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1
Bounaix, Nolwenn. "Repositionnement thérapeutique de molécules pharmacologiques dans les maladies mitochondriales : impact et mécanisme d'action". Electronic Thesis or Diss., Angers, 2024. http://www.theses.fr/2024ANGE0038.
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Les maladies mitochondriales (MM) liées à un déficit du complexe I de la chaîne respiratoire représente 30% des MM. Elles peuvent être responsables de nombreuses atteintes touchant principalement les tissus consommateurs d’énergie comme cerveau, cœur et muscle. Le complexe I comporte 44 sous-unités incluant en particulier la sous-unité clé NDUFV1 qui a fait l’objet de cette étude. Le développement d’une médecine mitochondriale est également fortement compromis par l’absence de thérapies efficaces. Dans un 1er temps, un des objectifs de ce travail a été de mieux comprendre les pathologies liées à NDUFV1, avec la mise en évidence des hotspots de mutations au sein de domaines clés de cette sous unité NDUFV1. Des modèles in vitro à l’aide de différents modèles cellulaires, lignées cellulaires mutantes NDUFV1 mais également à partir de cardiomyocytes NDUFV1 différenciés à partir de cellules souches induites ont permis de caractériser la dysfonction mitochondriale de sévérité variable. Une réduction de l’activité et assemblage du complexe I, une perturbation de la dynamique mitochondriale associée à une surproduction de ROS oxydant et phénomènes inflammatoires ont pu être mis en évidence. Dans un 2e temps, il a été entrepris un criblage de molécules visant à compenser la dysfonction mitochondriale utilisant des organismes simples notamment un modèle champignon Podospora Anserina portant une mutation pour le gène nuo-51A357V homologue de NDUFV1. Ce travail a permis de sélectionner 2 médicaments candidats Alvérine et Ebselen. L’exposition des modèles cellulaires NDUFV1 ou cardiomyocytes mutants a confirmé l’efficacité de ces molécules. Ces molécules ont permis une restauration des principaux paramètres mitochondriaux, incluant respiration mitochondriale, réduction de la production de ROS mais aussi des marqueurs de l’inflammation, mécanismes clefs de la maladie. La poursuite de cette étude avec un nouveau modèle murin NDUFV1 pourrait confirmer l’efficacité de ces molécules et la mise en place d’essais cliniques pour ces MMMitochondrial diseases (MD) related to a deficiency in complex I of the respiratory chain represent 30% of all MDs. They can lead to various impairments primarily affecting energy-demanding tissues such as the brain, heart, and muscle. Complex I consists of 44 subunits, notably including the key subunit NDUFV1, which is the focus of this study. The development of mitochondrial medicine is significantly compromised by the absence of effective therapies. Initially, one of the objectives of this work was to gain a better understanding of the pathologies associated with NDUFV1 by identifying mutation hotspots within key domains of this NDUFV1 subunit. In vitro models utilizing various cellular systems, mutant NDUFV1 cell lines, and NDUFV1 cardiomyocytes differentiated from induced pluripotent stem cells were employed to characterize the variable severity of mitochondrial dysfunction. A reduction in complex I activity and assembly, disturbances in mitochondrial dynamics, associated with overproduction of oxidative ROS and inflammatory phenomena, were demonstrated. Subsequently, a screening of compounds aimed at compensating for mitochondrial dysfunction was conducted using simple organisms, notably a fungal model, Podospora anserina, carrying a mutation in the nuo-51A357V gene homologous to NDUFV1. This work led to the selection of two candidate drugs, Alverine and Ebselen. Exposure of NDUFV1 cell models or mutant cardiomyocytes confirmed the efficacy of these compounds. These molecules restored key mitochondrial parameters, including mitochondrial respiration, reduced ROS production, and inflammatory markers—key mechanisms in the disease. The continuation of this study with a new NDUFV1 mouse model could confirm the efficacy of these compounds and facilitate the establishment of clinical trials for these MDs
2
Wohlgemuth, Eva-Maria [Verfasser]. "Funktionelle Charakterisierung neu identifizierter NDUFB3-Mutationen / Eva-Maria Wohlgemuth". Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2021. http://d-nb.info/1234982722/34.
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Lescuyer, Pierre. "Étude de l'expression des gènes nucléaires codant pour les sous-unités du complexe I mitochondrial humain". Phd thesis, Université Joseph Fourier (Grenoble), 2002. http://tel.archives-ouvertes.fr/tel-00175098.
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La NADH:ubiquinone oxydoréductase (complexe I) est le plus gros complexe enzymatique du système mitochondrial d'oxydation phosphorylante (43 sous-unités chez l'homme). Très peu de données sont disponibles concernant les mécanismes régulant l'expression de ces protéines. Cette étude a été initiée par l'étude des promoteurs de deux gènes du complexe I mitochondrial humain. Les résultats montrent que le gène NDUFS8 qui code pour la sous-unité 23 kDa (TYKY) est transcrit sous le contrôle des facteurs de transcription YY1 et Sp1 tandis que gène NDUFS7 codant pour la sous-unité 20 kDa (PSST) est régulé par NRF-1 et Sp1.
Dans la deuxième partie de ce travail, une méthode d'analyse du protéome mitochondrial humain par électrophorèse bidimensionnelle a été développée. Le but est d'aborder de manière globale et sans a priori l'expression des protéines du complexe I : déterminer qui est régulé et comment en réponse à un stimulus déterminé?
