Uncoupling proteins (UCPs) constitute a highly conserved subset of mitochondrial solute carriers. Discovered in small rodents in the early 1970’s, UCPs and their homologs have since been found in nematodes, plants, birds, and, most recently, in significant depots within humans (Krauss et al. 2005, Van marken Lichtenbelt 2009). Following activation by long chain fatty acids (LCFA, e.g. oleic acid) and reactive oxygen species (ROS, e.g. 4-hydroxynonenal (4HNE)), UCPs form a proton channel within the inner mitochondrial membrane and permit the influx of hydrogen ions from the inter membrane space into the mitochondrial matrix. UCPs effectively uncouple oxidative phosphorylation (OX-PHOS) from ATP generation, resulting in increasing oxygen consumption and dissipating the chemical energy in the form of heat. Found primarily in brown adipose tissue (BAT) of small hibernating mammals, the canonical role of uncoupling protein 1 (UCP1) in mammalian adaptive thermogenesis has been thoroughly studied. However, UCP1 is not the only member of the uncoupling family found within BAT. Also playing a key role in this tissue is uncoupling protein 3 (UCP3), which is a close homolog to UCP1. However, in spite of the fact that UCP3 shares more than 50% amino acid homology and tissue localization with UCP1, the true function UCP3 is very poorly elucidated. Part of the difficulty in determining this function lies in the expression levels of the UCP3 protein, which are hundreds of folds less than UCP1 in this tissue. In addition, their homologous structure makes teasing apart UCP3-specific phenomena from UCP1-mediated mechanisms very difficult using conventional techniques in cell and molecular biology. While UCP1 is almost exclusively found in BAT, UCP3 is expressed primarily in skeletal muscle (SKM), which lacks UCP1 completely (Krauss et al. 2005). Because UCP3 is so enriched in SKM, many studies have focused on its role in that tissue and have then tried to transpose these functions into BAT. As a result, UCP3 has been implicated in facilitating numerous biological processes, including non-adaptive facultative thermogenesis, affecting SKM oxidative capacity by modulating LCFA export, and ameliorating elevated levels of ROS-mediated stress within the tissue via glutathionine (GSH) interacting moieties. Ultimately, however, little consensus exists on the function of UCP3 within SKM, and subsequently, even less is known about its purpose in BAT. Previous data has shown that murine UCP1 has the capacity to bind to itself and form homo-tetramers when expressed in vitro in recombinant E. coli (Hoang T. et al. 2013). Here we show that UCP1 interacts with UCP3 in BAT in vivo, supporting Hoang’s research above by showing that UCP1 has the capacity to not only homodimerize but potentially oligomerize with other UCP homologs. While many groups using UCP3-null mice have reported no gross changes in physiologic responses, data previously published in the lab showed that mice lacking UCP3 were protected from potentially fatal hyperthermic effects when administered sympathomimetic agents such as 3,4-Methylenedioxymethamphetamine (MDMA), methamphetamine (METH), lipopolysaccharide (LPS), or norepinephrine (NE) (Mills et al. 2003, Kenaston et al. 2010). This implies that UCP3 plays an intimate role in sympathetic nervous system (SNS) mediated thermogenesis. Based upon the foregoing, the primary goal of the research discussed in this thesis was to elucidate the functions of UCP3 within BAT. In this study, we recapitulated results seen by other students in this lab: that global UCP3-null mice do indeed exhibit a blunted thermogenic response when treated with sympathomimetic agonists. In addition, despite the near-ubiquitous expression of UCP2 throughout the mammalian organism, this UCP is not involved in SNS-mediated thermogenesis (Arsenijevic et al. 2000). Our data shows that UCP3 is vital to the catecholamine-mediated thermogenic responses following sympathomimetic drug administration. When challenged by METH, UCP3-null mice were able to respond, albeit with a blunted increase in body temperature. Furthermore, when challenged by NE, a key neurotransmitter involved in