proteins: pyruvate dehydrogenase kinase and propionyl-CoA carboxylase. Loss of pyruvate dehydrogenase kinase and propionyl-CoA carboxylase activity correlated with protein thiol oxidation and was very sensitive to ROS, suggesting a plausible mechanism of redox regulation of these proteins in vivo. Lastly, glutathionylation of complex I was investigated in intact mitochondria exposed to a glutathione oxidant; two cysteine residues on the 75 kDa subunit of complex I were shown to become glutathionylated. The functional effect of glutathionylation of these two cysteine residues on complex I activity is currently under investigation.
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Li, Xinyuan. "Mitochondrial Reactive Oxygen Species Mediate Lysophosphatidylcholine-induced Endothelial Cell Activation." Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/320473.
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PharmacologyPh.D.
Lysophosphatidylcholines (LPCs) are a class of pro-inflammatory lipids that play important roles in atherogenesis. LPC activates endothelial cells (ECs) to upregulate adhesion molecules, cytokines and chemokines, which is the initiation step of atherogenesis. However, the mechanisms underlying LPC-triggered EC activation are not fully understood. Previously considered as the toxic by-products of cellular metabolism, mitochondrial reactive oxygen species (mtROS) are recently found to directly contribute to both the innate and adaptive immune responses. Here we tested a novel hypothesis that mtROS serve as signaling mediators for LPC-induced EC activation. Using electron spin resonance and flow cytometry with mtROS-specific fluorescence probe MitoSOX, we found that several LPC species including LPC 16:0, 18:0, and 18:1 induced mtROS in human primary aortic ECs (HAECs). Mechanistically, our analysis using confocal microscopy and Seahorse XF96 mitochondrial function analyzer showed that LPC induced mtROS via increasing mitochondrial calcium-mediated increase of mitochondrial respiration. In addition, we found that mtROS scavenger MitoTEMPO abolished LPC-induced EC activation by downregulating Intercellular adhesion molecule 1 (ICAM-1) in HAECs. Moreover, our analysis with mass spectrometer analysis of histone H3 lysine acetylation and electrophoretic mobility shift assay (EMSA) showed that MitoTEMPO acts by blocking LPC-induced histone H3 lysine 14 acetylation (H3K14ac) and nuclear translocation of pro-inflammatory transcription factor activator protein-1 (AP-1). Remarkably, all the above effects can be inhibited by anti-inflammatory cytokines interleukin (IL-35) and IL-10. Our results indicate that mtROS are responsible for LPC-induced EC activation, which can be inhibited by anti-inflammatory cytokines. MtROS targeting therapies and anti-inflammatory cytokines such as IL-35 may serve as novel therapeutic targets for vascular inflammation and cardiovascular diseases. The studies in this dissertation were supported by grants from the National Institutes of Health (NIH) funded to Dr. Xiao-Feng Yang.
Temple University--Theses
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Hinchy, Elizabeth. "How cellular ATP/ADP ratios and reactive oxygen species affect AMPK signalling." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/270029.
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Mitochondria are key generators of cellular ATP, vital to complex life. Historically, mitochondrial generation of reactive oxygen species (ROS) was considered to be an unregulated process, produced by dysfunctional mitochondria. More recently, mitochondrial ROS generated by complex I, particularly by the process of reverse electron transfer (RET), has emerged as a potentially biologically relevant signal that is tightly-regulated and dependent on mitochondrial status. ROS production by RET is reported to play a role in the innate immune response and lifespan extension in fruit flies. One way in which mitochondrial ROS may behave as a signal is by altering the activity of AMP-activated protein kinase (AMPK), a key metabolic sensor and regulator of cell metabolism, which is activated when cellular ATP levels decrease during energy demand. Mitochondria can signal to AMPK via the magnitude of the cellular ATP/AMP and ATP/ADP ratios, which alter in response to mitochondrial function. Our view is mitochondria may also signal to AMPK via ROS. Important studies have helped to clarify the role of exogenous or cytosolic ROS in AMPK regulation. However, the effects of mitochondrial ROS on AMPK activity, specifically that generated by complex I, remain unclear and is the main focus of this thesis. I characterized the effects of exogenous H2O2 on cellular AMPK activity, ATP/ADP ratios and cellular redox state in a cell model. I then compounded this with selective mitochondria generated ROS by the mitochondria-targeted redox-cycler, MitoParaquat (MPQ). AMPK activity appeared to correlate with decreasing cell ATP/ADP ratios, indicating that both sources of ROS primarily activate AMPK in an AMP/ADP-dependent mechanism. In parallel, I developed an approach for analyzing the redox state of candidate proteins, an important step in determining if a protein is directly regulated by ROS. I also initiated development of a cell model for studying the downstream effects of mitochondrial ROS production by RET, by expressing alternative respiratory enzymes in a mammalian cell line.
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Collins, Yvonne. "Regulation of pyruvate dehydrogenase kinase 2 by mitochondrial reactive oxygen species." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708470.
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Sanusi, Morufat Olayide Abisola. "Mitochondrial reactive oxygen species signalling and vascular smooth muscle cell senescence." Thesis, University of Leicester, 2016. http://hdl.handle.net/2381/37968.
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Ageing is a risk factor for the development of cardiovascular disease. In particular, senescent vascular smooth muscle cells (VSMCs) have been observed within atherosclerotic plaques. Oxidants are widely implicated in vascular ageing and cardiovascular disease with evidence of oxidative stress in cells undergoing senescence. Our previous data showed that Angiotensin II caused stress induced premature senescence (SIPS) in primary human VSMC via oxidant generation. Prevention of senescence with a mitochondria targeted antioxidant, Mito-TEMPO, suggested the mechanism was dependent on mitochondrial superoxide. The current study aimed to investigate if modulation of mitochondrial reactive oxygen species signalling is a general mechanism for senescence induction in human VSMC. The electron transport chain inhibitors Antimycin A and rotenone and the mitochondrial redox cycler, MitoParaquat all stimulated SIPS in VSMC. Interestingly, Antimycin A and rotenone also lead to a reduction in overall H₂O₂ levels suggesting a possible protective mechanism and highlighting the complexity of the signalling mechanism involving mitochondrial oxidants. qPCR Analysis suggested that changes in antioxidant gene expression do not account for the reduction in peroxide levels. Although there was no evidence that Angiotensin II induced senescence in human coronary artery SMC, there was evidence for enhanced mitochondrial hydrogen peroxide production. Senescent cells acquire a senescence associated secretory phenotype (SASP). To determine the composition of VSMC SASP, the tryptically digested secretome of conditioned media was analysed by LC-MS/MS. Bioinformatic analysis identified the NRF2-mediated oxidative stress response pathway and several endogenous antioxidants as amongst the affected responses in the aged VSMC secretome. These new data suggest that senescent VSMC produce a SASP that has multiple effects on neighbouring cell types including the induction of cell senescence and death; but also elements that might serve to preserve cell integrity and function and may limit the expression of a pro-inflammatory phenotype.
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Rogers, Kara Emilie. "Mitochondrial Antioxidants, Protection Against Oxidative Stress, and the Role of Mitochondria in the Production of Reactive Oxygen Species." Diss., The University of Arizona, 2006. http://hdl.handle.net/10150/194490.
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Mitochondria serve as the major source of reactive oxygen species (ROS) production in cells resulting in antioxidant systems and cell signaling pathways that are unique to mitochondria. Thioredoxin-2 (Trx-2) is the mitochondrial member of the thioredoxin superfamily, and acts specifically to reduce the mitochondrial peroxidase, peroxiredoxin-3. It has been proposed that Trx-2 associates with cytochrome c, which functions in mitochondrial respiration and apoptosis. Homozygous Trx-2 deletion in mice is embryonic lethal and it is hypothesized here that Trx-2 lethality is caused by loss of mitochondrial function and oxidative stress. Results of experiments investigating mitochondrial integrity, cell viability, and ROS levels in Trx-2(-/-) mouse embryonic fibroblasts (MEFs), and results from Trx-2 siRNA MEFs, are similar to findings of knockouts in previously reported proteins that function in mitochondrial respiration and support the involvement of Trx-2 in this process. Mitochondrial ROS have also been implicated as major secondary messengers in cell signaling. Results reported here using cancer cells and cancer cells depleted of mitochondrial DNA, which consequently produce few ROS, have indicated that mitochondrial ROS produced in hypoxia are necessary for HRE and ARE activation, and are fundamental in the activation of SP-1 during reoxygenation. However, mitochondrial ROS are not required for HIF-1α protein expression in hypoxia, indicating a unique relationship between HIF-1α, hypoxia, and mitochondrial ROS.
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Schwarzlander, Markus. "The Response to Mitochondrial Reactive Oxygen Species and Redox Status in Plants." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504582.
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Garlid, Anders Olav. "Mitochondrial Reactive Oxygen Species (ROS): Which ROS is Responsible for Cardioprotective Signaling?" PDXScholar, 2014. https://pdxscholar.library.pdx.edu/open_access_etds/1641.
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Mitochondria are the major effectors of cardioprotection by procedures that open the mitochondrial ATP-sensitive potassium channel (mitoKATP), including ischemic and pharmacological preconditioning. MitoKATP opening leads to increased reactive oxygen species (ROS), which then activate a mitoKATP-associated PKCε, which phosphorylates mitoKATP and leaves it in a persistent open state (Costa, ADT and Garlid, KD. Am J Physiol 295, H874-82, 2008). Superoxide (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (HO•) have each been proposed as the signaling ROS but the identity of the ROS responsible for this feedback effect is not known. Superoxide was excluded in earlier work on the basis that it does not activate PKCε and does not induce mitoKATP opening.To further examine the identity of the signaling ROS, respiring rat heart mitochondria were preincubated with ATP and diazoxide to induce the phosphorylation-dependent open state, together with agents that may interrupt feedback activation of mitoKATP by ROS scavenging or by blocking ROS transformations. Swelling assays of the preincubated mitochondria revealed that dimethylsulfoxide (DMSO), dimethylformamide (DMF), deferoxamine, trolox, and bromoenol lactone (BEL) each blocked the ROS-dependent open state but catalase did not interfere with this step. The lack of a catalase effect and the inhibitory effects of agents acting downstream of HO• excludes H2O2 as the endogenous signaling ROS and focuses attention on HO•. In support of the hypothesis that HO• is required, we also found that HO•-scavenging by DMF blocked cardioprotection by both ischemic preconditioning and diazoxide in the Langendorff perfused rat heart. HO• itself cannot act as a signaling molecule, because its lifetime is too short and it reacts immediately with nearest neighbor phospholipids and proteins. Therefore, these findings point to a product of phospholipid peroxidation, such as hydroperoxy-fatty acids. Indeed, this hypothesis was supported by the finding that hydroperoxylinoleic acid (LAOOH) opens the ATP-inhibited mitoKATP in isolated mitochondria. This effect was blocked by the specific PKCε inhibitor peptide εV1-2, showing that LAOOH activates the mitoKATP-associated PKCε. During ischemia, catabolism of mitochondrial phospholipids is accelerated, causing accumulation of plasmalogens and free fatty acids (FA) in the heart by the action of calcium independent phospholipases A2 (iPLA2). We first assessed the role of FAs and hydroxy FAs on mitoKATP opening and cardioprotection. Swelling assays of isolated rat heart mitochondria showed that naturally formed free FAs inhibit mitoKATP opening and that they are more potent inhibitors of the pharmacological open state of mitoKATP than the phosphorylation-dependent open state. That is, sustained mitoKATP opening induced by the phosphorylation-dependent feedback loop is more resistant to FA inhibition than direct mitoKATP opening by a potassium channel opener. Moreover, rat hearts perfused with micromolar concentrations of FA were resistant to cardioprotection by diazoxide or ischemic preconditioning. Racemic bromoenol lactone (BEL), a selective inhibitor of iPLA2, confers protection to otherwise untreated Langendorff perfused hearts by preventing ischemic FA release. To bring this story full circle, BEL blocks protection afforded by preconditioning and postconditioning by preventing the iPLA2-mediated release of FAOOH generated in the conditioned heart. HO• resulting from mitoKATP opening oxidizes polyunsaturated fatty acid components of the membrane phospholipids, resulting in a peroxidized side chain. FAOOH must be released in order to act on the mitochondrial PKCε, and this is achieved by the action of iPLA2. iPLA2 is essential for most modes of cardioprotection because it catalyzes the release of FAOOH. This fully supports the hypothesis that the second messenger of cardioprotective ROS-mediated signaling is hydroperoxy fatty acid (FAOOH), a downstream oxidation product of HO•.
