A obesidade está relacionada com uma grande variedade de disfunções, tais como as dislipidemias, resistência à insulina e diabetes tipo II. Estas patologias estão relacionadas com alterações da fisiologia mitocondrial. As mitocôndrias são o principal sítio do metabolismo oxidativo e são importantes fontes de espécies reativas de oxigênio. Alberici et al. (2006) mostraram que a atividade de canais de potássio sensíveis a ATP mitocondriais (mitoKATP), via dissipativa branda, estava aumentada em fígados de animais hipertrigliceridêmicos transgênicos. Este dado sugere um papel adaptativo importante para o mitoKATP como regulador do metabolismo e da massa corpórea. O principal objetivo deste trabalho foi investigar o papel deste canal em fígados de camundongos submetidos a uma dieta hiperlipídica, obtida por meio de suplementação de óleo de soja à única fonte de água dos animais. Foram estabelecidas correlações entre a atividade do canal e parâmetros da fisiologia mitocondrial ou parâmetros sorológicos clássicos como colesterolemia e trigliceridemia. Sendo realizados experimentos de espalhamento de luz, quantificação de peróxido de hidrogênio, potencial de membrana e consumo de oxigênio em mitocôndrias. Nossos resultados mostram aumento de massa corpórea sem dislipidemias e aumento da atividade do mitoKATP, dependente do tempo da suplementação, diretamente correlacionada com a colesterolemia. Nossos resultados apontam aumento da geração de EROs e diminuição da eficiência de conversão energética no grupo suplementado. Como conclusão: manipulações de dieta são eficazes para a indução da atividade do mitoKATP, sendo este um novo e interessante sítio de regulação do metabolismo oxidativo e da massa corpórea. / Obesity is associated with multiple dysfunctions including dyslipidemia, insulin resistance and diabetes. These malfunctions are associated with modifications in mitochondrial physiology. Furthermore, mitochondria are the most important site of energy metabolism and reactive oxygen species production. Alberici et al. (2006) demonstrated that hypertriglyceridemic transgenic mice display increased expression ad activity of mitochondrial ATP sensitive potassium channels (mitoKATP), a mild uncoupling pathway, in liver. This suggests that mitoKATP is an important adaptation to regulate body mass and metabolism. The aim of this work is to investigate the role of mitoKATP in a high fat diet induced by soy oil supplementation, correlating changes in channel activity with metabolic and mitochondrial parameters. Mitochondria were isolated from mouse livers and serological parameters were measured for each animal. Light scattering (to estimate mitoKATP activity), hydrogen peroxide generation, membrane potentials and oxygen consumption were measured in the mitochondrial suspensions. Our results indicate an increase in body mass, without dyslipidemia; and increases in mitoKATP activity, in time-dependent manner, directly correlated to cholesterol levels. In addition, we found increases in ROS generation and decreased capacity of energy conversion (ADP/O) in the high fat diet group. In conclusion, our results indicate that the activity of mitoKATP could be induced by high fat diets and that this is an novel site for metabolic and body mass control.