mediating the responses initiated by the SNS following METH exposure, UCP3-null mice were able to mount half the hyperthermic response seen in WT littermates. However, UCP1/UCP3 double-null animals exhibited an almost four-fold hypothermic effect compared to WT littermates when challenged with NE. In addition, UCP1/UCP3 double-null mice were unable to restore body temperatures back to baseline values, an effect seen in all the other genotypes. This implies that UCP3 plays an important role in restoring body temperatures to physiological norms. Therefore, while the mechanism underlying the decreased responsiveness to NE remains unclear, it is clear that whether localized to SKM or BAT, UCP3 is a major player in the mammalian response to SNS-mediated thermogenesis and global thermoregulation. / text

Orientador: Ivan de Godoy Maia / Resumo: Proteínas desacopladoras (UCPs; do inglês uncoupling proteins) são proteínas especializadas no transporte mitocondrial que desacoplam a respiração da síntese de ATP. UCPs geram um fluxo de prótons através da membrana mitocondrial interna, dependente de ácidos graxos e sensível a nucleotídeos purínicos (PN). No presente trabalho, foram realizados vários estudos empregando UCPs de plantas (pUCP). Inicialmente, foi empreendida a caracterização bioquímica de uma UCP de milho (Zea mays; ZmUCP), representativa de espécies monocotiledôneas. Essa proteína, quando expressa em Escherichia coli e reconstituída em lipossomos, foi capaz de induzir um fluxo de prótons dependente de ácido linoléico (LA) e sensível a ATP com valores de Km, Vmax e Ki similares àqueles observados para pUCPs de espécies dicotiledôneas. A ZmUCP foi utilizada para investigar a importância de um par de histidinas presente no segundo loop matricial da UCP1 de mamíferos e ausente nas pUCPs. A introdução do referido par de histidinas na ZmUCP (Lys155His e Ala157His) provocou um aumento na afinidade por LA enquanto que a sua atividade permaneceu inalterada. Em um estudo mais abrangente de estrutura-função, mutações pontuais nos resíduos Lys147, Arg155 e Tyr269, localizados nas chamadas assinaturas das UCPs, e Cys28 e His83, específicos para pUCPs, foram introduzidas na proteína AtUCP1 de Arabidopsis. Os efeitos de tais mutações nas propriedades bioquímicas da AtUCP1 foram examinados usando sistema de reconstituição em lipossomos. O resíduo Arg155 parece ser crucial para a afinidade da AtUCP1 por LA enquanto que His83 tem importante função na atividade de transporte. Os resíduos Cys28, Lys147, e também Tyr269, são importantes para a correta funcionalidade da AtUCP1, já que suas substituições... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins, which uncouple respiration from ATP synthesis. UCPs mediate a fatty acid (FA)-dependent, purine nucleotide (PN)-inhibitable proton flux across the inner membrane mitochondrial. In the present study, several assays on plant UCPs (pUCP) were performed. Firstly, we biochemically characterized an UCP from maize (Zea mays; ZmUCP), a representative uncoupling protein from monocot species. This protein was expressed in Escherichia coli and reconstituted in liposomes. ZmUCP was fully active and induced a linoleic acid-dependent proton flux that was sensitive to ATP. The obtained Km, Vmax and Ki values were similar to those observed for dicot pUCPs. ZmUCP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in pUCPs. ZmUCP with the introduced histidine pair (Lys155His and Ala157His) displayed increased LA-affinity while its activity remained unchanged. In a subsequent study using AtUCP1, point mutations were introduced in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and Cys28 and His83, specific for pUCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted liposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to LA whereas His83 plays an important role in transport activity. Residues Cys28, Lys147, and also Tyr269 are important for correct AtUCP1 function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to PN inhibitors. Furthermore, we analyzed the expression profiles of the six genes encoding pUCP in Arabidopsis thaliana (AtUCP1-6) in response to salt (NaCl) and osmotic (mannitol)... