Dissertations / Theses on the topic 'Mitocondrias'

Orientadores: Alicia J. Kowaltowski, Anibal E. Vercesi
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: o gradiente de prótons (AuH+) gerado através da membrana mitocondrial interna pela oxidação de substratos não é totalmente acoplado à síntese de ATP. Além de transportadores específicos que utilizam a energia do AuH+,a membrana mitocondrial interna possui sistemas dissipadores de energia, tais como a proteína desacopladora de plantas (PUMP) e a oxidase alternativa (AOX), que diminuem a eficiência da fosforilação oxidativa em mitocôndrias vegetais. Outro mecanismo potencialmente dissipativo recentemente descrito envolve um ciclo do íon K+,onde a captação do cátion é mediada por canais de K+ sensíveis a ATP (mitoKATP) e seu efluxo é balanceado através do trocador K+/H+...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: The proton gradient (AuH+) across the mitochondrial inner membrane generated by substrate oxidation is not totally coupled to ATP synthesis. In addition to specific uniporters that use the energy contained in AuH+, the mitochondrial inner membrane presents energy-dissipating systems such as the plant uncoupling protein (PUMP) and the alternative oxidase (AOX), which decrease the efficiency of oxidative phosphorylation in plant mitochondria. Another potential dissipative mechanism recently described involves a K+ cycle,where ion import is mediated by ATP-sensitive K+ channels (mitoKATP) and its efllux is obtained through a K+/H+exchanger...Note: The complete abstract is available with the full electronic digital thesis or dissertations
Mestrado
Ciencias Biomedicas
Mestre em Ciências Médicas

Orientador: Lucia Pereira da Silva
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O Dipiridamol (DIP) é um vasodilatador coronário, antiplaquetário que atua de maneira sinergística com várias drogas antitumorais, além de possuir atividade antioxidante em diferentes sistemas de membrana, interagindo com micelas, monocamadas de fosfolipídios, vesículas e membranas biológicas. O efeito antioxidante do DIP sobre a lipoperoxidação mitocondrial induzida por Fé2+ já foi estudado, observando-se boa correlacão com sua característica lipofílica (Nepomuceno et alii, 1997). Neste trabalho, utilizando mitocôndrias e mitoplastos o papel do DIP como inibidor da peroxidação lipídica induzida por Fé2+, foi melhor estudado. A utilização de baixas concentrações de DIP não provocou mudanças no estado respiratório 4 nem no estado respiratório 3 e portanto, a interação do DIP com os receptores periféricos de benzodiazepínicos foi descartada. A constante de associação do DIP com mitoplastos foi estimada em torno de 1,1 ± 0,2 (mg/mL)-1. Este valor é ligeiramente superior ao obtido para mitocôndrias, 0,8 ± 0,1 (mg/mL)-1. Observou-se para os mitoplastos que o DIP não foi totalmente recuperado após incubação com essas membranas, provavelmente devido à metabolização da droga formando um produto não fluorescente. Os estudos de consumo de oxigênio na presença de FeSO4 mostraram que o efeito antioxidante do DIP não provocou mudanças na oxidação do Fé2+. Nossos dados permitem-nos sugerir que o efeito antioxidante do DIP está relacionado com a sua partição na membrana e não à sua ligação específica a proteínas de membrana. A proteção contra a peroxidação lipídica pode ser devida à inibição direta das reações de propagação ou à ação do DIP em sequestrar as espécies radicalares que poderiam iniciar o processo peroxidativo
Abstract: Dipyridamole (DIP), a coronary vasodilator, presents activity for a number of antitumor drugs as well as antioxidant activity in membrane systems. DIP and derivatives interact with membrane systems such as micelles, phospholipid monolayers, vesicles and biological membranes. The antioxidant effect of DIP upon iron induced lipoperoxidation on mitochondria has been reported and a good correlation between the hydrophobicity and its protective effect was found (Nepomuceno et alii, 1997). In the present work an effort is made to betler understand the role of DIP as inhibitor of Fe2+induced lipid peroxidation in mitochondria and mitoplasts. At low concentration, no significant effect on either state IV or state III respiration was found, discarding a possible direct interaction of DIP with the peripheral benzodiazepine receptor. The association constants for DIP in mitoplasts were estimated, being 1.1 ± 0.2 (mg/mL)-1. This value is slight1y higher than that obtained for mitochondria, 0.8 ± 0.1 (mg/mL)-1. It was observed that under mitoplast was used, some of the drug was not recovered, probably due to DIP metabolization into a no fluorescent species. Oxygen consumption studies in the presence of FeSO4 showed that the antioxidant effect of DIP did not involved the initial step of Fé2+ oxidation. Our data strongly support the hypothesis that the antioxidant effect of DIP is related to its partition in the lipid phase of the mitochondrial or mitoplast membrane and not to a specific interaction with membrane proteins. This protection may be due either to a direct inhibition of the propagation steps or a scavenger effect, removing the radicalar species that would trigger the peroxidative process
Mestrado
Bioquimica
Mestre em Ciências Biológicas

Orientador: Anibal Eugenio Vercesi
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O dano oxidativo induzido por Ca2+e agentes prooxidantes em mitocôndrias é mediado pelo ataque de espécies reativas de oxigênio geradas pela própria mitocôndria. Estas espécies reativas de oxigênio atacam os grupos tióis de proteínas de membrana promovendo oxidação e ligações cruzadas que conduzem à abertura do poro de transição de permeabilidade mitocondrial (TPM). A TPM em mamíferos caracteriza-se por uma permeabilização não específica e dependente de ea2+que ocorre na membrana mitocondrial interna e é inibida por concentrações submicromolares de ciclosporina A (CsA). No presente estudo foi evidenciado que apesar de mitocôndrias isoladas de tubérculo de batata (Solanum tuberosum) não apresentarem captação e acúmulo significativo de Ca2+, sofrem permeabilização da membrana interna quando tratadas com Ca2+(> 0,2 mM). Esta permeabilização foi potencializada pela adição de diamida, um oxidante de grupos tiólicos que estabelece uma condição de estresse oxidativo devido à oxidação de nucleotídeos de piridina. Esta permeabilização foi inibida pela acidificação do pH do meio de reação, Mg2+ e pelos antioxidantes catalase e ditiotreitol, mas não foi prevenida por inibidores clássicos da TPM de mamíferos como: ADP, bongkrekato e CsA. A ausência de inibição da permeabilização mitocondrial de batata pela CsA contrasta com seu efeito inibitório sobre a atividade da enzima peptidilprolil cis-trans isomerase, que está relacionada com a proteína ciclofilina, um sítio de ligação para CsA. O mersalil, um reagente tiólico monofuncional induz um extenso inchamento insensível à CsA, mesmo na presença de baixas concentrações de Ca2+(> 0,01 mM). Com o objetivo de realizar um estudo comparativo utilizando mitocôndrias vegetais capazes de captar e acumular Ca2+presente no meio extramitocondrial, foram utilizadas mitocôndrias isoladas de batata doce (Impomoea batatas), as quais são capazes de captar Ca2+por um mecanismo eletroforético sensível ao vermelho de rutênio, o que caracteriza a presença de um sistema ativo de captação de Ca2+.O acúmulo de altas concentrações de Ca2+ por mitocôndrias isoladas de batata doce conduziu a uma permeabilização de membrana mitocondrial interna, demonstrada por inchamento da organela e por um lento efluxo de Ca2+. Esta permeabilização induzida por Ca2+também foi estimulada por diamida e inibida parcialmente por catalase. Assim como ocorreu com Solanum tuberosum, o bongkrekato não apresentou efeito inibitório sobre a permeabilização mitocondrial induzida por Ca2+, bem como a CsA não foi capaz de inibir o inchamento mitocondrial e o e fluxo de Ca2+em mitocôndrias de batata doce. Também na batata doce a atividade da enzima peptidilprolil cis-trans isomerase de mitocôndrias foi inibida por CsA. Considerando os resultados aqui apresentados, podemos concluir que foi identificada e caracterizada uma condição de transição de permeabilidade de membrana induzida por Ca2+e espécies reativas de oxigênio, insensível à CsA, em mitocôndrias isoladas de batata e de batata doce. É possível sugerir ainda que a possível existência de um sistema ativo de transporte de Ca2+nestes vegetais não está relacionada com a insensibilidade da transição de permeabilidade destas mitocôndrias vegetais a CsA
Abstract: Oxidative damage of mammalian mitochondria induced by Ca2+ and prooxidants is mediated by the atlack of mitochondria-generated reactive oxygen species on membrane proteins thiols, promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (MPT). In mammalian mitochondria, 1
Doutorado
Ciencias Biomedicas
Doutor em Ciências Médicas

Orientadores: Maria Alice da Cruz-Hofling, Anibal Eugenio Vercesi
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A veratrina, uma mistura de alcalóides obtidos da espécie de planta Shoenocaulon officinale, é uma toxina lipossolúvel que possui como sítio de ação primária os canais de sódiodependentes de voltagem. Recentemente, nossos estudos mostraram que a veratrina pode causar mionecrose e as evidências sugerem que as mitocôndrias e o retículo sarcoplasmático (RS) possam ser os alvos intracelulares da ação da veratrina. O objetivo deste trabalho foi investigar os efeitos tóxicos induzidos por diferentes concentrações de veratrina sobre o consumo de oxigênio, a atividade dos complexos da cadeia respiratória e a ultraestrutura das mitocôndrias através de análises bioquímicas, citoquímicas e morfométricas e de microscopia eletrônica de transmissão...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: The veratrine, a mixture of alkaloids obtained from plant Schoenocaulon officinale, is a lipid soluble toxin, which target voltage-gated Na+ channels for their primary action site. Recently, our studies showed that the veratrine may cause myonecrosis and evidences suggested mitochondria and sarcoplasmic reticuIum (SR) as intracellular cell targets. The aim of this work was to investigate the effects caused by variabIe concentration of veratrine on mitochondrial oxygen consumption, respiratory chain enzymes activities and ultrastructure, combining electron microscopy with biochemical, cytochemical and morphometrical analysis. Moreover, we investigated whether the interference on physiology of sodium channels by veratrine it alters the ultrastructure of SR on different types of muscles. Ultrastructural changes were observed on skeletal muscle mitochondria after intramuscular injection or incubation with veratrine...Note: The complete abstract is available with the full electronic digital thesis or dissertations
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural

O presente estudo tem por objetivo principal avaliar as alterações funcionais precoces de mitocôndrias hepáticas de ratos wistar submetidos ao estímulo de sepse através da técnica de ligadura cecal e punção (cecal ligation and puncture-CLP) e indução de tolerância ao lipopolissacarídeo (LPS) de Escherichia coli. As mitocôndrias exercem papel na alteração do metabolismo celular de pacientes sépticos. Os objetivos do presente trabalho foram: (1) padronizar a técnica de indução a tolerância para ratos wistar com LPS de E. coli (2) avaliar a função mitocondrial fosforilativa e oxidativa; (3) quantificar DNA mitocondrial em tecido hepático de animais submetidos à CPL e tolerância; (4) verificar a expressão dos genes responsáveis pela biogênese mitocondrial e replicação do DNA mitocondrial: nuclear respiratory factor (NRF-1), mitochondrial transcription factor A (TFAM) e peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alfa); (5) avaliar a função dos complexos respiratórios I e IV. Os resultados encontrados no presente estudo revelaram que: (a) a taxa de mortalidade dos animais submetidos a tolerância foi de 10% quando submetidos à dose letal de LPS, enquanto a taxa de mortalidade dos animais controle foi de 100% quando submetidos à dose letal de LPS; (b) observou-se que o grupo do controle respiratório que recebeu doses controladas de LPS e foi submetido à CLP apresentou razão igual ao grupo Controle, sugerindo que a fosforilação oxidativa se manteve igual ao basal, enquanto o grupo que foi submetido ao procedimento de CLP sem indução a tolerância apresentou piora da razão do controle respiratório em relação ao grupo controle; (c) a quantificação de DNA mitocondrial mostrou-se maior nos animais submetidos a CLP sem prévia indução a tolerância, com igual aumento da expressão dos fatores de biogênese mitocondrial em relação aos demais grupos; (d) houve diferença significativa na avaliação da funcionalidade dos complexo I, porém o complexo IV se manteve igual em todos os grupos. Concluiu-se que a indução a tolerância altera positivamente a função mitocondrial em animais submetidos à CLP
The aim of this study was to evaluate the early functional alterations of hepatic mitochondria of wistar rats submitted to the stimulation of sepsis through the technique of cecal ligation and puncture (CLP) and induction of tolerance to lipopolysaccharide (LPS) of Escherichia coli. Mitochondria play a role in altering the cellular metabolism of septic patients. The objectives of the present study were: (1) to standardize the tolerance induction technique for wistar rats with E. coli LPS (2) to evaluate the mitochondrial phosphorylation and oxidative function; (3) quantify mitochondrial DNA in hepatic tissue of animals submitted to CPL and tolerance; (4) to verify the expression of genes responsible for mitochondria biogenesis and mitochondrial DNA replication nuclear mitochondrial biogenesis (NRF-1), mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alpha); (5) to evaluate the function of respiratory complexes I and IV. The results found in the present study revealed that: (a) the mortality rate of the animals submitted to tolerance was 10% when submitted to the lethal dose of LPS, whereas the mortality rate of the control animals was 100% when submitted to the lethal dose of LPS; (B) it was observed that the group receiving controlled doses of LPS and submitted to CLP presented a ratio equal to the control group, suggesting that oxidative phosphorylation remained the same at baseline, whereas the group that underwent CLP procedure without induction of tolerance presented worsening of the respiratory control ratio in relation to the control group; (C) the mitochondrial DNA quantification was higher in the animals submitted to CLP without prior tolerance induction, with an equal increase in mitochondrial biogenesis factors expression in relation to the other groups; (D) there was significant difference in the evaluation of the functionality of complexes I, but no difference in complex IV in all groups. It was concluded that induction of tolerance positively alters mitochondrial function in animals submitted to CLP

Homeostasis of the hematopoietic stem/progenitor cell pool relies on a finely tuned balance between self-renewal, differentiation and proliferation. Recent work has revealed the importance of mitochondria during stem cell differentiation; however, it remains unclear whether mitochondrial content/function affects human hematopoietic stem versus progenitor function. We sorted cord blood-derived CD34+ cells on the basis of mitochondrial mass and examined their homeostasis and clonogenic potential in vitro and hematopoietic repopulation potential in vivo. CD34+ cells with high mitochondrial mass contained and expressed 2-fold high ATP levels and mitochondrial-specific genes than cells with low mitochondrial mass, however, HIF-1α and MEIS1 were high in the CD34+ cells with low mitochondria. We found that CD34+ cells with low mitochondrial content were enriched for hematopoietic stem cell function as demonstrated by significantly higher hematopoietic reconstitution potential in immunodeficient mice. By contrast, CD34+ cells with high mitochondrial content were enriched for hematopoietic progenitor function with high in vitro clonogenic capacity. Coenzyme Q10 (CoQ10) plays a critical role in mitochondria as an electron carrier within the electron transport chain (ETC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high energy demand including brain and skeletal muscle (SkM). A four-year-old girl was identified with a heterozygous mutation (c.483G>C; E161D) in COQ4, associated with a reduction in [CoQ10], CoQ10 biosynthesis and ETC activity affecting complexes I/II+III. Bona fide induced pluripotent stem cell (iPSC) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically corrected using CRISPR/Cas9 genome-editing (CQ4ed-iPSCs). Comprehensive differentiation and metabolic analysis of control-iPSCs, CQ4-iPSCs and CQ4ed-iPSCs faithfully reproduced the disease phenotype. Accordingly, the COQ4 mutation in iPSCs was associated with CoQ10 deficiency, metabolic dysfunction and impaired differentiation into SkM. Remarkably, differentiation of CQ4-iPSCs into dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation.

TP53INP2 es una proteína nuclear que activa la autofagia en moscas y células eucariotas. El propósito de esta tesis doctoral fue investigar si TP53INP2 modula la autofagia mitocondrial dependiente de Parkin. Aquí, se demuestra que en un modelo de sobreexpresión de TP53INP2 se reduce la actividad mitofágica en respuesta al agente despolarizante CCCP o a los inhibidores de la cadena respiratoria Oligomicina/Antimicina A, en células que expresan Parkin. Además, este modelo causa modificaciones en el metabolismo mitocondrial. Así por ejemplo, la sobreexpresión de TP53INP2 produjo un incremento de la masa mitocondrial y una mayor susceptibilidad frente al estrés oxidativo mientras que la pérdida de función de TP53INP2 causó una reducción de la masa mitocondrial y del potencial de membrana mitocondrial. Asimismo, se pudo determinar que no hay cambios en la expresión de reguladores de la biogénesis mitocondrial (PGC-1α), esto sin duda refuerza la hipótesis de que el efecto de TP53INP2 sobre el metabolismo mitocondrial es debido a cambios en la mitofagia. Posteriormente, con el propósito de analizar los mecanismos por los cuales TP53INP2 disminuye la mitofagia dependiente de Parkin, se analizó la localización celular de la proteína. Sorprendentemente, se observó que ciertos estresores mitocondriales causan el reclutamiento de TP53INP2 a la mitocondria y este efecto es acelerado por Parkin. Prosiguiendo estos resultados en células que expresan Parkin, se descubrió que TP53INP2 inhibe la acumulación de PINK1 en condiciones en las cuales la membrana mitocondrial se encuentra despolarizada. Al mismo tiempo, se encontró una reducción en la estimulación de la fosforilación de la ubiquitina en el residuo S65. Por otra parte, se determinó que TP53INP2 colocaliza con Parkin en el núcleo en condiciones basales o en condiciones de daño mitocondrial. Esta aparente interacción no requiere PINK1 dado que células deficientes en esta proteína también mostraron una colocalización positiva en ambas situaciones. De igual forma, TP53INP2 interactúa físicamente con Parkin, esta interacción es mantenida con una forma corta de Parkin que carece del dominio UBL, la cual a diferencia de la forma larga no colocaliza con la mitocondria en condiciones de despolarización de la membrana mitocondrial. Sin embargo, la interacción fue abolida por una forma mutante de Parkin que carece de la actividad ubiquitina ligasa. En suma, en esta tesis se demuestra que TP53INP2 participa en el mantenimiento de la función e integridad mitocondrial a través de un mecanismo que implica la inhibición de PINK1 en la mitocondria dañada y por consiguiente la disminución de la mitofagia a través de la vía PINK1/Parkin. Los resultados de esta tesis indican que TP53INP2 representa una especie de interruptor que activa la autofagia general o no selectiva pero previene un tipo de autofagia selectiva, la mitofagia dependiente de PINK1/Parkin, en células de mamífero.
TP53INP2/DOR is a nuclear protein that shuttles to the cytosol and stimulates autophagosomal formation and autophagy activity in mammalian cells and in flies. In this study we explored whether TP53INP2 also modulates mitochondrial autophagy. To this end, we analyzed the effects of TP53INP2 on Parkin-dependent mitophagy. TP53INP2 gain-of-function markedly reduced mitophagic activity in response to different stresses in Parkin-expressing cells. In keeping with these observations, genetic manipulation of TP53INP2 caused modifications of mitochondrial metabolism. Thus, TP53INP2 gain-of-function caused enhanced mitochondrial mass and high susceptibility to oxidative stress whereas TP53INP2 loss-of-function reduced mitochondrial mass and mitochondrial membrane potential. Again, in support of an effect of TP53INP2 mediated by changes in mitophagy, TP53INP2-dependent changes in mitochondrial metabolism were not a consequence of alterations in regulators of mitochondrial biogenesis (PGC-1α). In order to analyze the mechanisms by which TP53INP2 attenuates Parkin-dependent mitophagy, we analyzed the cellular localization of the protein. Surprisingly, mitochondrial stressors caused the recruitment of TP53INP2 to the mitochondria and this was accelerated by Parkin. In keeping with the mitochondrial localization of the protein, TP53INP2 inhibited PINK1 accumulation following mitochondrial depolarization, and in parallel it reduced the stimulation of ubiquitin Ser65 phosphorylation, and protein ubiquitination upon mitochondrial damage in Parkin-expressing cells. TP53INP2 showed a surprising property that is to colocalize with Parkin in the nucleus under basal conditions or in mitochondria upon mitochondrial damage. The colocalization of Parkin and TP53INP2 does not require PINK1 (PINK1-deficient cells also show the colocalization under basal or upon mitochondrial damage). Furthermore, TP53INP2 physically interacts with Parkin. This interaction is maintained under conditions in which mitochondrial clustering does not form by mutant forms of Parkin lacking the UBL domain. Furthermore, the interaction was prevented by Parkin mutant forms lacking ubiquitin ligase activity. In all, we document that TP53INP2 participates in the maintenance of mitochondrial integrity and function through a mechanism that entails the inhibition of PINK1 in damaged mitochondria, and in consequence the attenuation of the PINK1/Parkin pathway of mitophagy. Thus, TP53INP2 represents a switch that activates general macroautophagy but prevents PINK1/Parkin-dependent mitophagy in mammalian cells.