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Hansson, Anna. "Cellular responses to respiratory chain dysfunction /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-493-7/.
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Apostolova, Nadezda. "Mitochondrial role of Apoptosis-Inducing Factor (AIF): Oxidative Phosphorylation and Reactive Oxygen Species." Doctoral thesis, Universitat de València, 2008. http://hdl.handle.net/10803/9775.
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The apoptotic function of Apoptosis-inducing factor (AIF) is well documented in theliterature, but its physiological role in the mitochondrion is less certain. Using a smallinterfering RNA (siRNA) strategy, we studied whether modulation of AIF expression incultured cells influenced the production of reactive oxygen species (ROS). We foundthat siAIF-transfected cells had reduced AIF protein levels and this was paralleled by asignificant increase in ROS. We tested the generality of this response by using twodifferent human cell lines, the hepatoma cell line Hep3B and cervix carcinoma lineHeLa, and also by employing a mouse ES AIF-KO cell line. The increased ROS weremitochondrial in origin as a similar silencing strategy in cells devoid of a functioningmitochondrial electron transport chain (ETC) did not result in a ROS-increase. Theaugmented ROS levels were sufficient to activate Hypoxia-inducible factor 1α (HIF-1α),a ROS-sensitive transcription factor, and this effect could be reversed usingantioxidants, both the broad-range general antioxidant (N-acetyl cysteine) and aspecific mitochondrial-targeted antioxidant (MitoQ), proving the implication of ROS inthe HIF-1α stabilization. We also studied another two redox-sensitive transcriptionfactor and thus observed up-regulation in the expression of Nuclear factor (erythroidderived2)-like 2 (Nrf2), however without major changes in Nuclear factor-kappa B(NF-κB) levels. Examination of the cellular oxygen consumption rate revealed that AIFdepletedcells had a major impairment of respiration, at Complex I in the ETC. Westernblot analysis also showed a loss of Complex I 39 and 20 kDa subunits. Studies usingthe antioxidants mentioned above, revealed that the respiratory competence could beregained in AIF-silenced cells. However, neither of the antioxidant treatments we usedcould recover Complex I assembly. Studies of the energetic state of siAIF cells showedthat despite a 30% decrease in the overall intact cell respiration, these cells maintainnormal basal levels of ATP, presumably due to a higher glycolytic capacity and a lowerproliferation rate. Moreover, we analyzed the expression of another redox-activeprotein, thioredoxin, by Western blot and found that the mitochondrial isoform, Trx2,was significantly decreased when AIF was silenced. Preliminary co-immunoprecipitationanalyses and proteomic studies failed to show any direct correlation between AIF andTrx2 at the protein level.Our results lead us to the conclusion that the defect in respiration in siAIF cells isdownstream of Complex I protein loss and is presumably due to ROS-mediateddamage to the ETC. This suggests an integral mitochondrial function of AIF, as a redoxmodifier and chaperone-like molecule, necessary for Complex I assembly. Additionalstudies are required to define the detailed mechanism of the AIF enzymatic activity inthe mitochondrion and to establish its binding partners.La función proapoptótica del Factor Inductor de Apoptosis (AIF) está biendocumentada, sin embargo su papel fisiológico en la mitocondria es menos conocido.Empleando la metodología de interferencia por ARN, estudiamos si la modulación de laexpresión proteica de AIF en cultivo celular modifica la producción celular de especiesreactivas de oxígeno (ROS). Observamos que el silenciamiento de AIF estaba seguidopor un incremento significativo en los niveles de las ROS. Estas ROS fueronmitocondriales de origen, puesto que el silenciamiento de AIF en células que carecende la cadena de transporte electrónico funcional (ETC) en la mitocondria no llevó a unincremento de ROS. Este incremento fue suficiente para activar el Factor inducible porhipoxia (HIF-1α), efecto que se puede revertir usando los antioxidantes, N-AcetilCisteina y MitoQ, demostrando así la implicación de los ROS en la estabilización deHIF1-α. Los análisis del consumo de oxigeno celular mostraron que las células de AIFsilenciado sufren una disminución en la respiración celular, al nivel del Complejo I de laETC, acompañada por una disminución significativa en la expresión de sus subunidades39 y la 20kDa. Tratamientos con los antioxidantes previamente nombrados mostraronque la tasa de respiración se puede recuperar, no siendo así con la expresión delComplejo I de la ETC. Estudios del estado energético de las células siAIF mostraronque a pesar de la disminución de 30% en la tasa de la respiración celular, estas célulasmantienen niveles normales de ATP, como resultado de un incremento en la capacidadglucolítica y una reducción en la tasa de proliferación. Posteriormente, analizamos laexpresión de la proteína tioredoxina y observamos una disminución significativa en laisoforma mitocondrial, la tioredoxina 2 (Trx2), aunque los análisis preliminares de coinmunoprecipitacióny proteómica no mostraron la existencia de una correlacióndirecta entre las proteínas AIF y Trx2.Concluyendo, nuestros resultados sugieren que el defecto de la respiración celular esposterior al defecto en el Complejo I, probablemente como consecuencia al daño de laETC por ROS. Esta observación apunta a un papel integrador de AIF en la mitocondria,como modulador del estatus redox y necesario para el ensamblaje del Complejo I.
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Maranzana, Evelina Susana Beatriz <1971>. "Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from Complex I." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6601/.
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Evidence accumulated in the last ten years has demonstrated that a large proportion of the mitochondrial respiratory chain complexes in a variety of organisms is arranged in supramolecular assemblies called supercomplexes or respirasomes. Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain.
Following this line of thought we have decided to directly investigate ROS production by Complex I under conditions in which the complex is arranged as a component of the supercomplex I1III2 or it is dissociated as an individual enzyme. The study has been addressed both in bovine heart mitochondrial membranes and in reconstituted proteoliposomes composed of complexes I and III in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio.
The results of this investigation provide experimental evidence that the production of ROS is strongly increased in either model; supporting the view that disruption or prevention of the association between Complex I and Complex III by different means enhances the generation of superoxide from Complex I .
This is the first demonstration that dissociation of the supercomplex I1III2 in the mitochondrial membrane is a cause of oxidative stress from Complex I. Previous work in our laboratory demonstrated that lipid peroxidation can dissociate the supramolecular assemblies; thus, here we confirm that preliminary conclusion that primary causes of oxidative stress may perpetuate reactive oxygen species (ROS) generation by a vicious circle involving supercomplex dissociation as a major determinant.
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Brouillette, Marc James. "Mechanical stimulation of cartilage induces mitochondrial reactive oxygen species production mediating metabolic responses." Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/5428.
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This dissertation project is unique in that it seeks to link two historically independent concepts: mechanical loading of cartilage (1) induces reactive oxygen species (ROS) release from specific mitochondrial complexes, and (2) results in observable metabolic alterations. It is well known that ROS are released from certain loading conditions. It has also been shown that chondrocytes respond favorably to cyclic loading at moderate stresses, as determined metabolically by proteoglycan and collagen production. However, this study aims to demonstrate that these phenomena are interdependent, and in doing so, locates both the source(s) of load-induced ROS and the resultant molecule(s) responsible for metabolic stimulation.
To further this investigation, an osteochondral explant mechanical loading platform was built that allowed the imposition of physiological stresses on cartilage explants to further characterize cartilage metabolism. National Instruments hardware and LabVIEW controls a stepper motor driven platen, which when coupled with a load cell, allows for dynamic and static compression stimulation of articular cartilage.
Firstly, static stress (0.05 – 1.0 MPa for one hour) induces ROS release, which is mitochondrial in origin, relies on an intact cytoskeletal network, and tracks linearly with bulk tissue strain (r = 0.87). Dissolution of the cytoskeleton with cytochalasin B, blocking complex I of the mitochondria with rotenone, or addition of the cell-permeable SOD mimetic, manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) reduces this ROS release at 0.25 MPa.
Next, under dynamic stress (0.25 MPa/0.5 Hz for one hour), this mitochondrial ROS release was shown to be necessary for stimulating glycolytic energy production 24 hours after stress application. The ROS release from mechanical stimulation was blocked by the addition of rotenone or Mitoquinone (MitoQ10). These treatments also both blocked the increase in intracellular adenosine triphosphate (ATP) content, and therefore show that the ROS from the mitochondria are required for stimulating ATP production.
Probing the mitochondria directly with targeted inhibitors in unloaded conditions shows that forcing superoxide generation at ubiquinol: cytochrome c-oxidoreductase (complex III), and efficiently turning this superoxide into hydrogen peroxide, resulted in a dose-dependent increase in ATP content that resembles the response to loading. Here, ATP content increased with increasing doses of antimycin A, which, when accompanied with the SOD mimetic, Galera (m40401), is always higher than antimycin A alone.
Finally, if overloading proceeds for too long (three hours at 1.0 MPa or 0.25 MPa at 0.5 Hz for 7 days), ROS-related damage ensues, resulting in significantly impaired mitochondrial function and reduced intracellular ATP content. The damage and deleterious effects are negated by administration of the antioxidant, N-acetylcysteine (NAC).
Together, these results show that mechanical stimulation of cartilage produces mitochondrial ROS and resultant products, whose role in articular cartilage is complex. In short term mechanical stimulations, these ROS act to stimulate metabolism. At higher stresses, and over longer durations, ROS cause damage which results in mitochondrial dysfunction and suppressed ATP production. These findings have important implications for the progression of osteoarthritis, which has already been linked to mitochondrial dysfunction
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Liu, Bin. "P53 AND REACTIVE OXYGEN SPECIES: A CONVOLUTED STORY." UKnowledge, 2007. http://uknowledge.uky.edu/gradschool_theses/450.
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The tumor suppressor p53 has a close relation with reactive oxygen species (ROS). As an indispensable component of the cellular redox system, ROS not only have been established to be involved in p53-dependent apoptosis, but also regulate p53 activity. Recent studies revealed several novel actions of p53, such as transactivation of antioxidative proteins, mitochondria translocation and inhibition of glycolysis. The fate of cells where p53 signaling pathways are initiated is either survival or death. In this review, we examine the hypothesis that ROS regulate cell fate through p53, in a way that physiological ROS levels trigger the protective pathways, while p53 behaves more like a cell killer under cytotoxic oxidative stress.
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Yang, Wen. "Mitochondrial dysfunction and reactive oxygen species metabolism in the aging process of «Caenorhabditis elegans»." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=94989.