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2014-06-11T19:32:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-05-30Bitstream added on 2014-06-13T19:42:40Z : No. of bitstreams: 1 favaro_rg_dr_botib.pdf: 702142 bytes, checksum: 37419e284ee746774dfc59ce6ab27b1b (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Proteínas desacopladoras (UCPs; do inglês uncoupling proteins) são proteínas especializadas no transporte mitocondrial que desacoplam a respiração da síntese de ATP. UCPs geram um fluxo de prótons através da membrana mitocondrial interna, dependente de ácidos graxos e sensível a nucleotídeos purínicos (PN). No presente trabalho, foram realizados vários estudos empregando UCPs de plantas (pUCP). Inicialmente, foi empreendida a caracterização bioquímica de uma UCP de milho (Zea mays; ZmUCP), representativa de espécies monocotiledôneas. Essa proteína, quando expressa em Escherichia coli e reconstituída em lipossomos, foi capaz de induzir um fluxo de prótons dependente de ácido linoléico (LA) e sensível a ATP com valores de Km, Vmax e Ki similares àqueles observados para pUCPs de espécies dicotiledôneas. A ZmUCP foi utilizada para investigar a importância de um par de histidinas presente no segundo loop matricial da UCP1 de mamíferos e ausente nas pUCPs. A introdução do referido par de histidinas na ZmUCP (Lys155His e Ala157His) provocou um aumento na afinidade por LA enquanto que a sua atividade permaneceu inalterada. Em um estudo mais abrangente de estrutura-função, mutações pontuais nos resíduos Lys147, Arg155 e Tyr269, localizados nas chamadas assinaturas das UCPs, e Cys28 e His83, específicos para pUCPs, foram introduzidas na proteína AtUCP1 de Arabidopsis. Os efeitos de tais mutações nas propriedades bioquímicas da AtUCP1 foram examinados usando sistema de reconstituição em lipossomos. O resíduo Arg155 parece ser crucial para a afinidade da AtUCP1 por LA enquanto que His83 tem importante função na atividade de transporte. Os resíduos Cys28, Lys147, e também Tyr269, são importantes para a correta funcionalidade da AtUCP1, já que suas substituições... / Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins, which uncouple respiration from ATP synthesis. UCPs mediate a fatty acid (FA)-dependent, purine nucleotide (PN)-inhibitable proton flux across the inner membrane mitochondrial. In the present study, several assays on plant UCPs (pUCP) were performed. Firstly, we biochemically characterized an UCP from maize (Zea mays; ZmUCP), a representative uncoupling protein from monocot species. This protein was expressed in Escherichia coli and reconstituted in liposomes. ZmUCP was fully active and induced a linoleic acid-dependent proton flux that was sensitive to ATP. The obtained Km, Vmax and Ki values were similar to those observed for dicot pUCPs. ZmUCP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in pUCPs. ZmUCP with the introduced histidine pair (Lys155His and Ala157His) displayed increased LA-affinity while its activity remained unchanged. In a subsequent study using AtUCP1, point mutations were introduced in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and Cys28 and His83, specific for pUCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted liposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to LA whereas His83 plays an important role in transport activity. Residues Cys28, Lys147, and also Tyr269 are important for correct AtUCP1 function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to PN inhibitors. Furthermore, we analyzed the expression profiles of the six genes encoding pUCP in Arabidopsis thaliana (AtUCP1-6) in response to salt (NaCl) and osmotic (mannitol)... (Complete abstract click electronic access below)

