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Qaisiya, Mohammed Ali Hassan. « UNCONJUGATED BILIRUBIN MEDIATED OXIDATIVE STRESS, ER STRESS, AND ACTIVATION OF NRF2 PATHWAY ». Doctoral thesis, Università degli studi di Trieste, 2014. http://hdl.handle.net/10077/10137.
Texte intégralRésumé :
2012/2013Elevati livelli plasmatici di bilirubina non coniugata (UCB) sono responsabili dell’ittero neontale che è fisiologico nella maggior parte dei casi. L’iperbilirubinemia severa e prolungata nel tempo può causare encefalopatia da bilirubina e Kernicterus che, se non trattati, possono lasciare pesanti sequele neurologiche e nei casi più gravi condurre a morte. La neurotossicità da bilirubina è ancora una delle principali cause di malattie neurologiche nei paesi via di sviluppo ed è un problema riemergente nei paesi sviluppati a causa delle anticipate dimissioni dall’ospedale dei neonati. I meccanismi molecolari responsabili della neurotossicità da bilirubina non sono ancora completamente chiariti. Questo lavoro riporta i risultati ottenuti durante il mio progetto di dottorato volto a studiare il “molecular signalling” coinvolto nella neurotossicità da bilirubina. L’obiettivo principale è stato valutare gli effetti di concentrazioni pro-ossidanti di bilirubina sullo stato redox cellulare e sullo stress del reticolo endoplasmico (ER stress). Ci siamo focalizzati sulla pathway che coinvolge Nrf2, analizzando i geni indotti dalla bilirubina per effetto di Nrf2 e studiando il signalling a monte coinvolto nella sua attivazione. Parallelamente abbiamo anche studiato la cascata di segnali coinvolti nell’ER stress. Tutti gli esperimenti sono stati condotti nella linea cellulare di neuroblastoma umano SH-SY5Y, alcuni ripetuti anche nella linea di epatocarcinoma HepG2 e in colture primarie di astrociti dalla corteccia cerebrale di ratto. I nostri risultati mostrano che concentrazioni tossiche di bilirubina inducono un 40% di mortalità cellulare tra 1 e 4 ore di trattamento che si mantiene stabile fino alle 24 ore di trattamento. Le cellule trattate con UCB mostrano un incremento del livello dei ROS intracellulare dopo 1 ora seguito dall’accumulo nucleare dell’Nrf2 endogeno dopo 3 ore. La bilirubina aumenta l’induzione della trascrizione dell’ARE-GFP reporter gene associata ad una up-regolazione di diversi geni target di Nrf2. L’induzione dell’espressione genica può essere suddivisa in due categorie principali:la risposta precoce (4h-8h) e la risposta tardiva (16h-24h).La risposta precoce inizia con l’induzione dell’espressione di ATF3 dopo 4 ore di trattamento ed è seguita da i trasportatori di amminoacidi (xCT and Gly1) dopo 8h. Per la risposta tardiva abbiamo visto l’induzione dell’espressione genica degli enzimi coinvolti nella sintesi del glutatione. (γGCL and TNX1),nella risposta antiossidante e di detossificazione (HO-1, NQO1, FTH)e nell’omeostasi del NADPH (ME1, and G6PD). In seguito al silenziamento specifico di Nrf2, il trattamento con bilirubina diminuisce l’induzione dell’mRNA solo dell’HO-1 (75%), del NQO1 (56%) e della FTH (40%) Inoltre l’induzione dell’HO-1 è ridotta se le cellule vengono pretrattate con l’antiossidante NAC (65%) e con specifici inibitori per PKC (80%), P38α (40%) and MEK1/2 (25%). Risulta evidente che l’induzione di ATF3 è la prima risposta generata dal trattamento con UCB. Di seguito abbiamo osservato un’induzione sequenziale dei marker dell’ER stress: da quelli coinvolti nel signaling di PERK a 4h (PERK, ATF3, ATF4, CHOP), dalla diminuzione della proteina della ciclina D1 dopo 1 h e dall’induzione di IRE1 (XBP1), ATF6, e BiP dopo 8h di trattamento. Da notare però che il silenzia mento di PERK non riduce l’induzione dell’espressione dell’mRNA di ATFs/CHOP, ma induce l’espressione dell’mRNA di GCN2. Riassumendo noi abbiamo dimostrato che la bilirubina causa mortalità cellulare, produce la formazione di ROS, provoca l’accumulo di Nrf2 nel nucleo e induce la risposta antiossidante mediata dalle sequenze ARE. La bilirubina induce l’espressione di diversi geni coinvolti nella risposta antiossidante, tra tutti l’HO-1 e il NQO1 sono indotti dalla bilirubina in maniera dipendente da Nrf2. Abbiamo anche dimostrato che lo stress ossidativo (OS) e la PKC sono i principali fattori coinvolti nell’attivazione di Nrf2/HO-1. I risultati ottenuti dimostrano che l’induzione di ATFs/CHOP e di PERK sono uno dei primi eventi associati alla tossicità da bilirubina. Allo stesso tempo il silenziamento di PERK non influisce sull’induzione di ATFs/CHOP mentre induce GCN2, suggerendo un meccanismo di compensazione tra il signalling di PERK e GCN2. Concludendo i nostri dati dimostrano che lo stress ossidativo e lo stress del reticolo endoplasmico sono coinvolti nella neurotossicità indotta da UCB nella linea di neuroblastoma umano SH-SY5Y. Le cellule sviluppano una risposta adattativa alla bilirubina inducendo OS and ER stress e aumentando l’espressione dei geni coinvolti nella risposta antiossidante (in parte via Nrf2 pathway) e nello stress del reticolo endoplasmico (UPR).
Elevated levels of unconjugated bilirubin (UCB) are responsible for neonatal jaundice, and in some case, severe hyperbilirubinemia exposes babies to bilirubin encephalopathy and kernicterus with the risk of neurological sequela and death. Bilirubin neurotoxicity is still a major cause of neurological injury in the developing countries and is a re-emerged problem in the developed countries, due to the early hospital discharge of newborns after birth. The molecular mechanisms of UCB induced neurotoxicty are incompletely elucidated. Present thesis are reported the results obtained during my PhD course aimed to investigate the molecular signaling involved in UCB induced neurotoxicity .The main goal of this work was to evaluate the effects of the pro-oxidant concentration of UCB on cellular redox state and ER stress. We focused on Nrf2 pathway, analyzing the genes induced by UCB at Nrf2-dependent manner and the up-stream signaling involved in Nrf2 pathway activation. In parallel, we also studied the ER stress cascade signaling. All experiments were conducted in SH-SY5Y neuroblastoma cell line, with some performed in HepG2 cells and primary culture of cortical astrocytes. Our results showed that SH-SY5Y neuroblastoma cells incubated with toxic concentration of UCB suffer a 40% loss of cell viability between 1h to 4h, reaching a plateau until 24h after UCB treatment. Treated cells showed an increased level of intracellular ROS after 1h followed by the nuclear accumulation of endogenous Nrf2 after 3h. UCB enhanced the transcriptional activation of ARE-GFP reporter gene associated with an up-regulation of several Nrf2 target genes. Expression response could be divided into two main categories: early (4h-8h) and late response (16h-24h). As far as early genes, UCB mediates a sequential transcription starting with the ATF3 up-regulation at 4h and followed by the induction of amino acid transporters at 8h (xCT and Gly1). On the contrary, for late genes, we observed an up-regulation of the enzymes involved in GSH synthesis (γGCL and TNX1), antioxidant/detoxification (HO-1, NQO1, FTH), and NADPH homeostasis (ME1, and G6PD). Specific Nrf2 siRNA against Nrf2 decreased the induction only of HO-1 (75%), NQO1 (56%), and FTH (40%) upon UCB exposure. HO-1 induction was reduced in cells pre-treated with antioxidant NAC (65%) and with specific signaling inhibitors for PKC (80%), P38α (40%) and MEK1/2 (25%). It was evident that ATF3 up-regulation at 4h represents the earliest response to UCB exposure. We observed a sequential activation of UPR sensors starting with PERK signaling at 4h (up-regulation of PERK, ATF3, ATF4, CHOP at 4h, and loss of cyclin D1 protein at 1h), followed by IRE1 (XBP1), ATF6, and BiP at 8h after UCB treatment. Interestingly, PERK siRNA does not changed the induction of ATFs/CHOP while induced GCN2 mRNA upon UCB exposure. In summary, we demonstrated that UCB mediates loss of cell viability, ROS generation, Nrf2 nuclear accumulation and induction of ARE. Nrf2 pathway activation was associated with the induction of multiple antioxidant genes, among all, HO-1 and NQO1 are induced by UCB at Nrf2-dependent manner. We observed that OS and PKC are the major up-stream signaling involved in Nrf2/HO-1 activation. Results demonstrated ATFs/CHOP induction and ER stress (initiated by PERK signaling) as one of the earliest event associated with UCB toxicity. However, PERK siRNA does not affected ATFs/CHOP induction by UCB while induced GCN2, suggesting a compensatory mechanism between PERK and GCN2 signaling. In conclusion, our data demonstrate that OS and ER stress are involved in UCB induced neurotoxicity in SH-SY5Y cells. The cells undergo an adaptive response against UCB induced OS and ER stress, through activation of multiple antioxidant genes (in part via Nrf2 pathway), and activation of sequential UPR sensors
XXVI Ciclo
1985
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Lee, Sang C., Jack Zhang, Josh Strom, Danzhou Yang, Thai Nho Dinh, Kyle Kappeler et Qin M. Chen. « G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress ». AMER SOC MICROBIOLOGY, 2017. http://hdl.handle.net/10150/622753.
Texte intégralRésumé :
Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did post translational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress.
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Tao, Shasha, Pengfei Liu, Gang Luo, de la Vega Montserrat Rojo, Heping Chen, Tongde Wu, Joseph Tillotson, Eli Chapman et Donna D. Zhang. « p97 Negatively Regulates NRF2 by Extracting Ubiquitylated NRF2 from the KEAP1-CUL3 E3 Complex ». AMER SOC MICROBIOLOGY, 2017. http://hdl.handle.net/10150/623934.
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Activation of the stress-responsive transcription factor NRF2 is the major line of defense to combat oxidative or electrophilic insults. Under basal conditions, NRF2 is continuously ubiquitylated by the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex and is targeted to the proteasome for degradation ( the canonical mechanism). However, the path from the CUL3 complex to ultimate proteasomal degradation was previously unknown. p97 is a ubiquitin-targeted ATP-dependent segregase that extracts ubiquitylated client proteins from membranes, protein complexes, or chromatin and has an essential role in autophagy and the ubiquitin proteasome system ( UPS). In this study, we show that p97 negatively regulates NRF2 through the canonical pathway by extracting ubiquitylated NRF2 from the KEAP1-CUL3 E3 complex, with the aid of the heterodimeric cofactor UFD1/NPL4 and the UBA-UBX containing protein UBXN7, for efficient proteasomal degradation. Given the role of NRF2 in chemoresistance and the surging interest in p97 inhibitors to treat cancers, our results indicate that dual p97/NRF2 inhibitors may offer a more potent and long-term avenue of p97-targeted treatment.
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Maltagliati, Anthony, et Anthony Maltagliati. « Nrf2 : A Candidate Therapeutic Target to Dampen Oxidative Stress in Acute Myocardial Infarction ». Thesis, The University of Arizona, 2016. http://hdl.handle.net/10150/623086.
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This literature review posits that the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an attractive candidate therapeutic target in the setting of acute myocardial infarction (AMI). This transcription factor binds to antioxidant response elements (ARE) in the promoter region of a battery of genes that collectively encode an array of antioxidant, phase II drug metabolism, metabolically stabilizing, and overall cytoprotective enzymes, facilitating their transcription at basal levels and increasing transcription in response to various cellular stressors. Following a brief background tutorial on normal cardiac myocyte cellular physiology, key events that occur early in ischemia and reperfusion are outlined and integrated. These include ionic and metabolic dysregulation, electron transport chain uncoupling, mitochondrial depolarization, and the generation of reactive oxygen species (ROS). Abrupt changes in response to ischemia prime opening of the mitochondrial permeability transition pore (MPTP) and cardiac myocytes to generate a burst of ROS upon reperfusion–two key events that contribute to the umbrella term ischemia-reperfusion injury (IRI). How ROS damage cells is then outlined, and through a ROS-centric viewpoint, a case will be made as to how exogenous upregulation of Nrf2 could protect and/or salvage at-risk tissue immediately subjected to infarction and neighboring tissue in the peri-infarct zone (PIZ). The history of how Nrf2 came to be known as the "master regulator of oxidative stress" is reviewed, as well as the discovery of the canonical mechanism of Nrf2 regulation via Kelch-like ECH-associated protein 1 (Keap1) and other alternative mechanisms of endogenous Nrf2 regulation. Finally, compiling interdisciplinary evidence from research publications around the world, the benefits of therapeutically targeting Nrf2 are considered given the timescale and context of acute MI. Drug delivery methods, potential challenges, and limitations are then considered. Cardiac tissue is a dynamic substrate that exhibits changes for up to 90 days after AMI and patient outcomes are directly related to the extent of tissue lost following infarction/reperfusion. Targeting Nrf2 addresses an unmet need, as current clinical therapies focus on precluding occlusions and prompt reperfusion of infarcted tissue, but do not explicitly target at-risk tissue following infarcts and/or present-day reperfusion methodologies.
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Todorovic, Michael. « Assessing the Role of the Oxidative Stress Response ‘Master Regulator’ Nrf2 in Parkinson’s Disease ». Thesis, Griffith University, 2016. http://hdl.handle.net/10072/367349.
Texte intégralRésumé :
Parkinson’s disease (PD) is a complex neurodegenerative disorder influenced by a combination of genetic and environmental factors. The molecular mechanisms that underlie PD are unknown. However, oxidative stress and impairment of antioxidant defence mechanisms have been implicated as major contributors to disease pathogenesis. Previously, we have reported a PD patient-derived cellular model, generated from biopsies of the olfactory mucosa, termed hONS cells. These cells have demonstrated disease-specific differences in gene expression and metabolic activity associated with the Nrf2-mediated antioxidant defence pathway.
To date, few studies have examined the role of the Nrf2 encoding gene, NFE2L2, in PD. This thesis comprehensibly assessed whether rare and common NFE2L2 genetic variations modify susceptibility to PD using a large Australian case-control sample (PD=1,338; controls=1,379). We employed a haplotype-tagging approach that identified an association with the tagging SNP rs2364725 and PD (OR = 0.849 (0.760-0.948), P = 0.004). Further genetic screening for rare variants in patient-derived cell lines produced no obvious pathogenic variants in the coding regions of NFE2L2. In addition, we were able to identify some age-at-onset modifying SNPs and replicate an ‘early-onset’ haplotype that contains a previously identified ‘functional promoter’ SNP (rs6721961).Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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Strom, Joshua. « A Critical Role of Nrf2 In Protecting Cardiomyocytes Against Oxidative Stress and Ischemic Injury ». Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/333336.
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Coronary heart disease (CHD) remains the single leading cause of natural death worldwide. Despite significant advances in the diagnosis and treatment, CHD accounts for 1 out of every 6 deaths in the United States. Myocardial infarct (MI) as a result of CHD causes irreversible damage to the heart through the loss of viable myocardial tissue. Patients surviving the initial MI are at risk of developing heart failure due to lost contractile function and adverse cardiac remodeling. Improvement in the survival rates for MI have led to an increase in the incidence of heart failure, affecting approximately 5 million people in the United States. Although treatment of heart failure has improved, the mortality rates of heart failure remain high with 1 in 5 dying within the first year of diagnosis and 50% dying within 5 years. The cost of caring for heart failure patients ranks number one in Medicare. Oxidative stress plays an important role in the etiology and pathophysiology of CHD and heart failure. The transcription factor Nrf2 is a master regulator of cellular antioxidant defense mechanisms, controlling the expression of numerous antioxidant and detoxification genes through the Antioxidant Response Element (ARE) in the promoter regions. The cytoprotective effects of Nrf2 have been demonstrated in a variety of organs and disease states; however, the role of Nrf2 in the heart and heart disease has not been defined. The work presented here defines roles of Nrf2 in limiting cardiac injury and the progression to heart failure (Chapter II), protecting cardiac myocytes from oxidative stress through the preservation of mitochondria (Chapter III), and mediating the infarct reducing effects of statins, one of the most prescribed pharmacological agent (Chapter IV). In order to investigate a role of Nrf2 in the pathology of ischemic injury in the heart, a mouse model of ischemia and myocardial infarct by occlusion of the left anterior descending coronary artery was used. Nrf2 knockout mice subjected to ischemia/reperfusion injury experienced a larger infarct size than wild-type mice. Furthermore, mice lacking Nrf2 experienced a higher mortality rate and an accelerated progression to heart failure, indicated by severely compromised contractile function and reduced cardiac output, within 10 days following an MI. Morphological examination revealed maladaptive remodeling, including myocyte hypertrophy, heart enlargement, and dilated left ventricle, in Nrf2 KO mice that was absent in WT mice. Analysis of cardiac function by echocardiogram revealed increased left ventricular mass, increased systolic volume, decreased fraction shortening, reduced ejection fraction, and decreased cardiac output in Nrf2 KO mice. Nrf2 KO mice also demonstrated expression of biomarkers of heart failure, such as expression of fetal gene program, with elevated levels of β-MHC, ANF, and BNP mRNA in the myocardium. Interestingly, a lack of immune cell infiltrate and myofibroblasts as well as a deficiency in collagen deposition were observed in the infarcted region of hearts from Nrf2 KO mice. These data indicate that Nrf2 plays an important role in protecting the myocardium from ischemic injury and the progression to heart failure. Lack of Nrf2 response results in deficiency of wound healing and instead initiation of maladaptive remodeling, leading to heart failure. Mitochondria are key sources of reactive oxygen species (ROS) generation, as well as important targets for ROS-induced cell injury. Cardiac myocytes have the highest content of mitochondria among all cell types and can be particularly susceptible to mitochondrial dysfunction due to the high metabolic demand associated with the contractile function of the heart. With cardiomyocytes (CMCs) isolated from neonatal rats and kept under tissue culture conditions, mitochondria exist in elaborated networks. Such networks were replaced by predominately individual punctate mitochondria 24 hours after exposure to a sublethal dose of H₂O₂. Mitochondrial morphology was altered with membrane swelling and disorganization of inner cristae with areas of condensation. Disrupted mitochondrial morphology was associated with a loss of membrane potential and decreased expression of mitochondrial proteins involved in the electron transport chain, such as cytochrome b and cytochrome c. Nrf2 overexpression prevented H₂O₂ from inducing morphological changes in mitochondria and the reduction of cytochrome b and cytochrome c expresssion. Although Nrf2 is known as a transcription factor regulating antioxidant and detoxification genes, Nrf2 overexpression did not significantly reduce the level of protein oxidation as measured by carbonyl formation. Instead, we found that Nrf2 localizes to the outer mitochondrial membrane, suggesting a direct role of Nrf2 in mitochondrial protection. As further evidence of a direct role in mitochondrial protection, a cell-free system of mitochondria isolated from the myocardium of Nrf2 knockout mice were more sensitive to permeability transition, an indicator of mitochondrial dysfunction. Combined, these data suggest that Nrf2 protects mitochondria from oxidant injury likely through direct interaction with mitochondria. In the clinic, statins are now commonly administered for patients experiencing MI or CHD. Statins have become mainstays in the treatment of hypercholesterolemia and atherosclerosis as inhibitors of the rate limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. In addition, statins have been shown to elicit pleiotropic effects, including plaque stabilization, maintenance of endothelial function, anti-inflammatory actions, and antioxidant capabilities, independent of effects on cholesterol synthesis. Recently, these pleiotropic effects have been implicated in providing acute protection against ischemia and reperfusion injury, which has led to the use of high dose statins clinically before revascularization of an ischemic event. I have found that administration of atorvastatin in mice induced Nrf2 protein levels in the heart, brain, lung, and liver. While atorvastatin reduced infarct size following an MI in wild-type mice, this protective effect was lost in mice lacking Nrf2. Failure of atorvastatin to protect against MI in Nrf2 knockout mice indicates that Nrf2 plays a critical role in mediating the protective effects of acute statin treatment. Nrf2 induction by statins is a novel discovery. In order to understand the mechanism of such statin effect, I used an in vitro cell system, in which a variety of statins, atorvastatin, simvastatin, lovastatin, and pravastatin, were found to elevate Nrf2 protein levels. Elevation of Nrf2 by statins was independent of increased protein stability or transcriptional regulation. Instead, statins increased Nrf2 mRNA association with ribosomal complexes and induced Nrf2 protein through a translational mechanism. Recruitment of Nrf2 mRNA to ribosomes and induction of Nrf2 protein was dependent on activation of PI3 kinase. These studies provide evidence that Nrf2 plays a critical role in protecting cardiac myocytes and the heart from oxidative stress and MI. In the absence of Nrf2, mice experienced worse cardiac injury following MI and quickly advanced to heart failure. Mechanistically, this work has identified a novel role of Nrf2 in preserving mitochondrial morphology and integrity during oxidative stress through a direct interaction with the outer mitochondrial membrane. Finally, a newly defined role of Nrf2 induction by statins in mediating protection against MI by acute statin therapy indicates that modulation of Nrf2 may represent a viable pharmacological target for cardiac protection in humans.
