A oxidação de proteínas é um fenômeno metabólico e a degradação de proteínas oxidativamente modificadas confere uma proteção para a célula, evitando acúmulo e a agregação das mesmas. A ineficiência na remoção destas proteínas está relacionada ao processo de envelhecimento e ao aparecimento de doenças neurodegenerativas. A unidade catalítica do proteassomo, denominada de 20S (PT20S), é a principal via de degradação de proteínas danificadas pela oxidação sem que haja gasto de ATP, acoplamento de subunidades regulatórias ou poli-ubiquitinação do substrato proteico. A unidade PT20 por sua vez, pode sofrer modificação pós-traducional chamada de S-glutationilação, que aumenta a velocidade degradação proteica por processo independente de poli-ubiquitinação. Em levedura (Saccharomyces cerevisiae), foram identificados apenas dois resíduos de Cys glutationilados, ambos na subunidade α5 (α5-76 e α5-221). A S-glutationilação ocasiona a abertura da câmara catalítica e uma maior eficiência na degradação de proteínas. Mutações sítio-específicas foram realizadas nessas Cys pela substituição por Ser. As consequências estruturais e funcionais dessas mutações foram o aumento da frequência da conformação fechada da câmara catalítica no α5-76S-PT20S e α5-221S-PT20S. As linhagens que carregam essas mutações apresentaram menor tempo de vida cronológico. Uma dupla mutação randômica na subunidade α5 (S35P / C221S) induziu a abertura da câmera catalítica do 20SPT e esta linhagem apresentou tempo de vida cronológico significativamente aumentado e , aumento na resistência ao estresse oxidativo em paralelo ao aumento da atividade catalítica do 20SPT. O objetivo neste projeto de pesquisa foi realizar uma análise proteômica quantitativa no extrato celular das linhagens mutantes, com o objetivo de identificar proteínas que possam estar relacionadas com a regulação da longevidade celular. Foram selecionadas as linhagens que carregam as mutações: α5-76S e α5-S35P/C221S uma vez que apresentaram expectativa de vida oposta em relação à linhagem selvagem, além de queda e aumento da frequência da conformação aberta da câmera catalítica, respectivamente. A partir da quantificação sem marcação (Label-free quantification), foram identificadas 723-1000 proteínas nas amostras das linhagens selvagem e mutantes. Dentre elas, destacam-se as proteínas 3-isopropilmalato isomerase e argininossuccinato sintase, envolvidas na síntese de leucina e arginina, respectivamente, aumentadas na linhagem mutante C76S e reduzidas na linhagem S35P/C221S. O metabolismo de ambos os aminoácidos está relacionado com a via de sinalização TOR que, por sua vez, está envolvida com o tempo de vida cronológico em levedura. / The protein oxidation is a metabolic phenomenon and the degradation of oxidatively modified proteins confers a protection to cell, avoiding accumulation and aggregation of these proteins. The inefficiency in the removal of these proteins is related to aging process and neurodegenerative diseases. The catalytic unit of the proteasome, named 20S (PT20S) is the main degradative pathway of oxidized proteins in an ATP-independent manner, without proteasomal regulatory units assembly or protein poliubiquitination. The PT20 unit undergoes a post-translational modification named S-glutationilation, which increases the protein degradation by the ATP-independent process. In yeast, only two Cys residues were identified glutationilated, both in the α5 subunit (α5-C76 e α5-C221). The S-glutationilation causes opening of the catalytic chamber and higher efficiency of protein hydrolysis. Site-specific mutations were performed in those Cys residues by their replacement to Ser. The structural and functional consequences of mutations were the increasedfrequency of theclosed conformation of the catalytic chamber in the α5-76S-PT20S and α5-221S-PT20S. The strains carrying those mutations presented shorter chronological life span. A double random mutation in the α5 subunit (S35P / C221S) induced the opening of 20SPT catalytic chamber together toextended chronological life span and, increased resistant to oxidative stress in parallel to increased catalytic activity of the 20SPT. The goal of this project was to perform a label-free quantitative proteomic analysis in the mutant strains to identify proteins that could be related with the regulation of cellularlifespan. From that quantification, 723 - 1000 proteins were identified in the samples of the wild-type and mutant strains. Among these proteins, 3-isopropylmalate isomerase and argininesuccinate sintase, involved in the leucine and arginine biosynthesis, respectively, were found increased in the C76S mutant strain and reduced in the S35P/C221S mutant strain. The metabolism of both amino acids is related with TOR signallingthat, in turn,modulates chronological lifespan in yeast