A. fumigatus é um patógeno oportunista que causa infecções invasivas em hospedeiros imunocomprometidos. Estudos de respiração mitocondrial sugeriram a presença de componentes alternativos em sua cadeia respiratória envolvidos com processos de adaptação a ambientes adversos, como a proteína desacopladora (UCP). UCPs são proteínas mitocondriais cuja atividade dissipa o potencial de membrana gerado durante o transporte de elétrons. Um gene contendo características das três assinaturas moleculares das Proteínas Transferidoras de Energia foi clonado e sequenciado. O alinhamento das sequências genômica e de cDNA mostrou a presença de dois íntrons que, após o splicing, codifica uma proteína contendo 341 aminoácidos, com uma massa molecular de 37 kDa e um pI de 10,02. A fim de se avaliar as propriedades bioenergéticas da UCP-like, essa sequência foi clonada no vetor pYES2 e leveduras S. cerevisiae foram transformadas. Esferoplastos foram preparados e o potencial elétrico transmembrana mitocondrial foi estimado. Os resultados mostraram que o potencial de membrana de esferoplastos de leveduras expressando a proteína UCP-like foi ligeiramente menor e que o decréscimo momentâneo do potencial associado com a fosforilação do ADP foi mais lento quando comparado com o controle, indicando desacoplamento da respiração. Além disso, esse comportamento dos esferoplastos recombinantes foi similar ao controle quando GDP foi adicionado ao meio de reação, sugerindo uma inibição da proteína por esse composto. Para sua caracterização funcional em sistemas reconstituídos, a sequência foi clonada no vetor pET SUMO. A expressão foi realizada em E. coli e a proteína recombinante, purificada por cromatografia em resina de níquel, foi analisada por Western blot com anticorpos anti-(His)6-tag e anti-UCP2 e por espectrometria de massas. A formação dos lipossomos foi confirmada através de medidas de distribuição de partícula por espalhamento de luz dinâmico, as quais sugeriram a formação de vesículas estáveis. Em adição, foi investigada a participação da UCP-like na proteção do A. fumigatus contra danos oxidativos. O nível de mRNA foi determinado por PCR em tempo real na presença de paraquat e menadiona. Em A. fumigatus, a presença dessas drogas pró-oxidantes resultou em um aumento no nível de mRNA desse gene, sugerindo que essa proteína possa também fazer parte de um sistema de defesa antioxidante do fungo. / A. fumigatus is an opportunistic pathogen that causes invasive infections in immunocompromised hosts. Mitochondrial respiration studies suggested the presence of alternative components on its respiration chain, which are involved with the adaptation to hostile environments, such as the uncoupling protein (UCP). UCPs are mitochondrial proteins whose activity dissipates the membrane potential generated during electron transport. A gene containing features of three molecular signatures of Energy Carrier Protein was cloned and sequenced. The alignment between the cDNA and genomic DNA sequences revealed the existence of two introns which after splicing encodes a 341 amino acids protein with a molecular mass of 37 kDa and a pI of 10.02. In order to study bioenergetics properties of UCP-like, the cDNA sequence was cloned into pYES2 vector and transformed in S. cerevisiae. Spheroplasts were prepared and the mitochondrial electrical transmembrane potential was estimated. The results showed that, compared with control cells, mitochondrial electrical transmembrane potential of transformant spheroplasts was slightly smaller and the transient potential decrease associated with ADP phosphorylation was longer, indicating uncoupling of respiration. Moreover, this behavior of recombinant spheroplasts was similar to control cells when GDP was added to the reaction medium, suggesting the inhibition of uncoupling protein. For its functional characterization in reconstituted systems, the cDNA sequence was cloned into pET SUMO vector. The expression was carried out in E. coli and the recombinant protein, purified by chromatography on a nickel-chelating resin, was analyzed by Western blot using anti- (His)6-tag or UCP2 antibodies and by mass spectrometry. Liposome formation was confirmed by light scattering, suggesting the formation of stable vesicles. In addition, the participation of UCP-like in A. fumigatus protection against oxidative damage was investigated. mRNA level was determined by real time PCR in the presence of paraquat and menadione. In A. fumigatus, the presence of these pro-oxidants drugs resulted in increased mRNA level of this gene, suggesting that this protein might also be part of an antioxidant defense system of this fungus.

