The rampant growth of obesity worldwide has stimulated explosive research into human metabolism. Metabolic rate has been shown to be altered by diets differing in macronutrient composition, with low-carbohydrate, ketogenic diets eliciting a significant increase over other interventions. The purpose of this study was to determine the effects of the ketone β-hydroxybutyrate (βHB) on mitochondrial respiration and coupling status in adipose tissue. To explore this, we employed three distinct systems, namely cell, rodent, and human models. In every model, βHB robustly increased mitochondrial respiration. Furthermore, in cultured adipocytes and rodent adipose, we quantified the expression of genes involved in mitochondrial biogenesis and coupling status. We observed that genes involved in mitochondrial biogenesis and uncoupling were significantly higher in models exposed to ketone treatments. In conclusion, ketones increase mitochondrial respiration in cells and mammalian adipose tissue, but not ATP production, indicating greater mitochondrial uncoupling. These findings may partly explain the increased metabolic rate evident in states of elevated ketones and may facilitate the development of novel obesity interventions in the future.

Small molecule mitochondrial uncouplers are compounds that dissipate the proton motive force independent of ATP synthase that results in increased energy expenditure. Mild mitochondrial uncoupling has therapeutic potential in treating obesity, diabetes, neurological diseases, non-alcoholic steatohepatitis (NASH), and aging. Our group has previously reported the discovery of a small molecule mitochondrial uncoupler BAM15, which was efficacious in an obesity mouse model. Herein, we describe the design and synthesis of two scaffolds as well as their characterization as mitochondrial uncouplers through a series of in vitro and in vivo assays. Compounds that pass as bona fide mitochondrial uncouplers are administered in mice to determine pharmacokinetic properties and promising compounds are then tested in a mouse model of obesity. The first series of mitochondrial uncouplers are anilinopyrazines. By changing the substitution pattern and electronics on the aniline rings, our investigations reveal the importance of the proximity of aniline rings on the pyrazine core, with the 2,3-positions being crucial for uncoupling activity. We found that mitochondrial uncouplers 2.5g and 2.5l elicited a maximum oxygen consumption rate (OCR) of 260% and 343% with an EC50 of 2.5 and 5.9 µM, respectively. Utilizing the knowledge gained from the anilinopyrazine series, we designed a second novel chemical scaffold based on a related BAM15 analog 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol. The new series of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives have a pyridine instead of pyrazine core that is decorated with aniline substituents. We found that derivatives with electron withdrawing groups (EWG) substitutions in the 2,5-position on the aniline ring exhibited the greatest uncoupling activity compared to other structural isomers. Strong EWGs CF3/OCF3/SO2CF3 were well tolerated and demonstrated the highest uncoupling activity compared to other EWGs. Our studies indicated that placement of the hydroxyl group in the 2-position of the pyridine moiety was crucial for uncoupling activity. Several of the most promising compounds tested in vitro were examined in vivo and found to have good oral bioavailability in mice with ranges in Cmax of 10-90 µM and t1/2 of 0.9 to >24 hours. We found that analogs that have F/OCF3/SO2CF3 groups on the 4-position exhibited the longest t1/2 compared to other structural isomers, suggesting that this position is a site of metabolic lability. Among the 51 derivatives tested, SHM20519115 demonstrated mild uncoupling activity with 48% BAM15 OCR and an EC50 of 17.1 µM in L6 myoblast cells. SHM20519115 was found to have good oral bioavailability with a Cmax of 57 µM and a t1/2 of 4.4 hours. Additionally, SHM20519115 had significant distribution in adipose tissue where it can promote mitochondrial uncoupling. In a mouse model of obesity, SHM20519115 prevented fat mass gain by 59% compared to the western diet (WD) control group. Importantly, weight loss did not alter lean mass or food intake. Further characterization demonstrated that SHM20519115 prevented glucose and insulin intolerance in mice. Taken together, our investigations support the utility of mitochondrial uncouplers for the treatment of obesity and other metabolic disorders. / Doctor of Philosophy / Obesity is commonly considered a modern-day epidemic with more than 40% of adult Americans being classified as obese. The higher prevalence of obesity over the course of the last century has been attributed to a more sedentary lifestyle and high calorie diet. Obesity has been shown to negatively impact every organ system and increases the risk for heart disease, cancer, neurological diseases, non-alcoholic steatohepatitis (NASH), and diabetes. Moreover, obesity has further burdened the healthcare system with an estimated expenditure of $190 billion a year in the US. Although diet and exercise has shown excellent results in weight loss, long-term compliance with these regiments is extremely low. Current non-invasive treatments provide varying efficacies and a myriad of side-effects. Invasive procedures, which is restricted to those