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The nematode roundworm Caenorhabditis elegans has been extensively used to study the genetics of aging. The functions of many genes have been found to be able to affect this animal's lifespan. A large proportion of these genes are expressed in mitochondria, an organelle that plays important roles in energy metabolism and generates reactive oxygen species (ROS). These two characteristics of mitochondria have both been suggested to be crucial for lifespan determination. One hypothesis, called the oxidative stress theory of aging, proposes that high oxidative damage caused by ROS generated in mitochondria is the cause of aging. We found that the lifespan-increasing effect of electron transport chain (ETC) mutants (i.e. isp-1 and clk-1) as well as of several other long-lived mutants is not due to low oxidative damage, and that increasing oxidative damage fails to shorten their lifespan. I also identified a new long-lived mutant in a previously uncharacterized gene --nuo-6(qm200)--, which encodes the C. elegans orthologue of the B15 subunit of mammalian mitochondrial complex I. I found that this new mutation increases lifespan through the same pathway as isp-1(qm150) the previously characterized mutation in the iron sulphur subunit of complex III. Using RNA interference to knock down the level of expression of many mitochondrial genes has been found to increase the lifespan of C. elegans and of Drosophila by a mechanism that was assumed to be identical to that at work in mutations that altered the function of mitochondrial proteins. However, using the isp-1 and nuo-6 mutations I have shown that the mechanism that allows these mutants to be long-lived is, unexpectedly, completely distinct from the mechanism triggered by RNAi. Finally, I found that the generation of superoxide (a reactive oxygen species) is actually increased in these mutants, and that this elevation is necessary and sufficient for longevity. These studies challenge the traditional view about the roleLe nématode Caenorhabditis elegans est un organisme modèle qui a été extensivement étudié afin de mieux comprendre les bases de la génétique du vieillissement. Ces études ont mené à l'identification de plusieurs gènes jouant des rôles essentiels dans la détermination de la durée de vie de ce ver rond. La majorité de ces gènes sont exprimés au niveau des mitochondries, des organelles reconnues pour jouer un rôle primordial dans la régulation du métabolisme énergétique et pour être les principales sources des dérivés actifs de l'oxygène (reactive oxygen species, ROS). Ces deux caractéristiques propres aux mitochondries sont reconnues pour être cruciales dans la détermination de la durée de vie. Elles sont d'ailleurs à l'origine d'une hypothèse sur la cause du vieillissement, la théorie du stress oxydatif, qui propose que la génération de ROS par les mitochondries cause le vieillissement en induisant de façon graduelle des dommages oxydatifs aux divers constituants cellulaires. Dans cette étude, nous avons démontré que l'augmentation de la durée de vie observée chez des nématodes mutants pour des gènes mitochondriaux impliqués dans le transport des électrons (i.e. isp-1 and clk-1) ainsi que chez d'autres mutants reconnus pour leur longévité accrue n'est pas reliée à une diminution des dommages oxydatifs. D'autre part, nous avons montré que leur durée de vie n'est pas réduite suite à l'induction d'un stress oxydatif causant divers dommages. Cette étude nous a aussi amené à identifier un nouveau gène, nuo-6 (qm200), codant pour l'orthologue chez le ver de la sous-unité B15 du complexe I de la chaîne de respiration mitochondrial précédemment caractérisée chez les mammifères. La mutation de ce gène résulte en une augmentation de la durée de vie d'une manière similaire à ce qui est observé chez les vers mutants pour le gène isp-1(qm150), qui code pour une sous-unité du complexe III. L'utilisation d
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Deng, Ying. "ROLE OF THE REACTIVE OXYGEN SPECIES PEROXYNITRITE IN TRAUMATIC BRAIN INJURY." UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_diss/667.
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Reactive oxygen species (ROS) is cytotoxic to the cell and is known to contribute to secondary cell death following primary traumatic brain injury (TBI). We described in our study that PN is the main mediator for both lipid peroxidation and protein nitration, and occurred almost immediately after injury. As a downstream factor to oxidative damage, the peak of Ca2+-dependent, calpainmediated cytoskeletal proteolysis preceded that of neurodegeneration, suggesting that calpain-mediated proteolysis is the common pathway leading to neuronal cell death. The time course study clearly elucidated the interrelationship of these cellular changes following TBI, provided window of opportunity for pharmacological intervention.
Furthermore, we conducted a pharmacological study to solidify our hypothesis. First of all, we tested the potency of a membrane permeable, catalytic scavenger of PN-derived free radicals, tempol for its ability to antagonize PN-induced oxidative damage. Tempol successfully inhibited PNinduced protein nitration at dosages of 30, 100 and 300mg/kg. Moreover, early single dose of 300mg/kg was administered and isolated mitochondria were examined for respiratory function and oxidative damage level. Our data showed that tempol reduced mitochondrial oxidative damage, and maintained mitochondrial function within normal limits, which suggested that tempol is efficiently permeable to mitochondrial membrane and mitochondrial oxidative damage is essential to mitochondrial dysfunction. Next, we found that calpainmediated proteolysis is reduced at early treatment with a single dose of tempol. However, the effect of tempol on calpain is short-lived possibly due to systematic elimination. In our multiple dose study, tempol showed a significant inhibitory effect on SBDPs. Consequently, we measured neuordegeneration with the de Olmos aminocupric silver staining method at 7 days post-injury and detected a significant decrease of neuronal cell death.
Together, the time course study and pharmacological study strongly support the hypothesis that PN is the upstream mediator in secondary cell death in the CCI TBI mouse model. Moreover, inhibition of PN-mediated oxidative damage with the antioxidant, tempol, is able to attenuate multiple downstream injury mechanisms. However, targeting PN alone may be clinically impractical due to its limited therapeutic window. This limitation may be overcome in future studies by a combination of multiple therapeutic strategies.
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Ash, Catherine Elizabeth. "Functional and molecular mechanisms underlying reduced mitochondrial reactive oxygen species generation under dietary restricted feeding conditions." Thesis, University of Liverpool, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.494167.
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Dietary restriction (DR) across a wide phylogenetic range increases both mean and maximal lifespan by retarding the ageing process and delaying the onset and incidence of age-related pathologies. DR animals exhibit reduced tissue oxidative damage but anti-oxidant enzyme activities and other defence mechanisms against reactive oxygen species (ROS) are not consistently upregulated In such animals. As an alternative explanation ROS generation was Studied in isolated mitochondria from tissues of DR rats. About 90% of cellular ROS generation is thought to result from the single electron transfer to molecular oxygen within the mitochondria. Indeed mitochondria isolated from a range of tissues from DR rats had lower rates of H2O2 generation when compared to age matched control animals, whilst mitochondrial state III and IV respiration rates were unaltered.
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Raucci, Frank. "SPHINGOLIPID-INDUCED ACTIVATION OF THE VOLUME-SENSITIVE Cl− CURRENT IS MEDIATED BY MITOCHONDRIAL REACTIVE OXYGEN SPECIES." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/2016.
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Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in a number of models of cardiovascular disease. Previously we showed that angiotensin II (Ang II), endothelin-1 (ET-1), endothelial growth factor receptor (EGFR), and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and mitochondria are involved in the activation of ICl,swell by both osmotic swelling and Beta1 integrin stretch. Sphingolipid metabolism is modulated in several cardiopathologies and because sphingolipids are bioactive lipids involved in signaling cascades that overlap significantly with these modulators of ICl,swell, we investigated the role of sphingolipids in the regulation of ICl,swell in cardiac ventricular myocytes. Under isoosmotic conditions that isolate anions currents, addition of exogenous, cell permeant C2-ceramide (C2-Cer) elicited an outwardly-rectifying Cl− current that reversed near the Cl− equilibrium potential (ECl) in both physiological and symmetrical Cl− gradients. This current was inhibited by the ICl,swell-specific blockers DCPIB. Dihydro-C2-ceramide (C2-H2Cer), the inactive analogue of C2-Cer, failed to elicit current. These data strongly suggest that the identity of C2-Cer-induced Cl− current is ICl,swell and indicate that sphingolipid signaling pathways may be involved. Bacterial sphingomyelinase (SMase), which converts endogenous sphingomyelin in the outer leaflet of the sarcolemmal membrane to native chain-length ceramides, elicited a DCPIB-sensitive Cl− current. SMase-induced current is also suppressed by tamoxifen, which under conditions that isolate anion currents is a specific inhibitor of ICl,swell. SMase-induced ICl,swell was abrogated by ebselen, a membrane permeant glutathione peroxidase mimetic that dismutates H2O2 to H2O. This suggests that ROS are required mediators of SMase-induced activation of ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes and both have been implicated in modulating ICl,swell. Blocking NOX with apocynin or the NOX fusion peptide inhibitor gp91ds-tat had no effect on SMase-induced current. However, pretreatment of cardiomyocytes with gp91ds-tat reduced the maximum current amplitude of SMaseinduced ICl,swell, indicating that NOX may play a time-dependent role in this mechanism. By contrast, the mitochondrial Complex I blocker rotenone, which suppresses extramitochondrial ROS release by Complex III, completely suppresses SMase-induced ICl,swell. Additionally, SMase-induced ICl,swell is partially inhibited by blockade of mitochondrial KATP (mitoKATP) channels with 5-hydroxy-decanoic acid (5-HD). MitoKATP channels have been implicated as modulators of mitochondrial ROS release. Thus these data suggest that mitochondrial ROS generation is required for SMaseinduced activation of ICl,swell. Ceramides are metabolized to form several sphingolipids, including sphingosine-1-phosphate (S1P). We tested whether ceramide metabolites are responsible for eliciting ICl,swell. Under isosmotic conditions that isolate anion currents, SMase-induced ICl,swell was abrogated by blockade of ceramidase, which converts ceramide to sphingosine, with Derythro-MAPP. SMase-induced ICl,swell also was suppressed by inhibition of sphingosine kinase with DL-threo-dihydrosphingosine. These data suggested that the ceramide metabolite S1P is likely to stimulate ICl,swell. As expected, exogenous S1P elicited an outwardly rectifying Cl− current that was fully inhibited by DCPIB. As seen with SMaseinduced ICl,swell, S1P-induced ICl,swell was fully inhibited by rotenone. In contrast to results with SMase, S1P-induced current was partially inhibited by blockade of NOX with apocynin. These data indicate that S1P is a necessary component of SMase-induced ICl,swell activation and that the action of exogenous S1P involves ROS from both mitochondria and NOX. Importantly, the fact that exogenous C2-ceramide also activates ICl,swell even though C2-ceramide may not metabolized to S1P in native cells. Thus, it seems likely that ceramides can elicit ICl,swell via S1P and also by a distinct pathway and that both pathways converge at mitochondrial ROS. In order to determine the role of ERK in the proposed signaling pathway that regulates ICl,swell, we examined the effect of ERK inhibitors PD98059 and U0126 on the activation of ICl,swell. Both of these agents partially inhibited SMase-induced activation of ICl,swell, indicating SMase acts through both ERK-dependent and ERK-independent signaling pathways. HL-1 cells are derived from a murine atrial cell line that retains phenotypic characteristics of adult cardiomyocytes. Recently, ICl,swell has been observed in HL-1 cells with similar regulatory mechanisms to those seen in native cells. We showed that SMase elicits an outwardly-rectifying, DCPIB-sensitive Cl− current that reverses near ECl in HL-1 cells. Finally, we confirmed the production of ROS by SMase-induced signaling by flow cytometry in HL-1 cells using the nominally H2O2-selective fluorescent probe CH2DCFDA-AM. Exposure to SMase increased ROS production, as did the positive control H2O2. SMase-induced ROS generation was suppressed by pretreatment with rotenone but was unaffected by pretreatment with gp91ds-tat. These data indicate that exogenous and endogenous sphingolipids elicit ICl,swell in cardiomyocytes by stimulating mitochondrial ROS production. NOX may contribute to the ROS generation, but is not a required step in this mechanism. Sphingolipid signaling is likely to play an important role in stimulating ROS production and activating ICl,swell in a number of cardiovascular diseases.
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Duca, Edward. "Invadolysin, a conserved lipid droplet-associated protease interacts with mitochondrial ATP synthase and regulates mitochondrial metabolism in Drosophila." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5562.