A. fumigatus é um patógeno oportunista que causa infecções invasivas em hospedeiros imunocomprometidos. Estudos de respiração mitocondrial sugeriram a presença de componentes alternativos em sua cadeia respiratória envolvidos com processos de adaptação a ambientes adversos, como a proteína desacopladora (UCP). UCPs são proteínas mitocondriais cuja atividade dissipa o potencial de membrana gerado durante o transporte de elétrons. Um gene contendo características das três assinaturas moleculares das Proteínas Transferidoras de Energia foi clonado e sequenciado. O alinhamento das sequências genômica e de cDNA mostrou a presença de dois íntrons que, após o splicing, codifica uma proteína contendo 341 aminoácidos, com uma massa molecular de 37 kDa e um pI de 10,02. A fim de se avaliar as propriedades bioenergéticas da UCP-like, essa sequência foi clonada no vetor pYES2 e leveduras S. cerevisiae foram transformadas. Esferoplastos foram preparados e o potencial elétrico transmembrana mitocondrial foi estimado. Os resultados mostraram que o potencial de membrana de esferoplastos de leveduras expressando a proteína UCP-like foi ligeiramente menor e que o decréscimo momentâneo do potencial associado com a fosforilação do ADP foi mais lento quando comparado com o controle, indicando desacoplamento da respiração. Além disso, esse comportamento dos esferoplastos recombinantes foi similar ao controle quando GDP foi adicionado ao meio de reação, sugerindo uma inibição da proteína por esse composto. Para sua caracterização funcional em sistemas reconstituídos, a sequência foi clonada no vetor pET SUMO. A expressão foi realizada em E. coli e a proteína recombinante, purificada por cromatografia em resina de níquel, foi analisada por Western blot com anticorpos anti-(His)6-tag e anti-UCP2 e por espectrometria de massas. A formação dos lipossomos foi confirmada através de medidas de distribuição de partícula por espalhamento de luz dinâmico, as quais sugeriram a formação de vesículas estáveis. Em adição, foi investigada a participação da UCP-like na proteção do A. fumigatus contra danos oxidativos. O nível de mRNA foi determinado por PCR em tempo real na presença de paraquat e menadiona. Em A. fumigatus, a presença dessas drogas pró-oxidantes resultou em um aumento no nível de mRNA desse gene, sugerindo que essa proteína possa também fazer parte de um sistema de defesa antioxidante do fungo. / A. fumigatus is an opportunistic pathogen that causes invasive infections in immunocompromised hosts. Mitochondrial respiration studies suggested the presence of alternative components on its respiration chain, which are involved with the adaptation to hostile environments, such as the uncoupling protein (UCP). UCPs are mitochondrial proteins whose activity dissipates the membrane potential generated during electron transport. A gene containing features of three molecular signatures of Energy Carrier Protein was cloned and sequenced. The alignment between the cDNA and genomic DNA sequences revealed the existence of two introns which after splicing encodes a 341 amino acids protein with a molecular mass of 37 kDa and a pI of 10.02. In order to study bioenergetics properties of UCP-like, the cDNA sequence was cloned into pYES2 vector and transformed in S. cerevisiae. Spheroplasts were prepared and the mitochondrial electrical transmembrane potential was estimated. The results showed that, compared with control cells, mitochondrial electrical transmembrane potential of transformant spheroplasts was slightly smaller and the transient potential decrease associated with ADP phosphorylation was longer, indicating uncoupling of respiration. Moreover, this behavior of recombinant spheroplasts was similar to control cells when GDP was added to the reaction medium, suggesting the inhibition of uncoupling protein. For its functional characterization in reconstituted systems, the cDNA sequence was cloned into pET SUMO vector. The expression was carried out in E. coli and the recombinant protein, purified by chromatography on a nickel-chelating resin, was analyzed by Western blot using anti- (His)6-tag or UCP2 antibodies and by mass spectrometry. Liposome formation was confirmed by light scattering, suggesting the formation of stable vesicles. In addition, the participation of UCP-like in A. fumigatus protection against oxidative damage was investigated. mRNA level was determined by real time PCR in the presence of paraquat and menadione. In A. fumigatus, the presence of these pro-oxidants drugs resulted in increased mRNA level of this gene, suggesting that this protein might also be part of an antioxidant defense system of this fungus.

