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Nessa dissertação foi realizada uma scoping review com objetivo de buscar na literatura o que existe, até o presente momento, para explicar a relação entre o metabolismo mitocondrial com a síndrome da fadiga crônica (SFC). A SFC se apresenta de forma diferente para cada indivíduo, o que torna complexo seu entendimento, uma vez que não foram identificados biomarcadores específicos para auxiliar em um diagnóstico definitivo que favoreça uma intervenção adequada e tratamentos mais eficazes. Diferentes mecanismos biológicos são estudados, sendo que alterações no metabolismo mitocondrial têm sido foco de pesquisas recentes. Tais alterações podem ser a causa de fadiga severa e estudos sobre SFC mostraram que entre os principais indicadores da disfunção mitocondrial, envolvidos com a menor produção de ATP, está o comprometimento das vias de fosforilação oxidativa. O método utilizado nessa revisão, scoping review, é utilizado para investigar conceitos-chave subjacentes a uma nova área de pesquisa, bem como esclarecer definições de trabalhos, analisando o título e resumo de artigos para seleciona-los. Como critérios de inclusão ficaram determinados: (1) estudos clínicos que registram pacientes adultos (>= 18 anos de idade); (2) mostram uma relação entre questões do metabolismo e bioenergética mitocondrial com síndrome da fadiga crônica; (3) são escritos em português, inglês ou espanhol; (4) artigos publicados nos últimos 10 anos. E de exclusão: (1) utilizaram modelos animai; (2) relatos de casos, editoriais, cartas, revisões de literatura, resumos e dissertações de reuniões; (3) literatura cinzenta. Entre os descritores comuns, utilizados para realizar a busca nas bases de dados estão: mitochondria OR mitochondrial, fatigue, bioenergy OR bioenergetic OR energy metabolism. O estudo foi guiado pela seguinte questão: \"\"Alterações no metabolismo energético mitocondrial estão relacionados com a origem e prevalência da síndrome da fadiga crônica?\". Após utilizar a estratégia de busca, específica para cada uma das quatro bases de dados (PubMed, EMBASE, SCOPUS e Web of Science), foram encontrados 228 artigos, os quais foram exportados para o software Rayyan QCRI e removidos aqueles que se encontravam em duplicata. Este software permitiu que dois revisores executassem, de forma independente, a leitura dos títulos e resumos de 150 artigos e 27 foram selecionados para a leitura na íntegra, por atenderem aos critérios supracitados. Dentre esses últimos, apenas 10 relatavam alterações no metabolismo mitocondrial relacionadas à SFC. As alterações compreendem modificações nas vias de transporte mitocondrial e na cadeia respiratória; mutações no DNA mitocondrial e, até mesmo, disfunções energéticas em células do sistema imune, como as natural-killer. Foram encontrados dados de pesquisas em diversas áreas clínicas, tais como: cardiologia, oncologia e distúrbios musculares, os quais podem colaborar para trazer luz às causas biológicas dessa síndrome. Desta forma, tornou-se ainda mais evidente a conexão entre distúrbios na bioenergética mitocondrial, como uma menor capacidade de transporte de oxigênio por meio das vias de transporte, ou até mesmo, a insuficiência mitocondrial para produção de ATP, com a SFC. De acordo com os estudos que compuseram a amostra final desta revisão (n=10), o metabolismo mitocondrial e suas principais atividades, como a produção e transporte de ATP, são um alvo potencial para auxiliar na compreensão de incógnitas existentes sobre a SFC. Esses resultados são promissores para a enfermagem, sobretudo na área da ciência dos sintomas, com impacto na qualidade de vida e no manejo personalizado de sintomas em diferentes condições crônicas, especialmente na SFC / In this dissertation a scoping review was carried out with the objective of searching in the literature what exists to date to explain the relationship between mitochondrial metabolism and chronic fatigue syndrome (CFS). SFC presents itself differently