who are classified as 'morbidly obese' with a BMI > 40, have shown excellent results in facilitating weight loss but come with high cost and risks to patients. This excludes individuals in the BMI range of 30-40 unless they are qualified with additional comorbidities. In recent years, mitochondrial uncouplers have reemerged as a potential therapeutic treatment for obesity. This dissertation discusses the structure-activity relationship study of anilinopyrazines and 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives as mitochondrial uncouplers. Building on previous work on BAM15, we investigated uncoupling activity of anilinopyrazines. We discovered that although anilinopyrazines were previously found to be inactive, modifications to the aniline rings could result in uncoupling activity. We found that strong electron withdrawing groups placed in the meta and para positions were most favorable. We also determined that the 2,3-disubstitution on the aniline rings was crucial for uncoupling activity. From this study, we discovered 2.5g and 2.5l that elicited a maximum oxygen consumption rate (OCR) of 260 and 343% with EC50 of 2.5 and 5.9 µM, respectively. Furthermore, we recently reported a new series of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives and identified SHM20519115 as a mitochondrial uncoupler. Our studies determined that SHM20519115 demonstrated mild uncoupling activity with 48% BAM15 OCR with an EC50 of 17.1µM in L6 myoblasts cells. In a mouse model of obesity, SHM20519115 was found to be efficacious at a 130 mg/kg dose. Pharmacokinetic studies SHM20519115 showed greater overall distribution in adipose tissue in mice. Additionally, when examined in a mouse obesity prevention model, SHM20519115 successfully prevented 59% fat mass gain compared to the western diet (WD) control group. Finally, we found that SHM20519115 prevents glucose and insulin intolerance in mice. Taken together, these results support a role for mitochondrial uncouplers in the treatment of obesity.

The prototype uncoupling protein 1 (UCP1) mediates proton leak-dependent thermogenesis in mammals, but the physiological functions of the novel UCP2-5 are unclear. Nematodes only express one uncoupling protein that is most similar to UCP4 in the human brain, which is believed to be the most evolutionarily conserved of the uncoupling proteins. Consistent with reported UCP functions in mammals, we observed that ceUCP4-null nematodes had decreased metabolic rates and increased adiposity compared to wild type. Surprisingly, these phenotypes corresponded to decreased succinate-mediated mitochondrial respiration without apparent changes in mitochondrial uncoupling. ceUCP4-null mitochondria exhibited normal electron transport chain functions, but had a decreased capacity for succinate import. Supporting the functional importance of ceUCP4-dependent complex II regulation in vivo, ceUCP4 deficiency was demonstrated to result in a selectively lethal response to genetic and pharmacological inhibition of Complex I. Similarly, ceUCP4-deficiency significantly prolonged lifespan in the short-lived mev-1 mutant that generates deleterious complex II-derived reactive oxidants. These results define a new physiological function for the ancestral ceUCP4 in the regulation of complex II-mediated oxidative phosphorylation through an unexpected effect on mitochondrial succinate transport. The data described in this dissertation also describe a novel mechanism by which uncoupling proteins mediate mitochondrial bioenergetics. / text

Myocardial and skeletal muscle high energy phosphate metabolism is abnormal in heart failure, but the pathophysiology is not understood. Plasma non-esterified fatty acids (NEFA) increase in heart failure due to increased sympathetic drive, and regulate the transcription of mitochondrial uncoupling protein-3 (UCP3), through peroxisome proliferator-activated receptor-α. The aim of the work in this thesis was to determine whether cardiac PCr/ATP ratios and skeletal muscle PCr kinetics during exercise were related to cardiac and skeletal muscle UCP3 levels respectively, thus providing a mechanism for the apparent mitochondrial dysfunction observed in heart failure. Patients having cardiac surgery underwent pre-operative testing, including cardiac and gastrocnemius 31P magnetic resonance spectroscopy. Intra-operatively, ventricular, atrial and skeletal muscle biopsies were taken for measurement of mitochondrial protein levels by immunoblotting, along with mitochondrial function by tissue respiration rates. Fasting plasma NEFA concentrations increased in patients with ventricular dysfunction and with New York Heart Association (NYHA) class. Ventricular UCP3 levels increased and cardiac PCr/ATP decreased with NYHA class, however, demonstrated no relationship to each other. In skeletal muscle, maximal rates of oxidative ATP synthesis (Qmax) related to functional capacity. Skeletal muscle UCP3 levels increased with NYHA class but were unrelated to skeletal muscle Qmax. Tissue respiration experiments revealed no relationship between ventricular function and indices of mitochondrial coupling, furthermore, indices of mitochondrial coupling were unrelated to tissue UCP3 levels. No evidence was found to support mitochondrial uncoupling, mediated through UCP3, as a cause of the abnormalities in cardiac and skeletal muscle high energy phosphate metabolism.

