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Etudes structurales de la protéine ACAD9 et des facteurs d'assemblage du complexe 1 de la chaîne respiratoire mitochondriale pour établir leur implication dans les processus neurodégénératifs / Structural studies of ACAD9 and mitochondrial complex 1 assembly factors to investigate their role in neurodegenerationBouverot, Romain 27 February 2019 (has links)
Les mitochondries sont en charge de la bioénergétique cellulaire, tout particulièrement dans le cerveau humain, au sein duquel les neurones sont extrêmement demandeurs en énergie et hautement dépendant de la phosphorylation oxydative. En effet, celles-ci génèrent un potentiel énergétique grâce à une chaîne de transport d’électrons, ou chaîne respiratoire, composée de quatre complexes protéiques ancrés dans la membrane interne mitochondriale. La chaine respiratoire permet la production d’énergie via la phosphorylation oxydative d’ADP en ATP par l’ATP synthéase dans la matrice mitochondriale. Le premier complexe (CI) de la chaîne est composé de 45 sous-unités protéiques (dont 44 différentes). En tant que premier enzyme de la phosphorylation oxydative, il joue un rôle d’initiateur et est essentiel pour la production d'énergie cellulaire. Un défaut d’assemblage du CI se traduit par d’importantes conséquences sur la bioénergétique cellulaire et augmente la production d’espèces réactives de l’oxygène (ROS), pouvant être à l'origine de divers troubles mitochondriaux, parmi lesquels certains processus neurodégénératifs. La bonne intégration des sous-unités et cofacteurs composant le CI est par conséquent primordiales et requièrent la participation de facteurs d’assemblage jouant le rôle de chaperonnes afin de stabiliser les sous-unités et faciliter leur intégration au sein de l'enzyme complète. De plus, certaines fonctions additionnelles à leur rôle d’assemblage peuvent intervenir dans d’autres processus cellulaire régulant l’activité métabolique.Le fonctionnement des facteurs d'assemblage du CI au niveau moléculaire demeure encore obscur. Néanmoins, il est admis que la plupart des facteurs d'assemblages identifiés sont actifs dès le début de l'assemblage, particulièrement pour l'incorporation des sous-unités membranaires. Récemment un groupe de facteurs d’assemblage composés des protéines NDUFAF1 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex assembly factor 1), ACAD9 (Acyl-CoA dehydrogenase 9), ECSIT (Evolutionarily conserved signaling intermediate in Toll pathway), et potentiellement TMEM126B (Transmembrane protein 126B) and TIMMDC1 (Translocase of inner mitochondrial membrane domain-containing 1) est désigné sous l'appellation complexe d’assemblage du complexe mitochondrial I (MCIA). Cependant, la composition et la stœchiométrie de ce dernier restent inconnus, excluant ainsi toute compréhension satisfaisante de sa structure et de son importance dans les mécanismes à l'oeuvre dans l’assemblage du CI.Cette thèse a pour but les caractérisations des facteurs d’assemblage ACAD9, ECSIT and NDUFAF1 grâce à un ensemble d’approches biochimiques et biophysiques dans le but de déterminer les mécanismes moléculaires et la cartographie des interactions impliqués dans l’assemblage du complexe MCIA. / Mitochondria are responsible for bioenergetics, particularly critical in the human brain, where neurons are extremely energy demanding and highly dependent on the oxidative phosphorylation (OXPHOS) system. They generate energetic potential through the electron transport chain (ETC), also named the respiratory chain, which is composed of four protein complexes embedded into the mitochondrial inner membrane (MIM) to enable the phosphorylation of ADP into ATP by the ATP synthase in the mitochondrial matrix. Together these complexes form the OXPHOS system. Complex I (CI), the first enzyme of the respiratory chain, is composed of 45 protein subunits (of which 44 are different) and initiates the OXPHOS system, being essential in cellular energy production. Defects in CI assembly severally impair ATP production, increase the production of reactive oxygen species (ROS) and are implicated in several mitochondrial disorders, including neurodegenerative diseases. The integration of the 45 subunits and the insertion of cofactors into the nascent complex requires the help of assembly factors. Assembly factors may act as chaperones that stabilize the intermediate complexes or subunits and help to attach them to other intermediate assemblies to build the complete enzyme. However, they may also have additional functions besides their requirement for CI assembly, in line with the emerging evidence that mitochondria are involved with various (sub)cellular processes that regulate cell metabolic activity.How CI assembly factors function at the molecular level is currently unclear, with very little structural information available. Nevertheless, it is thought that most identified assembly factors are involved in early assembly, more specifically in the incorporation of hydrophobic membrane subunits. Recently, the CI assembly factors NDUFAF1 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex assembly factor 1), ACAD9 (Acyl-CoA dehydrogenase 9), ECSIT (Evolutionarily conserved signaling intermediate in Toll pathway), and potentially TMEM126B (Transmembrane protein 126B) and TIMMDC1 (Translocase of inner mitochondrial membrane domain-containing 1) were proposed to form the so-called mitochondrial complex I assembly (MCIA) complex. However, the composition and stoichiometry of the MCIA complex are unknown, which precludes a proper understanding of the structural and mechanistic bases for building-up assembly intermediates and how the MCIA complex achieves specificity.This thesis pursues the characterisation of the MCIA core components ACAD9, ECSIT and NDUFAF1, mapping their interactions and characterising their structures using a combination of biophysical and biochemical approaches in order to elucidate the molecular mechanisms underlying the MCIA complex formation.