Des cartes de référence ont été développées à partir de mitochondries extraites de placenta humain en utilisant deux types de gradient de pH : l'un est adapté aux protéines acides et neutres, l'autre aux protéines basiques. Sur ces cartes, 85 protéines mitochondriales ont été identifiées par spectrométrie de masse dont 17 sous-unités du complexe I. Cette technique d'analyse protéomique a ensuite été utilisée pour étudier la régulation de l'expression des protéines mitochondriales par le fer. Sur le plan technique, les premiers résultats sont encourageants : les gels d'électrophorèse bidimensionnelle préparés avec des mitochondries extraites de cellules en culture sont de bonne qualité et des variations reproductibles de l'expression de sous-unités du complexe I et d'autres protéines mitochondriales ont pu être détectées. Sur le plan fondamental, les données obtenues sont préliminaires. Il sera nécessaire de réaliser de nouvelles expériences pour confirmer les premières observations et analyser la cinétique des variations détectées.
4
Bris, Céline. "Influence de la génétique mitochondriale en pathologie : apport des techniques de séquençage haut débit Deep sequencing shows that oocytes are not prone to accumulate mtDNA heteroplasmic mutations during ovarian ageing Novel NDUFS4 gene mutation in an atypical late-onset mitochondrial form of multifocal dystonia". Thesis, Angers, 2017. http://www.theses.fr/2017ANGE0093.
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Les maladies mitochondriales sont des pathologies fréquentes du métabolisme caractérisées par une forte hétérogénéité clinique et génétique, notamment par la dépendance à 2 génomes, nucléaire (ADNn) et mitochondrial (ADNmt), et le concept d’hétéroplasmie (HT). L’objectif de ce travail de thèse a été de développer une stratégie d’analyse de l’ADNmt par séquençage haut-débit (NGS), puis de l’appliquer à l’étude des maladies mitochondriales et des pathologies liées au vieillissement : glaucome à angle ouvert (GPAO) et vieillissement ovarien précoce. Après validation des performances de notre stratégie NGS pour la détection et la quantification des variations de l’ADNmt, nous avons confirmé l’intérêt de l’analyse systématique de la totalité de l’ADNmt avec l’identification de nouveaux variants et l’utilisation de cellules uroépithéliales pour le diagnostic des maladies mitochondriales. Cependant, ces progrès génèrent de nouveaux défis dont l’interprétation des faibles HT et la priorisation des variants de signification inconnue. Pour les pathologies liées au vieillissement, nous avons mis en évidence le possible rôle protecteur des haplogroupes T et H chez les femmes, respectivement dans la survenue et la sévérité du GPAO, suggérant une modulation de l’influence de l’ADNmt par le genre et donc l’importance de la stratification par sexe dans les études d’association. En revanche, nous n’observons pas d’accumulation d’anomalies de l’ADNmt dans le vieillissement ovarien précoce. En perspective, nous rapportons l’identification d’une mutation de l’ADNn dans un phénotype atypique, rappelant la complexité de l’étude des pathologies mitochondriales, du fait de ce double génomeMitochondrial diseases are common metabolic disorders characterized by strong clinical and genetic heterogeneity, in particular due to the dependence on 2 genomes, nuclear (nDNA) and mitochondrial DNA (mtDNA), and the concept of mitochondrial heteroplasmy. The purpose of this work was to develop a strategy for the analysis of the mtDNA through next-generation sequencing (NGS), and then to apply it to the study of mitochondrial diseases and those related to aging: primary open-angle glaucoma (POAG) and ovarian aging. After validating the performances of our NGS strategy for the detection and quantification of mtDNA variations, we confirmed the power of systematic analysis of the whole mitochondrial genome with the use of uroepithelial cells for mitochondrial diseases diagnosis and the identification of novel mtDNA variants. However, these advances generate new challenges such as the interpretation of low percentages of mtDNA mutations or the prediction of the pathogenicity of new variants. For aging-related diseases, we have identified the possible protective role of the mitochondrial haplogroups T and H in women, respectively in the occurrence and severity of POAG, suggesting that mtDNA influence is drivenby gender, and thus the importance of gender stratification for association studies. By contrast, we did not observe any accumulation of mtDNA abnormalities in early ovarian aging. In perspective, we report the identification of a nDNA mutation in an atypical phenotype, highlighting the complexity of mitochondrial diseases diagnosis, due to this double genome
5
Chi, Tsung-Chen y 紀宗辰. "Functional study of NADH dehydrogenase (ubiquinone) flavoprotein 1 (NDUFV1) and its role in the assembly of respiratory complexes". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/2824j7.
