This thesis comprises of diverse projects all focused towards analysis of mitochondrial translation in unicellular parasites. As only two mitochondrially encoded genes are required during the life cycle stage when Trypanosoma brucei resides in the bloodstream of a mammalian host, this protist provides a simplified background in which to study mitochondrial translation termination phase. The leading project utilizes T. brucei to examine mitochondrial translation termination factor TbMrf1 by gene knockout. Subsequently, it is suggested that the peptidyl-tRNA hydrolase TbPth4 is able to abate the TbMrf1 knockout phenotype by its ability to rescue mitoribosomes that become stalled when TbMrf1 is absent. Additionally, modifying methyltransferase of TbMrf1, the TbMTQ1, was characterized. And finally, this work contributed to the development of the protein expression regulation method in Leishmania parasites, a protocol for measurement of proton pumping activity of FoF1 ATPase complex in native mitochondria, and optimization of purification protocol for hydrophobic recombinant proteins.

As mitocôndrias desempenham um papel fundamental na sobrevivência e morte celular. Alterações do DNA mitocondrial (DNAmt) - como, por exemplo, amplificação, mutação homoplásmica, deleção e depleção -, bem como suas implicações clínico-patológicas, tem sido analisadas em inúmeras neoplasias humanas. No intuito de se pesquisar alterações mitocondriais associadas à tumorigênese, o presente trabalho teve como objetivos analisar a expressão de genes implicados no metabolismo energético e envolvidos na replicação e transcrição mitocondriais, quantificar o número de organelas mitocondriais e de cópias de DNAmt e analisar a expressão dos genes em astrocitomas de diferentes graus de malignidade (23 OMS grau I, 26 grau II, 18 grau III e 84 grau IV ou GBM) em relação ao tecido cerebral não tumoral (22 amostras). As expressões relativas dos genes selecionados, bem como as quantificações relativa e absoluta do DNA mitocondrial, foram realizadas por PCR em tempo real. O aumento de expressão relativa de genes-chave da via glicolítica, alterações nos níveis de expressão dos genes do ciclo dos ácidos tricarboxílicos e hipoexpressão de genes da fosforilação oxidativa detectados corroboraram o efeito Warburg. Foi demonstrado que a redução do número de cópias do DNAmt está associada com o grau de malignidade dos astrocitomas difusamente infiltrativos, sendo GBM o mais depletado e independente do número de organelas. As médias observadas para tecido não tumoral, astrocitoma grau I, grau II, grau III e GBM foram, respectivamente, 1,28, 0,26, 0,45, 0,42 e 0,17. Níveis aumentados de expressão relativa dos genes dos fatores de transcrição mitocondriais A (TFAM), B1 (TFB1M), B2 (TFB2M) e da subunidade catalítica da polimerase mitocondrial (POLG) foram detectados em todos os graus de astrocitomas, exceto TFB2M em astrocitoma grau II. Embora exista forte correlação entre os fatores de transcrição mitocondriais, somente os níveis de expressão de POLG se correlacionaram inversamente com o número de cópias de DNAmt. A expressão elevada de TFAM está associada a uma maior sobrevida no grupo de pacientes com GBM, interpretada como compensatório. As hiperexpressões de TFAM e POLG estão relacionadas a um melhor prognóstico em pacientes com GBM. Embora nossos achados da disfunção do metabolismo intermediário e depleção do DNAmt em astrocitomas corroborem a literatura, ainda não está bem esclarecida sua implicação na iniciação e manutenção da transformação maligna. Investigações futuras são necessárias para o esclarecimento destas questões. / Mitochondria has a key role in cell survival and death. Mitochondrial DNA (mtDNA) alterations, for example, amplification, homoplasmic mutation, deletion and depletion, and their clinical and pathological implications have been analyzed in human malignancies. In order to search for mitochondrial alterations associated to tumorigenesis, this study aimed to analyze the expression levels of genes involved in energetic metabolism, and in mitochondrial replication and transcription, to quantify the number of mitochondrial organelle and mtDNA copy number in astrocytomas of different grades of malignancy (23 WHO grade I, 26 grade II, 18 grade III and 84 grade IV or GBM) related to non-neoplastic brain tissue (22 samples). The relative expression level of the selected genes as well as the relative and absolute quantification of mtDNA were performed by real-time PCR. Relative expression increase of glycolytic pathway key genes, change of citric acid cycle genes and hipoexpression of oxidative phosphorylation genes were detected, and confirmed the presence of Warburg effect. The reduced mtDNA copy number was associated to the grade of malignancy of diffusely infiltrating astrocytoma, being GBM the most depleted, and not related to parallel decrease in the number of organelle. The mean mtDNA copy number for non neoplastic tissue, astrocytoma grade I, grade II, grade III and GBM were respectively 1.28, 0.26, 0.45, 0.42 and 0.17. The increased relative gene expression of mitochondrial transcription factor A (TFAM), B1 (TFB1M), B2 (TFB2M) and the catalytic subunit of mitochondrial polymerase (POLG) were observed in all grades of astrocytoma, except TFB2M in grade II astrocytoma. Although a strong correlation was observed among the mitochondrial transcription factors, only the expression level of POLG correlated inversely to the mtDNA copy number. The overexpression of TFAM was associated with long-term survival in the GBM patients and interpreted as compensatory. TFAM and POLG overexpressions were related to better prognosis in GBM patients. Although our findings concerning the impairment of intermediary metabolism and depletion of mtDNA in astrocytomas confirmed previous reports, their role in initiation or maintenance of malignant transformation were not fully understood. Further investigations are needed to clarify these issues.