Due to two different mutations in the gene that encodes the a1A subunit of voltage-activated CaV 2.1 calcium ion channels, the compound heterozygous tottering/leaner (tg/tgla) mouse exhibits numerous neurological deficits. Human disorders that arise from mutations in this voltage dependent calcium channel are familial hemiplegic migraine, episodic ataxia-2, and spinocerebellar ataxia 6. The tg/tgla mouse exhibits ataxia, movement disorders and memory impairment, suggesting that both the cerebellum and hippocampus are affected. To gain greater understanding of the many neurological abnormalities that are exhibited by the 90-120 day old tg/tgla mouse the following aspects were investigated: 1) the morphology of the cerebellum and hippocampus, 2) proliferation and death in cells of the hippocampal dentate gyrus and 3) changes in basic biochemical parameters in granule cells of the cerebellum and hippocampus. This study revealed no volume abnormalities within the hippocampus of the mutant mice, but a decrease in cell density with the pyramidal layer of CA3 and the hilus of the dentate gyrus. Cell size in the CA3 region was unaffected, but cell size in the hilus of the dentate gyrus did not exhibit the gender difference seen in the wild type mouse. The cerebellum showed a decrease in volume without any decrease in cerebellar cellular density. Cell proliferation and differentiation in the subgranular zone of the hippocampal dentate gyrus remained normal. This region also revealed a decrease in cell death in the tg/tgla mice. Basal intracellular calcium levels in granule cells show no difference within the hippocampus, but an increase in the tg/tgla male cerebellum compared to the wild type male cerebellum. There was no significant difference in granule cell mitochondrial membrane potential within the wild type and mutant animals in either the hippocampus or cerebellum. The rate of reactive oxygen species (ROS) production in granule cells revealed no variation within the hippocampus or cerebellum. The amount of ROS was decreased in cerebellar granule cells, but not granule cells of the hippocampus. Inducing ROS showed no alteration in production or amount of ROS produced in the hippocampus, but did show a ceiling in the amount of ROS produced, but not rate of production, in the cerebellum.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease with an annual mortality rate of 15% despite current therapies. Uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) results in adverse vascular remodeling contributing to PAH. Understanding the mechanisms of PASMC proliferation may identify new targets for treatment. Chloride currents/channels (ICl) are expressed in PASMCs and their roles in proliferation have been suggested based on their importance in resting membrane potential and cell volume regulation. The present study explored the role of ICl in proliferation in rat and human PASMCs. We found that either nonspecific ICl inhibitors (DIDS or NPPB) or a putative specific blocker of swelling-activated ICl (ICl,swell) reduced proliferation of PASMCs cultured in serum-containing media. Patch-clamp studies showed that proliferating PASMCs had increased baseline ICl and ICl,swell in association with depolarized membrane potentials. Quantitative real-time RT-PCR studies identified expressions of CLC-3, a candidate gene of ICl,swell, and several other CLC genes in proliferating PASMCs. While selective knockdown of CLC-3 with lentiviral shRNA reduced PASMC proliferation, it had no effect on ICl,swell. These findings are consistent with the conclusion that ICl regulate proliferation of PASMCs and suggest that selective ICl inhibition may be useful in treating pulmonary arterial hypertension.

