<p>Cytochrome P450s are a super-family of heme containing proteins that are found in all domains of life and are involved in the synthesis and breakdown of steroids, xenobiotics, and pharmaceuticals. Using five heterologously expressed zebrafish (Danio rerio) CYP1s, an assay was developed for CYP activity in order to monitor the consumption of the cofactor NADPH, providing a label-free screening tool to determine function of novel CYP genes. Using well-established fluorogenic substrates, NADPH and NADP+ were separated by capillary electrophoresis (CE) from stopped CYP1 reactions and measured with UV absorbance detection as a surrogate to assess the rate of substrate metabolism. Product formation was confirmed by fluorometric detection of metabolites, giving rates of enzyme activity which could be compared to the rates of cofactor turn-over measured by CE. 17β-estradiol, four alkoxyresorufin and two coumarin based synthetic fluorogenic CYP substrates were screened for activity with recombinant zebrafish CYP1A, 1B1, 1C2, 1C2 and 1D1. Cofactor consumption was generally much larger than product formation for the majority of substrates and CYP1 isoforms, suggesting that the majority of metabolic events were uncoupled. Large uncoupling was seen in CYP1 when metabolizing estradiol, showing that endogenous compounds can also show severe uncoupling. Reactive oxygen species (ROS), a product of uncoupled events, were detected with 2,7- dichorofluorescein. Attempts for concomitant detection of ROS production and cofactor consumption with CE-UV detection were investigated, however, detection limits for 2,7-dichlorofluorescein were not adequate for detection of hydrogen peroxide production from CYP1 mediated reactions. Future work will be required to develop a single assay to quantitatively measure CYP activity by CE for functional determination of CYPs with unknown function.</p> / Master of Science (MSc)

Obesity is a rapidly growing epidemic in the USA and worldwide. While the molecular and cellular mechanism of obesity is incompletely understood, studies have shown that excess adiposity may arise from increased adipogenesis (hyperplasia) and adipocyte size (hypertrophy) . Emerging evidence underscores autophagy as an important mediator of adipogenesis and adiposity. We are interested in the upstream regulator of adipocyte autophagy and how it impacts adipocyte biology. Given that metabolic stress activates transcription factor FoxO1 in obesity, my dissertation project is designed to depict the role of FoxO1 in adipocyte autophagy and biology. We found that FoxO1 upregulation was concomitant with elevation of autophagy activity during adipogenesis. Inhibition of FoxO1 suppressed autophagy flux and almost completely prevented adipocyte differentiation. For the first time, we found that the kinetics of FoxO1 activation followed a series of sigmoid curves that showed multiple activation-inactivation transitions during adipogenesis. Our study provides critical evidence casting light on the controversy in the literature that either persistent inhibition or activation of FoxO1 suppresses adipogenesis. In addition, we identified two central pathways that FoxO1-mediated autophagy regulated adipocyte biology: (1) to control lipid droplet growth via fat specific protein 27 (FSP27) in adipocytes; and (2) to differentially regulate mitochondrial uncoupling proteins (UCP) that have been implicated in browning of white adipose tissue and redox homeostasis. Mechanistically, FoxO1 appears to induce autophagy through the transcription factor EB (Tfeb), which was previously shown to regulate both autophagosome and lysosome. Chromatin immunoprecipitation assay demonstrated that FoxO1 directly bound to the promoter of Tfeb, and inhibition of FoxO1 attenuated the binding, which resulted in reduced Tfeb expression. To investigate the role of FoxO1 in vivo, we have developed mouse models to modulate FoxO1 in adipose tissue using an inducible Cre-loxP system. Tamoxifen is widely used to activate the inducible Cre recombinase that spatiotemporally control target gene expression in animal models, but it was unclear whether tamoxifen itself may affect adiposity and confounds phenotyping. Part of my dissertation work was to address this important question. We found that tamoxifen led to reduced fat mass independent of Cre, which lasted for 4-5 weeks. Mechanistically, Tamoxifen induced reactive oxygen species (ROS) and augmented apoptosis. Our data reveals a critical period of recovery following tamoxifen treatment in the study of inducible knockout mice. Together, my dissertation work demonstrates FoxO1 as a critical regulator of adipocyte autophagy via Tfeb during adipogenesis. FoxO1-mediated autophagy controls FSP27, lipid droplet growth, and mitochondrial uncoupling proteins. Further study of FoxO1-autophagy axis in obese subjects is of physiological significance, and the investigation is under way. / Ph. D.