Orientador: Paulo Arruda / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T23:29:52Z (GMT). No. of bitstreams: 1 Barreto_PedroPaulo_D.pdf: 2593736 bytes, checksum: 667c2ed03e1e7e51ac393087c3acc7ae (MD5) Previous issue date: 2014 / Resumo: A proteína desacopladora mitocondrial 1 (UCP1) é uma proteína mitocondrial codificada pelo núcleo capaz de desacoplar o gradiente eletroquímico usado para a síntese de ATP, dissipando a energia na forma de calor. A descoberta de homólogos e ortólogos da UCP1, sugere outros papéis fisiológicos para estas proteínas. As UCPs podem servir como uma válvula de escape, diminuindo a força protonmotiva (PMF) e reduzindo a produção de ROS em condições desfavoráveis. Plantas superexpressando UCPs se desenvolvem melhor quando submetidas a estresses bióticos e abióticos. Estas plantas demonstraram diminuição na produção de ROS, alteração no estado redox celular, além de um aumento no metabolismo energético e na fotossíntese. Neste trabalho nós investigamos os mecanismos moleculares envolvidos no metabolismo energético celular e resposta a estresses em plantas de tabaco superexpressando a UCP1 de A. thaliana. Demonstramos, através de análises moleculares e genômicas, que a superexpressão da UCP1 é capaz de provocar o aumento na respiração desacoplada em mitocôndrias isoladas, diminuir o conteúdo de ATP intracelular, e desencadear um processo de sinalização retrógrada que resulta na indução de genes mitocondriais e genes responsivos a estresses. Esta sinalização retrógrada resultou na indução do processo de biogênese mitocondrial verificado pelo aumento no número e área mitocondrial por célula, além de alterações morfológicas nestas organelas. O processo de biogênese mitocondrial nestas plantas é acompanhado pelo aumento na expressão de um grande número de genes responsivos a estresses, o que resulta no melhor desempenho e reduzida produção de ROS mitocondrial quando submetidas a estresses abióticos. A análise detalhada do transcriptoma de plantas superexpressando UCP1 em comparação com plantas selvagens demonstrou uma forte conexão entre os metabolismos mitocondrial, citoplasmático e cloroplástico para compensar as alterações provocadas pelo aumento na atividade da UCP1. Um grande número de fatores de transcrição ainda não caracterizados foram identificados e podem representar bons alvos para investigações futuras a respeito da regulação da biogênese mitocondrial e do metabolismo energético em plantas. Os resultados contidos nesta tese nos permitem melhor compreender a flexibilidade do metabolismo energético em plantas e identificar possíveis reguladores do processo de biogênese mitocondrial e resposta a estresses em plantas / Abstract: The mitochondrial uncoupling protein 1 (UCP1) is a nuclear-encoded mitochondrial protein capable of uncouple the electrochemical gradient used for ATP synthesis, dissipating energy as heat. The discovery of UCP1 homologues, and its corresponding orthologues suggest diverse physiological functions for these proteins. UCPs may serve as an escape valve, decreasing the proton motive force (PMF) and preventing ROS production under unfavorable conditions. Plants overexpressing UCPs perform better under biotic and abiotic stresses. These plants show diminished ROS production, alteration of cell redox homeostasis, increased energy metabolism and photosynthesis. In this work we investigated the molecular mechanisms underlying cell energy metabolism and stress response in tobacco plants overexpressing an Arabidopsis thaliana UCP1. We demonstrated through molecular, cellular and genomic tools that UCP1 overexpressing plants is capable of increasing uncoupled respiration of isolated mitochondria, decrease intracellular ATP levels, and trigger a retrograde signaling that resulted in a broad induction of mitochondrial and stress response genes. The retrograde signaling resulted in the induction of mitochondrial biogenesis verified by increased mitochondrial number, area and alterations on mitochondrial morphology. The increased mitochondrial biogenesis in these plants accompanied by the broad increase in the expression of stress responsive genes, may be responsible for the diminished ROS production and