NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme in the mitochondrial electron transfer chain. It catalyses the oxidation of NADH and the reduction of ubiquinone, coupled to the translocation of protons across the mitochondrial inner membrane, maintaining the proton motive force used for ATP synthesis. Complex I is the least understood of the respiratory enzymes; although the mechanisms of NADH oxidation and intramolecular electron transfer are gradually becoming appreciated, the mechanisms of quinone binding and reduction and proton translocation remain unknown. Complex I dysfunction has been implicated in a wide range of pathologies including mitochondrial diseases such as Leigh's disease, as well as neurodegenerative diseases such as Alzheimer's and Parkinson's. The work described in the first part of this thesis is aimed at elucidating the structure of either a subcomplex of mitochondrial complex I, or of the intact enzyme itself. A comprehensive investigation revealed that hydrophilic subcomplexes of complex I from bovine heart mitochondria are not suitable for use as models of the intact enzyme. Attempts to prepare intact complex I of sufficient quality for structural work were successful; however, results from a large set of crystallization trials were disappointing. The second part of this thesis describes three studies of the function and mechanism of complex I from bovine heart mitochondria. First, the flavin mononucleotide, the site of NADH oxidation, was identified as the site of the 'inhibitor-insensitive' NADH:ubiquinone oxidoreduction reaction. The formation of semiquinones initiates redox cycling reactions with oxygen, producing vast amounts of reactive oxygen species; further studies revealed that other oxidants, such as paraquat, also react at the flavin site and initiate redox cycling reactions. Second, kinetic studies showed that the reaction between NADH and positively charged oxidants such as HAR (hexaammineruthenium (III)) proceeds by an unusual ternary reaction mechanism at the flavin site of complex I. Finally, double electron-electron resonance spectroscopy was used to show unambiguously that iron sulphur cluster 4Fe[TY]1 gives rise to electron paramagnetic resonance signal N4; the data provide an alternating potential energy profile for electron transfer along the cluster chain between the flavin and the quinone-binding site.

The main objective of this work is to determine the atomic structure of mammalian respiratory complex I. Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) is one of the central enzymes in the oxidative phosphorylation pathway. It couples electron transfer between NADH and ubiquinone to proton translocation across the inner mitochondrial membrane, contributing to cellular energy production. Complex I is the largest and most elaborate protein assembly of the respiratory chain with a total mass of 970 kilodaltons. It consists of 14 conserved ‘core subunits’ and 31 mitochondria-specific ‘supernumerary subunits’. Together they form a giant, Lshaped molecule, with one arm buried in the mitochondrial membrane and another protruding into the mitochondrial matrix. Here, a novel method for the purification of ovine (Ovis aries) complex I was developed and suitable conditions for cryo-EM imaging established, after extensive screening of detergents and additives. Cryo-EM images were acquired with the recently developed direct electron detector and processed using the latest software. This allowed the solution of the nearly complete atomic model of mitochondrial complex I at 3.9 Å resolution. The membrane part of the complex contains 78 transmembrane helices, mostly contributed by conserved antiporter-like subunits responsible for proton translocation. These helices are stabilized by tightly bound lipids (including cardiolipins). The hydrophilic arm harbours flavin mononucleotide and 8 iron–sulfur clusters involved in electron transfer. Supernumerary subunits build a scaffold around the conserved core, strongly stabilizing the complex. Additionally, subunits containing cofactors (NADPH, zinc ion and phosphopantetheine) may play a regulatory role. Two distinct conformations of the complex are observed, which may describe the active and deactive states or reflect conformations occurring during the catalytic cycle of the enzyme. Currently this is the most detailed model of this molecular machine, providing insight into the mechanism, assembly and dysfunction of mitochondrial complex I. It also allows molecular analysis of numerous disease-causing mutations, and so the structure may serve as a stepping-stone for future medical developments.

No description available.

The modulation of the electron transport during ischemia-reperfusion has been shown to be protective. We hypothesized that metformin, a Complex I inhibitor, may exhibit characteristics of a pharmacological agent that could achieve long-term therapeutic intervention against ischemia-reperfusion injury. Mitochondria were harvested from adult male mice and incubated with or without metformin at 30oC for 15 minutes, while being shaken at 300 rpm. Metformin decreased Complex I oxidative phosphorylation and Complex I activity. However, metformin also increased injury and decreased the maximum membrane potential. Even though there was a decrease in maximum membrane potential, the proton motive force (PMF) was still intact as the ADP/O ratio was not affected. In conclusion, metformin does exhibit some characteristics of a drug that could achieve long-term therapeutic benefit against ischemia-reperfusion.