for each individual, which makes complex their understanding, since no specific biomarkers were identified to aid in a definitive diagnosis that favors an appropriate intervention and more effective treatments. Different biological mechanisms are studied, and changes in mitochondrial metabolism have been the focus of recent research. Such alterations may be the cause of severe fatigue and studies on CFS have shown that among the main indicators of mitochondrial dysfunction, involved in the lower production of ATP, is the involvement of oxidative phosphorylation pathways. The method used in this review, scoping review, is used to investigate key concepts underlying a new research area, as well as clarifying definitions of papers, analyzing the title and abstract articles to select them. As inclusion criteria were determined: (1) clinical studies that register adult patients (>= 18 years of age); (2) show a relationship between metabolism and bioenergetic mitochondrial issues with chronic fatigue syndrome; (3) written in Portuguese, English or Spanish; (4) articles published in the last 10 years. The exclusion criteria: (1) used animal models; (2) case reports, editorials, letters, literature reviews, abstracts and dissertations; (3) gray literature. Among the common descriptors used to perform the search in the databases are: mitochondria OR mitochondrial, fatigue, bioenergy OR bioenergetic OR energy metabolism. The study was guided by the following question: \"\" Changes in mitochondrial energy metabolism are related to the origin and prevalence of chronic fatigue syndrome? \". After using the search strategy, specific to each of the four databases (PubMed, EMBASE, SCOPUS and Web of Science), 228 articles were found, which were exported to the Rayyan QCRI software and removed from those that were in duplicate. This software allowed two reviewers to independently perform the reading of the titles and abstracts of 150 articles and 27 were selected for reading in full, because they meet the aforementioned criteria. Among the latter, only 10 reported changes in mitochondrial metabolism related to CFS. The changes comprise modifications in mitochondrial transport pathways and respiratory chain; mutations in mitochondrial DNA, and even energy dysfunctions in cells of the immune system, such as natural killer. Research data have been found in several clinical areas, such as: cardiology, oncology and muscular disorders, which can collaborate to bring light to the biological causes of this syndrome. Thus, the connection between disturbances in mitochondrial bioenergetics, such as reduced oxygen transport capacity, or even mitochondrial insufficiency for ATP production, with CFS became even more evident. According to the studies that compose the final sample of this review (n = 10), mitochondrial metabolism and its main activities, such as the production and transport of ATP, are a potential target to aid in the understanding of existing unknowns about CFS. These results are promising for nursing, especially in the area of symptom science, with an impact on quality of life and personalized management of symptoms in different chronic conditions, especially CFS

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

This thesis primarily focuses on the FoF1-ATP synthase/ATPase complex in the parasitic protist, Trypanosoma brucei. Instead of its normal aerobic function to synthesize ATP, it is required to hydrolyze ATP to maintain the m in the infective bloodstream stage of T. brucei and the related parasite, T. b. evansi. To better understand the composition, structure and function of this druggable target, my work focused on deciphering the function of three of the unique Euglenozoa specific subunits that comprise this complex molecular machine. Furthermore, the ADP/ATP carrier, which provides substrates for the FoF1-ATP synthase/ATPase, was functionally characterized and evaluated if it is physically associated with the complexes of the oxidative phosphorylation pathway.