La mitocondria es un orgánulo de las células eucariotas en el cual se desarrollan multitud de procesos esenciales para la célula de los cuales se destacan la cadena de transporte de electrones, la síntesis de ATP, el ciclo de Krebs, regulación de la osmosis del calcio, apoptosis, etc. La mitocondria posee su propio genoma (mtADN) y su conservación es esencial para la viabilidad de la mitocondria y por tanto de la célula. El mtADN se transcribe y replica de modo independiente a la división celular y posee su propia maquinaria de replicación y transcripción. En este trabajo se expone el estudio de dos proteínas implicadas en el metabolismo del mtADN con funciones muy diferentes. La primera es Twinkle, es una helicasa y participa en la replicación del ADN mitocondrial. La segunda es TFAM, es una proteína implicada en múltiples procesos del metabolismo del mtADN, tales como transcripción mediante la interacción en las secuencias LSP y HSP, compactación del genoma mitocondrial, unión de las secuencias Site-X y Site-Y, reconoce estructuras dañadas del ADN, etc. El estudio de ambas proteínas se desarrolló principalmente en el marco de la biología estructural empleando técnicas como cristalografía de proteínas, microscopía electrónica y dispersión de bajo ángulo de rayos-X (SAXS). Además, los datos de obtenidos con estas técnicas fueron complementados con técnicas bioquímicas como calorimetría de titulación isotérmica (ITC), barrido diferencial de fluorescencia (DSF), ensayos enzimáticos y estudios de interacción proteína-ADN mediante técnicas electroforéticas y dispersión dinámica de la luz (DLS). El complejo proteína-ADN TFAM/ Site-X fue cristalizado y su estructura tridimensional resuelta. TFAM se compone de dos dominios HMGbox1 y 2 que contactan al ADN por el surco menor y además cada unos de ellos provoca que se doble 90 º el ADN. Además están conectados entre sí por un “linker” que posee una estructura tipo hélice que también interacciona con el surco menor. Finalmente TFAM envuelve al ADN doblado formando una estructura tipo “U-turn”. Los estudios termodinámicos no mostraron diferencias importantes entre distintos complejos TAFM/ADN con respecto a TFAM/Site-X. Todos ellos presentaron en todos los casos el típico perfil endotérmico de asociado a proteínas de unión al surco menor, excepto el complejo TFAM/poli-dA cuyo ADN es rígido y mostró un perfil exotérmico. Mediante estudios de dinámica molecular se evaluó la rigidez del ADN y se realizó una simulación de su estructura en disolución. Estos resultados mostraron que TFAM reconoce un patrón estructural del ADN definido por una región rígida y pre-doblada a 10pb de una región flexible también pre-doblada en las cuales contactan HMGbox1 y 2, respectivamente. En el segundo proyecto, Twinkle se logró expresar de modo recombinante en E.coli a gran escala de un modo soluble. Se estudió biofísicamente mediante DSF y DLS, y bioquímicamente se estudió su actividad helicasa y su interacción con el ADN. Además gracias a los estudios por crio-microscopía y SAXS, y con la ayuda de un programa de modelaje por predicción de estructura por homología de secuencia, se realizó un modelo del hexámero por microscopía, del heptámero por SAXS. Además mediante ambas técnicas de observo la gran flexibilidad de la proteína, principalmente del dominio N-terminal que se componer por dos subdominios ZDB y RDP los cuales se unen entre ellos por un “linker” desordenado e además tienen una interacción tipo trans entre los subdominios ZBD y RPD del monómero adyacente. Twinkle tiene otro “linker” flexible entre el RDP el dominio helicasa C-terminal. El domino C-terminal forma una estructura rígida de anillo y es responsable de la oligomerización, tanto para formar hexámeros como heptámero. En disolución Twinkle está en equilibrio entre los dos estados oligoméricos: hexámero y heptámero.
In this thesis are described two projects with two different DNA binding proteins: TWINKLE and TFAM in complex with DNA (Site-X). Both protein are human proteins and are involved into DNA mitochondrial metabolism, Twinkle is involved in mitochondrial DNA replication and TFAM in the DNA transcription and mitochondrial genome packaging. Crystallographic structure of TFAM/Site-X complex was solved using a molecular replacement method. TFAM is composed by two HMGbox, each one of them bind and bend the DNA Site-X in one point, bending the DNA 90º, and adopting a “U-turn” shape. From a structural point of view, all TFAM/DNA complexes did not show great differences between them. The TFAM/DNA complexes were analyzed by Isothermal Titration calorimetry and showed the same endothermic profile, typical from DNA-binding protein which bend the DNA as a consequence of minor groove interactions, and also similar thermodynamic values. However, TFAM-poly-dA complex showed an exothermic profile. In addition, molecular dynamic technique was used to study the rigidity and simulated the structure of Site-X in an unbounded state. This result showed that TFAM recognize a prebending Site-X DNA, and HMGbox1 bends in a rigid region and HMGbox2 in a flexible region, but both are prebended. This result may suggest this is a general recognition for others DNA sequences. The second project, consisted on the study of a human mitochondrial helicase: Twinkle. The structural data from this protein was obtained with a low resolution techniques: electron microscopy (EM) and Small Angle X-ray Scattering (SAXS). Despite the big difference between these two techniques, both showed similar results. With both techniques was seen that Twinkle is in equilibrium between a hexamer and heptamer in solution. Several 3D structure models were generated for both oligomeric states. In these model the C-terminal domain is responsible of oligomerization in a ring shape, even six or seven units for hexamer and heptamer respectively. The N-terminal domain is divided in two subdomains called ZBD and RPD. These domains are responsible of the Twinkle flexibility, both are connected by a disorded linker, and also the subdomains RDP are connected to the ring domain through other disorded linker. This flexibility was observed by EM and SAXS

Orientador : Anibal Eugenio Vercesi
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Com o objetivo de acrescentar dados que pudessem ajudar a esclarecer algumas particularidades da fisiologia de mitocôndrias de plantas, foram investigadas algumas das características do funcionamento de mitocôndrias isoladas de milho, batata e beterraba. Numa primeira parte de deste trabalho foram estudados alguns aspectos relacionados ao transporte de 'Ca IND. 2+¿, em mitocôndrias isoladas de coleóptiles de milho. Foi possível observar que o fosfato é estequiometricamente acumulado com o 'Ca IND. 2+¿, mantendo uma razão 'Ca IND. 2+¿/Pi próxima a 1,5 e que a atividade do transportador é independente da idade dos coleóptiles utilizados ... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: The first part of this work was dedicated to some aspects related to 'Ca IND. 2+¿ transport by corn coleoptile mitochondria. It was observed that the activity of the 'Ca IND. 2+¿ carrier is independent of the coleoptile age and that phosphate is accumulated together with 'Ca IND. 2+¿ keeping a 'Ca IND. 2+¿/Pi ratio near to 1.5 ... Note: The complete abstract is available with the full electronic digital thesis or dissertations
Doutorado
Doutor em Ciências Biológicas

Orientador : Anibal Eugenio Vercesi
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Mitocôndrias acop1adas isoladas de folhas brancas de repolho (Brassica oleracea L. varo capitata) apresentaram-se incapazes e captar 'Ca POT. 2+¿ quando energizadas por respiração, em contraste com mitocôndrias isoladas de coleoptiles ou raízes de milho (Zea mays L.) que apresentaram a habilidade de captar 'Ca POT. 2+¿ do meio, embora com uma atividade muito menor do que mitocôndrias de fígado de rato. A curva de velocidade inicial de captação de 'Ca POT. 2+¿ ou ejeção de H+ por mitocôndria de coleoptiles de milho em função da concentração de 'Ca POT. 2+¿ mostrou ser sigmoidal e sugeriu uma estequiometria de 2H+/'Ca POT. 2+¿ como ocorrem em mitocôndrias animais. Na presença de baixas concentrações de 'Ca POT. 2+¿ livre no meio de incubação as mitocôndrias de coleoptiles ou raízes de milho foram capazes de captar o íon atingindo uma concentração de ¿steady state" na faixa de 2 'mu¿M e de manter este equilíbrio por vários minutos. Perturbação deste equilíbrio pela adição de 'Ca POT. 2+¿ ou EGTA foi seguida pela captação ou liberação de 'Ca POT. 2+¿, respectivamente, até que o equilíbrio fosse novamente atingido com a concentração livre de 'Ca POT. 2+¿ extramitocondria1 original. A concentração de 'Ca POT. 2+¿ extramitocondrial na qual o "steady state" era atingido mostrou ser dependente do conteúdo de 'Ca POT. 2+¿ intramitocondrial e da presença de 'Mg POT. 2+¿ no meio de reação. Os resultados indicam que mitocôndrias de coleoptiles ou raízes de milho, assim como mitocôndrias animais, têm a habilidade de tamponar o 'Ca POT. 2+¿ externo e talvez estejam envolvidas na manutenção a homeostase do 'Ca POT. 2+¿ na célula
Abstract: Coupled mitochondria isolated from the white leaves of cabbage (Brassica oleracea L. var. capitata) were inactivein respiration-coupled 'Ca POT. 2+¿ accumulation, in contrast to mitochondria isolated from etiolated corn (Zea mays L.) which showed the ability to take up 'Ca POT. 2+¿ from the medium, although with a much lower activity than liver mitochondria. Abstract: The rate of 'Ca POT. 2+¿ uptake or H+ ejection by corn mitochondria plotted versus 'Ca POT. 2+¿ concetration showed a sigmoidal curve and the H+/'Ca POT. 2+¿ ratio was very close to 2.0 as occurs in animal mitochondria. The addition of corn coleoptile or root mitochondria to a medium containing a low free 'Ca POT. 2+¿ concentration resulted in 'Ca POT. 2+¿ uptake with a decrease in free 'Ca POT. 2+¿ concentration until a steady state of about 2.0 'mu¿M was reached and maintained constant for several minutes. Perturbation of this steady state by the addition of 'Ca POT. 2+¿ or EGTA was followed by 'Ca POT. 2+¿ uptake or release, respectively, until the steady state was attained at the original extramitochondrial free 'Ca POT. 2+¿ concentratlon. The steay state free 'Ca POT. 2+¿ concentration was dependent of the intramitochondrial 'Ca POT. 2+¿ content and external 'Mg POT. 2+¿.These results indicate that corn but not cabbage mitochondria, as some animal mitochondria, have the ability to buffer external 'Ca POT. 2+¿ and may be involved in the maintenance of 'Ca POT. 2+¿ homeostasis in the cell
Doutorado
Doutor em Ciências Biológicas

OPA1 is a dynamin-related protein that is responsible for the fusion of the internal mitochondrial membrane and essential to control the morphology of the mitochondrial cristae. Thus, it directly impacts the efficiency of OxPhos and the stability of mitochondrial DNA. In this study, we have explored the effects of hepatic deletion of OPA1 on mitochondrial function and metabolism. We have shown that the ablation of OPA1 in the liver produces a mitochondrial dysfunction characterized by alterations in mitochondrial cristae structure, concomitant with a reduced respiratory capacity and mtDNA copy number, and perturbed mitochondrial proteostasis. The mitochondrial dysfunction caused by the ablation of OPA1 in liver triggers the activation of a mitochondrial stress response, including mitochondrial unfolded stress response (UPRmt) that is probably mediated by the transcription factor ATF5. Interestingly, we have observed that OPA1 deficiency in the liver causes better glucose tolerance and protects against diet-induced obesity and insulin resistance concomitant to a high circulating levels of the metabolic modulator FGF21. Here we suggest that the systemic protective effects associated to OPA1 ablation are due to the action of FGF21, probably mediated by the mitochondrial stress response associated to OPA1 loss- of-function via ATF5 activation.
OPA1 es una proteína relacionada con la dinamina que es responsable de la fusión de la membrana mitocondrial interna y esencial para controlar la morfología de las crestas mitocondriales, afectando directamente la eficiencia de OxPhos y la estabilidad del DNA mitocondrial. En este estudio, hemos explorado los efectos de la eliminación hepática de OPA1 sobre la función mitocondrial y el metabolismo. Hemos demostrado que la ablación de OPA1 en el hígado produce una disfunción mitocondrial caracterizada por alteraciones en la estructura de las crestas mitocondriales, concomitante con una capacidad respiratoria reducida y menor número de copias de DNA miocondrial, y perturbación en la proteostasis mitocondrial. La disfunción mitocondrial causada por la ablación de OPA1 en el hígado desencadena la activación de una respuesta al estrés mitocondrial, incluyendo la respuesta a las proteínas mal plegadas de la mitocondria, que probablemente es mediada por el factor de transcripción ATF5. Curiosamente, hemos observado que la deficiencia de OPA1 en el hígado causa una mejor tolerancia a la glucosa y protege contra la obesidad y resistencia a la insulina inducida por la dieta, en paralelo con un aumento de los niveles de FGF21 circulante, un factor involucrado en la modulación metabólica. Con este estudio proponemos que los efectos sistémicos protectores asociados a la ablación de OPA1 se deben a la acción de FGF21 que probablemente es mediada por la respuesta al estrés mitocondrial asociada a la pérdida de función de OPA1 a través de la activación de ATF5.

Las mitofusinas 1 y 2 son unas GTPasas que se encuentran en la membrana mitocondrial externa, donde llevan a cabo el proceso de fusión mitocondrial. La existencia de mutaciones en Mfn2 es la principal causa de la enfermedad de Charcot- Marie-Tooth tipo 2A, un grupo de distintas neuropatías caracterizadas por degeneración axonal. Por otro lado, otras alteraciones en Mfn2 también han sido descritas en múltiples enfermedades neurodegenerativas crónicas, en episodios traumáticos agudos como accidentes cerebrovasculares e incluso otras enfermedades no neurológicas como las cardiometabólicas. Un gran número de estudios han demostrado que Mfn2 está relacionada con la regulación del metabolismo y la bioenergética mitocondrial, ya que su supresión afecta negativamente ambos procesos y su sobreexpresión los estimula. Dado el papel del metabolismo mitocondrial en la fisiopatología de estas enfermedades, entender el mecanismo por el que Mfn2 regula la bioenergética mitocondrial resulta de gran importancia. En esta tesis se demostró que la activación de la bioenergética mitocondrial por parte de Mfn2 requiere su localización en el retículo endoplasmático (RE) y es independiente a su función como proteína de fusión mitocondrial. A diferencia de Mfn1, Mfn2 se encuentra presente en el RE, concretamente en las membranas asociadas con las mitocondrias. Los contactos entre ambos orgánulos son de vital importancia en las células, ya que, entre otras funciones esenciales, crean micro-dominios que permiten la transferencia de Ca2+ del RE hacia las mitocondrias. Por su parte, el Ca2+ captado por las mitocondrias estimula diferentes enzimas del metabolismo mitocondrial y complejos encargados de mantener la bioenergética mitocondrial. En esta tesis, se observó que células carentes de Mfn2 también manifiestan defectos en la homeostasis del Ca2+ y en el mantenimiento de los contactos RE-mitocondrias. Mediante el uso de un enlazador artificial que mimetiza la función enlazadora de Mfn2 o diferentes formas de Mfn2 dirigidas a ambos orgánulos, se determinó que Mfn2 actúa como complejo enlazador de RE y mitocondrias, propiciando la formación de contactos óptimos para la transferencia de Ca2+ del RE a las mitocondrias. A su vez, el restablecimiento de la captación de Ca2+ por las mitocondrias gracias a la función enlazadora de Mfn2 fue capaz de estimular la bioenergética mitocondrial, definiendo de este modo un mecanismo por el cual Mfn2 regula la bioenergética mitocondrial a través de los contactos RE-mitocondria. La relevancia fisiológica de estos resultados se mostró en dos procesos neuronales. Por un lado, la falta de Mfn2 provocó alteraciones en el crecimiento neurítico y la formación de espinas dendríticas durante el periodo de desarrollo neuronal en modelos in vivo de ratón knockout para Mfn2. Estas alteraciones, fueron rescatadas in vitro mediante el enlazador artificial de RE-mitocondrias. Usando distintas formas de Mfn2 dirigidas específicamente a RE o mitocondria, se determinó que el mecanismo por el cual Mfn2 regula la bioenergética a través de los contactos RE-mitocondrias podría ser esencial durante el desarrollo neuronal para un correcto crecimiento y ramificación de sus neuritas. Por otro lado, el restablecimiento de los contactos RE-mitocondria protegió contra la excitotoxicidad. Mfn2 destaca también por su capacidad neuroprotectora frente diferentes tipos de lesiones como daño al ADN, estrés oxidativo o privación de iones K+. Frente a una sobreactivación persistente de los receptores de NMDA, acontece un aumento continuo y acusado de Ca2+ intracelular que desencadena en muerte neuronal. Durante este proceso, los niveles de Mfn2 decrecen, hecho que correlaciona con una mayor predisposición neuronal a la apoptosis. En este estudio, se determinó que la función enlazadora de Mfn2 podría estar relacionada con el reclutamiento de Bax hacia las mitocondrias, hecho típicamente definitorio de un proceso apoptótico. De este modo, se propone este mecanismo como una potencial diana terapéutica en procesos patológicos como los accidentes cerebrovasculares isquémicos, en los cuales aparecen eventos excitotóxicos y los niveles de Mfn2 se ven reducidos.

Sporadic inclusion body myositis (sIBM) is the most common myopathy in elderly. This disease causes muscle wasting with both distal and proximal affectation. Quadriceps and finger flexors are muscle typically affected. At pathological level, three different features are present in muscle biopsies: inflammation, mitochondrial abnormalities and degeneration. The presence of T-cell infiltrate, ragged-red fibers and rimmed vacuoles are some features linked to these pathological processes. Protein misfolding and aggregation lead to the formation of the mentioned rimmed vacuoles, composed by many different misfolded proteins: β-amyloid, caveolin, phosphor-Tau among others. The accumulation of β-amyloid is also a hallmark of Alzheimer’s disease (AD), which presents some parallelisms with sIBM. In this work, we aimed to evaluate the mitochondrial state in muscle from sIBM patients but also in peripheral blood mononuclear cells (PBMC) to describe the molecular abnormalities that mitochondria could present in this disease. In addition, we aimed to measure plasmatic molecules related to inflammation, mitochondria and degeneration, in search for plasmatic biomarkers in sIBM patients but also in dermatomyositis and polymyositis, both diseases from the inflammatory myopathy group, like sIBM. Regarding mitochondrial analysis, muscle biopsies from 23 sIBM patients were analyzed, as well as 18 controls free of muscle disease. In addition, PBMC from 14 sIBM patients and from 20 controls were assessed as well. MtDNA levels and also mitochondrial respiratory chain complex IV (COX) activity were significantly decreased in muscle from sIBM patients compared to controls. Interestingly, when analyzing PBMC, dysfunction in COX activity was also found. In this tissue, a deregulation in mitochondrial protein synthesis was also found. As 57% of the sIBM patients presented mtDNA deletions, we aimed to evaluate if the presence of mtDNA deletions correlate with impaired mitochondrial parameters. sIBM patients with mtDNA deletions presented the lowest amount of mtDNA, and those patients without deletions showed values more similar to controls. A similar pattern was found when correlating the presence of MFN-2, a protein involved in mitochondrial dynamics. Again, patients with mtDNA deletions presented the lowest amount of this protein, and patients without deletions showed an intermediate values between patients with deletions and controls. Regarding the analysis of plasmatic molecules related to sIBM pathological features, inflammatory, mitochondrial and amyloidogenic molecules were analyzed in plasma samples from 21 sIBM, 20 controls and also in 14 plasma samples from dermatomyositis (DM) and polymyositis (PM) patients, which constitute an inflammatory myopathy different from sIBM group. Inflammatory (IL-6 and TNF-α) and mitochondrial-related (circulating mtDNA, FGF-21 and CoQ) molecules did not show significant differences between groups. However, amyloidogenic molecules (BACE-1, PS-1 and sAPPβ) were increased in sIBM patients respect to controls but also respect to the DM and PM group confirming its implication in sIBM pathogenesis. Sensitivity and specificity test showed that BACE-1 would be the most suitable biomarker for sIBM diagnosis. This thesis describes at molecular level the mitochondrial implication in the disease, and also reinforces the amyloidogenic component in sIBM. In addition, it proposes a plasmatic and non-invasive biomarker that could help in the sIBM diagnosis, especially in discriminating between other inflammatory myopathies, like polymyositis.
La miositis per cossos d’inclusió en la seva forma esporàdica (MCI) és la miopatia més comú en individus de més de 50 anys tot i ser una malaltia rara. Cursa amb atròfia muscular progressiva distal i proximal i actualment no es coneix cura. A nivell histopatològic presenta un component inflamatori, un component mitocondrial i un component degeneratiu. Degut al seu component degeneratiu i a la similitud de les proteïnes que formen aquests cossos d’inclusió, s’ha establert un possible paral·lelisme amb la malaltia d’Alzheimer. Els objectius d’aquesta tesi doctoral són explorar a nivell molecular les alteracions mitocondriales en la MCI en múscul, però també en cèl·lules mononuclears de sang perifèrica (CMSP), ja que és un teixit menys invasiu. A més, com a segon objectiu principal pretén d’estudiar mol·lècules relacionades amb la inflamació, amb el mitocondri i amb la degeneració en plasma d’aquests pacients per tal de demostrar la seva implicación amb la etiopatogènia i a més per establir nous marcadors menys invasius que permetin diagnosticar la malaltia i diferenciarla d’altres malalties similars com la dermatomiositis i la polimiositis. Fent referència a l’estudi mitocondrial, tant la quantitat de DNA mitocondrial com l’activitat del complex IV de la cadena mitocondrial (COX) es van trobar disminuïts en músculs dels pacients amb MCI. D’altra banda, amb l’estudi de les CMSP, també vam trobar disminuïda l’activitat de la COX, i a més una desregulación de la síntesis de proteïnes mitocondrials. Donat que un 57% dels pacients va presentar delecions múltiples al DNA mitocondrial, la presència d’aquestes delecions correlacionava amb una menor quantitat de DNA mitocondrial i a més amb un decrement de proteïna MFN-2, implicada en la dinàmica mitocondrial. Amb l’estudi de les molècul·les plasmàtiques, es van analitzar en plasma de pacients amb MCI, en controls però també en pacients amb dermatomiositis i polimiositis mol·lècules relacionades amb la inflamació (IL-6 i TNF-α), amb el mitocondri (DNA mitocondrial circulant, FGF-21 i enzim CoQ) i amb la amiloidogènesi (BACE-1, PS-1 i sAPPβ). Les mol.lècules amiloidogèniques es trobaven incrementades en els pacients amb MCI respecte controls i altres miopatíes inflamatòries, demostrant la seva impliació en la etiopatogènia i obtenint un cert valor diagnòstic. Amb aquesta tesi, s’ha demostrat la implicació mitocondrial en la etiopatogènia de la MCI, i s’han trobat alteracions en plasma de mol·lècules amiloidogèniques que, a més, tenen potencial diagnòstic per diferenciar aquesta malaltia d’altres miopatíes inflamatòries com la polimiositis.

The mitochondrial Seryl-tRNA Synthetase (SerRS2) is a member of the class II tRNA synthetase family. The mature enzyme catalyses the ligation of serine to tRNASer in mitochondria. During the process of constructing a model for human disorders caused by mitochondrial tRNA aminoacylation deficiencies in Drosophila melanogaster, a previously uncharacterized paralogue of SerRS2 named Seryl-tRNA synthetase-Like Insect Mitochondrial Protein (SLIMP) was identified. SLIMP is a new type of aminoacyl tRNA synthetase-like protein that has acquired an essential function in insects. This fast evolving paralogue is a mitochondrial RNA-binding protein which lacks tRNA aminoacylation activity. It has been previously demonstrated that mitochondrial SLIMP interacts with its homologue SerRS2 and also with LON protease. We confirmed these interactions and we described the function of SLIMP by depleting its protein levels in Drosophila melanogaster S2 cells, which led to severe defects in mitochondrial function and cell cycle arrest. We found that SLIMP simultaneously acts as a regulator of DNA replication and translation in the mitochondria and, as regulator of cell cycle progression. We show that SLIMP activates mitochondrial protein synthesis through its interaction with SerRS2 and regulates mitochondrial DNA levels by stimulating TFAM digestion by the protease LON. SLIMP was previously reported to be required for correct cell cycle progression. We showed that the depletion of a non-mitochondrial pool of SLIMP causes cell cycle arrest in G2 and the activation of E2F-related and G2/M check-point genes. Our results indicate that SLIMP activity provides an important protein for the communication between mitochondrial anabolism and cell cycle regulation.
La Seril-ARNt Sintetasa mitocondrial (SerRS2) és membre de la família de ARNt sintetases de classe II. Aquest enzim es responsable de la lligació de l’aminoàcid serina al corresponent ARNtSer a la mitocòndria. En el procés de desenvolupament d’un model de malalties mitocondrials causades per deficiències en l’aminoacilació de ARNt en Drosophila melanogaster, es descobrí una proteïna paràleg de SerRS2 no caracteritzada fins el moment, anomenada Seril-ARNt sintetasa-Like Insect Mitochondrial Protein (SLIMP). SLIMP representa una nova classe de proteïna semblant a les ARNt sintetases que ha adquirit funcions essencials en insectes. Aquest paràleg ha evolucionat en poc temps i constitueix una proteïna amb unió a ARN però sense capacitat d’aminoacilació. Prèviament s’havia caracteritzat que SLIMP interacciona amb el seu homòleg SerRS2 i també amb la proteasa mitocondrial LON. Ara hem confirmat aquestes interaccions i hem descrit les funcions de SLIMP, caracteritzant l’efecte de la depleció dels seus nivells proteics en cèl·lules S2 de Drosophila melanogaster, que comportà severs defectes mitocondrials i un arrest del cicle cel·lular. Hem definit que SLIMP actua simultàniament com un regulador de la replicació del ADN i la traducció a la mitocòndria i, com a regulador de la progressió del cicle cel·lular. SLIMP activa la síntesis proteica mitocondrial gràcies a la interacció amb SerRS2, i a més regula els nivells de ADN mitocondrial, estimulant la degradació de TFAM per la proteasa LON. Anteriorment, es descrigué que SLIMP és necessari per la correcta progressió del cicle cel·lular. Hem trobat que la depleció d’una isoforma no mitocondrial de SLIMP arresta el cicle cel·lular en la fase G2 i activa la transcripció de gens relacionats amb E2F i amb el punt de control de G2/M. Aquests resultats indiquen la important tasca de SLIMP per la comunicació entre l’anabolisme mitocondrial i la regulació del cicle cel·lular.