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.

Les pegmatites à éléments rares de type LCT sont depuis longtemps étudiées et exploitées pour leurs gemmes et les métaux rares qu’elles contiennent. Malgré de nombreuses études réalisées sur les processus d’enrichissement en éléments rares, ou les mécanismes à l’origine de leurs textures, très peu d’études ont été dédiées aux mécanismes de mise en place des pegmatites et à leur répartition spatiale à l’échelle du champ. Afin de déterminer les mécanismes moteurs à l’origine de l’ascension de ces magmas, une étude multidisciplinaire a été menée sur 3 champs de pegmatites à éléments rares, répartis à l’échelle de la chaîne Varisque. Les résultats démontrent la proximité entre la localisation des pegmatites minéralisées et l’intensité de la déformation encaissante. De plus, la mise en place de ces magmas semble facilitée par un certain mode de fracturation. La modélisation numérique de la mise en place des magmas dans ces zones crustales fragilisées indique que l’ascension est facilitée par leurs faibles viscosité et densité, mais aussi par des perméabilités crustales très élevées (> 10⁻¹² m²), à des profondeurs de l’ordre de 10 km. Enfin, d’après les signatures isotopiques du Li mesurées sur des micas pegmatitiques, le lithium ne fractionne pas depuis le granite voisin, jusqu’aux pegmatites les plus différenciées, puisque les valeurs δ⁷Li (‰) sont toutes comprises dans une gamme de - 2 à + 2 ‰, similaire à celle rencontrée dans les granites orogéniques. Nous suggérons donc que la genèse des magmas pegmatitiques est commune à celle des granites hyperalumineux. Les mécanismes de fracturation et d’attraction (« magma-pumping ») sont envisagés pour avoir favorisé l’ascension de ces magmas résiduels, enrichis en éléments rares, de faibles volumes, au cours de transitions brutales et de courtes durées (~ 10³ ans), de la perméabilité. L’ensemble de ces résultats permet de remettre en question le modèle du granite parent classiquement utilisé pour la prospection de ces gisements, et de proposer un modèle revisité couplant la genèse et la mise en place de ces magmas. / LCT-type rare-element pegmatites have long been studied and exploited for their gems and rare metals they contain. Despite many studies about the rare-element enrichment, or about the mechanisms leading their exotic textures, very few studies have been dedicated to the mechanisms controlling their emplacement and their spatial distribution at the scale of the pegmatite field. To better investigate the origin of ascent-driving mechanisms of these magmas, a multidisciplinary study was conducted on 3 rare-element pegmatite fields across the Variscan belt. The results demonstrate the spatial proximity of the rare-metals-rich pegmatites with the intensity of deformation of the hosting rocks. In addition, spatial statistical analyses suggest that the emplacement of such magmas has been facilitated by fracture-controlled model. According to numerical models, the rise of these pegmatite-forming melts along weakened crustal zones would be facilitated by their peculiar physico-chemical properties (low viscosity and density), but also by very high crustal permeability (> 10⁻¹² m²) at depths around 10 km. Finally, accordingly to Li isotope signatures measured on pegmatitic micas, lithium does not fractionate from neighbouring granite up to the more differentiated pegmatites, since all δ⁷Li (‰) fall within a range of - 2 to + 2 ‰, as for orogenic granites. Therefore, we suggest that the genesis these pegmatite-forming melts is common to that of peraluminous granites. Mechanisms of fracturation and magma-pumping may have favoured the rise of these low volumes of residual melts, enriched in rare-elements, during short periods (~ 10³ yrs) of strong permeability increase. These results question the granitic model commonly used for the exploration of this type of mineral deposits. We suggest a revisited model accounting for both genesis and emplacement controlling mechanisms of the pegmatite-forming melts.