Non-canonical bioenergetics concerns with those physiological and pathophysiological situations under which ATP synthesis is suppressed. This thesis brings an outcome of three types of studies within the field of the non-canonical bioenergetics, investigating specific bioenergetic phenotypes of cancer cells, on one hand; and a role of mitochondrial uncoupling proteins as deduced from their transcript distribution in various tissues and organs; plus a role of a novel and likely pro-apoptotic factor CIDEa in mitochondria. Cancer cells generally present abnormal bioenergetic properties including an elevated glucose uptake, a high glycolysis and a poorly efficient oxidative phosphorylation system. However, the determinants of cancer cells metabolic reprogramming remain unknown. The main question in this project was how environmental conditions in vivo can influence functioning of mitochondrial OXPHOS, because details of mitochondrial bioenergetics of cancer cells is poorly documented. We have combined two conditions, namely glucose and oxygen deprivation, to measure their potential interaction. We examined the impact of glucose deprivation and oxygen deprivation on cell survival, overall bioenergetics and OXPHOS protein expression. As a model, we have chosen a human breast carcinoma (HTB-126) and appropriate control (HTB-125) cultured cells, as large fraction of breast malignancies exhibit hypoxic tumor regions with low oxygen concentrations and poor glucose delivery. The results demonstrate that glucose presence or absence largely influence functioning of mitochochondrial oxidative phosphorylation. The level of mitochondrial respiration capacity is regulated by glucose; by Crabtree effect, by energy substrate channeling towards anabolic pathways that support cell growth and by mitochondrial biogenesis pathways. Both oxygen deprivation and glucose deprivation can remodel the OXPHOS system, albeit in opposite directions. As an adaptative response to hypoxia, glucose inhibits mitochondrial oxidative phosphorylation to the larger extent than in normoxia. We concluded that the energy profile of cancer cells can be determined by specific balance between two main environmental stresses, glucose and oxygen deprivation. Thus, variability of intratumoral environment might explain the variability of cancer cells´ bioenergetic profile. Mitochondrial uncoupling proteins are proteins of inner mitochondrial membrane that uncouple respiration from ATP synthesis by their protonophoric activity. Originally determined tissue distribution seems to be invalid, since novel findings show that UCP1 is not restricted exclusively to brown fat and that originally considered brain-specific isoforms UCP4 and UCP5 might have wider tissue distribution. Hence, in second part of this thesis, I discuss consequences of findings of UCPn transcripts in the studied mouse and rat tissues. We have shown that mRNA of UCPn varies up to four orders of magnitude in rat and mouse tissues with highest expression in rat spleen, rat and mouse lung, and rat heart. Levels of the same order of magnitude were found for UCP3 mRNA in rat 100 and mouse skeletal muscle, for UCP4 and UCP5 mRNA in mouse brain, and for UCP2 and UCP5 mRNA in mouse white adipose tissue. Further, we have shown that expression pattern of UCPn varies between animal species, rat versus mouse, such as the dominance of UCP3/UCP5 vs. UCP2 transcript in mouse heart and vice versa in rat heart; or UCP2 (UCP5) dominance in rat brain contrary to 10-fold higher UCP4 and UCP5 dominance in mouse brain. spontaneous apoptosis due to CIDEa overexpression in HeLa cells, adapted for a tetracycline-inducible CIDEa expression, a portion of mitochondria-localized CIDEa molecules migrates to cytosol or nucleus. / Résumé non disponible

Orientador: Ivan de Godoy Maia / Banca: Paulo Eduardo Martins Ribolla / Banca: Marcelo Menossi Teixeira / Resumo: As proteínas desacopladoras pertencem à família de carreadores aniônicos mitocondriais. De maneira geral, as proteínas desacopladoras dissipam o gradiente eletroquímico de prótons gerados na respiração na forma de calor, sendo dependentes de ácidos graxos e sensíveis aos nucleotídeos purínicos. O presente estudo visou investigar o comportamento de plantas transgênicas de tabaco que expressam de forma constitutiva o gene AtUCP1, frente aos estresses osmótico e salino, bem como analisar a atividade das regiões promotoras dos genes AtUCP1 e AtUCP2 de Arabidopsis thaliana, em resposta aos estresses osmótico e de baixa temperatura, e ao ácido abscísico. Numa primeira