Les mécanismes cellulaires et moléculaires qui sous-tendent la mémoire et l’apprentissage chez les mammifères sont incomplètement compris. Le rythme thêta de l’hippocampe constitue l’état « en ligne » de cette structure qui est cruciale pour la mémoire déclarative. Dans la région CA1 de l’hippocampe, les interneurones inhibiteurs LM/RAD démontrent des oscillations de potentiel membranaire (OPM) intrinsèques qui pourraient se révéler importantes pour la génération du rythme thêta. Des travaux préliminaires ont suggéré que le courant K+ I(A) pourrait être impliqué dans la génération de ces oscillations. Néanmoins, peu de choses sont connues au sujet de l’identité des sous-unités protéiques principales et auxiliaires qui soutiennent le courant I(A) ainsi que l’ampleur de la contribution fonctionnelle de ce courant K+ dans les interneurones. Ainsi, cette thèse de doctorat démontre que le courant I(A) soutient la génération des OPM dans les interneurones LM/RAD et que des protéines Kv4.3 forment des canaux qui contribuent à ce courant. De plus, elle approfondit les connaissances sur les mécanismes qui régissent les interactions entre les sous-unités principales de canaux Kv4.3 et les protéines accessoires KChIP1. Finalement, elle révèle que la protéine KChIP1 module le courant I(A)-Kv4.3 natif et la fréquence de décharge des potentiels d’action dans les interneurones. Nos travaux contribuent à l’avancement des connaissances dans le domaine de la modulation de l’excitabilité des interneurones inhibiteurs de l’hippocampe et permettent ainsi de mieux saisir les mécanismes qui soutiennent la fonction de l’hippocampe et possiblement la mémoire chez les mammifères. / Cellular and molecular mechanisms underlying learning and memory in mammals are incompletely understood. The theta rhythm in the hippocampus constitutes the « on-line » state of this structure which is crucial for declarative memory. In the CA1 hippocampal area, LM/RAD inhibitory interneurons exhibit intrinsic membrane potential oscillations (MPOs) that could be important for the generation of theta rhythm. Preliminary work suggested that K+ current I(A) could be involved in the generation of these oscillations. Nevertheless, little is known about the identity of the principal and auxiliary protein subunits underlying I(A) current and the extent of the functional contribution of this K+ current in hippocampal interneurons. Thus, this Ph.D. thesis shows that I(A) current underlies MPO generation in LM/RAD interneurons and that Kv4.3 proteins form channels that contribute to this current. Also, it deepens the knowledge on the mechanism controlling the interactions between Kv4.3 channel-forming principal subunits and KChIP1 auxiliary proteins. Finally, it reveals that KChIP1 modulates native I(A)-Kv4.3 current and the action potential discharge frequency in interneurons. Our work takes part in advancing the knowledge on the field of modulation of excitability in hippocampal inhibitory interneurons and allows a better understanding of the mechanisms underlying the function of the hippocampus and possibly memory in mammals.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease with an annual mortality rate of 15% despite current therapies. Uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) results in adverse vascular remodeling contributing to PAH. Understanding the mechanisms of PASMC proliferation may identify new targets for treatment. Chloride currents/channels (ICl) are expressed in PASMCs and their roles in proliferation have been suggested based on their importance in resting membrane potential and cell volume regulation. The present study explored the role of ICl in proliferation in rat and human PASMCs. We found that either nonspecific ICl inhibitors (DIDS or NPPB) or a putative specific blocker of swelling-activated ICl (ICl,swell) reduced proliferation of PASMCs cultured in serum-containing media. Patch-clamp studies showed that proliferating PASMCs had increased baseline ICl and ICl,swell in association with depolarized membrane potentials. Quantitative real-time RT-PCR studies identified expressions of CLC-3, a candidate gene of ICl,swell, and several other CLC genes in proliferating PASMCs. While selective knockdown of CLC-3 with lentiviral shRNA reduced PASMC proliferation, it had no effect on ICl,swell. These findings are consistent with the conclusion that ICl regulate proliferation of PASMCs and suggest that selective ICl inhibition may be useful in treating pulmonary arterial hypertension.