L’infarctus du myocarde constitue la première cause de mortalité cardiovasculaire. Ainsi, toute stratégie permettant de moduler la vulnérabilité du coeur à l’ischémie-reperfusion (IR) peut représenter un intérêt majeur de santé publique. L’exercice en endurance est reconnu comme une stratégie de cardioprotection efficace dont les mécanismes cellulaires restent néanmoins peu connus. Les objectifs de ce travail de thèse sont donc i) d’évaluer le rôle préventif de l’exercice sur le développement d’un phénotype sensible à l’IR myocardique, et ii) de tenter de mieux comprendre le rôle de la eNOS dans la radioprotection par l’exercice. Dans la première partie de ce travail, nous avons mis en évidence que l’exercice permet de prévenir le développement d’un phénotype pathologique cardiomyocytaire,par une amélioration du statut antioxydant et un maintien de l’homéostasie calcique cellulaire, et ainsi permet de normaliser la sensibilité du coeur à l’IR chez une population à risque. Dans un second temps, les travaux réalisés ont permis de mettre en avant le rôle majeur de la eNOS dans la cardioprotection par l’exercice. Cette cardioprotection est associée à une diminution du niveau de phosphorylation (Ser1177) et surtout de l’état de découplage de cette enzyme au cours des premières minutes de reperfusion. Ces modifications, associées à l’amélioration du statut antioxydant cardiaque par l’exercice, sont à l’origine d’une diminution du stress nitro-oxydant au cours de la reperfusion,expliquée par une moindre synthèse de NO et une meilleure capacité à éliminer l’O2.-, permettant ainsi de limiter la synthèse de ONOO-. L’ensemble de ce travail de thèse a ainsi permis de mettre en évidence la complexité de la cardioprotection par l’exercice, nécessitant l’interaction entre différents mécanismes cellulaires tels que l’amélioration du statut enzymatique antioxydant, le découplage de la eNOS au cours de la reperfusion précoce et la régulation de l’homéostasie calcique intracellulaire. Ce travail à d’autre part permis de mieux appréhender le rôle complexe de la voie de synthèse du NO parla eNOS dans la modulation de la vulnérabilité du coeur à un stress tel que l’IR / Exercise training is recognized as an efficient way to protect the myocardium against ischemiareperfusion(IR). However, mechanisms responsible for such cardioprotection remain still unclear. Theaims of this work were then i) to evaluate the preventive effect of exercise on a model highly sensitiveto myocardial IR, and ii) to investigate the role of eNOS in exercise-induced cardioprotection. In a firstpart we showed that regular boots of exercise, by its beneficial effects on calcium handling andenzymatic antioxidant status, prevents the highly sensitive phenotypical remodeling of the heart andthen normalized heart vulnerability to IR. Then, in a second part of this work, we showed that exerciseinducedcardioprotection was associated with a decrease of eNOS phosphorylation at Ser1177 andespecially its uncoupling during early reperfusion. Such phenomenon, associated with increased heartantioxidant capacity was responsible for reduced nitro-oxidative stress. Indeed, reduced NOSdependentNO synthesis associated with the improved capacity to scavenge O2.- contribute to preventthe formation of ONOO-. Altogether, these results showed that exercise-induced cardioprotection is acomplex mechanism requiring interactions between antioxidant capacity improvement, eNOSuncoupling during reperfusion and intracellular calcium homeostasis. Finally, this work opens newperspectives regarding the role of NO synthesis modulation to impact heart sensitivity to IR

Made available in DSpace on 2015-03-26T13:36:35Z (GMT). No. of bitstreams: 1 texto completo.pdf: 318019 bytes, checksum: f14201c1f1fe349bcc7b8771be868f8e (MD5) Previous issue date: 2006-02-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The effects of aluminum on growth, respiratory activities and on some enzymes related to reactive oxygen species (ROS) elimination were examined in roots of two corn seedlings cultivated in nutrient solution, by comparing a susceptible (BR 106) and a tolerant cultivar (BR 206). Growth of the main root decreased in both cultivars, mainly in the susceptible one, at aluminum concentration of 50 and 100 µM. Only in the tolerant cultivar