Abstract The mitochondrial oxidative phosphorylation system is composed of five multisubunit enzyme complexes. Complex I is the first and largest of these, containing 46 subunits, seven encoded by mitochondrial DNA (mtDNA) and the rest by nuclear DNA. Isolated complex I deficiency is a major cause of metabolic errors in infancy and childhood, presenting as encephalomyopathies or multisystem disorders. Due to the bigenomic origin of complex I, the genetic causes of these defects can be either mitochondrial or nuclear. The object of the present work was to identify the underlying genetic cause in cases of children with complex I deficiency and to obtain more information on the structurally and functionally important sites of complex I subunits. The complete coding region of mtDNA was analysed by conformation-sensitive gel electrophoresis and subsequent sequencing. In addition, nine nuclear genes encoding conserved subunits of complex I were sequenced. The structural and functional consequences of the new sequence variants were further elucidated using mutagenesis of homologous residue in bacterial NDH-1 or by studying complex I assembly and expression in patient cell lines. Analysis of the mtDNA coding region in 50 children revealed four definitely pathogenic mutations, 3460G>A, 10191T>C, 11778G>A and 14487T>C, in seven patients. In addition, two novel mtDNA base pair substitutions were identified, 3866T>C in a patient with muscle weakness and short stature and 4681T>C in a patient with Leigh syndrome. The latter mutation causes a Leu71Pro amino acid exchange in the ND2 subunit. Cybrid clones harbouring this mutation retained the complex I defect, and reduced amounts of fully assembled complex I were detected in patient cell lines. The 3866T>C mutation leads to a Ile187Thr amino acid substitution in the ND1 subunit, and functional studies of the homologous amino acid substitution in E. coli showed that this had an effect on the assembly or stability of the NDH-1 holoenzyme. Sequencing of the nine nuclear-encoded complex I genes revealed only one novel base pair substitution with pathogenic potential. Further studies are needed, however, to establish the role of the Arg18Cys substitution in the mitochondrial leading peptide of the TYKY subunit. The above findings emphasize the contribution of mtDNA mutations to the aetiology of pediatric patients with complex I deficiency. Furthermore, two LHON primary mutations were identified in the present cohort of patients, although the clinical signs differed considerably from the classical symptoms of LHON. This suggests that the phenotype caused by primary LHON mutations is more variable than has so far been thought.

Introduction: Recent evidence in isolated mitochondria and permeabilized muscle fibers in ex vivo using simultaneous measurements of O2 consumption and ATP production suggest that mitochondrial efficiency provides an additional mechanism to fine-tune oxidative phosphorylation rate to ATP demand in skeletal muscle. However, in the absence of a direct measurement of both VO2 and ATP synthesis from the same region of the contracting muscle, whether this mechanism plays a role in the skeletal muscle in vivo is still unknown. Purpose: Using a noninvasive approach combining phosphorus and proton magnetic resonance spectroscopy (31P/1H-MRS), the present study aimed to determine skeletal muscle ATP synthesis rate and muscle VO2 during a graded dynamic plantar flexion exercise to determine mitochondrial efficiency in contracting skeletal muscle contraction. Method: To measure mitochondrial efficiency under physiological conditions, we applied a recently developed methodological approach in ex vivo to human gastrocnemius muscle in vivo using 31P/1H-MRS noninvasive techniques. We conducted a series of constant workloads and ischemic protocols to assess oxidative ATP synthesis (ATPox) rate and Myoglobin-derived oxygen consumption (Mb-derived VO2). Specifically, during two separate visits, in 12 healthy, sedentary to recreationally active young male adults, we determined the ATPox rate by measuring the initial phosphocreatine (PCr) resynthesis rate during recovery and Mb-derived VO2 during 30 seconds of occlusion at the end of each given exercise workload. Results: The calculated mean power output during constant load performed by all subjects increased linearly at each incremental workload for both 1H and 31P visits. The mean percent coefficient of variation (CV%) at all exercise workloads (25% of WRmax= 6.0 ± 6.6, 50% of WRmax = 4.5 ± 2.7, 75% of WRmax = 4.9 ± 3.9, 