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Hintsala, H. R. (Hanna-Riikka). « Oxidative stress and cell adhesion in skin cancer ». Doctoral thesis, Oulun yliopisto, 2016. http://urn.fi/urn:isbn:9789526212692.
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Abstract Skin is the largest organ in our body protecting us from ultraviolet radiation and xenobiotics. UV-radiation is a common cause of squamocellular carcinoma and melanoma of the skin that cause morbidity and mortality world wide. Reactive oxygen species are constantly formed by, for example, cellular respiration and UV-radiation, and they can readily react with virtually any macromolecule within cell structures causing damage to DNA, proteins and lipids. Oxidative stress (OS) is a homeostatic process that is dysregulated in cancer cells to their benefit. Nuclear factor erythroid-2-related factor 2 (Nrf2) is the main regulator of antioxidant response and it has been shown to be upregulated in various cancers enabling their survival and growth. By using immunohistochemistry we studied the change and prognostic significance of OS markers in melanoma from paraffin embedded patient samples. Nrf2 expression is increased in melanoma, associating with deeper invasion and a worse melanoma-specific outcome. In addition, epithelial-to-mesenchymal transition markers Slug, Twist and Zeb1 showed altered expression levels in relation to invasion and metastasis associating also with Nrf2. With the help of target inhibition molecules Vemurafenib and MEK-inhibitor CI-1040, In vitro study showed that BRAF- and NRAS-mutations might activate Nrf2. Furthermore, Nrf2-regulated antioxidant enzyme peroxiredoxin I showed decreased expression in malignant melanomas and metastases compared to benign naevi. Intriguing findings were made from the surrounding structures of melanomas e.g. loss of expression of an oxidative lesion marker 8-hydroxy-2’-deoxyguanosine in adjacent endothelial cells associated with worse melanoma-specific survival. Changes in the expression of adhesion molecules claudins 1-5 and 7 were studied in the progression of cutaneous squamous cell carcinomas and preneoplastic lesions. Change in claudin composition can alter epidermal permeability and cell polarity. Efficiency of oncological treatment modalities is frequently based on oxidative stress damage. Nrf2-inhibition could offer the means to increase the sensitivity of cancerous tissue to oxidative insults and hinder proliferative and survival signalling. Later research should focus on the relation of Nrf2 with other signalling and observations made from the tumour microenvironmentTiivistelmä Iho on elimistön suurin elin, ja se suojaa meitä auringon ultravioletti (UV)-säteilyltä ja muilta ulkoisilta tekijöiltä. UV-säteily on yhteinen etiologinen tekijä ihon levyepiteelikarsinoomalle ja melanoomalle, jotka aiheuttavat maailmanlaatuisesti paljon sairastavuutta ja kuolleisuutta. Reaktiivisia happiradikaaleja muodostuu esimerkiksi soluhengityksestä ja UV-säteilystä, ja ne voivat reagoida minkä tahansa makromolekyylin kanssa aiheuttaen vaurioita solun perimäainekseen, proteiineihin ja lipidirakenteisiin. Oksidatiivisen stressin (OS) säätely on tärkeä homeostaattinen prosessi, joka vinoutuu syöpäsolujen hyödyksi. Nuclear factor erythroid-2-related factor 2 (Nrf2) on antioksidanttivasteen pääsäätelytekijä, ja sen ilmentyminen on lisääntynyt useissa syövissä lisäten syöpäsolun selviytymistä ja kasvua. Tutkimme potilasaineiston ja immunohistokemian avulla OS:n merkkiaineiden muutoksia melanoomassa ja niiden merkitsevyyttä taudin ennusteelle. Nrf2:n ilmentyminen on lisääntynyt melanoomassa liittyen syvempään invaasioon ja huonompaan tautispesifiseen ennusteeseen. Lisäksi epiteliaali-mesenkymaalitransition merkkiaineiden, Slug, Twist ja Zeb1 ekspression muutoksia havaittiin syvyyskasvun ja metastasoinnin yhteydessä assosioituen myös Nrf2 ilmentymiseen. In vitro- tutkimus osoitti spesifisten inhibiittoreiden avulla, että BRAF- ja NRAS-mutaatiot saattavat aktivoida Nrf2 melanoomassa. Myös Nrf2:n säätelemän entsyymin peroksiredoksiini I:n ilmentyminen on vähentynyt melanoomassa ja metastaaseissa verrattuna hyvänlaatuisiin pigmenttiluomiin. Merkittäviä muutoksia havaittiin myös melanoomaa ympäröivistä rakenteista, esimerkiksi OS:n vauriomarkkerin 8-hydroksi-2’-deoksiguanosiinin vähentynyt ilmentyminen endoteelisoluissa liittyi huonompaan tautispesifiseen ennusteeseen. Lisäksi tutkimme soluväliliitosproteiinien klaudiinien 1–5 sekä 7 ilmentymistä levyepiteelikarsinoomissa ja niiden esiasteissa. Klaudiinien muutokset voivat vaikuttaa ihon permeabiliteettiin ja solujen polarisaatioon. Onkologisten hoitomuotojen teho perustuu usein happiradikaalien aiheuttamiin vaurioihin. Nrf2-inhibitio voisi tarjota keinon lisätä syöpäkudoksen herkkyyttä näille vaurioille sekä estää syöpäsolun selviytymissignalointia. Tulevat tutkimukset tulisivat keskittyä Nrf2 signaloinnin ja muun solusignaloinnin välisiin suhteisiin sekä havaintoihin kasvaimen mikroympäristön muutoksista
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Woolridge, Cooper JàNay K. B. S. « Galactomyces Ferment Filtrate Suppresses Melanization and Oxidative Stress in Epidermal Melanocytes ». University of Cincinnati / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1511799237125245.
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Amin, Ahmed [Verfasser]. « NRF2 mediated oxidative stress response activity during early in vitro bovine embryo development / Ahmed Amin ». Bonn : Universitäts- und Landesbibliothek Bonn, 2015. http://d-nb.info/107726920X/34.
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Edwards, Heather Gray. « Protection from oxidative stress in the cardiac H9C2-cell line by the transcription factor NRF2 ». Auburn, Ala., 2007. http://repo.lib.auburn.edu/07M%20Dissertations/GRAY-EDWARDS_HEATHER_53.pdf.
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Zgheib, Elias. « Bioinformatic and modelling approaches for a system-level understanding of oxidative stress toxicity ». Thesis, Compiègne, 2018. http://www.theses.fr/2018COMP2464/document.
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Avec les nouvelles avancées en biologie et toxicologie, on constate de plus en plus la complexité des mécanismes et le grand nombre de voies de toxicité. Les concepts de ‘biologie systémique’ (SB) et de ‘voies des effets indésirables’ (adverse outcome pathway, AOP) pourraient être des outils appropriés pour l’étude de la toxicologie à ces niveaux de complexité élevés. Le point central du travail de cette thèse est le développement d’un modèle de SB du rôle de la voie de signalisation Nrf2 dans le contrôle du stress oxydant. Pour la calibration de ce modèle avec des données expérimentales (exposition des cellules rénales RPTEC/TERT1 à différentes doses de bromate de potassium), plusieurs cycles de proposition/vérification d’hypothèses ont progressivement contribué à l’ajout de nouvelles réactions. Ces nouvelles hypothèses (par exemple : action directe du bromate de potassium sur le DCF, atténuation de la fluorescence du DCF avec le temps, etc.) devraient être confirmées par de futures expérimentations. Ce modèle de SB a été ensuite utilisé pour la quantification d’un AOP de l’insuffisance rénale chronique et comparé à deux autres approches: l’utilisation de modèles statistiques empiriques et celle d’un réseau Bayésien dynamique. Les calibrations des paramètres ont été effectuées par chaînes de Markov simulées MCMC avec le logiciel GNU MCSim avec une quantification des incertitudes associées aux prédictions. Même si la mise au point du modèle SB a été une tâche complexe, la compréhension de la biologie qu’offre ce modèle n’est pas accessible aux deux autres approches. Nous avons aussi évalué les interactions entre Nrf2 et deux autres voies de toxicité, AhR et ATF4, dans le cadre d’une analyse utilisant des données de toxico-génomique provenant de trois projets différents. Les résultats de cette dernière analyse suggèrent d’ajouter au modèle SB de Nrf2 la co-activation par AhR de plusieurs gènes (par exemple, HMOX1, SRXN1 et GCLM) ainsi que d’associer (au moins partiellement) à ce modèle la voie ATF4. Malgré leur complexité, les modèles SB constituent un investissement intéressant pour le développement de la toxicologie prédictiveNew understanding of biology shows more and more that the mechanisms that underlie toxicity are complex and involve multiple biological processes and pathways. Adverse outcome pathways (AOPs) and systems biology (SB) can be appropriate tools for studying toxicology at this level of complexity. This PhD thesis focuses on the elaboration of a SB model of the role of the Nrf2 pathway in the control of oxidative stress. The model’s calibration with experimental data (obtained with RPTEC/TERT1 renal cells exposed to various doses of potassium bromate) comprised several rounds of hypotheses stating/verification, through which new reactions were progressively added to the model. Some of these new hypotheses (e.g., direct action of potassium bromate on DCF, bleaching of DCF with time, etc.) could be confirmed by future experiments. Considered in a wider framework, this SB model was then evaluated and compared to two other computational models (i.e., an empirical dose-response statistical model and a dynamic Bayesian model) for the quantification of a ‘chronic kidney disease’ AOP. All parameter calibrations were done by MCMC simulations with the GNU MCSim software with a quantification of uncertainties associated with predictions. Even though the SB model was indeed complex to conceive, it offers insight in biology that the other approaches could not afford. In addition, using multiple toxicogenomic databases; interactions and cross-talks of the Nrf2 pathway with two other toxicity pathways (i.e., AhR and ATF4) were examined. The results of this last analysis suggest adding new AhR contribution to the control of some of the Nrf2 genes in our SB model (e.g., HMOX1, SRXN1 and GCLM), and integrating in it description of the ATF4 pathway (partially at least). Despites their complexity, precise SB models are precious investments for future developments in predictive toxicology
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Purdom-Dickinson, Sally Elizabeth. « Early Responses to Oxidative Stress In Heart Cells : Signals From The Cell Membrane To The Nucleus and Beyond ». Diss., Tucson, Arizona : University of Arizona, 2005. http://etd.library.arizona.edu/etd/GetFileServlet?file=file:///data1/pdf/etd/azu%5Fetd%5F1310%5F1%5Fm.pdf&type=application/pdf.
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Claverie, Damien. « Marqueurs et mécanismes de la vulnérabilité à la dépression ». Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066727.
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La vulnérabilité à la dépression est un état pour lequel le risque d’évolution vers la dépression après un stress est accru. L'exposition de rats à 4 défaites sociales (DS) induit 42% d’animaux vulnérables (V) identifiés par un faible taux sérique de BDNF 1 mois post-DS. Ces derniers développent un phénotype dépressif après exposition à un second stress (stress chronique léger (CMS)). Une première étude a révélé l’existence d’une signature EEG identifiant les rats V et prédisant cet état avant exposition aux DS. Cette observation pose le problème de la diathèse sur laquelle se développe la vulnérabilité. Le remplacement du CMS par des injections d’acide kaïnique entraine chez les rats V, outre une épilepsie, un phénotype dépressif, suggèrant que l’induction pathologique ne dépend pas du stresseur. Les rats V présentent lors de la phase de vulnérabilité des altérations neuroanatomiques de l'hippocampe, conséquence d'un stress oxydant. Ainsi, une baisse du BDNF dans l’hippocampe, entraine un déficit de translocation vers le noyau de Nrf2, facteur de transcription activant l’expression d’enzymes antioxydantes, et in fine une diminution des défenses antioxydantes cellulaires. La correction de la translocation de Nrf2 ou du stress oxydant par un mimétique du BDNF, le composé 7,8-DHF ou un antioxydant, le Tempol, préviennent le phénotype dépressif sans modifier les taux de BDNF. Ces profils "haut BDNF" vs. "haut stress oxydant" sont retrouvés chez l'homme sain exposé à des contraintes militaires, donnant à la vulnérabilité à la dépression un cadre plus généralVulnerability to depression is a state for a subject for which depression onset risk’s increases following a stress. 4 Social Defeats (SD) exposure induces 42% of vulnerable rats (V) identifiable by a lack of serum BDNF 1 month post-SD. These latter develop a depressive-like phenotype after exposure to a Chronic Mild Stress (CMS). We observed in a first study an EEG pattern identifying V but also predicting this state before SD exposure. This observation highlights a diathesis predisposing vulnerability. Then, we observed that depressive-like phenotype is induced by kainic acid injection instead of CMS, suggesting that pathological onset is independent of the stressor. V were characterized during vulnerability stage by neuroanatomical altered hippocampus, due to an oxidative stress state. A BDNF decrease into hippocampus, leads to a deficit of Nrf2 translocation into the nucleus, since Nrf2 is a transcription factor inducing antioxidant enzymes expression, and therefore induced a decreased in cellular antioxidant defenses. Correction of Nrf2 translocation or oxidative stress with administration of, a BDNF mimetic: the 7,8-DHF compound or an antioxydant : Tempol, prevent depressive-like phenotype without changing BDNF levels. These patterns “high oxidative stress” vs. “high BDNF levels” are observed in healthy human exposed to military constraint, suggesting a more general framework for vulnerability to depression
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Lines, Simon. « The influence of vitamin E bonded haemodialysis membranes on erythropoiesis stimulating agent requirements, oxidative stress, inflammation, haemostasis and outcomes ». Thesis, University of Leeds, 2013. http://etheses.whiterose.ac.uk/5871/.
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Introduction: Haemodialysis (HD) patients have high rates of cardiovascular (CV) disease and mortality yet the reasons for this have not been fully elucidated. High doses of erythropoiesis stimulating agents (ESAs), increases in oxidative stress and inflammation, and alterations to the fibrin clot phenotype are all possible contributors. Vitamin E (VE)- bonded dialysis membranes are purported to have favourable effects on a number of these parameters which were tested here in the setting of a randomised controlled trial. Methods: Patients were randomised to HD with VE-bonded polysulfone membranes or non-VE-bonded equivalents and followed for 12 months. Data on anaemia parameters were collected monthly and blood tests were performed at baseline, 6 and 12 months for the measurement of oxidative stress (oxidatively modified-low density lipoprotein, thiobarbituric acid reactive species), inflammation (C-reactive protein, complement components C3, SC5b-9, factor D, properdin) and fibrin clot properties. Contemporaneous data were collected on CV events and death. Results: Of the 260 patients enrolled, 123 were randomised to dialysis with VE-bonded membranes. Analysis of the full dataset revealed no differential effects of the VEmembranes on ESA requirements, oxidative stress, inflammation, fibrin clot structure or clinical outcomes. Key findings included a potential ESA-sparing effect of the VEmembranes in ESA resistant patients, a progressive decline in C3 levels over 12 months and associations between the levels of C3 and SC5b-9 at baseline and the subsequent risks of dying or experiencing a CV event. Conclusions: There were no benefits in switching prevalent HD patients to dialysis with VE-bonded dialysis membranes with the exception of possible utility in ESA-resistant patients. The novel finding suggesting a link between the complement system and poor outcomes in HD patients may provide further insights to explain the high rates of CV disease and mortality for this patient group and merits further study.
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di, BELLO GIORGIA. « Nrf2 Inhibition Is Required To Activate Hepatic Progenitor Cells ». Doctoral thesis, Università degli studi di Foggia, 2019. http://hdl.handle.net/11369/382255.
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L'attuale trattamento dell'insufficienza epatica è il trapianto di organo. Tuttavia, i costi elevati, la mancanza di donatori, la mortalità correlata al trattamento e l'immunosoppressione a lungo termine rendono questa opzione possibile solo per un numero limitato di pazienti. Il trapianto di cellule staminali del fegato è stato recentemente proposto come trattamento alternativo. L'identificazione dei principali regolatori nella differenziazione delle cellule progenitrici epatiche è determinante per la rigenerazione dell’ organo e può migliorare il trapianto di cellule staminali per la malattia epatica allo stadio terminale. Questo lavoro si basa sullo studio del ruolo che il fattore di trascrizione Nrf2 può avere nella regolazione del destino delle cellule progenitrici epatiche. I nostri dati mostrano che Nrf2 è costitutivamente attivato nelle nicchie delle cellule staminali epatiche per il mantenimento delle stesse, ma è down-regolato nelle lesioni croniche del fegato. L'inibizione in vitro di Nrf2 induce modificazioni morfologiche, fenotipiche e funzionali tipiche degli elementi differenziati. Abbiamo quindi inibito Nrf2 tramite il modulatore di espressione ARE 1 (AEM1) nella linea cellulare umana HepaRG; queste cellule sono state trapiantate in topi SCID/beige somministrati con anticorpi anti-Fas per indurre apoptosi epatocellulare, con conseguente ripopolamento efficace da parte degli epatociti umani e ripristino della funzionalità epatica. Per concludere, questo studio mostra che l'inibizione di Nrf2 porta all'attivazione e alla differenziazione delle cellule progenitrici del fegato. Questo fattore di trascrizione redox-dipendente può dunque rappresentare un potenziale bersaglio per regolare il processo di attivazione e differenziazione in linee specifiche di cellule progenitrici epatiche indifferenziate.The current treatment of liver failure is organ transplantation. Nevertheless, the high costs, lack of donors, treatment-related mortality and long-term immunosuppression make this option possible only for a limited number of patients. Liver stem cell transplantation has been recently proposed as an alternative treatment. The identification of key regulators in hepatic progenitor cell differentiation is determinant for organ regeneration and may improve stem cell transplantation for end-stage liver disease. The present investigation studied the role of the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the regulation of hepatic progenitor cell fate. Our data show that Nrf2 is constitutively activated in the hepatic stem cell niches to maintain progenitor stemness, but it is down-regulated in chronic liver injury. The in vitro inhibition of Nrf2 induces morphological, phenotypical and functional modifications typical of differentiated elements. We thus inhibited Nrf2 via ARE expression modulator 1 (AEM1) in the human-derived HepaRG cell line; these cells were transplanted into SCID/beige mice administered with anti-Fas antibody to induce hepatocellular apoptosis, resulting in effective human hepatocyte repopulation with restoration of liver function. To conclude, the present study shows that Nrf2 inhibition leads to the activation and differentiation of liver progenitor cells. This redox-dependent transcription factor may represent a potential target to regulate the commitment of undifferentiated hepatic progenitor cells into specific lineages.