Orientadores: Anibal Eugenio Vercesi, Nadja Cristhina de Souza Pinto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T03:45:12Z (GMT). No. of bitstreams: 1 Ravagnani_FelipeGustavo_D.pdf: 7335132 bytes, checksum: 7043cdada57a4c0242520d7fcb95daeb (MD5) Previous issue date: 2013 / Resumo: O número de indivíduos que sofrem com patologias associadas ao consumo abusivo de etanol tem aumentado significativamente no último século. Como consequência desse fato, os custos associados ao tratamento do alcoolismo, bem como das doenças associadas a ele também têm aumentado, onerando o sistema de saúde e se tornando um problema de saúde pública de grande relevância atualmente. Os mecanismos moleculares que desencadeiam muitas dessas doenças não estão completamente esclarecidos. O tecido hepático é o mais afetado pelo etanol e as mitocôndrias têm sido apontadas como alvos cruciais na toxicidade hepática induzida pelo álcool. Logo, o objetivo desse trabalho foi investigar como o consumo de etanol afeta o estado redox e o metabolismo mitocondriais no fígado. Ratos Wistar machos adultos jovens e de meia-idade receberam ad libitum solução alcoólica 25% (v/v) como única fonte de líquido. Os grupos controle receberam somente água. Ambos os grupos receberam ração ad libitum. Mitocôndrias hepáticas foram isoladas usando técnicas padrão. O consumo de ração e de líquidos foi significativamente menor em animais que ingeriram álcool, resultando em menor ganho de massa corpórea nos protocolos utilizados. As mitocôndrias dos animais que consumiram etanol apresentaram menores níveis de respiração em condição basal e quando energizadas com substratos respiratórios. A atividade e os níveis protéicos de citocromo c oxidase foi menor nos grupos tratados com etanol. Independente da duração do período de tratamento, mitocôndrias hepáticas de animais que ingeriram álcool foram menos susceptíveis à transição de permeabilidade mitocondrial induzida por cálcio, quando comparadas às mitocôndrias dos animais do grupo controle. Esse efeito foi revertido pela adição de oxidantes de nucleotídeos de piridina (acetoacetato, diamida ou tert butil-hidroperóxido) ou em mitocôndrias desacopladas. Também houve aumento em nucleotídeos de piridina na forma reduzida e aumento na razão NAD(P)H/NAD(P)+ em mitôndrias hepáticas de ratos consumidores de etanol. Em concordância a esses dados, houve aumento na capacidade de retenção de cálcio, processo que é dependente do estado redox intramitocondrial. Por outro lado, não houve diferença na produção de espécies reativas de oxigênio entre os grupos controle e tratados com álcool. A atividade de glutationa peroxidase e as quantidades de GSH e de GSSG também não sofreram alterações. Entretanto, houve redução nos níveis de DNA mitocondrial nos tratamentos agudos, porém com tendência para retornar aos níveis normais nos tratamentos crônicos, indicando uma resposta adaptativa à injúria induzida pelo etanol. Em conjunto, nossos resultados indicam que o consumo de etanol modula o estado redox mitocondrial e de sistemas antioxidantes, prevenindo a abertura do poro de transição de permeabilidade mitocondrial. A presença desse xenobiótico no fígado também altera significativamente os níveis de NADP reduzido, agente redutor final para o sistema glutationa redutase/peroxidase que detoxifica H2O2 na matriz mitocondrial. Além disso, a resposta adaptativa ao álcool observada no DNA mitocondrial pode contribuir para compreender melhor os mecanismos envolvidos no reparo de lesões a biomoléculas e os estágios iniciais