100% = 7.0 ± 4.5) demonstrated high reliability and reproductivity in power output between each visit. The mean concentration of PCr consumption at the steady state remained constant between 25 and 50% of WRmax (28.6 ± 1.7 and 28.2 ± 1.4, respectively) but increased linearly beyond 50% of WRmax (75% = 22.9 ± 1.4 mM and 100%= 18.0 ± 1.4 mM, respectively). Mean intracellular pH was not significantly different between 25 and 50% of submaximal workloads (6.98 ± 0.02 and 6.97 ± 0.02, respectively). Intracellular pH dropped to 6.94 ± 0.02 during the last min of exercise at 75% of WRmax and further decreased at 100% of WRmax, (6.87 ± 0.03). The percentage of Mb oxygenation level and partial pressure of oxygen(PO2) at the steady state exhibited a consistent linear decline with increasing workload. Accordingly, a distinct and strong linear relationship was found between the MbO2 and workload (r2 = 0.71). Similarly, as the exercise workload increased, the ATPox synthesis rate also increased linearly throughout all exercise workloads (r2 = 0.45). In contrast, there was no significant change in Mb-derived VO2 with increased exercise workload averaged over a 30 s (r2 = 0.36) and 10 s time frame (r2 = 0.17). As a result, there was no significant correlation between ATPox synthesis rate and Mb-derived VO2 across exercise intensities. However, at rest, the P/O ratio in the gastrocnemius muscle was 1.95 ± 0.68, consistent with theoretical values and previous studies in mice. Conclusion: Despite the decrease in MbO2 (%), Mb-derived VO2 from both 30 and 10 s averages remained relatively constant during the ischemic protocol, likely due to O2 availability limitation induced by the prolonged occlusion and the slow time-resolution for measuring the dMb signal, which precluded the quantification of mitochondrial efficiency during exercise. However, mitochondrial efficiency calculated at rest was in agreement with previously documented values using other methodologies and thus can provide an additional parameter to more comprehensively evaluate mitochondrial function in vivo.

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Apesar da melhoria no sistema de produção e cultivo dos embriões in vitro, cerca de 60% dos oócitos que entram no sistema, não atingem o estágio de blastocisto e a qualidade dos embriões obtidos é bastante variável quando comparadas com embriões produzidos in vivo. Este bloqueio pode ser afetado por íons inorgânicos, tampões, aminoácidos e composição da atmosfera gasosa. Partindo-se da premissa que há influência da mitocôndria sobre a ativação da morte celular programada levou-nos a formular a hipótese que ausência de fragmentação nuclear nos embriões antes das 72 hpi está relacionada com a ausência do potencial de membrana mitocondrial e a inibição da OXPHOS pela utilização de bloqueadores leva a manutenção de baixos níveis de potencial de membrana mitocondrial e baixas taxas de fragmentação nuclear nos embriões. Embriões foram produzidos in vitro mediante maturação durante 22 horas, fecundação e cultivo 18 horas após a inseminação (hpi). Decorridas 24hpi realizou-se o cultivo com 0% de oxigênio, a fim de bloquear o processo de OXPHOS. Após 48 hpi realizou-se o feeding do meio de cultivo (SOF) com inibidores da OXPHOS (antimicina A e/ou oligomicina, cianeto de potássio) em diferentes doses. O número de embriões 8 células foi determinado às 80 hpi, mesmo momento em que foram realizadas as técnicas de JC-1 e TUNEL. Verificou-se as 168 hpi o efeito dos tratamentos no desenvolvimento embrionário. Os resultados obtidos com a inibição da OXPHOS após 48 hpi com oligomicina e/ou antimicina A nas doses utilizadas não alterou a capacidade do embrião atingir o estádio de 8 células. Entretanto, esta inibição inviabilizou o desenvolvimento até o estádio de blastocisto. O tratamento com KCN permitiu o desenvolvimento até o estádio de 8 células e a blastocisto em taxas semelhantes ao controle. A inibição do cultivo na ausência do O2 inviabilizou o processo de cultivo. Já os resultados obtidos quanto ao Ψmm e TUNEL evidenciam que os tratamentos dos embriões antimicina e/ou oligomicina levaram a um aumento do Ψmm e fragmentação nuclear na maioria dos embriões testados. Portanto, não foi possível testar a hipótese de que o Ψmm é necessário para o estabelecimento da MCP, todavia, foi observada uma correlação positiva entre Ψmm e fragmentação nuclear. / Although in vitro embryo production has been improved in the last 2 decades, about 60% of the oocytes do not reach the blastocyst stage and embryo quality is very variable