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Sun, Zheng. « Mechanistic Study of Nucleocytoplasmic Trafficking and Reversible Acetylation in Modulating the NRF2-Dependent Antioxidant Response ». Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/194904.
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To maintain intracellular redox homeostasis, genes encoding many endogenous antioxidants and phase II detoxification enzymes are transcriptionally upregulated upon deleterious oxidative stress through the cis- antioxidant responsive elements (AREs) in their promoter regions. Nrf2 has emerged as the pivatol transcription factor responsible for ARE-dependent transcription, and has been shown to play critical roles in hepatotoxicity, chemical carcinogenesis, pulmonary inflammatory diseases, neurodegenerative diseases and aging. Therefore, understanding the molecular mechanism of the Nrf2-dependent cytoprotective system is important for development of drugs for therapeutic intervention.Nrf2 is targeted by Keap1 for ubiquitin-mediated degradation under basal conditions. Upon oxidative stress, distinct cysteine residues of Keap1 are alkylated, leading to inhibition of Keap1 and activation of Nrf2. However, it was not clear how Nrf2 is re-entered into the repression status when redox homeostasis is re-achieved. In this dissertation, we establish that the post-induction repression of Nrf2 is controlled by the nuclear export function of Keap1 in alliance with the cytoplasmic ubiquitination/ degradation machinery. We show that a nuclear export sequence (NES) in Keap1 is required for termination of Nrf2 signaling; ubiquitination of Nrf2 is carried out in the cytosol; Keap1 nuclear translocation is independent of Nrf2; and the Nrf2-Keap1 complex does not bind the ARE. Collectively, these results suggest that Keap1 translocates into the nucleus to dissociate Nrf2 from the ARE and mediates nuclear export of Nrf2 followed by ubiquitination and degradation of Nrf2 in the cytoplasm.In addition to Keap1-mediated negative regulation, we identified a novel positive regulatory mechanism of Nrf2 mediated by transcription co-activator p300/CBP. We show that p300/CBP directly binds and acetylates Nrf2 in response to oxidative stress. We have identified multiple acetylated lysine residues within the Nrf2 Neh1 DNA-binding domain. Combined lysine-to-arginine mutations on the acetylation sites, with no effects on Nrf2 protein stability, compromised the DNA-binding activity of Nrf2 in a promoter-specific manner both in vitro and in vivo. These findings demonstrated that acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 and established acetylation as a novel regulatory mechanism that functions in concert with Keap1-mediated ubiquitination in modulating the Nrf2-dependent antioxidant response.
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Khadrawy, Omar Zainelabdeen Shehata [Verfasser]. « Modulation of Nrf2-mediated oxidative stress response in bovine granulosa cells and preimplantation embryos / Omar Zainelabdeen Shehata Khadrawy ». Bonn : Universitäts- und Landesbibliothek Bonn, 2019. http://d-nb.info/1200098145/34.
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Harris, Jessica Lynn. « INVESTIGATIONS INTO MODULATION OF BRAIN OXIDATIVE STRESS BY VARIOUS INTERVENTIONS ». UKnowledge, 2012. http://uknowledge.uky.edu/chemistry_etds/12.
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In this thesis study we examined glycogen synthase kinase-3β (GSK-3β) and its effects over Nrf2 and Pin 1 as it relates to Alzheimer’s disease (AD). AD is a neurodegenerative disease characterized by a prolonged high oxidative environment. Transcription factor Nrf2 is vital in the brain’s defense against oxidative insults through its up-regulation of over 100 antioxidants. Depletion of the brain’s antioxidant defense system results in intolerance to an oxidative environment, contributing to the progression of AD. The regulatory Pin 1 protein promotes cellular homeostasis, and when down-regulated results in increased deposits of neurofibrillary tangles (NFTs) and amyloid-β (Aβ) plaques, the two pathological hallmarks of AD.
Using aged SAMP8 mice treated with antisense oligonucleotide (AO) directed at GSK-3β and random AO, the data presented here demonstrate decreased oxidative stress and increased Nrf2 transcriptional activity and Pin 1 levels as a result of the down-regulation of GSK-3β. Collectively, these results implicate GSK-3β activity in the increased oxidative stress of AD and support its inhibition as a possible therapeutic treatment for the disease. Further, we elucidate a possible mechanism connecting GSK-3β to the loss of tolerance to an oxidative environment and increased deposits of NFTs and Aβ plaques observed in AD.
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Mubarak, Bashayer Rashed A. « Control of anti-apoptotic and antioxidant pathways in neural cells ». Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8057.
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Oxidative stress is a feature of many chronic neurodegenerative diseases as well as a contributing factor in acute disorders including stroke. Fork head class of transcription factors (Foxos) play a key role in promoting oxidative stress-induced apoptosis in neurons through the upregulation of a number of pro-apoptotic genes. Here I demonstrate that synaptic NMDA receptor activity not only promotes Foxos nuclear exclusion but also suppresses the expression of Foxo1 in a PI3K-dependent fashion. I also found that Foxo1 is in fact, a Foxo target gene and that it is subject to a feed-forward inhibition by synaptic activity, which is thought to result in longerterm suppression of Foxo downstream gene expression than previously thought. The nuclear factor (erythroid 2-related) factor 2 (Nrf2) is another transcription factor involved in oxidative stress and the key regulator of many genes, whose products form important intrinsic antioxidant systems. In the CNS, artificial activation of Nrf2 in astrocytes has been shown to protect nearby neurons from oxidative insults. However, the extent to which Nrf2 in astrocytes could respond to endogenous signals such as mild oxidative stress is less clear. The data presented herein, demonstrate for the first time that endogenous Nrf2 could be activated by mild oxidative stress and that this activation is restricted to astrocytes. Contrary to the established dogma, I found that mild oxidative stress induces the astrocytic Nrf2 pathway in a manner distinct from the classical Keap1 antagonism employed by prototypical Nrf2 inducers. The mechanism was found to involve direct regulation of Nrf2's transactivation properties. Overall these results advance our knowledge of the molecular mechanism(s) associated with the control of endogenous antioxidant defences by physiological signals.
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Corenblum, Mandi J., Sneha Ray, Quentin W. Remley, Min Long, Bryan Harder, Donna D. Zhang, Carol A. Barnes et Lalitha Madhavan. « Reduced Nrf2 expression mediates the decline in neural stem cell function during a critical middle-age period ». WILEY-BLACKWELL, 2016. http://hdl.handle.net/10150/622598.
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Although it is known that the regenerative function of neural stem/progenitor cells (NSPCs) declines with age, causal mechanisms underlying this phenomenon are not understood. Here, we systematically analyze subventricular zone (SVZ) NSPCs, in various groups of rats across the aging spectrum, using in vitro and in vivo histological and behavioral techniques. These studies indicate that although NSPC function continuously declines with advancing age, there is a critical time period during middle age (13-15 months) when a striking reduction in NSPC survival and regeneration (proliferation and neuronal differentiation) occurs. The studies also indicate that this specific temporal pattern of NSPC deterioration is functionally relevant at a behavioral level and correlates with the decreasing expression of the redox-sensitive transcription factor, Nrf2, in the NSPCs. When Nrf2 expression was suppressed in 'young' NSPCs, using short interfering RNAs, the survival and regeneration of the NSPCs was significantly compromised and mirrored 'old' NSPCs. Conversely, Nrf2 overexpression in 'old' NSPCs rendered them similar to 'young' NSPCs, and they showed increased survival and regeneration. Furthermore, examination of newborn Nrf2 knockout (Nrf2-/-) mice revealed a lower number of SVZ NSPCs in these animals, when compared to wild-type controls. In addition, the proliferative and neurogenic potential of the NSPCs was also compromised in the Nrf2-/- mice. These results identify a novel regulatory role for Nrf2 in NSPC function during aging and have important implications for developing NSPC-based strategies to support healthy aging and to treat age-related neurodegenerative disorders.
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Spitzer, Alexander Jonathan. « Endotoxin Increases Oxidative Stress And Oxygen Tension While Reducing Milk Protein Gene Expression In The Mammary Gland ». ScholarWorks @ UVM, 2019. https://scholarworks.uvm.edu/graddis/1123.
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Mastitis, the inflammation of the mammary gland by bacterial infection, is one of the costliest diseases to the dairy industry primarily due to a loss in milk production. The aim of this study was to investigate the mechanisms underlying reduced milk production during mastitis. We hypothesized that bacterial endotoxin induces cell apoptosis, oxidative stress and increases hypoxia while inhibiting milk gene expression in the mammary gland. To test this hypothesis, mice were bred to pregnancy, and 3 days post-partum the left and right sides of the 4th pair of mammary glands were alternately injected with either the endotoxin liposaccharide (LPS, E. coli 055:B5, 100 ul of 0.2 mg/ml) or sterile PBS through the teat meatus. At 10.5 and 22.5 h post-injection, pimonidazole HCl, a hypoxyprobe, was injected intraperitoneally. At 12 or 24 h after the LPS injection, the fourth glands were individually collected (n=8 pairs) and analyzed for hypoxia, gene expression and oxidative stress. LPS treatment induced mammary gland inflammation as shown by increases in inflammatory cytokine expression (P < 0.001) and neutrophil recruitment at 12 and 24 h. LPS promoted cell apoptosis in a transient manner; an abundance of cleaved caspase 3 was evident only at 12 h after LPS challenge (P = 0.02). Increased H2O2 content was seen at 12 h (P < 0.001) but decreased dramatically after 24 h of LPS treatment (P < 0.001). Total antioxidative capacity tended to decrease at both 12 and 24 h (P = 0.067 and 0.061, respectively). In agreement with these findings, LPS activated Nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidative signaling in the mammary gland, demonstrated by increased expression of its target gene Nqo1 at 12 h (P = 0.05) and xCT at 24 h (P = 0.076). Hypoxyprobe staining, indicative of hypoxia, was greater in the alveoli of PBS-treated glands than LPS-treated glands at both 12 and 24 h. This suggests oxygen tension rises in response to LPS treatment. Conversely, milk expression genes, β-casein gene (CSN2) and α-lactalbumin (LALBA), were inhibited by LPS treatment across time. Expression of α-S1 casein (CSN1S1) mRNA increased with LPS treatment at 24 h, but protein expression was reduced at this same time point (P < 0.05). In summary, intramammary LPS challenge incurs inflammation, augments cell apoptosis, induces oxidative stress and activation of the Nrf2 antioxidation pathway, increases oxygen tension, and inhibits milk protein expression in the mammary gland. This study provides functional insight into mechanisms of reduced milk production during mastitis and provides possible approaches to combat reduction in milk production, such as enhancing the Nrf2-antioxidative signaling pathway and reducing inhibition of milk protein expression.
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Helou, Doumet. « Rôle du facteur de transcription Nrf2 dans la régulation des fonctions du neutrophile in vitro et dans l’allergie cutanée ». Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS305/document.
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Les neutrophiles constituent une première ligne de défense contre les agents infectieux. En revanche, leur activation incontrôlée peut exacerber certaines pathologies inflammatoires telles que les allergies cutanées. Notre équipe a montré précédemment que le facteur de transcription Nrf2 connu pour son rôle anti-oxydant, régulait l’inflammation cutanée dans l’hypersensibilité de contact (HSC). Ainsi ce travail a été mené pour évaluer in vitro l’implication de la voie Nrf2 dans les fonctions des neutrophiles et pour identifier son rôle dans le recrutement et l’activation des neutrophiles dans l’HSC.In vitro, nous montrons que la protéine Nrf2 est fortement exprimée dans les neutrophiles de la moelle osseuse. Nrf2 est fonctionnelle dans les neutrophiles stimulés : il active la transcription de gènes cibles cytoprotecteurs et diminue celle des gènes de l’inflammation. Ainsi, le prétraitement des neutrophiles avec un activateur de Nrf2 tel que le sulforaphane, réduit la production des formes réactives de l’oxygène (FRO)en réponse à une stimulation. En parallèle, l’absence de Nrf2 ne semble pas affecter la phagocytose et la nétose, deux fonctions clés du neutrophile. Enfin, Nrf2 est indispensable pour une migration optimale des neutrophiles en réponse aux chimiokines.Au cours de l’HSC induite par le dinitrochlorobenzène (DNCB), Nrf2 régule indirectement le recrutement des neutrophiles, en contrôlant le stress oxydant cutané et les voies inflammatoires impliquées dans la production de chimiokines, notamment CCL2, CCL4 et CCL11. En outre, Nrf2 induit l’augmentation d’expression du scavenger CD36 dans les macrophages et augmente ainsi leur capacité à éliminer les neutrophiles apoptotiques pour initier la résolution de l’inflammation.En conclusion, l’activation de Nrf2 dans les neutrophiles participe au contrôle de la production des FRO et la migration. En outre, Nrf2 émerge comme un effecteur clé dans le contrôle du recrutement et de la clairance des neutrophiles au cours de la réponse inflammatoire cutanée aux molécules allergisantes. La mise en évidence de ces mécanismes protecteurs de Nrf2 nous permet de proposer cette protéine comme nouvelle cible thérapeutique dans le contrôle d’inflammations cutanées chroniquesNeutrophils form the first line of defense against infectious agents. However, their uncontrolled activation may exacerbate certain inflammatory conditions such as cutaneous allergies. Our team has previously shown that Nrf2 transcription factor known for its antioxidant role, regulates skin inflammation in contact hypersensitivity (CHS). Thus, our work was carried out to evaluate in vitro the involvement of Nrf2 pathway in neutrophil functions and to identify Nrf2 role in neutrophil recruitment and activation in CHS.In vitro, we showed that the protein Nrf2 was highly expressed in bone marrow neutrophils. Nrf2 is functional in stimulated neutrophils: it activates the transcription of cytoprotective genes and downregulates that of inflammatory genes. Thus, pretreatment of neutrophils with an Nrf2 activator such as sulforaphane reduces the production of reactive oxygen species (ROS) in response to stimulation. In parallel, Nrf2 does not affect two key functions of neutrophil, phagocytosis and netosis.Finally, Nrf2 is essential for optimal migration of neutrophils toward chemokines. In CHS induced by the dinitrochlorobenzene (DNCB), Nrf2 indirectly regulates the recruitment of neutrophils, through regulation of skin oxidant stress and inflammatory pathways that are involved in chemokines production, including CCL2, CCL4 and CCL11. In addition, Nrf2 induces the up-regulation of scavenger CD36 in macrophages and thus increases their ability to eliminate apoptotic neutrophils leading to the resolution of inflammation.In conclusion, Nrf2 activation in neutrophils participates in the control of ROS production and migration. In addition, Nrf2 emerges as an important effector in the control of neutrophil recruitment and clearance during the skin inflammatory response to allergenic molecules. The demonstration of Nrf2 protective mechanisms leads us to suggest this protein as a new therapeutic target in the control of chronic skin inflammations
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Prudencio, Mariana Baldini. « Impacto dos ácidos graxos dietéticos no perfil lipídico, inflamatório, oxidativo e na ativação dos fatores de transcrição NF-KB e Nrf2 em pacientes com epilepsia submetidos à dieta cetogênica ». Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/6/6138/tde-31102018-105019/.
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Introdução: a epilepsia é uma doença neurológica, cuja prevalência é de 0,5 a 1% na população, sendo que 20 a 30% dos pacientes apresentam crises refratárias ao tratamento medicamentoso. A dieta cetogênica (DC) composta por alto teor de gorduras, baixo teor de carboidratos e quantidade proteica adequada tem emergido como um tratamento adjuvante e eficaz no controle de crises. Apesar de sua eficácia ser bem descrita na literatura, em humanos pouco se sabe sobre influência da DC em marcadores oxidativos e na modulação de fatores de transcrição como o NF-kB e Nrf2. Além disso, não se sabe sobre o impacto que diferentes tipos de ácidos graxos dietéticos ofertados na DC poderiam ocasionar nesses marcadores. Objetivo: avaliar o impacto da modificação do perfil de ácidos graxos dietéticos ofertados na DC SAFA sobre o perfil lipídico, oxidativo e ativação de fatores de transcrição NF-kB e Nrf2 em crianças e adolescentes com epilepsia refratária submetidos ao tratamento com dieta cetogênica. Metodologia: tratase de um ensaio clínico com seguimento de 6 meses, no qual o grupo DC SAFA recebeu uma dieta rica em colesterol e gordura saturada, e o grupo DC NSAFA recebeu uma dieta com redução de pelo menos 20% de gorduras saturadas; aumento em > 50% da oferta de ácido graxo monoinsaturados e ácidos graxos poli-insaturados quando comparados ao grupo DC SAFA. Foram avaliadas características socioeconômicas e clínicas e marcadores: antropométricos; bioquímicos de adesão a dieta; de consumo alimentar; de estresse oxidativo; e de capacidade antioxidante em ambos os grupos. Esses parâmetros foram avaliados em três momentos: antes do início da DC (T0), três meses (T1) e seis meses (T2) após o início do tratamento. As análises estatísticas foram realizadas através do software SPSS. Resultados: Foram incluídas 26 crianças e adolescentes no grupo DC clássica e 26 no grupo DC modificada. A DC NSAFA conferiu alterações menos intensas no perfil lipídico com menor percentual de aumento de colesterol total, LDL-C, APO B, razão LDL-C por HDL-C, razão HDL-C por APO A, razão LDL-C por APO B, colesterol não HDL e ácidos graxos não esterificados em comparação com a DC SAFA. Em ambas as dietas houve aumento significativo da concentração de LDLox. Foi observado o aumento significativo do NFkB na DC SAFA, sem diferenças significativas no percentual de mudança dessa variável entre as intervenções, além disso na DC SAFA houve redução de Nrf2 e o mesmo se comportou de forma distinta entre as intervenções, sendo que na DC SAFA houve redução significativa desse marcador. Conclusão: a DC SAFA demonstrou apresentar pior perfil de resposta lipídica, inflamatória e oxidante em comparação a DC NSAFA, a adoção da DC NSAFA para o tratamento da epilepsia refratária poderia minimizar os efeitos negativos observados na DC SAFA e atenuar o principal efeito adverso observado que são as dislipidemias.Introduction: epilepsy is a neurological disease, its prevalence is estimated about 0,5 to 1 % in the population and 20 to 30% are refractory to the drug treatment. The ketogenic diet (KD) composed by high content of fat, low quantity of carbohydrate and adequate content of protein it is an adjuvant treatment with high efficacy in seizure control. Although it is well known about the efficacy of the diet, in humans there are few studies about the influence of the diet in oxidative biomarkers and its modulation in transcription factors such as NF-kB and Nrf2. Moreover it is not known about the impact of different types of dietetic fatty acids offers in KD could be influence in oxidative biomarkers and transcription factors. Objective: to evaluate and compare the impact of two different ketogenic diet, one composed by high content of saturated fatty acids (KD SAFA) and another one composed by high content of monounsaturated fatty acids and polyunsaturated fatty acids (KD NSAFA), on lipid and oxidative profile and activations of NF-kB and Nrf2 transcriptions factors in children and adolescents with refractory epilepsy on dietetic treatment with KD. Methods: clinical study, with 6 months of follow up, the patients was divided in two groups: one received a KD rich in saturated fatty acids and cholesterol (KD SAFA) and other received a KD with 20% less content of saturated fatty acids, increase in 50% of monounsaturated and polyunsaturated fatty acids comparing to KD SAFA (KD NSAFA). It was evaluated socioeconomic and clinical characteristics, anthropometric measure, food intake and lipids, oxidative and inflammatory biomarkers. The patients was evaluated in three different moments: before start the diet (T0), three months after start the KD (T1) and six months after start the KD (T2). The statistical analysis was performed using the software SPSS. Results: It was included 26 children and adolescents in KD SAFA and 26 in KD NSAFA. The participants treated with KD NSAFA had less modifications on classical lipid profile with less increase of: total cholesterol, LDL-C, APO B, LDL-C/HDL-C, HDL-C/APO A, LDL-C/APO B, non HDL cholesterol and non esterified fatty acids comparing to the participants treated with KD SAFA. In both diets there was a significant increase in oxidized LDL. It was observed increase of NFkB in the group treated with KD SAFA, without differences on the percentage of change of this biomarker between the interventions, in addition on the KD SAFA there was decrease in Nrf2 and the percentage of change it was different between the interventions with more reduction of this biomarker on KD SAFA. Conclusion: the KD SAFA showed worst profile in lipid, oxidative and inflammatory parameters comparing to KD NSAFA, suggesting that the use of KD NSAFA could be attenuate one of the main negative effect that are dyslipidemias.