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Biological and Synthetic Studies of Mitochondrial Respiratory Chain Inhibitors / ミトコンドリア呼吸鎖阻害剤に関する生物および合成化学的研究Tsuji, Atsuhito 23 March 2023 (has links)
京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24555号 / 薬科博第172号 / 新制||薬科||19(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 小野 正博, 教授 掛谷 秀昭 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM
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Avaliação da função mitocondrial muscular e sua repercussão na capacidade funcional nos pacientes com distrofia muscular de Duchenne / Assessment of mitochondrial function in muscle and its relation to functional capacity in patients with Duchenne muscular dystrophyOkama, Larissa de Oliveira 10 August 2018 (has links)
A distrofia muscular de Duchenne (DMD) é uma doença hereditária, degenerativa e progressiva dos músculos esqueléticos. É causada pela ausência da proteína distrofina e caracterizada pela perda progressiva da força muscular e deterioração da capacidade funcional. Alterações na regulação da homeostase do cálcio, proteólise e alterações metabólicas, especialmente mitocondriais, são parte da patogênese da doença. A coenzima Q (CoQ10), potente antioxidante que participa da atividade da cadeia respiratória, tem sido utilizada em ensaios clínicos, entretanto, não há estudos que evidencie seu comprometimento na DMD. O objetivo deste estudo foi avaliar a CoQ10 e a da atividade da cadeia respiratória em fragmentos de biópsia muscular de pacientes com DMD e sua correlação com parâmetros clínicos e a capacidade funcional. O estudo constitui de uma etapa retrospectiva, onde foram analisadas 22 biópsias musculares de pacientes com DMD, e outra prospectiva, onde foram avaliados dez pacientes com DMD. Dez pacientes controles foram utilizados nas duas etapas do estudo. A concentração da CoQ10 foi realizada através da técnica de cromatografia líquida de alta performance de fase reversa. As atividades das enzimas da cadeia respiratória foram medidas através de técnicas espectrofotométricas. A capacidade funcional foi mensurada através das escalas Medida da Função Motora (MFM) e Escala de Avaliação para deambulantes North Star (NSAA), e dos testes cronometrados: tempo para percorrer 10 metros (T10), tempo para realizar a manobra de Gowers (TGowers) e teste da caminhada dos 6 minutos (TC6min). Fase retrospectiva: a média de idade dos pacientes com DMD foi de 6,9 anos, (DP ±2,4) e controles de 8 anos, (DP ±2,69). A dosagem média de CoQ10 nos fragmentos de pacientes com DMD foi de 8,6 µg/g de tecido (DP ±3,9) e nos fragmentos dos controles foi de 31,6 µg/g de tecido (DP ±6,9). A média da área ocupada por fibras musculares nos pacientes com DMD foi de 27,3% (DP ±14,2%) e nos controles foi de 89,2% (DP ±3,3%). Evidenciou-se alta correlação entre aquantidade de CoQ10 e a área relativa ocupada por fibras musculares (r= 0,767 e p= 0,016). As atividades dos complexos enzimáticos da cadeia respiratória dos pacientes com DMD não demonstraram deficiência. Já o resultado do ensaio conjunto dos complexos II+III, encontra-se significativamente reduzido nos pacientes com DMD. Etapa prospectiva: a média de idade dos pacientes com DMD foi de 6,5 anos, (DP ±2,4). A dosagem média de CoQ10 nos fragmentos de pacientes com DMD foi de 12,6 µg/g de tecido (DP ±5,1). A média da área ocupada por fibras musculares nos pacientes com DMD foi de 40,3% (DP ±20,4%). Houve alta correlação entre a quantidade de CoQ10 e a área relativa ocupada por fibras musculares (r= 0,690 e p= 0,058). A correlação da dosagem da CoQ10 com os instrumentos de avaliação da capacidade funcional foi alta com o TGowers e moderada com MFM total e dimensões 1 e 2, NSAA, T10 e TC6min. Em relação à área relativa de fibras musculares, houve moderada correlação com a