Andrologische Forschungsarbeiten der letzten Jahre beweisen, dass einzelne, aus somatischen Zellen bekannte Apoptose-typische Veränderungen bei humanen Spermien einen negativen Einfluss auf die Fertilität des Mannes haben. Umstritten ist, ob es sich dabei nur um einen abortiven Zelltod als Zeichen einer Reifungsstörung während der Spermatogenese handelt oder ob Apoptose auch in reifen Spermien induzierbar ist. Ziel der vorliegenden Arbeit war es, durch Untersuchungen zur Induzierbarkeit der Apoptose in reifen und unreifen Spermatozoen die vollständige Funktionalität verschiedener Signalkaskaden sowie deren Umsetzung in morphologische Veränderungen aufzudecken. Darüber hinaus sollte die Rolle des intrazellulären Calciumspiegels als möglicher Interaktionspartner zwischen Akrosomreaktion und Apoptose geklärt werden, um Informationen über die Zukunft der nicht fertilisierenden Spermien im weiblichen Genitaltrakt zu erlangen. In den vorliegenden Versuchsreihen konnte erstmals die gezielte Induktion der Apoptose in reifen und unreifen Spermatozoen anhand biochemischer und elektronenmikroskopischer Untersuchungen nachgewiesen werden. Dabei wurde ausführlich die erfolgreiche Aktivierung mehrerer intrinsischer Apoptosesignalwege in reifen Spermien gezeigt, deren Initiation entweder auf den Zusammenbruch innerer Mitochondrienmembranen, auf eine veränderte intrazelluläre Calciumkonzentration oder auf das Einwirken von oxidativem Stress zurückzuführen war. Zudem konnten Erkenntnisse zum antioxidativen Schutzmechanismus von Spermien gewonnen werden, welcher die Spermien gegenüber einer spezifischen Menge an H2O2 vor oxidativem Stress-bedingter Apoptose bewahrt. Sowohl die Apoptose als auch die Akrosomreaktion waren durch die Zugabe eines Calciumchelators blockierbar. Die Initiation des programmierten Zelltodes in Spermien durch einen Anstieg der intrazellulären Calciumkonzentration erklärt zudem eine weitere wichtige Funktion dieses Prozesses: das Absterben von akrosomenreagierten Spermien bei nicht erfolgter Fertilisation im weiblichen Genitaltrakt. Die Theorie einer rein abortiven Apoptose als Folge einer Spermatogenesestörung ist damit widerlegt.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mycotoxins are secondary metabolites, produced by fungi species of the genus Aspergillus, Fusarium, Penicillium and Claviceps. The high incidence of toxicity is by ingestion of contaminated plant products and by consuming products derived from food. Among the 400 different mycotoxins; there are the fumonisins, especially fumonisin B1 (FB1), which corresponds to about 70% of total fumonisins. It has been demonstrated that toxicity of this mycotoxin may be related to toxic effects on the central nervous system, engaging in inflammatory processes and seizures. The mechanism of toxicity of fumonisins is related to the metabolism of sphingolipids in which the breakdown of these sphingolipids complexes and accumulation of sphingoid bases alter important cellular functions, favoring oxidative stress, inhibition of protein secondary and excitotoxicity. Thus, this work tried to investigate whether FB1 is able to enhance the inductive effect of the model of pentylenetetrazole (PTZ) seizures. We also evaluated the mechanism of action responsible for this effect, in order to study strategies that make it possible to prevent or minimize their toxic effects. So, C57BL/6 mice were exposed to FB1 (8 mg/kg i.p.) or vehicle (1.6% DMSO + 0.9% NaCl i.p.) and elapsed 30 minutes were injected with PTZ (30 mg/kg i.p.) or vehicle (0,9% NaCl i.p.). Behind 15 minutes observation, mice were euthanized and cerebral cortex and hippocampus were collected for analysis of Na+, K+-ATPase activity, membrane potential and mitochondrial complex I and II activities. FB1 reduced latency for myoclonic jerks and increased myoclonus number. Even, Na+, K+-ATPase total activity as well as subunit �1of this enzyme were increased in cerebral cortex of animals treated with FB1, but in hippocampus it was reduced. FB1 elevated mitochondrial membrane potential in cerebral cortex however mitochondrial complex I and II activity weren t changed in both structures with this treatment. PTZ was able to enhance Na+, K+-ATPase �2/�3 subunit activity in hippocampus. Thus, we showed that FB1 induces neurotoxicity, since this mycotoxin FB1 facilitates seizures induced by PTZ, altered mitochondrial membrane potential and the enzyme Na+, K+ - ATPase activity, which are possibly mediating the mechanism of toxicity. / As micotoxinas são metabólitos secundários, produzidas por espécies de