As doenças cardiovasculares ainda são as principais causas de mortalidade e morbidade em todo o mundo. E a aterosclerose é uma das principais precursoras de vários desfechos clínicos como isquemias e infarto do miocárdio. As placas ateroscleróticas se desenvolvem preferencialmente em regiões de bifurcação ou curvatura dos vasos, onde o shear stress (SS) encontra-se diminuído ou perturbado. A expressão de proteínas pró-aterogênicas em regiões de baixo SS e ateroprotetoras em regiões de SS alto foram relatadas na literatura, porém o mecanismo completo carece de elucidação. Este trabalho teve por objetivo integrar proteômica e metabolômica para um melhor entendimento das alterações moleculares que acontecem nas células endoteliais em situações de alto e baixo SS, que podem resultar no desenvolvimento de lesões e placas ateroscleróticas. Para esta finalidade, células endoteliais foram submetidas a alto e baixo SS em sistema cone plate, seguido de análise proteômica e metabolômica por espectrometria de massas. Nossos dados demonstraram que o metabolismo de lipídio e metabolismo de modificações pós-traducionais de proteínas (N-glicosilações) estavam diminuídos em baixo SS. Em relação ao metabolismo de lipídio, foi identificada diminuição na concentração de ácidos graxos e na expressão de enzimas e proteínas transportadoras de lipídios em células sob baixo SS. O receptor de LDL, proteína importante para a homeostase do colesterol, foi identificado em menor concentração na membrana, bem como com alteração no seu perfil de glicosilação em células após baixo SS. As células submetidas a baixo SS e, portanto, aquelas com perfil pró-aterogênico, quando tratadas com estatina para o aumento da expressão de LDLR, aproximaram seu fenótipo ao de células submetidas a alto SS, adquirindo parte de um fenótipo ateroprotetor, com recuperação dos níveis de aminoácidos, lipídios, açúcares e ácidos carboxílicos. Os dados deste trabalho sugerem que o metabolismo de lipídios é um processo importante na manutenção do perfil ateroprotetor de células submetidas a alto SS. Além disso, as evidências demonstraram que estatinas apresentam uma atividade protetora, não apenas sistêmica, com diminuição do LDL circulante, mas também no microambiente vascular, contribuindo para o bom funcionamento das células endoteliais / Cardiovascular diseases are the main cause of the mortality and morbidity worldwide. Atherosclerotic plaque development is closely associated to the hemodynamic forces applied to endothelial cells (EC). Among these, shear stress (SS) plays a key role in disease development since changes in flow intensity and direction could stimulate an atheroprone or atheroprotective phenotype. EC under low and/or oscillatory SS (LSS) have upregulation of inflammatory proteins, adhesion and cellular permeability molecules. On the contrary, cells under high/laminar SS (HSS) increase their expression of protective and anti-inflammatory factors. The mechanism behind the SS regulating an atheroprotective phenotype is not completely elucidated. Here we used proteomics and metabolomics to better understand the changes suffered by endothelial cells under LSS and HSS that promote the atheroprone and atheroprotective profile and how these modifications can be connected to atherosclerosis development. Our data showed that lipid metabolism and post translational modification protein metabolism were downregulated in cells under LSS. About lipid metabolism, we found the LDLR, one important protein in cholesterol homeostasis, showed significant alterations both at the quantitative expression level, as well as regarding post-translational modifications. Under LSS, LDLR was seem at lower concentrations and with a different glycosylation profile. Finally, modulating LDLR with atorvastatin led to the recapitulation of an HSS metabolic phenotype in EC under LSS. The phenotype was recovery based on increasing of amino acids, lipids, sugars and carboxylic acids. Altogether, our data suggest lipid metabolism is important in atheroprotective phenotype of endothelial cells under HSS. Statins showed benefits not only systemic, decreasing cholesterol level in blood, but also in vascular environment, contributing for protector phenotype of endothelial cells