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Macoch, Mélinda. « Impact de l’arsenic inorganique sur la physiologie in vitro des cellules dendritiques humaines ». Thesis, Rennes 1, 2013. http://www.theses.fr/2013REN1B013/document.
Texte intégralRésumé :
L’arsenic inorganique est un contaminant environnemental, cancérogène pour l’homme, mais également un métalloïde étudié, aujourd’hui, dans le traitement de maladies inflammatoires chroniques. Il possède des propriétés immunosuppressives pouvant déréguler les mécanismes physiologiques de défense ou bloquer l’exacerbation de réponses inflammatoires chroniques. L’arsenic inorganique altère principalement les fonctions des lymphocytes T et des macrophages. En revanche, l’impact du métalloïde sur la physiologie des cellules dendritiques (DCs) est peu connu. Pourtant, ces cellules présentatrices d’antigène jouent un rôle fondamental dans les processus d’immunosurveillance et sont très impliquées dans la physiopathologie des maladies inflammatoires chroniques. Dans ce contexte, les objectifs de mon travail de thèse étaient d’étudier les effets de l’arsenic inorganique sur la différenciation et la maturation in vitro de DCs générées à partir de monocytes humains. Nos résultats démontrent principalement que des concentrations de métalloïde, compatibles avec les taux plasmatiques d’arsenic mesurés chez les individus exposés, répriment la capacité des DCs à sécréter différentes cytokines pro-inflammatoires jouant un rôle essentiel dans l’activation et la polarisation des lymphocytes T. En particulier, l’arsenic inhibe l’expression et la sécrétion de l’interleukine-12 par un mécanisme moléculaire impliquant le facteur de transcription Nrf2. Au total, ces travaux de thèse démontrent que l’arsenic inorganique possède des propriétés immunosuppressives sur la physiologie in vitro des DCs humaines. Cette immunotoxicité pourrait contribuer à la toxicité du métalloïde chez l’homme exposé par voie environnementale, et être prise en compte pour déterminer les effets de l’arsenic dans le traitement de certaines maladies inflammatoires chroniquesInorganic arsenic is an environmental human carcinogen, but is also studied these days because of its potential effectiveness in curing chronic inflammatory disease. Indeed, this metalloid possesses immunosuppressive properties which can dysregulate physiological mechanisms involved in immune defense, or reduce inflammation associated with those inflammatory diseases. Inorganic arsenic is known mainly to alter functions of T cells and macrophages. However, it is unknown whether arsenic targets dendritic cells (DCs). Yet, this antigen presenting cells plays a major role in the immunosurveillance, and is involved in the physiopathology of chronic inflammatory diseases. So, the aim of my thesis work was to study the effects of inorganic arsenic on in vitro differenciation and maturation of dendritic cells from human monocytes. Our results mainly shows that concentrations corresponding to those measured in environmentally exposed people, inhibits DCs secretion of proinflammatory cytokines, which plays a major role in activation and polarization of T cells. Particularly, arsenic strongly impairs expression and secretion of interleukine 12 (IL-12) by an underlying molecular mechanism involving Nrf2. Finally, this work shows that inorganic arsenic has immunosuppressive properties on the physiology in vitro of human dendritic cells. Immunotoxicity may then contribute to the metalloid toxicity in environmentally exposed people. This element could be taken into account when determining arsenic effects in curing some chronic inflammatory diseases
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Page, Audrey. « Etude de la modulation de la réponse cellulaire au stress oxydatif par les protéines VP24 des virus Marburg et Ebola ». Phd thesis, Ecole normale supérieure de lyon - ENS LYON, 2012. http://tel.archives-ouvertes.fr/tel-00671994.
Texte intégralRésumé :
Les virus Ebola (EBOV) et Marburg (MARV) causent des fièvres hémorragiques chez les primates, y compris l'homme. Le taux de létalité peut atteindre 90% et il n'existe ni vaccin ni traitement contre ces virus. En raison de leurs caractéristiques moléculaires communes, EBOV et MARV sont regroupés au sein de la famille des Filoviridae. Le virion est composé de 7 protéines, dont la VP24, qui joue un rôle important dans l'assemblage et la condensation des nucléocapsides, et pour EBOV, elle est également responsable de l'inhibition de la réponse à l'IFN. Des mutations dans la séquence protéique de VP24 sont impliquées dans le processus d'adaptation chez un nouvel hôte. La protéine VP24 d'EBOV est donc multifonctionnelle. Pour MARV, cette protéine ne semble pas porter les fonctions décrites pour la VP24 d'EBOV. Afin de comprendre le rôle de la VP24 de MARV, nous avons identifié ses partenaires cellulaires par un crible double-hybride en levures. Nous avons mis en évidence l'interaction entre Keap1 et la VP24 de MARV, et confirmé ce résultat en cellules mammifères. Keap1 est une protéine impliquée dans le contrôle de la réponse au stress oxydatif, car elle inhibe le facteur de transcription Nrf2, qui régule l'expression d'enzymes impliquées dans la réduction des ERO. Nos résultats montrent que le domaine de Keap1 liant la VP24 est le même que celui liant Nrf2, et que la VP24 de MARV active Nrf2 pour la synthèse de molécules anti-oxydantes. Nous avons enfin évalué l'impact de la VP24 de MARV sur ERR, une autre cible de Keap1, et mesuré l'activité Nrf2 au cours de l'infection par EBOV. Nos résultats montrent des effets opposés des VP24 d' EBOV et de MARV sur l'activité de Nrf2.
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Murphy, Kelsey E. « BBB-bypassing polysaccharide mini-GAGR activates the neuronal Nrf2- mediated antioxidant defense system for the treatment of Alzheimer’s disease ». University of Toledo Health Science Campus / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=mco1576192220098119.
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Farage, Najla Elias. « Expressão dos fatores transcricionais Nrf2 e Nf-κB e associação com estado nutricional em pacientes renais crônicos em hemodialise ». Niterói, 2016. https://app.uff.br/riuff/handle/1/4615.
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Universidade Estadual de Campinas. Instituto de Filosofia e Ciências Humanas. Departamento de Antropologia
Pacientes renais crônicos, principalmente os que estão em diálise, possuem alta prevalência da chamada síndrome protein energy wasting e, dentre os fatores de risco destacam-se a inflamação e o estresse oxidativo. A ativação do Fator Nuclear- kappa B (NF-κB) está associada com a síntese de citocinas inflamatórias e, recentemente neste cenário, em contraste ao NF-κB, o sistema fator nuclear eritróide 2-relacionado ao fator 2-Kelch-like ECH proteína 1 (Nrf2-Keap1) tem surgido como importante mecanismo de defesa contra o estresse oxidativo e inflamação, promovendo a síntese de enzimas antioxidantes. Assim, o presente estudo teve como objetivo avaliar a expressão dos fatores de transcrição, NF-κB e Nfr2, em pacientes renais crônicos em hemodiálise (HD) e verificar possíveis associações com estado nutricional. Foram avaliados 83 pacientes em HD (47 homens, 52,3 ± 14,4 (22-84) anos, 24,7 ± 3,9 Kg/m2 e 6,2 ± 3,8 anos em tratamento dialítico). Após a coleta de sangue as células mononucleares foram isoladas (PMBC) a expressão de NF-κB e Nfr2 foi analisada pela Reação em cadeia de polimerase em tempo real. A análise da concentração plasmática de proteína C reativa ultrassensível foi realizada por ELISA. O estado nutricional foi avaliado por antropometria (% gordura corporal e Índice de Massa Corporal - IMC), pela estimativa de massa muscular (índice de creatinina - IC) e avaliação Subjetiva Global (ASG) de 7 pontos. De acordo com o IMC, 49,4% dos pacientes apresentaram sobrepeso ou obesidade e apenas um paciente apresentou baixo peso (IMC< 18,5 Kg/m2). De acordo com os níveis de proteína C reativa, 47,6% apresentaram inflamação (> 3mg/dL) e 62% apresentou baixos valores de IC (<22mg/Kg/d). A expressão do NF-κB foi positivamente correlacionada com a idade (r =0,33, p=0,02) e negativamente com o IC (r= -0,54, p=0,0001), albumina (r= -0,32, p= 0,02) e % GC (r = -0,61, p= 0,001). De acordo com a ASG, os pacientes desnutridos apresentaram maiores valores da expressão de NF-κB (1,7 ± 0.9, p=0,03), quando comparados com os eutróficos (1,11 ± 0.6). A expressão de Nrf2 não se correlacionou com os parâmetros antropométricos. Os dados parecem mostrar que a ativação do NF-κB contribui para a perda de massa muscular e desnutrição nos pacientes em HD, entretanto, estudos que avaliem melhor essa relação são necessários.
Protein energy wasting (PEW) is a common outcome in chronic kidney disease patients undergoing hemodialysis (HD) and oxidative stress and inflammation are common risk factors to PEW. Nuclear transcription factors are involved in these processes as Fator Nuclear-kappa B (NF-κB) which plays important role in coordinate expression of inflammatory genes and in contrast, the transcription nuclear E2-related factor 2 (Nrf2) operates in the genes promoter region coding for detoxifying enzymes stage II or antioxidant enzymes. The aim of this study was to evaluate the NF-kB and Nrf2 expression in patients undergoing HD and the association with nutritional status. Eighty three HD patients (47 men, 52.2 ± 14.4 (22-84) years, 24.7 ± 3.8 Kg/m2 and dialysis vintage 6.2 ± 3.8 years) were enrolled in this study. Blood samples were collected and peripheral blood mononuclear cells were isolated, and analyses of real time polymerase chain reaction was performed to evaluate the Nrf2 and NF-kB mRNA expression levels. The C-reactive protein (CRP) plasma levels were analyzed by ELISA. Nutritional status was assessed by anthropometry (% body fat and body mass index - BMI), estimation of muscle mass (creatinine index - CI) and subjective global assessment (SGA- 7-point scale version). According to BMI, 49.4% of patients were overweight or obese and only one patient had low weight (BMI <18.5 kg/m2). According to C-reactive protein levels, 47.6% had inflammation (> 3 mg/dL) and 62% had low CI values (<22mg/Kg/d). The expression of NF-κB was positively correlated with age (r = 0.33, p = 0.02), and negatively with CI (r = -0.54, p = 0.0001), albumin (r = - 0.32, p = 0.02) and % body fat (r = -0.61, p = 0.001). According to SGA, malnourished patients showed higher values of NF-κB expression (1.7 ± 0.9, p = 0.03) compared to eutrophic patients (1.11 ± 0.6). Nrf2 eas not correlated with nutritional status. In conclusion, the activation of NF-κB seems contribute to muscle loss and malnutrition in HD patients, however the mechanisms that lead to this mechanism remain unknown.
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Medvedev, Regina [Verfasser], Rolf [Akademischer Betreuer] [Gutachter] Marschalek et Eberhard [Gutachter] Hildt. « Impact of oxidative stress by hepatitis C virus-mediated Nrf2 inhibition on autophagy and viral particle release / Regina Medvedev ; Gutachter : Rolf Marschalek, Eberhard Hildt ; Betreuer : Rolf Marschalek ». Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2017. http://d-nb.info/1138276901/34.
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Fiorin, Fernando da Silva. « Efeito protetor do exercício físico nas alterações bioquímicas e cognitivas iniciais e tardias induzidas pelo traumatismo cranioencefálico em ratos ». Universidade Federal de Santa Maria, 2014. http://repositorio.ufsm.br/handle/1/11230.
Texte intégralRésumé :
Conselho Nacional de Desenvolvimento Científico e TecnológicoTraumatic brain injury (TBI) is a major cause of morbidity and mortality in industrialized countries leading to the motor and cognitive deficits. Evidence demonstrated that exercise is neuroprotective in traumatic brain injury. However, the effects of exercise before of the TBI at the cognitive function are unknown. Role of excitotoxicity and oxidative damage in secondary damage of TBI, however, until this moment, were not demonstrated if exists a relationship between early phase of damage and the late cognitive deficit. In the current study, we proposed that improvement cognitive response induced by exercise prior in rats after a TBI can be associated with the neuroprotection of early phase after injury. To demonstrate this hypotheses, adult rats practice swimming exercise during 6 weeks followed for TBI operation. We assessed the motor alterations of early phase, the glutamate uptake and antioxidant defense in twenty four hours (24 h) and 15 days after TBI. Acquisition of memory was assessed by recognition object task on days 15 post TBI. Moreover, we evaluated the brain-derived neurotrophic factor (BDNF) to assessement the synaptic plastic. In the present study, we showed that TBI induced by fluid percussion injury (FPI) in adult male Wistar rats induced early motor impairment 24 h, followed by learning retention deficit (2 weeks after neuronal injury). Previous swimming training improved the memory in object recognition task per se and protected against FPI-related disabilities. Although the FPI did not alter hippocampal expression of glutamate transporters (EAAT1 / EAAT2) and brain-derived neurotrophic factor (BDNF), the alterations in the redox status, herein characterized by DCFH-DA oxidation and SOD activity inhibition, led to marked impairment of protein functionally (Na+, K+-ATPase activity inhibition) and glutamate uptake inhibition 24 h after neuronal injury in sedentary injured rats. Indeed, the early increase of nuclear factor erythroid 2-related factor (pNRF2/NRF2 ratio) followed by a repair mechanism (protein HSP70 expression), 24 h and 2 weeks after neuronal injury, suggests that FPI-induced signal transduction may exert compensatory effect on pathophysiological processes. In this report we showed that previous physical exercise induced the increase of immune content of glutamate transporters (EAAT1/ EAAT2), pNrf2/Nrf2 ratio, SOD enzyme and HSP70 per se besides preventing against FPI-induced Na+, K+ - ATPase activity, glutamate uptake inhibition DCFH-DA oxidation 24 h after neuronal injury. The enhancement of hippocampal pNrf2/Nrf2 and HSP70 immune content in trained injured when compared with sedentary rats suggest that protein expression modulation associated to antioxidant defense elicited by previous physical exercise prevent against toxicity induced by TBI. The significant increase of BDNF levels in trained injured rats 24 h and 2 weeks strongly reinforce the idea that physical activity alters neuronal functions and thus delays or prevents secondary cascades that leave the neurobehavioral disability after TBI.
O traumatismo cranioencefálico (TCE) é uma das maiores causas de morte e morbidade nos países industrializados podendo levar ao comprometimento motor e déficits cognitivos. Evidências demonstram que o exercício físico é neuroprotetor na recuperação após o TCE. Porém, os efeitos do exercício físico antes do TCE na função cognitiva não são totalmente conhecidos. Sabe-se da participação da excitotoxicidade e do estresse oxidativo na cascata do dano secundário após o TCE, entretanto até o momento não foi demonstrado qual a relação da fase inicial após o TCE com os déficits cognitivos tardios. Portanto, no presente estudo, nós propomos que a melhora cognitiva tardia induzida pelo exercício prévio em ratos após o TCE pode estar associada com a neuroproteção da fase inicial após o dano. Para demonstrar esta hipótese, ratos adultos praticaram treinamento de natação durante 6 semanas e posteriormente foram submetidos a cirurgia para o TCE. Nós avaliamos as alterações motoras iniciais, a captação de glutamato e a defesa antioxidante em 24 horas (24 h) e 15 dias após o TCE. Aquisição da memória foi avaliada pela tarefa de reconhecimento de objetos em 15 dias após o TCE. Além disso, nós avaliamos o fator neurotrófico derivado do encéfalo (BDNF) para avaliar a plasticidade sináptica. No presente estudo, nós mostramos que o TCE induzido pela lesão de percussão de fluido (LPF) em ratos Wistar machos adultos induziu déficit motor inicial 24 h, seguido por déficit de aprendizagem (15 dias após o dano neuronal). O treinamento de natação prévio melhorou a memória na tarefa de reconhecimento de objeto per se e protegeu contra desabilidades relacionadas ao LPF. Embora o LPF não tenha alterado a expressão dos transportadores de glutamato (EAAT1/EAAT2) e de BDNF, causou uma alteração no estado redox, caracterizado pela oxidação de DCFH-DA e inibição da atividade da SOD. O LPF também causou prejuízo acentuado da funcionalidade de proteínas (inibição da atividade da enzima Na+, K+-ATPase) e inibição da captação de glutamato 24 h após o dano neuronal em ratos sedentários lesionados. De fato, o aumento inicial do fator de transcrição Nrf2 (relação pNrf2/Nrf2), 24 h após o TCE, seguido por um mecanismo de reparo (expressão da proteína Hsp70), 24 h e 15 dias após o dano neuronal, sugerem que a transdução de sinal induzida pelo LPF pode exercer um efeito compensatório em processos patofisiológicos. Neste trabalho, nós mostramos que o exercício físico prévio induziu o aumento do imunoconteúdo dos transportadores de glutamato (EAAT1/EAAT2), relação pNrf2/Nrf2, enzima SOD e a proteína Hsp70 per se, além de prevenir contra inibição da atividade da Na+, K+-ATPase, inibição da captação de glutamato e oxidação de DCFH-DA induzida pelo LPF, 24 h após o dano neuronal. O aumento do imunoconteúdo hipocampal de pNrf2/Nrf2 e Hsp70 em ratos treinados e lesionados quando comparado com ratos sedentários, sugerem que a modulação da expressão das proteínas associadas às defesas antioxidantes induzidas pelo exercício físico prévio preveniu contra a excitotoxicidade induzida pelo TCE. O significante aumento nos níveis de BDNF em ratos treinados e lesionados 24 h e 15 dias, reforçam fortemente a ideia que a atividade física altera a função neuronal e assim retarda ou previne as cascatas do dano secundário que levam a desabilidade neuronal após o TCE.
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Berthe, Julie. « Rôle de la protéine immuno-régulatrice PD-L1 sur le métabolisme des cellules tumorales ». Thesis, Lille, 2018. http://www.theses.fr/2018LIL2S006.