fungos dos gêneros Aspergillus, Fusarium, Claviceps e Penicillium. Geralmente as intoxicações se dão pela ingestão de produtos vegetais contaminados, bem como pelo consumo de alimentos derivados. Dentre os 400 diferentes tipos de micotoxinas, destacam-se as fumonisinas, em especial a fumonisina B1 (FB1) que corresponde a cerca de 70% das fumonisinas totais. Tem sido demonstrado que a toxicidade desta micotoxina pode estar relacionada com efeitos tóxicos no sistema nervoso central, envolvendo-se em processos inflamatórios e crises convulsivas. O mecanismo de toxicidade das fumonisinas está relacionado ao metabolismo dos esfingolipídios no qual o esgotamento desses esfingolipídios complexos e o acúmulo das bases esfingóides alteram funções celulares importantes, favorecendo estresse oxidativo, a inibição secundária de proteínas e excitotoxicidade. Deste modo, este estudo investigou se a FB1 é capaz de facilitar o efeito indutor de convulsões do modelo do pentilenotetrazol (PTZ). Avaliamos também o mecanismo de ação responsável por tal efeito, a fim de estudar estratégias que tornem possível a prevenção ou minimização dos seus efeitos tóxicos. Assim, camundongos machos C57BL/6 foram expostos a FB1 (8 mg/kg i.p.) ou veículo (1,6% DMSO + 0,9% NaCl i.p.) e após 30 minutos injetados com PTZ (30 mg/kg i.p.) ou veículo (0,9% NaCl i.p.). Após 15 minutos de observação, os camundongos foram eutanasiados e o córtex cerebral e o hipocampo foram coletados para a análise das enzimas Na+, K+-ATPase e o potencial de membrana mitocondrial e as atividades dos complexos I e II mitocondriais. A FB1 reduziu a latência para mioclonias e aumentou o número de mioclonias. Da mesma forma, a atividade total e da subunidade �1 da Na+, K+-ATPase foi aumentada no córtex cerebral de animais tratados com FB1, mas no hipocampo foi reduzida. A FB1 elevou o potencial de membrana mitocondrial no córtex cerebral, porém a atividade dos complexos I e II da cadeia respiratória não foram alteradas em ambas estruturas com este tratamento. O PTZ foi capaz de aumentar a subunidade �2/�3 da Na+, K+-ATPase no hipocampo. Assim, mostramos que a FB1 induz a neurotoxidade, pois esta micotoxina facilitou as convulsões induzidas por PTZ, alterou o potencial de membrana mitocondrial e a atividade da enzima Na+, K+-ATPase, que possivelmente estão mediando o mecanismo da toxicidade.

Orientador: Tereza Cristina Cardoso / Coorientadora: Katia Denise Saraica Bresciani / Banca: Roberto Gameiro de Carvalho / Banca: Ana Carolina Borsanelli / Banca: Andréa Fontes Garcia / Banca: Jamila Cristina Baptistella / Resumo: O Brasil é o segundo maior produtor mundial de carne bovina. Entretanto diversas patologias podem acometer o rebanho bovino e causar significativas perdas econômicas. Diante deste cenário, a infecção por Herpesvirus bovino 5 (BHV5) possui grande importância epidemiológica neste setor, sendo o BHV5 conhecido pelo desenvolvimento de meningoencefalite. Esse vírus possui como características a rápida replicação lítica em cultura de células e a capacidade de estabelecer latência nos gânglios sensoriais do hospedeiro. Assim, essa cepa viral está relacionada à habilidade do BHV5 de invadir, replicar-se no sistema nervoso central (SNC) e causar enfermidade neurológica. É de conhecimento científico que infecções virais estimulam ou inibem a resposta imunológica com objetivo de impedir a replicação e disseminação viral. Entretanto, pouco se sabe sobre o desenvolvimento da resposta imunológica do BHV5 no curso da infecção aguda ou latente. Assim, muitos conceitos sobre a patogênese do BHV5 acabam sendo extrapolados do Herpesvirus bovino 1 (BHV1). Diante deste contexto, faz-se necessário a busca pelo conhecimento da relação entre os vírus e as células de defesa do hospedeiro. Esses achados irão contribuir para um melhor entendimento da patogênese da doença, bem como para o possível desenvolvimento de vacinas mais específicas e eficazes. / Abstract: Brazil is the second largest producer of beef in the world. However several pathologies can affect the bovine herd and cause significant economic losses. In view of this scenario, bovine herpesvirus 5 infection (BHV5) is of great epidemiological importance in this sector, and BHV5 is known for the development of meningoencephalitis. This