Genetically tractable models such as Drosophila melanogaster may help to identify new approaches to halt malignant growth. During my doctoral thesis, I performed a screen to find mitochondrial genes required for the growth of lethal (3) malignant brain tumor. I discovered that several testis-mitochondrial genes, i.e. genes with a mitochondrial function and a maximum expression in testes, were required for the growth of l(3)mbt tumors, rescuing both viability and brain anatomy traits. Nevertheless, inhibition of testis-mitochondrial genes did not affect either wild-type or brat tumor development. One of these testis-mitochondrial genes, ttm2, was found to be expressed both in wild-type and l(3)mbt brains. In addition, when overexpressed in an otherwise wild-type background, Ttm2 produces hyperplasia specifically in the neuroepithelial cells of the medial outer proliferative center (OPC). Yet, ectopic expression of Ttm2 in either central brain or medulla neuroblasts does not affect neuroblast division or number, suggesting that the brain cell types are differentially sensitive to mitochondrial function alteration. The data presented in this thesis project provides novel information about the critical role of mitochondria controlling both cell fate and carcinogenesis.
Durante los últimos años, el uso de Drosophila melanogaster como organismo modelo ha servido para identificar nuevos mecanismos moleculares implicados en el crecimiento tumoral. Durante mi tesis doctoral, descubrí que varios genes mitocondriales son necesarios para el crecimiento del tumor cerebral causado por la falta de función del gen lethal (3) malignant brain tumor (l(3)mbt). En concreto, varios genes mitocondriales testiculares, es decir, genes con función mitocondrial y máxima expresión en el testículo de machos adultos, son imprescindibles para el desarrollo de tumores l(3)mbt. Sin embargo, la función de estos genes mitocondriales testiculares es prescindible tanto para el desarrollo de cerebros “wild-type” como del tumor cerebral brat. Uno de estos genes mitocondriales testiculares, ttm2, se expresa tanto en cerebros normales como en cerebros tumorales l(3)mbt. Además, cuando ttm2 es sobreexpresado en el neuroepitelio del cerebro larvario, produce hiperplasia. Esta hiperplasia es específica de las células mediales del “outer proliferative center” (OPC), sin afectar ni al “inner proliferative center” (IPC) ni a los neuroblastos del cerebro. Estos resultados sugieren que los diferentes tipos celulares del cerebro de la larva de Drosophila tienen requerimientos metabólicos distintos y no reaccionan de igual manera a la alteración de la función mitocondrial. Los resultados presentados en esta tesis doctoral proporcionan nuevos datos que confirman el papel crítico de la mitocondria tanto en el proceso de carcinogénesis como en el mantenimiento de la identidad celular.

Tolueno y xileno son compuestos químicos presentes en varios solventes y otros productos industriales y de laboratorio; su toxicidad para el sistema nervioso central y el hígado ha sido bien documentada. En el presente trabajo, se han estudiado los efectos in vitro de tolueno y de xileno sobre la respiración de mitocondrias aisladas de hígado de rata energizadas con succinato evaluada por medición del consumo de oxígeno, el potencial de membrana usando safranina O como indicador, la liberación de Ca2+ usando Calcium Green 5N, la producción de especies reactivas de oxígeno (EROs) con ácido homovanílico, y los cambios en el nivel de ATP utilizando el sistema luciferina-luciferasa. El hinchamiento mitocondrial, dependiente de Ca2+, sensible a ciclosporina A, un indicador de transición de permeabilidad de la membrana (TPM) mitocondrial, fue monitoreado con la medición de la disminución de la absorbancia aparente a 540 nm. Tolueno y xileno, a concentraciones 0.5-2.5 y 0.25-1 mM, respectivamente, estimularon la respiración de estado 4 en asociación aparente con la disipación del potencial de membrana y la liberación de Ca2+; estos efectos de ambos solventes indican desacoplamiento mitocondrial. A concentraciones mayores (2.5 y 5 mM, respectivamente), tolueno y xileno también inhibieron el estado 3 de respiración. A concentraciones 0.1-1 mM, xileno ocasionó una producción significante de EROs y un hinchamiento mitocondrial parcialmente dependiente de Ca2+ y parcialmente sensible a ciclosporina A. A una concentración 1mM, tolueno o xileno causaron depleciones del ATP mitocondrial hasta niveles del 66.3% y 40.3%, respectivamente; las depleciones fueron sólo ligeramente dependientes de Ca2+. Se concluyó que el desacoplamiento mitocondrial causante de la depleción de ATP puede ser responsable de la toxicidad celular de tolueno y en particular, de xileno, descrita por otros investigadores. En el último caso, parecen también estar involucrados la TPM y la generación de EROs. Palabras Clave: Tolueno, Xileno, Mitocondria, Desacoplamiento, Especies Reactivas de Oxígeno.
--- Toluene and xylene are chemicals present in various solvents and other industrial and laboratory products; their toxicity to the nervous system and to the liver has been well documented. In the present work, we have studied in vitro effects of toluene and xylene on the respiration of succinate-energized isolated rat liver mitochondria, evaluated by measuring oxygen consumption, membrane potential using safranine O as indicator, Ca2+ release using calcium green 5N, reactive oxygen species (ROS) by homovanillic acid, and ATP level changes using the luciferin-luciferase system. Ca2+-dependent, cyclosporine A-sensitive mitochondrial swelling, an indicator of mitochondrial permeability transition (MPT), was followed by measuring the decrease of apparent absorbance at 540 nm. At 0.5-2.5 and 0.25-1 mM concentrations respectively, toluene and xylene stimulated state 4 respiration in apparent association with mitochondrial membrane potential dissipation and Ca2+ release; these effects of both solvents indicate mitochondrial uncoupling. At higher concentrations (2.5 and 5 mM respectively) toluene and xylene also inhibited state 3 respiration. At 0.1-1 mM concentrations, xylene elicited significant ROS generation and partly Ca2+-dependent and partly cyclosporine A-sensitive mitochondrial swelling. At 1 mM concentration, toluene or xylene caused depletions of mitochondrial ATP, amounting to respectively 66.3% and 40.3%; depletions were only slightly dependent on Ca2+. It was concluded that mitochondrial uncoupling via ATP depletion migth be responsible for the earlier described cell toxicity of toluene and in particular, of xylene. In the latter case, mitochondrial generated ROS and MPT also appear to be involved. Key Words: Toluene; Xylene; Mitochondria; Uncoupling; Reactive oxygen species.
Tesis

One of the most fundamental events in the history of life is the origin of mitochondria. It was firstly proposed by Ivan Wallin, and later popularized by Lynn Margulis, as part of the endosymbiotic theory, that mitochondria initially arose from free-living bacteria that invaded eukaryotic cells (Wallin, 1922; Sagan, 1967). As a consequence, a mutually beneficial relationship was formed, where the eukaryote delivered protection and nutrients to the prokaryote and, in return, the prokaryote provided additional energy to its host by encoding the gene products essential for the energy-generating process known as oxidative phosphorylation (OXPHOS). However, as this relationship became permanent over time, OXPHOS became indispensable to its host and the mitochondrion lost its autonomy, causing many of the mitochondrial genes to be transferred to the nuclear genome. The mammalian mitochondria has retained only a small subset of 37 genes in the form of a circular DNA molecule with a size of approximately 16.6 kb, including 13 essential subunits of the electron transport chain (ETC) (Anderson et al, 1981). Thus, these mitochondrial DNA (mtDNA)-encoded genes rely on nuclear encoded proteins for their transcription, processing and translation. Defects in the production or stabilization of these mtDNA-encoded subunits can lead to OXPHOS dysfunction, contributing to diverse types of human diseases, including different types of cancer, cardiomyopathies and neurodegenerative conditions (Chandra and Singh, 2011; Breuer et al, 2013). Therefore, it has become of vital importance to understand and explore the regulatory mechanisms involved in controlling mitochondrial function. The identification and study of novel proteins that might enable particular mitochondrial functions will allow for the identification of new candidate genes for the molecular diagnosis of mitochondrial disorders. Exonuclease 3’-5’ domain containing 2 (EXD2) is a nuclear encoded gene that has been described to promote homologous recombination by facilitating DNA end resection (Broderick et al, 2016). Here we report that EXD2 is targeted to the mitochondria and it’s critical for normal metabolism. We found that EXD2 interacts with Complex I of the ETC and the mitoribosome and its depletion impairs mitochondrial translation, leading to a defective OXPHOS, accumulation of ROS and reduced ATP production. In Drosophila melanogaster, we observed that EXD2 deficiency leads to premature stem cell attrition in the female germline and an extension in lifespan that can be rescued by an antioxidant diet. Together, our results define EXD2 as a mitochondrial regulator of translation and OXPHOS activity, that is required for germline stem cell maintenance and suggest that it could be a candidate gene for human metabolic disorders.
Las mitocondrias son orgánulos celulares con una función bioenergética, biosintética y de señalización. A pesar de tener su propio genoma, las mitocondrias requieren genes codificados en el núcleo para su correcto funcionamiento. Nuestro trabajo se centra en el estudio de una proteína no caracterizada llamada Exonuclease 3’-5’ domain like 2 (EXD2), codificada por un gen nuclear que ha sido previamente asociado con la recombinación y reparación del daño en el DNA. Curiosamente, observamos que EXD2 localiza en la mitocondria y que su reducción en los niveles de expresión resulta en un consumo de oxígeno más bajo, defectos en el metabolismo y mtDNA reducido. EXD2 no parece asociarse con proteínas de replicación conocidas y su reducción no afecta la replicación del mtDNA. En cambio, encontramos que EXD2 interacciona con diferentes subunidades del Complejo I de la cadena de transporte electrónico y el mitoribosoma y que su reducción afecta la traducción mitocondrial causando anormalidades metabólicas. Un mal funcionamiento de EXD2 en Drosophila conlleva a defectos en el desarrollo, un desgaste prematuro de las células madre germinales (GSCs) y problemas de fertilidad, los cuales son acompañados de un incremento significante de esperanza de vida. Todos los fenotipos pueden ser revertidos con una dieta anti- oxidante, apoyando la idea de que la reducción de EXD2 genera estrés oxidativo. Finalmente, observamos que una deficiencia en EXD2 afecta al crecimiento de tumores de mama en xenoinjertos, posiblemente por la rendición de células dependientes de glutamina o sensibles a hipoxia. Nuestra hipótesis es que EXD2 afecta a la traducción mitocondrial, y su inhibición resulta en una deficiencia en la fosforilación oxidativa y una acumulación de especies reactivas de oxígeno (ROS). Proponemos que las mutaciones en EXD2 podrían subyacer a una enfermedad metabólica sin diagnosticar y ello podría tener una aplicación como biomarcador o diana en cáncer humano.

Mitochondria are well known for their role as bioenergetic and biosynthetic organelles. Recently, they also have emerged as one of the main regulators of innate immune responses, mostly for its ability to modulate several signaling pathways through mechanisms such ROS production. Mitofusin-2 (Mfn2) is a GTPase located in the external mitochondrial and ER membranes. It is responsible for the fusion between mitochondria and the ER-mitochondria contacts, both necessary for the correct functioning of both organelles. Even that, the role of Mfn2 in immune responses. Here we demonstrate that Mfn2 is crucial for the pro-inflammatory activation of macrophages. Murine Mfn2-/- macrophages show a disruption in the mitochondrial network morphology that leads to loss in the mitochondrial membrane potential and in mitochondrial respiration. These two defects in the mitochondrial function do not affect their ability to normally generate ATP. However, the production of ROS in the mitochondria from Mfn2-/- macrophages is markedly decreased. This defect in ROS generation leads to a severe dysfunction in macrophage responses, particularly in the inflammatory activation, phagocytosis, and the processing of proteins. First, the decrease in ROS in Mfn2-/- macrophages results in a defective activation of p38, ERK, and NF-kB signaling pathways in response to LPS. The decrease in these signaling cascades leads to a reduction in the production of pro- inflammatory cytokines, severely impairing their ability to undergo pro- inflammatory activation. Second, in addition to show reduced ROS levels, Mfn2-/- also show an accumulation of autophagosomes due to a Mfn2-dependent defect in the autophagosome-lysosome fusion. As a result to both increased autophagy and decreased ROS levels, Mfn2-/- macrophages show decreased expression of type- A scavenger receptors. We demonstrate that these alterations lead to a widespread defect in the phagocytic capabilities of Mfn2-/- macrophages, showing defective phagocytosis of bacteria (both gram positive and gram negative) and apoptotic bodies. Thirdly, Mfn2-/- macrophages also show a defect in the bactericidal activity of phagocyted bacteria, as well as a defective proteolysis, being unable to process antigens to present them to CD4+ cells in a MHC-II context, and therefore, potentially impairing their ability to initiate adaptive immune responses. Finally, we demonstrated that Mfn2 is relevant in in vivo models of inflammation or infection. Myeloid-conditional Mfn2-/- mice were infected with either Listeria monocytogenes or Mycobacterium tuberculosis. In both models, Mfn2-/- mice showed a severe decrease in their survival, when compared to their WT counterparts. Furthermore, the colony counts in selected organs (spleen and liver for listeria, spleen and lung for tuberculosis) was significantly increased in Mfn2-/- mice, indicating that Mfn2 in macrophages is required to effectively control bacterial infections. In addition, we performed a model of sterile inflammation using the irritant DNFB on the mice’s ear. Confirming the in vitro results, Mfn2-/- mice show decreased inflammation in the ear, as confirmed by the decrease in size and weight, and the reduced expression of inflammatory cytokines. All these findings suggest that Mfn2 is a crucial regulator of macrophage pro- inflammatory responses, including production of pro-inflammatory cytokines, phagocytosis, and antigen presentation, through modulation of mitochondrial ROS production, autophagy, and protein processing.
Apart del seu rol en la regulació del metabolisme, és cada cop més acceptat els mitocondris són un dels principals controladors de les respostes immunes. En aquesta tesi ens centrem en l’estudi de Mitofusina 2 (Mfn2), una GTPasa que es troba a la membrana mitocondrial externa i que promou la fusió entre mitocondris. Degut a que Mfn2 controla aspectes clau de la fisiologia mitocondrial com la respiració, la producció de ROS i l’apoptosi, tots ells intrínsecament lligats en el funcionament de les respostes immunes, decidim estudiar el paper que juga aquesta proteïna en els macròfags, cèl·lules clau en la inflamació. Els macròfags Mfn2-/- presenten una xarxa mitocondrial completament fragmentada, així com una pèrdua del potencial de membrana mitocondrial i una disminució de la respiració. Això fa que les mitocòndries d’aquestes cèl·lules, tot i generar ATP de forma normal, siguin incapaces de produir nivells fisiològics de ROS. Aquesta disminució de ROS provoca que vies de senyalització com ERK, p38 i NF-κB no puguin ser activades per LPS, portant els macròfags Mfn2-/- a ser incapaços de sintetitzar citocines inflamatòries com el TNF-α o la IL-1β, i per tant de generar una resposta inflamatòria eficient. Per altra banda, la deficiència de Mfn2 provoca una acumulació d’autofagosomes en els macròfags. Això genera per una banda un increment de l’apoptosi en aquestes cèl·lules, i per altra un defecte en la seva capacitat fagocítica. Tot això a més és combina amb un defecte en la degradació de bacteris fagocitats i el processament de proteïnes necessari per la presentació antigènic, probablement degut també a la falta de ROS. Finalment demostrem que Mfn2 és crucial en les respostes inflamatòries usant 3 models in vivo. En dos d’ells mostrem com els macròfags necessiten Mfn2 per controlar les infeccions de listèria i tuberculosis, mentre que el l’últim demostrem que Mfn2 també és necessària en la inflamació asèptica. Per concloure, en aquesta tesi demostrem que la Mfn2 és crucial per l’activació pro- inflamatòria del macròfag, afectant tres processos clau: la producció de citocines inflamatòries, la fagocitosi i la presentació antigènica.

Tolueno y xileno son compuestos químicos presentes en varios solventes y otros productos industriales y de laboratorio; su toxicidad para el sistema nervioso central y el hígado ha sido bien documentada. En el presente trabajo, se han estudiado los efectos in vitro de tolueno y de xileno sobre la respiración de mitocondrias aisladas de hígado de rata energizadas con succinato evaluada por medición del consumo de oxígeno, el potencial de membrana usando safranina O como indicador, la liberación de Ca2+ usando Calcium Green 5N, la producción de especies reactivas de oxígeno (EROs) con ácido homovanílico, y los cambios en el nivel de ATP utilizando el sistema luciferina-luciferasa. El hinchamiento mitocondrial, dependiente de Ca2+, sensible a ciclosporina A, un indicador de transición de permeabilidad de la membrana (TPM) mitocondrial, fue monitoreado con la medición de la disminución de la absorbancia aparente a 540 nm. Tolueno y xileno, a concentraciones 0.5-2.5 y 0.25-1 mM, respectivamente, estimularon la respiración de estado 4 en asociación aparente con la disipación del potencial de membrana y la liberación de Ca2+; estos efectos de ambos solventes indican desacoplamiento mitocondrial. A concentraciones mayores (2.5 y 5 mM, respectivamente), tolueno y xileno también inhibieron el estado 3 de respiración. A concentraciones 0.1-1 mM, xileno ocasionó una producción significante de EROs y un hinchamiento mitocondrial parcialmente dependiente de Ca2+ y parcialmente sensible a ciclosporina A. A una concentración 1mM, tolueno o xileno causaron depleciones del ATP mitocondrial hasta niveles del 66.3% y 40.3%, respectivamente; las depleciones fueron sólo ligeramente dependientes de Ca2+. Se concluyó que el desacoplamiento mitocondrial causante de la depleción de ATP puede ser responsable de la toxicidad celular de tolueno y en particular, de xileno, descrita por otros investigadores. En el último caso, parecen también estar involucrados la TPM y la generación de EROs. Palabras Clave: Tolueno, Xileno, Mitocondria, Desacoplamiento, Especies Reactivas de Oxígeno.
--- Toluene and xylene are chemicals present in various solvents and other industrial and laboratory products; their toxicity to the nervous system and to the liver has been well documented. In the present work, we have studied in vitro effects of toluene and xylene on the respiration of succinate-energized isolated rat liver mitochondria, evaluated by measuring oxygen consumption, membrane potential using safranine O as indicator, Ca2+ release using calcium green 5N, reactive oxygen species (ROS) by homovanillic acid, and ATP level changes using the luciferin-luciferase system. Ca2+-dependent, cyclosporine A-sensitive mitochondrial swelling, an indicator of mitochondrial permeability transition (MPT), was followed by measuring the decrease of apparent absorbance at 540 nm. At 0.5-2.5 and 0.25-1 mM concentrations respectively, toluene and xylene stimulated state 4 respiration in apparent association with mitochondrial membrane potential dissipation and Ca2+ release; these effects of both solvents indicate mitochondrial uncoupling. At higher concentrations (2.5 and 5 mM respectively) toluene and xylene also inhibited state 3 respiration. At 0.1-1 mM concentrations, xylene elicited significant ROS generation and partly Ca2+-dependent and partly cyclosporine A-sensitive mitochondrial swelling. At 1 mM concentration, toluene or xylene caused depletions of mitochondrial ATP, amounting to respectively 66.3% and 40.3%; depletions were only slightly dependent on Ca2+. It was concluded that mitochondrial uncoupling via ATP depletion migth be responsible for the earlier described cell toxicity of toluene and in particular, of xylene. In the latter case, mitochondrial generated ROS and MPT also appear to be involved. Key Words: Toluene; Xylene; Mitochondria; Uncoupling; Reactive oxygen species.
Tesis

La transferencia nuclear de células somáticas (SCNT) en la especie porcina se ha convertido en una herramienta muy útil para para la elaboración de modelos genéticos de enfermedades humanas y para el uso en xenotransplantes. Aunque el número de cerdos clonados aumenta cada año, la eficiencia total de esta tecnología es todavía muy baja. Uno de los pasos más difíciles de la SCNT en porcino es la enucleación del ovocito, principalmente debido a que su citoplasma contiene numerosas gotas lipídicas. El principal objetivo de la tesis fue evaluar el efecto de diversas técnicas para visualizar la placa metafásica y el corpúsculo polar sobre la capacidad de desarrollo de ovocitos porcinos madurados in vitro.
Somatic cell nuclear transfer (SCNT) technology in porcine has become a very useful tool for the elaboration of genetic models for human diseases and the use in xenotransplantation. The efficiency of SCNT is still very low, although the number of cloned pigs increases each year. One of the hardest steps of porcine SCNT is the enucleation of the oocyte because its cytoplasm contains many lipid droplets. The main objective of this thesis was to assess the effect of several approaches to visualize the metaphase II plate and the first polar body on the developmental ability of in vitro mature porcine oocytes.