de adaptação a esse xenobiótico, etapas que precedem a morte celular, hepatite alcoólica ou carcinogênese em tecido hepático exposto cronicamente ao etanol / Abstract: The number of people suffering from alcoholism has increased significantly over the last century. As a result, costs associated with treating the addiction itself as well as the associated pathologies have also increased, such that this is considered as public health issue. Furthermore, the molecular events leading to several of these diseases are not yet clearly understood. Hepatic tissue is the most affected by alcohol, and mitochondria have been suggested to be a crucial target in alcohol-induced liver toxicity. Thus, the aim of our study was to investigate how ethanol consumption affects the redox state and mitochondrial metabolism in the liver. Young adult and middle-aged male Wistar rats were given a 25 % (v/v) ethanol solution as the only source of drinking water. Control groups received water only. Liver mitochondria were isolated using standard techniques. Food and water intake was significantly lower in alcohol-drinking rats, resulting in lower weight gain during the treatment regimes. Mitochondria from the alcohol-drinking group had lower respiration under levels in basal condition, when energized by substrates feeding electrons into complexes I and IV. Cytochrome c oxidase activity and protein levels were lower in the alcohol group as well. Additionally, regardless of the length of the treatment, liver mitochondria from the alcohol-treated animals were more resistant to Ca2+-induced mitochondrial permeability transition (MPT), when compared to mitochondria from control animals. This effect was abrogated by oxidizing agents of pyridine nucleotides (acetoacetate, diamide or tert butylhydroperoxide) or in uncoupled mitochondria. We also found that liver mitochondria from the alcohol-drinking rats had a more reduced pyridine nucleotide pool and higher NAD(P)H/NAD(P)+ ratios. In addition, Nampt (an enzyme of the NAD+ synthetic pathway) protein levels did not differ after alcohol consumption. Accordingly, the calcium retention capacity of the isolated mitochondria, which is dependent upon intramitochondrial redox state, was higher in the alcohol group. On the other hand, levels of reactive oxygen species showed no differences between the control and alcohol groups, both in mitochondria and in splenic lymphocytes. Glutathione peroxidase activity and the amounts of GSH and GSSG were also not changed. However, mitochondrial DNA levels were decreased in the short term treatments, but tended to go back up to normal levels in the chronic treatments, indicating an adaptative response to ethanol-induced injury. Together, our results indicate that ethanol consumption modulates the mitochondrial redox state and the antioxidant systems, protecting against Ca2+-induced mitochondrial pore transition permeability opening. The presence of this xenobiotic can significantly change the levels of reduced NADP, the ultimate reducing agent in the gluthatione reductase/peroxidase system that detoxifies H2O2 in the mitochondrial matrix. In addition, the adaptative response to ethanol, seen in mitochondrial DNA, may contribute to further understand the mechanisms related to lesions in biomolecules and the initial steps that preceed cell death, alcoholic hepatitis or carcinogenic process in hepatic tissue exposed chronically to ethanol / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

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.