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Invadolysin (inv) is a member of the M8 class of zinc-metalloproteases and is conserved throughout metazoans. It is essential for development and invadolysin homozygous Drosophila mutants are third instar larval lethal. These larvae exhibit a reduced larval brain size and an absence of imaginal discs. Detailed analysis showed that inv mutants exhibit pleiotropic effects, including defects with chromosome architecture, cell cycle progression, spindle assembly, nuclear envelope dynamics, protein turnover and problems with germ cell migration. These findings indicated that Invadolysin must have a critical role in Drosophila. In order to better understand these roles, I set out to identify genetic interactors of invadolysin. I performed a genetic screen scoring for enhancer/suppressor modification of a ‘rough eye’ phenotype induced by invadolysin overexpression. Screening against the Drosdel ‘deficiency kit’ identified numerous genetic interactors including genes linked to energy regulation, glucose and fatty acid pathways. Immunofluorescence experiments in cultured cells showed that H. sapiens Invadolysin localises to the surface of lipid droplets (LD), and subcellular fractionation confirmed its enrichment to these structures. Lipid droplets are highly dynamic organelles involved not only in energy storage but also in protein sequestration, protein and membrane trafficking, and cell signaling. Drosophila fat bodies are enriched in LDs and therefore important energy stores. In addition, they are nutritional sensors and regulators, which are proposed to be the ortholog of vertebrate liver and adipose tissue. Mutant inv fat bodies appeared smaller and thinner than wild type fat body, and accumulated lower levels of triacylgylcerides. This indicated that the loss of invadolysin might be affecting lipid metabolism and storage, confirming the genetic data. However, it was not clear whether these effects were due to the direct action of Invadolysin. Hence, transgenic fly lines expressing either HA, RFP or FLAG tagged forms of Invadolysin were generated to identify physical interactors of Invadolysin. Subsequent mass spectrometry analysis detected ATP synthase-α, -β and -d as interactors. This result suggested that Invadolysin might play a role in regulating mitochondrial function, which might then be manifest in the fat body as the defects previously observed. Energy levels are known to affect the cell cycle, cell growth, lipid metabolism and inevitably development. Further in vivo and in vitro experiments confirmed this hypothesis. Genetic crosses confirmed the interaction of invadolysin with ATP-synthase subunit-α, whilst staining of mitochondria in mutant third instar larval fat bodies suggested decreased mitochondrial activity. Mutants also showed lower ATP levels and an accumulation of reactive oxygen species, hence indicating the possibility of a dysfunctional electron transport chain. Lipid droplets are known to interact with mitochondria, whilst ATP synthase has been found on lipid droplets by proteomic studies in Drosophila. Therefore, based on these data, we propose that Invadolysin is found, with ATP synthase, on lipid droplets, where Invadolysin (likely acting as a protease) could be aiding the normal processing or assembly of ATP synthase. This interaction is vital for the proper functioning of ATP synthase, and hence mitochondria. In this scenario, cellular ATP needs are not met, energy levels drop which results in an inhibition of fatty acid synthesis, cell and organismal growth defects.
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Heyno, Eiri. "Production of reactive oxygen species in plasma membranes, mitochondria and chloroplasts." Paris 11, 2009. http://www.theses.fr/2009PA112192.
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Les formes réactives de l’oxygène (FRO) ont été analysées dans différents compartiments cellulaires en utilisant des méthodes spectroscopiques (UV/VIS, fluorescence, infrarouge, résonance paramagnétique électronique). L’identité et les mécanismes catalytiques des enzymes qui produisent les FRO dans les membranes plasmiques (MP) et les mitochondries ont été étudiés, ainsi que le rôle protectif de l’oxydase terminale plastidiale (PTOX) des chloroplastes. Cd2+ s’est révélé être un inhibiteur de la NADPH oxydase des MP. In vivo Cd2+ inhibait la production extracellulaire de O2. - mais stimulait l’accumulation de H2O2. Dans des mitochondries isolées, Cd2+ a augmenté la production de FRO. Antimycin A a entrainé une élévation du H2O2 extracellulaire, confirmant que la mitochondrie est le site principal de production de l’H2O2 extracellulaire induite par Cd2+ in vivo. Une quinone réductase (QR) génératrice de FRO a été isolée des MP. La déprotonation PH dépendante du quinole a produit des formes intermédiaires instables qui génèrent des FRO par réaction avec O2. Des espèces quinoniques ont été détectées dans la MP et pourraient servir de substrat aux QR in vivo. La protection de la chaine photosynthétique de transfert d’électron par la plastoquinol ; O2 oxydoréductase a été étudiée chez des plantes PTOX+ surexprimant PTOX. En raison de leur réponse altérée en conditions de faible et de forte intensité lumineuse, il a été proposé que pour fonctionner comme enzyme protectrice, PTOX est couplée à une SOD. Chez les lignées PTOX+, le niveau de SOD chloroplastique n’était pas plus élevé, limitant probablement leur capacité à détoxifier les taux élevés de O2. - généréProduction of reactive oxygen species (ROS) was studied in different subcellular compartments using spectroscopic methods (UV/VIS, fluorescence, infrared and electron paramagnetic resonance). The identities and catalytic mechanisms of ROS-producing enzymes in the plasma membrane (PM) were studied as well as mitochondrial ROS accumulation in response to cadmium (Cd2+) and the protectrice role of the plastid terminal oxidase (PTOX). Cd2+ was shown to be an inhibitor of the PM superoxide (O2. -) –producing NADPH oxidase in vitro. In vivo Cd2+ inhibited the extracellular production of O2. - but stimulated the accumulation of hydrogen peroxide (H2O2 ). Cd2+ induced ROS production in isolated mitochondria. Mitochondrial ROS- inducing inhibitors increased extracellular H2O2 production confirming these organelles the main source of Cd2+ induced extracellular H2O2 generation in vivo. A ROS-producing quinone reductase(QR) was isolated from PMs. ROS production occurred via the pH –dependent deprotonation of the end product menadiol leading to its intermediate forms that react with O2 forming O2. - and H2O2. A potential naphthoquinose species in PMs was identified that could serve as a QR substrate in vivo. The protection of the photosynthetic electron transfer chain by the PTOX, a plastoquinol : O2 oxidoreductase, was studied in PTOX-overexpressing plants (PTOX+ ). Based on the altered response to low and high light conditions in PTOX+ it was proposed that PTOX is coupled to an SOD in order to function as a protective enzyme. The absence of additional chloroplastic SOD in PTOX+ lines might have limited the plant’s ability to detoxify O2. - produced by elevated levels of PTOX+
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Kramer, Adam Hildyard. "An investigation of the role of mitochondrial STAT3 and modulation of Reactive Oxygen Species in adipocyte differentiation." Thesis, Rhodes University, 2014. http://hdl.handle.net/10962/54632.
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Stem cells have the ability to differentiate into a myriad of different cell types. The understanding of the differentiation process is of paramount importance if we are to use these cells in the lab as well as in therapeutics. Here, the levels and localization of the signal transducer and activator of transcription 3 (STAT3), with particular attention focused on the mitochondrial serine 727 phosphorylated form of STAT3 (pSTAT3S727) during differentiation, was investigated. Using the murine preadipocyte progenitor cell line 3T3-L1, as well as adipose derived human mesenchymal stem cells (HMSC-ad) as differentiation models, the relative levels of Reactive Oxygen Species (ROS) and the levels and localization of STAT3 were investigated during the differentiation process. ROS is known to play an important signalling role during differentiation and is well reported during the events of adipogenesis. ROS are generated as a by-product in the Electron Transport Chain (ETC), and it has recently been reported that pSTAT3S727 plays an important role at complex I of the ETC. Various techniques including fluorescence confocal microscopy, flow cytometry and Western blots were utilized to investigate the non-canonical role STAT3 plays during adipogenesis. Mitochondrial isolations were performed to investigate the levels of STAT3 in the mitochondria during differentiation. Further to this, an impedance based real time differentiation assay was developed using the xCELLigence Real Time Cell Analyser to monitor differentiation and the affects various compounds, including a STAT3 inhibitor, have on differentiation. Results indicate that upon induction of differentiation, levels of mitochondrial pSTAT3S727 dramatically decrease and leave the mitochondria. This corresponds to increasing levels of ROS. The canonical active form of STAT3 following phosphorylation on tyrosine 705 (pSTAT3Y705) was found to decrease and lose its nuclear localization. These initial results indicate that STAT3 plays an important non-canonical role in the mitochondria during differentiation.
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Costa, Rute Alves Pereira e. 1984. "Avaliação das funções mitocondriais de células deficientes na proteína XPC, envolvida na via de reparo por excisão de nucleotídeos (NER) = Evaluation of mitochondrial functions of XPC protein deficient cells, involved in nucleotide excision repair (NER) pathway." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/311361.
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Orientadores: Nadja Cristhina de Souza Pinto, Anibal Eugenio VercesiTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Xeroderma Pigmentosum (XP) é uma doença rara, autossômica recessiva, caracterizada por fotossensibilidade, mudanças pigmentares, envelhecimento precoce da pele e incidência elevada de neoplasias de pele. XP é causada por mutações em, pelo menos oito genes, que caracterizam sete diferentes grupos de complementação genética (XP-A a XP-G) e um tipo variante (XP-V). Mutações em cada em dos genes envolvidos resultam em diferentes graus de severidade da doença, principalmente quanto ao comprometimento neurológico. Pacientes XP-C apresentam mutações no gene Xpc, que resultam, geralmente, em proteínas truncadas e instáveis. XPC é uma proteína envolvida na via de reparo de DNA por excisão de nucleotídeos (NER) e sua função é reconhecer a lesão na fita de DNA e dar início ao reparo. Recentemente, a participação indireta de XPC no reparo por excisão de bases (BER) foi sugerida, através de sua interação física e funcional com a DNA glicosilase OGG1. Uma vez que OGG1 é essencial para a remoção de purinas oxidadas do DNA mitocondrial, nós hipotetizamos que o DNAmt, e consequentemente a função mitocondrial, estariam comprometidas em células deficientes em XPC. Desta forma, este trabalho se propôs a investigar alterações bioenergéticas mitocondrias em células obtidas de pacientes XP-C. Nossos resultados revelaram que linhagens celulares XP-C apresentavam menor função mitocondrial, apesar de não apresentarem alterações no número de cópias de DNAmt. O consumo de oxigênio pelo complexo I estava significativamente diminuído em células XP-C quando comparado à células controle, enquanto que o consumo de O2 via os complexos II, III e IV foi maior em células XP-C. A capacidade de captar cálcio também se mostrou alterada nas células XP-C, uma vez que essa célula era incapaz de captar e reter concentrações fisiológicas desse íon. A produção de espécies reativas de oxigênio foi significativamente maior em células XP-C comparadas a células controle. Em acordo, a atividade das enzimas antioxidantes superóxido dismutase e glutationa peroxidase foi menor em células XP-C, indicando um desbalanço redox nessas células. A análise da expressão de genes relacionados à biogênese mitocondrial revelou que um regulador transcricional fundamental, o coativador PGC1?, estava significativamente reduzido em células XP-C transformadas e primárias. Resultados de Western blotting e imunofluorescência revelaram que as alterações bioenergéticas e genômicas observadas em células XP-C eram via sinalização e não por efeito direto, uma vez que nas condições experimentais utilizadas neste trabalho, XPC não está presente na mitocôndria. Nossos resultados demonstram, pela primeira vez, que a proteína XPC exerce um papel indireto na manutenção da integridade funcional da mitocôndria, provavelmente através de seu papel no controle da expressão de genes envolvidos na biogênese mitocondrial
Abstract: Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by photosensitivity, pigmentary changes, premature skin aging and increased incidence of skin cancer. XP is caused by mutations in at least eight genes, which characterize seven different genetic complementation groups (XP-A to XP-G) and variant type (XP-V). Mutations in each gene result in varying degrees of severity, mostly regarding the presence or not of neurodegeneration. XP-C is caused by mutations in the Xpc gene, resulting, mostly, in a truncated and unstable protein. The XPC protein is involved in the nucleotide excision repair pathway (NER), where it functions as a damage recognition factor. Recently, a role for XPC in the base excision repair (BER) pathway has been proposed, through its physical and fucntional interaction with the DNA glycosylase OGG1. Since OGG1 has a major function in repairing oxidized purines in the mitochondrial DNA (mtDNA), we hypothesized that XPC played a role in maitaining mtDNA integrity, and consequently, mitochondrial function. Thus, this study proposes to investigate mitocondrial function in XP-C cell. Our results showed that XP-C cells had less mitochondrial function, although without changes in mtDNA copy number. Oxygen consumption through complex I was lower in XP-C cells compared to control cells, while respiration through complexes II, III and IV was higher in XP-C cells. Calcium uptake and retention by mitochondria was also decreased in XP-C cells, as these cells were unable to retain even physiological spikes in calcium concentration. Reactive oxygen species production was significantly higher in XPC cells compared to controls. In agreement to that, the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase was significatly decreased in XP-C cells, indicating that these cells are under a severe redox signaling inbalance. The analysis of the expression of genes related to mitochondrial biogenesis revealed that the key transcriptional regulator PGC1? was significantly lower in both transformed and primary XP-C cells. The results of Western blotting and imunofluorescence revealed that the bioenergetic impairment observed in XP-C cells is likely the result of changes in expression and signaling pathwyas, since, under the experimental conditions used here, XPC is not present in mitochondria. Our results indicate, for the first time, that XPC plays an important role in mitochondrial maintenace, likely via its role in transcription regulation of mitochondrial biogenesis
Doutorado
Fisiopatologia Médica
Doutora em Ciências
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Ho, Hsiang-Ting. "The Role of Reactive Oxygen Species in Arrhythmogenicity of Cardiac Glycoside." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1373401702.