Le premier acide aminé incorporé dans une chaîne polypeptidique naissante est toujours la méthionine. On identifie donc toujours ce premier résidu à la méthionine N-terminale. Cependant, les deux tiers des protéines accumulées à l’état stationnaire ne présentent plus leur méthionine initiatrice. Cet enlèvement résulte essentiellement d’une maturation protéolytique affectant chaque protéine. Ainsi, l’Excision de la Méthionine N-terminale (NME) concerne la majorité des protéines et ce dès que les premiers résidus émergent du ribosome. Ce mécanisme est retrouvé dans tous les compartiments cellulaires où une synthèse protéique a lieu : le cytoplasme, les plastes et les mitochondries. Les enzymes responsables du clivage de la méthionine initiatrice sont les METhionine AminoPeptidases (METAPs) ; les METAPs sont conservées dans le Règne vivant. Des études fonctionnelles de délétions géniques ont montré le caractère létal du maintien de la première méthionine dans tous les organismes. Il y a plus de dix ans, les METAPs ont été identifiées comme étant la cible de composés naturels ayant des effets anticellulaires. Aujourd’hui un nombre croissant d’études rapportent que la NME est une cible prometteuse pour le traitement de nombreuses pathologies. Néanmoins, les bases moléculaires qui expliquent le caractère essentiel de la NME restent très peu comprises, en particulier dans le cytoplasme des eucaryotes supérieurs. Grâce à un système inductible permettant de moduler finement la NME cytoplasmique dans la plante modèle Arabidopsis thaliana et différentes approches incluant des analyses protéomiques et métabolomiques, j’ai pu étudier les événements moléculaires précoces associés à l’inhibition de la NME cytoplasmique. J’ai également caractérisé la contribution relative des deux types de METAP cytoplasmiques au processus. Dans ce contexte, j’ai pu démontrer chez A. thaliana que la NME cytoplasmique agit sur deux voies de signalisation fréquemment dérégulées lors de conditions pathologiques : le statut des composés thiolés et la protéolyse. La diminution de la NME cytoplasmique induit une protéolyse accrue principalement via une augmentation du nombre de protéines destinées à une dégradation rapide. Ainsi, l’activité de la NME, en modulant la sensibilité de nombreuses protéines à subir la protéolyse, est un élément fondamental de la régulation de la demi-vie protéique. Finalement, mes résultats simialires obtenus également chez les Archées, levures et les lignées de cellules humaines suggèrent l’existence d’un mécanisme ubiquitaire associé à la NME. / The first amino acid incorporated in nascent polypeptide chain is always methionine so called N-terminale methionine. However, in a given proteome, more than fifty percent of proteins have not this first methionine. Indeed, the early proteolytic event affecting a majority of proteins is N-terminal Methionine Excision (NME) as soon as few residues exit from the ribosome. Enzymes ensuring NME process are conserved along species. This mechanism takes place in all compartments where protein synthesis occurs including cytoplasm, plastids and mitochondria and the enzymes responsible of N-methionine excision are METhionine AminoPeptidases (METAP). Early functional studies of gene deletion has quickly showed that NME is an essential process. Ten years ago, METAPs have been identified as the molecular target of natural compounds with anticancer activities. Now, a growing number of studies suggest that NME is a promising target for treatment of various deseases. Nevertheless, molecular mechanisms making NME an essential process is poorly understood in particular in higher eukaryote cytoplasms.Using a dedicated inducible system in the model organism Arabidopsis thaliana and multiple approaches, including proteomics and metabolomics, I examined the earliest molecular events associated with the inhibition of this process and the contribution of both METAP to NME process. In this context, I demonstrated that cytoplasmic NME in A. thaliana orchestrates a cross-talk between two fundamental signaling pathways frequently deregulated in pathological conditions: thiol status and proteolysis. In these studies, we demonstrated that developmental defects induced by cytoplasmic NME inhibition are associated with an increase of the proteolytic activity due to an increase of the proteins available for rapid degradation. Thus, NME activity that modifies the availability of several proteins for degradation is an integral and fundamental element protein turnover regulation. Finally my preliminary results obtained in Archea, Fungi and human cells seem to suggest the existence of a ubiquitous mechanism associated with NME process.