Les mitochondries sont des organites qui possèdent leur propre ADN (ADNmt), codant pour des gènes de la chaine respiratoire. La réparation des dommages dus aux ROS, une réplication défectueuse ou d’autres sources exogènes tels des agents chimiothérapeutiques ou des irradiations ionisantes peuvent générer des cassures double-brin (CDB) de l’ADNmt. L’ADNmt code pour des protéines essentielles à la production d’énergie, et des systèmes de maintenance de l’intégrité de ce génome efficaces sont donc nécessaires pour la viabilité des cellules. En effet des mutations de l’ADNmt sont présentes dans de nombreuses pathologies comme les myopathies mitochondriales, les cancers et les maladies neurodégénératives. Cependant les processus responsables de la maintenance de l’ADNmt suite à des CDB restent controversés.Pour élucider les mécanismes impliqués, nous avons généré des CDB mitochondriales en utilisant une lignée cellulaire humaine exprimant de manière inductible l’enzyme de restriction PstI liée à une séquence d’adressage mitochondrial. Nos résultats montrent, dans notre système, une première phase de dégradation de l’ADNmt lésé avec une cinétique rapide, n’impliquant pas l’autophagie ou l’apoptose, suivie de la ré-amplification d’ADNmt intact dans un deuxième temps. Contrairement à d’autres études nous n’avons pas pu détecter d’évènements de réparation des CDB mitochondriales générées. Nous avons ensuite cherché à identifier les protéines impliquées dans la dégradation de l’ADNmt lésé que nous observons, mais aucune nucléase testée ne semble responsable de ce processus. Des approches plus globales sont mises au point pour identifier de nouveaux acteurs, notamment un crible RNAi à grande échelle. Parallèlement nous nous intéressons aussi à une famille de phosphohydrolases, les Nudix, et à leur rôle protecteur en assainissant le réservoir de nucléotides libres. / Mitochondria are organelles that possess their own genome, the mitochondrial DNA (mtDNA). Repair of oxidative damages, defective replication, or various exogenous sources, such as chemotherapeutic agents or ionizing radiations, can generate double-strand breaks (DSBs) in mtDNA. MtDNA encodes for essential proteins involved in ATP production and maintenance of integrity of this genome is thus of crucial importance. Mutations in mtDNA are indeed found in numerous pathologies such as mitochondrial myopathies, neurodegenerative disorders or cancers. However, the mechanisms involved in mtDNA maintenance after DSBs remain unknown.To elucidate this question, we have generated mtDNA DSBs using a human inducible cell system expressing the restriction enzyme PstI targeted to mitochondria. Using this system, we could not find any support for DSBs repair of mtDNA. Instead we observed a loss of the damaged mtDNA molecules and a severe decrease in mtDNA content, followed by reamplification of intact mtDNA molecules. We have demonstrated that none of the known mitochondrial nucleases are involved in mtDNA degradation and that DNA loss is not due to autophagy, mitophagy or apoptosis but to a selective mechanism. Our study suggests that a still uncharacterized pathway for the targeted degradation of damaged mtDNA in a mitophagy/autophagy-independent manner is present in mitochondria, and might provide the main mechanism used by the cells to deal with DSBs. Global approaches are ongoing to identify proteins involved in degradation of damaged mtDNA following DSBs, mainly an RNAi screen targeting 80 nucleases. In parallel we are interested in a family of phosphohydrolases named Nudix and their putative protective role in sanitizing the nucleotides pool in mitochondria.

L'ADN mitochondrial (mtDNA) des plantes est caractérisé par les activités de recombinaison qui modulent sa structure. Ces activités sont nécessaires à son maintien et contribuent à son évolution rapide. Des facteurs contrôlant la recombinaison sont donc indispensables à la stabilité du mtDNA des plantes. Au cours de ma thèse j'ai identifié et caractérisé deux ADN hélicases présentes dans les organelles d'Arabidopsis thaliana. L'une d'entre elles est homologue à une hélicase bactérienne impliquée dans la réparation couplée à la transcription. Son rôle précis dans les organelles des plantes reste à déterminer. La deuxième hélicase, l'hélicase RECG1, a des rôles dans la réparation par recombinaison, la surveillance de la recombinaison ectopique impliquant des courtes séquences répétées, mais aussi dans la ségrégation du mtDNA. En effet, nous avons observé qu'en absence de RECG1 il y a une perte du contrôle de la recombinaison qui a pour conséquence la création de versions alternatives du mtDNA par recombinaison. L'analyse de leur ségrégation, induite par RECG1, nous a permis de modéliser comment de nouvelles configurations stables du mtDNA sont générées par le changement de stoechiométrie entre sous-génomes. Ce travail a permis de mieux comprendre les mécanismes de recombinaison et de ségrégation du mtDNA d'Arabidopsis. / The mitochondrial DNA (mtDNA) of flowering plants is characterized by the recombination activities that modulate its structure. These activities are required for the mtDNA maintenance, and drive its rapid structural evolution. The factors that control recombination are therefore essential for plant mtDNA stability. During my PhD, I identified and characterized two DNA helicases that are present in the organelles of Arabidopsisthaliana. One is the homologue of a bacterial helicase involved in transcription-coupled repair. Its role in the plant organelles is still not determined. The other one, the RECG1 helicase, has roles in recombination dependent repair, the surveillance of ectopic recombination involving short repeated sequences, and also the segregation of the mtDNA. We have found that in the absence of RECG1 there is loss of recombination control resulting in the occurrence of alternative versions of the mtDNA generated by recombination. The analysis oftheir segregation, induced by RECG1, allowed us to build a model to how new stable mtDNA configurations are generated by the stoichiometric shift of mtDNA sub-genomes. This work allowed us to better understand the recombination and segregation mechanisms that modulate the Arabidopsis mtDNA.