abordagem foram utilizadas sementes selvagens e de duas linhagens transgênicas, germinadas em meio MS adicionados ou não de NaCl e Manitol. O teste de germinação revelou que as linhagens transgênicas apresentam uma maior tolerância aos referidos estresses. Quando o crescimento radicular foi analisado, uma maior inibição foi constatada no controle não transgênico em relação às duas linhagens transgênicas testadas. Adicionalmente, quando submetidas aos estresses, uma maior acumulação de ânion superóxido foi verificada nas folhas de plântulas não transgênicas em relação às plântulas das linhagens transgênicas. Quanto à quantificação de GUS nas plantas transformadas com os promotores dos genes AtUCP1 e AtUCP2, nenhuma alteração significativa foi observada em nenhum dos tratamentos testados / Abstract: The uncoupling proteins belong to the mitochondrial anion carrier family. In general, the uncoupling proteins dissipate the proton electrochemical gradient generated in respiration as heat, being dependent on fatty acids and sensitive to purine nucleotides. In the present study, we investigated the behavior of transgenic tobacco plants that overexpress the AtUCP1 gene when subjected to osmotic and saline stress, as well as the activity of the promoters of the AtUCP1 and AtUCP2 genes of Arabidopsis thaliana, in response to osmotic and cold stress, and abscisic acid. In the first approach, seeds from wild type and two transgenic lines were germinated in MS medium containing (or not) NaCl and mannitol. The germination test showed that the transgenic lines have a higher stress tolerance. When root growth was analyzed, a greater inhibition was observed in non-transgenic control seedlings as compared to seedlings of the two transgenic lines tested. Additionally, when subjected to stress, a greater superoxide anion accumulation was detected in leaves of non-transgenic seedlings as compared to seedlings of transgenic lines. Quantification of GUS activity in the plants transformed with the tested promoters, revealed no treatmentspecific differences / Mestre

<p>The aim of the present study was to investigate the biosynthesis of the ATP synthase in various tissues, and to test hypotheses about possible models of activation of several mitochondrial proteins, the ATP/ADP translocase and UCPs, that could utilize the proton gradient, thus bypassing the ATP synthase. </p><p>We have examined the role of the expression of the P1 isoform of the c-F_o subunit in the biogenesis of ATP synthase in brown adipose tissue. Our findings point to a role for the c-F_o subunit in defining the final content of the ATP synthase in brown adipose tissue.</p><p>We have analyzed sequences in the 3’UTR of the β subunit F₁-ATPase mRNA that are important for formation of RNA-protein complexes. We could detect protein complexes that bind to two different sequence regions of the 3’UTR, one being the poly(A) tail and an adjacent region), and the other being a sequence stretch at the 3’ end of the 3’UTR able to form a stem-loop structure, which is evolutionarily conserved throughout mammalian species. </p><p>We investigated a role of the ATP/ADP carrier (ANT) in fatty acid-induced uncoupling in brown-fat mitochondria. We conclude that the ANT cannot substitute for UCP1 in fatty acid uncoupling in brown-fat mitochondria from mice lacking UCP1. We propose that the two ANT isoforms mediate proton translocation under different conditions.</p><p>We have investigated a role of UCP1 in defence against oxidative stress. We found that products of oxidative stress (4-HNE) could neither reactivate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1 in brown-fat mitochondria from UCP1(+/+) and UCP1(-/-) mice. We conclude that UCP1 is not involved in defence against oxidative stress. </p><p>We evaluated possible uncoupling activity of UCP3 in skeletal muscle from warm- and cold-acclimated UCP1(+/+) and UCP1(-/-) mice. We conclude that no evidence exists for a higher UCP3-mediated uncoupling activity; a high UCP3 content in cold-acclimated UCP1(-/-) mice could possibly be linked to improved fatty acid oxidative capacity.</p>