La Fusariose de l’épi (FHB) est une maladie importante des céréales et en particulier du blé tendre. Elle est causée par deux genres fongiques, le genre Fusarium et le genre Microdochium. L’espèce Fusarium graminearum est l’agent principal de cette maladie. Elle affecte non seulement les rendements et la qualité des grains chez le blé, mais elle cause un sérieux problème sanitaire via la production des mycotoxines. L’établissement de cette maladie requiert l’expression de gènes végétaux (facteurs de sensibilité) qui restent encore méconnus. Afin d’étudier les événements moléculaires qui participent à la mise en place de cette maladie sur un grain de blé tendre en développement et sensible au FHB, une cinétique d’infection de cinq points correspondant aux principaux stades développementaux du grain a été réalisée. Ensuite, deux approches, la protéomique comparée et la transcriptomique comparée à l’aide de puces à ADN, ont été utilisées pour répondre à ces questions. L’analyse protéomique a permis d’identifier 73 protéines différentiellement régulées appartenant à 5 grands groupes fonctionnels alors que l’approche transcriptomique a mis en évidence 1309 gènes répartis dans 16 groupes fonctionnels différents. Ces deux approches ont montré que l’infection ne bloque pas le développement du grain, mais elle induit des changements importants dans le métabolisme primaire notamment sur la synthèse de l’amidon et des protéines de réserve. Elle a également montré que la réponse du grain au FHB est liée au stade développemental du grain. Cette étude apporte de nouveaux éléments nécessaires à la compréhension de la sensibilité du blé tendre à la Fusariose de l’épi. Cette sensibilité du grain de blé se caractérise principalement par l’induction des mécanismes de détoxification des mycotoxines, la mise en place des mécanismes du détournement du métabolisme carboné de l’hôte par l’agent pathogène et le contrôle de la mort cellulaire programmée des cellules végétales. Enfin, l’étude a permis d’établir une liste d’au moins 100 gènes candidats potentiellement impliqués dans la sensibilité du blé au FHB. / Fusarium head blight (FHB) is an important disease of cereals, particularly of wheat. It is caused by two fungal genera, the genus Fusarium and genus Microdochium. The species Fusarium graminearum is the principal agent of this disease. This disease affects not only yield and grain quality in wheat, but it causes serious health problem through the production of mycotoxins. The establishment of this disease requires the expression of plant genes (susceptibility factors) that are still unknown. To study the molecular events involved in the development of this disease in the susceptible wheat grain during its development, time course infection of five points corresponding to the main developmental stages of grain was performed. Then two approaches, proteomics and transcriptomics using DNA microarrays were used to answer to this question. Proteomic analysis identified 73 differentially regulated proteins belonging to five major functional groups while the transcriptomics data revealed 1309 genes involved in 16 distinct functional groups. Both approaches have shown that infection does not interrupt grain development, but induces significant changes in primary metabolism, mainly on the synthesis of starch and storage proteins. It also showed a link between FHB response and the grain development. This study provides new evidence necessary to understand the susceptibility response of wheat to FHB. The wheat grain susceptibility is mainly characterized by the induction of detoxifying mechanisms of mycotoxins, the diversion ofcarbon metabolism of the host by the pathogen and control of Programmed Cell Death (PCD) of plant cells. Finally, the study allowed the establishment of a list of at least 100 candidate genes potentially involved in the wheat susceptibility to FHB.