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Silva, José Pablo. "The pathophysiology of respiratory chain dysfunction /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-234-9/.
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Blein, Sophie. "Étude de la variabilité du génome mitochondrial comme facteur de susceptibilité au cancer du sein." Thesis, Lyon 1, 2014. http://www.theses.fr/2014LYO10240/document.
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Une large part de la composante génétique du risque de cancer du sein est encore inexpliquée. J'ai ainsi étudié dans quelle mesure les variants observés sur le génome mitochondrial pourraient en partie expliquer ce risque. En effet la mitochondrie, en tant que source d'énergie cellulaire, est un organite impliqué dans la synthèse des espèces oxygénées réactives ou radicaux libres, éléments contribuant à l'instabilité génomique et au développement tumoral. Un premier axe de recherche m'a conduit à étudier une interaction potentielle entre des variants du génome mitochondrial et du génome nucléaire, en conjonction avec la consommation d'alcool. J'ai ensuite analysé les haplogroupes mitochondriaux peuvent être considérés en tant que potentiels modificateurs de l'association entre le risque de cancer du sein et les mutations causales portées par les gènes BRCA1 et BRCA2. L'haplogroupe T1a1 a été identifié comme modificateur du risque conféré par les mutations pathogènes localisées sur le gène BRCA2. Enfin, j'ai caractérisé par séquençage à haut débit le génome mitochondrial de 436 femmes ayant un cancer du sein et de forts antécédents familiaux, mais n'étant porteuses d'aucune mutation causale sur BRCA1 et BRCA2. Plusieurs variants ont été prédits comme dommageables. Deux gènes en particulier MT-ATP6 et MT-CYB, sont spécifiquement enrichis à la fois en nombre de variants portés, et de par le nombre d'individus porteurs de ces variants dans notre étude. L'ensemble du travail réalisé a ainsi contribué à enrichir les connaissances sur les potentielles associations entre les variations du génome mitochondrial et le risque du cancer du seinA large part of the genetic component of breast cancer risk (BCR) is still unexplained. Therefore I studied if variants of the mitochondrial genome (mtDNA) might explain a part of this risk. In fact, mitochondria is the main source of reactive oxygen species (ROS), which contribute to genomic instability and tumor development. As a first axis of research, I studied potential interactions between some nuclear and mitochondrial variants, in conjugation to alcohol consumption. Despite the large dimensions of our dataset, the lack of statistical significant interaction in our data might reveal that former published results that show such interactions were not robust. I also studied if mitochondrial haplogroups could be considered as modificators of known association between BCR and pathogenic mutations in the BRCA1/2 genes. I identified haplogroup T1a1 such as modificator for individuals carrying a mutated BRCA2. Finally, I characterized by NGS mitochondrial genome of women diagnosed for a familial breast cancer, but tested negative for known pathogenic BRCA1/2 mutations. Several variants were identified as potentially damaging. Two genes, MT-ATP6 and MT-CYB are specifically enriched both in terms of distinct variants and in the number of individuals carrying these variants. They are both essential structural components of the mitochondrial respiratory chain, the main ROS production source in the cell. All these analyses contribute to enrich the knowledge about associations between BCR and variability of mtDNA, by integrating questions linked to interactions between genomic variants, environmental exposure, and effect modifications related to mitochondrial haplogroups
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Zuo, Li. "Molecular Mechanisms of Stress-induced Reactive Oxygen Species Formation in Skeletal Muscle." The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1038853894.
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Lucas, Stephen Marc. "Valproic Acid Leads to an Increase in ROS Generation by Inhibiting the Deacetylation of Mitochondrial SOD." BYU ScholarsArchive, 2020. https://scholarsarchive.byu.edu/etd/9247.
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Valproic Acid Promotes Acetylation of Superoxide Dismutase-2 During Neurogenesis. Valproic acid (VPA) is a known developmental toxicant associated with a high prevalence of neural tube defects (NTD). The mechanism of VPA-induced NTD is unclear, but oxidative stress may be implicated. To understand how embryotoxic oxidative stress may occur, we measured superoxide dismutase (SOD) activity following VPA treatment in the embryonic pluripotent P19 mouse carcinoma cell line. In undifferentiated P19 cultures treated with VPA (5 mM), dichlorofluorescein fluorescence increased 15% compared to untreated controls over 20 min, indicating a modest, yet statistically significant increase in ROS generation. Undifferentiated P19 cells were treated with VPA for 6 h, after which total SOD and mitochondrial SOD (SOD2) activities were measured. VPA treatment decreased total SOD activity by approximately 20% but SOD2 activity was undetectable; but this was not a consequence of changes to SOD (SOD1 or SOD2) protein concentrations. Interestingly, glutathione redox state increased from -262 mV to -245 mV after a 6 h treatment with VPA, indicating significant oxidation of the cellular redox environment. Measurement of mitochondrial superoxide levels showed an increase following VPA treatments. While it is unlikely that VPA works directly as an oxidant, these data suggest that VPA may promote oxidative stress through an alternative means, such as via the inhibition of SOD activity and thus, allow for an increase in ROS. Importantly, VPA is a known deacetylase inhibitor, and SOD2 function is regulated by acetylation. As such, we evaluated the acetylation state of SOD2 to determine potential disruption via acetylation. Treated undifferentiated P19 cells showed a significant increase in SOD2 acetylation. However, in fully differentiated P19-derived neurons, cells showed no such SOD2 acetylation. Additionally, pretreatment with dithiole-3-thione (D3T), a Nrf2 activator of the antioxidant response, attenuated VPA-induced mitochondrial ROS production and SOD2 acetylation and improved SOD2 activity, suggesting Nrf2 as a potential means to reduce VPA-mediated oxidative stress. To evaluate the effects in the embryo proper, gestational day 8 mouse embryos were treated with VPA in culture for 6 h. Similar to P19 cells, VPA-treated neurulating embryos showed significant SOD2 acetylation and a concomitant decrease in total SOD activity. These data support a similar consequence of VPA-induced oxidative stress in embryos as is demonstrated in our cellular model. Since no SOD2 acetylation is observed in differentiated neurons and VPAinduced SOD2 acetylation occurs more prevalently in undifferentiated/differentiating cells, these data purport means by which VPA preferentially induces oxidative stress in developing systems.
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Bowling, Benjamin D. "Inhibition of mitochondrial protein translation sensitizes melanoma cells to arsenic trioxide cytotoxicity via a reactive oxygen species dependent mechanism." [New Haven, Conn. : s.n.], 2008. http://ymtdl.med.yale.edu/theses/available/etd-11212008-111938/.
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Barlow, Jonathan. "Mitochondrial involvement in pancreatic beta cell glucolipotoxicity." Thesis, University of Plymouth, 2015. http://hdl.handle.net/10026.1/3314.
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High circulating glucose and non-esterified free fatty acid (NEFA) levels can cause pancreatic β-cell failure. The molecular mechanisms of this β-cell glucolipotoxicity are yet to be established conclusively. In this thesis by exploring mitochondrial energy metabolism in INS-1E insulinoma cells and isolated pancreatic islets, a role of mitochondria in pancreatic β-cell glucolipotoxicity is uncovered. It is reported that prolonged palmitate exposure at high glucose attenuates glucose-stimulated mitochondrial respiration which is coupled to ADP phosphorylation. These mitochondrial defects coincide with an increased level of mitochondrial reactive oxygen species (ROS), impaired glucose-stimulated insulin secretion (GSIS) and decreased cell viability. Palmitoleate, on the other hand, does not affect mitochondrial ROS levels or cell viability and protects against the adverse effects of palmitate on these phenotypes. Interestingly, palmitoleate does not significantly protect against mitochondrial respiratory or insulin secretion defects and in pancreatic islets tends to limit these functions on its own. Furthermore, strong evidence suggests that glucolipotoxic-induced ROS are of a mitochondrial origin and these ROS are somehow linked with NEFA-induced loss in cell viability. To explore the mechanism of glucolipotxic-induced mitochondrial ROS and associated cell loss, uncoupling protein-2 (UCP2) protein levels and activity were probed in NEFA exposed INS-1E cells. It is concluded that UCP2 neither mediates palmitate-induced mitochondrial ROS production and the related cell loss, nor protects against these deleterious effects. Instead, UCP2 dampens palmitoleate protection against palmitate toxicity. Collectively, these data shed important new light on the area of glucolipotoxicity in pancreatic β-cells and provide novel insights into the pathogenesis of Type 2 diabetes.
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Julienne, Cloé Mimsy. "Altérations du métabolisme énergétique mitochondrial lors de la cachexie cancéreuse." Thesis, Tours, 2012. http://www.theses.fr/2012TOUR3318/document.
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La cachexie est un syndrome complexe caractérisé par une balance énergétique négative. Le rôle joué par le métabolisme énergétique mitochondrial dans ce syndrome est peu connu. Nos précédents travaux montraient une diminution de la synthèse de l’ATP dans les mitochondries hépatiques en stade de cachexie cancéreuse sévère. Dans ce travail, nous démontrons, in vitro, que l’augmentation de la production d’espèce réactive de l’oxygène et du contenu en cardiolipine dans des mitochondries hépatiques saines, mime partiellement les mécanismes observés lors d’un stade cachexie sévère. Nous observons cependant que l’altération du métabolisme mitochondrial hépatique apparait à un stade tardif du développement de la cachexie. En stade sévère les mitochondries musculaires ne développent pas d’altération de leur efficacité de synthèse d’ATP mais une diminution des leurs capacités oxydativesCancer cachexia is a composite syndrome, characterized by a negative energetic balance. The role played by mitochondrial energetic metabolism in this syndrome is poor known. Our past work showed a decrease of ATP synthesis efficiency in hepatic mitochondria in severe state of cancer cachexia. In this work, we demonstrate, in vitro, that increase of reactive oxygen species and cardiolipine content, in healthy mitochondria, can partly mimic the mechanisms observed in severe state of cancer cachexia. We observe that alteration of hepatic mitochondrial metabolism appear last during the development of cancer cachexia. In sever state of cancer cachexia, skeletal muscle mitochondria don’t develop this alteration but demonstrated a decrease of oxidative capacities
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Lashin, Ossama M. "Functional modification of cardiac mitochondria in type-I diabetes." Connect to text online, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1093467515.
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Thesis (Ph. D.)--Case Western Reserve University, 2005.[School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.
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Barnes, Robert. "A nuclear role for the respiratory enzyme CLK-1 in regulating reactive oxygen species, the mitochondrial unfolded protein response and longevity." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/a-nuclear-role-for-the-respiratory-enzyme-clk1-in-regulating-reactive-oxygen-species-the-mitochondrial-unfolded-protein-response-and-longevity(9a41f8d6-d11d-4102-b250-7b20091f4ded).html.