aluminum induced a decrease in the leak of electrolytes. On the contrary, the susceptible cultivar showed significant increase in lipid peroxidation. Aluminum promoted higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), only in the tolerant cultivar. No difference was observed in the activities of catalase (CAT), peroxidases (POX) and glutathione reductase (GR) in both cultivars. Ascorbate content was not altered, but the ascorbate redox state (ratio ascorbate/dehydroascorbate) increased in the tolerant cultivar. In absence of aluminum, respiratory oxygen consumption was higher in the tolerant cultivar, both in states 3 and 4. These respiratory activities were decreased by aluminum in the susceptible cultivar, but were increased in the tolerant one. ADP/O ratios were decreased by aluminum, in both cultivars at a similar extent. The cytochrome c pathway was not altered by aluminum in the tolerant cultivar, but decreased in the susceptible one. Without aluminum, the susceptible cultivar showed a higher alternative oxidase (AOX) activity than the tolerant one. Aluminum promoted a decrease in AOX activity in the susceptible cultivar, the opposite response being observed in the tolerant one. In both cultivars aluminum induced an increase above 120% in the residual oxygen consumption. Activity of the plant uncoupling mitochondrial protein (PUMP) was decreased by aluminum in the susceptible cultivar and enhanced in the tolerant one. These results suggest that the tolerant cultivar possesses a more efficient enzymatic system for ROS removal than the sensitive one. Furthermore, the partial mitochondrial uncoupling, resulting from higher AOX and PUMP activities should have contributed to the higher aluminum stress tolerance showed by cultivar BR 206. / Os efeitos do alumínio sobre o crescimento, a respiração e as atividades de algumas enzimas envolvidas na eliminação de espécies reativas de oxigênio foram avaliadas em plântulas de duas cultivares de milho, uma sensível (BR 106) e outra tolerante ao alumínio (BR 206), cultivadas em solução nutritiva, pH 4,0. O alumínio, nas concentrações de 50 e 100 µM, reduziu o crescimento da raiz principal das duas cultivares, especialmente da cultivar sensível. O alumínio reduziu o extravasamento de eletrólitos, apenas nas raízes da cultivar tolerante e aumentou a peroxidação de lipídios, apenas na cultivar sensível. O tratamento com alumínio resultou em aumentos significativos nas atividades da dismutase do superóxido (SOD) e da peroxidase do ascorbato (APX), apenas na cultivar tolerante. Entretanto, não foram observadas diferenças significativas nas atividades da catalase (CAT), das peroxidades (POX) e da redutase da glutationa (GR), em ambas as cultivares. Os teores de ascorbato também não apresentaram variação significativa, mas foi observado aumento no estado redox (razão ascorbato/desidroascorbato), porém apenas na cultivar tolerante. No tratamento controle, as taxas respiratórias de mitocôndrias isoladas da cultivar tolerante se apresentaram mais elevadas, tanto no estado 3 como no estado 4. Estas taxas foram reduzidas pelo alumínio na cultivar sensível, e aumentadas na cultivar tolerante. As razões ADP/O foram reduzidas pelo tratamento com alumínio, nas duas cultivares, na mesma proporção. A rota do citocromo c não foi alterada pelo alumínio, na cultivar tolerante, mas foi reduzida na cultivar sensível. Na ausência de alumínio, a cultivar sensível apresentou atividade da oxidase alternativa (AOX) mais elevada, que foi reduzida pela presença de alumínio. Ao contrário, o consumo de oxigênio pela rota alternativa foi aumentado, pelo alumínio, na cultivar tolerante. Nas duas cultivares, o alumínio promoveu acréscimos superiores a 120% no consumo residual de oxigênio. A atividade da proteína desacopladora de plantas (PUMP) foi diminuída pelo alumínio, na cultivar sensível, e aumentada, na tolerante. Estes resultados sugerem que a cultivar tolerante possua um mecanismo enzimático mais eficiente de remoção ou neutralização de espécies reativas de oxigênio que a cultivar sensível. Além disso, o parcial desacoplamento mitocondrial observado, resultante do aumento das atividades da oxidase alternativa e da proteína desacopladora, deve contribuir para a maior tolerância da cultivar BR 206 ao estresse por alumínio.