the better performance of these plants when submitted to several abiotic stresses. We also performed a detailed analysis of the transcriptome expression of the UCP1 overexpressing plants as compared with the wild type plants. We verified that the UCP1 overexpressing plants exhibited a tight connection between mitochondrial, cytoplasm and chloroplast energy metabolism to accommodate the alterations caused by the increased UCP1 activity. A number of uncharacterized transcription factors seem to be good targets for future investigations on the regulation of plant mitochondrial biogenesis and energy metabolism. The results presented in this work allowed a better understanding of the flexibility of energy metabolism in plants, and the use of this mechanism to identify possible regulators of plant mitochondrial biogenesis and stress response / Doutorado / Bioinformatica / Doutor em Genetica e Biologia Molecular

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-24T12:31:54Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Raphael Silva da Cruz - 2014.pdf: 2331298 bytes, checksum: 48f73362fb99734396ab1734092aeb86 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-24T14:11:11Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Raphael Silva da Cruz - 2014.pdf: 2331298 bytes, checksum: 48f73362fb99734396ab1734092aeb86 (MD5) / Made available in DSpace on 2015-03-24T14:11:20Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Raphael Silva da Cruz - 2014.pdf: 2331298 bytes, checksum: 48f73362fb99734396ab1734092aeb86 (MD5) Previous issue date: 2014-09-29 / The increase of the elderly population requires the development of strategies to minimize the negative effects of advancing chronological age in the body. The sarcopenia is one of the main changes observed in aging that causes loss of muscle strength. The assessment of hand grip strength (HGS) is an important variable to estimate the impairment of overall muscular strength of a person and is used as an indicator in several countries. Has been highlighted the genetic susceptibility studies underlying the aging phenomenon. In this context, the objective of this study was to evaluate the possible relationship between the SNPs rs1800849 and rs15763 of UCP3 gene and the HGS in adults. This study was a cross-sectional that included 161 individuals from Goiás population, who underwent an evaluation of HGS, and the collection of biological samples for genotyping by qPCR of polymorphic sites rs15763 and rs1800849 of UCP3 gene. Of the participants, 69.9% were women. The mean age was 50.5 (± 19.9) years and the HGS was 29.7 (± 14.6) kgf. We observed a negative and statistically significant correlation between FPP and age (r = -0.55; p≤0,0001). Regarding the polymorphism CC individuals were stronger than those individuals TT + CT for both SNPs. The maximum of HGS was between 30 to 50 years. The greatest decrease of HGS/ year was associated with genotypes that had the T allele for both SNPs studied. Individuals who presented the T allele in rs18949 had 1,684 times more likely to reduce the HGS in relation to genotype rs15763, the OR was 1.876. The UCP3 appears to be an important variable to modulate muscle strength and therefore may be a useful marker to monitor the aging process. This data from will contribute to the specialized attention to health, especially for the elderly population group. / O aumento da população de idosos requer o desenvolvimento de estratégias que possam minimizar os efeitos negativos do avanço da idade cronológica no organismo. A sarcopenia é uma das principais alterações observada no envelhecimento que causa perda de força muscular. A avaliação da força da preensão palmar (FPP) é uma variável importante para se estimar o comprometimento da força muscular global de uma pessoa, sendo usada como indicador em diversos países. Tem merecido destaque os estudos de susceptibilidade genéticas subjacente ao fenômeno do envelhecimento. Neste contexto, o objetivo do presente estudo foi avaliar a possível relação entre os SNPs rs15763 e rs1800849 do gene UCP3 e a FPP em adultos. O presente estudo foi do tipo transversal, composto por161 participantes da população goiana, que foram submetidos a uma