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As the major source of energy in the cell, mitochondria need to be able to effectively communicate their status to the nucleus. Defects in this process can have profound effects on the health of an organism and may affect its lifespan. The mitochondrial enzyme CLK-1 is required to produce ubiquinone for respiration and CLK-1 loss of function mutants have been shown to increase lifespan in both Caenorhabditis elegans and mammals. In this thesis, it is demonstrated that in addition to its mitochondrial role, CLK-1 also localises to the nucleus in C. elegans. This nuclear localisation is mediated by reactive oxygen species (ROS) and can be blocked using anti-oxidant treatment. In the nucleus CLK-1 regulates gene expression to suppress ROS production, suppresses the mitochondrial unfolded protein response and regulates lifespan. The importance of CLK-1 enzymatic activity for its nuclear role was also tested and it appears that enzymatic function is required to regulate ROS homeostasis but not for lifespan regulation. Interestingly, in fertile adult hermaphrodites nuclear CLK-1::GFP was only detected during the reproductive period. This suggests that there is a second mechanism regulating its localisation. This research indicates that CLK-1 may be part of a homeostatic regulatory mechanism that acts to suppress activation of stress responses in response to minor fluctuations in ROS levels.
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MacLellan, James Darcy. "Effects of the mitochondrial uncoupling protein 3 on fuel substrate oxidation and reactive oxygen species formation in rat L6 muscle cells." Thesis, University of Ottawa (Canada), 2007. http://hdl.handle.net/10393/27533.
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Uncoupling protein 3 (UCP3) is an integral mitochondrial membrane protein thought to disassociate fuel substrate oxidation by allowing proton re-entry into the mitochondrial matrix. Expression of UCP3 has been correlated with fatty acid and glucose metabolism, and reactive oxygen species (ROS) formation. To improve our understanding of the potential involvement of UCP3 in such pathways we investigated the effects of a UCP3 overexpression (2.2-2.5 fold) in the L6 muscle cell line. These findings were compared to those of UCP2 overexpression and DNP exposure. Palmitate oxidation was significantly increased by overexpressing UCP3 but unaffected by the other treatment conditions. Both glucose oxidation and oxygen consumption were unaffected by UCP2 and UCP3 overexpression but were significantly increased by DNP treatment. ROS production was decreased by UCP2, UCP3 and DNP treatment. These findings suggest a role for UCP3 in the regulation of fatty acid oxidation and ROS formation but not in glucose oxidation.
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Endoh, Yasumi Medical Sciences Faculty of Medicine UNSW. "New mechanisms modulating S100A8 gene expression." Publisher:University of New South Wales. Medical Sciences, 2008. http://handle.unsw.edu.au/1959.4/42942.
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S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.
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Herb, Marc [Verfasser], Björn [Gutachter] Schumacher, and Martin [Gutachter] Krönke. "Mitochondrial reactive oxygen species license pro-inflammatory signaling in infected macrophages via disulfide linkage of NEMO / Marc Herb ; Gutachter: Björn Schumacher, Martin Krönke." Köln : Universitäts- und Stadtbibliothek Köln, 2019. http://d-nb.info/1178671852/34.
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Jang, Kyoung-Jin. "Mitochondrial function provides instructive signals for activation-induced B cell fates." Kyoto University, 2015. http://hdl.handle.net/2433/199208.
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Merabet, Nadège. "Modélisation mathématique de la production d'espèces actives de l'oxygène par la chaîne respiratoire mitochondriale : vers une meilleure compréhension de l'atrophie optique dominante de type 1." Thesis, Toulouse 3, 2019. http://www.theses.fr/2019TOU30026.
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L’ATP est synthétisée par les mitochondries à partir de réactions d’oxydoréduction catalysées les complexes de la chaîne respiratoire. Ces réactions impliquent des transferts d’électrons intra-protéine. Une capacité de production de l’anion superoxyde, formé par la réaction de l’oxygène avec un électron, a été identifiée pour les complexes I et III. Les espèces actives de l’oxygène (EAOs) sont des molécules dérivées de l’anion superoxyde. Si elles ne sont pas correctement régulées par les défenses antioxydantes de la cellule, ces EAOs peuvent réagir avec les composants de la cellule et nuire à son fonctionnement : ce déséquilibre est appelé stress oxydatif. L’altération d’un ou plusieurs complexes respiratoires associée à un stress oxydatif cellulaire est un mécanisme commun à de nombreuses maladies neurodégénératives. Dans ce travail nous nous intéressons plus particulièrement à l’atrophie optique autosomique dominante de type 1 (ADOA-1). L’ADOA-1 est une maladie neurodégénérative principalement causée par des mutations du gène codant la protéine mitochondriale OPA1 impliquée dans la dynamique mitochondriale. Les tableaux cliniques et l’âge de début de la maladie sont variables. Il n’existe pas de corrélation claire entre génotypes et phénotypes permettant d’expliquer cette variabilité ni de traitement à cette pathologie. L’hypothèse d’un stress oxydatif a été proposée pour expliquer la variabilité de ces symptômes. C’est pourquoi notre objectif est d’améliorer la compréhension des mécanismes physiopathologiques impliqués dans cette maladie en développant des modèles mathématiques de la production des EAOs par la chaîne respiratoire. Nous avons utilisé deux méthodes de modélisation. Dans le premier cas, nous modélisons l’activité des complexes respiratoires et la production d’anion superoxyde par les complexes I et III par des équations de vitesse que nous construisons en trois étapes. Nous analysons d’abord les données biochimiques disponibles dans la littérature. Nous proposons ensuite des interprétations physiques à ces comportements et les traduisons sous forme de règles floues. Nous modélisons enfin ces règles en utilisant des fonctions données par le formalisme de Michaelis-Menten. Les équations de vitesse sont fonction de variables chimiques telles que la concentration des espèces chimiques impliquées dans les réactions des complexes respiratoires et ne prennent pas en compte le détail des réactions intra-protéine impliquées dans le fonctionnement des complexes. Cette méthode permet de construire un modèle simple, permettant de simuler l’activité des complexes I et III et leur production de superoxyde dans différentes conditions, et qui est facilement modifiable ou intégrable dans un modèle plus complet de la mitochondrie. Le modèle du complexe I que nous avons créé, est capable de simuler l’activité catalytique et la production des EAOs en mode direct par le complexe I pour différentes configurations et concentrations de substrats et produitsMitochondria are cellular organelles involved in ATP (adenosine triphosphate) supply to cells. Mitochondrial ATP is produced by the oxidative phosphorylation which involves redox reactions catalysed by the four protein complexes of the mitochondrial respiratory chain. These redox reactions require intra-protein electron transfers. The complex I and complex III of the respiratory chain are able to generate superoxide anion, which is formed by the reaction of oxygen with one electron. Reactive oxygen species (ROS) are molecules derived from the superoxide anion. ROS which are not regulated by cellular antioxidant defences can react with the components of the cells and disturb its functioning: this imbalance between ROS and antioxidant defences has been termed “oxidative stress”. Dysfunctions of one or several respiratory complexes associated to an oxidative stress is a mechanism common to numerous neurodegenerative diseases. In this work, we focus on autosomal dominant optic atrophy 1 (ADOA-1 or DOA-1). DOA-1 is a neurodegenerative pathology mainly caused by mutations in the gene OPA1 which codes for a mitochondrial protein involved in mitochondrial dynamics. The symptoms and ages of onset of the disease are variable. There is no clear correlation between genotypes and phenotypes which can explain this variability and to date, there is no established medical treatment for the disease. The hypothesis of an oxidative stress has been proposed to explain the variability of symptoms observed in patients. Indeed, the mitochondrial energetic metabolism is altered in biological models (cell cultures and animal models) of DOA-1 and low levels of antioxidant defences have been measured in cells from patients suffering from severe forms of the pathology. Hence, our objective is to improve the understanding of the physio-pathological mechanisms involved in this disease by developing mathematical models of ROS production by the respiratory chain. We use two modelling methods. The first method consists in modelling the activities of respiratory complexes and the superoxide production by complexes I and III with rate equations that we build in three steps. We first analyse the biochemical data available in the literature. We subsequently interpret this data physically and translate them in the form of fuzzy rules. We then model these rules with mathematical functions provided by the formalism of Michaelis-Menten. The rate equations depend on chemical variables such as the concentrations of chemical species involved in the reactions catalysed by the respiratory complexes. They do not include the details of intra-protein electron transfers, occurring during the catalysis performed by the complexes. This method enables us to build a simple model simulating the activities and superoxyde productions of complexes I and III in different conditions and that can easily be modified or integrated in a more comprehensive model of the mitochondrion. Our model of complex I can simulate the forward and reverse activities and ROS productions of the enzyme for different concentrations of substrates and products
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Belikov, Aleksey Vitalyevich [Verfasser], and Luca [Akademischer Betreuer] Simeoni. "The role of reactive oxygen species and mitochondria in T-cell activation / Aleksey Vitalyevich Belikov. Betreuer: Luca Simeoni." Magdeburg : Universitätsbibliothek, 2016. http://d-nb.info/1100055479/34.
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Desmoulins, Lucie. "Détection hypothalamique du glucose chez le rat soumis à un régime gras enrichi en saccharose : rôle de la dynamique mitochondriale et des espèces actives de l'oxygène d'origine mitochondriale." Thesis, Dijon, 2016. http://www.theses.fr/2016DIJOS024/document.