Orientador: Lucia Pereira da Silva
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: É geralmente aceito que o processo de peroxidação lipídica causa distúrbios nas funções mitocondriais (Vladimirov et alii, 1980) e que tal processo é desencadeado pelo ataque de espécies ativas de oxigênio aos lipídeos de membrana (Bindoli, 1988; Carbonera e Azzone, 1988). A geração de espécies ativas na mitocondria a nível de cadeia respiratória, leva a formação da espécie ativa de oxigênio mais agressiva, o radical hidroxila, de vida extremamente curta e que reage praticamente com todos os compostos orgânicos, oxidando grupos sulfidrílicos, degradandoDNA e causando peroxidação lipídica(Halliwell e Gutteridge, 1988). Por outro lado, o aumento na permeabilidade da membrana mitocondrial, conhecido como transição de permeabilidade, pode ser induzido por vários agentes, entre eles Ca2+ e Pi ou Ca2+ e hidroperóxidos, sendo que estes últimos podem conduzir a um aumento na geração de espécies ativas causando danos à membrana mitocondrial interna devido ao ataque dessas espécies aos lipídios e proteínas componentes de membrana. Neste trabalho estudamos o possível envolvimento do processo de peroxidação lipídica com alterações na permeabilidade da membrana mitocondrial interna e subseqüente liberação de íons e moléculas de baixa massa molecular. Nossos resultados mostraram claramente a independência entre esses mecanismos, pois em condições onde a transição de permeabilidade foi prevenida pela presença de inibidores específicos, mesmo assim foi possível observar ocorrência de peroxidação lipídica. Embora ambos os processos mostrassem ser dependentes de Ca2+, nossos resultados mostraram que a transição de permeabilidade pode ser revertida pela simples remoção do íon pelo quelante EGTA porém a peroxidação lipídica, uma vez iniciada, não sofreu interrupção com a remoção do Ca2+.Também a trifluoperazina e a ciclosporina A, conhecidos inibidores da transição de permeabilidade não impediram ou bloquearam ocorrência de peroxidação lipídica. Por outro lado, alterações na fluidez de membrana só foram detectada quando o processo oxidativo foi provocado por FeSO4 , um clássico indutor da peroxidação lipídica. E interessante ressaltar que este modelo oxidativo mostrou-se ineficaz na indução de transição de permeabilidade, mesmo observando-se a formação de altos níveis de TBARS. Este conjunto de resultados permitiu-nos descartar uma correlação direta entre a peroxidação lipídica e a transição de permeabilidade. o mecanimo do processo peroxidativo parece ocorrer após a transição de permeabilidade (decorrente da abertura de um poro protéico dependente de Ca2+) em situação onde a geração de espécies ativas de oxigênio supera a capacidade dos mecanismos de defesa
Abstract: It is generally accepted that lipid peroxidation leads to mitochondrial disfunction (Vladimirov et alii, 1980) and that this process is triggered by oxygen reactive species on membrane lipids (Bindoli, 1988;Carbonera e Azzone, 1988). The generation of such reactive species, at the level of respiratory chain, leads to formation of radicals among which the hydroxyl radical, a short-lived but extremely reactive one. This radical reacts with nearly all organic compounds, oxidizing sulphydryl groups, breaking DNA and casing lipid peroxidation ( Halliwell and Gutteridge, 1988). On the other hand, mitochondrial permeabilization can be induced by several agents, as Ca2+ and inorganic phosphate (Pi) Of Ca2+ and hydroperoxides. This later condition would increase the generation of oxygen reactive species, leading to damage at the mitochondrial inner membrane level, due to the attack of these species to lipids and proteins of this membrane. We studied the involvement of the lipid peroxidation process with alterations on the mitochondrial inner membrane permeability and, as consequence, the release of ions and low molecular mass compounds, Our results clearly showed that these two processes are independent. Since under conditions where the permeability transition process was. Prevented by specific inhibitors, it was observed the occurrence of lipid peroxidation. Although both processes were dependent on the presence of Ca2+, our reults showed that the permeability transition could be reverted by Ca2+ Chelation with EGTA, but the lipoperoxidation, once initiated , was not stopped by EGTA addition. Two other drugs, cyclosporin A and trifluoperazine, well known permeability transition inhibitors were inefficient in preventing or stopping the lipid peroxidation process. On the other hand, membrane fluidity alteratians evaluated by changes an the sparameter arder, were only detected when the oxidative process was greater, as that induced by a FeS04. lnterestingly this condition did not Ied to permeability transition, even when very high levels of TBARS were observed. These data allowed to discard any direct correlation between lipid peroxidation and permeability transition. The first process seems to occur after the later one, and is probably consequence of the opening of a Ca2+ dependent pore, under conditions where the generation of oxygen reactive species exceeds the capacity of the antiaxidant endogenous systems
Doutorado
Bioquimica
Doutor em Ciências Biológicas

Actualment en el nostre medi només l’1-2% dels fills de mares infectades pel VIH contreuen la infecció, gràcies a les mesures preventives aplicades, fonamentades sobretot en l'ús de fàrmacs antiretrovirals. Malgrat que el benefici que aporten aquests fàrmacs és inqüestionable, se sap que generen una càrrega d'efectes secundaris en els fills, ja que els reben durant la seva vida fetal i les primeres setmanes de vida i se'n desconeix la seva potencial repercussió a llarg termini. La tesi consisteix en 5 articles científics que pretenen objectivar què determina aquesta toxicitat i indagar en els mecanismes mitjançant els quals es genera. La toxicitat hematològica en els pacients pediàtrics exposats no infectats consisteix principalment en anèmia macrocítica les primeres setmanes de vida, habitualment subclínica i sempre autolimitada; i neutropènia. En els treballs realitzats observem que ni la reducció de la durada de la profilaxi neonatal amb zidovudina de sis a quatre setmanes ni l’ús durant la gestació de fàrmacs antiretrovirals de nova generació no disminueixen de forma significativa la incidència ni la gravetat de l’anèmia, que sembla relacionar-se sobretot a l´ús de zidovudina durant la gestació, molt habitual en tots els subgrups estudiats. D’altra banda, varis treballs apunten que la clínica derivada de disfunció mitocondrial en aquest col·lectiu de pacients podria ser més freqüent que en la població general. Fins l’actualitat escassos treballs han aportat dades sobre el metabolisme mitocondrial en pacients exposats no infectats, amb resultats contradictoris. En la nostra activitat de recerca, determinem per primera vegada de forma longitudinal els paràmetres mitocondrials, tant genètics com funcionals en els pacients exposats no infectats, observant una clara i persistent disfunció (disminució de l’activitat del complex IV de la cadena respiratòria mitocondrial), que és inversament proporcional al nivell d’ADN mitocondrial fins als sis mesos de vida. Observem també una disminució de les activitats enzimàtiques combinades (CII+CIII i CI+CIII+CIV de la cadena respiratòria mitocondrial) tant en les gestants com en els seus nadons a les sis setmanes de vida, i una correlació positiva entre els paràmetres materns i dels seus fills. Finalment, en analitzar conjuntament les dades de metabolisme mitocondrial i toxicitat hematològica, no observem una correlació clara entre elles. A les sis setmanes de vida, els pacients amb anèmia moderada o greu presenten menor activitat del CIV de la cadena respiratòria mitocondrial, fet que suggereix que la disfunció mitocondrial podria explicar almenys parcialment aquesta anèmia.
Nowadays in developed countries, only 1-2% of infants born of HIV-infected women become infected thanks to preventive measures, mainly to antiretroviral drugs use. Although the benefits of these drugs are unquestionable, the burden of side effects they produce in otherwise healthy children is of concern. This doctoral thesis consists of five papers about the mechanisms and determinants of mitochondrial and hematological toxicity among HIV and antiretrovirals exposed uninfected infants. Several relevant results are obtained. Regarding mitochondrial disturbances, enzymatic dysfunction of complex IV of mitochondrial respiratory chain is observed longitudinally during the first year of life, negatively correlated to mitochondrial DNA content the first 6 months. Lower CII+CIII and CI+CIII+CIV activities are observed among HIV-infected mothers and their infants, with positive correlation between maternal and neonatal parameters. Respecting haematological data, no reduction on incidence of anemia or neutropenia is observed when shorter neonatal prophylaxis or new generation antiretroviral drugs are used. Zidovudine is the drug most strongly related to haematological toxicity. At 6 weeks of life, lower CIV activity is observed among infants with moderate to severe anemia, suggesting that mitochondrial impairment could partially explain this observed anemia.

Liver diseases resulting from the toxicity induced by frequent pharmacological drug consumption are among the main health problems of modern western societies. On the other hand, healthy life-style-based behaviors including physical exercise are critical for counteracting, by preventing and/or treating, the drug-associated deleterious consequences for the hepatic tissue. The present thesis aimed to study, in a rat model, the effects of two chronic physical exercise regimens on liver morphological, biochemical and functional features centered on mitochondria, as these subcellular network compartments are known as dynamic structures closely involved in important mechanisms related to both the physiopathology of the disease and the beneficial adaptations of tissues afforded by exercise. Functional alterations in liver mitochondria were measured in in vitro: respiratory-driven endpoints, susceptibility to permeability transition pore opening. Additionally, enzymatic activities and the expression of proteins involved in redox response, apoptotic cell death, mitochondrial biogenesis, dynamic and autophagic markers were analyzed throughout the experimental work comprised in this thesis. Basal mitochondrial responses to toxic drugs exposure, both after in vitro (diclofenac) and in vivo (doxorubicin) stimulation were determined. It was overall concluded that chronic physical exercise induced liver mitochondrial alterations suggestive of positive remodeling, which were translated in a resultant more resistant phenotype against the in vitro toxicity of diclofenac and the in vivo harmful effects of doxorubicin. The observed mitigation effects were associated with favorable modifications in functional endpoints of mitochondrial respiration and in key signaling proteins related to oxidative stress and damage, apoptosis, mitochondrial biogenesis and dynamics, and auto(mito)phagy-related quality control mechanisms.

Tot i que el fetge és considerat com el principal lloc de producció del FGF21 sistèmic, sobretot en condicions de dejuni i sota control de PPARα, darrerament s’han acumulat diverses evidències que indiquen que FGF21 també pot actuar com a mioquina, un factor hormonal que pot ser produït i alliberat a la circulació pel múscul esquelètic. Concretament, s’ha observat que pacients que pateixen malalties neuromusculars causades per alteracions en la funció mitocondrial, com ara mutacions i deplecions al DNA mitocondrial, presenten valors d’expressió d’FGF21 incrementats, així com també els de la seva secreció a la circulació. Per tant, alguns indicis mostren que el múscul pot ser un lloc de producció i secreció d’FGF21 associat a l’estrès mitocondrial muscular. Durant la realització d’aquesta tesi, hem trobat que l’expressió i la secreció d’FGF21 en un context de cèl•lula muscular estan íntimament associades amb la diferenciació miogènica tant en rosegadors com en models humans. Per contra, els nivells dels seus receptors es mantenen bastant estables, mentre que pel que fa al cofactor β-Klotho, necessari pel seu procés de senyalització, no es detecta expressió del transcrit. Aquest fet suggereix, que la cèl•lula muscular podria ser una font d’expressió i secreció d’FGF21, però no un teixit diana d’aquesta. A part, s’ha identificat el factor miogènic MyoD com a un regulador molt potent de la transcripció del gen FGF21, així com també se n’ha mapat la regió del promotor responsable de vehicular aquest efecte. D’altra banda, s’han intentat descriure altres factors de transcripció i coreguladors que poden actuar com a activadors o inhibidors, els quals poden estar involucrats en el control de la transcripció gènica d’FGF21, com ara els PPARs, PGC-1α o Sirt1, així com també esbrinar els efectes de diversos activadors naturals i sintètics. En aquest sentit, val la pena destacar que dins d’un context en presència del factor miogènic MyoD s’ha identificat a PPARα, juntament amb el seu activador, com a un clar activador de l’activitat transcripcional d’aquest promotor. PGC-1α, sembla actuar de la mateixa manera, potenciant-ne l’efecte en presència de MyoD. Per contra, Sirt1 s’ha revelat com a un inactivador de l’activitat transcripcional del gen FGF21 en presència del factor MyoD. Altres experiments indiquen que el tractament amb diferents àcids grassos no sorgeixen cap efecte sobre l’expressió ni la secreció del gen en el context muscular, sinó més aviat el contrari, malgrat que la cèl•lula muscular es mostrava sensible a l’acció dels àcids grassos sobre altres gens. De la mateixa manera que ja indicaven determinats indicis bibliogràfics, vam poder comprovar que pacients de patologia MNGIE (miopatia associada a alteracions del DNA mitocondrial), mostraven la presència d’alts nivells d’FGF21 circulants. Així doncs, intentant mimetitzar una disfunció mitocondrial experimentalment, utilitzant inhibidors de la cadena respiratòria/fosforilació oxidativa, es van poder confirmar aquests increments d’expressió i secreció d’FGF21 per part de les cèl•lules musculars. Investigant una mica més a fons la mecanística de tot el procés, es va veure que l’increment de la producció d’espècies reactives d’oxigen derivades d’aquesta disfunció, provocava una inducció de la p38-MAP cinasa i conseqüentment l’activació ATF2, el qual és capaç d’interaccionar amb el seu lloc d’unió en la regió proximal del promotor del gen FGF21, provocant així aquest efecte sobre el gen FGF21 a les cèl•lules miogèniques. Alhora, s’ha descrit que la presència de MyoD és imprescindible per tal que es doni la resposta de la transcripció del gen FGF21 a la disfunció mitocondrial induïda experimentalment, justificant la resposta d’increment de la secreció d’FGF21 per part de les cèl•lules musculars en resposta a aquesta disfunció. Aquests canvis en la secreció d’FGF21 per part de les cèl•lules miogèniques en resposta a les afectacions a nivell mitocondrial, també poden ser un reflex del mecanisme fisiològic pel qual es detecten canvis a nivell d’estatus energètic a nivell muscular. D’aquesta manera, un increment de l’alliberació d’FGF21 podria desencadenar diferents respostes metabòliques adaptatives a nivell sistèmic. Aquest procés posa de manifest la gran capacitat que té la funció mitocondrial, per tal d’influenciar en el metabolisme sistèmic a través de la senyalització mitocondrial retrògrada. Així doncs, aquest mecanisme de senyalització permet l’expressió i alliberació de molècules d’acció endocrina com ara FGF21 i reforça la idea de considerar el múscul esquelètic com a una important font d’FGF21, la qual podem considerar com a una mioquina.
Although the liver is generally considered the main production site for fibroblast growth factor-21 (FGF21), high FGF21 levels have been found to be associated with neuromuscular mitochondrial genetic diseases, and there are indications that shows muscle as a source of FGF21 production under conditions of muscular mitochondrial stress. In this thesis we describe that FGF21 expression and release is associated with myogenic differentiation in different muscular cell lines. However, FGFRs transcription levels don’t change across differentiation and β-Klotho is undetectable, suggesting that muscle cells as a source but not as a target of FGF21. Furthermore we have identified MyoD as a major controller of FGF21 gene transcription, as well as we have mapped the most important region in the promoter responsible for MyoD-dependent regulation. Moreover, we determined the role of some transcription factors and co-regulators potentially involved in the control of FGF21 gene transcription, such as PPARs, PGC-1α or Sirt1, as well as several natural and synthetic agents (e.g. fatty acids) On the other hand, mimicking mitochondrial dysfunction by the use of respiratory chain/oxidative phosphorylation inhibitors resulted in enhanced expression and release of FGF21 by muscle cells. Increased production of reactive oxygen species, subsequent induction of p38-MAP kinase and activation of an ATF2-binding site at the proximal promoter region of the FGF21 gene were found to comprise the major mechanism connecting mitochondrial dysfunction and enhanced FGF21 gene transcription in myogenic cells. Furthermore, we show that MyoD is required for the responsiveness of FGF21 gene transcription to experimentally induced mitochondrial dysfunction, which explains the preferential response of muscle cells to enhanced FGF21 secretion in response to mitochondrial alterations. FGF21 release by muscle cells in response to mitochondrial alterations may reflect a physiological mechanism by which the sensing of internal energetic status by muscle tissue results in the release of FGF21 to favor systemic metabolic adaptations. This process highlights the capacity of mitochondrial function to influence systemic metabolism by retrograde signaling, which leads to the expression and release of molecules with endocrine action, such as FGF2,1 and reinforces the concept of considering the skeletal muscle as an important source of the FGF21, as well as to consider FGF21 as a myokine.

Orientador : Quivo S. Tahin
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Devido ao fato de que a manipulação dietária altera a composição de ácidos graxos (AG) os lipídeos dos tecidos e inclusive de organelas celulares dos organismos, podendo alterar funções hormonais, enzimáticas, estruturais das membranas celulares, investigamos as possíveis alterações causadas pela administração de três dietas diferentes: óleo de semente de soja 12%, óleo de soja 20% e óleo de sardinha 12%, sendo que a dieta de semente de soja é rica em AGPI da série n-6 e óleo de semente de sardinha é rica em AGPI da série n-3, em ratos machos da raça Sprague-Dawley com cerca de 37 dias de idade pseudo-operados e tireoidectomizados durante 30 dias. Durante o período experimental com as três dietas os animais foram pesados e ainda se determina a ingestão de alimento diária. Após 30 dias determinou-se a captação de I131 nos animais controles, a seguir animais pseudo-operados e tireoidectomizados foram sacrificados. O sangue foi retirado para análise dos hormônios tireoideanos T3 e T4 (apenas dos animais pseudo-operados) e do conteúdo de AG de lipídeos daquelas organelas celulares. Demonstramos que os ratos tireoidectomizados quando comparados com sues respectivos controles apresentaram um ganho do peso corpóreo e dos órgãos estudados (fígado e encéfalo) e em relação à ingestão de alimentos os animais tireoidectomizados ingeriram menos alimento que os respectivos controles nos três grupos dietários... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: ¿Lipid dierary effect on mitochondrial AT Pase and fatty acid composition of lipids of serum and liver and brain mitochondria of normal and thyroidectomized rats¿. Normal and thyroidectomized Sprangue ¿ Dawley male rats of 37 days old, were fed three different lipid diets during 30 days. Two diets contained soybeam seed oil. Rich in n-6 polyunsaturatede (PUFA), but differente concentrations, one of 12% other 20%. The third diet contained 12% of sardine (sea fish) oil, rich in n-3 PUFA. Thyroidectomized rats had lesser gain of body, liver and brain weight and food ingestion. Normal animals had thyroid activity alterated in fuction of lipid diets. Sardine oil group shown different T3 and T4 serum leves, but similar I131 uptake in comparison to 12% soybeam oil group. Howrever 12% soybean oil group shown different T3 and T4 seryn levels and I131 uptake in comparison to 20% soybeam oil group. The thyroidectomia affected the fatty acid (FA) contents of serum lipids in function of lipid diet. Thyroidectomized animals had similar saturated FA (SFA) content in 12% soybeam oil group, but higher in 20% soybeam oil and 12% sardine oil groups , in comparison to respective controls animals... Note: The complete abstract is available with the full electronic digital thesis or dissertations
Doutorado
Biologia Celular
Doutor em Ciências Biológicas

Orientador : Quivo S. Tahin
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Ratas Sprague-Dawley virgens de 50-60 dias de idade, quando inoculadas i.p. com 20 mg/ml de DMBA e alimentadas com dietas semi-sintéticas e isocalóricas, contendo 12% de lipídios, provenientes do óleo de semente de girassol (rica em AGPI n-6) ou do óleo de sardinha (rica em AGPI n-3), não apresentaram diferenças importantes no ganho de peso corpóreo nos tempos de 30 ou 63 dias. O tecido mamário das ratas alimentadas com a dieta óleo de semente de girassol, apresentou alterações histológicas mais severas do que o das alimentadas com a dieta óleo de sardinha, após o tratamento com o DMBA nos tempos estudados. A atividade específica da ATPase mitocondrial do tecido mamário dos animais controle e tratados com o DMBA, apresentou alterações mais significativas naqueles animais alimentados com a dieta óleo de semente de girassol, quando comparada com os que se alimentaram com a dieta óleo de sardinha nos tempos estudados. No grupo óleo de semente de girassol, as atividades especificas da ATPase foram de 17,2¿+ ou ¿' 6,4 e 40,3 ' + ou ¿' 12,0 unidades de atividade ATPásica (p<0,01), respectivamente aos dias 30 ou 63 de experimento para as ratas controles e de 33,1 : 21,4 e 70,0: 19,2 unidades especifica de AT Pase (p< 0,01), respectivamente após os 30 ou 63 dias de experimento com DMBA. Neste grupo dietário, o tratamento com o DMBA causou aumento na atividade específicas da ATPase de 92% (p<0,01) e 88% (p<0,01), respectivamente após 30 ou 63 dias. No grupo de óleo de sardinha, as atividades específicas da ATPase foram de 37,6¿ + o0u ¿' 9,2 e 50,3 '+ ou ¿' 25,8 unidades de ATPase especifica (N.S.) para as ratas controles e de 40,2 '+ ou ¿' 16,0 e 59,2 '+ ou ¿' 15,3 unidades específicas de atividade ATPásica (p<0,01) para as ratas tratadas com DMBA, respectivamente após 30 ou 63 dias. Neste grupo dietário o DMBA também causou o aumento na atividade ATp.ásica, porém não foi estatistidaIrente' significante. Quanto a incorporação dos ácidos graxos pelos lipídios das mitocôndrias, ressaltamos que nos animais controles não foi detectado alterações importantes na incorporação dos AGS totais e AGMI totais frente às duas dietas e ao tempo de experimento; que os AGPI n-6 foram mais incorporados pelos animais alimentados com a dieta óleo de semente de girassol, quando comparados com os que se alimentaram com a dieta óleo de sardinha nos tempos estudados, o mesmo acontecendo com os AGPI n-3. O tratamento com o DMBA,diminui a incorporação pelos lipídios das mitocôndrias dos AGS totais, aumentou a incorporação dos AGMI totais naqueles animais alimentados com a dieta óleo de semente de girassol, aumentou a incorporação dos AGPI n-6, principalmente do ácido linoléico e eicosatrienóico, sem contudo aumentar a incorporação do ácido araquidônico nos animais alimentados com a dieta óleo de semente de girassol, ocorrendo o efeito contrário nos animais alimentados com a dieta óleo de sardinha e aumentou a incorporação dos AGPI n-3 pelos lipídios mitocondriais dos animais alimentados com a dieta óleo de sardinha, quando comparados com os que se alimentaram com a dieta óleo de semente de girassol nos tempos estudados
Abstract: Female virgin rats Sprague-Dawley (50-60 days old), were injected, i.p., with 20 mg/ml of 7,12-dimethylbenzenanthracene (DMBA) and fed semipurified and isocaloric diets, containing 12% of sunflower seed oil (rich in AGPI n-6) or 12% of sardine oil (rich in AGPI n-3), during 30 or 63 days. During the experimental time alI groups of rat, present few alterations on the gain of body weights. Rats fed sunflower seed oil showed, more severe histological alterations in the mammary tissues after DMBA treatment in comparasion to controls and to sardine oil. ATPase activity mammary mitochondria were a1ter in function of time, diet and DMBA treatment , after 30 or 63 days, respectively. ATPase of control animaIs of sunflower seed oil diet were 17,2 i 6,4 and 40,3 i 2,0 units of ATPase activity (p<0,01), and after DMBA tre atment we found 33,1 i 21,4 and 70,0 i 19,2 units of ATPase activity (p<0,01) after 30 or 63 days, respectivelYi the specificNP ase of control animaIs fed ardine oil, were 37,6 i 9,2 and 50,3 -98-f 25,8 units of specific ATPase (N.S.), and after DMBA treatment, were 40,2 f 16,0 and 59,2 f 15,3 units of ATPase (p<0,01)." In sun flower seed oil group, after 30 or 63 days of DMBA treatment we bbserved an increase of 92% (p<0,01) and 88% (p<0,01), respective ly in comparisionto controls. In sardine oil group the alterations caused by DMBA treatment after 30 or 63 days in comparasion of controls group was not significant. There was not impoEtant alterations on total saturated fatty acid (SFA), and total monounsaturated fatty acid (MUFA) contents on mammary mitochondriaI lipids of two diets group. The total n-6 polyunsaturated fatty acid (PUFA) and n-3 fatty acids of mitochondrial lipids sunflower seed oil were higher than sardine oil group in function of time. DMBA treatment cause an increase in the SFA contents, and on the n-6 PUFA contents in sunflower seed oil group, mainly increasing linoleic acid and eicosatrienoic acid but noarachildonic acid. However, in the sardine oil group it was observed the reversal event, increasing n-3 PUFA after DMBA treatment
Mestrado
Doutor em Ciências Biológicas

Orientadores: Anibal Eugenio Vercesi, Helena Coutinho Franco de Oliveira
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Oxigênio singlete molecular (1O2) gerado por decomposição térmica do 3,3-(1,4-naphthylidene) dipropionate endoperoxide (NDPO2), inibiu a respiração de mitocôndrias isoladas de fígado de rato sustentada por substratos dependentes de NADH ou por succinato, mas não por N,N,N,N-tetramehyl-p-phenylene-diamine (TMPD)/ascorbato. Nesta última condição, as mitocôndrias tratadas com NDPO2 exibiram uma diminuição do potencial elétrico transmembrânico (DY) de maneira dependente do tempo de exposição ao NDPO2. Este processo foi sensível aos inibidores da transição de permeabilidade mitochondrial (TPM) como EGTA, ditiotreitol, ADP e ciclosporina A. A presença de óxido de deutério (D2O), que aumenta a meia vida do 1O2, aumentou significativamente a permeabilização promovida por NDPO2. Além disso, a permeabilização mitocondrial induzida por NDPO2 foi acompanhada por oxidação tiólica de proteínas de membrana sensível à DTT ou ADP. Estes resultados indicam que a TPM induzida por oxigênio singlete gerado quimicamente é mediada por oxidação de tióis de proteínas de membrana. Verificou-se ainda neste trabalho que mitocôndrias isoladas de fígado de camundongos hipercolesterolêmicos por inativação do gene do receptor de LDL, apresentaram controle respiratório e razão ADP/O similares às mitocôndrias isoladas a partir de camundongos controles, mas exibiram maior suscetibilidade a desenvolver TPM induzida por Ca2+. Esta maior susceptibilidade à TPM parece ser conseqüência do estado mais oxidado de NADP mitocondrial nos camundongos hipercolesterolêmicos
Abstract: Pure singlet molecular oxygen (1O2) generated by thermal decomposition of the 3,3-(1,4-naphthylidene) dipropionate endoperoxide (NDPO2), inhibited respiration of isolated rat liver mitochondria supported by NADH-linked substrates or succinate, but not by N,N,N,N-tetramehyl-p-phenylene-diamine (TMPD)/ascorbate. Under the latter condition, mitochondria treated with 2.7mM NDPO2 exhibited a decrease in transmembrane potential (DY) in manner dependent on NDPO2 exposure time. This process was sensitive to the mitochondrial permeability transition (MPT) inhibitors EGTA, dithiothreitol, ADP, and cyclosporin A. The presence of deuterium oxide (D2O), that increases 1O2 lifetime, significantly enhanced NDPO2-promoted mitochondrial permeabilization. In addition, NDPO2-induced mitochondrial permeabilization was accompanied by DTT or ADP-sensitive membrane protein thiol oxidation. Taken together, these results provide evidence that mitochondrial permeability transition induced by chemically generated singlet oxygen is mediated by the oxidation of membrane protein thiols. In this work it was also observed that liver mitochondria isolated from hypercholesterolemic LDL receptor knock out mice present respiratory control and ADP/O ratio comparable to control mitochondria, but exhibit higher susceptibility to develop Ca2+ induced MPT. This higher susceptibility to develop MPT seems to be the consequence of a more oxidized state of mitochondrial NADP
Doutorado
Ciencias Biomedicas
Doutor em Ciências Médicas