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碩士國立清華大學
分子醫學研究所
102
NADH dehydrogenase (ubiquinone) flavoprotein 1 (NDUFV1) is a nuclear-encoded subunit of mitochondrial Complex I. It provides a conserved FMN binding site and is responsible for transferring electrons from NADH to FMN to facilitate the entrance of electrons into the electron transport chain (ETC). NDUFV1 also has an iron sulfur cluster [4Fe-4S] (N3) that is the first of that kind in the Complex I. The defect in NDUFV1 could cause neural disease like psychomotor retardation, and some point mutations on FMN binding site have been found in patients with Leigh syndrome. The assembly of intact OXPHOS complexes (complex I~V) is very important in mitochondrial energy production. In addition, the assembly of several respiratory complexes to form a larger supercomplex may improve the efficiency of electron transport. The supercomplex composed by complex I, complex III and complex IV has been confirmed and the defect in supercomplex assembly is associated with encephalomyopathies and neurodegenerative disorders. In present study, we applied a RNA interference system to suppress the NDUFV1 expression in human embryonic kidney cell line (HEK293), and generated three knockdown cell lines A9, B5 and E12 with 55~70% reduction in NDUFV1 protein level. We also performed oxygen consumption assay, dynamic complex I activity assay, ATP determination assay and cell growth rate measurement to evaluate the effects of NDUFV1 suppression. The results showed that the metabolic activity and growth rate were significantly decreased in NDUFV1 knockdown cell lines. In the respiratory complex assembly study, we solubilized mitochondria and applied a high resolution clear native electrophoresis (HrCNE) approach to investigate whether NDUFV1 knockdown affects individual respiratory complex assembly. The results III indicated that NDUFV1 knockdown would significantly decrease the level of complex I, III and IV. Further investigation of OXPHOS supercomplex formation also showed that while NDUFV1 was suppressed, CI/CIII2/CIVn supercomplexes were significantly reduced in cells. These finding suggests that NDUFV1 might play an important role in the assembly/stability of mitochondrial OXPHOS complexes and supercomplexes.
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施德玉. "The research on Ban Chiang Ti and Chu Pai Ti". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/ndufdh.
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Liu, Hsin-Yu y 劉欣瑜. "The functional and mitochondrial targeting signal studies of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) subunit in mitochondrial complexⅠ". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/03608895926813533123.
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碩士國立清華大學
分子醫學研究所
97
Mammalian NADH-ubiquinone oxidoreductase (complex I) is the first, largest and most complicated respiratory complex in mitochondria. Seven subunits of complex I, including ND1-6 and ND4L, are encoded by mitochondrial DNA (mtDNA), and the other thirty-eight subunits are encoded by nuclear DNA (nDNA). NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) is one of the core nucleus-encoded subunits existing in human mitochondrial complex I. It contains one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), which may play a role in the prevention of oxidative damage. The defect of NDUFV2 subunit is associated with neurodegenerative diseases, including Parkinson disease, Alzheimer’s disease, Bipolar disorder and Schizophrenia. In this study, we applied the RNA interference (RNAi) technology in human T-REx293 cells to investigate the function of NDUFV2 subunit. We found that suppression of NDUFV2 expression in the cells would cause a slowing growth cell rate in galactose medium, decreasing oxygen consumption rate, reducing mitochondrial membrane potential (MMP) and increasing reactive oxygen species (ROS) generation, but did not affect complex I assembly. These observations provided the evidences that NDUFV2 plays an essential role for energy production in cells. In addition, we designed various truncation constructs to investigate the mitochondrial targeting mechanism of NDUFV2. We identified that the cleavage site of NDUFV2 was located around amino acid residue 32 and the first 22 residues of NDUFV2 was enough to function as a mitochondrial targeting sequence (MTS) to carry the passenger protein, enhanced green fluorescent protein (EGFP), into mitochondria successfully. Furthermore, we used the site-directed mutagenesis to study the basic, hydrophobic and hydroxylated residues in this identified N-terminal MTS. We found that the basic and hydrophobic residues were important for the MTS of NDUFV2, but the hydroxylated residues were not. In a recent study, the patients of the hypertrophic cardiomyopathy and encephalomyopathy were found to contain 4 bp deletion in the second intron of NDUFV2 (IVS2+5_+8delGTAA) to cause the exon 2 losing. To dissect the pathogenetic mechanism caused by this mutation, we established the human disease model and found that lost of this exon 2 cause NDUFV2 to lose its mitochondrial targeting ability. In this report, we proved that the NDUFV2 plays an important role for energy production in mammalian cells and identified the location of mitochondrial targeting sequence in this protein.
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Chang, Fu-Ming y 張富茗. "The functional study of NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) subunit". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/42951432985652303444.
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碩士國立清華大學
分子醫學研究所
101
NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) is one of the nuclear-encoded and highly conserved core subunits of mitochondrial complex I. This protein houses the tetranuclear iron-sulfur cluster N2, which is the terminal receptor of electrons and the reducer for quinone in complex I peripheral arm. NDUFS7 protein plays a critical role in complex I assembly and activity, according to the results of studies from simple organisms and human disease models. Mutations of NDUFS7 have been associated with Leigh Syndrome and bipolar disorder. To detailedly investigate the role of NDUFS7 in mitochondrial complex I of human cells, NDUFS7 was suppressed in human T-REx293 cells by RNA interference system in this study. In the NDUFS7 knockdown cells, the growth rate, ATP generation, oxygen consumption and complex I activity were reduced. These results provided the evidence of the essential role of NDUFS7 for complex I activity and energy production in human cells.
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Yang, Jia shin y 楊佳欣. "Phosphorylation of Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) by c-Src". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/35833250274081088716.