O objetivo desse trabalho foi observar a evolução do modelo de jogo entre as categorias de base do voleibol feminino, analisando como cada categoria conquista o ponto e a como as variantes das ações de jogo que antecedem o ataque influenciam no seu desempenho no complexo I. Foram analisados três jogos das quatro melhores equipes classificadas da categoria infantil, infanto-juvenil e juvenil feminino, participantes do quadrangular final do Campeonato Metropolitano de São Paulo em 2009 organizado pela FPV. A coleta de dados foi realizada pelo sistema de vídeo. A análise dos dados comportou a estatística descritiva, pela obtenção de frequências e porcentagens, e a estatística inferencial pelo recurso ao Qui-quadrado e ao Phi de Cramer, no sentido de verificar a possível relação de dependência entre as vaiáveis do estudo. Através da análise descritiva observamos que a categoria infantil conquista mais pontos com o erro do adversário e as categorias infanto-juvenil e juvenil conquistam mais pontos com a ação de ataque. Com o avanço de categoria observamos uma tendência de aumento na recepção perfeita, na utilização do levantamento com toque em suspensão, do ataque contra um bloqueio duplo quebrado, na realização do ataque pela zona 2 bem como, um aumento na ocorrência dos ataques de 1º e 2º tempo e do ataque ponto. Assim como, uma diminuição na utilização do toque sem salto para o levantamento, dos ataques contra um bloqueio duplo compacto, da utilização dos ataques de 3º tempo e dos ataques que proporcionam a continuação do rally. Pela análise inferencial observamos uma relação de dependência estatisticamente significante do resultado do ataque com o resultado da recepção, a zona de levantamento, a técnica de levantamento, o resultado do levantamento, bem como com a zona de ataque, o tempo de ataque e tipo de ataque para todas as categorias / The main purpose of this study was to determine game model evolution on female volleyball under-age divisions. It was analyzed how each category wins the point and how the game actions that occur before the attack affects the performance on complex I. We analyzed three games of the four best ranked female teams in infantil, infanto-juvenil and juvenil divisions, participants of the city of São Paulo Championship in 2009. The data base was captured using video system. Data analysis included descriptive statistics, by obtaining frequencies and percentages, and inferential statistics by using chi- square and Phi Cramer, in order to verify the possible relationship of dependency between the study variables. Descriptive analysis revealed that the infantil division obtain more points with the errors of the opponent and the infanto-juvenil and juvenil divisions obtain more points with the attack. As players advance through the age divisions, there is a trend toward increasing the occurrence of perfect receptions, the use of jump sets, the attack against a broken double block, the attack in zone 2 as well as an increase in the occurrence of attacks of 1st and 2nd tempo and the attack point. Moreover, we observed a decrease in the use of overhead set without jump, attack against a compact double block, the use of 3rd tempo attack and attack that provide continued rally. For the inferential analysis we observed a statistically significant relationship of dependency between attack result and the serve reception result, the set zone, set technique, the result of the set, as well as the attack zone, the attack tempo and type of attack for all categories