Orientador : Anibal Eugenio Vercesi
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Os resultados apresentados neste trabalho indicam que as alterações no fluxo de Ca2+,causadas por oxidantes de nucleotídeos de piridina na presença deste íon, secundárias ao aumento de permeabilidade da membrana mitocondrial interna, são influenciadas por vários fatores, tais como: tração de acetato, pH do meio de reação, mudanças no potencial de membrana e regentes tiólicos. Observamos que este efluxo transiente de Ca2+ que acompanha a oxidação transitória dos nucleotídeos de piridina por t-butil hidroperóxido ou diamida é potencializado pelo aumento da concentração de acetato na faixa de O a 20 mM ou pelo decréscimo do pH de do meio, de 7,4 para 6,8. Os diferentes potenciais de membrana foram obtidos pela energização das mitocôndrias com succinato na presença de ATP ou pela hidrólise de ATP apenas. Além disso, a suspensão mitocondrial foi pré-incubada na presença de Ca2+ antes da energização, isto é, na ausência de alto potencial de membrana ou o potencial de membrana foi colapsado pela adição de antimicina A após o acúmulo do cátion. Todos estes procedimentos mostraram que as mitocôndrias são mais sensíveis aos efeitos deletérios do Ca2+ na presença destes agentes oxidantes em potenciais de membrana mais baixos. Observou-se também que mitocôndrias de cérebro, cuja atividade da enzima fosfolipase A2 dependente de Ca2'+ é menor do que em mitocôndrias hepáticas, são mais sensíveis aos efeitos do Ca2+ na presença destes agentes, sugerindo que esta enzima não deve exercer um papel central neste mecanismo. Por outro lado, os efeitos inibitórios de NEM e DTT, ambos reagentes tiólicos, observados em nossos experimentos, corroboram resultados anteriores de nosso laborat6rio indicando que a liberação transiente de Ca2+(aumento transiente na permeabilidade da membrana) induzido pela oxidação transiente dos nucleotídeos de piridina ocorre em paralelo à transições sulfe to-dissulfeto na membrana mitocondrial
Abstract: Ca2+are influenced by several factors such as: acetate concentration, medium pH,changes in membrane potential and thiol reagents. It was observed that the extension of the reversible Ca2+ efflux-influx cycle that accompanies the transient oxidation of pyridine nucleotides by diamide or t-butyl hidroperoxide is augmented by increasing the acetate concentration from O to 20mM or by decreasing the medium pH from 7.4 to 6.8. The different membrane potentials were obtained by energizing mitochondria with succinate in the presence of ATP or by ATP alone. Moreover, the mitochondria were preincubated in the presence of prior to energization, that is, in the absence of an appreciable membrane potential or the membrane potential collapsed by the addition of Antimycin A after the cation had been accumulated. AlI these procedures have shown that mitochondria are more sensitive to the deleterious effects of Ca2+ plus an oxidant of pyridine nucleotides at lower membrane potentials. In addition it was shown that rat brain mitochondria, whose activity of the Ca2+ estimulated phospholipase A2 is much lower than in liver mitochondria, are more sensi tive to the effects of Ca2+ plus these oxidants indicating that this enzyme may not play a major role in this mechanism. On the other hand, the inhibitory effects of NEM.and D'l'T, both thiol reagents, observed in our experiments strongly supports previous data from this laboratory indicating that the transient Ca2+ release (transient increase in membrane permeability) induced by transent oxidation of pyrid1ne nucleotides is paralleled by membrane sulfhydril ¿ disulfide transitions
Mestrado
Bioquimica
Mestre em Ciências Biológicas

Orientador : Anibal Eugenio Vercesi
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Estudos recentes deste laboratório indicaram que o baixo grau de acoplamento entre a respiração e a fosforiIação oxidativa em mitocôndrias isoladas de tubérculos de batata parece estar relacionado à existência de uma proteína de aproximadamente 35 KDa presente na membrana interna destas mitocôndrias, com a propriedade de transportar H+ por um mecanismo sensível a nucleotídeos de purina. No presente trabalho, obtivemos mitocôndrias de batata com controle respiratório mais elevado que em estudos anteriores, graças à modificações introduzidas no procedimento de isolamento. Ainda assim, a velocidade de respiração destas mitocôndrias, no estado IV, foi reduzida pela adição de ATP e ADP, mesmo na presença de carboxiatractilato (CAT). Experimentos de ligação de nucleotídeos de purina mostraram que estas mitocôndrias apresentaram a habilidade de ligar 1,3 nmol ATP.mg proteína; 1,1 nmol -1 . ¿1 ADP. mg proteína; 0,7 nmol GTP. mg ¿1 proteína. Experimentos de competição mostraram ainda: a maior afinidade de ligação destas mitocôndrias por nucleotídeos de adenina. A construção de um potencial de membrana mais elevado por mitocôndrias de batata e de cole6ptiles de milho é dependente da presença de BSA e nucleotídeos de purina. A presença destes não afeta o potencial construído por mitocôndrias de feijão, beterraba e fígado de rato. Anticorpos contra esta proteína de 35KDa isolada de mitocôndrias de batata foram obtidos e utilizando a técnica de Western Blotting foi verificado que as outras mitocôndrias vegetais em estudo neste laboratórIo como as de coleóptiles 'de milho, feijão e beterraba possuem esta proteína ou similar como mostrado pela reação cruzada com estes anticorpos. Contrariamente, uma série de mitocôndrias não vegetaIs, taIs como as de fígado de rato, coração bovino e T.cruzi epimastigotas não mostraram reação com estes anticorpos. Experimentos de reconstituição desta proteína isolada de batata e da UCP isolada de tecido adiposo marrom foram realizados e demonstraram que, no sistema por nós utilizado, elas apresentam comportamento similar, isto é, nas vesículas com proteína incorporada, a velocidade de efluxo de H + foi maior que em vesículas controle, preparadas na ausência de proteína
Abstract: Early studles from thls laboratory have lndicated that the low degree of coupling between respiration and oxidative phosphorylation in potato mitochondria seems to be related to a 35 KDa protein, present in the inner membrane of those mitochondria, with the ability to translocate H with a: mechanism sensitive to purine nucleotides. In this work, we obtained potato mitochondria with better respiratory control, thanks to modificatlons made in the isolation procedure. In spite of the lsolation method, the respiration rate of these mitochondria ln state IV decreased after additltion of ADP or ATP nucleotides ln the presence of carboxyatractylate (CAT). Binding experiments showed that the mitochondria have the ability to bind 1,3 nmol ATP.mg ¿1 protein 1,1 nmol ADP. Mg ¿1 protein; 0,7 nmol GTP. Mg -1proteln. Competition experiments showed the largest affinity of binding of these mltochondria towards adenlne nucleotides. The increase of potato and corn mitochondria membrane potential is dependent on the presence of BSA and purine nucleotides. The presence of BSA and nucleotides dld not affect the membrane potential of red beet roots (Beta vulgaris); bean (Phaseolus vulgaris) and rat liver mltochondria. Antibodies against a 35 KDa protein isolated from potato mitochondria were obtained and, using the Western blotting techique, we verified that the other plant mitochondria in studies in this laboratory, such as corn, red beet roots and bean, have this or a similar protein as shown by cross reactivity wlth these antibodies. In contrast a series of non-plant mitochondria, also studied in thls laboratory, such as those from rat liver, beef heart and T. cruzi epimastigotes did not show cross reactivity with these antibodies. Reconstitution experiments of this protéln and of UCP isolated from brown adipose tissue were performed and showed that, in the system used, they have similar behavior. In the vesicles with protein incorporated, the rate of H+ efflux was larger than in control vesicles, prepared without either protein
Mestrado
Bioquimica
Mestre em Ciências Biológicas

Parkinson's disease (PD) is the second most common neurodegenerative disease, and the most common movement disorder in the world population. In most cases its aetiology is still unknown, however, mitochondrial alterations and autophagy deregulations are some of the molecular mechanisms that are altered in this disease. These molecular alterations of PD are not limited only to the destruction of dopaminergic neurons in the substantia nigra pars compacta, but they have also been described in the peripheral nervous system and the organs that it innervates. There is also evidence of the presence of other molecular alterations in diverse tissues, such as dysfunction of Complex I of the mitochondrial respiratory chain and accumulation of alpha synuclein in fibroblasts of patients with PD. One of the great difficulties the research and understanding of the mechanisms that lead to PD is the inaccessibility of the target tissue of the disease. In the best of cases, autopsy tissue from patients with advanced PD is available, leaving a question mark about the molecular processes of prodromal and early stages of the disease. Animal models have helped to unravel some questions, but the development of accessible and replicable cell models, preferably at low cost, is much needed. It is in this context that the cellular models obtained from PD patients and from asymptomatic carriers of genes associated with the disease are of great importance and require validation. The present thesis consists of the study of two cell models obtained from patients with PD associated with the LRRK2 mutation (G2019S), asymptomatic carriers of LRRK2 (G2019S) and homozygous and heterozygous carriers of GBA (N370S); which are the genes most frequently associated with familial PD and the most important genetic risk factor for PD, respectively. First, the mitochondrial and autophagic profile of fibroblasts derived from the skin of asymptomatic carriers of the LRRK2 (G2019S) mutation and with PD were analysed. The analysis was carried out under two conditions, keeping the fibroblasts in a standard culture medium (DMEM with 25mM glucose) and after subjecting them to a mitochondrial challenge for 24 hours (DMEM with 10mM galactose), in order to simulate the oxidative environment of neurons. dopaminergic. In this study, a genotype-phenotype correlation was confirmed in fibroblasts obtained from asymptomatic carriers of the LRRK2 (G2029S) mutation and patients with PD linked to this same mutation, and it was demonstrated that a mitochondrial and autophagic function profile allows to differentiate between groups. The second study explored the genotype-phenotype correlation in a cellular model characterized by neurospheres, a conglomerate of cells obtained from the dedifferentiation of human adipocytes into neuronal stem cells, and its relationship with the onset of macroautophagy in subjects carrying the mutation GBA (N370S). The main finding of this study is that mitochondrial dysfunction preceded alterations of macroautogphagic flux in subjects carrying the GBA (N370S) mutation. In conclusion, the study of asymptomatic subjects carrying mutations associated with PD represents a relevant study method that shows initial molecular alterations and the presence of compensatory mechanisms that can be studied for the development of preventive strategies and treatments in early stages of the disease.
La enfermedad de Parkinson (EP) es el trastorno de movimiento más frecuente en la población mundial. Considerada mayoritariamente idiopática y multifactorial, alteraciones mitocondriales y en la regulación autofagica son algunos de los mecanismos moleculares que se han encontrado alterados en la etiopatología de la enfermedad. El descubrimiento de genes relacionados a formas familiares de EP, del cual LRRK2 es el más frecuente, y los genes que aumentan el riesgo de padecer la enfermedad, como GBA, han abierto un campo de estudio en el cual se pueden analizar los mecanismos moleculares que llevan a la neurodegeneración en formas genéticas de la EP. La presente tesis consiste en el estudio de dos modelos celulares obtenidos a partir de portadores asintomáticos de LRRK2(G2019S) (NMLRRK2(G2019S)), pacientes con EP asociada a la mutación LRRK2(G2019S) (PDLRRK2(G2019S)), así como de portadores homozigotos y heterozigotos de GBA(N370S). El primer estudio analizó el perfil mitocondrial y autofágico de fibroblastos NMLRRK2(G2019S) y PDLRRK2(G2019S). El análisis se realizó en dos condiciones, en un medio de cultivo estándar (DMEM, glucosa 25mM) y tras someterlos 24 horas a un reto mitocondrial (DMEM, galactosa 10mM), simulando el ambiente oxidativo de las neuronas dopaminérgicas. En este estudio se confirmó una correlación genotipo-fenotipo en fibroblastos obtenidos de ambos grupos y una función mitocondrial y autofágica que permite diferenciarlos entre ellos. El segundo estudio exploró la correlación genotipo-fenotipo en un modelo celular caracterizado por neuroesferas, un conglomerado de células obtenido a partir de la desdiferenciación de adipocitos humanos en células madres neuronales, y su relación con el inicio de la macroautofagia en sujetos portadores de la mutación GBA(N370S). El hallazgo principal de este segundo estudio es que la disfunción mitocondrial precede a las alteraciones del flujo macroautofágico en sujetos portadores de la mutación GBA(N370S). El estudio de sujetos asintomáticos portadores de mutaciones asociadas a PD representa un relevante método de estudio que evidencia alteraciones moleculares iniciales y la presencia de mecanismos compensatorios que pueden ser estudiados para el desarrollo de estrategias preventivas y tratamientos en lateabas tempranas de la enfermedad.

Brown and beige adipose tissues are mediators of adaptive energy expenditure in mammals, in contrast with the energy storage role of white fat. Mitochondrial activity, including uncoupled respiration, and the release of the so-called “batokines” in brown and beige adipocytes account for the majority of local and systemic adaptations to energy expenditure requirements mediated by these cells. One of these batokines is fibroblast growth factor 21 (FGF21), which has been recently proposed to be an antiaging hormone. Aging is associated with a decline in brown adipose tissue (BAT) activity and in browning of white adipose tissue (WAT). It has been proposed that the extent of BAT decline in aging may play a causative role in the enhanced propensity to age-associated metabolic conditions, and it has even been speculated that BAT reactivation may reverse them. On the other hand, aging is associated with increased amount of WAT. A loss of mitochondrial homeostasis by defective mitochondrial quality control as a result of decreased biogenesis but also to decreased degradation through mitophagy has been proposed as an underlying cause of aging in multiple tissues. Recently, Parkin has been identified as a key component of adipose tissue plasticity in response to thermogenic requirements, associated with the adaptive control of mitophagy. In the present study we investigated the impact of aging on adipose tissues in mice and humans with special focus on the status of the FGF21 system. We also tried to establish the role of Parkin in our mice model at distinct stages of aging. Parkin transcript and protein levels were up-regulated in relation to aging in mice adipose tissues, which may point to a compensatory homeostasis against diminished Parkin- independent mitophagy. Middle-aged wild-type mice presented an age-associated phenotype reminiscent of obesity and signs of BAT malfunction in middle-aged mice, which did not affect thermogenic response at this specific aging stage. Thus, our data would not support a massive effect of BAT in systemic derangements associated with aging. Parkin-KO mice were protected against age-associated obesity found in control animals. BAT activity does not seem to conduct the protection of Parkin-KO mice against aging-associated adiposity, as Parkin-KO mice do not present enhanced BAT- or browning-mediated energy expenditure. This phenomenon might rather be attributable to diminished food intake and risen energy expenditure-related processes in Parkin-KO mice. Our data denote that Parkin is possibly involved in the in the metabolic flexibility of lipid versus carbohydrate metabolism. FGF21 expression and its secretion in mice may be induced as stress hormone by mitochondrial dysfunctions in aged tissues. However, the chronic metabolic and stress-related disorders might elicit an FGF21 resistance, menacing healthy aging. Middle-aged mice lacking Parkin abolish increased levels of FGF21, which might be associated to the prevention of obesity occurring in those animals. In humans, serum FGF21 levels were increased in parallel with indicators of mildly deteriorated glucose homeostasis. FGF21- responsiveness machinery was not disrupted in subcutaneous adipose tissue from elderly individuals relative to those from young controls. Therefore, in contrast to what is observed in chronic metabolic pathologies or mice aging, high levels of FGF21 in healthy aging are not associated with repressed FGF21-responsiveness machinery in adipose tissue.

Hi ha evidències que impliquen la funció mitocondrial en alguns trastorns del neurodesenvolupament (TN) com el trastorn de l'espectre autista (TEA), la discapacitat intel·lectual (DI) i l'esquizofrènia (ESQ). A més, en aquests trastorns és freqüent la presència de característiques clíniques associades a les malalties mitocondrials (CCAMMs). És per aquests antecedents que aquesta tesi doctoral ha volgut posar el focus en aquest orgànul i en el paper que podria tenir en l'etiologia d'alguns TN. Concretament, s'han avaluat diversos aspectes clínics, genètics i metabòlics en un grup de pacients amb DI (N=115), pacients amb DI i TEA (DI-TEA=122), ESQ (N=57) i en persones control (N=33). Segons els resultats obtinguts, les persones amb DI, DI-TEA i ESQ presenten més freqüència de determinades CCAMMs que les persones control. A més, també presenten un menor número de còpies d'ADN mitocondrial i determinades variants de canvi de nucleòtid, que podrien ser patogèniques. Els pacients amb ESQ van mostrar nivells de lactat més elevats que les persones control durant la realització d'exercici físic. Finalment, també s'han identificat diverses alteracions mitocondrials en una família amb els diagnòstics d'ESQ i síndrome de fatiga crònica. Aquest treball de tesi doctoral suggereix que el mitocondri pot tenir un paper rellevant en l'etiopatogènia d'algunes malalties psiquiàtriques.
Hay evidencias que implican la función mitocondrial en algunos trastornos del neurodesarrollo (TN) como el trastorno del espectro autista (TEA), la discapacidad intelectual (DI) y la esquizofrenia (ESQ). Además, en estos trastornos es frecuente la presencia de características clínicas asociadas a las enfermedades mitocondriales (CCAEMs). Es por estos antecedentes que esta tesis doctoral ha querido focalizar en este orgánulo y en el papel que podría tener en la etiología de algunos TN. Concretamente, se han evaluado varios aspectos clínicos, genéticos y metabólicos en un grupo de pacientes con DI (N=115), pacientes con DI y TEA (DI-TEA=122), ESQ (N=57) y personas control (N=33). Según los resultados obtenidos, las personas con DI, DI-TEA y ESQ presentan más frecuencia de determinadas CCAEMs que las persones control. Además, también presentan un menor número de copias de ADN mitocondrial y determinadas variantes de cambio de nucleótido que podrían ser patogénicas. Los pacientes con ESQ mostraron niveles de lactato más elevados que las persones control durante la realización de ejercicio físico. Finalmente, también se han identificado varias alteraciones mitocondriales en una familia con los diagnósticos de ESQ y síndrome de fatiga crónica. Este trabajo de tesis doctoral sugiere que la mitocondria puede tener un papel relevante en la etiopatogenia de algunas enfermedades psiquiátricas.
There are evidences that involve the mitochondrial function as a key factor in some neurodevelopmental disorders (ND) such as autism spectrum disorders (ASD), intellectual disability (ID) and schizophrenia (ESQ). Besides, in these disorders, the presence of clinical characteristics associated with mitochondrial diseases (CCAMDs) is frequently observed. These two premises set the basis of this doctoral thesis, which focuses on the study of this organul and the role it plays in the etiology of some ND. Specifically, several clinical, genetic and metabolic aspects were evaluated in a group of patients with ID (N=115), with ID and ASD (ID-ASD=122), ESQ (N=57) and a control group (N=33). The results showed that, people with DI, DI-ASD and ESQ present higher frequency of certain CCAMDs compared with the control group. In addition, smaller number of mitochondrial DNA copies was found in the aforementioned patients. Furthermore, some variants of nucleotic change that could be pathogenic are observed as well. Patients with ESQ also presented higher lactate levels during physical exercise. Finally, several mitochondrial alterations have also been identified in a family with SCZ and chronic fatigue syndrome. This doctoral thesis suggests that mitochondria plays a relevant role in the etiopathogeny of some psychiatric diseases