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碩士國立清華大學
分子醫學研究所
103
Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7), participating in the process of oxidative phosphorylation (OXPHOS), is one of the most conserved core subunits of mitochondrial complex I. In addition to containing a mitochondrial targeting sequence (MTS), a functional nuclear localization signal (NLS) and a nuclear export signal (NES) are both demonstrated to be present in NDUFS7. Deficiency of NDUFS7 is associated with Leigh syndrome (LS) and patients suffering from this disease develop movement disorders and other serious complications. Thus, clearing out the controlling mechanism of NDUFS7 subcellular distribution and its functions at the molecular level is important. Previous studies conducted in our laboratory indicated that NDUFS7 could be located in mitochondria, the cytosol and the nucleus. However, the mechanism of regulating its localization is still unknown. Post–translational modifications are well recognized as the candidates for controlling protein localization and functions. The main purpose of this project is to study the influence of phosphorylation on the subcellular localization of NDUFS7 and/or its involvement in the regulation of NDUFS7 functions. The current results showed that the precursor form of NDUFS7 is phosphorylated at tyrosine residues and enhanced by ATP treatment. In addition, the tyrosine phosphorylation of NDUFS7 could be enhanced by c-Src kinase both in vivo and in vitro. However, the subcellular localization of NDUFS7 is not significantly affected by the expression of c-Src as compared to that of the control groups. Moreover, one of the sites involved in the phosphorylation of NDUFS7 is identified to be tyrosine-160, and the stability of NDUFS7 protein is positively regulated by phosphorylation at this residue. Further studies are on the way to explore the effect of NDUFS7 phosphorylation on mitochondrial functions.
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Wu, Jian Shian y 吳建賢. "Sumoylation of Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) by SUMO-1". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/10504655345694324867.
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碩士國立清華大學
分子醫學研究所
101
Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) is one of the most conserved core subunits of mitochondrial complex I. NDUFS7 has a bound iron-sulfur cluster N2 (tetranuclear) which is the terminal redox center in the electron transport chain (ETC) of complex I. NDUFS7 protein is encoded by the nuclear genome and is incorporated in the peripheral segment of complex I. Most mitochondrial matrix proteins are synthesized in the cytosol and imported into mitochondria by mitochondrial targeting sequences (MTSs). We previously defined the N terminus of the first 60 amino acids of NDUFS7 is an effective MTS. We also identified that there is a nuclear localization signal (NLS) and a nuclear export signal (NES) located in the C-terminus of NDUFS7. Sumoylation has been recognized to play an important role in protein nucleocytoplasmic transportation. Small ubiquitin-related modifiers (SUMOs) could be conjugated to target proteins by E1 (SAE1/SAE2), E2 (UBC9) and E3 enzymes after translation. Using sequence predication software, NDUFS7 protein was found to have several potential sumoylation sites. In this study, we co-transfected plasmids expressed NDUFS7, SUMO-1 and UBC9 into HEK293 cells, and detected the conjugation of NDUFS7 with SUMO-1 by western blotting with different antibodies. The results indicated that NDUFS7 could indeed be sumoylated at the current experimental conditions in vivo. To confirm these findings, SUMO-specific protease (SENP) was co-expressed with NDUFS7, SUMO-1 and UBC9 proteins in some experiments. The results showed that overexpression of SENP could reduce the level of NDUFS7 and SUMO-1 interaction. These results suggested that NDUFS7 can be sumoylated by SUMO-1 in cells. In addition, we also identified one of the sumoylation sites of NDUFS7 to be Lys 202, which is consistent with the consensus sumoylation motif and is required for NDUFS7 protein stability. Further studies are needed and on the way to investigate the functional detail of NDUFS7 sumoylation.
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Chou, Ting-Fang y 周定芳. "Human mitochondrial complex I NDUFS7 subunit has a dual distribution both in mitochondria and nuclei". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/65608741390037035540.
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碩士國立清華大學
分子醫學研究所
97
Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7), one of the most conserved core subunits of mitochondrial complex I, plays an important role in the oxidative phosphorylation system (OXPHOS). This protein binds one iron-sulfur cluster N2 (tetranuclear) which is the terminal redox center in the electron transport process of complex I. Three types of mutations in this subunit have been associated with Leigh syndrome (LS). NDUFS7 protein is encoded by nuclear genome and incorporated in the peripheral segment of complex I facing the mitochondrial matrix. The NDUFS7 was suppressed in human T-REx-293 cells using the RNA interference (RNAi) technology. The reduction in the NDUFS7 expression caused a slow growth rate in galactose containing medium and increased H2O2 generation. These results indicated that NDUFS7 may play an important role in cell energy production and survival. Most mitochondrial matrix proteins are synthesized in the cytoplasm and imported into mitochondria by TIM/TOM complexes recognizing the mitochondrial targeting sequences (MTSs). Using predication softwares, the N-terminal fragment was suggested to be the MTS of NDUFS7. In this study, we fused NDUFS7 containing N-terminal deletions, C-terminal deletions or different portions of the protein to enhanced green fluorescent protein (EGFP), and used these obtained constructs to study their intracellular localization in T-REx-293 cells. We found that the chimeric NDUFS71-60-EGFP was colocalized with mitochondria, demonstrating that this N-terminal fragment contained an effective MTS. We then used site-directed mutagenesis analyses to study the role of basic and hydrophobic residues in the first 60 amino acids of NDUFS7. These results suggested that both two types of amino acids are important for the mitochondrial import of the MTS in NDUFS7. In addition, we also demonstrated that there is a nuclear localization signal (NLS) and a nuclear export signal (NES) located in the C-terminus of NDUFS7. These results suggested that NDUFS7 has a dual distribution both in mitochondria and nuclei.