dimensão 1 da MFM e com o TGowers. Não houve correlação da CoQ10 e da área relativa ocupada por fibras musculares com os parâmetros clínicos: idade no momento da biópsia, idade do início dos sintomas e tempo de evolução da doença. No presente estudo, concluímos que existe uma deficiência secundária de CoQ10 em pacientes com DMD, a qual contribui para entender a fisiopatologia da doença e com grande relevância para as propostas terapêuticas. / Duchenne muscular dystrophy (DMD) is a hereditary, degenerative and progressive skeletal muscles disease. It is caused by the absence of the protein dystrophin and characterized by progressive loss of muscle strength and deterioration of functional capacity. Alterations in the regulation of calcium homeostasis, proteolysis and metabolic abnormalities, especially mitochondrial dysfunction, are part of the pathogenesis of the disease. Coenzyme Q (CoQ10), a potent antioxidant that participates in respiratory chain activity, has been used in clinical trials, however, there are no studies showing its involvement in DMD. The purpose of this study was to investigate CoQ10 content and respiratory chain activity in muscle biopsy of patients with DMD and its correlation with clinical parameters and functional capacity. The study consisted of a retrospective phase, in which 22 muscle biopsies from patients with DMD were analyzed, and a prospective phase, where ten patients with DMD were evaluated. The same control group of ten patients were used in the two phases of the study. The concentration of CoQ10 was measured using the reverse phase high performance liquid chromatography technique. Activities of the respiratory chain enzymes were measured by spectrophotometry. The functional capacity was evaluated using the Motor Function Measurement (MFM) and North Star Ambulatory Assessment (NSAA) and the following timed tests: to run 10 meters (T10), to perform the Gowers maneuver (TGowers) and the 6-minute walk test (6MWT). Retrospective phase: the mean age of patients with DMD was 6.9 years (SD ± 2.4) and of controls was 8 years (SD ± 2.69). The mean CoQ10 content in fragments from patients with DMD was 8.6 ?g / g tissue (DP ± 3.9) and in fragments from controls was 31.6 ?g / g tissue (DP ± 6.9). The mean area occupied by muscle fibers in patients with DMD was 27.3% (SD ± 14.2%) and in controls was 89.2% (SD ± 3.3%). There was a high correlation between the amount of CoQ10 and the relative area occupied by muscle fibers (r= 0.767 and p= 0.016). The activities of respiratory chain enzymes from patients with DMD were not deficient. On the other hand, the results of the combined analysis of complexes II + III were significantly reduced in patients with DMD. Prospective phase: the mean age of patients with DMD was 6.5 years (SD ± 2.4). The mean CoQ10 content in fragments from patients with DMD was 12.6 ?g / g tissue (SD ± 5.1). The mean area occupied by muscle fibers in patients with DMD was 40.3% (SD ± 20.4%). There was a high correlation between the amount of CoQ10 and the relative area occupied by muscle fibers (r= 0.690 and p= 0.058). The correlation between the amount of CoQ10 and the functional capacity assessment instruments was high forTGowers and moderate for MFM total and dimensions 1 and 2, NSAA, T10 andd TC6min. Regarding the relative area of muscle fibers, there was a moderate correlation with MFM dimension 1 (standing position and transfers) and TGowers. There was no correlation between CoQ10 and relative area occupied by muscle fibers with clinical parameters: age at time of biopsy, age of onset of symptoms and time of disease progression. In the present study, we conclude that there is a secondary deficiency of CoQ10 in patients with DMD, which contributes for the understanding of its physiopathology and is relevant for therapy.