Texte intégralRésumé :
Lorsque les cellules normales évoluent vers un état néoplasique, elles acquièrent de nombreuses caractéristiques. Par exemple, ces cellules exhibent des voies métaboliques anormales et possèdent la capacité d’échapper à la destruction par les cellules de l’immunité, notamment en exploitant des points de contrôles immunitaires ou « immune checkpoints ». La molécule PD-L1 (Programmed Death-Ligand 1) appartient à la famille de protéines immuno-régulatrices B7 et a tout d’abord été décrite comme impliquée dans l’immuno-échappement tumoral suite à son interaction avec PD-1, récepteur exprimé à la surface des lymphocytes T. Associée à un mauvais pronostic, une expression aberrante de PD-L1 est retrouvée dans les hémopathies malignes ainsi que dans de multiples tumeurs solides. De manière intéressante, il a été montré que PD-L1 possède également des fonctions pro-tumorales intrinsèques. En effet, cette protéine joue un rôle dans la prolifération des cellules cancéreuses et leur résistance aux chimiothérapies, sans interagir avec PD-1. Toutefois, les mécanismes moléculaires modulés par PD-L1 et impliqués dans ces fonctions sont encore inconnus. Des voies métaboliques anormales ont été décrites comme pouvant contribuer à la croissance tumorale et la résistance aux thérapies. Ainsi, les objectifs de ma thèse ont été d’explorer le potentiel rôle de la protéine PD-L1 dans le métabolisme des cellules tumorales. En utilisant la méthode d’édition du génome avec les Zinc Finger Nucleases, nous avons invalidé le gène CD274 codant la protéine PD-L1 dans les cellules cancéreuses de sein MDA-MB-231 et investigué les fonctions métaboliques de cette molécule après surexpression dans ces mêmes cellules. Nous avons observé que PD-L1 induit un shift de la phosphorylation oxydative vers la glycolyse, correspondant à l’effet Warburg. Afin de valider cette reprogrammation métabolique, nous avons analysé le profil métabolique de ces cellules et mis en évidence une élévation des niveaux des intermédiaires de la glycolyse tels que le F-6-P, le F-1,6-P, le GAP, le DHAP, le PEP et le pyruvate dans la lignée surexprimant PD-L1, confirmant nos précédents résultats. D’autre part, et en accord avec nos observations quant à une augmentation de la production de ROS (Reactive Oxygen Species), nos données transcriptomiques suggèrent une répression de la voie de réponse au stress oxydatif NRF2 suite à l’expression de PD-L1 et notamment de ses gènes cibles tels que NQO2, GSTM3 et ABCC2. En outre, l’analyse in silico de bases de données de cohortes de patients atteints de cancer du sein a révélé une corrélation entre l’expression du gène PD-L1/CD274 et l’expression des gènes de la voie du stress oxydant (GSTM3 ; CYBB) ou des gènes codant les transporteurs de glucose (SLC2A1/GLUT1 ; SLC2A3/GLUT3), ces données supportant nos résultats obtenus in vitro. Par ailleurs, le glucose étant principalement utilisé par les cellules cancéreuses pour favoriser la biosynthèse de diverses biomolécules nécessaires à la prolifération cellulaire, ces résultats pourraient expliquer la tumorigénicité augmentée dans la lignée surexprimant PD-L1 lors des expériences de xénogreffe de cellules de cancer du sein humain chez des souris Nude. Ainsi, les travaux présentés dans cette thèse mettent en évidence de nouvelles fonctions intrinsèques de PD-L1 promouvant le développement tumoral, suggérant l’utilisation d’agents thérapeutiques inhibant ces mécanismes seraient prometteurs pour le traitement du cancer du seinEvolving to a neoplastic state, normal cells acquire many characteristics; indeed, tumor cells follow abnormal metabolic pathways and exhibit the ability to avoid immune destruction, partly by exploiting immune checkpoints. Many of these are currently under clinical investigation for new cancer treatments, notably the PD-1/PD-L1 axis.Programmed Death-Ligand 1 (PD-L1) molecule belongs to the B7 immunoregulatory proteins family and was originally described as mediating tumor immuno-escape through interaction with its receptor PD-1 on T cells. Associated with poor cancer outcome, aberrant PD-L1 expression has been observed in hematologic malignancies and in multiple solid tumor types. Actually, this protein has been shown to regulate tumor cell proliferation and resistance to chemotherapy through apoptosis inhibition, without interacting with PD-1. However, cellular mechanisms modulated by PD-L1 and involved in these functions are still unclear. Abnormal metabolic pathways are known for contributing to tumor growth and therapy resistance; therefore, the objective of my PhD thesis was to investigate the impact of PD-L1 in breast cancer cell metabolic reprogramming.Using genome editing, we knocked-out the CD274 gene encoding PD-L1 in breast cancer cell line MDA-MB-231 and investigated metabolic functions after PD-L1 overexpression in the same cells. We observed that PD-L1 induces a shift from oxidative phosphorylation to glycolysis, indicating this molecule promotes the Warburg effect in these tumor cells. To validate PD-L1 metabolic reprogramming, we performed metabolomic profiling that highlighted significantly increased levels of glycolysis intermediated such as F6P, F1,6P, GAP, DHAP, PEP and pyruvate in PD-L1-expressing cells, confirming our latter results. Moreover, in agreement with an increasing mitochondrial reactive oxygen species (ROS) production, transcriptomic study suggested that PD-L1 represses NRF2-mediated oxidative stress response pathway, especially NQO2, GSTM3 and ABCC2 genes. Furthermore, in silico analysis of breast cancer patients databases highlighted a correlation between PD-L1/CD274 gene and oxidative stress gene signature (GSTM3; CYBB) or glucose transporters genes (SLC2A1; SLC2A3) expressions, supporting our results. Besides, glucose is mostly used by cancer cells to favor biosynthesis of diverse biomolecules required for cellular proliferation; the above results could explain our human breast cancer cells xenograft experiments in Nude mice demonstrating that PD-L1 increases tumoreginicity.Thus, the work presented in this thesis evidences novel PD-L1 intrinsic tumor-promoting functions, suggesting that therapeutic agents inhibiting these mechanisms would be promising for breast cancer treatment
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Camus, Duboc Marine. « Chimiosensibilisation de l’adénocarcinome canalaire du pancréas par la perturbation du microenvironnement tumoral et l’augmentation de la biodisponibilité dans la cellule tumorale : effets de la cavitation ultrasonore et de l’inhibition de nrf2 ». Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB113.
Texte intégralRésumé :
L’adénocarcinome pancréatique (AP) connaît une forte augmentation d’incidence, qui en fait la quatrième cause de mortalité par cancer avec un pronostic extrêmement sombre, moins de 5% des patients étant en vie à 5 ans. De nombreuses avancées dans la compréhension de l’oncogénèse pancréatique notamment sur les aspects génétiques, immunitaires et sur les interactions cellulaires du stroma tumoral ont permis d’envisager le développement de nouvelles stratégies de traitement. Cependant malgré des résultats pré-cliniques très encourageants aucune de ces stratégies n’a encore permis l’émergence d’un traitement plus efficace que la chimiothérapie standard. Ce travail de thèse a abordé 2 approches thérapeutiques innovantes distinctes mais potentiellement complémentaires dans le traitement de l’adénocarcinome pancréatique en étudiant in vitro (cultures cellulaire 2D et 3D) et in vivo (modèles ectopiques et orthotopiques) les effets sur la croissance tumorale de l’inhibition d’un acteur important du stress oxydant (la voie Nrf2) d’une part, de la combinaison d’une chimiothérapie liposomale et d’un agent physique, la cavitation ultrasonore, d’autre part. La cavitation ultrasonore est un effet mécanique des ultrasons permettant d’augmenter l’internalisation de molécules ou de gènes dans les cellules. Dans cette thèse, la faisabilité et l'efficacité de la combinaison d’une chimiothérapie liposomale ciblée par la cavitation ultrasonore a été évaluée dans des modèles murins orthotopiques d’AP. Un système de délivrance d’ultrasons a été adapté afin d’appliquer une cavitation inertielle focalisée sur des xénogreffes d’AP créées après l'injection de doxorubicine liposomale (L-DOX) selon une étude pharmacocinétique préliminaire réalisée dans un modèle murin. La L-DOX, conçue à base de phospholipides non saturés de dioleoylphosphatidyléthanolamine, connue pour être stable dans la circulation sanguine, a été choisie afin de maximiser son accumulation et le relargage du principe actif lors de la délivrance des ultrasons. Nous montrons que l’association de la L-DOX à la cavitation inertielle permet de réduire in vivo le volume tumoral dans un modèle orthotopique d’AP chez la souris nude. La cavitation inertielle peut donc augmenter l'effet antitumoral des liposomes porteurs de chimiothérapie avec un effet mécanique minimal sur le tissu environnant la tumeur.Des études récentes suggèrent que Nrf2 est une cible de choix pour vaincre la chimiorésistance de l’AP. Des méthodes in vitro et in vivo ont été utilisées afin d’examiner l'effet du brusatol associé à des agents chimiothérapeutiques de référence sur la mort cellulaire et son impact sur le stress oxydant. Nous montrons que l’inhibition de la voie Nrf2 par le brusatol, un composé naturel issu de Fructus Bruceae, potentialise les effets de la chimiothérapie et permet l’inhibition de la croissance tumorale in vitro sur des lignées cellulaires d’AP cultivées en 2D et 3D. Cette inhibition s’accompagne d’une modulation du stress oxydant dont témoignent l’augmentation des espèces réactives de l’oxygène (ROS) et la diminution du glutathion (GSH). In vivo, la combinaison du brusatol et de l'oxaliplatine a réduit le volume tumoral dans deux modèles murins de xénogreffe d’AP. Ces résultats suggèrent l'efficacité du brusatol pour lutter contre la chimiorésistance et renforce l’hypothèse d’un rôle clinique potentiel de l’inhibition de Nrf2 comme adjuvant à la chimiothérapie dans l’AP. Un travail clinique a également été mené en parallèle sur une modalité de traitement physique innovante, la radiofréquence endobiliaire, dans la prise en charge endoscopique de l’ampullome vatérien, tumeur rare à la croisée entre le tube digestif et le système biliopancréatique. Les résultats de cette étude prospective multicentrique seront également présentés dans cette thèsePancreatic ductal adenocarcinoma (PDAC) has increased in incidence over the past decade, leading it to be the fourth lethal cause of cancer in the world with a very poor prognosis, since less than 5% of patients are alive at 5 years. Many advances in the understanding of pancreatic tumorigenesis, notably on the genetic, immune and cellular stroma interactions of the tumor, have led to the development of new treatment strategies in the last decade. However, despite very encouraging pre-clinical results, none of these strategies has yet led to the emergence of a truly effective treatment in comparison with standard chemotherapy. This thesis focused on two innovative therapeutic modalities in the treatment of PDAC at a preclinical stage by studying in vitro (2D and 3D cell cultures) and in vivo (ectopic, orthotopic xenografts) the effects on the tumor growth of an inhibitor of the Nrf2 pathway (involved in oxidative stress), on the first hand, and of a physical element, ultrasound cavitation associated with liposomal chemotherapy, on the second hand. Ultrasound cavitation is a mechanical effect of ultrasound to increase the uptake of molecules or genes in cells. The feasibility and effectiveness of the combination of liposomal chemotherapy targeted by ultrasonic cavitation was evaluated in murin orthotopic models of PDAC. An ultrasound delivery system has been adapted to apply focused inertial cavitation to PDAC xenografts created after the injection of liposomal doxorubicin (L-DOX) according to a preliminary pharmacokinetic study carried out in the murine model. L-DOX, designed on unsaturated phospholipids of dioleoylphosphatidylethanolamine, was known to be stable in the bloodstream and to maximize its accumulation and release of the active drug during ultrasound delivery. This thesis shows that this therapeutic combination (L DOX and inertial cavitation) makes it possible to reduce the tumor volume in vivo in a nude mouse orthotopic model of PDAC. Inertial cavitation may be generated to increase the therapeutic effect of chemotherapybearing liposomes accumulated in the tumor with minimal mechanical effect on the surrounding tissue. Recent studies strongly suggest that Nrf2 is an ideal target against chemoresistance of PDAC. In vitro and in vivo methods were combined to examine the effect of brusatol associated with chemotherapeutic agents on cell death in addition to its impact on oxidative stress (reactive oxygen species and gluthation levels). This thesis demonstrates that the inhibition of the Nrf2 pathway via brusatol, a natural compound derived from Fructus Bruceae, potentiates the effects of chemotherapy and allows the inhibition of tumor growth in vitro on PDAC cell lines. This inhibition is accompanied by a modulation of oxidative stress by brusatol, with increasing ROS and decreasing GSH. In vivo, the combination of brusatol and oxaliplatin reduced tumor volume in two mouse models of PDAC xenograft. These results suggest the efficacy of using brusatol to combat chemoresistance and reinforce the idea that brusatol could be developed as an adjuvant to chemotherapy in PA. Clinical work was also carried out in parallel on an innovative physical treatment modality, endobiliary radiofrequency, in the management of adenoma of the ampoule of Vater, a rare tumor located between the digestive and the bilio-pancreatic systems. The results of this work will also be presented in this thesis
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Ebabe, Elle Etienne Raymond. « Le double aspect des nanoparticules manufacturées sur les métabolismes oxydatifs et inflammatoires : effets délétères et effets protecteurs ». Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT008/document.
Texte intégralRésumé :
On étudie les effets des nanoparticules (d'argent et de silice) manufacturées sur les métabolismes oxydatifs et inflammatoire. La première partie étudie la toxicité in vivo de l'ingestion de nanoparticules d'argent, pendant 11 semaines, sur un modèle animal - rat Sprague Dawley. Nous y avons mis en évidence l'action toxique des nanoparticules d'argent notamment une hausse de la production d'anion superoxyde par les NADPH oxydases hépatiques et cardiaques, des dyslipidémies, une cytolyse hépatique, une augmentation de cytokines pro-inflammatoires et une tendance à la baisse de l'activité d'enzymes antioxydantes. Ceci nous a conduit à aborder l'étude in vitro, sur des modèles cellulaires intestinaux (Caco-2) et cutanés (HaCaT). Au cours de cette étude, des nanoparticules de silice, fonctionnalisées ou non avec des antioxydants, ont été incubées pendant 24 H en présence des cellules. Nous montrons que la modification de la surface des nanoparticules réduit considérablement leur toxicité en limitant la production d'espèces radicalaires et la mortalité cellulaire. D’autre part, le couplage avec un antioxydant permet d’augmenter la stimulation de voie de signalisation du facteur Nrf2. Cette voie est impliquée dans la protection de l’organisme contre les troubles liés aux espèces radicalaires. En somme, ce travail met en avant les potentialités de la vectorisation d’antioxydants avec des nanoparticules à des fins thérapeutiquesThe purpose of this study is to explore the effects of nanoparticles (silver and silica) manufactured on oxidative and inflammatory metabolism. In the first part of this work, we explored the in vivo toxicity from ingestion of silver nanoparticles, for 11 weeks, in an animal model - Sprague Dawley rat. This enabled us to demonstrate the toxic properties of silver nanoparticles including superoxide anion production by hepatic and cardiac NADPH oxidases, dyslipidemia, hepatic cytolysis, an increase in proinflammatory cytokines and a downward trend the activity of antioxidant enzymes. This led us to address the in vitro study on intestinal cell models (Caco-2) and cutaneous (HaCaT). During this study, silica nanoparticles, functionalized or not with anti-oxidants, were incubated for 24 hours in the presence of the cells. We show that the modification of the surface of the nanoparticles significantly reduces their toxicity limiting the production of free radical species and cell death. Furthermore, the coupling with an anti-oxidant increases the stimulation of Nrf2 factor that involves the protection of the body against disorders associated with radical species. In summary, this work highlights the potential of vectorization of antioxidants with nanoparticles for therapeutic purposes
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Frias, Daniela Perroni. « Participação do Nrf2 no processo de autofagia de células de brônquios humanos expostas ao material particulado de diesel ». Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/5/5160/tde-20032019-101342/.
Texte intégralRésumé :
As partículas eliminadas na exaustão do diesel (DEP) são importantes fontes diárias de partículas inaladas, responsáveis por gerar espécies reativas de oxigênio no sistema respiratório, fazendo com que as células ativem mecanismos de defesa, como o sistema Keap1-Nrf2 e a autofagia. Para investigar o papel do Nrf2 no processo de autofagia induzida pelas DEPs, BEAS-2B foram expostas às DEP, coletadas diretamente de um motor a diesel. BEAS-2B foram tratadas com sulforafano, bafilomicina e EBSS para testar a relação entre as vias autofágica e antioxidante. A quantidade relativa de mRNA foi verificada por RT-PCR para os seguintes genes: Nrf2, NQO1, HO-1, p62, Atg5 e LCB3. A seguir, BEAS-2B foram transfectadas com RNA silenciador (siRNA) para Nrf2, expostas ou não às DEPs (10 e 50 micro g/mL por 1h e 2 h), e mRNA detectado por RT-PCR e Western blot para proteínas. Bafilomicina (inibidor de autofagia) mostrou uma diminuição significativa nos marcadores antioxidantes Nrf2 (p = 0,024), HO-1 (p = 0,002) e NQO1 (p = 0,003), enquanto sulforafano (ativador de Nrf2) aumentou os marcadores autofágicos LC3B (p = 0,004) e Atg5 (p = 0,007). BEAS-2B expostas às DEP na concentração de 50 micro g/mL por 2hs mostraram um aumento significativo nos genes autofágicos LC3B (p = 0,018) e p62 (p = 0,007) e nos genes da via antioxidante Nrf2 (p = 0,007) e NQO1 (p = 0,025). Houve uma diminuição significativa no mRNA de LC3B (p < 0,001), p62 (p = 0,001) e Atg5 (p = 0,024) nas células transfectadas com siRNA, expostas ou não à DEP. Western blotting mostrou uma redução das proteínas Nrf2, p62 e LC3II nas BEAS-2B siRNA, indicando que a exposição ao silenciamento de Nrf2 modificou a expressão de marcadores de autofagia (R < 1). Os resultados deste estudo mostram que, em células brônquicas expostas às DEP, o sistema Nrf2 e a autofagia trabalham em conjunto para tentar manter a homeostase celularDiesel Exhaust Particles (DEPs) are main sources of daily inhaled particles, responsible for generating reactive oxygen species in the respiratory system, and causing the cells to activate defense mechanisms, such as the Keap1-Nrf2 system and autophagy. In order to investigate the role of Nrf2 in Dep-induced autophagy, BEAS-2B cells collected directly from a diesel engine were exposed to DEP and treated with sulforaphane, bafilomycin and BESS to test the relationship between autophagic and antioxidant pathways. The relative amount of mRNA was verified by RT-PCR for the following genes: Nrf2, NQO1, HO-1, p62, Atg5 and LCB3. Next, BEAS-2B cells were transfected with silencer RNA (siRNA) specific to Nrf2, exposed or not to DEPs (10 and 50 micro g/mL 1h and 2hs), and mRNA detected by RT-PCR and Western blotting for protein. Bafilomycin ( autophagy inhibitor) showed a significant decrease in the antioxidant markers Nrf2 (p=0.024), HO-1 (p = 0.002) and NQO1 (p = 0.003), whereas sulforaphane (Nrf2 activator) increased the expression levels of autophagic markers LC3B (p=0.004) and Atg5 (p=0.007). BEAS-2B exposed to DEP at a concentration of 50 micro g/mL for 2hs showed a significant increase in autophagic genes LC3B (p=0.018) and p62 (p=0.007),and in the antioxidant pathway markers Nrf2 (p=0.007) and NQO1 (p=0.025). There was a significant decrease in mRNA of the LC3B (p < 0.001), p62 (p=0.001) and Atg5 (p=0.024) in cells transfected with siRNA, exposed or not to DEP. Western blotting showed a reduction of Nrf2, p62 and LC3II proteins in BEAS-2B transfected with siRNA, indicating that Nrf2 silencedexposed to DEP modulated the expression of autophagy markers (R < 1). The results of this study show that, in bronchial cells exposed to DEP, the Nrf2 system and autophagy work together in order to try to maintain cellular homeostasis
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Ali, Malika. « Effets des activateurs pharmacologiques de Nrf2 sur la réponse antioxydante et anti-inflammatoire dans le macrophage humain Comparative effectiveness of 5 natural and chemical activators of Nrf2 on inflammation, oxidative stress, macrophage polarization, and bactericidal activity in an in vitro macrophage infection model Sulforaphane reduces intracellular survival of Staphylococcus aureus in macrophages through inhibition of JNK and p38 MAPK-induced inflammation ». Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLV100.