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As proteÃnas desacopladoras de planta (pUCP) estÃo localizadas na membrana mitocondrial interna e facilitam o transporte de prÃtons do espaÃo intermembranar para a matriz mitocondrial, desviando a passagem de H+ atravÃs da F1-ATPase, afetando assim a eficiÃncia da fosforilaÃÃo oxidativa, isto Ã, diminuindo a sÃntese de ATP acoplada ao funcionamento da cadeia transportadora de elÃtrons. Portanto, essas proteÃnas sÃo responsÃveis pela dissipaÃÃo do gradiente eletroquÃmico de H+, gerado pela respiraÃÃo, liberando calor para o ambiente. Tais proteÃnas pertencem a FamÃlia de Carreadores de Ãnions Mitocondriais (FCAM) e sÃo codificadas por famÃlias multigÃnicas. Sua funÃÃo ainda nÃo està completamente elucidada, mas a literatura sugere participaÃÃo na adaptaÃÃo a situaÃÃes de estresses biÃticos e abiÃticos e na proteÃÃo da cÃlula evitando a produÃÃo de espÃcies reativas do oxigÃnio (EROs). Sua participaÃÃo na termogÃnese adaptativa à questionÃvel. O objetivo do presente trabalho foi caracterizar a famÃlia multigÃnica da pUCP em Vigna unguiculata (L.) Walp bem como sua regulaÃÃo atravÃs da expressÃo gÃnica em resposta a diferentes estresses abiÃticos. Sementes de Vigna unguiculata foram germinadas em papel umedecido com Ãgua, no escuro e apÃs 3 dias as plÃntulas foram transferidas para soluÃÃo de Hoagland durante 3 dias antes da aplicaÃÃo dos estresses. As raizes e as folhas foram coletadas com 0, 6, 12 e 24 horas, apÃs respectivas adiÃÃes de NaCl 100 mM, PEG 200,67 g/L, H2O2 10 mM e Ãcido salicÃlico 5 mM, para caracterizar a famÃlia multigÃnica e o perfil de expressÃo dos genes da pUCP por RT- PCR semiquantitativa e por PCR em tempo real (qPCR). Primers especÃficos foram desenhados com base nas sequÃncias de cDNAs/genes de pUCPs identificadas em Vigna unguiculata usando a ferramenta PerlPrimer. A amplificaÃÃo do cDNA da actina foi usada para a normalizaÃÃo dos dados de RT-PCR semi-quantitativa e trÃs genes constitutivos foram usados na normalizaÃÃo dos dados da qPCR: 2 genes para actina (actinas 4 e 5) e 1 gene para o fator de alogamento 1-&#945; (EF1&#945;). AnÃlise in silico revelou que a pUCP na ordem Fabales à codificada por pelo menos seis genes com duplicaÃÃo do gene pUCP do tipo 1 (1a e 1b) e deleÃÃo do gene pUCP6. A famÃlia multigÃnica da pUCP, constituÃda de 6 genes, entÃo foi identificada em Vigna unguiculata (VuUCP1a, VuUCP1b, VuUCP2, VuUCP3, VuUCP4 e VuUCP5 ). O alinhamento de sequÃncias nucleotÃdicas e de aminoÃcidos das xiii. espÃcies da ordem Fabales incluindo as de Vigna unguiculata, revelou 3 sequÃncias conservadas denominadas Sinal ProtÃico de TransferÃncia de Energia (SPTE), alÃm de 4 domÃnios especÃficos que caracterizam existÃncia de pUCPs verdadeiras em Vigna unguiculata. O gene VuUCP1a foi expresso constitutivamente em folhas e raÃzes, contrastando com VuUCP1b, cuja expressÃo foi modulada em funÃÃo dos estresses e de tecidos. Em raÃzes a expressÃo do VuUCP1b aumentou em resposta a todos os tratamentos (PEG, NaCl, H2O2 e Ãcido salicÃlico) enquanto que em folhas a expressÃo nÃo aumentou em reposta ao NaCl. VuUCP2 teve a expressÃo inibida em resposta ao PEG em folhas. VuUCP4 apresentou expressÃo constitutiva em resposta aos estresses em ambos os tecidos, enquanto que VuUCP3 e VuUCP5 apresentaram induÃÃo de expressÃo por vÃrios estresses dependente do tecido. A identificaÃÃo da famÃlia multigÃnica das pUCPs em Vigna unguiculata e seu perfil de expressÃo gÃnica diferencial, em funÃÃo do estresse aplicado e do tecido estudado, pÃe em evidÃncia um possÃvel papel dessa proteÃna nos mecanismos de ajustamento das plantas aos estresses ambientais. / The plant uncoupling proteins (pUCP) are located in the inner mitochondrial membrane and facilitate the proton translocation across the intermembrane space into the mitochondrial matrix, deflecting the passage of H + by F1-ATPase activity, thus affecting the efficiency of oxidative phosphorylation, i.e decreasing the synthesis of ATP coupled to the operation of the electron transport chain. Therefore, these proteins are responsible for dissipating the electrochemical gradient of H+, generated by respiration, releasing heat to the environment. These proteins belong to family of carriers Mitochondrial Anion (FCAM) and are encoded by multigene