when compared with in vivo produced embryo. This developmental block can be affected by inorganic ions, buffers, aminoacids and gaseous atmosphere composition. The knowledge that there influence of mitochondria on the activation of the programmed cellular death led to formulate the hypothesis that nuclear fragmentation absence in embryos before 72 post insemination is related with absence of mitochondrial membrane potential and OXPHOS blockage by inhibiting agents, would cause the maintenance of low levels of mitochondrial membrane potential and low rates of embryo nuclear fragmentation. Embryos were cultured in vitro for 18 hours post insemination (hpi) and after 24 hpi, they were submitted to 0% oxygen culture, in order to block the OXPHOS process. At 48 hpi, feeding was performed with SOF medium containing OXPHOS inhibitors (antimycin A and/or oligomycin, potassium cyanide) in different concetrations. The numbers of 8 cell embryos were estimated at 80 hpi, the same moment that they were submitted to JC-1 probes and TUNEL for mitochondrial membrane potential and DNA damages evaluation, respectively. At 168 hpi the effect of the treatments was verified on embryonic development. The results obtained with the OXPHOS inhibition after 48 after hpi using oligomycin and/or antimycin A did not modify embryo capacity to reach 8 cell stage. However, this inhibition prevented development to the blastocyst stage. KCN treatment allowed development up to the 8 cell stage and blastocyst similar to controls. The absence of O2 prevented embryo development. The Ψmm and TUNEL results showed that antimycin and/or oligomycin treatment increased Ψmm and nuclear fragmentation in the majority of the embryos tested. In conclusion, it was not possible to test the hypothesis that Ψmm is necessary to the establishment of MCP, but a positive correlation between Ψmm and nuclear fragmentation was observed.

Les stéroïdes sexuels ne sont pas impliqués uniquement dans la reproduction, ils sont également actifs dans le système nerveux où ils exercent des effets neuroprotecteurs. La mitochondrie a un rôle central dans la synthèse de l’énergie cellulaire et le contrôle du stress oxydant. Ces fonctions mitochondriales seraient une cible potentielle des effets des stéroïdes sexuels dans le cerveau. Deux approches ont été développées au cours de ce travail de thèse : une approche physiologique avec l’étude de l’influence des stéroïdes endogènes sur la fonction mitochondriale cérébrale et une approche thérapeutique en utilisant le modèle expérimental de l’ischémie cérébrale et du traitement par la progestérone. Pour analyser l’influence des stéroïdes endogènes, nous avons comparé le fonctionnement de la phosphorylation oxydative (consommation d’oxygène ou « respiration » et activités enzymatiques) ; le niveau du stress oxydant (pool de glutathion mitochondrial et inactivation oxydative de l’aconitase mitochondriale) et les taux cérébraux de stéroïdes dans des groupes de souris mâles et femelles soit jeunes adultes intactes ou gonadectomisées (3 mois) ; soit âgées (20 mois). Nous avons montré que la respiration NADH-dépendante est plus importante et que le stress oxydant mitochondrial est moins important chez les femelles que chez les mâles jeunes. Cette différence n’existe plus chez les souris âgées et est abolie après ovariectomie mais pas après orchidectomie, ce qui démontre l’influence des stéroïdes ovariens. Les taux cérébraux importants de prégnènolone et de progestérone chez les souris jeunes femelles par rapport aux jeunes mâles pourraient être impliqués dans le dimorphisme sexuel observé.Les modifications de la respiration mitochondriale induites par l’ischémie cérébrale sont également différentes entre les mâles et les femelles dans notre modèle. La respiration NADH-dépendante est diminuée dans les deux sexes, mais la respiration FADH2-dépendante est diminuée spécifiquement chez les femelles. Le stress oxydant mitochondrial est augmenté dans les deux sexes. L’administration de progestérone permet de restaurer la respiration FADH2-dépendante chez les femelles et la respiration NADH-dépendante ainsi que le pool de glutathion mitochondrial dans les deux sexes. Ce travail a permis de mettre en évidence des différences de fonctionnement mitochondrial cérébral chez les souris mâles et femelles jeunes et d’identifier la phosphorylation oxydative et le stress oxydant mitochondrial comme cibles d’action des effets neuroprotecteurs de la progestérone lors de l’ischémie cérébrale. / Besides the reproduction control, sex steroids also act on nervous