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Avaliação quantitativa da função mitocondrial no músculo esquelético em pacientes com distrofia muscular de cinturas / Quantitative assessment of mitochondrial function in skeletal muscle of patients with limb-girdle muscular dystrophyBená, Marjory Irineu 18 October 2018 (has links)
As distrofias musculares de cinturas (DMC) representam um grupo heterogêneo de desordens hereditárias e degenerativas da musculatura esquelética, com evolução progressiva, caracterizadas principalmente pelo acometimento predominante das cinturas escapular e/ou pélvica. São classificadas de acordo com o padrão de herança e o gene envolvido, podendo ser autossômicas dominantes ou autossômicas recessivas. Embora as disfunções mitocondriais tenham sido pouco descritas nas DMC, alguns estudos apresentaram evidências morfológicas e bioquímicas de alterações secundárias na cadeia respiratória mitocondrial. As razões envolvidas em tais disfunções na DMC permanecem pouco compreendidas. Portanto, é imprescindível uma caracterização detalhada da disfunção mitocondrial nos pacientes com DMC para uma melhor compreensão dos processos fisiopatológicos envolvidos na doença. Os objetivos do estudo foram: quantificar a CoQ10 em fragmentos do músculo esquelético de pacientes com DMC; quantificar a atividade enzimática de cada complexo da cadeia respiratória isoladamente; quantificar a atividade dos complexos II+III em conjunto como avaliação indireta da CoQ10; verificar a relação entre a área relativa de fibras musculares no fragmento de biópsia e o grau de disfunção mitocondrial nesses fragmentos; correlacionar a função mitocondrial com parâmetros clínicos de pacientes com DMC. Participaram do estudo 21 pacientes com DMC e 9 controles saudáveis. Os parâmetros clínicos incluídos foram idade no momento da biópsia, idade de início dos sintomas e tempo de evolução da doença. A análise do metabolismo mitocondrial foi realizada por: quantificação da coenzima Q10 nos fragmentos de biópsia muscular e quantificação da atividade dos complexos enzimáticos mitocondriais I, II, III, II+III e IV da cadeia respiratória e da enzima da matriz mitocondrial citrato sintase. Foram correlacionados os parâmetros clínicos, a proporção de fibras na biópsia muscular e os resultados das análises bioquímicas para a caracterização da função mitocondrial. A média dos pacientes com DMC em relação à idade no momento da biópsia, início dos sintomas e tempo de evolução da doença foi de 28,9 anos (DP ±11,7), 16,7 anos (DP ±10,3) e 12,1 anos (DP ±10,9), respectivamente. O grupo controle apresentou média de idade no momento da biópsia de 29,6 anos (DP ±10,3). A dosagem média de CoQ10 nos fragmentos de biópsia de pacientes com DMC foi de 17,9 µg/g de tecido (DP ±9,2) e nos fragmentos dos controles foi de 29,5 µg/g de tecido (DP ±4,4). Não foi observada alteração das atividades isoladas dos complexos enzimáticos da cadeia respiratória. Houve deficiência da atividade do conjunto de complexos II+III nos pacientes com DMC. A média da área ocupada por fibras musculares nos pacientes com DMC foi de 58,5% (DP ±26,1%) e nos controles foi de 76,35% (DP ±1,9%). Observou-se correlação moderada entre a quantidade de CoQ10 e a área relativa de fibras musculares (r= 0,57 e p= 0,007). Não houve correlação da CoQ10 com os parâmetros clínicos. No presente estudo, concluímos que existe uma deficiência secundária de CoQ10 em pacientes com DMC, sendo um achado importante para melhor entendimento do acometimento mitocondrial nessa doença e uma abordagem terapêutica mais eficaz. / The limb-girdle muscular dystrophy (LGMD) is a group of inherited and degenerative disorders of the skeletal muscle, with a progressive clinical course and predominant involvement of the scapular and/or pelvic girdles. They are classified according to the pattern of inheritance (autosomal dominant or autosomal recessive) and the gene involved. Mitochondrial dysfunction has been reported in the LGMD, some studies have described morphological and biochemical evidences of secondary mitochondrial respiratory chain alterations in patients with LGMD. The reasons involved in such dysfunctions in the LGMD remain poorly understood. Therefore, a detailed characterization of the mitochondrial dysfunction in patients with LGMD allows a better understanding of the pathophysiological processes involved in the disease. Therefore, a detailed characterization of mitochondrial dysfunction in patients with LGMD is essential for a better understanding of the pathophysiological processes involved in the