Le NAD+ apparaît comme un régulateur majeur du fonctionnement mitochondrial. En effet, ce cofacteur régule non seulement l’activité de nombreuses enzymes impliqués dans le métabolisme énergétique (enzymes de la β-oxydation des acides gras, du cycle de Krebs) mais joue également un rôle dans la production d’espèces réactives de l’oxygène (ROS). Le NAD+ est aussi le cofacteur des sirtuines, des enzymes déacétylases régulatrices notamment du métabolisme mitochondrial. De plus, la mitochondrie est l’organite au sein duquel la concentration en NAD+ est la plus élevée (jusqu’à 70% du NAD cellulaire). Le complexe I, qui possède une activité NADH déshydrogénase, pourrait être l’un des régulateurs majeurs du rapport NADH/NAD+ mitochondrial. L’objectif de ce travail de thèse a été d’étudier le rôle du rapport NADH/NAD+ mitochondrial dans le métabolisme énergétique et l’implication du complexe I dans les pathologies mitochondriales. Nous avons mis en évidence qu’une modulation du rapport NADH/NAD+ mitochondrial (augmentation par un activateur pharmacologique ou diminution consécutive à une mutation touchant une sous-unité du complexe I, modifie de manière drastique le métabolisme énergétique notamment en activant ou inhibant la protéine SIRT3, isoforme mitochondriale des sirtuines. Le complexe I semble jouer un rôle majeur dans cette modulation. Le resveratrol, ciblant le complexe I, ainsi que le NMN, un précurseur du NAD+, permettent de restaurer ce rapport et d’améliorer ainsi le métabolisme mitochondrial. Nos résultats suggèrent donc que le rapport NADH/NAD+ pourrait être une cible thérapeutique particulièrement intéressante dans les déficits du complexe I. / NAD+ appears as a main regulator of the mitochondrial function. Indeed, this compound not only regulates the enzymatic activity of enzymes involved in energetic metabolism (fatty acid oxidation, tricarboxylic acid cycle) but is also involved in ROS production. NAD+ is also the cofactor of sirtuins, deacetylase enzymes, in particular regulating the mitochondrial function. Moreover, mitochondria sequester most of the cellular NAD+ (up to 70 %). The complex I, which possesses an NADH dehydrogenase activity, is thought to be the most important regualtor of the mitochondrial NADH/NAD+ ratio. The work presented here aimed at studying the role of the mitochondrial NADH/NAD+ ratio in mitochondrial metabolism and to test the involvement of the complex I in mitochondrial disorders. We show that a modulation of the mitochondrial NADH/NAD+ ratio (increase by a pharmacological agent or decrease in complex-I mutated fibroplasts) severely affects the mitochondrial energetic function especially by interacting with SIRT3 a mitochondrial sirtuin isoform. The NADH/NAD+ ratio is highly regulated by complex I activity. Resveratrol, which targets the complex I, as well as NMN, a NAD+ precursor, improves the mitochondrial NADH/NAD+ ratio and consequently increases the mitochondrial metabolism. Our results strongly suggest that the mitochondrial NADH/NAD+ ratio could be an interesting therapeutic target especially in complex I- deficient patients.