Texte intégralRésumé :
Etat de la question : Les infections respiratoires jouent un rôle important dans les exacerbations au cours des pathologies respiratoires chroniques (bronchopneumopathie chronique obstructive, asthme, insuffisance respiratoire des maladies neuromusculaires). Ces exacerbations entraînent une augmentation de la morbidité et de la mortalité, une dégradation de la qualité de vie et une majoration des dépenses de santé. En contact direct avec l’environnement extérieur, les voies respiratoires et le poumon sont la première cible des aérocontaminants tels que les oxydants issus de polluants atmosphériques et les agents microbiens. Les cellules inflammatoires (macrophages et polynucléaires neutrophiles) réagissent à l’agression microbienne en augmentant leur production d’oxydants. L’excès d’oxydants a un effet bactéricide bien connu mais peut conduire à la mort des cellules du système respiratoire, notamment par apoptose. Pour faire face à ce stress oxydant, les cellules de l’hôte activent de nombreuses défenses antioxydantes dont l’une est la cascade signalétique contrôlée par le facteur de transcription Nrf2 (Nuclear factor Erythroid 2-related factor 2). Nrf2 joue un rôle crucial dans l’induction de l’expression de nombreux gènes codant pour les enzymes de phase II telles que : hème oxygénase 1, glutathion-S-transférases, catalase, superoxyde dismutase, NADPH quinone oxydoréductase et époxyde hydrolase. Ces enzymes peuvent être impliquées dans les réponses anti-inflammatoires, antioxydantes et cytoprotectrices. En conditions normales, Nrf2 est séquestré dans le cytoplasme par son inhibiteur Keap1 (Kelch-like Ech-associated protein 1) où il est rapidement dégradé par le protéasome. En présence d’un stress oxydant, Nrf2 est libéré et transloque dans le noyau, où il hétérodimérise avec ses co-facteurs et se fixe sur les séquences régulatrices ARE (pour antioxidant responsive element) situées dans les promoteurs de plus d’une centaine de gènes dont ceux codant pour les enzymes de phase II. Nous avons démontré un lien entre la diminution de Nrf2 activée et la diminution de l’expression des enzymes de phase II dans les macrophages alvéolaires de patients atteints d'emphysème pulmonaire post-tabagique. Ce déficit pourrait être associé à une plus grande sensibilité des cellules aux infections microbiennes. Cependant, plusieurs études ont montré que la voie de signalisation Nrf2 pouvait jouer un rôle aussi bien bénéfique que délétère au cours des infections. Récemment, nous avons mis en évidence une augmentation de la bactéricidie des macrophages dérivés de la lignée THP1 après traitement par un activateur de Nrf2. Dans ces conditions, une activation des voies signalétiques contrôlées par Nrf2 et p38 MAPK conduisait à une apoptose cellulaire et une diminution de la prolifération bactérienne dans les macrophages. L’objectif principal de ce projet de thèse est de déterminer l’intérêt thérapeutique de la modulation de la voie de signalisation Nrf2 en pathologie respiratoire, en utilisant des activateurs pharmacologiques et non pharmacologiques. Objectifs du projet : - Caractériser in vitro et in vivo des effets moléculaires, cellulaires et physiologiques de la modulation de Nrf2 par des traitements pharmacologiques et non pharmacologiques ; - Comprendre les mécanismes impliqués dans la réponse immunitaire des macrophages après activation de la voie de signalisation Nrf2. Stratégies : 1. Utilisation de modèles cellulaires et murins (souris Nrf2-/-) après infection bactérienne 2. Utilisation de techniques moléculaires, cellulaires et physiologiquesState of the question: Respiratory infections are important in exacerbations in chronic respiratory diseases (COPD, asthma, respiratory failure of neuromuscular diseases). These exacerbations lead to increased morbidity and mortality, a deterioration in the quality of life and an increase in health spending. In direct contact with the external environment, the respiratory tract and lungs are the primary target of airborne contaminants such as oxidants from air pollutants and microbial agents. Inflammatory cells (macrophages and neutrophils) react to microbial attack by increasing their production of oxidants. Excess oxidants well known bactericidal effect but can lead to cell death of the respiratory system, including apoptosis. To address this oxidative stress, the host cells activate many antioxidant defenses of which is the signaling cascade controlled by the Nrf2 transcription factor (Nuclear factor Erythroid 2-related factor 2). Nrf2 plays a crucial role in the induction of the expression of numerous genes encoding phase II enzymes such as heme oxygenase 1, glutathione S-transferase, catalase, superoxide dismutase, NADPH quinone oxidoreductase and epoxide hydrolase. These enzymes may be involved in anti-inflammatory responses, antioxidant and cytoprotective. Under normal conditions, Nrf2 is sequestered in the cytoplasm by the inhibitor Keap1 (Kelch-like Ech-associated protein 1) where it is rapidly degraded by the proteasome. In the presence of oxidative stress, Nrf2 is released and translocates into the nucleus, where it heterodimerizes with its co-factors and binds to the regulatory sequences ARE (for antioxidant responsive element) located in the promoters of more than one hundred genes including those coding for the phase II enzymes. We have shown a link between the activated reduction of Nrf2 and the decrease in the expression of phase II enzymes in alveolar macrophages from patients with post-smoking pulmonary emphysema. This deficit may be associated with a greater cell sensitivity to microbial infections. However, several studies have shown that Nrf2 signaling pathway may play a beneficial role as well as deleterious during infections. Recently, we have demonstrated an increase in macrophage bactericidal derivatives THP1 line after processing by Nrf2 activator. Under these conditions, activation of the signaling pathways controlled by Nrf2 and p38 resulted in cellular apoptosis and a decrease in bacterial growth in macrophages. The main objective of this thesis project is to determine the therapeutic value of the modulation of Nrf2 signaling pathway in respiratory diseases, using pharmacological and non-pharmacological activators. The project's objectives : - Characterize in vitro and in vivo effects of molecular, cellular and physiological modulation of Nrf2 by pharmacological and non-pharmacological treatments; - Understanding the mechanisms involved in the immune response of macrophages after activation of the Nrf2 signaling pathway. strategies: 1. Use of cell and mouse models (mouse Nrf2 - / -) after bacterial infection 2. Using molecular techniques, cellular and physiological
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Alencar, Luciane Luca de. « Estudo dos polimorfismos Pro198Leu no gene da glutationa peroxidase 1 e -617C/A no gene do fator de transcrição Nrf2 com relação ao estresse oxidativo e ao estado nutricional relativo ao selênio de pacientes com diabetes mellitus tipo 1 ». Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/9/9132/tde-27052015-142459/.
Texte intégralRésumé :
Estudos têm mostrado que a atividade da enzima glutationa peroxidase (GPx) se encontra reduzida na presença do polimorfismo de nucleotideo único (SNP) Pro198Leu no gene que codifica para a GPx1. Associado a isso, polimorfismos na região promotora do gene do fator de transcrição Nrf2, o qual se liga ao elemento de resposta antioxidante na via de expressão de genes de enzimas antioxidantes, também pode alterar a expressão gênica da GPx1. Como o mineral selênio faz parte do sítio catalítico desta enzima antioxidante, muitos estudos têm associado o estado nutricional relativo a este nutriente com doenças relacionadas ao estresse oxidativo, como o diabetes mellitus tipo 1 (DM1). Nesse sentido, este estudo visou avaliar a presença dos SNPs Pro198Leu e -617 C/A no Nrf2, bem como da expressão gênica da GPx1 em pacientes com diabetes mellitus tipo 1 e relacioná-los com marcadores do estado nutricional relativo ao selênio e de estresse oxidativo, comparados com um grupo controle sem a doença. Este é um estudo caso e controle, constituído por dois grupos experimentais, um grupo composto por 77 pacientes com diabetes mellitus tipo 1 (DM1) atendidos no Setor de Endocrinologia do Hospital das Clínicas, com idade entre 10 e 19 anos, de ambos os gêneros, e um grupo controle (GC) constituído por 74 indivíduos da mesma faixa etária, os quais relataram ausência de doenças crônicas. Foi realizada avaliação antropométrica e da ingestão alimentar. Além disso, foram determinados parâmetros bioquímicos de status de selênio, controle glicêmico (glicose sérica, HbA1c), atividade enzimática, concentração de malondialdeído e 8-isoprostanos. A determinação dos SNPs e da expressão gênica foi realizada por PCR em tempo real. O grupo DM1 apresentou média de idade de 15,9 anos e o GC de 13,4 anos. A concentração de glicose sérica e HbA1c foi significativamente diferente entre os grupos (p<0,001). As frequências dos genótipos do SNP da GPx1 para o grupo DM1 e GC foram, respectivamente, 60% e 61% (CC), 30% e 32% (CT), 10% e 7% (TT),estando em equilíbrio gênico. A concentração de selênio no plasma foi significativamente maior no grupo DM1 e, ao avaliar essa concentração de acordo com genótipos, observou-se menor concentração de selênio no plasma no genótipo TT no grupo controle (p<0,05). A expressão gênica da GPx1 não apresentou diferença estatística entre os grupos nem entre os genótipos. O mesmo foi observado quanto à concentração de 8-isoprostanos. No entanto, a atividade das enzimas GPx e SOD assim como a concentração de MDA foram significantemente maiores no grupo DM1 (p<0,05). O estado nutricional de todos participantes em relação ao selênio estava deficiente. Foi observada correlação entre a concentração de selênio no plasma e nos eritrócitos e a atividade da GPx, assim como foi observada maior atividade enzimática e concentração de MDA no grupo DM, sem apresentar diferença na distribuição segundo os alelos estudados. Desta forma, pode-se concluir que independentemente da doença todos os indivíduos apresentaram deficiência de selênio. Em ambos os grupos, foi observada maior peroxidação lipídica, na presença do alelo variante T, o que pode indicar alteração da proteção antioxidante. No entanto, a presença do alelo variante nos SNPs avaliados não apresentaram influencia sobre a expressão gênica.Studies have shown that the activity of glutathione peroxidase (GPx) is reduced in the presence of single nucleotide polymorphism (SNP) in Pro198Leu encoding GPx1 gene. Beyond that, polymorphism of the transcription factor Nrf2 gene promoter, which binds to the antioxidant response element in the pathway of antioxidant enzymes gene expression, may also alter gene expression of GPx1. As mineral selenium is part of the catalytic site of this antioxidant enzyme, many studies have associated the nutritional status of this nutrient with oxidative stress diseases, such as type 1 diabetes mellitus (DM1). Thus, this study aimed to evaluate the presence of Pro198Leu and -617 C / A in Nrf2 SNPs, as well as GPx1 gene expression in patients with type 1 diabetes mellitus, and to associate them with nutritional status of selenium and stress oxidative markers, comparing with a control group without the disease. This is a case-control study, compound of two groups, one group containing 77 patients with type 1 diabetes mellitus (DM1) from the Service of Endocrinology of Hospital das Clinicas, aged between 10 and 19 years, of both genders, and a control group (CG) was composed for 74 individuals of the same age, who reported no chronic diseases. Anthropometric and dietary intake assessment was performed. In addition, the biochemical parameters of selenium status, glycemic control (serum glucose, HbA1c), enzyme activity, concentration of malondialdehyde and 8-isoprostane were determined. The determination of SNPs and gene expression was performed by real-time PCR. The DM1 group had a mean age of 15.9 years and 13.4 years for the CG. The concentration of serum glucose and HbA 1c were significantly different between groups (p <0.001). The genotype frequencies of the GPx1 SNP for DM1 and control group were, respectively, 60% and 61% (CC), 30% and 32% (CT), 10% and 7% (TT), which is in genetic equilibrium. The selenium concentration in plasma was significantly higher in DM1 group. To assess the selenium concentration according to genotypes, we observed lower plasma concentration in TT genotype in the control group (p <0.05). The GPx1 gene expression showed no statistical difference between groups or between genotypes. The same result was observed for the 8-isoprostane concentration. However, GPx and SOD activity and MDA concentration were significantly higher in DM1 (p <0.05). The nutritional status of all participants in relation to selenium was deficient. Correlation between selenium concentration in plasma and erythrocytes and GPx activity, as well as higher enzyme activity and MDA concentration in the DM1 group were observed, with no significant difference in the distribution according to studied alleles was observed. The presence of the variant allele in the SNPs evaluated showed influence neither on gene expression, nor on the activity of GPx. Thus, it can be concluded that, regardless of the disease, all subjects had low nutritional status of selenium, without genotype influence, and, as expected, oxidative stress was increased in individuals with DM1, as demonstrated by laboratory tests.
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Goven, Delphine. « Régulation de l’hème oxygénase-1 dans les macrophages au cours des pathologies pulmonaires liées à l’exposition de la fumée de cigarette ». Thesis, Paris Est, 2009. http://www.theses.fr/2009PEST0051.
Texte intégralRésumé :
L’intoxication tabagique, source d’oxydants, est un facteur de risque important de développement de l’emphysème pulmonaire et du pneumothorax spontané primitif. Les macrophages alvéolaires contribuent pour une large part à l’inflammation pulmonaire au cours de ces pathologies en produisant des métalloprotéases et des espèces réactives de l’oxygène à l’origine du déséquilibre des balances protéase/anti-protéase et oxydant/antioxydant. L'hème oxygénase-1 (HO-1), exprimée principalement par les macrophages, est une enzyme clé des défenses anti-oxydantes pulmonaires. Nous avons initialement étudié l’expression et la localisation cellulaire de l’HO-1 et de ses régulateurs potentiels (Nrf2, Keap1, Bach1 et HIF-1a) dans les macrophages alvéolaires au cours de l’emphysème pulmonaire post-tabagique et du pneumothorax spontané primitif. Les voies de régulation de l’expression de ces protéines ont été analysées in vitro sur des macrophages dérivés de la lignée THP-1 exposés ou non au condensat de fumée de cigarette et à l’hypoxieréoxygénation visant à mimer une partie des effets de l’atélectasie-réexpansion observée lors de la prise en charge thérapeutique des pneumothorax récidivants. Les travaux présentés dans cette thèse nous ont permis de mettre en évidence une altération de l’expression de la voie Nrf2/Keap1-Bach1 associée à une diminution de l’expression des enzymes anti-oxydantes, dont l’HO-1, dans les macrophages alvéolaires au cours de l’emphysème pulmonaire sévère post-tabagique, malgré un stress oxydant important. In vitro, ces altérations pourraient être liées à une activation spécifique des MAPKinases ERK1/2 et JNK par le condensat de fumée de cigarette. Nous avons également montré que la stimulation du système de l’HO-1 était probablement orchestrée par la voie du facteur HIF-1a, et non par celle de Nrf2, dans les macrophages alvéolaires au cours du pneumothorax spontané primitif récidivant du sujet fumeur. Ces résultats pourraient contribuer à une meilleure connaissance de la physiopathologie de l’emphysème pulmonaire et permettre d’envisager de nouvelles approches thérapeutiques basées sur la préservation et/ou la restauration de l’équilibre Nrf2/Keap1-Bach1. Nos travaux suggèrent également que la physiopathologie du pneumothorax spontané primitif est différente chez les patients fumeurs et non fumeurs. Le pneumothorax du sujet fumeur est associé à un stress oxydant pulmonaire et à une induction de l’HO-1 probablement orchestrée par HIF-1a. Ces résultats, confirmés in vitro, mettent en évidence une interaction potentielle entre le stress oxydant et l’hypoxie-réoxygénationChronic cigarette smoking, a source of oxidants, is an important risk factor for lung emphysema and primary spontaneous pneumothorax development. Alveolar macrophages are mainly involved in lung inflammation observed in these pathologies through the production of metalloproteases and reactive oxygen species resulting to protease/anti-protease and oxidant/anti-oxidant imbalances. Heme oxygenase-1 (HO-1), mainly expressed in macrophages, is a key enzyme in pulmonary anti-oxidant defences. Therefore, the first aim of our studies was to investigate the expression and cellular localisation of HO-1 and its potential regulators (Nrf2, Keap1, Bach1 and HIF-1a) in alveolar macrophages from smoking related lung emphysema and primary spontaneous pneumothorax. Regulation pathways involved in expression of these proteins were assessed in vitro in macrophage cell line THP-1 exposed or not to cigarette smoke condensate and with or without hypoxia-reoxygenation mimicking parts of events induced by atelectasia-reexpansion during recurrent pneumothorax constitution and treatment. In these studies, we showed an altered expression of Nrf2/Keap1- Bach1 pathway associated with a reduced expression of anti-oxidants enzymes, like HO-1, in alveolar macrophages from smoking related lung emphysema patients, despite an important oxidative stress. These alterations might be related to cigarette smoke condensate activated ERK1/2 and JNK MAPKinases as observed in THP-1 cells. Furthermore, we showed that HO- 1 system induction was mediated by HIF-1a instead of Nrf2 pathway in alveolar macrophages from smoking related recurrent primary spontaneous pneumothorax. These findings may contribute to a better knowledge of the pathophysiology of lung emphysema and could provide new therapeutic approaches based on preservation and/or restoration of Nrf2/Keap1-Bach1 equilibrium. Our results also suggest that the pathophysiology of primary spontaneous pneumothorax could be different in smokers and non smokers. Spontaneous pneumothorax in smokers is associated with lung oxidative stress and the orchestrated induction of HO-1 probably via HIF-1a. These results provide a new link between oxidative stress and hypoxia/reoxygenation
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Corssac, Giana Blume. « Efeitos do sulforafano em parâmetros de estresse oxidativo em cultura de cardiomiócitos adultos ». reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2017. http://hdl.handle.net/10183/165287.