families. Their function is not yet fully elucidated, but the literature suggests involvement in adapting to biotic and abiotic stresses and cell protection by avoiding the production of reactive oxygen species (ROS). Their participation in adaptive thermogenesis is unclear. The aim of this work was to characterize the multigene family of pUCP in Vigna unguiculata (L.) Walp and your regulation through gene expression in response to different abiotic stresses. Vigna unguiculata seeds were germinated on paper imbibed water in the dark. Three days after germination the seedlings were transferred to Hoagland solution for 3 days before application of stress. Roots and leaves were collected at 0, 6, 12 and 24 hours after addition of the respective 100 mM NaCl, 200.67 g / L PEG, 10 mM H2O2 and 5 mM salicylic acid to characterize the profile of multigene family expression of genes for pUCP by semiquantitative RT-PCR and real-time PCR (qPCR). Specific primers were designed based on the sequences of cDNA / gene identified in pUCPs Vigna unguiculata using the PerlPrimer tool. Amplification of cDNA of actin was used to normalize the data for RT-PCR semi-quantitative and three constitutive genes were used to normalize the data of qPCR: 2 to actin gene (actin 4 and 5) and a gene for factor alogament 1-&#945; (EF1&#945;). In silico analysis revealed that in Fabales order pUCP is encoded by at least six pUCPs genes presenting a duplication of the gene type 1 (1a, 1b) and a pUCP6 gene deletion. The multigene family of pUCP, consisting of six genes was then identified in Vigna unguiculata (VuUCP1a, VuUCP1b, VuUCP2, VuUCP3, VuUCP4 and VuUCP5). The alignment of amino acid and nucleotide sequences of the species of Fabales order including Vigna unguiculata, revealed three xv. conserved sequences called signal Energy Transfer Protein (SPTE), and four domains (or regions or sites) existence of specific true pUCPs in Vigna unguiculata. VuUCP1a gene was constitutively expressed in leaves and roots, contrasting with VuUCP1b, whose expression was modulated in a stress and tissue-dependent manner. The VuUCP1b expression increased in response to all treatments (PEG, NaCl, H2O2 and salicylic acid) in roots, whereas the expression in leaves did not increase in response to NaCl. VuUCP2 expression was inhibited in response to PEG in leaves. VuUCP4 showed constitutive expression in response to stresses in both tissues, while VuUCP3 and VuUCP5 showed induction of expression by various stresses depending on the tissue type. The identification of the multigene family of pUCPs in Vigna unguiculata and its gene expression profile in different tissues and stress conditions highlights a possible role of this protein in the adjustment of plants to environmental stresses.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / As proteÃnas desacopladoras de planta (pUCPs) sÃo proteÃnas integrais de membrana, localizadas na membrana mitocondrial interna, pertencentes a FamÃlia de Carreadores de Ãnions Mitocondriais (FCAM), responsÃveis pela dissipaÃÃo do gradiente eletroquÃmico de prÃtons, gerado pela respiraÃÃo, como calor. Na presenÃa de Ãcidos graxos livres (AGL), as pUCPs facilitam a reentrada de prÃtons do espaÃo intermembranar para a matriz, desviando-os da ATP Sintase, inibindo assim a fosforilaÃÃo oxidativa. A funÃÃo dessas enzimas ainda nÃo està completamente elucidada, mas a literatura sugere a sua participaÃÃo em processos de amadurecimento de frutos, adaptaÃÃo a situaÃÃes de estresses biÃticos e abiÃticos e proteÃÃo da cÃlula evitando a produÃÃo de espÃcies reativas de oxigÃnio (EROS). Sua participaÃÃo na termogÃnese adaptativa à questionÃvel. O objetivo do presente trabalho foi caracterizar a atividade enzimÃtica da pUCP de mitocÃndrias de hipocÃtilos de Vigna unguiculata (L.) Walp atravÃs de ensaios polarogrÃficos, identificar o(s) gene(s) das pUCPs, estudando a sua expressÃo em diferentes situaÃÃes de estresses abiÃticos. A atividade da pUCP foi avaliada em presenÃa de Ãcidos graxos (palmÃtico, linolÃico, mirÃstico e lÃurico) e BSA tendo succinato e malato como substratos e distintos pHs (6,5, 7,2 e 7,8). As maiores atividades foram obtidas com Ãcido linoleico na presenÃa de succinato em pHs mais alcalinos (7,2 e 7,8). Primers degenerados a partir de dez diferentes pUCPs, denominados