disease. The purposes of the study were: to quantify CoQ10 in fragments of muscle biopsy of patients with LGMD; quantify the enzymatic activity of each respiratory chain complex isolated; quantify the activity of the II + III complexes together as an indirect measure of CoQ10; to verify the relation between the relative area of muscular fibers in the fragment of biopsy and the degree of mitochondrial dysfunction in these fragments; correlate mitochondrial function with clinical parameters of patients with LGMD. Twenty-one patients with LGMD and nine healthy controls participated in the study. The clinical parameters included were age at the time of biopsy, age of onset of symptoms and time of disease progression. The analysis of mitochondrial metabolism was performed by: quantification of CoQ10 in the muscle biopsy fragments and quantification of the activity of the mitochondrial enzyme complexes I, II, III, II + III and IV of the respiratory chain and the enzyme of the mitochondrial matrix citrate synthase. We analyzed the correlation of the clinical parameters, the proportion of fibers in the muscle biopsy and the results of the biochemical analyzes for the characterization of the mitochondrial function. For the LGMD groups, the mean age at the time of biopsy, onset of symptoms and disease duration was 28.9 years (SD ± 11.7), 16.7 years (SD ± 10.3) and 12.1 years (SD ± 10.9), respectively. The control group presented a mean age at the time of biopsy of 29.6 years (SD ± 10.3). The mean CoQ10 dosage in the biopsy specimens of patients with LGMD was 17.9 ?g / g tissue (SD ± 9.2) and in the fragments of the controls was 29.5 ?g / g tissue (SD ± 4, 4). No alterations were observed in the isolated activities of the enzymatic complexes of the respiratory chain. There was a deficiency of the activity of complexes II + III in patients with LGMD. The mean area occupied by muscle fibers in patients with LGMD was 58.5% (SD ± 26.1%) and in controls it was 76.35% (SD ± 1.9%). A moderate correlation was observed between the amount of CoQ10 and the relative area of muscle fibers (r = 0.57 and p = 0.007). There was no correlation of CoQ10 with clinical parameters. In the present study, we conclude that there is a secondary deficiency of CoQ10 in patients with LGMD, which has important implications in the understanding of mitochondrial involvement in this group of diseases and the development of a more effective therapeutic approach.
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Autoxidação de 1,4-dihidronicotinamidas promovida por N,N,N\',N\'-tetrametil-p-fenilenodiamina: Modelo de síntese de ATP no sítio I da cadeia respiratória / 1,4-Dihidronicotinamidas autoxidation promoted by N, N, N \', N\'-tetramethyl-p-phenylenediamine: ATP synthesis template in site I of the respiratory chainBechara, Etelvino Jose Henriques 07 March 1972 (has links)
N,N,N\',N \'-tetrametil-p-fenilenodiamina (TMPD) catalisa a autoxidação de coenzimas piridínicos (NADH, NADPH) e modelos (ClBCH ,ClPCH ) ao cátion piridínico com rendimentos de 80-100%. A velocidade destas reações mostrou dependência de primeira ordem com respeito à concentração da 1,4-dihidronicotinamida e de meia ordem em relação às concentrações de O2 e TMPD. Estes dados cinéticos e testes com captadores de ion superóxido e superóxido dismutase indicam que os radicais HO•2 oriundos da autoxidação lenta do TMPD promovem a oxidação da dihidronicotinamida numa reação em cadeia; no término os radicais HO•2 se aniquilam por dismutação. O mecanismo proposto também é confirmado (1º) pela razão kC-H/kC-D=2,3 quando se substitui um dos hidrogênios do C4 de ClBCH por deutério, (2º) pelas idênticas velocidades iniciais em H2O e D2O, (3º) pelo valor da Ea = 10 kcal/mol na autoxidação do NADH e (4º) pelo aumento da velocidade de pH = 7,8 a pH 6,5. TMPD também promove a autoxidação do derivado 5, 6-hidratado (PHTN) da dihidronicotinamida ao cátion piridínico (ClPC+) apenas de fosfato ou arsenato estão presentes. O ClPC+ nã o se forma a partir do ClPCH em equilíbrio com o PHTN. Muito provavelmente se forma a partir do intermediário fosforilado no C6 por oxidação no C4 seguida de eliminação de fosfato. Quando PHTN e ClPCH foram oxidados pelo sistema O2/TMPD na presença de fosfato de piridínio ou de tetra-n-butilamônio em meio piridínico houve formação de pirofosfato, isolado por cromatografia de papel e por resina de troca aniônica. Adicionando-se ADP de tetra-n-butilamôneo ao sistema, constatou-se a formação de pirofosfato e de ATP com rendimentos mínimos de 5% e 3% , respectivamente. Por outro lado se a