Abstract Finding the genetic causes leading to phenotypes of mitochondrial diseases is challenging because of heterogeneity of the disorders and variety of the underlying biochemical defects. In adults, many of the manifestations of mitochondrial diseases cannot be distinguished from the neurodegenerative processes associated with old age. A single mutation or mutations within the same gene can result in a broad range of disorders. Conversely, clinically similar, monogenic disorders may be caused by genes which are governing entirely different cellular pathways. This study investigated the genetic etiology underlying certain symptoms which are characteristic for mitochondrial syndromes, or mimics of the mitochondrial ones. In the first project, we presented the contribution of genetic variation in the Wolfram Syndrome 1 gene to the risk of diabetes mellitus and sensorineural hearing impairment. We also estimated the frequency of a rare pathogenic variation in WFS1. The second project detected a link between the complex phenotype of age-related hearing impairment and the WFS1 gene. Monogenic forms of ARHI are extremely rare and we succeeded in recognizing one Mendelian form of the trait. The third project confirmed the Mitofusin 2 gene causality in the outlier phenotype of Charcot-Marie-Tooth disease. The fourth project described a Finnish family with two affected siblings with adult-onset ataxia, diabetes mellitus, and hypergonadotropic hypogonadism. The found novel mutation in mtDNA, m.8561C>G, was located in the overlapping region of two mitochondrial genes and resulted in an impaired assembly and dysfunctional energy production of mitochondrial ATP synthase. This thesis expands our knowledge about complex neurological phenotypes and identifies not only some causative genes but also outlier phenotypes, which should be noted in clinical practice. / Tiivistelmä Perintötekijät mitokondriaalisten ja niiden kaltaisten tautien taustalla ovat vaikeasti tunnistettavissa. Tautien kirjo on valtava, ja niihin johtavat biokemialliset syyt ovat moninaisia. Aikuisten mitokondriotaudit voivat jäädä diagnosoimatta, koska oireet voivat peittyä vanhenemiseen liittyviin neurodegeneratiivisiin prosesseihin. Sama mutaatio tai eri mutaatiot samassa geenissä voivat johtaa kliinisesti täysin erilaisiin ilmiasuihin. Toisaalta, kliinisesti samankaltaiset taudit voivat olla geneettisesti ja solubiologiallisesti kirjavia. Tässä tutkimuksessa selvitetään geneettistä etiologiaa tiettyjen mitokondriaalisille ja niiden kaltaisille taudeille tyypillisten oireiden taustalla. Ensimmäisessä osajulkaisussa tunnistetaan geneettisiä riskivariantteja Wolfram Syndrome 1 -geenissä diabeteksen ja kuulonaleneman taustalta. Lisäksi tutkimuksessa estimoidaan harvinaisen tautia aiheuttavan variaation määrää kyseisessä geenissä. Toinen projekti esittelee suomalaisen perheen, jossa myöhään alkaneen kuulonaleneman, ikäkuulon, geneettinen syy paljastuu WFS1-geenistä, jota ei aiemmin ole liitetty kyseiseen ilmiasuun. Yhden geenin aiheuttamat ikäkuulotapaukset ovat todella harvinaisia, koska ikäkuulo on monimutkainen kokonaisuus, johon ympäristötekijöillä on suuri vaikutus. Kolmas osajulkaisu kuvaa potilastapauksia, joiden ilmiasu on epätyypillinen Charcot-Marie-Toothin neuropatia. Tautigeeni on tunnettu Mitofusin 2, mutta sen aiheuttaman taudinkuvat ovat yleensä vakavampia ja varhain alkaneita. Viimeinen osajulkaisu kuvaa suomalaisen perheen, jonka kahden oireisen sisaruksen taustalta löytyy mitokondriaalisen DNA:n uusi mutaatio, joka sijaitsee kahden geenin alueella muuttaen niiden molempien lopputuotetta. Mutaation, m.8561C>G, osoitetaan vaikuttavan mitokondriaalisen ATP-syntaasin rakentumiseen ja energiatuotantoon. Tämä väitöskirja laajentaa geneettistä tietoisuutta neurologisten tautien taustalla ja esittelee uusia geneettisiä syitä ja ilmiasuja, jotka tulisi huomioida kliinisessä työssä terveydenhuollossa.