[ES] La familia de proteínas Bcl-2 regula la apoptosis a través de una compleja red de interacciones. Las células tumorales suelen presentar mutaciones que afectan a su expresión o sus interacciones para mejorar la progresión tumoral. Además, alteraciones en su regulación también promueven la migración de células cancerígenas, la invasión y la metástasis. Para llevar a cabo sus funciones, las proteínas Bcl 2 interaccionan entre sí tanto en el citoplasma como en las membranas intracelulares. Los equilibrios de interacción de los dominios Bcl citosólicos se han investigado ampliamente y recientemente, se han propuesto como dianas terapéuticas. Sin embargo, el interactoma de los dominios transmembrana (TMD, del inglés transmembrane domains) sigue siendo poco conocido. Por ello, un conocimiento profundo de la biología de las proteínas Bcl-2 es necesario para explotar eficientemente sus superficies de unión en el tratamiento del cáncer. Para llevar a cabo este objetivo, nos hemos centrado en tres áreas: 1. La comprensión detallada de la contribución del TMD de Mcl-1 a su interactoma en membrana y su función. 2. El descubrimiento de nuevos inhibidores de Mcl-1 que actúen sobre su TMD y que permitan desarrollar una clase de drogas anticancerígenas aún por explorar. 3. La caracterización molecular de mutaciones relacionadas con el cáncer descritas en los TMD de Bcl-2 y Bcl-xL y sus implicaciones en la supervivencia de las células tumorales. La proteína antiapoptótica Mcl-1 inhibe a los miembros proapoptóticos Bak, Bax, Bok, Noxa, etc. Aunque se ha estudiado en detalle su actividad promoviendo la supervivencia celular, el mecanismo molecular por el cuál previene la apoptosis mediada por Bok aún no está claro. Además, el conocimiento de las actividades de Mcl-1, descritas hasta ahora, se basa exclusivamente en las estructuras resueltas de las regiones solubles en agua y en estudios centrados en los dominios citosólicos. Por primera vez, hemos demostrado la relevancia del TMD de Mcl-1 en su equilibrio de interacción. En este trabajo describimos su capacidad específica para homo- y hetero-oligomerizar con el TMD de Bok. También ponemos de manifiesto la influencia de estas interacciones en la modulación de apoptosis y resaltamos la relevancia clínica de los mutantes del TMD de Mcl-1 identificados en pacientes con cáncer. Muchos tumores hematológicos y sólidos sobre-expresan Mcl-1 como mecanismo para adquirir quimiorresistencia. Se han desarrollado miméticos de BH3 específicos para modular su actividad antiapoptótica en células cancerosas. Sin embargo, aún no disponemos de datos científicos que informen sobre su toxicidad y eficacia en humanos. En este trabajo, proponemos la novedosa interacción de los TMDs de Mcl-1 y Bok como un nuevo sitio de acción de fármacos quimioterapéuticos. Hemos identificado tres inhibidores de esta interacción con características que los hacen prometedores candidatos para el desarrollo farmacéutico, así como buenas herramientas moleculares para estudiar la interacción de los TMDs de Mcl-1 y Bok. Para modular la apoptosis, las células tumorales también presentan versiones mutadas de las proteínas antiapoptóticas Bcl-2 y Bcl-xL. En nuestro conocimiento, este es el primer estudio que analiza mutaciones somáticas de sus TMDs. Nuestro trabajo demuestra cómo estas mutaciones alteran el equilibrio en membrana de las proteínas. Además, nuestros resultados explican la influencia que algunos mutantes somáticos ejercen en la regulación de la apoptosis. En general, los resultados científicos que aparecen en esta tesis resaltan el papel de los Bcl TMDs en el interactoma de las proteínas Bcl-2. Estos hallazgos corroboran que las interacciones laterales entre los TMDs son específicas y contribuyen activamente a la funcionalidad de la proteína. Por lo tanto, comprender los Bcl TMDs puede proporcionar nuevos conocimientos sobre la biología de las proteínas Bcl.
[CA] La família de proteïnes Bcl-2 regula l'apoptosi a través d'una complexa xarxa d'interaccions. Les cèl·lules tumorals solen presentar mutacions que afecten la seua expressió o les seues interaccions per a millorar la progressió tumoral. A més, alteracions en la seua regulació també promouen la migració de cèl·lules cancerígenes, la invasió i la metàstasi. Per a dur a terme les seues funcions, les proteïnes Bcl-2 interaccionen entre si tant en el citoplasma com en les membranes intracel·lulars. Els equilibris d'interacció dels dominis Bcl citosòlics s'han investigat àmpliament i recentment, s'han proposat com a dianes terapèutiques. No obstant això, l'interactoma dels dominis transmembrana (TMD, de l'anglés transmembrane domains) continua sent poc conegut. Per això, un coneixement profund de la biologia de les proteïnes Bcl-2 és necessari per a explotar eficientment les seues superfícies d'unió en el tractament del càncer. Per a dur a terme aquest objectiu, ens hem centrat en tres àrees: 1. La comprensió detallada de la contribució del TMD de Mcl-1 al seu interactoma en membrana i la seua funció. 2. El descobriment de nous inhibidors de Mcl-1 que actuen sobre el seu TMD i que permeten desenvolupar una classe de drogues anticanceroses encara per explorar. 3. La caracterització molecular de mutacions relacionades amb el càncer descrites en els TMD de Bcl-2 i Bcl-xL i les seues implicacions en la supervivència de les cèl·lules tumorals. La proteïna anti apoptòtica Mcl-1 inhibeix als membres pro apoptòtics Bak, Bax, Bok, Noxa, etc. Encara que s'ha estudiat detalladament la seua activitat promovent la supervivència cel·lular, el mecanisme molecular pel qual prevé l'apoptosi mediada per Bok encara no és clar. A més, el coneixement de les activitats de Mcl-1, descrites fins ara, es basa exclusivament en les estructures resoltes solubles en aigua i en estudis centrats en els dominis externs a la membrana. Per primera vegada, hem demostrat la rellevància del TMD de Mcl-1 el seu equilibri d'interacció. En aquest treball descrivim la seua capacitat específica per a unir-se amb si mateix i per a hetero-oligomeritzar amb el TMD de Bok. També expliquem la influència d'aquestes interaccions en l'apoptosi i ressaltem la rellevància clínica dels mutants del TMD de Mcl-1 identificats en pacients amb càncer. Molts tumors hematològics i sòlids sobre-expressen Mcl-1 com un mecanisme per a adquirir quimioresistència. S'han desenvolupat mimètics de BH3 específics per a modular la seua activitat anti apoptòtica en cèl·lules canceroses. No obstant això, encara no disposem de dades científiques que informen sobre la seua toxicitat i eficàcia en humans. Per això, proposem la nova interacció dels TMDs de Mcl-1 i Bok com un lloc d'actuació de fàrmacs quimioterapèutiques. Hem identificat tres inhibidors d'aquesta interacció amb característiques que els fan prometedors candidats per al desenvolupament farmacèutic, així com bones eines moleculars per a estudiar la interacció dels TMDs de Mcl-1 i Bok. Per a modular l'apoptosi, les cèl·lules tumorals també presenten versions mutades de les proteïnes anti apoptòtiques Bcl-2 i Bcl-xL. En el nostre coneixement, aquest és el primer estudi que analitza mutacions somàtiques de les seues TMDs. El nostre treball demostra com aquestes mutacions alteren l'equilibri en membrana de les proteïnes. A més, els nostres resultats expliquen la influència que alguns mutants somàtics exerceixen en la regulació de l'apoptosi. En general, els resultats científics que apareixen en aquesta tesi ressalten el paper dels Bcl TMDs en l'interactoma de les proteïnes Bcl-2. Aquestes troballes corroboren que les interaccions laterals entre els TMDs són específiques de la seqüència i contribueixen activament a la funcionalitat de la proteïna. Per tant, comprendre els Bcl TMDs pot proporcionar nous coneixements sobre la biologia de les proteïnes Bcl
[EN] The family of the Bcl-2 proteins modulates the apoptotic pathway by a complex network of interactions. Tumor cells frequently present mutations that affect Bcl-2 proteins expression or interactions to enhance cancer progression. Dysregulation of these proteins also promotes cancer cell migration, invasion, and metastasis. To execute their functions, Bcl-2 proteins interact in both the cytosol and intracellular membranes. Binding equilibria of Bcl extramembrane domains has been largely investigated and recently proposed as chemotherapeutic targets. However, the interactome of transmembrane domains (TMDs) remains poorly understood. In this scenario, a deep knowledge of the biology of Bcl-2 proteins is needed to exploit efficiently their binding surfaces for cancer treatment. To address this aim, our research focuses on three areas: 1. The detailed comprehension of the TMD contribution to both the Mcl-1 membrane interactome and protein functionality. 2. The discovery of new Mcl-1 inhibitors that target the transmembrane surface to develop a class of anticancer drugs currently unexplored. 3. The molecular characterization of cancer-related mutations within the Bcl-2 and Bcl-xL TMDs and their implications for the survival of cancer cells. Antiapoptotic Mcl-1 protein inhibits the proapoptotic members Bak, Bax, Bok, and Noxa, among others. Although its prosurvival activity has been well studied, the molecular mechanism to prevent Bok-mediated apoptosis remains unclear. Furthermore, understanding of Mcl-1 activities described to date is only based on water-soluble structures and studies focused on extramembrane domains. For the first time, we uncover the relevance of the Mcl-1 TMD in the interaction equilibria of the protein. In the present work, we describe its specific capacity to self-associate and hetero-oligomerize with the Bok TMD. We also explain the influence of these interactions in the apoptotic pathway and highlight the clinical relevance of Mcl-1 TMD mutants identified in tumor patients. Many hematological and solid malignancies overexpress Mcl-1 as an acquired chemoresistance mechanism. To modulate its antiapoptotic activity in cancer cells, specific BH3 mimetics have been developed; however, there is no scientific data yet regarding human toxicity and efficacy. In this work, we propose the novel Mcl-1 and Bok TMDs interaction interface as a drugging site in the development of chemotherapeutics. We identify three potential inhibitors of such molecular interface with promising features to become both drug candidates for pharmaceutical development and research toosl for the molecular study of the Mcl-1 and Bok TMDs interaction. To take advantage of apoptosis modulation, tumor cells also present mutated versions of the antiapoptotic members Bcl-2 and Bcl-xL. To our knowledge, this is the first study that analyzes patient-derived mutations within Bcl-2 and Bcl-xL TMDs and demonstrates how said mutations alter the membrane equilibria of these proteins. The results presented here also explain the functional influence of some somatic mutants in apoptosis regulation. Overall, the scientific results exhibited in this Thesis highlight the role of Bcl TMDs in the interactome of Bcl-2 proteins. These findings corroborate that lateral interactions between TMDs are sequence-specific and actively contribute to protein functionality. Therefore, understanding of Bcl transmembrane segments may provide new insights into the biology of Bcl 2 proteins for their pharmaceutical modulation in antitumoral therapy.
The student has been granted with a PhD fellowship and a short-term fellowship from the Generalitat Valenciana (Subvenciones para la contratación de personal investigador de carácter predoctoral, 2016-2019, and Grant for predoctoral stays out of the Comunitat Valenciana, 2019). This work has been supported by the Spanish Ministry of Economy and Competitiveness (projects SAF2014-52614-R and SAF2017-84689-R
Lucendo Gutiérrez, E. (2020). Understanding and drugging the Bcl-2 transmembrane interactome for tumor treatment [Tesis doctoral]. Universitat Politècnia de València. https://doi.org/10.4995/Thesis/10251/155914
TESIS

Orientador: Lucia Pereira da Silva
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Neste trabalho estudamos o papel dos íons de ferro na peroxidação lipídica induzida por FeSO4 em mitocôndrias isoladas de fígado de rato (MFR). Na literatura aparecem duas teorias distintas para explicar o papel dos íons de ferro na iniciação da peroxidação lipídica. A primeira propõe que o ferro é o catalisador das reações de Haber-Weiss e de Fenton que produzem o radical hidroxila (OR) que atacaria os ácidos graxos polinsaturados das membranas. Uma segunda hipótese contesta a primeira, por considerar que o 'OH é muito reativo para difundir-se dos sítios onde é formado até a parte hidrofóbica da membrana, onde estão os ácidos graxos passíveis de ataque. Neste caso, a proposta é que a peroxidação lipídica seria induzida por complexos radicalares fonnados por Fe2+, Fe3+ e O2. Estas espécies teriam reatividade suficiente para abstrair átomos de hidrogênio das pontes metilênicas entre as duplas ligações dos ácidos graxos polinsaturados. Nossos resultados. mostram que a peroxidação lipídica induzida por FeSO4 causa alterações na fluidez das membranas mitocôndriais e é dependente de uma relação Fe2+/Fe3+ equimolar para atingir seu efeito máximo. Fatores que levem a um aumento na velocidade de oxidação do Fe2+ a Fe3+ pelo O2 contribuem para um aumento significativo da lipoperoxidação. Entre esses estão a presença de H2O2, o vazamento de elétrons quando as mitocôndrias estão energizadas, e íons como Pi presentes no meio de reação. Os experimentos em que a peroxidação lipídica foi inibida pelo sequestrador de radicais butilhidroxitolueno (BHT) permitem afirmar que a iniciação do processo peroxidativo depende da geração de uma espécie radicalar além da presença de FeSO4. O fato do manitol, conhecido sequestrador do radical hidroxila, não ter apresentado efeito inibitório sobre essa peroxidação, somado à observação anterior, permite descartar a hipótese de ser o radical hidroxila o iniciador do processo. Os resultados aqui apresentados, corroboram a hipótese de que complexos radicalares de valências mistas de ferro e 02 seriam os iniciadores da peroxidação lipídica induzida por FeSO4 em mitocôndrias isoladas, nas condições por nós estudadas
Abstract: Here we have studied the role of iron ions on the mitochondrial lipid peroxidation induced by FeSO4 in isolated rat liver mitochondria. There are two hypothesis to explain the role of iron ions on the initiation of this peroxidative process. The first one suggests that iron catalyses Haber Weiss and Fenton reactions generating the hydroxyl radical (OR). This later would attack the polly saturated fatty acids of the membrane phospholipids. The second explanation discards a direct participation of OIL as it is a very reactive species to difuse from its generating site to the hydrophobic part of the membrane, where are situated the fatty acids. In this case, the proposal is that the peroxidative process would be initiated by complexes of Fe2+ and Fe3+ and O2. These species would be reactive enough to react with the hydrogen from the methylenic bridges between the polly saturated fatty acid double bonds. Our results show that the FeS04-induced lipid peroxidation causes alterations on the mitochondrial membrane fluidity and the maximum effect is reached when an equimolar Fe2+/Fe3+ relation is achieved. Conditions favouring an increase on the Fe2+ to Fe3+ oxidation by O2 contribute to a significant increase on lipid peroxidation. Among these are the presence of H2O2, the electron leak occuring when energized mitochondria were used and phosphate ions in the reaction medium. The experiments showing that the peroxidation is inhibited by butylhydroxytoluene (BHT), a known radical scavenger, lead to the conclusion that to initiate the peroxidative process a radical should be generated besides the presence of FeSO4. As mannitol was found unable to inhibit this process and it is known that mannitol scavenges OH, the hypothesis that hydroxil is the radical species needed to initiate this process can be discarded by our results. Moreover, our data support the proposal that complexes of iron mixed valences and O2 would be the real initiators of the lipid peroxidation induced by FeSO4 in isolated mitochondria
Mestrado
Bioquimica
Mestre em Ciências Biológicas

El documento digital no refiere asesor
El objetivo de este estudio de caso fue conocer el tratamiento odontológico integral bajo anestesia general de un paciente con encefalopatía necrosante subaguda o enfermedad mitocondrial ó síndrome de Leigh, con el fin de restablecer la salud oral en el paciente, brindar orientación y promoverle motivación a la madre/cuidador principal. Se reportó a un paciente de género femenino de 12 años portador de esta patología, se analizó sus antecedentes médicos y odontológicos, se realizó la exploración física y clínica, se revisó los exámenes médicos. Fue una paciente renuente a la consulta odontológica, manifestada por el dolor espontáneo en las primeras molares inferiores (1MP) cuyo estado era de deterioro dental y dolor provocado en los incisivos centrales superiores (ICS) que evidenciaba cambio de coloración. El diagnóstico odontológico fue enfermedad gingival debido a acúmulo de placa bacteriana, remanentes radiculares de 1MP inferiores, necrosis pulpar de ICS, presencia de lesiones de caries y anomalías dentomaxilofaciales. Debido a su cuadro clínico estomatológico, su conducta y su condición sistémica, se la consideró como un paciente negativo por lo que se optó por el abordaje farmacológico. La patogénesis de la enfermedad fue discutida, se estudiaron reportes del cuidado operativo y se hizo revisión de literatura. Los datos sugieren el cuidado y el mantenimiento de la salud oral luego de la intervención odontológica que responde a la atención primaria renovada, no sólo, como programa preventivo-promocional en odontología para pacientes con compromiso neuro-psico-motor y en este caso particular de Síndrome de Leigh. Aunque existe controversia respecto a elegir qué tipo de tratamiento farmacológico debido a factores condicionantes, en el presente reporte se utilizó la anestesia general como alternativa de abordaje anestésico seguro porque no se consiguió el grado de cooperación por el cuadro de alta complejidad odontológica con calidad de atención y aceptación de los padres.
Trabajo académico

Las abejas son insectos sociales de gran interés económico y ecológico, la disminución de su población ha sido registrada durante las últimas tres décadas. Dentro de este grupo de insectos existen tres tipos de abejas melíferas: reina, obreras y zánganos, ellas tienen diferentes papeles en una colonia, siendo de mayor interés en este estudio la evaluación de espermatozoides de los zánganos pre y post criopreservación para su uso en programas de inseminación artificial (IA) de abejas reina. El objetivo de la investigación fue acondicionar dos métodos de evaluación de los parámetros espermáticos que permitan seleccionar de forma rigurosa grupos de abejas con mejores cualidades reproductivas. Se evaluaron los parámetros espermáticos convencionales (movilidad, integridad de membrana y concentración espermática) pre y post criopreservación de la temporada artificial y natural a partir de las vesículas seminales de 489 zánganos pertenecientes a la Asociación de Apicultores Agroecológicos “Abejas de Pichanaki”. Se registró una correlación positiva entre los parámetros convencionales y la actividad mitocondrial, p<0.05 y una correlación negativa entre los parámetros convencionales y los niveles de estrés oxidativo, p>0.05 en ambas temporadas. Los espermatozoides mostraron diferencias luego de la criopreservación en la movilidad y la integridad de membrana (p<0.05) aunque no en la actividad mitocondrial y el estrés oxidativo. Se reporta una diferencia significativa de las muestras pre criopreservadas entre las temporadas en la movilidad, actividad mitocondrial y nivel de estrés oxidativo (p<0.05) aunque no en la integridad de membrana. Se concluye entonces que es posible considerar a la actividad mitocondrial como una prueba para caracterizar mejor la calidad espermática.
Tesis

Esta tesis se basa en diagnóstico de pacientes con deficiencia de 2 metil 3 hidroxibutiril CoA deshidrogenasa (MHBD o HSD10) y en el estudio de la fisiopatología de esta enfermedad y de su implicación en la Enfermedad de Alzheimer (EA). El desarrollo de un método cuantitativo de metabolitos clave en orina y un método de análisis enzimático de la proteína HSD10 en fibroblastos cultivados, así como el análisis molecular, de los niveles de HSD10 y de los estudios patogenicidad de las mutaciones encontradas, nos ha permitido el diagnóstico de 6 pacientes y 8 portadoras, que representan el 37% de los pacientes descritos en la literatura. La herencia de esta enfermedad se halla ligada al cromosoma X. Se había descrito que el gen HSD17B10, que codifica para la proteína HSD10, se escapaba de la inactivación del cromosoma X. Sin embargo, el fenotipo bioquímico y clínico de nuestros pacientes no apoyaban este hallazgo. Mediante estudios de expresión de HSD17B10 por PCR cuantitativa se demostró que HSD17B10 no escapaba a la inactivación del cromosoma X. HSD10 es una proteína mitocondrial multifuncional que además de su papel en el metabolismo de la isoleucina y de los ácidos grasos, desempeña otras funciones en el metabolismo de hormonas esteroideas sexuales, esteroides neuroactivos, en la detoxificación de aldehídos citotóxicos y en la metabolización de cardiolipina peroxidada. La clínica neurológica de los pacientes con deficiencia de HSD10 difiere de otras deficiencias de la vía metabólica de la isoleucina. Por ello, realizamos estudios de microarrays de expresión. Obtuvimos una expresión significativamente diferente en 31 genes. Los resultados se confirmaron mediante PCR cuantitativa. La diferencia de expresión de estos genes podrían explicar los síntomas clínicos de los pacientes con deficiencia de HSD10. La proteína HSD10 parece estar implicada en la EA, debido a su interacción con el péptido β amiloide (pßA), habiéndose sugerido que dicha interacción es una de las causas de la disfunción mitocondrial en la EA. Para determinar la implicación de HSD10 en la EA se realizaron una serie de estudios en homogenados de cerebro de pacientes con EA y controles. Encontramos una disminución de la actividad de HSD10 en homogenados de cerebro de pacientes con EA y esta disminución no se debía a una disminución de los niveles de proteína, ya que el wester blot no reveló diferencias entre pacientes y controles. Se observó también que la actividad HSD10 disminuye con la progresión de la enfermedad. Por otro lado, comprobamos que la incubación de homogenados de cerebro control con pßA provocaba una inhibición de la actividad HSD10, que se rescataba al incubar con elevadas concentraciones de NAD +. Esta observación también fue corroborada en la EA. Este hecho se podía explicar por una interacción competitiva entre NAD+ y pßA, ya que ambos interaccionan con HSD10 en la misma posición aminoacídica. Por ello, al incubar con elevadas concentraciones NAD+ se inhibe la interacción pßA HSD10. Por tanto, la medición de la actividad HSD10 podría ser utilizada como diana terapéutica para la EA. Con esta finalidad, obtuvimos un sistema “in vitro” con la valoración de la proteína recombinante HDSD10 isoforma 1, que es la que expresa mayoritariamente en cerebro humano, antes y después de la incubación con pßA, con y sin adición de NAD+. Los resultados obtenidos fueron los mismos que al utilizar homogenados de cerebro humano. Por lo tanto, quedaba validado un sistema para cribar moléculas terapéuticas que pudieran inhibir la interacción pßA HSD10 y restablecer la actividad HSD10. Se probaron 117 compuestos de una librería peptídica, Coenzima Q10 y otros antioxidantes, pero no se consiguió rescatar la actividad HSD10. La actividad enzimática sólo se consiguió rescatar con NAD+ . Así, proponemos que los precursores de NAD+ podrían ser considerados una terapia coadyuvante en la EA. Estudios recientes apoyan nuestros resultados, ya que la administración de precursores de NAD+ a ratones modelo para la EA, mejoran la disfunción mitocondrial así como la cognición.
This thesis is based on diagnosis of patients with 2 methyl 3 hydroxybutyryl CoA dehydrogenase (MHBD or HSD10) deficiency and the study of its pathophysiology and its involvement in Alzheimer's Disease (AD). The development of different methods for quantification of key metabolites in urine and for enzymatic analysis in cultured fibroblasts, as well as the molecular studies performed, has allowed us the diagnosis of 6 patients and 8 carriers, which represents 37% of the patients described in the literature. Expression studies of HSD17B10 demonstrated that this gen not escapes inactivation of the X chromosome, contrary to that published by other authors. The neurological symptoms of patients with deficiency HSD10 differs from other deficiencies in the isoleucine's metabolic pathway, maybe due to the alteration of other functions involved in both, brain and mitochondrial homeostasis. The expression "microarray" studies performed revealed significantly different expression in 31 genes, which could explain the clinical symptoms of the patients. The HSD10 protein appears to be involved in AD, due to their interaction with β amyloid peptide (Aβ). Studies in brain homogenates of AD patients showed decreased HSD10 activity from the early stages of the disease, which could explain the early mitochondrial dysfunction observed in AD. Furthermore, the Aβ incubation caused inhibition of HSD10 activity in control brain homogenates, which was rescued by incubating with high NAD+ concentrations. This observation was also corroborated in AD. This fact could be explained by a competitive interaction that occurs between NAD+ and Aβ. Therefore, the measurement of HSD10 activity could be used as a therapeutic target for AD. To this end, we obtained "in vitro" system to the evaluation of the HDSD10 isoform 1 recombinant protein, which is the mainly expressed in human brain, before and after incubation with Aβ, with and without addition of NAD+. The results were the same as when using human brain homogenates. Therefore, the system was validated for screening therapeutic molecules that could inhibit the Aβ HSD10 interaction and restore HSD10 activity. Among 117 compounds tested, only NAD+ was able to rescue HSD10 activity. Thus, we propose that the precursors of NAD+ could be considered an adjunctive therapy in AD. Recent studies support our results, since administration of NAD precursors mice model for AD, improve mitochondrial dysfunction and cognition.