12
Lee, Chao-Chang y 李兆昌. "Developing a TAT –mediated protein transduction system to rescue mitochondrial complex I deficiency caused by the defect of NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2)". Thesis, 2018. http://ndltd.ncl.edu.tw/handle/dy3986.
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碩士國立清華大學
分子醫學研究所
106
NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) is a nuclear-encoded subunit of human mitochondrial complex I. It contains a binuclear iron sulfur cluster N1a, and may play a role in temporary storage of excess electrons to prevent free radical production. Defects of NDUFV2 have been associated with Alzheimer's disease and Parkinson's disease. Cell-penetrating peptide derived from HIV-1’s transactivator of transcription (TAT) has been successfully applied as a carrier to bring fusion proteins into cells by crossing plasma membranes without compromising the biological function of proteins. In this study, we tried to develop a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFV2 defects. Two fusion proteins (TAT-NDUFV2 and NDUFV2-TAT) were exogenously expressed and purified from E. coli for transduction of human cells. In addition, similar constructs were also generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFV2 and NDUFV2-TAT could be delivered into mitochondria and correctly assembled in complex I. Interestingly, the mitochondrial import of TAT-containing NDUFV2 was independent of mitochondrial membrane potential. To explore the therapeutic application of the developed system, a NDUFV2 knockdown cell line (IF4) generated in previous studies was applied for rescuing studies. Treating with TAT-NDUFV2 not only significantly improve the assembly of complex I in IF4 cells, but also partially rescue complex I functions both in the in-gel activity assay and the complex I enzymatic activity assay. In addition, the oxygen consumption rate and mitochondrial membrane potential of IF4 cells were also greatly increased. Similar results were also observed while IF4 cells were treated with NDUFV2-TAT. Our current findings suggest a great potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency and other mitochondrial disease.
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GALLO, GIOVANNA. "Investigation of the molecular mechanisms underlying left ventricular hypertrophy in the presence of Complex I deficiency-dependent mitochondrial dysfunction". Doctoral thesis, 2022. http://hdl.handle.net/11573/1627551.
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Several studies have demonstrated that a dysfunction of the mitochondrial Complex I (CI) and of the sirtuins pathway is associated with the development of left ventricular hypertrophy (LVH). A deficiency of Ndufc2 (a subunit of NADH dehydrogenase [ubiquinone]) has been previously shown to cause an altered CI activity with severe mitochondrial dysfunction.
We examined the role of Ndufc2 deficiency and of subsequent CI dysfunction on the development of cardiac hypertrophy both in an in-vitro model and in humans.
We performed Ndufc2 gene silencing in H9c2 cardiomyocytes. We found that Ndufc2-silenced cardiomyocytes, compared to not silenced cells, showed a significant degree of hypertrophy by fluorescence microscopy (p<0.01), with a significant increase of the hypertrophy markers ANP and beta-myosin heavy chain (p<0.01). In parallel, sirtuin 3 and its downstream molecules (AMPK, phosphoAMPK, FOXO3a) were significantly reduced (p<0.05), with an increase of the mTOR pathway (p<0.05). The administration of nicotinamide, restoring CI function, was able to reduce the cellular volume and the gene expression level of both hypertrophy markers. We confirmed these results also in rat neonatal cardiomyocytes.
With regard to the human study, we included 246 hypertensive subjects (147 male, 59.7%) with a mean age of 59 ± 15 years. Seventy-nine individuals (32%) presented LVH. Genomic DNA was isolated from venous peripheral blood by using the FlexiGene kit (Qiagen). We studied 2 tag SNPs in NDUFC2: rs641836 and rs11237379
The analysis of possible associations with echocardiographic parameters showed that hypertensive patients who were carriers of the mutant A allele at NDUFC2 rs641836 had a significantly increase in septal thickness (p=0.001), posterior wall thickness (p=0.001), relative wall thickness (RWT) (p=0.005), LV mass (p=0.001), LV mass/body surface area (BSA) (p=0.001) and LV mass/height2.7 (p=0.002) compared to wild-type homozygotes. To better separate out the genetic effect, a covariate ANOVA was performed for each cardiac variable, considering age, gender, body mass index (BMI), office blood pressure (BP), antihypertensive treatment with a combination of 2 or more drugs and the number of BP-lowering agents as covariates. The analysis results were significant for septal thickness (p=0.017), posterior wall thickness (p=0.011), LV mass (p=0.003), LV mass/BSA (p=0.002) and LV mass/height2.7 (p=0.010).
With regard to the NDUFC2 rs11237379 polymorphism, hypertensive patients who were carriers of the TT genotype had a significantly increase in septal thickness (p=0.001), posterior wall thickness (p=0.003), RWT (p=0.01), LV mass/BSA (p=0.012) and LV mass/height2. (p=0.0033) compared to subjects who were carriers of both the CC genotype and CT genotype. After the adjustment for covariates, significant differences were maintained for septal thickness (p=0.07), posterior wall thickness (p=0.008), RWT (p=0.021) and LV mass/BSA (p=0.03).
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Yao, Li Hsin y 姚莉歆. "Sumoylation of human mitochondrial NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) and its association with various stress responses". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/08302812121819730701.