mistura de reação contém AMP de tetra-n-butilamôneo pôde-se verificar a formação de pirofosfato, ADP e ATP com rendimento total de 28% de \"ligações ricas\". A reação estudada foi proposta como modelo para síntese de ATP no sítio I da cadeia respiratória. / N,N,N\',N\'-tetramethyl-p-phenylenediamine (TMPD) catalyses the autoxidation of the pyridine coenzymes and of their models to the pyridinium form (80-100% yields). The first arder dependence of the rate upon the dihidronicotinamide concentration and half order upon both the O2 and TMPD concentrations, indicates that the relatively slow autoxidation of TMPD is the source of free radicals: dihydronicotinamide autoxidizes by the HO•2 chain mechanism and in the termination step the HO•2 radicals decay by dismutation. Such a mechanis is also supported by the inhibitory effects of cathecol, a scavenger of the HO•2 radical, and of superoxide dismutase, an enzyme which accelerates the dismutation of the O-2/ HO•2 species. The mecanism is further supported by (1) kC-H/kC-D=2,3 for substitution in 1-benzyl-1,4-dihydronicotinamide (ClBCH), (2) identical rates in H2O and D2O buffers, (3) Ea = 10 kcal/mole in the autoxidation of NADH and (4) the increase in rate from pH 7,8 to 6,5. TMPD promotes also the autoxidation of 1-n-propyl-6-hydroxy-1,4,5,6 - tetrahydronicotinamide (PHTN) to the 1-propyl-3-carboxamidopyridinium cation (ClPC+) provided phosphate ar arsenate are present. ClPC+ originates not from 1-n-propyl-1,4-dihydronicotinamide (ClPCH) in equilibrium with PHTN but most certainly from a C6 phosphorylated intermediate by oxidation at C4 and loss of phosphate. When PHTN an ClPCH were oxidated by the system O2/TMPD in the presence of pyridinium phosphate or tetra-n-butylammonium phosphate in pyridineas solvent, formation of pyrophosphate occurred. Pyrophosphate was isolated and identified by paper and ionic exchange resin chromatography. If tetra-n-butylammonium ADP is also present in the system, one can observe the formation of both pyrophosphate and ATP (5% and 3% minimum yields, respectively). In the presence of tetra-n-butylammonium AMP there, formation of pyrofosphate, ADP and ATP occurs. The total yield of energy rich bond is 28%. We suggest that the reaction is a model for the generation of the first ATP in the respiratory chain.
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The role of nuclear-encoded subunit genes in mitochondrial complex 1 deficiencyWorgan, Lisa Catherine, Women & Children's Health, UNSW January 2005 (has links)
BACKGROUND: Mitochondrial complex I deficiency often leads to a devastating neurodegenerative disorder of childhood. In most cases, the underlying genetic defect is unknown. Recessive nuclear gene mutations, rather than mitochondrial DNA mutations, account for the majority of cases. AIM: Our aim was to identify the genetic basis of complex I deficiency in 34 patients with isolated complex I deficiency, by studying six of the 39 nuclear encoded complex I subunit genes (NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS7 and NDUFS8). These genes have been conserved throughout evolution and carry out essential aspects of complex I function. METHODS: RNA was extracted from patient fibroblasts and cDNA made by reverse transcription. Overlapping amplicons that together spanned the entire coding area of each gene were amplified by PCR. The genes were screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC). Patient samples with abnormal dHPLC profiles underwent direct DNA sequencing. RESULTS: Novel mutations were identified in six of 34 (18%) patients with isolated complex I deficiency. Five patients had two mutations identified and one patient had a single mutation in NDUFS4 identified. All patients with mutations had a progressive encephalopathy and five out of six had Leigh syndrome or Leigh like syndrome. Mutations were found in three nuclear encoded subunit genes, NDUFV1, NDUFS2 and NDUFS4. Three novel NDUFV1 mutations were identified (R386H, K111E and P252R). The R386H mutation was found in two apparently unrelated patients. Four novel NDUFS2 mutations were identified (R221X, M292T, R333Q and IVS9+4A<G). The novel NDUFS4 mutation c.221delC was found in two patients - one in homozygous form and the other heterozygous. Specific genotype and phenotype correlations were not identified. CONCLUSIONS: Nuclear encoded complex I subunit gene mutations are an important contributor to the aetiology of isolated complex I deficiency in childhood. Screening of these genes is an essential part of the investigation of complex I deficiency.