Orientador: Benedito de Oliveira Filho
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A crotoxina, isolada e cristalizada em 1938 por Slotta e Fraenkel-Conrat a partir do veneno total de Crotalus durissus terrificus mostrou ser um complexo resultante da interação quaternária de duas sub-unidades representadas por uma proteína ácida com massa molecular de 9 kDa denominada crotapotina e um componente básico, com massa molecular de 14,5 kDa que foi identificado como sendo uma fosfolipase A2 (PLA2 ). Esta última é considerada o principal componente responsável pelo desencadeamento dos efeitos farmacológicos induzidos pelo complexo crotoxina. Estudos mais recentes demonstraram a ocorrência de várias isoformas de PLA2, presentes em diferentes lotes de veneno coletados de várias serpentes, sendo tais isoformas decorrentes da expressão de diferentes RNAs mensageiros. As PLA2 (EC 3.1.1.4.) são enzimas que catalisam especificamente a reação de hidrólise da ligação acil-ester na posição sn-2 de fosfoglicerídeos numa reação dependente de álcio, liberando quantidades equimolares de ácidos graxos livres e lisofosfolipídeos. Já foi demonstrado que trações puras de PLA2 apresentam efeitos inibitórios marcantes sobre várias funções mitocondriais, reduzindo as atividades da NADH oxidase, das succinato e NADH-desidrogenase e da capacidade de fosforilação. Além disso, as PLA2 podem modificar a microviscosidade da fase lipídica da bicamada hidrofóbica de membranas, afetando a atividade funcional de enzimas ligadas à membrana, e, através desse mecanismo, regular vários processos metabólicos. No presente trabalho purificamos e caracterizamos, a partir da crotoxina, três isoformas de PLA2 (F1, F2 e F3) com elevado grau de pureza. As isoformas apresentaram massa molecular aparente com valor igual a 15 kDa e alta homologia seqüencial dos 20 resíduos N-terminais em comparação a isoformas já descritas na literatura. o efeito de cada isoforma de PLA2 purificada sobre mitocôndrias isoladas de fígado de rato foi determinado através de consumo de oxigênio durante o estado respiratório 4 e de inchamento mitocondrial. FI apresentou um estímulo dose dependente do consumo de oxigênio enquanto F2 e F3 causaram estímulo apenas em baixas concentrações e inibição em quantidades maiores. Esses efeitos foram completamente abolidos quando soro albumina bovina a 0,1% ou EGTA a 0,5mM estavam presentes no meio de incubação. Usando-se o inchamento mitocondrial como um parâmetro comparativo, todas as isoformas apresentaram o mesmo comportamento com intensidades diferentes, levando à permeabilização da membrana mitocondrial. Neste caso, a adição de EGTA preveniu o inchamento enquanto a soro albumina bovina foi ineficiente, indicando que o microambiente lipídico foi realmente afetado. Esses resultados sugerem que ácidos graxos livres liberados pela ação das isoformas são diretamente responsáveis pelos efeitos observados nos experimentos de consumo de oxigênio. A proteção oferecida pela CSA contra o inchamento causado pelas isoformas, principalmente quando estas estavam presentes em baixas concentrações, sugere que a ligação da CSA a um sítio da membrana mitocondrial protege esta última contra o ataque das PLA2. Já a pequena proteção oferecida pelo CAT, bem como o prevalecimento do pequeno efeito protetor do CAT quando utilizamos simultaneamente CSA e CAT, sugerem que a ligação do CAT ao seu sítio no carreador ADP/ATP impede a proteção conferida pela CSA
Abstract: Crotoxin, isolated and cristalized in 1938 by Slotta and Fraenkel-Conrat from Crotalus durissus terrificus venom showed to be a complex resultant from the quaternary association between two sub-units: an acidic protein with a molecular mass of 9 kDa named crotapotin and a basic component, with a molecular mass of 14.5 kDa which was identified as a phospholipase A2 (PLA2). This last one is considered to be the main component responsible for the pharmacological effects induced by the crotoxin complex. Recent studies have demonstrated the existence of several PLA2 isoforms which were present in different venom batches collected from several snakes. These isoforms result from the expression of different menssager RNAs. The PLA2 (EC 3.1.1.4) are enzimes that specifically catalyse the hydrolysis of the acyl-ester bond at the sn-2 position of phosphoglycerides in a calcium dependent reaction, producing equimolar amounts of ftee fatty acids and Iysophospholipids. It has already been demonstrated that purified PLA2 fractions show marked inhibitory effects on several mitochondrial features, decreasing NADH-oxidase, succinate and NADH-dehydrogenase activities and phosphorylation capacity. It is also known that PLA2 can modify the microviscosity of the lipid phase of the hydrophobic membrane bilayer, affecting the functional activity of membrane-bound enzymes, and through this mechanism regulate various metabolic processes. In the present work, we have purified and characterized, from crotoxin, three PLA2 isoforms (F1, F2 and F3) with high degree of purity. The isoforms presented an apparent molecular mass of 15 kDa and a high degree of homology regarding the 20 N terminal aminoacid residues when compared to other isoforms already described in the literature. The effect of each purified phospholipase A2 isoform on isolated rat liver mitochondria was determined through mitochondrial swelling and Oz consumption during respiratory state 4. FI showed a dose-dependent stimulation of O2 consumption while F2 and F3 caused stimulation only at low doses and inhibition at higher amounts. These effects were completely suppressed by the presence of 0.1% bovine serum albumin or 0.5mM EGTA in the incubation medium. Taking the mitochondrial swelling as a comparative parameter, alI of them presented the same behaviour at different intensities, leading to permeabilization of the mitochondrial membrane. In this case, addition of EGTA prevented it whereas bovine serum albumin was ineffective, indicating that the lipid microenvironrnent was actualIy,affected. These results suggest that free fatty acids must be directly responsible for the observed effects induced by hospholipase A2 isoforms on oxygen consumption experiments. The protection confered by cyclosporin-A on swelling induced by the isoforms, mainly when they were present in low concentrations, suggests that cyclosporin-A binding to a mitochondrial membrane site protects the membrane against the phospholipase A2 attack. On the other hand, the smalI protection confered by CAT, as well as the prevailment of the small protective CAT effect when we used both CSA and CAT, suggest that the CAT binding to the ADP/ ATP carrier inhibits the protection confered by CSA
Mestrado
Bioquimica
Mestre em Ciências Biológicas

L'etiologia de l'esquizofrènia és encara desconeguda però hi ha evidències que l'ADN mitocondrial (ADNmt), que s'hereta exclusivament a través de la mare, està implicat en el desenvolupament d'aquesta malaltia. La present tesi doctoral es va realitzar basant-se en aquesta hipòtesi. En el primer treball es van recollir totes les evidències d'herència materna, disfunció mitocondrial i alteracions de l’ADNmt associades a l'esquizofrènia i es va identificar la presència de simptomatologia psicòtica en pacients amb un trastorn mitocondrial causat per una mutació en l'ADNmt. El segon treball va analitzar el risc de presentar esquizofrènia i altres malalties psiquiàtriques en familiars, considerant si compartien o no l'ADNmt amb el pacient. Els familiars que compartien l'ADNmt amb un pacient tenien més risc de presentar esquizofrènia i, a més, les dones tenien més risc de presentar depressió, trastorns d'ansietat i característiques clíniques associades a trastorns mitocondrials. El tercer estudi va comparar les característiques clíniques freqüentment presents en les malalties mitocondrials entre un grup de pacients amb esquizofrènia i un grup control. Es va observar que la fatiga crònica i les crisis epilèptiques eren significativament més freqüents en el grup de pacients. Les conclusions d'aquesta tesi doctoral són les següents: 1) Hi ha evidències de disfunció mitocondrial i d'herència materna en l'esquizofrènia, i la simptomatologia psicòtica es pot presentar en pacients amb un trastorn mitocondrial causat per una mutació en l'ADNmt; 2) Els familiars que comparteixen l'ADNmt amb un pacient d'esquizofrènia tenen més risc de presentar la malaltia, i en dones, compartir l'ADNmt amb un pacient d'esquizofrènia confereix risc per desenvolupar altres malalties psiquiàtriques com la depressió i els trastorns d'ansietat; i 3) Característiques clíniques associades a les malalties mitocondrials són més freqüents en els pacients amb esquizofrènia que en la població control.
La etiología de la esquizofrenia es aún desconocida pero hay evidencias de que el ADN mitocondrial (ADNmt), que se hereda exclusivamente a través de la madre, está implicado en el desarrollo de esta enfermedad. La presente tesis doctoral se realizó basándose en esta hipótesis. En el primer trabajo se recogieron todas las evidencias de herencia materna, disfunción mitocondrial y alteraciones del ADNmt asociadas a la esquizofrenia y se identificó la presencia de sintomatología psicótica en pacientes con un trastorno mitocondrial causado por una mutación en el ADNmt. El segundo trabajo analizó el riesgo de presentar esquizofrenia y otras enfermedades psiquiátricas en familiares, considerando si compartían o no el ADNmt con el paciente. Los familiares que compartían el ADNmt con un paciente tenían más riesgo de presentar esquizofrenia y, además, las mujeres tenían más riesgo de presentar depresión, trastornos de ansiedad y características clínicas asociadas a trastornos mitocondriales. El tercer estudio comparó las características clínicas frecuentemente presentes en las enfermedades mitocondriales entre un grupo de pacientes con esquizofrenia y un grupo control. Se observó que la fatiga crónica y las crisis epilépticas eran significativamente más frecuentes en el grupo de pacientes. Las conclusiones de esta tesis doctoral son las siguientes: 1) Existen evidencias de disfunción mitocondrial y de herencia materna en la esquizofrenia, y la sintomatología psicótica puede presentarse en pacientes con un trastorno mitocondrial causado por una mutación en el ADNmt; 2) Los familiares que comparten el ADNmt con un paciente de esquizofrenia tienen más riesgo de presentar la enfermedad, y en mujeres, compartir el ADNmt con un paciente de esquizofrenia confiere riesgo para desarrollar otras enfermedades psiquiátricas como la depresión y los trastornos de ansiedad; y 3) Características clínicas asociadas a las enfermedades mitocondriales son más frecuentes en los pacientes con esquizofrenia que en la población control.
The etiology of schizophrenia is unknown but there is evidence that mitochondrial DNA (mtDNA), which is inherited exclusively from the mother, is involved in the development of this disease. This thesis was conceived based on this assumption. In the first study all evidence of maternal inheritance, mitochondrial dysfunction and mtDNA alterations associated with schizophrenia were collected, and the presence of psychotic symptoms in patients with mitochondrial dysfunction caused by mutations in mtDNA were identified. The second study analyzed the risk of schizophrenia and other psychiatric disorders in relatives, considering if they shared or did not share mtDNA with the patient. Family members who shared mtDNA with a patient had a higher risk of developing schizophrenia and, moreover, women were more likely to develop depression, anxiety disorders and clinical features associated with mitochondrial disorders. The third study compared the clinical features often present in mitochondrial diseases between schizophrenia patients and control subjects. It was observed that chronic fatigue and seizures were significantly more frequent in the group of patients. The conclusions of this thesis are: 1) There is evidence of mitochondrial dysfunction and maternal inheritance in schizophrenia and psychotic symptoms can occur in patients with a mitochondrial disorder caused by a mtDNA mutation; 2) Family members who share mtDNA with a schizophrenic patient have higher risk of developing the disease than those who do not share mtDNA and, in women, to share mtDNA with a schizophrenic patient confers risk for other psychiatric illnesses such as depression and anxiety disorders; and 3) Clinical features associated with mitochondrial disorders are more common in patients with schizophrenia than in the control population.

El síndrome de Down constituye la cromosomopatía más frecuente que ocurre en uno de cada 700 a 1000 nacimientos y está causado por la trisomía completa o por una parte del cromosoma 21 humano (HSA21). Aún se desconoce cómo la presencia del cromosoma 21 extra da lugar al fenotipo característico de este síndrome. En este sentido la participación de las especies reactivas de oxígeno (ROS) ha sido propuesta como uno de los mecanismos que intervienen en la patogénesis del mismo. Dicho mecanismo se fundamenta en la sobreexpresión de al menos 16 genes del HSA21 relacionados con el metabolismo de las especies reactivas de oxígeno (ROS) y con la generación de energía mitocondrial. Uno de estos genes es el que codifica una importante enzima del sistema antioxidante celular, el gen SOD1, propuesto como potencial culpable del estrés oxidativo inusual en los individuos con SD. En condiciones normales, los ROS, producidos in vivo principalmente por la respiración aeróbica, se eliminan de la célula por la acción de las enzimas antioxidantes, superóxido dismutasa (SOD), catalasa (CAT) y glutatión peroxidasa (GPx). La Cu/Zn superóxido dismutasa (SOD1) convierte el radical superóxido a peróxido de hidrógeno, el cual es eliminado por la glutatión peroxidasa y/o catalasa a agua y oxígeno. La sobreexpresión de la SOD1 puede producir un desequilibrio en la relación de las enzimas antioxidantes (SOD1, GPx y CAT) generando estrés oxidativo y podría resultar en el daño oxidativo a biomoléculas tales como ácidos grasos poliinsaturados en los lípidos de las membranas, proteínas esenciales y el DNA, ya que existe una variabilidad en los niveles de enzimas antioxidantes dentro de la población con SD, lo que puede estar relacionado con una desregulación compleja que afecte no sólo a los genes del HSA21 sino también en otros cromosomas. Así, el daño celular puede ser inducido por los ROS y asociarse a algunas de las alteraciones celulares en el SD, causando diversas patologías y conducir a un envejecimiento prematuro. Se obtuvieron 18 muestras de fibroblastos primarios fetales humanos, 9 de ellos con síndrome de Down (FT21) y 9 controles (FC), en los cuales se evaluó la disminución de la capacidad endógena antioxidante debido a la sobreexpresión de la SOD1, causando un exceso en la producción intracelular de ROS y el origen prematuro de estrés oxidativo asociado al daño oxidativo a lípidos y proteínas, así como a una disfunción mitocondrial. Se analizaron varios marcadores de senescencia celular con la finalidad de contribuir al conocimiento de un nuevo aspecto de la patología de este síndrome, el envejecimiento prematuro. Estos mecanismos fisiopatológicos podrían estar relacionados con la aparición y establecimiento de la senescencia celular prematura en los fibroblastos con trisomía del cromosoma 21 (FT21).
Down syndrome is the most common chromosomal disorder that occurs in 1 in 700 to 1,000 births and is caused by trisomy full or part of human chromosome 21 (HSA21). It is still unknown how the presence of the extra chromosome 21 results in the phenotype of this syndrome. In this sense the involvement of reactive oxygen species (ROS) has been proposed as one of the mechanisms involved in the pathogenesis of same. This mechanism is based on the overexpression of at least 16 genes related HSA21 metabolism of reactive oxygen species (ROS) and mitochondrial energy generation. One of these genes encoding is an important cellular antioxidant enzyme system, the SOD1 gene, proposed as a potential culprit unusual oxidative stress in individuals with DS. Under normal conditions, the ROS produced in vivo mainly by aerobic respiration of the cells are removed by the action of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). The Cu/Zn superoxide dismutase (SOD1) converts the superoxide radical to hydrogen peroxide, which is eliminated by glutathione peroxidase and/or catalase into water and oxygen. SOD1 overexpression can produce an imbalance in the ratio of antioxidant enzymes (SOD1, GPx and CAT) generating oxidative stress and may result in oxidative damage to biomolecules such as polyunsaturated fatty acids in the membrane lipids, proteins and essential DNA, since there is a variability in the levels of antioxidant enzymes in the DS population, which can be related to a complex deregulation affects not only Hsa21 genes but also on other chromosomes. Thus, cell damage can be induced by ROS and associated with some of the cellular changes in the DS, causing various diseases and lead to premature aging. Eighteen samples were obtained from primary human fetal fibroblasts, 9 with Down syndrome (TF21) and 9 normal (NF), which was evaluated in decreasing endogenous antioxidant capacity due to overexpression of the SOD1, causing excess in intracellular production of ROS and oxidative stress origin associated premature oxidative damage to lipids and proteins, as well as mitochondrial dysfunction. We analyzed several markers of cellular senescence in order to contribute to the knowledge of a new aspect of the pathology of this syndrome, premature aging. These pathophysiological mechanisms may be related to the emergence and development of premature cellular senescence in fibroblasts with trisomy 21 (FT21).

Intrauterine growth restriction (IUGR) is an obstetric complication characterized by placental insufficiency and secondary cardiovascular remodeling that may lead to cardiomyopathy in adulthood. Its etiology and potential therapeutics are poorly understood. Mitochondrial bioenergetics pathways are mainly regulated by nuclear effectors such as sirtuins and are essential for embryonic development and cardiovascular function. Members of our group developed a rabbit model of IUGR and cardiovascular remodeling, in which heart, mitochondrial alterations were observed by microscopic and transcriptomic analysis. We aimed to evaluate if such alterations are translated at a functional mitochondrial level to establish the ethiopathology and potential therapeutic targets for this obstetric complication. For that aim, heart and placenta from the rabbit model was included as well as placenta from human pregnancies together with maternal and neonatal blood. At delivery, peripheral blood and cord blood mononuclear cells (PBMC and CBMC, respectively) were isolated. For the mitochondrial characterization, we assessed: oxygen consumption of the mitochondrial respiratory chain (MRC) by polarography using endogen cellular substrates and substrates for complex I. Also, enzymatic activity of complex I, II, IV, I+III and II+III of MRC, subunit protein expression of some of the MRC complexes (CII-SDHA, CII-SDHB and CIV-COX5A), Coenzyme Q levels, mitochondrial content (through citrate synthase activity, Tom20 expression or mitochondrial DNA (mtDNA) levels), oxidative stress (by lipid peroxidation and SOD2 activity) and ATP levels. Finally, Sirtuin 3 protein expression was measured by Western Blot. In the IUGR offspring from the rabbit model, we found a significant decrease of MRC function: enzymatic activity of complexes II, IV and II+III in IUGR hearts (p<0.05) and complexes II and II+III in IUGR placentas (p<0.05 and p<0.01, respectively). This was occurring with a not significant reduction in CI-stimulated oxygen consumption in both tissues and a significant decrease of complex II SDHB subunit expression in placenta (p<0.001). Additionally, levels of mitochondrial content, Coenzyme Q and cellular ATP were conserved. Lipid peroxidation significantly decreased in IUGR hearts (p<0.001), but not significantly increased in IUGR placentas. Finally, Sirtuin3 protein expression significantly increased in IUGR hearts (p<0.05). In human pregnancies, IUGR placental tissue showed an altered mitochondrial phenotype with a significant decrease of CI-stimulated oxygen consumption (p<0.05) and MRC complex I enzymatic activity (p<0.05). The enzymatic activities of the others MRC complexes and CS were preserved. In blood cells, conserved cellular oxygen consumption and trends to decrease CI-stimulated oxygen consumption was observed in maternal PBMC, but trends to decrease both cellular and CI-stimulated oxygen consumption were evidenced in neonatal CBMC, pointing out that IUGR newborns presented higher mitochondrial deficits compared to mothers. Moreover, no differences in MRC enzymatic activities in maternal PBMC or in neonatal CBMC were observed. Conserved CS activity was present in maternal PBMC but was significantly decreased in neonatal CBMC. So, in front of unaltered mtDNA levels in neonatal CBMC, alterations in neonatal CS would be related to Krebs cycle imbalances rather than to mitochondrial content. All these changes did not affect oxidative stress or ATP production in any tissue. Finally, Sirtuin3 protein expression also showed a relevant increase in human IUGR placenta (p=0.05). The relevance of this thesis relies on the description of mitochondrial impairment in the offspring of a rabbit model of IUGR but also in newborns from pregnancies complicated by IUGR. This mitochondrial imbalance is widely present in the different studied tissues, including the heart and the placenta from the rabbit model and the placenta and neonatal blood cells from human pregnancies. The mitochondrial characterization of this obstetric complication could help to greater understand the pathophysiologic mechanisms underlying cardiac remodelling and IUGR.
Els nounats amb creixement intrauterí restringit (CIR) desenvolupen un remodelat cardiovascular fetal i idiopàtic que pot portar a cardiopatia durant l’etapa adulta. La bioenergètica mitocondrial, essencial pel desenvolupament embrionari i la funció cardíaca, està regulada per diferents proteïnes, entre elles la Sirtuina 3. Es tracta d’una proteïna deacetilasa d’alt interès terapèutic, ja que es pot modular a través de la dieta. Els cors de cries amb CIR d’un model animal de conill mostren alteracions transcriptòmiques i ultraestructurals a nivell mitocondrial. L’objectiu de l’estudi ha sigut determinar la implicació d’una possible disfunció mitocondrial i de la Sirtiuna 3 en el CIR. Les troballes demostren una alteració mitocondrial de la cadena respiratòria en el cor i la placenta de les cries amb CIR del model animal (sobretot a nivell de l’activitat enzimàtica dels complexes II i IV; p<0.05) i també a la placenta de gestants humanes amb CIR (especialment del complex I; p<0.05). A més a més, aquesta alteració mitocondrial s’ha evidenciat en els nounats amb CIR a través de la reducció de l’activitat de l’enzim citrat sintasa (p<0.05), suggerint alteracions a nivell del cicle de Krebs. L’ATP cel·lular i el dany oxidatiu es troba preservat en tots els teixits estudiats, excepte en el cor de les cries del model animal de CIR, on el trobem disminuït significativament (p<0.001). Aquest desajust mitocondrial va acompanyat d’un augment significatiu de l‘expressió de la proteïna Sirtuina 3 en el cor de les cries del model animal de CIR i també a la placenta de les gestants humanes amb CIR (p<0.05). Les troballes derivades d’aquest estudi permeten associar la disfunció mitocondrial al desenvolupament del CIR i el remodelat cardiovascular associat, donant lloc al disseny d’estratègies dietètiques destinades a modular l’esmentat desbalanç bioenergètic a través de la regulació de la Sirtuina 3.

With the most absolute purpose of understanding what the “simple” concept of “growth regulation” implies, the present thesis is a compilation of original works that aims to deepen in how the growth and metabolism of fish, and most concretely, its muscle, behaves in different metabolically challenging conditions. Hopefully, the knowledge generated in these works and integrated in this multi-perspective thesis, can contribute to somehow help to improve the Aquaculture sector in the Mediterranean Sea and abroad. This ambitious goal is structured in different objective: 1) To establish the regulation of the ghrelinergic system in bream through the fasting and feedback model: a) Characterization of the sequences of Preproghrelin, Ghsr1a and Ghsr1b in gilthead sea bream (Article 1). b) Characterizing Preproghrelin and its two receptors in acute and long-term feeding responses and its interaction with the Gh/Igf1 axis (Article 1). 2) To deepen in the beneficial effects of exercise on growth and its modulation through changes in the dietary protein/lipid ratio: a) Determining how this dietary modification alters different growth parameters, muscular proximal composition and reserves turnover (Article 3). b) Determining how the mitochondrial proteins in red and white muscle and in the liver respond to this dietary modification in order to infer changes in the nutritional efficiency and growth improvement (Article 3). 3) To make a comparative study about the response of the main musculoskeletal growth regulation systems under two different challenging experimental models, the fasting and refeeding and the sustained exercise under two different diets. a) Studying the gene expression of specific proteins of the ontogenetic and growth pathways (myogenesis and osteogenesis), as well as the degradation pathways (proteolysis and osteoclastogenesis) in white muscle and bone (Articles 2 and 4). b) Determining its effects on white muscle growth (myogenesis/proteolysis balance) and meat quality (Article 4). 4) To complete the characterization of the new Myomaker and Myomixer fusogens in rainbow trout and gilthead sea bream and its involvement in the myogenic process in vivo and in vitro and in the in vivo muscle regeneration. a) Characterization of the sequence of Myomixer in rainbow trout (Article 5) and Muomaker and Myomixer in gilthead sea bream (Article 6). b) To study the involvement of Myomaker (in gilthead sea bream) and Myomixer (in rainbow trout and gilthead sea bream) in the myogenic process in vivo and in vitro as well as during the muscle regeneration in vivo (Article 5 and 6).

Durante el ictus, los niveles elevados de glutamato extracelular, el principal neurotransmisor excitador en el sistema nervioso central, aumentan y se unen al receptor de NMDA promoviendo la excitotoxicidad. Esta situación genera que un flujo excesivo de Ca2+ pase a través del receptor de NMDA aumentando sus concentraciones intracelulares activando toda una serie de cascadas de señalización que pueden actuar directamente sobre las mitocondrias, promoviendo varios programas de muerte celular. Las mitocondrias son orgánulos muy dinámicos que constantemente se fusionan y dividen, cambiando de forma y localización. El equilibrio entre la fisión y la fusión es importante para la función mitocondrial y es un regulador clave de la progresión de la muerte celular. Las proteínas que conforman el proceso de fusión mitocondrial y de fisión están formadas por un grupo de GTPasas. La fusión de la membrana mitocondrial interna está mediada por OPA1. Dos mitofusinas (Mfn1 y 2) median la fusión de la membrana externa mitocondrial. Por otro lado, la fisión mitocondrial está mediada por Drp1, una proteína citoplasmática que es reclutada a la superficie mitocondrial después de ser modificada a nivel post-traduccional. Los problemas relacionados con la dinámica mitocondrial se han descrito en múltiples enfermedades neurodegenrativas así como también en cáncer y sida. En esta Tesis hemos demostrado que Mfn2 juega un papel muy importante en el mantenimiento de la funcionalidad mitocondrial y la supervivencia neuronal. En excitotoxicidad es la única proteína de la maquinaria de fusión/fisión que disminuye su expresión en dos modelos in vitro distintos y también en un modelo in vivo. Hemos identificado dos fases de fragmentación mitocondrial en excitotoxicidad. La primera y que cursa en poco tiempo depende de la activación y reclutamiento de Drp1 mientras que la fase tardana que se inicia 4 horas después del insulto, parece depender de la reducción de los niveles proteicos de Mfn2. Esta fase tardía es irreversible y parece condenar a la neurona. La disminución de Mfn2 genera alteraciones en la homeóstasis del Ca2+, disminuye el potencial de membrana mitocondrial, empeora la comunicación entre las mitocondrias y el retículo endoplasmático, aumenta la traslocación de Bax a las mitocondrias y favorece la liberación del citocromo c. Hemos encontrado que la reducción en la expresión de Mfn2 durante la excitotoxicidad se da a nivel transcripcional y depende del factor de transcripción MEF2 que regula a Mfn2 a nivel basal en neuronas. En excitotoxicidad, MEF2 es degradado y causa la disminución de Mfn2. Este contexto hace pensar que Mfn2 podría ser una diana a tener en cuenta para futuros desarrollos de drogas y poder, de este modo, incrementar la pequeña ventana terapéutica que existe actualmente para el ictus.
Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented, but the mechanism underlying this process is poorly understood. Here, we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures, Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeo- stasis, and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons and that excitotoxicity- dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke.