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碩士國立清華大學
分子醫學研究所
103
Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) is one of 44 subunits in mitochondrial complex I. The N-terminal 1-60 amino acids of NDUFS7 have been defined as a mitochondrial targeting sequence (MTS) and the C-terminus contains a nuclear localization signal (NLS) and a nuclear export signal (NES). The sequence of NDUFS7 is highly conserved in the motif: CCXXE(X)60C(X)30CP. It can bind to an iron-sulfur cluster (a [4Fe-4S] cluster) called N2, a redox center in the terminus of complex I electron transfer pathway. In previous study, we identified NDUFS7 as a protein substrate of sumoylation. Posttranslational modifications of proteins by the small ubiquitin-like modifier (SUMO) have been found to be associated with various cellular processes. The SUMO protein can be conjugated to a target protein by sequential actions of enzyme E1 (SAE1/2), enzyme E2 (UBC9) and E3 ligases. The proteins conjugated by SUMO could be involved in subcellular localization, function or responding to stresses. In this study, NDUFS7, SUMO-1 and UBC9 were co-expressed in HEK293 cells to clarify its subcellular localization by cell fractionation. The result showed that the sumoylated NDUFS7 were present in both the nuclear and cytosol fractions. Then, we established a new construct which can express the NDUFS7-SUMO-1 fusion protein, and found that the fusion protein can block the mitochondrial import. Furthermore, the effect of different stress response on sumoylation of NDUFS7 was explored by treating cells with various stress inducers, such as CoCl2, H2O2, starvation and apoptosis reagents. The results suggested that CoCl2-induced hypoxia increases the sumoylation of NDUFS7. The effects of NDUFS7 sumoylation on the subcellular localization of the protein and its physiological consequence would be further explored.
15
HSIEH, JUNG-SHENG y 謝鎔聲. "Sumoylation of human mitochondrial NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) and the effect on its subcellular localization". Thesis, 2019. http://ndltd.ncl.edu.tw/handle/d52k8k.
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Lee, Yen-Ling y 李彥陵. "Association of human 8-oxoguanine DNA glycosylase 1 beta isoform with NDUFB10 and its potential role in mitochondrial DNA repair". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/31306611064364281137.
Texto completoResumen
碩士國立成功大學
醫學檢驗生物技術學系碩博士班
97
The human 8-oxoguanine-DNA glycosylase 1 (hOGG1) is an essential base excision repair (BER) for oxidative DNA damage. hOGG1 yields two alternatively spliced isoforms, designated alpha- and beta-hOGG1. The alpha-hOGG1 is localized in nucleus and mitochondria (mt); whereas the beta-hOGG1 is mainly localized in mitochondria. In nucleus, the function and regulation of alpha-hOGG1 is well known. However, the potential functions of alpha- or beta-hOGG1 in mitochondrial DNA repair were not extensively investigated yet. In order to understand the possible roles of alpha- and beta-hOGG1 in mtDNA repair, we previously used yeast two-hybrid screening assays to screen for the proteins that are associated with them. We found that beta- but not alpha-hOGG1 directly interacts with NADH dehydrogenase (ubiquinone) 1 beta subcomplex 10 (NDUFB10). NDUFB10 is a subunit of the NADH:ubiquinone oxidoreductase complex I, which is located on mitochondrial inner membrane. We also confirmed the in vivo interaction between beta-hOGG1 with NDUFB10 by co-immunoprecipitation and immunofluorescence assays. By co-IP studies, it was found that association of ���{hOGG1 with NDUFB10 was enhanced after treatment of hydrogen peroxide, suggesting that beta-hOGG1 repairs mtDNA damages through its interaction with NDUFB10 upon oxidative stress. Furthermore, alpha-hOGG1 also interacts with beta-hOGG1 and might form a repair complex with beta-hOGG1 and NDUFB10 for mtDNA repair. Although, we tried to look for the interaction region of beta-hOGG1 that directly binds with NDUFB10. However, the co-IP result validated either helix-hairpin-helix motif or predictive transmembrane region (C terminus) is not the binding domain. The results of these studies will help us to understand the role of beta-hOGG1 on mitochondrial DNA repair or other relevant functions.
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Chaung, Meng Fan y 莊夢凡. "SUMO3 conjugation of human mitochondrial complex I subunit NDUFS7 is contributed to its subcellular localization and response to various cellular stresses". Thesis, 2016. http://ndltd.ncl.edu.tw/handle/61815368304144793695.