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L’hypothalamus participe au contrôle de l’homéostasie énergétique en détectant les signaux circulants tels que le glucose. L’hypothalamus médiobasal (MBH) en particulier, est capable de détecter l’hyperglycémie afin d’initier des réponses physiologiques adaptées, comme par exemple la sécrétion d’insuline via le système nerveux autonome (par un contrôle vagal). Notre équipe a récemment montré que la détection du glucose nécessite la production d’espèces actives de l’oxygène d’origine mitochondriale (mROS), fortement dépendante de la dynamique mitochondriale (fusion et fission). Récemment, l’étude de modèles génétiques ont permis de faire un lien entre ces évènements dynamiques dans le MBH et le développement de pathologies métaboliques. L’objectif de ma thèse a été tout d’abord été de mettre en place un modèle expérimental présentant uniquement une altération de la détection hypothalamique du glucose induite par l’exposition à un régime gras enrichi en saccharose (HFHS) chez le rat. Après avoir caractérisé ce modèle, nos objectifs ont été de déterminer si l’exposition à ce régime hypercalorique avait un impact sur la dynamique mitochondriale ainsi que la signalisation mROS, via la fonction respiratoire de la mitochondrie dans l’hypothalamus. Nous avons finallement réversé quelques acteurs métaboliques dérégulés, potentiellement impliqués dans la dynamique mitochondriale, dans le but de réverser le phénotype observé chez les rats HFHS. Nos résultats montrent qu’après 3 semaines d’exposition au régime HFHS, les rats ont un poids corporel normal malgré l’augmentation de leur masse grasse, comparés aux rats contrôles. Les rats HFHS présentent aussi une intolérance au glucose et une augmentation de la glycémie basale sans modification de leur insulinémie. La sécrétion d’insuline en réponse à la détection hypothalamique du glucose, mesurée après une injection intra-carotidienne de glucose en direction du cerveau qui induit une hyperglycémie uniquement cérébrale, a été fortement diminuée. Cependant, la capacité sécrétoire des îlots pancréatiques est normale chez les rats HFHS. Ces défauts sont associés à une diminution de la production de ROS dans le MBH en réponse au glucose, sans modification du status redox. L’efficacité de la respiration mitochondriale hypothalamique a été mesurée par oxygraphie, et les résultats montrent une déficience de la respiration mitochondriale chez les rats HFHS. La translocation de la protéine de fission DRP1 à la mitochondrie est diminuée en réponse au glucose, suggérant une diminution de la fission mitochondriale. L’augmentation de l’activation de l’AMPK dans l’hypothalamus n’est pas responsable de l’altération de la détection hypothalamique du glucose car sa réversion avec une injection intracérébroventriculaire (ICV) de composé C, n’a pas permis de restaurer la sécrétion d’insuline en réponse à l’hyperglycémie cérébrale. De même, une injection ICV de leptine induisant l’activation de STAT3 n’a pas permis de restaurer la sécrétion d’insuline en réponse à l’hyperglycémie cérébrale. Enfin, la diminution de l’activation d’AKT suggère une résistance centrale à l’insuline. Ces résultats démontrent pour la première fois que l’altération hypothalamique de la signalisation ROS, de la fission et de la respiration mitochondriale, sont présent chez les rats exposés pendant 3 semaines à un régime HFHS. Ces défauts précoces hypothalamiques pourraient ainsi participer à un défaut primaire du contrôle de la sécrétion d’insuline, et finallement, à l’installation d’un phénotype diabétiqueThe hypothalamus participates in the control of energy homeostasis by detecting circulating nutrients, such as glucose. The mediobasal hypothalamus (MBH), in particular, senses hyperglycemia and initiates physiological responses, e.g., insulin secretion via the autonomous (vagal) nervous system. We have recently demonstrated that glucose sensing requires mitochondrial reactive oxygen species (mROS) signaling heavily dependant on mitochondrial fusion and fission (dynamics). Recently, genetic models have associated some of these dynamics within the MBH to their obesogenic susceptibility. The aims of my thesis were first to establish a model that only presents a hypothalamic glucose sensing defect induced by a high fat high sucrose (HFHS) feeding in rats. After caracterizing this model, our objectives were to determine whether modulating the diet affects mitochondrial dynamics, and thus, mROS signaling, through the mitochondrial respiratory function in the hypothalamus. We finally reversed some dysregulated metabolic signalings potentially involved in mitochondrial dynamics in order to reverse the phenotype observed in HFHS fed rats. Our results demonstrate that after 3 weeks of HFHS feeding, rats had a normal body weight despite an increase in the fat mass compared to control rats. HFHS fed rats displayed also a glucose intolerance, increased fasting glycemia but no modification of fasting insulinemia. Hypothalamic glucose sensing induced insulin secretion, measured after an intra-carotid glucose injection towards the brain that only increases brain glycemia without alteration in peripheral glycemia, was drastically decreased. However, glucose stimulated insulin secretion in isolated islets was not different compared to controls. These defects correlate with a decrease of MBH ROS production in response to glucose, with no modification in the redox status. Efficiency of hypothalamic mitochondrial respiration was evaluated using oxygraphy, and results showed mitochondrial respiratory deficiencies in HFHS fed rats. The fission protein DRP1 exhibited decreased mitochondrial translocation in the MBH in response to glucose, suggesting decreased mitochondrial fission. The increase of AMPK activation in the hypothalamus was not responsible for the alteration of hypothalamic glucose sensing since its reversal with an intracerebroventricular (ICV) injection of compound C failed to restore brain hyperglycemia induced insulin secretion. Likewise, an ICV injection of leptin that induced STAT3 activation also failed to restore brain hyperglycemia induced insulin secretion. Finally, the decrease in AKT activation suggested a central insulin resistance. These results demonstrate for the first time that hypothalamic alteration of mitochondrial ROS signaling, fission and respiration were present in rats exposed to a 3 weeks HFHS diet. Such hypothalamic glucose sensing defects are early events preceding those in islets. These early but drastic hypothalamic modifications could participate in a primary nervous defect of the control of insulin secretion, and finally, the etablishment of a diabetic phenotype
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Aitken, Gillian Roxburgh. "Investigation of UV-induced reactive oxygen and nitrogen species in human skin cells and its association with the individual complexes of the mitochondrial electron transport chain." Thesis, University of Newcastle Upon Tyne, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424156.
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Bagsiyao, Pamela. "Role of brain uncoupling proteins in energy homostasis and oxygen radical metabolism." Honors in the Major Thesis, University of Central Florida, 2007. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1025.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology
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Dehina, Leila. "Influence de l’ischémie et de la cinétique de reperfusion myocardique sur la structure et le fonctionnement des mitochondries chez le porc : effets de la trimétazidine, de la ranolazine et du propranolol." Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10006.
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La production de radicaux libres oxygénés (ROS), la surcharge calcique cytosolique et l’ouverture des pores de transition membranaires mitochondriales (mPTP) consécutives à l’ischémie myocardique (IM) sont aggravées lors de la reperfusion. Dans cette thèse, nous nous sommes intéressés : 1) à l’évaluation des effets de la trimétazidine sur le seuil électrique de fibrillation ventriculaire (VFT) et sur les lésions structurales et fonctionnelles des mitochondries lors de l’IM (étude 1, N=26 porcs); 2) à la détermination de la cinétique d’évolution des lésions d’ischémie/reperfusion (I/R) (étude 2a, N=30 porcs) ;3) à l’étude de l’impact de la ranolazine, du propranolol et de leur association dans la préventions des lésions d’I/R (étude 2b, N=30 porcs). Ces études qui ont été réalisées sur le cœur de porcs anesthésiés, ont permis de suivre l’évolution des paramètres électrophysiologiques et hémodynamiques cardiaques et, à l’échelle cellulaire, l’évolution de la structure et de la fonction des mitochondries. Les résultats montrent : 1) dans l’étude 1 : que la TMZ prévient la chute du VFT et l’ensemble des altérations structurales et fonctionnelles mitochondriales observées lors de l’IM ; 2) dans l’étude 2a : que les lésions observées durant l’IM sont significativement aggravées dans les premières dizaines de secondes de la reperfusion alors qu’une certaine amélioration est observée après 10 et surtout 45 min de reperfusion; 3) dans l’étude 2b : qu’un prétraitement par de la ranolazine, du propranolol et par leur association réduit la sévérité de ces lésions d’I/R. Les mécanismes moléculaires et cellulaires d’action des produits utilisés dans cette étude seraient en rapport avec l’amélioration des lésions de l’I/RThe generation of reactive oxygen species (ROS), the cytosolic calcium overload and the opening of mitochondrial permeability transition pores (mPTP) resulting from myocardial ischemia (MI) are aggravated during reperfusion. In the present work, the following points have been addressed: 1) the evaluation of trimetazidine effects on the electrical threshold of ventricular fibrillation (VFT) and both structural and functional alterations of mitochondria during MI (study 1, N=26 pigs); 2) the determination of the kinetics of ischemia/reperfusion (I/R) lesions (study 2a, N=30 pigs); 3) the protective effects of ranolazine and propranolol, alone or combined on I/R lesions (study 2b, N=30 pigs). All studies were performed in anesthetized pigs. They allowed to follow changes in cardiac electrophysiological and hemodynamic parameters and, at the cellular level, changes in the structure and function of mitochondria. The obtained results show: 1) in study 1, that TMZ can prevent the drop in VFT and all structural and functional alterations of mitochondria noticed during MI; 2) in study 2a, that the lesions seen during MI are significantly aggravated within the first seconds of reperfusion whereas some improvement is observed after 10 minutes and more markedly after 45 minutes of reperfusion; 3) in study 2b, that pretreatment with ranolazine or propranolol, alone or combined can reduce the severity of I/R lesions. The molecular and cellular mechanisms of action of both agents are thought to be involved in this improvement of I/R lesions
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Pan, Minglin, Ying Han, Rui Si, Rui Guo, Ankit Desai, and Ayako Makino. "Hypoxia-induced pulmonary hypertension in type 2 diabetic mice." SAGE PUBLICATIONS INC, 2017. http://hdl.handle.net/10150/623894.
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Hypoxia-induced pulmonary hypertension (HPH) is a progressive disease that is mainly caused by chronic exposure to high altitude, chronic obstructive lung disease, and obstructive sleep apnea. The increased pulmonary vascular resistance and increased pulmonary arterial pressure result in increased right ventricular afterload, leading to right heart failure and increased morbidity. There are several clinical reports suggesting a link between PH and diabetes, insulin resistance, or obesity; however, it is unclear whether HPH is associated with diabetes as a progressive complication in diabetes. The major goal of this study is to examine the effect of diabetic ''preconditioning'' or priming effect on the progression of HPH and define the molecular mechanisms that explain the link between diabetes and HPH. Our data show that HPH is significantly enhanced in diabetic mice, while endothelium-dependent relaxation in pulmonary arteries is significantly attenuated in chronically hypoxic diabetic mice (DH). In addition, we demonstrate that mouse pulmonary endothelial cells (MPECs) isolated from DH mice exhibit a significant increase in mitochondrial reactive oxygen species (ROS) concentration and decreased SOD2 protein expression. Finally, scavenging mitochondrial ROS by mitoTempol restores endothelium-dependent relaxation in pulmonary arteries that is attenuated in DH mice. These data suggest that excessive mitochondrial ROS production in diabetic MPECs leads to the development of severe HPH in diabetic mice exposed to hypoxia.
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Savu, Octavian. "Mechanisms of chronic complications of diabetes with focus on mitochondria and oxygen sensing." Stockholm, 2010. http://diss.kib.ki.se/2010/978-91-7409-764-1/.
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Giedt, Randy James. "Real-Time Acquisition and Analysis of Endothelial Mitochondrial Superoxide Radical Production and Membrane Potential During In Vitro Ischemia/Reperfusion." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1243541457.
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Giedt, Randy James. "Mitochondrial Network Dynamics in Vascular Endothelial Cells Exposed to Mechanochemical Stimuli: Experimental and Mathematical Analysis." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1333985787.
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Semont, Audrey. "Implication des ROS mitochondriaux dans le couplage excitation contraction cardiaque." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0426.
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L’activation électrique des cardiomyocytes, via le courant de dépolarisation qu’elle induit, est primordiale dans la contraction cardiaque qui requiert l’adéquation de la production d'énergie par les mitochondries et des besoins énergétiques de l’appareil contractile. Les espèces radicalaires de l'oxygène (ROS) ont été récemment impliquées dans la régulation de nombreux acteurs du couplage excitation-contraction cardiaque. L’objectif de ce travail est d’explorer l'implication des ROS d'origine mitochondriale dans la régulation du couplage excitation-contraction au niveau du cardiomyocyte, en conditions physiologiques et pathologiques. C’est à cette fin qu’un modèle de surproduction endogène de ROS par addition de succinate - substrat énergétique mitochondrial - dans des cardiomyocytes isolés de cœur de rat a été mis au point. Différents protocoles pharmacologiques utilisant différents antioxydants (Trolox, MitoTEMPO et EUK) nous ont permis d’établir l'origine mitochondriale des ROS produits en présence de succinate. Enfin une étude pharmacologique utilisant la molécule OP2113 (inhibiteur spécifique de la production mitochondriale de ROS au niveau du complexe I de la chaine respiratoire- Brevet Philippe Diolez) nous a permis d'établir qu'environ 80% de cette production provenait du complexe I de la chaîne respiratoire. Notre travail s’est alors attaché à étudier, sur des cardiomyocytes isolés battants, les effets des ROS mitochondriaux sur différents paramètres du couplage excitation-contraction. Le succinate, via une surproduction de ROS mitochondriaux, provoque une baisse de 50% de l'amplitude de la contraction. L’amplitude de la contraction initiale est rétablie en présence de Trolox, MitoTEMPO ou OP2113, ce qui indique l'implication des ROS mitochondriaux et plus spécifiquement ceux produits au niveau du site IQ. Cette surproduction de ROS induit également une diminution de l’amplitude du transitoire calcique impliquant une diminution de la concentration en calcium systolique au cours de la contraction. Ces effets sont annulés en présence de Trolox et OP2113. Nous nous sommes alors attachés à moduler la concentration mitochondriale de calcium grâce à la cyclosporine A (inhibiteur du pore de transition mitochondrial) et au Ru360 (inhibiteur de l'entrée de calcium dans la mitochondrie). Il s’est avéré que ces inhibiteurs induisent une production de ROS insensible au Trolox et à OP2113 dont l'origine reste à établir. En conclusion une augmentation de la production mitochondriale de ROS se traduit par une diminution de la contraction intracytosolique et du transitoire calcique. L'ensemble de nos résultats illustre donc l’importance majeure de la mitochondrie dans le couplage excitation-contraction. Nos résultats ouvrent de nouvelles perspectives thérapeutiques dans le contexte de l’insuffisance cardiaque aigue ou chroniqueThe electrical activation of the cardiomyocyte through a generated depolarisation current is essential in the cardiac contraction, which requires the adequacy of the mitochondrial energy production and the energy needs of the contractile system. Radical oxygen species (ROS) have recently been involved in the regulation of many actors of the excitation-contraction coupling. The aim of this study was to explore the involvement of mitochondrial ROS in the regulation of the excitation-contraction coupling in cardiomyocytes, under physiological and pathological conditions. A model of endogenous ROS overproduction with the use of succinate was developed in isolated rat cardiomyocytes. Different pharmacological protocols, using various antioxidants (Trolox, Mito-Tempo,EUK and OP2113) allowed us to establish the mitochondrial origin of ROS production. Finally, the use of OP2113, (a specific inhibitor of mitochondrial ROS production, Patent P.Diolez) enabled us to establish that approximately 80 % of ROS production came from complex I in the respiratory chain. To start with, isolated cardiomyocytes were used to study the effects of mitochondrial ROS on different excitation-contraction coupling parameters. Succinate induced an overproduction of mitochondrial ROS, which lead to a drop of 50% of the contraction amplitude. The initial amplitude of contraction wasrecovered with addition of Trolox, Mito Tempo or OP2113, which demonstrates the implication of mitochondrial ROS produced at the site of Iq. Secondly, the overproduction of ROS leadsto a decrease of the calcium transient amplitude, due to a decrease of systolic calcium concentration during contraction. These effects were inhibited by Trolox and OP2113. Finally, mitochondrial calcium concentration was modulated with the use of Cyclosporin A (mitochondrial transition pore inhibitor) and Ru360 (mitochondrial calcium entry inhibitor). The inhibitors induced a ROS production unresponsive to Trolox and OP2113. The origin of which remains to be established. To conclude, an increase of mitochondrial ROS production results in a decrease of contraction amplitude and calcium transients. Our overall results demonstrate the critical significance of the mitochondrion in the excitation-contraction coupling. Our results open new therapeutic perspectives in the context of acute or chronic heart failure
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Assaly, Rana. "Protection du myocarde ischémique et pore géant mitochondrial : applications pharmacologiques." Phd thesis, Université Paris Sud - Paris XI, 2011. http://tel.archives-ouvertes.fr/tel-00734466.