avaliação da FPP, e a coleta de amostras biológicas para a genotipagem através da qPCR dos sítios polimórficos rs15763 e rs1800849 do gene UCP3. Dos participantes, 69,9% eram mulheres. A média da idade foi 50,5 (±19,9) anos, e a da FPP foi de 29,7 (±14,6) kgf. Observou se correlação negativa e estatisticamente significativa entre FPP e idade (r=-0,55; p≤0,0001). Quanto ao polimorfismo os indivíduos CC eram mais fortes dos que os indivíduos CT+TT para os dois SNPs. O pico de FPP dos participantes foi entre 30 a 50 anos. O decréscimo maior de FPP/ano foi associado ao genótipos que apresentavam o alelo T (CT+TT) para ambos os SNPs estudados. Os indivíduos que aprestaram o alelo T em rs18949 tiveram 1,684 (p=0,001) vezes mais chance de reduzir a FPP e em relação ao genótipo rs15763, o OR foi de 1,876 (≤0,0001). A avaliação da qualidade de vida indicou valores altos nos domínios do SF-36, o domínio aspecto social apresentou maior escore com 84,1 e o domínio aspecto físico o menor com 66,1. A desacoplação mediada por UCP3 parece ser uma variável importante para modular a força muscular e, portanto, pode ser um marcador útil para se acompanhar o processo do envelhecimento. Nesse contexto, os dados deste estudo contribuirão para a atenção especializada à saúde, sobretudo para o grupo populacional de idosos.

A atividade biológica de aldeídos α,Β-insaturados tem sido associada a diversos processos incluindo regulação gênica, envelhecimento Alzheimer e disfunções mitocondriais. Neste trabalho investigamos a formação de adutos do trans,trans-2,4-decadienal (DDE), um aldeído produzido endogenamente e presente como contaminante em alimentos e água, em lisina, histidina e citocromo c. Avaliamos também alterações na função de mitocôndrias de fígado de rato expostas ao aldeído. As análises por espectrometria de massas, LC-ESI/MS, indicaram a formação de diversos tipos de adutos de DDE nos aminoácidos lisina e histidina, entre eles bases de Schiff e enaminas. Os resultados obtidos por espectrometria de massas, MALDI-Tof, indicaram a formação de adutos de DDE formados via base de Schiff de maneira concentração do aldeído, tempo e pH dependentes. As análises da proteína digerida por ESI-Q-ToF, indicaram que os adutos foram formados nos resíduos H-33, K-39, 72 e 100, localizados em regiões ricas em resíduos básicos, cuja interação com membranas e citocromo e oxidase tem sido postulada. Observamos também o deslocamento da banda Soret (λ = 409 nm) e o desaparecimento da banda em λ = 695 nm, relativa a coordenação do sexto ligante do grupo heme (M-80). Este fenômeno está associado a abertura da cavidade do grupo heme e o deslocamento do ligante, indicando alterações nas estruturas secundária e terciária da proteína. Os experimentos realizados com mitocôndrias isoladas indicaram que DDE promove danos à membrana interna mitocondrial, demonstrando i pelo aumento no consumo de O2 em mitocôndrias em estado 4. Em decorrência destas lesões observamos também o intenso inchamento mitocondrial, indicado por experimentos de espalhamento de luz e microscopia eletrônica de transmissão. O inchamento não foi bloqueado por ciclosporina A, um inibidor do poro de transição mitocondrial. DDE também induziu a perda do potencial de membrana da organela, demonstrado pelo monitoramento do indicador fluorescente safranina O e aumento da peroxidação lipídica atestado pela quantificação de malondialdeido (MDA). Estes resultados indicam que DDE promove alterações estruturais no citocromo e podendo levar ao comprometimento da atividade da proteína, além de promover alterações em parâmetros mitocondriais, indicando um possível envolvimento na disfunção mitocondrial promovida por estresse oxidativo. / Lipid hydroperoxide-derived α,β-unsaturated aldehydes are involved in several cellular processes such as gene expression, aging, Alzheimer disease and mitochondrial dysfunction. In this work we have investigated adduct formation in lysine, histidine and cytochrome c by trans,trans-2,4-decadienal (DDE), an endogenously lipoperoxidation product. DDE is also a widespread environment aldehyde found, for example, in food and as a contaminant in water. Alterations in rat liver mitochondrial parameters such as oxygen