Texto completoResumen
碩士國立清華大學
分子醫學研究所
104
Mitochondrial respiratory complex I (NADH:ubiquinone oxidoreductase) is essential for energy production. Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) is a well-conserved subunit and contains an iron-sulfur cluster N2 (4Fe-4S tetranuclear) as the finial electron transport center in complex I. As a nuclear-encoded protein and translated in cytosol, we previously demonstrated NDUFS7 is imported into mitochondria by a mitochondrial targeting sequence (MTS). In addition, we also identified that NDUFS7 possesses a nuclear export signal (NES) and a nuclear localization signal (NLS) near the C-terminus. SUMOylation, the process of protein conjugation with a small ubiquitin-like modifier (SUMO), is one type of reversible modification and has been considered to play a key role in modulating many important cellular processes. In previous studies, we have shown that NDUFS7 could be modified by SUMO1 and the importance of this modification has been discussed. In this study, we tried to explore other involvement of SUMO3, another SUMO paralogue, in NDUFS7 modification and function. To map the major SUMOylation sites, we first mutated the lysine residue at position 11 on SUMO3 to arginine, which was used to study the mono-SUMOylation and abolished the poly-SUMOylated chain. We also constructed a series of NDUFS7 mutants with mutation(s) at seven lysine residues or removing the predicated SUMO-interacting motif (SIM). According to the result of mutation-scanning analyses, SUMOylation of NDUFS7 couldn’t be fully abolished in any of the mutant construct. Therefore, multiple lysine residues and SIMs might be involved in the SUMOylation of NDUFS7. In addition, the change of NDUFS7 translocation was observed in NDUFS7_SUMO3AA fusion protein. In contrast to SUMO1 modification, NDUFS7 conjugation with SUMO3 facilitated the import of NDUFS7 into mitochondria and nearly not in the nucleus. The biological meaning of NDUFS7 modification with SUMO3 was investigated by introducing various treatments such as dissipation of mitochondrial membrane potential, etoposide-induced III apoptosis, hypoxia, oxidative stress and deprivation of nutrition, respectively. Among them, we uncovered that SUMOylation of NDUFS7 with SUMO3 was up-regulated in the CoCl2-induced hypoxia condition but down-regulated under H2O2-triggered oxidative stress. The detailed mechanism about how SUMOylation influences NDUFS7 subcellular localization and its contribution to physiologic consequences deserves further exploration.
18
吳珮玉. "The functional analysis of NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) subunit and its iron-sulfur clusters in human mitochondrial complex I". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/85222579117160475992.
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Huang, Zi Xuan y 黃子軒. "Functional study of NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) and the effect of its deficiency on the assembly of mitochondrial OXPHOS complexes". Thesis, 2016. http://ndltd.ncl.edu.tw/handle/33125566436403079182.
Texto completoResumen
碩士國立清華大學
分子醫學研究所
104
NADH-dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7), a highly conserved protein encoded by the nuclear DNA, is one of the core subunits in the mitochondrial NADH-coenzyme Q oxidoreductase (Complex I). It contains a tetranuclear iron-sulfur cluster N2 that assists electron transfer and serves as the final electron donor for ubiquinone and thus contributes to energy generation in the oxidative phosphorylation (OXPHOS) system. Mutations in NDUFS7 have been reported in patients suffering from neurodegenerative diseases such as Leigh syndrome and might also been assoicated with bipolar disorder. In addition to being present as an individual complex, the assembly of component OXPHOS complexes (Complex I~V) into several larger supercomplexes is considered to have functional significance not only for maintaining the stability of complexes but also for increasing the efficiency of electron transfer. In this study, we applied the RNA interference technique to suppress NDUFS7 expression in T-REx293 cells and then analyzed the obtained knockdown cell lines O10 and U21 with a series of functional assays. Our results showed that knockdown of NDUFS7 caused a significant decrease in the cellular oxygen consumption rate, mitochondrial membrane potential, ATP generation and Complex I activity. In addition, the amount of harmful reactive oxygen species was apparently increased in mitochondria. On the other hand, by conducting the high resolution clear native electrophoresis (HrCNE) analyses, we found that the deficiency of NDUFS7 or NDUFV2 influenced individual complex assembly and reduced the stability of Complex I subunits. By applying a milder condition for extraction of protein complexes from the mitochondrial samples, we also found that the deficiency of NDUFS7 could hamper supercomplex formation. Based on these findings, we conclude that NDUFS7 plays an indispensable role in maintaining the functions of electron transport chain and the assembly of individual Complex I, III, and IV and the supercomplex.
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Tsai, Ai-Ling y 蔡艾凌. "人類粒線體酵素複合體 I 內NDUFV2次單元蛋白質及其鐵硫中心在氧化壓力下之功能研究". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/21305621358478755748.
Texto completo
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Leshabane, Nompumelelo. "Development of selected sulphur compounds and oxygenated volatile organic compounds refernce gas mixtures for air quality monitoring". Diss., 2005. http://hdl.handle.net/10500/27315.
Texto completoResumen
Highly accurate analysis for the quantification of sulphur compounds and
oxygenated volatile organic compounds are crucial for the adherence of the
legislation in different environmental sectors. The sulphur compounds and
oxygenated volatile organic compounds measurements are challenging, due to
various factors such as molecules being adsorbed on the inner surfaces of
cylinders. It is therefore important to produce accurate and reliable reference gas
mixtures with mole fraction at ambient levels for the air quality monitoring and field
of gas sensing in South Africa. The challenges in producing sulphur compounds
and oxygenated volatile organic compounds reference gas mixtures are that the
overall process from gravimetric preparation steps until the comparison analysis
process and the stability of mixture in the gas cylinder, results in the large
measurement uncertainties. In order to produce reference gas mixtures of the
highest level, three important steps are followed: purity assessment of starting
material, gravimetric preparation, and verification/validation of prepared gas
mixtures. The purity analysis of high purity starting materials was determined using
gas chromatography coupled with various detectors and Karl Fischer for
determination of moisture content in high purity chemicals.