Orientadores: Tiago Rezende Figueira, Roger Frigério Castilho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T03:45:16Z (GMT). No. of bitstreams: 1 Chweih_Hanan_M.pdf: 1684363 bytes, checksum: edae156378f90e7315bca30c16544071 (MD5) Previous issue date: 2015 / Resumo: Algumas das características das mitocôndrias, incluindo as suas funções de transporte de Ca2+, podem apresentar dimorfismo sexual e especificidades teciduais. No entanto, as mensurações do transporte de Ca2+ em mitocôndrias isoladas estão sujeitas a artefatos secundários a abertura do poro de transição de permeabilidade mitocondrial (PTP) induzido pelo acúmulo excessivo de Ca2+ nesta organela. Neste estudo, o objetivo inicial foi avaliar se a inibição do PTP pela ciclosporina A (CsA) afeta a mensuração de diversas variáveis que descrevem o transporte de Ca2+por mitocôndrias isoladas de fígado de rato. Os resultados obtidos indicam que as concentrações de estado estável do Ca2+ externo a mitocôndria e as taxas deefluxo mitocondrial de Ca2+através de trocadores seletivos foram superestimados em até 4 vezes quando o PTP não foi inibido farmacologicamente pela CsA. O objetivo subsequente foi analisar o transporte de Ca2+ em mitocôndrias isoladas de fígado, de músculo esquelético, de coração e de cérebro de ratos machos e fêmeas sob condições experimentais específicas (i.e. meio de incubação contendo inibidores TPM, substratos energéticos ligados a NAD e níveis relevantes de Ca2+, Mg2+e Na+). Os dados indicaram que a taxa de influxo de Ca2+em mitocôndrias de fígado foi ~4 vezes superior a dos outros tecidos, as quais foram semelhantes entre si. Em contrapartida, as taxas de efluxo de Ca2+ apresentaram uma maior diversidade entre tecidos, especialmente na presença de Na+. Curiosamente, o efluxo de Ca2+na ausência de Na+foi significativamente mais elevado nas mitocôndrias cardíacas (~4nmol/mg/min) em relação às taxas observadas nos outros tecidos, contrariando a concepção de que o efluxo de Ca2+de mitocôndrias de coração é dependente, quase que exclusivamente, de um trocador que requer Na+. A especificidade em relação ao sexo só foi observada em dois índices relacionados a homeostase mitocondrial de Ca2+(i.e. cinética geral normalizada da captação de Ca2+ e a concentração de estado estável do Ca2+ externo a mitocôndria) em mitocôndrias isoladas de coração (mais lentos ou maiores na fêmea) e na respiração estimulada por ADP em mitocôndrias de fígado (~20% maior na fêmea). O presente estudo demonstrou a importância metodológica de se prevenir a abertura do PTP para a análise das propriedades e da variabilidade fisiológica do transporte de Ca2+por mitocôndrias isoladas. Adicionalmente, concluímos que sob as condições experimentais aqui utilizadas, o efluxo de Ca2+ mitocondrial apresenta grandes especificidades teciduais e que alguns achados desafiam conceitos estabelecidos em estudos anteriores sob condições arguivelmente menos controladas / Abstract: The characteristics of mitochondria, including their Ca2+ transport functions, may exhibit tissue specificity and sex dimorphism. Because the measurements of the Ca2+ handling by isolated mitochondria may be biased by dysfunction secondary to Ca2+-induced mitochondrial permeability transition (MPT) pore opening, this study evaluates the extent to which MPT inhibition by cyclosporine-A affects the measurement of Ca2+ transport in isolated rat liver mitochondria. The results indicate that the steady-state levels of external Ca2+ and the rates of mitochondrial Ca2+ efflux through the selective pathways can be overestimated by up to 4-fold if MPT pore opening is not prevented. Then, we analyzed the Ca2+ transport in isolated mitochondria from the liver, skeletal muscle, heart and brain of male and female rats under incubation conditions containing MPT inhibitors, NAD-linked substrates and relevant levels of free Ca2+, Mg2+ and Na+. Except for the liver mitochondria displaying values4-fold higher, the Ca2+ influx rates were similar among the other tissues. In contrast, the Ca2+ efflux rates exhibited more tissue diversity, especially in the presence of Na+. Interestingly, the Na+-independent Ca2+ efflux was highest in the heart mitochondria (~4 nmol/mg/min), thus challenging the view that heart mitochondrial Ca2+ efflux relies almost exclusively on a Na+-dependent pathway. Sex specificity was only observed in two kinetic indexes (i.e. the normalized overall kinetics of Ca2+ uptake and the steady-state levels of external Ca2+) of heart mitochondrial Ca2+ homeostasis (slower or higher in female)and in the ADP-stimulated respiration of liver mitochondria (~20% higher in females). The present study shows the methodological importance of preventing MPT when measuring the properties and the physiological variability of the Ca2+ handling by isolated mitochondria. Moreover, we conclude that mitochondrial Ca2+ efflux exhibits great tissue specificity under our conditions, which may challenge some concepts raised in previous studies that employed experimental conditions that are arguably not well controlled / Mestrado / Fisiopatologia Médica / Mestra em Ciências