consumption, membrane potentials, swelling and lipoperoxidation were also investigated. LC-ESI/MS experiments indicated that DDE react with aminoacids lysine and histidine producing adducts. In addition, MALDI-TOF experiments indicated increases in the molecular weight of cytochrome c consistent with the formation of DDE adducts via the Schiff base mechanism. Our data shows that the protein modification was time, pH and DDE-concentration dependent, leading to the formation of at least six adducts after 2 h incubation at pH 7.4. ESI-Q-TOF MS analysis of tryptic digests indicated that H-33, K-39, K-72 and K-100 were modified by DDE. These adducts are present in clusters of basic amino acid residues, which are believed to participate in the interaction of cytochrome c with mitochondrial membrane and cytochrome c oxidase. A blue shift in the Soret band from 409 to 406 nm was also observed, indicating heme crevice opening and displacement of heme sixth ligand (Met-80) coordination in modified protein. DDE (180 µM) treatment increases the rate of mitochondrial oxygen consumption, suggesting a partial mitochondrial uncoupling. Moreover, extensive mitochondrial swelling upon treatment with DDE (900 nM-162 &#181M) was observed by light scattering and transmission electron microscopy experiments. These effects were not prevented by the mitochondrial permeability transition inhibitor cyclosporin A. A DDE concentration-dependent loss in the inner mitocondrial membrane potential, monitored by safranin O fluorescence and an increase in lipoperoxidation were also observed. All together, these results suggest that reactive aldehydes can induce inner mitochondrial membrane damage playing a role in mitochondrial dysfunction associated with oxidative stress.

A atividade biológica de aldeídos α,Β-insaturados tem sido associada a diversos processos incluindo regulação gênica, envelhecimento Alzheimer e disfunções mitocondriais. Neste trabalho investigamos a formação de adutos do trans,trans-2,4-decadienal (DDE), um aldeído produzido endogenamente e presente como contaminante em alimentos e água, em lisina, histidina e citocromo c. Avaliamos também alterações na função de mitocôndrias de fígado de rato expostas ao aldeído. As análises por espectrometria de massas, LC-ESI/MS, indicaram a formação de diversos tipos de adutos de DDE nos aminoácidos lisina e histidina, entre eles bases de Schiff e enaminas. Os resultados obtidos por espectrometria de massas, MALDI-Tof, indicaram a formação de adutos de DDE formados via base de Schiff de maneira concentração do aldeído, tempo e pH dependentes. As análises da proteína digerida por ESI-Q-ToF, indicaram que os adutos foram formados nos resíduos H-33, K-39, 72 e 100, localizados em regiões ricas em resíduos básicos, cuja interação com membranas e citocromo e oxidase tem sido postulada. Observamos também o deslocamento da banda Soret (λ = 409 nm) e o desaparecimento da banda em λ = 695 nm, relativa a coordenação do sexto ligante do grupo heme (M-80). Este fenômeno está associado a abertura da cavidade do grupo heme e o deslocamento do ligante, indicando alterações nas estruturas secundária e terciária da proteína. Os experimentos realizados com mitocôndrias isoladas indicaram que DDE promove danos à membrana interna mitocondrial, demonstrando i pelo aumento no consumo de O2 em mitocôndrias em estado 4. Em decorrência destas lesões observamos também o intenso inchamento mitocondrial, indicado por experimentos de espalhamento de luz e microscopia eletrônica de transmissão. O inchamento não foi bloqueado por ciclosporina A, um inibidor do poro de transição mitocondrial. DDE também induziu a perda do potencial de membrana da organela, demonstrado pelo monitoramento do indicador fluorescente safranina O e aumento da peroxidação lipídica atestado pela quantificação de malondialdeido (MDA). Estes resultados indicam que DDE promove alterações estruturais no citocromo e podendo levar ao comprometimento da atividade da proteína, além de promover alterações em parâmetros mitocondriais, indicando um possível envolvimento na disfunção mitocondrial promovida por estresse oxidativo. / Lipid hydroperoxide-derived α,β-unsaturated aldehydes are