pump1 e pump2, foram desenhados para a amplificaÃÃo dos fragmentos gÃnicos por RT-PCR e PCR. Isolou-se o RNA total de folhas de plÃntulas de V. unguiculata submetidas a diferentes condiÃÃes de estresses (NaCl 100mM, H2O2 1mM e PEG 200,67g/L) que foram amplificados por RT-PCR, purificados e clonados no plasmÃdio pCR4-TOPO. Sete fragmentos gÃnicos de aproximadamente 760 pb foram seqÃenciados, apresentando 100% de identidade entre si. A comparaÃÃo da seqÃÃncia deduzida de aminoÃcidos desse fragmento de cDNA com a soja revelou 94% de homologia com GmUCP1a e 90% de homologia com GmUCP1b. Os resultados sugerem que o gene do feijÃo identificado em V. unguiculata (VuUCP1a) à ortÃlogo ao gene GmUCP1a de soja (Glycine max). A expressÃo de VuUCP1a em feijÃo em condiÃÃes de estresses abiÃticos (salino, oxidativo e osmÃtico) atravÃs de anÃlise por xv. RT-PCR revelou um perfil diferencial, sugerindo induÃÃo de expressÃo de VuUCP1a apenas em resposta ao estresse salino. Isolou-se o DNA genÃmico de V. unguiculata e dois fragmentos gÃnicos (1700 e 1900pb) foram amplificados por PCR. A amplificaÃÃo de dois fragmentos distintos a partir do DNA genÃmico sugere a existÃncia de pelo menos dois genes codificando pUCPs em feijÃo, sendo assim, a pUCP à codificada por uma famÃlia multigÃnica. / The uncoupling proteins of plants (pUCPs) are integral proteins of membrane, located in the mitochondrial inner membrane, belong the Mitochondrial Anion Carrier Family (MACF). They are responsible for the dissipation as heat of the electrochemical gradient of protons, generated during respiration. In the presence of free fatty acid acids (FFA), the UCPs facilitate the re-entry of protons from intermembrane space for the matrix and these protons are deviated from the influence of the ATP sintase what leads to the inhibition the oxidative phosphorilation. The function of these enzymes completely is still not elucidated, but literature suggests its participation in processes of fruit maturation, in the adaptation to stress conditions and in cell protection by avoiding the production of reactive species of oxygen (EROS). The involvement of this enzyme in adaptive thermogenesis is questionable. The objective of the present work was to characterize the enzymatic activity of pUCP of mitochondria from hypocotyls of Vigna unguiculata (L.) Walp through polarographic assays and to identify the(s) gene(s) of pUCPs, through its expression in different conditions of abiotic stress. The activity of pUCP was evaluated in the presence of fatty acids (palmitic, linoleic, myristic and lauric) and BSA, having succinate and malate as oxidizable substrates at different pHs (6,5, 7,2 and 7,8). The higuest activities were obtained in the presence of linoleic acid, succinate as substrate and with more alkaline pHs (7,2 and 7,8). Degenerate primers, obtained from ten different pUCPs, called pump1 and pump2, has been designed for amplification of the gene fragments through RT-PCR and PCR. The total RNA was isolated from plants leaves of V. unguiculata submitted to different stress conditions (100 mM NaCl, 1 mM H2O2 and 200,67 g/L PEG) and cDNAs fragments amplified by RT-PCR had been purified and cloned in the plasmid PCR4-TOPO. Seven cDNA fragments of approximately 760 pb had been sequenced and presented 100% of identity among themselves. The comparison of the deduced amino acid sequence of this cDNA fragment with those of soybean disclosed 94% of identity with the gene GmUCP1a and 90% of identity GmUCP1b soybean gene. These results suggest that the pUCP gene identified in V. unguiculata (VuUCP1a) is ortologous to the GmUCP1a gene from soybean (Glycine max). The expression of genes of pUCP in beans, under abiotic stress xvii. conditions (salinity, oxidative and osmotic conditions) analyzed through RT-PCR disclosed a differential profile what suggests induction of expression of VuUCP1a only in response to salt stress. The genomic DNA of V. unguiculata was isolated and two gene fragments (1700 and 1900 pb) had been amplified by PCR. The amplification of two fragments from the genomic DNA suggests the existence of at least two pUCPs genes in beans what leads to the conclusion of a pUCP multigenic family.

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.

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