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Inhibition of the Toxoplasma gondii replication by inhibition of the mitochondrial respiratory chain / Inhibierung der Toxoplasma-gondii-Replikation durch Hemmung der mitochondrialen AtmungsketteNaujoks, Britta 12 December 2008 (has links)
No description available.
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Etude de la toxicité des nanoparticules d'oxyde de fer (Fe3O4) chez le rat : analyses mitochondriales et du stress oxydant / Study of the toxicity of the iron oxide nanoparticles (Fe304) in rats : analysis of mitochondrial activities and oxidative stressBaratli, Yosra 06 June 2015 (has links)
L’objectif de notre travail consiste à caractériser des nanoparticules d’oxyde de fer (Fe3O4) et étudier leur toxicité aiguë chez le rat Wistar. Nos résultats ont montré que l’administration orale aiguë des Fe3O4, entraîne une altération dose- et temps-dépendante des paramètres du stress oxydant ainsi qu’une atteinte hépatique. En ce qui concerne l’étude in vitro sur des mitochondries isolées, nos résultats ont montré que ces nanoparticules n’altèrent ni les différents complexes de la chaîne respiratoire mitochondriale ni le couplage mitochondrial, et ceci dans aucun des organes étudiés (cerveau, cœur, poumon, foie et reins) et quelle que soit la concentration utilisée (100, 200, 300 et 500 μg/ml), alors que les mitochondries hépatiques isolées des rats âgés (18 mois), une altération est observée au niveau de tous les complexes de la chaîne respiratoire mitochondriale hépatique ainsi que pour le couplage mitochondrial quelle que soit la concentration utilisée (250, 300 et 350 μg/ml) alors que pour les rats jeunes (3mois) on n’observe aucune altération. / The objective of our work is to characterize iron oxide nanoparticles (Fe3O4) and study their acute toxicity in Wistar rats. Our results showed that acute oral administration of Fe3O4, results in a dose and time-dependent alteration of oxidative stress parameters as well as liver damage. Regarding the in vitro study on isolated mitochondria, our results showed that these nanoparticles do not adversely affect the various complexes of the mitochondrial respiratory chain or mitochondrial coupling in any of the organs studied (brain, heart, lung, liverand kidneys) and regardless of the concentration used (100, 200, 300 and 500 μg/ml) while the isolated liver mitochondria from aged rats (18 months), an alteration is observed at all the complexes of the liver mitochondrial respiratory chain as well as the mitochondrial coupling regardless of the concentration used (250, 300 and 350 μg/ml), whereas for the young rats (3 months) no change is observed.
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Autoxidação de 1,4-dihidronicotinamidas promovida por N,N,N\',N\'-tetrametil-p-fenilenodiamina: Modelo de síntese de ATP no sítio I da cadeia respiratória / 1,4-Dihidronicotinamidas autoxidation promoted by N, N, N \', N\'-tetramethyl-p-phenylenediamine: ATP synthesis template in site I of the respiratory chainEtelvino Jose Henriques Bechara 07 March 1972 (has links)
N,N,N\',N \'-tetrametil-p-fenilenodiamina (TMPD) catalisa a autoxidação de coenzimas piridínicos (NADH, NADPH) e modelos (ClBCH ,ClPCH ) ao cátion piridínico com rendimentos de 80-100%. A velocidade destas reações mostrou dependência de primeira ordem com respeito à concentração da 1,4-dihidronicotinamida e de meia ordem em relação às concentrações de O2 e TMPD. Estes dados cinéticos e testes com captadores de ion superóxido e superóxido dismutase indicam que os radicais HO•2 oriundos da autoxidação lenta do TMPD promovem a oxidação da dihidronicotinamida numa reação em cadeia; no término os radicais HO•2 se aniquilam por dismutação. O mecanismo proposto também é confirmado (1º) pela razão kC-H/kC-D=2,3 quando se substitui um dos hidrogênios do C4 de ClBCH por deutério, (2º) pelas idênticas velocidades iniciais em H2O e D2O, (3º) pelo valor da Ea = 10 kcal/mol na autoxidação do NADH e (4º) pelo aumento da velocidade de pH = 7,8 a pH 6,5. TMPD também promove a autoxidação do derivado 5, 6-hidratado (PHTN) da dihidronicotinamida ao cátion piridínico (ClPC+) apenas de fosfato ou arsenato estão presentes. O ClPC+ nã o se forma a partir do ClPCH em equilíbrio com o PHTN. Muito provavelmente se forma a partir do intermediário fosforilado no C6 por oxidação no C4 seguida de eliminação de fosfato. Quando