system and exert neuroprotective effects. The mitochondria are centrally involved in cellular energy synthesis and oxidative stress regulation and constitute a potential target of steroids effects on brain. The aim of our study was twofold: (1) to study the influence of endogenous steroids on brain mitochondrial function in physiological conditions ; (2) to determine the effects of progesterone on mitochondrial function when used as therapeutic agent in an experimental model of cerebral ischemia. To analyze the influence of endogenous sex steroids, the oxidative phosphorylation system (oxygen consumption or “respiration” and enzymatic activities) and mitochondrial oxidative stress (glutathione pool and mitochondrial aconitase oxidative inactivation) were analyzed in brain mitochondria of young adult male and female mice (3-month-old), intact and after gonadectomy, and of aged male and female mice (20-month-old). Our results showed that young adult females have lower oxidative stress and a higher NADH-linked respiration rate as compared to young adult males. This sex difference was suppressed by ovariectomy but not by orchidectomy and no longer existed in aged mice. Concomitant analysis of brain steroids suggest that the major male/female differences in brain pregnenolone and progesterone levels may contribute to the sex differences observed in brain mitochondrial function.We have also shown that the decrease of brain mitochondrial respiration induced by ischemia is different according to sex in our experimental model. The NADH-linked respiration decreased after ischemia in males and female but a decrease of FADH2-linked respiration only occurred in females. Ischemia induced oxidative damages in both males and females. Progesterone restored NADH-linked respiration in both sexes and FADH2-linked respiration in females. Progesterone also preserved mitochondrial glutathione pool in both sexes. Our findings point to a sex difference in brain mitochondrial function of young male and female mice and identify the oxidative phosphorylation system and the mitochondrial oxidative stress as targets of the neuroprotective effects of progesterone.

<p>Numerous studies on mammalian spermatozoa have reported large variations in the dimensions of the main sperm structural components, namely the head, midpiece and flagellum. These variations in sperm architecture are believed to be adaptations for functioning of spermatozoa in complex environments outside the male reproductive system. The midpiece of the mammalian&nbsp / permatozoon contains a varied number of mitochondria, but the reason for the marked difference in the size and structure of this sperm component is not clear. This study&nbsp / confirmed the variations in the sperm morphometry of seven selected mammalian species and revealed unique features of the sperm midpiece and sperm mitochondria of these seven species. Evaluation of several sperm kinematic parameters revealed the unique swimming characteristics of the different spermatozoa. The importance of using standardized motility&nbsp / parameters was highlighted as well as the assessment of different subpopulations of spermatozoa in order to produce more reliable and comparable data. Investigating the role of sperm mitochondria in human sperm&nbsp / metabolism indicated that these organelles are related to sperm function in terms of sperm motility. Furthermore, it was suggested that glycolysis and mitochondrial respiration are linked processes and that both are important for the maintenance of human sperm motility. By optimizing and employing standardized experimental procedures and analysis techniques, this study was&nbsp / able to confirm the species specificity of almost all the sperm parameters evaluated, while also elucidating the phylogenetic relatedness of the non-human primate species. In conclusion, the present study has confirmed that the various midpiece morphometry parameters are related to the remaining sperm morphometry parameters as well as to the sperm kinematic parameters.&nbsp / These proposed associations between the various sperm parameters were used to explain the sperm velocity of two hypothetical and morphologically different sperm structures. Therefore, the results of the current study support the idea of co-evolution between sperm components in mammalian spermatozoa and propose that the midpiece morphometry parameters that are selected for in these spermatozoa are midpiece volume, total number of mitochondrial gyres, thickness of the mitochondrial sheath and mitochondrial height.</p>