involved in several cellular processes such as gene expression, aging, Alzheimer disease and mitochondrial dysfunction. In this work we have investigated adduct formation in lysine, histidine and cytochrome c by trans,trans-2,4-decadienal (DDE), an endogenously lipoperoxidation product. DDE is also a widespread environment aldehyde found, for example, in food and as a contaminant in water. Alterations in rat liver mitochondrial parameters such as oxygen consumption, membrane potentials, swelling and lipoperoxidation were also investigated. LC-ESI/MS experiments indicated that DDE react with aminoacids lysine and histidine producing adducts. In addition, MALDI-TOF experiments indicated increases in the molecular weight of cytochrome c consistent with the formation of DDE adducts via the Schiff base mechanism. Our data shows that the protein modification was time, pH and DDE-concentration dependent, leading to the formation of at least six adducts after 2 h incubation at pH 7.4. ESI-Q-TOF MS analysis of tryptic digests indicated that H-33, K-39, K-72 and K-100 were modified by DDE. These adducts are present in clusters of basic amino acid residues, which are believed to participate in the interaction of cytochrome c with mitochondrial membrane and cytochrome c oxidase. A blue shift in the Soret band from 409 to 406 nm was also observed, indicating heme crevice opening and displacement of heme sixth ligand (Met-80) coordination in modified protein. DDE (180 µM) treatment increases the rate of mitochondrial oxygen consumption, suggesting a partial mitochondrial uncoupling. Moreover, extensive mitochondrial swelling upon treatment with DDE (900 nM-162 &#181M) was observed by light scattering and transmission electron microscopy experiments. These effects were not prevented by the mitochondrial permeability transition inhibitor cyclosporin A. A DDE concentration-dependent loss in the inner mitocondrial membrane potential, monitored by safranin O fluorescence and an increase in lipoperoxidation were also observed. All together, these results suggest that reactive aldehydes can induce inner mitochondrial membrane damage playing a role in mitochondrial dysfunction associated with oxidative stress.

L’attention scientifique s’est récemment portée sur l’identification des mécanismes proximaux sous-tendant les compromis évolutifs;tels que les compromis existant entre croissance/reproduction et longévité. La production d’espèces réactives de l’oxygène (ROS )a été suggérée comme un candidat potentiel ,de par sa liaison étroite au métabolisme énergétique (sous-­produits du fonctionnement mitochondrial) et son caractère inévitable. Si la production de ROS excède le niveau des défenses antioxydantes, une situation de stress oxydant va en résulter et a été associé au vieillissement . Puisque la mitochondrie n’est pas uniquement la centrale énergétique de la cellule mais aussi le principal producteur de ROS, cette thèse s’est attachée à clarifier les relations entre métabolisme énergétique , fonctionnement mitochondrial et stress oxydant ; avec des études concernant l’impact d’activités coûteuses en énergie (croissance, reproduction, thermogénèse) sur l’équilibre de la balance oxydative. / In recent years, scientific attention has turned towards the identification of the mechanisms underlying the trade-­‐offs occurring between growth rate/reproductive investment and longevity. Amongst these mechanisms, the production of reactive oxygen species (ROS) appears to be a key factor due both to its universal and inevitable nature. ROS are by-­‐products of energy processing by the mitochondria. If ROS production exceeds the capacity of the various antioxidant systems, oxidative stress will occur, and the accumulation of oxidative damage over time is thought to be a potential cause of ageing. Since mitochondria are not only the powerhouse of animal cells but also the main producer of ROS, this PhD thesis aimed to clarify the relationships between mitochondrial uncoupling state (i.e. efficiency to produce ATP), energy metabolism and oxidative stress. I investigated the impact of energy-­‐demanding activities such as thermogenesis, reproduction and growth on oxidative homeostasis.