O objetivo desse trabalho foi observar a evolução do modelo de jogo entre as categorias de base do voleibol feminino, analisando como cada categoria conquista o ponto e a como as variantes das ações de jogo que antecedem o ataque influenciam no seu desempenho no complexo I. Foram analisados três jogos das quatro melhores equipes classificadas da categoria infantil, infanto-juvenil e juvenil feminino, participantes do quadrangular final do Campeonato Metropolitano de São Paulo em 2009 organizado pela FPV. A coleta de dados foi realizada pelo sistema de vídeo. A análise dos dados comportou a estatística descritiva, pela obtenção de frequências e porcentagens, e a estatística inferencial pelo recurso ao Qui-quadrado e ao Phi de Cramer, no sentido de verificar a possível relação de dependência entre as vaiáveis do estudo. Através da análise descritiva observamos que a categoria infantil conquista mais pontos com o erro do adversário e as categorias infanto-juvenil e juvenil conquistam mais pontos com a ação de ataque. Com o avanço de categoria observamos uma tendência de aumento na recepção perfeita, na utilização do levantamento com toque em suspensão, do ataque contra um bloqueio duplo quebrado, na realização do ataque pela zona 2 bem como, um aumento na ocorrência dos ataques de 1º e 2º tempo e do ataque ponto. Assim como, uma diminuição na utilização do toque sem salto para o levantamento, dos ataques contra um bloqueio duplo compacto, da utilização dos ataques de 3º tempo e dos ataques que proporcionam a continuação do rally. Pela análise inferencial observamos uma relação de dependência estatisticamente significante do resultado do ataque com o resultado da recepção, a zona de levantamento, a técnica de levantamento, o resultado do levantamento, bem como com a zona de ataque, o tempo de ataque e tipo de ataque para todas as categorias / The main purpose of this study was to determine game model evolution on female volleyball under-age divisions. It was analyzed how each category wins the point and how the game actions that occur before the attack affects the performance on complex I. We analyzed three games of the four best ranked female teams in infantil, infanto-juvenil and juvenil divisions, participants of the city of São Paulo Championship in 2009. The data base was captured using video system. Data analysis included descriptive statistics, by obtaining frequencies and percentages, and inferential statistics by using chi- square and Phi Cramer, in order to verify the possible relationship of dependency between the study variables. Descriptive analysis revealed that the infantil division obtain more points with the errors of the opponent and the infanto-juvenil and juvenil divisions obtain more points with the attack. As players advance through the age divisions, there is a trend toward increasing the occurrence of perfect receptions, the use of jump sets, the attack against a broken double block, the attack in zone 2 as well as an increase in the occurrence of attacks of 1st and 2nd tempo and the attack point. Moreover, we observed a decrease in the use of overhead set without jump, attack against a compact double block, the use of 3rd tempo attack and attack that provide continued rally. For the inferential analysis we observed a statistically significant relationship of dependency between attack result and the serve reception result, the set zone, set technique, the result of the set, as well as the attack zone, the attack tempo and type of attack for all categories

Complex I (NADH:ubiquinone oxidoreductase), located in the mitochondrial inner membrane, is a major electron entry point to the respiratory chain. It couples the energy released from electron transfer (from NADH to ubiquinone) to the concomitant pumping of protons across the membrane, to generate an electrochemical proton motive force. Mammalian complex I is composed of 45 subunits, 14 of which comprise its simpler bacterial homologues. It is encoded by both the mitochondrial and nuclear genomes, and pathological mutations in both sets of subunits result in severe neuromuscular disorders such as Leigh syndrome. Several structures of mammalian complex I from various organisms have been determined, but the limited resolutions of the structures, which typically refer to poorly characterised enzyme states, has hampered detailed analyses of mechanistic features. The first part of this thesis describes development of a method for purifying complex I from the genetically amenable and medically relevant model organism Mus musculus (mouse), in a pure, stable and active state. The enzyme from mouse heart mitochondria was then comprehensively characterised, to ensure the presence of all the expected subunits and co-factors, and to define its kinetic properties. The second part of this thesis describes structural studies by single particle electron cryomicroscopy (cryo-EM) on the purified mouse enzyme in two distinct states, the 'active' and 'de-active' states. The active state was determined to 3.3 Å resolution, the highest resolution structure of a eukaryotic complex I so far. Subsequently, comparison of the two mouse structures, together with previously determined mammalian and bacterial structures, revealed variations in key structural elements in the membrane domain, which may be crucial for the catalytic mechanism. Moreover, in the high-resolution active mouse complex I structure a nucleotide co-factor was observed bound to the nucleoside kinase subunit NDUFA10. Finally, complex I from the Ndufs4 knockout mouse model, which recapitulates the effects of a human mutation that causes Leigh syndrome, was purified and subjected to kinetic and proteomic analyses. Following cross-linking and preliminary structural studies, it was concluded that the detrimental effects of deleting NDUFS4 are due to lack of stability of the mature complex.

Thesis (M.Sc.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references (leaves 79-89).

Dissertation (Ph.D.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.