The sulphur compounds and oxygenated volatile organic compounds to be
developed in this study were hydrogen sulphide, sulphur dioxide, acetone,
methanol, ethanol, isopropanol, and n-butanol. These components were produced
following the International Organisation for Standardisation documents at mole
fraction of 10 µmol/mol for sulphur compounds and 5 µmol/mol for oxygenated
volatile organic compounds. The preparation of sulphur compounds reference gas
mixtures was done with a static gravimetric method using a direct method where a
target component was transferred directly into the cylinder. The preparation of
oxygenated volatile organic compounds used an indirect method whereby a target
liquid component from high purity chemicals was transferred into a cylinder using a
gas-tight syringe.The comparison between the reference gas mixtures was
validated using Non-Dispersive Ultra-Violet analysers (NDUV), gas chromatograph
coupled with pulsed discharge helium ionisation detector (GC-PDHID, UV
fluorescence analysers for sulphur compounds and gas chromatograph coupled
with flame ionisation detector (GC-FID) for the oxygenated volatile organic compounds. A multi-point calibration method was used to analyse sulphur dioxide
and hydrogen sulphide on the NDUV analyser, and the single-point calibration
method was used for analysis on the gas chromatography and UV fluorescence
where a sample mixture is analysed against a reference mixture with a similar mole
fraction. The statistical data considered during analysis included calculation of the
instrument drift and percentage relative standard deviation to check measurements repeatability, reliability, and measurement uncertainty. The gravimetric results of
prepared sulphur compounds at 10 µmol/mol gave a percentage relative expanded
uncertainty of 0.041 % REU for hydrogen sulphide, 0.12 % REU for sulphur dioxide.
The gravimetric results of prepared oxygenated volatile organic compounds at 5
µmol/mol showed a percentage relative expanded uncertainty 0.068 to 0.35 % REU
for isopropanol and ethanol respectively and less than 2.4 % REU for multi component of oxygenated volatile organic compounds. Finally, the primary standard
gas mixtures of sulphur compounds and oxygenated volatile organic compounds
were developed with the highest metrological measurement uncertainty level of
(k=2).Environmental Sciences
M. Sc. (Environmental Sciences)
22
Leshabane, Nompumelelo. "Development of selected sulphur compounds and oxygenated volatile organic compounds reference gas mixtures for air quality monitoring". Diss., 2020. http://hdl.handle.net/10500/27315.
Texto completoResumen
Highly accurate analysis for the quantification of sulphur compounds and
oxygenated volatile organic compounds are crucial for the adherence of the
legislation in different environmental sectors. The sulphur compounds and
oxygenated volatile organic compounds measurements are challenging, due to
various factors such as molecules being adsorbed on the inner surfaces of
cylinders. It is therefore important to produce accurate and reliable reference gas
mixtures with mole fraction at ambient levels for the air quality monitoring and field
of gas sensing in South Africa. The challenges in producing sulphur compounds
and oxygenated volatile organic compounds reference gas mixtures are that the
overall process from gravimetric preparation steps until the comparison analysis
process and the stability of mixture in the gas cylinder, results in the large
measurement uncertainties. In order to produce reference gas mixtures of the
highest level, three important steps are followed: purity assessment of starting
material, gravimetric preparation, and verification/validation of prepared gas
mixtures. The purity analysis of high purity starting materials was determined using
gas chromatography coupled with various detectors and Karl Fischer for
determination of moisture content in high purity chemicals.
The sulphur compounds and oxygenated volatile organic compounds to be
developed in this study were hydrogen sulphide, sulphur dioxide, acetone,
methanol, ethanol, isopropanol, and n-butanol. These components were produced
following the International Organisation for Standardisation documents at mole
fraction of 10 µmol/mol for sulphur compounds and 5 µmol/mol for oxygenated
volatile organic compounds. The preparation of sulphur compounds reference gas
mixtures was done with a static gravimetric method using a direct method where a
target component was transferred directly into the cylinder. The preparation of
oxygenated volatile organic compounds used an indirect method whereby a target
liquid component from high purity chemicals was transferred into a cylinder using a
gas-tight syringe.The comparison between the reference gas mixtures was
validated using Non-Dispersive Ultra-Violet analysers (NDUV), gas chromatograph
coupled with pulsed discharge helium ionisation detector (GC-PDHID, UV
fluorescence analysers for sulphur compounds and gas chromatograph coupled
with flame ionisation detector (GC-FID) for the oxygenated volatile organic compounds. A multi-point calibration method was used to analyse sulphur dioxide
and hydrogen sulphide on the NDUV analyser, and the single-point calibration
method was used for analysis on the gas chromatography and UV fluorescence
where a sample mixture is analysed against a reference mixture with a similar mole
fraction. The statistical data considered during analysis included calculation of the
instrument drift and percentage relative standard deviation to check measurements repeatability, reliability, and measurement uncertainty. The gravimetric results of
prepared sulphur compounds at 10 µmol/mol gave a percentage relative expanded
uncertainty of 0.041 % REU for hydrogen sulphide, 0.12 % REU for sulphur dioxide.
The gravimetric results of prepared oxygenated volatile organic compounds at 5
µmol/mol showed a percentage relative expanded uncertainty 0.068 to 0.35 % REU
for isopropanol and ethanol respectively and less than 2.4 % REU for multi component of oxygenated volatile organic compounds. Finally, the primary standard
gas mixtures of sulphur compounds and oxygenated volatile organic compounds
were developed with the highest metrological measurement uncertainty level of
(k=2).Environmental Sciences
M. Sc. (Environmental Sciences)