Texte intégralRésumé :
O sulforafano (SFN) é um composto natural que possui propriedades antioxidantes, estimulando, principalmente, o sistema antioxidante endógeno celular. Este composto está associado a uma via clássica de ativação, a via do fator eritroide nuclear tipo 2 (Nrf2). Entretanto, estudos mais recentes têm demonstrado que a ação do SFN também pode se dar pela via do coativador 1-alfa do receptor ativado por proliferador do peroxissoma (PGC-1α). A diferença da via de ativação pelo SFN parece ter relação com o tempo de exposição das células a este composto. Visto que o SFN é uma importante estratégia terapêutica no combate ao estresse oxidativo, que está relacionado ao desenvolvimento de diversas doenças cardiovasculares, a investigação do seu mecanismo de ação é necessária. A análise in vitro é uma ferramenta importante para a investigação das vias e tempos de incubação envolvidos na ação antioxidante do SFN. Sendo assim, a cultura primária de cardiomiócitos de ratos adultos é um dos modelos que pode ser utilizado, sendo a sua principal vantagem, o fato da fisiologia destas células se aproximar mais das condições fisiológicas in vivo. O objetivo deste estudo, então, foi analisar a estimulação de defesas antioxidantes feita pelo SFN, através das vias do Nrf2 e do PGC-1α, em tempos diferentes, utilizando a técnica de cultura de cardiomiócitos adultos. Ratos Wistar machos foram eutanasiados, para que seus corações fossem retirados e submetidos ao processo de isolamento de células cardíacas, em aparelho de Langendorff modificado. As células foram isoladas através da perfusão do coração com solução de Krebs e colagenase tipo II, por um período de 30 minutos. Após isso, as células isoladas foram plaqueadas e mantidas em incubadora a 37°C e 5% de CO2. Foi realizado o tratamento com 5 μM de SFN e/ou 5 μM de peróxido de hidrogênio (H2O2). As células foram divididas nos seguintes grupos experimentais: Controle, SFN, H2O2 e SFN+H2O2. Os grupos foram subdivididos em dois tempos de incubação: 1 e 24 horas. Foram realizadas as análises dos níveis totais de espécies reativas de oxigênio (ROS) e de lipoperoxidação (LPO); atividade das enzimas antioxidantes superóxido dismutase (SOD), catalase (CAT) e glutationa s-transferase (GST); expressão proteica das isoformas citosólica (SOD-1) e mitocondrial (SOD-2) da SOD, e dos fatores Nrf2 e PGC-1α. Os resultados do trabalho mostram que, em relação ao tempo de 1 hora, o SFN incubado por 24 horas aumentou em 59% a atividade da SOD, 55% a expressão proteica da SOD-1, 24% a expressão proteica da SOD-2 e 69% a expressão proteica do PGC-1α. A expressão do Nrf2 foi 17% maior no tempo de 1 hora, em relação a 24 horas. Em relação à atividade da catalase e aos níveis de ROS e de LPO, houve diferença somente nos grupos incubados por 1 hora, nos quais a atividade da CAT foi menor no grupo H2O2, os níveis de ROS estavam diminuídos no grupo SFN, e os níveis de LPO estavam maiores no grupo H2O2. Não foram encontradas diferenças em relação à atividade da GST. Como conclusão, o SFN demonstrou um papel protetor nos grupos 1 hora, impedindo a geração de ROS e de dano a lipídeos, apesar de não apresentar um efeito expressivo sobre as enzimas antioxidantes. O efeito dos tempos de incubação na expressão do Nrf2 (aumentada em 1 hora) e do PGC-1α (aumentada em 24 horas) mostrou que realmente há uma relação temporal entre a sinalização destas duas vias, ativadas pelo SFN. Este resultado é instigante para que futuras análises dessa relação temporal das vias do SFN sejam realizadas.Sulforaphane (SFN) is a natural compound that has antioxidant properties, mainly stimulating the endogenous cellular antioxidant system. This compound is associated with a classical pathway of activation, the nuclear erythroid factor 2 (Nrf2) pathway. However, more recent studies have shown that the action of SFN can also occur through the peroxisome proliferator-activated receptor coactivator 1-alpha (PGC-1α). The difference in the pathway of activation by SFN seems to be related to the time of exposure of the cells to this compound. Since SFN is an important therapeutic strategy in the fight against oxidative stress, which is related to the development of various cardiovascular diseases, the investigation of its mechanism of action is necessary. In vitro analysis is an important tool for investigating the pathways and incubation times involved in the antioxidant action of SFN. Thus, a primary culture of adult mouse cardiomyocytes is one of the models that can be used, the main advantage being that the physiology of these cells are closer to the physiological conditions in vivo. The objective of this study was to use adult cardiomyocyte culture technique to analyze the stimulation of antioxidant defenses by SFN through Nrf2 and PGC-1α pathways at different times. Male Wistar rats were euthanized, so that their hearts were removed and submitted to the process of isolation of cardiac cells, in modified Langendorff apparatus. Cells were isolated by perfusion of the heart with Krebs solution and type II collagenase for a period of 30 minutes. After that, the isolated cells were plated and incubated at 37°C and 5% CO2. Treatment was performed with 5μM SFN and/or 5μM hydrogen peroxide (H2O2). Cells were divided into the following experimental groups: Control, SFN, H2O2 and SFN+H2O2. The groups were subdivided into two incubation times: 1 and 24 hours. Analyzes of total oxygen reactive species (ROS) and lipoperoxidation (LPO) levels were performed; activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione s-transferase (GST); protein expression of citosolic (SOD-1) and mitochondrial (SOD-2) isoforms of SOD, as well as Nrf2 and PGC-1α factors. The results of this work show that, compared to 1 hour time, SFN incubated for 24 hours increased SOD activity by 59%, SOD-1 protein expression by 55%, SOD-2 protein expression by 24%, and 69% PGC-1α protein expression. Expression of Nrf2 was 17% higher at 1 hour, over 24 hours of incubation. Regarding catalase activity and ROS and LPO levels, there were differences only in the groups incubated for 1 hour, in which the CAT activity was lower in H2O2 group, the ROS levels were decreased in SFN group, and levels of LPO were higher in H2O2 group. No differences were found in relation to GST activity. In summary, SFN demonstrated a protective role in 1 hour groups, preventing generation of ROS and lipid damage, although it does not present an expressive effect on the expression of antioxidant enzymes. The effect of incubation times on expression of Nrf2 (increased by 1 hour) and PGC-1α (increased by 24 hours) showed that there is actually a temporal relationship between the signaling of these two pathways, activated by SFN. This result is instigating for future analyzes of this temporal relationship of SFN pathways to be performed.
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MBIANDJEU, TOYA SERGE CEDRICK. « Functional Characterization of Nrf2 in erythroid cells : from erythropoiesis to mature red cells ». Doctoral thesis, 2019. http://hdl.handle.net/11562/995084.
Texte intégralRésumé :
Erythropoiesis is a dynamic and multistep process where early erythroid progenitors undergo differentiation into matured red cells. Nrf2 is a transcription factor that participates in acute response to oxidative stress and controls the expression of anti-oxidant and cytoprotective systems. Mice genetically lacking Nrf2 show a mild chronic hemolytic anemia due to erythrophagocytosis. Here, we show that Nrf2-/- mice display an age-dependent anemia characterized by accelerated senescence of circulating erythrocytes and reduced reticulocyte count, suggesting a perturbation of erythroid maturation process. Indeed, we found ineffective erythropoiesis in 12 months-old Nrf2-/- mice as supported by extramedullar erythropoiesis, increased ROS levels and cell apoptosis. In agreement, Nrf2-/- mice showed a blunted response to stress erythropoiesis induced by either PHZ or Doxo, suggesting an impairment of cellular back up mechanisms against oxidative stress such anti-oxidants and cytoprotective systems. The persistent oxidation promoted activation of UPR system and autophagy, which are unable to fully counteract oxidation re-directing cells towards apoptosis as supported by the increased caspase 3 activity. As a proof of concept, we used Astaxanthin as powerful anti-oxidant administrated in PLGA loaded nanoparticles (ATS-NP). In Nrf2-/- mice, ATS-NP ameliorated the age-dependent anemia and improved ineffective erythropoiesis with inactivation of UPR system and autophagy. In conclusion, we propose Nrf2 as key transcriptional factor against aged related oxidation to ensure erythroid maturation and growth. Future studies should be designed to evaluate the impact of Nrf2 activators as well as of ATS-NP administration in models of pathologic erythropoiesis.
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Conteh, Abass M. « The Role of Lipoxygenase and Interleukin-6 on Islet β-cell Oxidative Stress and Dysfunction ». Diss., 2019. http://hdl.handle.net/1805/19948.
Texte intégralRésumé :
Indiana University-Purdue University Indianapolis (IUPUI)Type 1 and Type 2 diabetes (T1D/T2D) share a common etiology that involves an increase in oxidative stress that leads to dysfunction and subsequent β cell death. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids to form lipid metabolites involved in a variety of biological functions including cellular oxidative stress response. On the other hand, Interleukin 6 (IL-6) signaling has been demonstrated to be protective in islets. In this study, we explored the effect of lipoxygenase enzymes 12-Lipoxygenase, 12/15 Lipoxygenase and IL-6 on β cell function and survival in mice using both STZ and high-fat diet (HFD) models of diabetes. Alox12-/- mice showed greater impairment in glucose tolerance following STZ and HFD compared to wild-type mice (WT), whereas Alox15-/- were protected against dysglycemia. These findings were accompanied by evidence of islet oxidative stress in Alox12-/- mice and reduced oxidative stress in Alox15-/- mice, consistent with alterations in the expression of antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12-/- mice showed a compensatory increase in Alox15 gene expression and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. IL-6 was able to significantly attenuate the generation of reactive oxygen species by proinflammatory cytokines in human pancreatic islets. Furthermore, we find that IL-6 regulates the master antioxidant response protein NRF2. Collectively these results show that loss of Alox12 activates a compensatory increase in Alox15 that sensitizes β cells to oxidative stress and signaling by IL-6 is required for maximal antioxidant response under conditions of increased ROS formation, such as obesity.
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Brennan, Melanie Shackett. « The Nrf2 transcriptional target, OSGIN1, contributes to the cytoprotective properties of dimethyl fumarate ». Thesis, 2014. https://hdl.handle.net/2144/15345.
Texte intégralRésumé :
Understanding how defense signaling pathways regulate neuronal protection in the compromised central nervous system (CNS) is essential for combating neurodegenerative disorders. This is apparent in the intrinsic activation of the transcription factor Nrf2 during periods of oxidative stress, a hallmark of neurodegeneration. This regulator of the antioxidant response induces the transcription of genes essential for protecting against oxidative stress-induced damage and is a prime target for drug discovery. Delayed-release dimethyl fumarate (DMF), currently approved for the treatment of relapsing-remitting forms of multiple sclerosis (MS), is believed to mediate its effect via the Nrf2 pathway; however, the exact mechanisms of action are unknown. The primary aim of the studies outlined in this dissertation was to identify the molecular mechanisms of Nrf2 regulation and subsequent cellular protection conferred by DMF and its bioactive metabolite, monomethyl fumarate (MMF). For this thesis study, transcriptional profiling studies following oral administration of DMF were conducted to characterize DMF pharmacodynamic responses in the central nervous system (CNS) and peripheral tissues to understand the functional effects of DMF in vivo as well as explore the necessity of Nrf2 in this process. Data from these studies confirm earlier findings that DMF activates transcription of Nrf2 target genes in the CNS and periphery; however, tissue-specific gene expression was also observed, indicating additional levels of transcriptional control beyond Nrf2 activation. These findings suggest that there may be unique cytoprotective and immunomodulatory capabilities of DMF within specific tissues. In the CNS, a novel Nrf2 transcriptional target gene OSGIN1 was identified to be significantly upregulated following DMF treatment in vivo; however, the contribution of this gene to the pharmacodynamic properties of DMF or MMF has not been previously described. Therefore, the in vitro effects of MMF on OSGIN1 expression were characterized, and the necessity of OSGIN1 in mediating cytoprotective effects against toxic oxidative stress in human astrocytes was evaluated. These data identify a potential mechanism for MMF-mediated cytoprotection in human astrocytes that function in an OSGIN1 and p53-dependent manner. Overall, the experiments described in this dissertation allow for a broader understanding of endogenous cellular protection and how it can be used to combat CNS disorders.
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Kraft, Andrew D. « The role of the Nrf2-antioxidant response element pathway in neuronal support cells during degeneration ». 2006. http://www.library.wisc.edu/databases/connect/dissertations.html.
Texte intégral
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Wu, Yi-Ci, et 巫逸琦. « NFE2-related factor 2 (Nrf2) in neurodegenerative disease : promoter polymorphism and therapeutic strategy targeting oxidative stress ». Thesis, 2012. http://ndltd.ncl.edu.tw/handle/52764583766979550531.
Texte intégralRésumé :
碩士國立臺灣師範大學
生命科學研究所
100
Nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) is a member of the basic leucine zipper transcription factors that regulate the expression of many antioxidant pathway genes and maintains cellular redox homeostasis. Increased oxidative stress is involved in the pathogenesis of many neurodegenerative diseases. For example, oxidative stress has been implicated as a major contributing factor in Parkinson’s disease (PD) and varying efficiency in the oxidative protection by Nrf2 may influence PD pathogenesis. In polyQ-mediated spinocerebellar ataxias, the expansions of translated CAG repeats in the disease genes result in long polyQ tracts in the respective proteins, leading to accumulation of aggregated polyQ proteins and increased oxidative stress. In this study, PCR-RFLP test was developed to examine the frequency of Nrf2 -653 A/G, -651 G/A and -617 C/A promoter polymorphisms in a larger cohort of PD (n = 480, 49.2% female, age at onset 61.8±11.2 years) and age- and gender-matched controls (n = 526, 50.5% female, age 60.3±13.1 years). No association between polymorphic genotype, allele or haplotype and PD was observed. In addition, Flp-In SH-SY5Y cells with ATXN3/Q14~75 expression in an inducible fashion were established. In retinoic acid-induced differentiated SH-SY5Y cells, the expressed ATXN3/Q75 formed aggregates, accompanying with reducing neurite outgrowth. By combining high content image analysis and immunoblotting, treatment of ATXN3/Q75 cells with Chinese herbs NH004, NH008, NH021 and NH008 derivative NH008-1 activate Nrf2 expression, accompanying decreasing ATXN3/Q75 aggregates. Thus NH004, NH008, NH021 and NH008-1 may be potential therapeutic strategies for polyQ-mediated disease.
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Afzal, Sualiha. « Identification of novel and potent Nrf2 activators from medicinal plants ». Thesis, 2022. http://hdl.handle.net/1959.7/uws:68946.
Texte intégralRésumé :
Oxidative stress has been implicated in the pathogenesis of various age-related chronic diseases (cancer, cardiovascular disease, chronic obstructive pulmonary disease and neurodegenerative disease). The antioxidant response pathway, which is regulated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), shields cells from oxidative stress by enhancing the expression of cytoprotective genes and enzymes. Transcriptional regulator Nrf2 induces the expression of detoxification enzymes (Phase II detoxifying and antioxidant enzymes) such as glutathione S-transferase (GST) and metabolic pathway enzymes for glutathione synthesis. Antioxidant therapy with the Nrf2 activation is a strategy to prevent cells from exposure to the oxidants and correct cellular redox homeostasis. Plant-derived Nrf2 activators might be used to stimulate the body's antidefense mechanisms. Our study was focused on the discovery of novel Nrf2 activators as therapeutic agents from medicinal plants. A variety of commercially available ethanolic extracts were screened for Nrf2 activity using an Nrf2-luciferase reporter cell line (AREc32), and Valeriana officinalis (root), Cynara scolymus (leaves) and Salix alba (bark) were identified to be part of the most potent samples. Sequential extraction and bioassay-guided fractionation of these plants led to the isolation of compounds identified as Nrf2 activators. NMR and LC/HRMS analysis confirmed the structures. As GST is among the Nrf2 upregulated genes, changes in GST activity upon incubation with the isolated Nrf2 activators were determined in a HepG2 cell line resulted in an increased GST activity. These compounds augmented the intracellular glutathione (GSH) and cysteinylglycine (CysGly) levels by promoting the nuclear translocation of Nrf2 that was determined by the HPLC fluorescence detection method. This thesis describes the screening of ethanolic extracts of 91 medicinal herbal samples from Integria Health care, Ballina, NSW, Australia. This study led to the isolation of 8 pure compounds that are known, but the Nrf2-ARE related pharmacological activity is reported first time with the sequential extraction of these plants. Natural products undoubtedly fulfill irreplaceable drug discovery roles and are an invaluable source for drug candidates and leads. The Nrf2-activity of these compounds has demonstrated their potential as a therapeutic agent against oxidative stress-related diseases (e.g., COPD, diabetes, cardiovascular diseases). These Nrf2 activators have been shown to activate the Nrf2-ARE pathway and its regulated genes and enzymes to protect the cells from oxidative damage. Future directions call for a further biological evaluation to illustrate the detailed mechanisms by which these Nrf2 active compounds activate the Nrf2-ARE pathway and a sufficient pharmacological investigation in vivo to confirm the prevention of oxidative stress-induced diseases.
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Cesário, Rute Sofia Rodrigues. « The effects of ionizing and UVA radiation induced oxidative stress in glioblastoma stem cells silenced for NRF2 ». Master's thesis, 2021. https://hdl.handle.net/10216/138583.
Texte intégral
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Doszpoly, Agnes. « The Roles of Danio Rerio Nrf2 Paralogs in Response to Oxidative Stress in the Pancreatic Beta Cell ». Thesis, 2020. http://hdl.handle.net/1805/23183.
Texte intégralRésumé :
Indiana University-Purdue University Indianapolis (IUPUI)Oxidative stress can disrupt cellular homeostasis, leading to cellular dysfunction and apoptosis. The Nrf2 transcription factor regulates the antioxidant response in cells by binding to antioxidant response elements (ARE) in DNA and activating genes of enzymes that combat oxidative stress. During the pathogenesis of diabetes mellitus (DM), β-cells are exposed to increased amounts of reactive oxygen species (ROS) that cause oxidative stress. Zebrafish (ZF) are excellent models for studying the dynamic mechanisms associated with DM pathogenesis, and we recently developed a ZF model of β-cell apoptosis caused by ROS. Two paralogs of Nrf2 have been identified in ZF, Nrf2a and Nrf2b, but their roles in pancreas development and/or β-cell survival are unknown. To investigate their roles, Nrf2a and Nrf2b antisense morpholinos (MO) were injected into Day 0 ZF embryos and analyzed over time. While Nrf2a MO showed no obvious phenotypes compared to WT, Nrf2b MO exhibited reduced pancreas size and islets with disrupted morphology. Ins:NTR Nrf2a MO showed reduced β-cell loss upon exposure to Metronidazole (MTZ) under generation of ROS compared to WT. Sequence analysis of ZF nrf2b in 3-day post-fertilization (dpf) embryos revealed a novel splice variant containing an additional exon that has not been described. Further investigation of Nrf2a and Nrf2b is likely to yield additional insights regarding the function and regulation of the NRF2-signaling pathway and their roles in β-cell protection under oxidative stress.
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MANCA, SONIA. « STUDY OF THE MOLECULAR ALTERATIONS AND TREATMENT OF HAILEY-HAILEY DISEASE ». Doctoral thesis, 2014. http://hdl.handle.net/11573/917467.
Texte intégralRésumé :
Hailey-Hailey Disease (HHD) is a chronic, recurrent blistering rare disorder, clinically
characterized by erosions occurring primarily in intertriginous regions and histologically
by suprabasal cutaneous cells separation (acantholysis). Despite a strong relationship
between mutations in ATP2C1 gene, encoding for hSPCA1 calcium pump,
manifestations of the disease occurs with high variability between patients. In our
previous studies we used primary keratinocytes derived from lesions or normal skin of
patients with HHD, reporting several molecular changes related to manifestation of the
disease. We reported that oxidative stress has a specific role in the pathogenesis of
HHD by regulating the expression of important factors, which play a crucial role in
keratinocyte homeostasis. In this study, we analyzed the miRNA expression profile of
keratinocytes derived from lesional skin of HHD-patients, and we found that miR-125b,
in particular, is upregulated. Furthermore, we identified both, Notch1 and p63, as direct
targets of mir-125b. In silico analysis of regulatory motifs in miR-125b promoter
revealed TF-binding sites of genes known to function in the oxidative-stress response,
such as ARNT, MARE or NF-KB. Additionally, we found that miR-125b expression is
increased by an oxidative stress-dependent mechanism. Moreover, we saw that Nrf2, a
transcription factor that plays a key role in cell response to oxidative stress, is
downregulated in keratinocytes derived from lesional skin. Furthermore, our results
show that Nrf2 is an important target of α-MSH-analogue, NIe4-D-phe7-α-MSH
(afamelanotide) and treatment of keratinocytes with NIe4-D-phe7-α-MSH restored the
proliferative capability of lesions-derived keratinocytes. Therefore, to assess the clinical
potential of this compound, we conducted a phase II open-label pilot study on two
patients with several long-standing skin lesions. Both patients treated had experienced
100% clearance of HHD lesions 60 days after the first injection, independently of the
lesion localization, demonstrating that NIe4-D-phe7-α-MSH may be effective and safe for
the treatment of Hailey-Hailey disease.
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Gomes, Barbara Silva. « Gene expression of NFE2L2 in myelodysplastic syndrome patients – clinical implications ». Master's thesis, 2017. http://hdl.handle.net/10316/81942.