O infarto do miocárdio é atualmente considerado a etiologia que mais contribui para o aparecimento de insuficiência cardíaca (IC) em humanos. Em detrimento a hiperativação de fatores neuro-humorais, a progressão da IC é caracterizada por uma série de anormalidades celulares associadas à disfunção ventricular. Dentre estas anormalidades, alterações na função e dinâmica mitocondrial merecem destaque, uma vez que a homeostase da organela é essencial para a viabilidade celular e o bom funcionamento da bomba cardíaca. No presente estudo, caracterizamos em modelo animal de disfunção cardíaca associada ao infarto do miocárdio: a) fenótipo cardíaco; b) função mitocondrial; c) equilíbrio redox; e d) dinâmica mitocondrial. Nossos resultados nos permitem afirmar que doze semanas após a cirurgia de infarto do miocárdio, os animais desenvolveram importantes alterações fenotípicas como aumento da massa cardíaca, dilatação ventricular, hipertrofia do cardiomiócito e maior deposição de tecido fibroso cardíaco, que contribuíram para o estabelecimento da disfunção ventricular. Além disso, foi possível confirmar a instalação do quadro de disfunção mitocondrial cardíaca, representada pela redução na capacidade respiratória e perda da homeostase redox. Por fim, encontramos um aumento no número de mitocôndrias cardíacas com menor diâmetro, alterações que vieram acompanhadas de uma menor atividade das enzimas relacionadas à fusão mitocondrial. Uma vez caracterizada a função e a dinâmica mitocondrial na disfunção cardíaca, avaliamos o efeito do treinamento físico aeróbico (TF) nessas variáveis. O TF, atualmente utilizado como um adjuvante no tratamento das doenças cardiovasculares, foi eficaz em promover o remodelamento cardíaco reverso e melhorar a função cardíaca nos animais infartados. Além disso, melhorou a capacidade respiratória e reduziu o estresse oxidativo, restaurando a função mitocondrial. Aliado a esses achados, o TF normalizou a atividade das enzimas relacionadas à dinâmica mitocondrial, fato associado à normalização do número e tamanho da organela. Esses resultados demonstram que a disfunção cardíaca induzida por infarto do miocárdio está associada à um quadro de mitocondriopatia em ratos, com alterações tanto na função quanto estrutura mitocondrial, e que o TF desencadeia efeitos benéficos na manutenção da integridade/função mitocondrial e melhora da função contrátil cardíaca / Myocardial infarction is considered the etiology that most contributes to the onset of heart failure in humans. Among the ventricular dysfunction-associated cellular abnormalities, changes in mitochondrial function and dynamics are critical, since the organelle homeostasis is crucial in maintaining the metabolic, electrical and mechanical properties of the heart. In the present study, we characterized in cardiac dysfunction- induced myocardial infarction in rats: a) cardiac phenotype; b) mitochondrial metabolism; c) redox balance, and d) mitochondrial dynamics. Our results show that twelve weeks after myocardial surgery, the animals developed pathological cardiac remodeling-associated ventricular dysfunction. Furthermore, we observed a reduced mitochondrial respiratory capacity and loss of redox homeostasis. Finally, we found a lower activity of enzymes related to mitochondrial fusion, these changes were accompanied by an increase in the number of small mitochondria. Once characterized mitochondrial function and dynamics, we evaluated the effect of exercise training in these variables in rats with cardiac dysfunction. The exercise training, currently established as an important non-pharmacological treatment for cardiovascular diseases, reversed the pathological cardiac remodeling and minimized the ventricular dysfunction in infarcted animals. Furthermore, exercise training restored the mitochondrial function by increasing respiratory capacity and reducing oxidative stress. Finally, exercise training restored the activity of mitochondrial dynamics-related enzymes and morphology. Taken together, our findings uncover the potential benefits of exercise training in reversing the cardiac mitochondriopathy observed in failing hearts, reinforcing the importance of this intervention as a non-pharmacological tool for heart failure therapy