PHTN e ClPCH foram oxidados pelo sistema O2/TMPD na presença de fosfato de piridínio ou de tetra-n-butilamônio em meio piridínico houve formação de pirofosfato, isolado por cromatografia de papel e por resina de troca aniônica. Adicionando-se ADP de tetra-n-butilamôneo ao sistema, constatou-se a formação de pirofosfato e de ATP com rendimentos mínimos de 5% e 3% , respectivamente. Por outro lado se a mistura de reação contém AMP de tetra-n-butilamôneo pôde-se verificar a formação de pirofosfato, ADP e ATP com rendimento total de 28% de \"ligações ricas\". A reação estudada foi proposta como modelo para síntese de ATP no sítio I da cadeia respiratória. / N,N,N\',N\'-tetramethyl-p-phenylenediamine (TMPD) catalyses the autoxidation of the pyridine coenzymes and of their models to the pyridinium form (80-100% yields). The first arder dependence of the rate upon the dihidronicotinamide concentration and half order upon both the O2 and TMPD concentrations, indicates that the relatively slow autoxidation of TMPD is the source of free radicals: dihydronicotinamide autoxidizes by the HO•2 chain mechanism and in the termination step the HO•2 radicals decay by dismutation. Such a mechanis is also supported by the inhibitory effects of cathecol, a scavenger of the HO•2 radical, and of superoxide dismutase, an enzyme which accelerates the dismutation of the O-2/ HO•2 species. The mecanism is further supported by (1) kC-H/kC-D=2,3 for substitution in 1-benzyl-1,4-dihydronicotinamide (ClBCH), (2) identical rates in H2O and D2O buffers, (3) Ea = 10 kcal/mole in the autoxidation of NADH and (4) the increase in rate from pH 7,8 to 6,5. TMPD promotes also the autoxidation of 1-n-propyl-6-hydroxy-1,4,5,6 - tetrahydronicotinamide (PHTN) to the 1-propyl-3-carboxamidopyridinium cation (ClPC+) provided phosphate ar arsenate are present. ClPC+ originates not from 1-n-propyl-1,4-dihydronicotinamide (ClPCH) in equilibrium with PHTN but most certainly from a C6 phosphorylated intermediate by oxidation at C4 and loss of phosphate. When PHTN an ClPCH were oxidated by the system O2/TMPD in the presence of pyridinium phosphate or tetra-n-butylammonium phosphate in pyridineas solvent, formation of pyrophosphate occurred. Pyrophosphate was isolated and identified by paper and ionic exchange resin chromatography. If tetra-n-butylammonium ADP is also present in the system, one can observe the formation of both pyrophosphate and ATP (5% and 3% minimum yields, respectively). In the presence of tetra-n-butylammonium AMP there, formation of pyrofosphate, ADP and ATP occurs. The total yield of energy rich bond is 28%. We suggest that the reaction is a model for the generation of the first ATP in the respiratory chain.
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Vliv proliferace endoteliálních buněk na jejich citlivost k mitochondriálně řízené apoptóze a oxidačnímu stresu. / The effect of endothelial cell proliferation on susceptibility to mitochondrially controlled apoptosis and oxidative stress.Blecha, Jan January 2014 (has links)
Mitochondria are multifunctional organelles playing a key role in energy metabolism and cell death induction. Mitochondria, and specifically their respiratory chain, are also the main producers of reactive oxygen species (ROS) in cells. Metabolism can be affected by the state of cellular proliferation and certain ROS-inducing agents have an antiangiogenic effect based on the preferential elimination of proliferating endothelial cells (EC). Therefore, in this work we investigated, whether mitochondria could be responsible for different sensitivity of proliferation and confluent EC to cell death. We mainly focused on systems that regulate ROS level and apoptosis: respiratory chain (ROS production), antioxidant defense (ROS detoxification) and Bcl-2 family of proteins (apoptosis regulation). First, we treated EC with functional and nonfunctional respiratory chain with various oxidative stress- and apoptosis-inducing agents and determined ROS production and susceptibility to apoptosis in proliferating and confluent cells. Our results show that functional respiratory chain greatly increases the susceptibility of proliferating cells to ROS induction and apoptosis, whereas in qiescent cells it protects against cell death. Given these findings, we assessed the activity of respiratory chain in proliferating...
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