virus is characterized by rapid lytic replication in cell culture and the ability to establish latency in host sensory ganglia. Thus, this viral strain is related to the ability of BHV5 to invade, replicate in the central nervous system (CNS) and cause neurological disease. It is scientifically known that viral infections stimulate or inhibit the immune response in order to prevent viral replication and dissemination. However little is known about the development of the BHV5 immune response in the course of acute or latent infection. Thus, many concepts about the pathogenesis of BHV5 end up being extrapolated from bovine herpesvirus 1 (BHV1). In this context, it is necessary to search for knowledge about the relationship between viruses and host defense cells. These findings will contribute to a better understanding of the pathogenesis of the disease, as well as to the development of more specific and effective vaccines. / Doutor

A obesidade está relacionada com uma grande variedade de disfunções, tais como as dislipidemias, resistência à insulina e diabetes tipo II. Estas patologias estão relacionadas com alterações da fisiologia mitocondrial. As mitocôndrias são o principal sítio do metabolismo oxidativo e são importantes fontes de espécies reativas de oxigênio. Alberici et al. (2006) mostraram que a atividade de canais de potássio sensíveis a ATP mitocondriais (mitoKATP), via dissipativa branda, estava aumentada em fígados de animais hipertrigliceridêmicos transgênicos. Este dado sugere um papel adaptativo importante para o mitoKATP como regulador do metabolismo e da massa corpórea. O principal objetivo deste trabalho foi investigar o papel deste canal em fígados de camundongos submetidos a uma dieta hiperlipídica, obtida por meio de suplementação de óleo de soja à única fonte de água dos animais. Foram estabelecidas correlações entre a atividade do canal e parâmetros da fisiologia mitocondrial ou parâmetros sorológicos clássicos como colesterolemia e trigliceridemia. Sendo realizados experimentos de espalhamento de luz, quantificação de peróxido de hidrogênio, potencial de membrana e consumo de oxigênio em mitocôndrias. Nossos resultados mostram aumento de massa corpórea sem dislipidemias e aumento da atividade do mitoKATP, dependente do tempo da suplementação, diretamente correlacionada com a colesterolemia. Nossos resultados apontam aumento da geração de EROs e diminuição da eficiência de conversão energética no grupo suplementado. Como conclusão: manipulações de dieta são eficazes para a indução da atividade do mitoKATP, sendo este um novo e interessante sítio de regulação do metabolismo oxidativo e da massa corpórea. / Obesity is associated with multiple dysfunctions including dyslipidemia, insulin resistance and diabetes. These malfunctions are associated with modifications in mitochondrial physiology. Furthermore, mitochondria are the most important site of energy metabolism and reactive oxygen species production. Alberici et al. (2006) demonstrated that hypertriglyceridemic transgenic mice display increased expression ad activity of mitochondrial ATP sensitive potassium channels (mitoKATP), a mild uncoupling pathway, in liver. This suggests that mitoKATP is an important adaptation to regulate body mass and metabolism. The aim of this work is to investigate the role of mitoKATP in a high fat diet induced by soy oil supplementation, correlating changes in channel activity with metabolic and mitochondrial parameters. Mitochondria were isolated from mouse livers and serological parameters were measured for each animal. Light scattering (to estimate mitoKATP activity), hydrogen peroxide generation, membrane potentials and oxygen consumption were measured in the mitochondrial suspensions. Our results indicate an increase in body mass, without dyslipidemia; and increases in mitoKATP activity, in time-dependent manner, directly correlated to cholesterol levels. In addition, we found increases in ROS generation and decreased capacity of energy conversion (ADP/O) in the high fat diet group. In conclusion, our results indicate that the activity of mitoKATP could be induced by high fat diets and that this is an novel site for metabolic and body mass control.

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