Perú. Universidad Nacional Mayor de San Marcos. Vicerrectorado de Investigación y Posgrado. Proyecto N° B18100531 Perú. Fondo Nacional de Ciencia y Tecnología-Fondecyt. Contrato N° 101-2018-FONDECYT-BM-IADT-AV
Evalúa el estado taxonómico de la víbora sudamericana Bothrops atrox “jergón” mediante el uso de marcadores mitocondriales y enzimáticos. Los marcadores mitocondriales elegidos fueron la subunidad I de la citocromo oxidasa (COI) y citocromo b (Cytb). Por su parte, los marcadores enzimáticos elegidos por su importancia en el envenenamiento ofídico fueron los siguientes: metaloproteasa tipo III (SVMP-III), L-aminoácido oxidasa (LAAO), hialuronidasa (Hyal), fosfolipasa A2 (PLA2) y enzima similar a trombina (TLE). Se emplearon las secuencias de B. atrox depositadas en la base de datos del GenBank de distintas distribuciones geográficas, adicionando información de individuos peruanos de la localidad de Pucallpa, así como de especímenes con los que presenta una mayor cercanía evolutiva. El análisis de estas secuencias se realizó mediante la determinación de las posiciones variables, preferencia de uso de codón y análisis filogenético para determinar la distancia génica entre las distintas poblaciones de B. atrox. Los resultados obtenidos para los marcadores mitocondriales evidenciaron que los especímenes de Perú son cercanos a la especie brasileña Bothrops leucurus y con algunas poblaciones de B. atrox de Brasil y Colombia. Por otro lado, los marcadores enzimáticos demostraron que existen marcadas variaciones tanto a nivel venómico como genómico entre las poblaciones de B. atrox de Perú y Brasil. Adicionalmente, se encontró que B. atrox de Perú mantiene una elevada afinidad enzimática con Bothrops moojeni. En conjunto, estos resultados, por primera vez, sientan las bases, para establecer la singularidad de la población peruana de B. atrox y poder discutir en un futuro la identidad taxonómica de esta especie en el continente sudamericano.

Este trabajo, pretende ofrecer nuevos conocimientos acerca de los mecanismos moleculares que provocan afectación celular en los desordenes mitocondriales provocados por las mutaciones m.14487T>C, m.8993T>G, m.3243A>G y m.8344A>G del ADN mitocondrial. Para conseguir nuestro objetivo, como modelo de estudio se obtuvieron líneas de cíbridos transmitocondriales para las diferentes mutaciones de estudio, las cuales fueron cultivadas en unas condiciones de cultivo que permitiesen manifestar el defecto en el sistema OXPHOS (sistema de fosforilación oxidativa) manteniendo la viabilidad, incluso en aquellas mutaciones que afectan a ARNt. En estas condiciones de cultivo, se estudiaron parámetros que nos permitieron valorar la función mitocondrial como la concentración de lactato, succinato, ATP, ADP, AMP, Adenosina y el potencial de membrana mitocondrial, evidenciando el gran defecto en el sistema OXPHOS causado por las mutaciones que afectan a ARNt no siendo tan evidente en aquellas mutaciones que afectan a subunidades del sistema OXPHOS. Además los estudios de expresión génica realizados nos permitieron identificar la posible activación de procesos autofágicos y apoptóticos como posibles mecanismos moleculares responsables de los desordenes mitocondriales causados por las mutaciones m.3243A>G y m.8344A>G. Así como, evidenciar la no inducción de biogénesis mitocondrial en ninguna de las cuatro mutaciones estudiadas, incluso en aquellas mutaciones que afectan a ARNt con una mayor depleción en el contenido de ATP. Toda la información que se derivó de los estudios de expresión génica, no solo mostró una respuesta transcripcional diferencial entre mutaciones que afectan a ARNt y subunidades sino que además se observó una respuesta nuclear específica para cada una de las mutaciones del ADNmt estudiadas. Poniendo de manifiesto la complejidad de la fisiopatología de las enfermedades mitocondriales. Nosotros en este estudio intentamos poner de manifiesto esta complejidad y a la vez intentar esclarecer los mecanismos fisiopatológicos implicados en las mutaciones estudiadas, en algunos casos, como en las mutaciones en ARNt (gracias a las condiciones de cultivo utilizadas) hemos propuesto posibles mecanismos (que deben ser ratificados) que podrían explicar la degeneración celular causada por estas mutaciones y en otros, como en las mutaciones que afectan a subunidades hemos identificado aspectos a tener en cuenta en la fisiopatología de estas mutaciones.
This work aims to provide new insights into the molecular mechanisms that cause cell involvement in mitochondrial disorders caused by mutations m.14487T>C, m.8993T>G, m.3243A>G and m.8344A>G in mitochondrial DNA. To achieve our goal, as a study model lines cybrids were obtained to study the different mutations, which were grown in culture conditions that allowed the defect state in the OXPHOS system (oxidative phosphorylation system) maintaining the viability, even in those mutations that affect tRNA. In these culture conditions were studied parameters were allowed assess mitochondrial function as the lactate concentration, succinate, ATP, ADP, AMP, adenosine and mitochondrial membrane potential, demonstrating the great defect in the OXPHOS system caused by mutations tRNA affecting not be so apparent in those subunits mutations affecting OXPHOS system. Furthermore gene expression studies allowed us to identify made possible activation of apoptotic and autophagic processes as potential molecular mechanisms responsible for mitochondrial disorders caused by mutations m.3243A>G and m.8344A>G. So as not show the induction of mitochondrial biogenesis in any of the four mutations studied, even those mutations that affect tRNA in a greater depletion of ATP content. All information derived from studies of gene expression, not only showed a differential transcriptional response mutations that affect tRNA and subunits but also showed a specific nuclear response to each of the mitochondrial DNA mutations studied. Noting the complexity of the pathophysiology of mitochondrial diseases. We in this study we manifest this complexity while trying to clarify the pathophysiological mechanisms involved in the mutations studied, in some cases, such as mutations in tRNA (thanks to the culture conditions used) have proposed possible mechanisms (which should be ratified) that may explain the cellular degeneration caused by these and other mutations, such as mutations affecting subunits have identified aspects to consider in the pathophysiology of these mutations.

Mitochondria dysfunction is documented in the substantia nigra in Parkinson's disease (PD), a region which plays a central role in the characteristic motor manifestations of the disease. Mitochondria! dysfunction is a cause of neuron energy exhaustion, oxidative stress and dopaminergic cell death. However, little is known about mitochondria! dysfunction in other brain regions in PD, the impairment of which is causative of other symptoms such as cognitive impairment and, in some cases, dementia. The present study was undertaken to analyse mitochondria! function in the frontal cortex area 8 and angular gyrus. On the other hand, protein synthesis has also been shown to play role in neurodegenerative diseases which leads to neuronal atrophy. It is reported that significant percentage of protein synthesis results in the generation of defective ribosomal products, occurring as the result of faulty coding/or transfer within the ribosomal complex. The present study was undertaken to analyse impairment of ribosomal subunits in substantia nigra, frontal cortex area 8, angular gyrus, and precuneus. The frozen human post-mortem brain samples obtained following generous donation after informed consent, and stored at the Institute of Neuropathology Brain Bank (a branch of the HUB-ICO-IDIBELL brain bank). The study contemplates different stages of disease progression, according to the findings of the neuropathological study, and includes expression levels of mRNA and protein of selected subunits of the mitochondria! complexes and ribosomal subunits, and also enzymatic activities of each one of these complexes compared to those seen in age-matched controls processed in parallel. Results show altered activity of selected complexes of the respiratory chain thus supporting the concept that mitochondria! alterations in PD are not restricted to the substantia nigra but they rather represent a more widespread region-dependent alteration in PD. On the other hand the results of ribosomal proteins show disease-dependent differences in ribosomal protein gene expression.

G proteins transduce a myriad of signals from receptors at the plasma membrane. Recent reports point to a novel localization of G proteins at the mitochondria and other endomembranes where they regulate the physiology of these organelles. In particular, the Gαq subfamily is required to keep the proper balance between mitochondria fusion and fission acting at both outer and inner membrane, among other functions. In order to unveil the putative effectors of Gαq that mediate those effects at the mitochondria, our group has undertaken a mass-spectrometry analysis based on Gαq immunoprecipitates from cellular endomembranes. The “mito- interactome” study provided evidence of Gαq interaction with the armadillo domain-containing proteins Alex3 and Armc10. Subsequent immunoprecipitation and pull-down studies demonstrated a specific interaction of Gαq with the mitochondrial Rho GTPase 1 (Miro1) and both milton adaptor proteins TRAK1 and 2, that couple mitochondria to kinesin and dynein motor proteins and constitute the main regulators of mitochondrial transport in neurons. To analyze the physiological role of those interactions, we have performed tracking analysis of mitochondria along the axons of hippocampal neurons overexpressing Gαq or its constitutive-active mutant, GαqR183C, as well as activating a Gαq-specific GPCR (DREADD) with its specific agonist. The results of these studies reveal a significant increase in anterograde movement upon Gαq expression, whereas Gαq activation by either expressing the active-mutant or activating the Gαq-specific GPCR induces mitochondrial arrest. In contrast, depletion of Gαq using short-hairpin RNAs increases the number of motile mitochondria and their speed and promotes retrograde transport. Both activation of Gαq or its depletion alter mitochondrial dynamics including fusion/fission events, whereas expression of active-Gαq also alters neuronal physiology by reducing their complexity and dendritic branching. In summary, our group postulates a new non-canonical mitochondrial function of Gαq acting as a molecular switch through its association with Alex3, Miro1 and TRAK2. Gαq would associate to Alex3 and Miro1 to allow mitochondrial movement, whereas its GTP-bound conformation would associate to TRAK2 to halt motility. This process would be regulated by Alex3, which could play crucial roles as an adaptor for the protein complex and Gαq transactivation.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
As doenças mitocondriais são o resultado de mutações herdadas ou espontâneas do DNA mitocondrial (DNAmt) ou DNA nuclear (DNAn) levando à função anormal do sistema de fosforilação oxidativa. Um dos defeitos no DNAn mais freqüentemente descritos neste grupo de doenças são as deficiências isoladas do Complexo I, que é o maior complexo enzimático da cadeia respiratória com 42 subunidades (7 codificadas pelo DNAmt). Propõe-se que a subunidade de 39kDa (CI-39kDa) apresente estreita correlação com a atividade enzimática do complexo I. Sendo assim, poderíamos interrogar se a diminuição da expressão de CI-39kDa, detectada por Western blotting, poderia ser utilizada como um método diagnóstico para as deficiências do Complexo I, além deste método ter como vantagem a utilização de pequena porção de tecido congelado obtido por biópsia. Este trabalho teve como objetivo avaliar a freqüência da diminuição da proteína mitocondrial CI-39kDa no músculo esquelético de pacientes com suspeita de doença mitocondrial, e verificar se mutações conhecidas em genes codificadores de subunidades do Complexo I estariam associadas à diminuição da expressão desta proteína. As proteínas foram obtidas através do lisado de músculo esquelético de 56 pacientes e 21 controles. As proteínas foram separadas por eletroforese em gel de poliacrilamida denaturante, transferidas para membrana PVDF, que foi incubada com um anticorpo anti-CI-39kDa e SDH-Fp (succinato desidrogenase-fração flavoproteína). As bandas foram obtidas por autoradiografia e quantificadas por densitometria. A diminuição da expressão da proteína CI-39kDa, encontrada nos casos com diagnóstico possível e provável para doença mitocondrial, foi de 6%. O estudo por seqüenciamento mostrou somente uma alteração suspeita: mutação de sentido ainda não descrita em NDUFV2. Nossos resultados mostraram que: (1) a freqüência da diminuição da expressão de CI-39kDa foi baixa nos casos estudados com suspeita de doença mitocondrial; (2) a avaliação molecular desses casos mostrou que mutações em genes codificados pelo DNAmt, e mutações conhecidas em genes nucleares para subunidades do complexo I, não foram a causa do defeito destes pacientes; (3) a alteração de NDUFV2 pode ser a causa de uma disfunção do complexo I, a qual deverá ser melhor investigada para confirmar a sua patogenicidade. A ampliação deste estudo poderá confirmar a utilidade deste método para o diagnóstico das deficiências do Complexo I.
Mitochondrial diseases are caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA), leading to abnormal function of the oxidative phosphorylation. One of the most frequent nDNA defects described in this group of diseases are Complex I deficiency. Complex I is the largest enzymatic complex of the respiratory chain, contains 42 subunits (7 are mtDNA encoded). It is proposed that a 39kDa subunit (CI-39kDa) expression closely correlates with the enzymatic activity of complex I. Thus we could hypothesize that a decrease in the expression of CI-39kDa, detected by Western blotting, would be a good diagnostic method for complex I deficiencies, considering the requirement of low amounts of frozen biopsied tissue as a good advantage of this method. The aim of this study was to evaluate the frequency of decreased expression of the CI- 39kDa in skeletal muscle of patients with a suspicion for mitochondrial disease and to verify whether known mutations in Complex I subunit genes are associated with a decrease in the expression of this protein. Total proteins were obtained by skeletal muscle lysates of 56 patients and 21 controls. Proteins were electrophoresed through a denaturing polyacrylamide gel, transferred to a PVDF membrane and incubated with antibodies against CI-39kDa and SDH-Fp (succinate dehydrogenase-flavoprotein subunit). After autoradiography the bands were quantified by densitometry. A decreased expression of CI-39kDa was found in 6 % of the cases with a possible or probable diagnosis of mitochondrial disease. Sequencing studies demonstrated only one abnormality: a not yet described missense mutation in NDUFV2. Our results show that: (1) the frequency of a decreased expression in CI-39kDa was low in the studied cases with a suspicion of mitochondrial disease; (2) molecular studies of these cases showed that mutations in mtDNA encoded genes and known mutations in nuclear encoded genes for complex I subunits were not the cause of the defects observed in these patients; (3) the mutation in NDUFV2 can be the cause of a complex I defect, but further investigations are still needed to confirm its pathogenicity. Confirmation of the utility of this method for the diagnosis of complex I deficiency will probably be achieved by a continuation of this study.

Our research group focuses on protein translation and more specifically on the mechanism of transfer RNA (tRNA) aminoacylation by a family of essential and universal enzymes called aminoacyl-tRNA synthetases (aaRSs). aaRS are essential and universal components of the genetic code and their long evolutionary history explains the growing number of functions being discovered for aaRS and for aaRS homologues, beyond their canonical role in gene translation. During the process of constructing a model for human disorders caused by mitochondrial tRNA aminoacylation deficiencies in Drosophila melanogaster in the laboratory has been identified a previously uncharacterized paralog of a seryl-tRNA synthetase (SerRS) named SLIMP. Given the conservation of SLIMP in all available insect genomes, a characterization of the phenotype upon SLIMP depletion was conducted in Drosophila melanogaster. We analyzed the specific effect of SLIMP knockdown in muscles and glia, and we observed a dramatic reduction of the lifespan in both mutants. Interestingly, SLIMP knockdown in the glia causes neurodegeneration visualized as vacuolization in the brain of mutant flies. The sequence identity that SLIMP shares with SerRS allowed to predict that this protein has an identical fold of a seryl-tRNA synthetase. It was previously shown that SLIMP does not posses tRNA aminoacylation activity, but it retains the property to bind mitochondrial tRNASer isoacceptors as a possible reflection of the evolutionary origin of the protein. We characterized the RNA-and DNA-binding property of SLIMP through in vitro and in vivo methodologies. We found that SLIMP binds in vitro all RNA sequences that are encoded in the mitochondrial genome. We suggest that the composition of these sequences (AT enrichment) and the respective folding energy (low .G) are the main determinants for SLIMP binding to RNA. We performed ribonucleoprotein immunoprecipitation assay (RIP) in cells to study SLIMP-RNA interaction in vivo and we showed that SLIMP interact with almost all mitochondrial transcripts. Pull-down experiments combined to mass spectrometry analysis revealed at least two SLIMP protein interactors: SRS2 and LON protease. We demonstrated that SLIMP and SRS2 are interdependent, as the knockdown or the overexpression of one protein reduced or increase respectively the level of the other. We suggest that SLIMP and SRS2 might form a functional complex that is maintained at a given SLIMP:SRS2 ratio. LON was the other identified SLIMP interactor. Our data shows that LON and SRS2 do not interact, but SRS2 might be a substrate of LON proteolytic activity. We also found OPA1 as a potential interactor of LON. OPA1 is a dynamin-like GTPase that is responsible for inner membrane fusion. Our results indicated that the overexpression of LON protease results in an increase of the smaller OPA1 isoforms that correlates with mitochondrial stress. Given the proposed role of SLIMP in RNA binding, we aimed to determine whether SLIMP has an effect on mitochondrial transcription. The results obtained by Northern blot screening of some mitochondrial mRNAs revealed that knockdown of SLIMP significantly reduced the steady-state levels of COX2 and COX3 mRNA, and 12S and 16S rRNAs whereas tRNA levels were found constant. Our results suggest that the defect in transcription upon SLIMP depletion, might be limited to mature mRNAs and may reveal a specific function for SLIMP in the binding and stabilization of processed mitochondrial mRNAs rather than a role in the control of their transcriptional rate or processing. We showed that knockdown of SLIMP affects also cellular growth and cell cycle progression, in fact, upon SLIMP depletion, we observed a dramatic increase of cells in G2/M phase. This result suggests that the mitochondrial role of SLIMP, or a consequence of its function, may be acting as a crosstalk between mitochondria and nuclear transcription factors that regulate cell proliferation. Collectively, the work described in this thesis has contributed to the characterization of a mitochondrial seryl-tRNA synthetase paralog that has acquired an essential function in insects despite a relatively modest divergence from a canonical SerRS structure.
El nostre grup de recerca es centra en la traducció de proteïnes i més específicament en el mecanisme d’aminoacilació dels àcids ribonucleics (ARNs) de transferència (ARNt) per una família d’enzims essencials i universals anomenats aminoacil-ARNt sintetases (aaRSs). Al laboratori s’han analitzat el paper de les aaRSs en la traducció proteica, les seves funcions no canòniques, la seva evolució, així com la seva implicació en malalties humanes. Les aaRSs són components universals i essencials del codi genètic. La seva llarga historia evolutiva explica el creixent número de funcions que s’estan descobrint, tant per a elles com per a proteïnes paràlogues, més enllà del seu paper canònic en traducció genètica. Al laboratori, durant el procés d’obtenció d’un model a Drosophila melanogaster per a l’estudi de malalties humanes degudes a deficiències en l’aminoacilació d’ARNt, es va identificar un nou gen, paràlog de la seril-ARNt sintetasa (SeRS) mitocondrial, anomenat SLIMP. La proteïna SLIMP representa un nou tipus de proteïna similar a aaRS que ha adquirit una funció essencial a insectes, tot i la relativament baixa divergència respecta a una estructura d’SeRS canònica. Tot i amb això, són necessaris estudis addicionals per a identificar el paper biològic de SLIMP. Per aconseguir aquesta fita, s’ha portat a terme el projecte descrit en aquest manuscrit, el qual consisteix en anàlisis addicionals del fenotip resultant de la depleció de SLIMP in vivo, seguits d’estudis detallats de les interaccions moleculars amb àcids nucleics i proteïnes, per acabar amb un estudi dels efectes de SLIMP en la fisiologia cel•lular. En conjunt, els nostres resultats demostren que SLIMP s’uneix específicament a ARNs mitocondrials in vivo i in vitro. SLIMP interacciona amb SerRS2 i les dues són interdependents a nivell d’estabilitat proteica. La depleció de SLIMP o de SerRS2 redueix els nivells basals d’alguns ARNm mitocondrials, però la transcripció d’ARNt és manté inalterada. Es proposa un rol en la regulació post-transcripcional o en l’estabilitat dels ARNm madurs. Hem observat també que la depleció de SLIMP indueix l’aturada del cicle cel•lular en la transició G2/M. Aquests resultats suggereixen que SLIMP, o una conseqüència de la seva funció, podria tenir un paper d’enllaç entre els mitocondris i els factors de transcripció nuclears que regulen la proliferació cel•lular.

L’atàxia de Friedreich és una malaltia rara neurodegenerativa causada pel dèficit d’una proteïna mitocondrial anomenada frataxina. La miocardiopatia és freqüent en els pacients i sol ser la principal causa de mort. Actualment no existeix cap model cel•lular cardíac per estudiar la patofisiologia en aquestes cèl•lules. És per això que hem desenvolupat un model utilitzant miòcits ventriculars de rates neonatals i RNA d’interferència. Utilitzant aquest model hem observat que el dèficit de frataxina en aquestes cèl•lules causa una alteració de la xarxa mitocondrial i estrès oxidatiu. A més, els miòcits cardíacs pateixen un canvi en el perfil metabòlic i acumulen una gran quantitat d’àcids grassos en gotes lipídiques. Aquest model ens permetrà en un futur estudiar els mecanismes de la malaltia cardíaca a l’atàxia de Friedreich, descobrir nous biomarcadors i provar possibles tractaments.
La ataxia de Friedreich es una enfermedad rara neurodegenerativa causada por el déficit de una proteína mitocondrial denominada frataxina. La miocardiopatía es frecuente en los pacientes y suele ser la principal causa de muerte. Actualmente no existe ningún modelo celular cardíaco para estudiar la patofisiología en estas células. Es por ello que hemos desarrollado un modelo utilizando miocitos ventriculares de ratas neonatales y RNA de interferencia. Utilizando este modelo hemos observado que el déficit de frataxina en estas células causa una alteración de la red mitocondrial y estrés oxidativo. Además, los miocitos cardíacos sufren un cambio en el perfil metabólico y acumulan una gran cantidad de ácidos grasos en gotas lipídicas. Este modelo nos permitirá en un futuro estudiar los mecanismos de la enfermedad cardíaca en la ataxia de Friedreich, descubrir nuevos biomarcadores y probar posibles tratamientos.
Friedreich's ataxia is a neurodegenerative rare disease caused by a deficiency of the mitochondrial protein frataxin. Cardiomyopathy is frequent in patients and is usually the main cause of death. Currently there is no cardiac cell model to study the pathophysiology in these cells. Under these circumstances we have developed a model using neonatal rat ventricular myocytes and RNA interference. Frataxin deficiency in these cells causes an alteration of the mitochondrial network and oxidative stress. Furthermore, cardiac myocytes undergo a change in the metabolic profile and accumulate large amounts of fatty acids in lipid droplets. This model will favor the study of the mechanisms of cardiac disease in Friedreich ataxia, discover new biomarkers and test potential treatments.