The aim of the present study was to investigate the biosynthesis of the ATP synthase in various tissues, and to test hypotheses about possible models of activation of several mitochondrial proteins, the ATP/ADP translocase and UCPs, that could utilize the proton gradient, thus bypassing the ATP synthase. We have examined the role of the expression of the P1 isoform of the c-Fo subunit in the biogenesis of ATP synthase in brown adipose tissue. Our findings point to a role for the c-Fo subunit in defining the final content of the ATP synthase in brown adipose tissue. We have analyzed sequences in the 3’UTR of the β subunit F1-ATPase mRNA that are important for formation of RNA-protein complexes. We could detect protein complexes that bind to two different sequence regions of the 3’UTR, one being the poly(A) tail and an adjacent region), and the other being a sequence stretch at the 3’ end of the 3’UTR able to form a stem-loop structure, which is evolutionarily conserved throughout mammalian species. We investigated a role of the ATP/ADP carrier (ANT) in fatty acid-induced uncoupling in brown-fat mitochondria. We conclude that the ANT cannot substitute for UCP1 in fatty acid uncoupling in brown-fat mitochondria from mice lacking UCP1. We propose that the two ANT isoforms mediate proton translocation under different conditions. We have investigated a role of UCP1 in defence against oxidative stress. We found that products of oxidative stress (4-HNE) could neither reactivate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1 in brown-fat mitochondria from UCP1(+/+) and UCP1(-/-) mice. We conclude that UCP1 is not involved in defence against oxidative stress. We evaluated possible uncoupling activity of UCP3 in skeletal muscle from warm- and cold-acclimated UCP1(+/+) and UCP1(-/-) mice. We conclude that no evidence exists for a higher UCP3-mediated uncoupling activity; a high UCP3 content in cold-acclimated UCP1(-/-) mice could possibly be linked to improved fatty acid oxidative capacity.

Made available in DSpace on 2014-06-11T19:26:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-07Bitstream added on 2014-06-13T19:53:55Z : No. of bitstreams: 1 nunes_av_me_botib.pdf: 662812 bytes, checksum: e1faf5a1bccf83ade3dc24706edbc278 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / As proteínas desacopladoras pertencem à família de carreadores aniônicos mitocondriais. De maneira geral, as proteínas desacopladoras dissipam o gradiente eletroquímico de prótons gerados na respiração na forma de calor, sendo dependentes de ácidos graxos e sensíveis aos nucleotídeos purínicos. O presente estudo visou investigar o comportamento de plantas transgênicas de tabaco que expressam de forma constitutiva o gene AtUCP1, frente aos estresses osmótico e salino, bem como analisar a atividade das regiões promotoras dos genes AtUCP1 e AtUCP2 de Arabidopsis thaliana, em resposta aos estresses osmótico e de baixa temperatura, e ao ácido abscísico. Numa primeira abordagem foram utilizadas sementes selvagens e de duas linhagens transgênicas, germinadas em meio MS adicionados ou não de NaCl e Manitol. O teste de germinação revelou que as linhagens transgênicas apresentam uma maior tolerância aos referidos estresses. Quando o crescimento radicular foi analisado, uma maior inibição foi constatada no controle não transgênico em relação às duas linhagens transgênicas testadas. Adicionalmente, quando submetidas aos estresses, uma maior acumulação de ânion superóxido foi verificada nas folhas de plântulas não transgênicas em relação às plântulas das linhagens transgênicas. Quanto à quantificação de GUS nas plantas transformadas com os promotores dos genes AtUCP1 e AtUCP2, nenhuma alteração significativa foi observada em nenhum dos tratamentos testados / The uncoupling proteins belong to the mitochondrial anion carrier family. In general, the uncoupling proteins dissipate the proton electrochemical gradient generated in respiration as heat, being dependent on fatty acids and sensitive to purine nucleotides. In the present study, we investigated the behavior of transgenic tobacco plants that overexpress the AtUCP1 gene when subjected to osmotic and saline stress, as well as the activity of the promoters of the AtUCP1 and AtUCP2 genes of Arabidopsis thaliana, in response to osmotic and cold stress, and abscisic acid. In the first approach, seeds from wild type and two transgenic lines were germinated in MS medium containing (or not) NaCl and mannitol. The germination test showed that the transgenic lines have a higher stress tolerance. When root growth was analyzed, a greater inhibition was observed in non-transgenic control seedlings as compared to seedlings of the two transgenic lines tested. Additionally, when subjected to stress, a greater superoxide anion accumulation was detected in leaves of non-transgenic seedlings as compared to seedlings of transgenic lines. Quantification of GUS activity in the plants transformed with the tested promoters, revealed no treatmentspecific differences