Texte intégralRésumé :
Trabalho de Projeto do Mestrado Integrado em Medicina apresentado à Faculdade de MedicinaIntrodução: A Síndrome Mielodisplásica (SMD) é uma doença clonal que é caracterizada por hematopoiese ineficaz, citopenias periféricas e está associada a elevado risco de progressão para Leucemia Mieloide Aguda (AML). A patogénese da SMD é complexa, estando envolvidos múltiplos eventos genéticos e epigenéticos e, apesar do enorme progresso realizado na ultima década em torno da melhor compreensão dessas anomalias genéticas, continua sem ser esclarecida. O stress oxidativo (SO), que resulta de um desequilíbrio entre a produção de espécies reativas de oxigénio (ROS) e de defesas antioxidantes, contribui para o dano e proliferação celulares, assim como para a apoptose e a hematopoiese ineficaz características das SMD. O nuclear factor-erythroid 2-related factor 2 (NRF2), codificado pelo gene NFE2L2, é um dos mais importantes fatores de transcrição envolvidos na resposta antioxidante que tem sido identificado como anticarcinogénico. No entanto, a sobre expressão de NRF2 tem sido observada num grande número de tumores sólidos e hematológicos, que tem sido relacionada com um papel procarcinogénico. Objetivos: Avaliar os níveis de expressão do gene NFE2L2 nas SMD e compará-los com vários parâmetros clínicos e laboratoriais na SMD, explorando a sua importância como biomarcador no diagnóstico e prognóstico, nomeadamente como preditor de progressão para LMA. Materiais e Métodos: Amostras de sangue periférico foram colhidas de 55 doentes diagnosticados com SMD e 44 controlos saudáveis. RNA total foi isolado de leucócitos derivados de sangue periférico e transcrito em cDNA. A expressão de NFE2L2 foi quantificada por real-time PCR. A comparação entre grupos de doentes e controlos foi realizada através de testes não paramétricos de Mann-Whithney e Kruskal-Wallis. A análise de sobrevivência foi efetuada recorrendo ao método de Kaplan-Meier e as curvas ROC foram elaboradas. Resultados e Discussão: Os níveis de expressão do NFE2L2 não apresentaram diferenças quando comparados entre os indivíduos com SMD e os indivíduos controlos (SMD: mediana 2,29; amplitude interquartil 6,037; CTL: mediana 3,40; amplitude interquartil 3,40; p=0,816). No entanto, quando os pacientes com SMD foram estratificados segundo os diferentes subtipos de SMD da classificação da WHO, foi possível observar níveis inferiores de expressão de NFE2L2 na citopenia refratária com displasia multilinhagem (CRDM) quando comparados com os restantes subgrupos de SMD (CRDM: mediana 1,48; amplitude interquartil 1,41; p<0,05), o que parece sugerir uma maior participação do NRF2 neste subtipo de SMD. Foi também possível observar a sobre expressão do NFE2L2 nos pacientes com SMD que progrediram para LMA (LMA: mediana 8,57; amplitude interquartil 13,57; Não-LMA: mediana 2,12; amplitude interquartil 4,03; p=0,018), com uma sensibilidade de 100% e uma especificidade de 76,5%, quando utilizado um valor de cut-off de 5,44 (p=0,021). Assim, a expressão de NFE2L2 poderia ser usada como possível biomarcador na identificação dos pacientes com maior risco de progressão para LMA. Não foram observadas quaisquer relações entre o padrão de expressão do NFE2L2 e qualquer parâmetro laboratorial, IPSS ou sobrevivência. Conclusão: O NFE2L2 poderá a vir a ser usado como potencial biomarcador de evolução para LMA nos doentes com SMD. Ao longo da última década, progressos significativos na compreensão das anomalias genéticas têm sido desenvolvidos, no entanto são ainda necessários mais estudos para compreender a verdadeira importância do NFE2L2/NFR2 na patogénese das SMD, particularmente nos doentes do subtipo CRDM e nos doentes de alto risco.
Introduction: Myelodysplastic syndromes (MDS) are clonal stem-cell disorders that are characterized by ineffective haematopoiesis, peripheral blood cytopenias, and a higher progression to acute myeloid leukaemia (AML). The pathogenesis of MDS is complex and involves multiple genetic and epigenetic events, and although the significant progress in understanding the molecular genetics aberrations in MDS over the last decade its pathogenesis is not yet clear. Oxidative stress (OS), resulting from an imbalance between reactive oxygen species (ROS) production and antioxidant defences, contributes to cell proliferation and damage, as well as to apoptosis and dysfunctional haematopoiesis. Nuclear factor-erythroid 2-related factor 2 (NRF2), encoded by the NFE2L2 gene, is a key transcriptional activator of the antioxidant response pathway that has been identified as a protector of tumorigenesis. However, enhanced NRF2 activity has been found in a great number of solid and hematologic tumours and has been related with higher survival of neoplastic cells. Objectives: Evaluate the expression levels of NFE2L2 gene in MDS patients and correlate it with clinical and analytical parameters, exploring its potential role as diagnostic and prognostic biomarker, namely as a predictor of AML transformation. Materials and Methods: Peripheral blood samples were collected from 55 MDS patients and 44 healthy controls. Total RNA was isolated from peripheral blood leukocytes and transcribed to cDNA. NFE2L2 expression was quantified by real-time PCR. Comparison between groups of patients and controls was performed using nonparametric Mann-Whithney and Kruskal-Wallis tests. The ROC curves analysis were performed. Survival analysis was completed using Kaplan-Meier method. Results and Discussion: Our results show no differences in the expression levels of NFE2L2 between the MDS patients and the control group (MDS: median 2,29; interquartile range 6,037; CTL: median 3,40; interquartile range 3,40; p=0,816). However, when patients were stratified according to MDS subtypes and compared among them, we found that refractory cytopenia with multilineage dysplasia (RCMD) patients had lower expression levels of NFE2L2 when compared with the others subtypes (RCMD: median 1,48; interquartile range 1,41; p<0,05), which might suggest a higher participation of NFE2L2/NRF2 in the pathogenesis of this MDS subgroup. We also observed that NFE2L2 is overexpressed in MDS patients who progress to AML (AML: median 8,57; interquartile range 13,57; Non-AML: median 2,12; interquartile range 4,03; p=0,018), with a sensitivity of 100% and a specificity of 76,5% at a cut-off value of 5,44 (p=0,021), therefore it could be used as a potential biomarker to identify MDS patients at high risk of progression to AML. No relations were observed between NFE2L2 expression pattern and any laboratorial parameter, neither IPSS nor survival. Conclusion: In summary, our results suggest that NFE2L2 could be used as a new potential biomarker for prediction of AML progression in MDS patients. Over the last decade, significant progress in understanding the molecular genetics aberrations in MDS has been made, however, further studies are needed in order to understand the importance of NFE2L2/NFR2 in MDS pathogenesis, particularly in RCDM patients and in high-risk patients.
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Zhao, Shuiling. « The physiological role of Nrf2 in diabetic kidney disease ». Thesis, 2020. http://hdl.handle.net/1866/25552.
Texte intégralRésumé :
La néphropathie diabétique (DN) est l’une des premières causes de maladie rénale en
phase terminale (ESKD). L’ESKD est un important facteur de risque d'insuffisance cardiaque et
d'accidents vasculaires cérébraux. La dysfonction du système rénine-angiotensine intrarénal
(iRAS) est considérée comme étant l'une des principales causes du développement de la DN. Tous
les composants du iRAS sont identifiés dans les cellules épithéliales des tubules rénaux proximaux
(RPTCs), y compris l'angiotensinogène (Agt), le seul précurseur de toutes les angiotensines. Notre
laboratoire a rapporté précédemment que la surexpression spécifique de l’Agt dans les RPTCs
provoque l’hypertension, la protéinurie, la fibrose rénale, l’apoptose et des lésions rénales.
Nrf2 (Nuclear factor erythroid 2-related factor 2) est un facteur de transcription qui est
exprimé de façon abondante dans les RPTCs et a été considéré comme étant un régulateur central
de l'équilibre redox dans les réponses cytoprotectrices cellulaires. Le rôle de l’activation du Nrf2
dans la DN, toutefois, est controversé. L’objectif général de cette thèse est de comprendre le rôle
physiologique du Nrf2 dans la DN et d’étudier le(s) mécanisme(s) moléculaire(s) de l’action de
Nrf2.
Premièrement, nous avons démontré que la délétion génétique de Nrf2 ou l’inhibition
pharmacologique de Nrf2 avec de la trigonelline chez les souris Akita diabétiques de type 1 régule
à la hausse la voie Ace2/MasR et supprime l’expression de Agt/ACE dans les RPTCs, ce qui a pour
effet d'atténuer l’hypertension systémique et les lésions rénales. Conformément, dans les cellules
immoratalisées de tubule proximal de rat (IRPTC) en culture, la transfection de ARNsi ou le
traitement à la trigonelline empêche la régulation positive de Agt/ACE induite par le HG, avec une
baisse subséquente de l’expression des gènes Ace2/MasR. Ces données identifient un nouveau
mécanisme dans lequel l’activation de Nrf2 stimule l’expression et l’activation des gènes du iRAS,
menant au développement de l’hypertension et de la néphropathie dans le diabète.
Deuxièmement, nous avons généré des souris Nrf2 transgéniques qui surexprime
spécifiquement Nrf2 dans les RPTCs (souris Nrf2RPTC Tg), sous le contôle du promoteur KAP (kidney
specific androgen-regulated protein). Nous avons ensuite croisé les souris Nrf2RPTC Tg avec les 6
souris Akita Nrf2-/- pour générer des souris Akita Nrf2-/-
/Nrf2RPTC Tg. Nous avons trouvé que la
surexpression de Nrf2 dans les RPTCs des souris Akita Nrf2-/- augmentait significativement
l’expression du gène SGLT2, entraînant une élévation du glucose sanguin, du taux de filtration
glomérulaire, du rapport albumine/créatinine urinaire et de la fibrose tubulo-interstitielle. Dans
les cellules tubulaires proximales humaines immortalisées (HK2), le traitement à l’oltipraz ou la
transfection de l’ADNc du NRF2 stimule l’expression de l’ARNm du SGLT2 et l’activité de son
promoteur. De plus, des tests de retard sur gel et d’immunoprécipitation de chromatine ont
montrés que NRF2 se lie au NRF2-RE du promoteur du SGLT2. En outre, une expression plus
élevée de NRF2 et SGLT2 est observée dans les RPTCs de reins de patients diabétiques que dans
les reins de patients non diabétiques. Ces données ont établi un nouveau mécanisme de la
régulation du NRF2 sur l’expression et l’activation du gène SGLT2, menant à une exacerbation du
glucose sanguin, de l’hyperfiltration et des lésions rénales dans le diabète.
En somme, cette thèse a démontré que le stress oxidatif (hyperglycémie) induisait
l’activation du Nrf2 qui stimulait le iRAS et l’expression de SGLT2, contribuant ainsi à la
progression de la DN. Ces études suggèrent que le Nrf2 pourrait être une cible thérapeutique
potentielle dans le traitement de la DN et pourront fournir de valabless données pré-cliniques
pour les essais cliniques en cours avec le bardoxolone méthyle (un activateur de Nrf2).Diabetic nephropathy (DN) is one of the leading causes of end-stage kidney disease (ESKD). ESKD is a major risk factor for heart failure and stroke. Dysfunction of intrarenal renin angiotensin system (iRAS) is considered as one of the main reasons that caused the DN. All components of the iRAS are identified in the renal proximal tubule cells (RPTCs), including angiotensinogen (Agt), the sole precursor of all angiotensins. Our lab has previously reported that specific overexpression of Agt in RPTCs induces hypertension, proteinuria, kidney fibrosis, apoptosis and kidney injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that abundantly expresses in RPTCs and has been considered as a master regulator of redox balance in cellular cytoprotective responses. The role of Nrf2 activation in DN, however, is not clear. The overall aim of this study is to understand the physiological role of Nrf2 in DN and investigate the molecular mechanism(s) of Nrf2 action. First, we have demonstrated that genetic deletion of Nrf2 or pharmacological blockade of Nrf2 with trigonelline in type 1 diabetic Akita mice effectively upregulates Ace2/MasR and suppresses Agt/ACE expression in isolated RPTCs, resulting in attenuation of systemic hypertension and kidney injury. Consistently, in cultured IRPTCs, Nrf2 siRNA transfection or trigonelline treatment prevents high glucose-induced upregulation of Agt/ACE with downregulation of Ace2/MasR gene expression. These data identified a novel mechanism in which Nrf2 activation stimulates iRAS gene expression and activation, leading to the development of hypertension and nephropathy in diabetes. Second, we have generated Nrf2 transgenic mice under the kidney specific androgen regulated protein (KAP) promoter which specifically overexpress Nrf2 in RPTCs (Nrf2RPTC Tg mice). We further crossbred the Nrf2RPTC Tg mice with Akita Nrf2-/- mice to generate Akita Nrf2-/- /Nrf2RPTC Tg mice. We have found that overexpression of Nrf2 in RPTCs of Akita Nrf2-/- mice significantly unregulated sodium-glucose transporter-2 (SGLT2) expression, resulting in elevation of blood glucose, glomerular filtration rate, albumin-creatinine ratio and tubulointerstitial fibrosis. In 8 immortalized human proximal tubular cells (HK2), oltipraz treatment or NRF2 cDNA transfection stimulated SGLT2 mRNA expression and its promoter activity. Furthermore, NRF2 bound to NRF2- RE of SGLT2 promoter were identified by gel mobility shift assay and chromatin immunoprecipitation assay. Moreover, human diabetic kidneys exhibited higher expression of NRF2 and SGLT2 in RPTCs than non-diabetic kidneys. These data established a novel mechanism of NRF2’s regulation on SGLT2, leading to exacerbation of blood glucose, hyperfiltration and kidney injury in diabetes. In summary, this study documented that activation of Nrf2 in hyperglycemia contributed to the progression of DN via regulation of iRAS and SGLT2, suggesting that Nrf2 might be a potential therapeutic target in the treatment of DN.
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Berg, James Michael. « A Big Response to a “Small” Problem : Identifying the Oxidative Potential of Nanomaterials and the Physicochemical Characteristics That Play a Role ». Thesis, 2011. http://hdl.handle.net/1969.1/ETD-TAMU-2011-12-10234.
Texte intégralRésumé :
Nanotechnology as a science is emerging rapidly. As materials are synthesized and utilized at the nanometer size scale, concerns of potential health and safety effects are arising. In an effort to elucidate the physicochemical characteristics of nanoparticles influential in toxicological studies, surface properties of metal oxide and carbonaceous nanoparticles were measured. These properties include zeta potential, dissolution and surface-bound chemical components. Subsequently, the role of these properties in oxidative stress was examined in vitro.
This work identifies the influence that pH has on the zeta potential of nanoparticles. The zeta potential has the ability to alter colloidal stability, as the largest nanoparticle agglomerate is seen at or near the isoelectric point for each of the particles tested. Furthermore, it was observed that metal oxide nanoparticles which exhibit a charged surface at physiological pH, lead to decreased in vitro cellular viability as compared to those that were neutral. Thus, nanoparticle zeta potential may be an important factor to consider when attempting to predict nanoparticle toxicity.
Real world exposure to nanoparticles is a mixture of various particulates and organics. Therefore, to simulate this particle mixture, iron oxide (Fe2O3) and engineered carbon black (ECB) were utilized in combination to identify potential synergistic reactions. Following in vitro exposure, both nanoparticle types are internalized into endosomes, where liberated Fe3+ reacts with hydroquinone moieties on the ECB surface yielding Fe2+. This bioavailable iron may then generate oxidative stress through intracellular pathways including the Fenton reaction.
As oxidative stress is common in particulate toxicology, a comparison between the antioxidant defenses of epithelial (A549) and mesothelial (MeT-5A) cell lines was made. The A549 cell line exhibits alterations in the NRF2-KEAP1 transcription factor system and therefore retains high basal levels of phase II antioxidants. Both cell types were exposed to 33 nm silica where intracellular oxidant generation coupled with markers of oxidative stress were observed. While the MeT-5A cells exhibited a decrease in cell viability, the A549 cell line did not. Therefore, proper characterization of both material and biological systems prior to toxicity testing will help to further define the risks associated with the use of nanotechnology.
50
Laplante-El, Haïli Youri. « Les effets de l’Angiopoietin-like 2 sur la voie cytoprotectrice antioxydante Nrf2 ». Thèse, 2015. http://hdl.handle.net/1866/13453.
Texte intégralRésumé :
L’angiopoietin-like 2 (angptl2) est une glycoprotéine de 64 kDa pro-inflammatoire et pro-athérogénique associée à divers maladies inflammatoires chroniques. Il est probable que l’angptl2 possède également un effet pro-oxydant, puisqu’elle stimule la production aigüe de dérivés réactifs oxygénés (DRO). Le facteur de transcription « nuclear factor (erythroidderived 2)-like 2 » (Nrf2) fait partie d’un mécanisme antioxydant majeur permettant de maintenir l’équilibre redox via l’induction de plusieurs gènes de l’élément de réponse antioxydante (ERA). En présence de DRO, la protéine Keap-1 se dissocie de Nrf2 et cesse de promouvoir sa dégradation protéasomale. Cette dissociation est stimulée par la protéine DJ-1 qui favorise la translocation nucléaire de Nrf2. La p38MAPK peut phosphoryler Nrf2 et promouvoir son interaction avec Keap-1. Nous avons posé l’hypothèse selon laquelle l’angptl2 causait un stress oxydant chronique, d’abord en stimulant en aigu la production de DRO, puis en inhibant la voie antioxydante Nrf2. Nous avons étudié, par immunobuvardage de type Western sur des cellules endothéliales en culture (HUVEC), les effets de l’angptl2 recombinante (100 nM) sur les niveaux protéiques nucléaires de Nrf2, les niveaux de Keap-1 dans le cytosol et de DJ-1 dans le noyau, en absence et en présence de l’antioxydant NAC (10 μM). Nous avons également étudié l’activation de la p38MAPK. Les niveaux nucléaires de Nrf2 n’ont pas été affectés par la stimulation aigüe (10 min) à l’angptl2 recombinante, mais ont été diminués par la stimulation chronique (24 h). L’ajout d’un agent antioxydant n’a pas altéré l’effet chronique, indiquant que les DRO ne sont pas directement impliqués. Les niveaux protéiques cytosoliques de Keap-1, protéine inhibitrice de Nrf2, et les niveaux nucléaires de DJ-1, protéine stabilisatrice de Nrf2, n’ont pas été affectés par l’angptl2 de manière significative. La phosphorylation de la p38MAPK n’a pas été non plus affectée par la stimulation aigüe ou chronique à l’angptl2. Ces données suggèrent que l’angptl2 n’a pas d’effet aigu, mais a un effet chronique inhibiteur sur la voie Nrf2, qui n’est pas associé à un effet sur Keap-1, DJ-1 ou p38MAPK.Angiopoietin-like 2 (angptl2) is a 64 kDa pro-inflammatory and pro-atherogenic glycoprotein associated with various chronic inflammatory diseases. It is likely that angptl2 also has a pro-oxidant property, since it stimulates the acute production of reactive oxygen species (ROS). The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) constitutes a major antioxidant mechanism, responsible for maintaining redox balance within the cell via the induction of various genes composing the antioxidant response element (ARE). In the presence of ROS, cytosolic protein Keap-1 dissociates from Nrf2 and is thus no longer able to target Nrf2 for proteasomal degradation. The interaction between Nrf2 and Keap-1 is inhibited by DJ-1, another regulator of Nrf2, but is favoured by phosphorylation of Nrf2 by p38MAPK. Our hypothesis was that angptl2 caused a chronic oxidative stress, at first by stimulating an acute ROS production, and later by inhibiting the Nrf2 antioxidant pathway. We studied, by Western Blot on cultured endothelial cells (HUVEC), the effects of recombinant angptl2 (100 nM) on nuclear protein levels of Nrf2, as well as cytosolic levels of Keap-1 and nuclear levels of DJ-1 in the absence and presence of the antioxidant NAC (10 μM). We also measured the activation of p38MAPK in response to angptl2 stimulation. Nuclear protein levels of Nrf2 were not affected by acute simulation (10 min) by recombinant angptl2, but were diminished after chronic stimulation (24 h). The addition of an antioxidant did not alter angptl2’s chronic effect on Nrf2, indicating that ROS were not directly implicated. Cytosolic levels of Keap-1 and nuclear levels of DJ-1 were not significantly affected by angptl2. Similarly, angptl2 did not affect p38MAPK phosphorylation. These data suggest that angptl2 has no acute effect on Nrf2, but has an inhibitory chronic effect, which is not likely to involve Keap-1, DJ-1 or p38MAPK.