Full textAbstract:
La maladie coronaire d'origine ischémique reste l'une des principales causes de mortalité dans le monde industrialisé. Le traitement de l'ischémie aiguë du myocarde est cependant entré dans une nouvelle ère où la mortalité peut être diminuée de moitié en utilisant des procédures qui permettent un retour rapide du débit sanguin dans la zone ischémique du myocarde, c'est-à-dire la revascularisation. Toutefois, cette reperfusion entraîne par elle-même des complications appelées lésions de la reperfusion qui ont été décrites pour la première fois par Jennings et al., en 1960. Par conséquent, le développement de stratégies cardioprotectrices associées à la reperfusion constitue un besoin majeur en clinique afin d'améliorer la fonction myocardique, de diminuer l'incidence des arythmies, de retarder l'apparition de la mort cardiomyocytaire et de limiter la taille de l'infarctus du myocarde lors de l'ischémie/reperfusion (I/R). La découverte de deux formes principales de mécanismes cardioprotecteurs endogènes, qui consistent en la réalisation de brefs cycles d'I/R appliqués avant l'ischémie (pré-conditionnement ischémique) ou après une longue période d'ischémie au début de la reperfusion (post-conditionnement ischémique), a encouragé la recherche de nouveaux moyens pharmacologiques capables de protéger le myocarde ischémié/reperfusé et a développé nos connaissances sur les bases moléculaires des lésions et de la survie cellulaire au cours des processus d'I/R.L'étude des mécanismes responsables de l'induction de la mort cellulaire a permis de mettre en évidence le rôle prépondérant joué par la mitochondrie et l'augmentation de la perméabilité de ses membranes, induite notamment par la formation/ouverture d'un pore au niveau des points de contacts entre les membranes mitochondriales ; ce pore a été appelé " pore de transition de la perméabilité mitochondriale " (mPTP). L'inhibition de l'ouverture de ce pore apparaît comme une stratégie privilégiée pour protéger le myocarde.De nombreuses études ont montré que les espèces réactives d'oxygène (EROs) jouent un rôle majeur dans les lésions de l'I/R et dans l'ouverture du mPTP. En revanche, il existe peu d'informations claires sur le seuil et la période de production (ischémie et/ou reperfusion) des EROs qui conduisent à l'ouverture du mPTP.Par conséquent, nous avons mis au point un modèle cellulaire d'hypoxie/réoxygénation (H/R) afin d'établir une relation causale entre la production d'EROs, l'ouverture du mPTP et la mort cellulaire tout en explorant le rôle de différents types d'EROs.Ce modèle d'H/R développé sur des cardiomyocytes de rats adultes fraîchement isolés nous a permis de mesurer en temps réel et simultanément la production des EROs, l'ouverture du mPTP et la mort cellulaire. Nous avons montré que la production des EROs débute pendant la période d'hypoxie et que cette production est directement liée à l'augmentation du temps d'hypoxie. Cette production d'EROs à l'hypoxie, plus particulièrement de radicaux hydroxyles et de peroxyde d'hydrogène, a été directement relié, et ceci pour la première fois, à l'ouverture du mPTP et à la mort cellulaire lors de l'H/R.Nous avons utilisé ce modèle pour étudier le mécanisme d'action de deux stratégies pharmacologiques cardioprotectrices, un nouveau ligand de la protéine translocatrice mitochondriale (TSPO), le TRO 40303, et l'activation de la voie RISK (Reperfusion Injury Salvage Kinase) par la morphine. Nous avons ainsi montré que (1) les propriétés cardioprotectrices du TRO40303 sont associées à une inhibition de l'ouverture du mPTP, ce qui n'avait pas pu être démontré au moyen d'expériences réalisées ex vivo et (2) l'activation de la voie RISK par la morphine, qui aboutit à une limitation de la taille d'infarctus associée à une amélioration des fonctions respiratoires mitochondriales, entraîne également une inhibition de l'ouverture du mPTP et un retard de la mort cellulaire des cardiomyocytes isolés soumis à une H/R.La suite logique de ce travail sera de rechercher si l'inhibition du stress oxydant peut constituer un mécanisme commun aux deux stratégies pharmacologiques cardioprotectrices que nous avons décrites en utilisant notre modèle d'H/R. Pour cela, il serait possible d'étendre notre modèle à des animaux génétiquement modifiés pour appréhender plus précisément les phénomènes impliqués dans cette activité antioxydante.A plus long terme, il sera nécessaire d'approfondir nos connaissances sur la production d'EROs pendant l'I/R en recherchant plus spécifiquement l'origine de cette production, notamment le rôle joué par la mitochondrie et l'effet d'autres espèces réactives dans le but de cibler le traitement et de développer de nouvelles stratégies cardioprotectrices.
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Silva, Carolina Solon da 1982. "Apoptose induzida por palmitato em células HEPG2 depende da produção de TNF-Alfa." [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/309382.
Full textAbstract:
Orientador: Gabriel Forato AnhêDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A prevalência de esteato hepatite não alcólica (NASH) aumenta de 20% em indivíduos magros para 80% em pacientes obesos com inflamação hepática caracterizada por elevados níveis de TNF-alfa. Um dos eventos que caracteriza a evolução para NASH é a marcante morte de hepatóciotos resultante da ação do excesso de ácidos graxos livres circulantes. O mecanismo pelo qual o palmitato induz a apoptose é dependente, entre outros parâmetros, do aumento dos níveis de espécies reativas de oxigenio (EROS). O objetivo do presente trabalho foi avaliar se a apoptose de hepatócitos induzida pelo palmitato é dependente do aumento da produção de TNF-alfa. Para testar tal hipótese, utilizamos o Infliximabe, um anticorpo monoclonal específico anti-TNF-alfa, como ferramenta farmacológica para reverter as injúrias provocadas pelo palmitato. Foi observado que após 6 horas de tratamento com o palmitato houve um aumento de expressão de mRNA de TNF-alfa levando a um aumento de apoptose 24 horas após à exposição com o ácido graxo. Este fenômeno concordou temporalmente com um aumento na fosforilação das proteínas IkK, IKbeta e JNK, indicativo de ativação da via de sinalização do TNF-alfa. A apoptose induzida pelo palmitato foi revertida pela adição de um inibidor geral de síntese proteica (Ciclohexamida) ou de um anticorpo neutralizante para o TNF-alfa circulante. Além disso, a produção de EROs e a disfunção mitocondrial induzidas pelo palmitato também foram revertidos por estas estratégias farmacológicas. Com base em tais resultados, concluímos que a apoptose, o acúmulo de EROs e da disfunção mitocondrial induzidas pelo palmitato em células HepG2 são dependentes da produção de TNF-alfa
Abstract: In the last three decades, the prevalence of overweight and obesity has been continuously increasing. Obesity is a risk factor for developing a series of diseases such as whole-body insulin resistance and type 2 diabetes mellitus. Adipose tissue, originally considered merely energy storage, today is recognized as an endocrine organ able of secreting a variety of cytokines, hormones and other substances with specific biological activities, such as saturated fatty acids. Both long chain saturated fatty acids, like palmitate, and the proinflammatory cytokines, as TNF-alfa, are known to activate signaling pathways that promote apoptosis. The mechanism by which the palmitate induces apoptosis is dependent on cell type, for example, human hepatocellular carcinoma line (HepG2) treated with palmitate led lipotoxicity and to increased levels of reactive oxygen species (ROS). Thus, the objective of this study was to evaluate whether apoptosis in HepG2 cells is dependent on increased production of TNF-alfa induced by treatment with palmitate. To test this hypothesis, we used the Infliximab, a monoclonal antibody anti-TNF-alfa, as a pharmacological tool to reverse injuries caused by palmitate. We observed that palmitate increased the mRNA for TNF-alfa and phosphorylation of IkK, Ikbeta and JNK, all indicative of activation of inflammatory signaling pathways. Apoptosis induced by palmitate was suppressed by simultaneous treatment with cycloheximide or infliximab. Furthermore, the production of ROS and mitochondrial dysfunction induced by palmitate were also suppressed by these two pharmacological strategies. Based on these results, we conclude that apoptosis and related events such as increased ROS production and mitochondrial dysfunction induced by palmitate in HepG2 cells are dependent on autocrine action of TNF--alfa
Mestrado
Farmacologia
Mestra em Farmacologia
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Semete, Boitumelo. "Analysis of metallothionein gene expression in oxidative stress related disorders / by Boitumelo Semete." Thesis, North-West University, 2004. http://hdl.handle.net/10394/51.
Full textAbstract:
Increased reactive oxygen species (ROS) have been reported to be at the centre of various diseases. Although several reports have implicated elevated levels of ROS in the
pathogenesis of diabetes mellitus, the early detection of ROS is still not attainable. This limitation causes difficulty in the early diagnosis of ROS related disorders. The presence of high levels of ROS was reported to result in differential expression of antioxidant genes involved in protecting cells from their deleterious effects. Among the antioxidant genes that are expressed, it was postulated that expression of metallothioneins (MTs) are also induced. MTs are low molecular weight, cysteine-rich proteins involved in metal homeostasis and reported to harbour antioxidant function. The aim of this investigation was to explore MTs as biomarkers for elevated levels of ROS in
whole blood of type 2 diabetic (T2D) individuals. The level of ROS in diabetic, non-diabetic as well as individuals at risk of developing T2D was determined via the use of biochemical assays. Real-Time PCR was utilised to analyse the expression of MTs and the presence of MT proteins was analysed via the ELISA. In this study it was observed that diabetic individuals had elevated levels of ROS. However, no significant difference in the expression of MTs and the presence of MT proteins between the diabetic and non-diabetic individuals was observed. In vitro experimental conditions indicated that MT expression is induced by elevated levels of ROS. In pathological conditions the ROS-dependent induction of MT expression needs to be elucidated further. It therefore can be suggested that MTs can not yet be utilised as biomarkers for the detection of elevated levels of ROS in pathological conditions with ROS aetiology. This investigation also highlights the fact that blood is not an optimal medium in which this objective can be attained.Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2005.