A hipótese desta pesquisa foi que a maturação epididimária dos espermatozoides caninos envolve modificações nas proteínas e ácidos graxos do fluido sintetizado pelos distintos segmentos epididimários, além de alterações no perfil de ácidos graxos da membrana plasmática, organização celular e morfológica dos espermatozoides. Para tanto, este projeto foi realizado com 20 cães em idade reprodutiva e negativos para Brucella canis. Após a orquiectomia bilateral, os testículos e epidídimos foram acondicionados a 5°C por no máximo 24 horas, em seguida, as amostras foram colhidas por incisões na cauda, corpo e cabeça do epidídimo. As amostras foram imediatamente processadas por avaliação subjetiva e automática da motilidade e vigor espermático, concentração, morfologia espermática, integridade da membrana plasmática, acrossomal e atividade mitocondrial. Foi possível observar determinar maior número de espermatozoides dentro da normalidade na cauda do epidídimo, especialmente, referindo-se à motilidade, integridade de membrana e atividade mitocondrial. A avaliação das modificações ultraestruturais dos espermatozoides permitiu observar a migração da gota citoplasmática e alterações acrossomais. No perfil de ácidos graxos observaram-se variações na quantidade e presença dos ácidos durante o traje epididimário, destacando o acréscimo do DHA na região da cauda epididimária. Ainda, no perfil protéico do plasma epididimário canino, foi possível identificar um padrão regional de secreção de proteínas, maior nas regiões da cabeça e corpo em relação à cauda do epidídimo. Apesar das importantes informações geradas a partir do presente trabalho, mais estudos são necessários para a compreensão da fisiologia reprodutiva, especialmente da maturação espermática epididimária na espécie canina. / The hypothesis of this research is that the canine maturation of epididymal spermatozoa involves progressive changes in the protein and fatty acid content of the fluid synthesized by the different epididymal segments, as well as changes in the fatty acid profile of the sperm plasma membrane, cell organization and morphology of the canine sperm. Therefore, this experiment was conducted with 20 dogs in reproductive age and free of Brucella canis. After bilateral orchiectomy, testes and epididymis were stored at 5°C for up to 24 hours and then the samples were harvested through incisions of the tail, corpus and caput of the epididymis. The samples were immediately processed for subjective and automatic motility and sperm vigor, sperm count, morphology, plasma membrane integrity, acrosomal and mitochondrial activity. It was possible to observe a greater number of mature sperm in the epididymal tail compared to the corpus and caput, especially referring to motility, membrane integrity and mitochondrial activity. The evaluation of ultrastructural changes of the spermatozoa allowed to observed the migration of the cytoplasmic droplets and acrosomal changes. In the fatty acid profile was observed variations in the amount and presence of acids during epididymal maturation, highlighting the addition of DHA in the epididymal tail region. Moreover, in the protein profile of the canine epididymal plasma, it was possible to identify a regional pattern of protein secretion, higher in the caput and corpus in relation to the tail epididymis. In spite of the important data generated from this study, further studies are needed for understand the reproductive physiology, especially the epididymal sperm maturation in dogs.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-04-26T14:05:04Z No. of bitstreams: 1 2012 - Patricia Gon?alves Galv?o.pdf: 5289496 bytes, checksum: 8dcb41bc971793cc8b4cdf383401ade4 (MD5) / Made available in DSpace on 2017-04-26T14:05:05Z (GMT). No. of bitstreams: 1 2012 - Patricia Gon?alves Galv?o.pdf: 5289496 bytes, checksum: 8dcb41bc971793cc8b4cdf383401ade4 (MD5) Previous issue date: 2012-03-01 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The objective of this study was to evaluate the protein profile expression of G. diazotrophicus PAL5 and its defective mutant in the indole compound production (lao-) grown in the presence or absence of tryptophan through 2DE-PAGE technique. The spectrometric analysis allowed the identification of 24 differentially expressed proteins. The majority of the proteins down regulated in the wild type PAL5 cultivated with tryptophan as compared to the cultivation without the amino acid belonged to the category of transductional modification, protein turnover and chaperones. For the mutant lao- grown in the same conditions, the majority of the proteins that presented differential expression belonged to the category of production and conversion of energy. In addition, the majority of the protein differentially expressed in the mutant lao- as compared to the wild-type PAL5 strains belonged to carbohydrates metabolism and transport. On the other hand, no proteins related to the tryptophan biosynthesis were detected in any condition, possibly due to the low yield of the proteins during the spectrometric analysis. Furthermore, mutants lao- and nif- of G. diazotrophicus were used for inoculation of micropropagated sugarcane plants in order to determine the influence of auxins produced by the bacteria in the plant growth promotion in comparison with PAL5. The first experiment, carried out in hydroponic conditions for 10 days showed a significant inoculation effect of the wild type on plant shoot. The other experiment was conducted in a period of 120 days in pots containing sand:vermiculite substrate fertilized with 30 and 60 ppm with ammonium sulphate enriched with 15N. The plants were inoculated in vitro with the wild type and mutants lao- and nif-, and the results showed a visual difference in the roots inoculated with PAL5 that showed higher volume suggesting a higher number of secondary roots and root hairs. On the other hand, the plants inoculated with the lao- mutant were ticker and showed lower number of secondary roots and root hairs. The shoot biomass of plants inoculated with PAL5 was higher than those inoculated with the mutant strains for both N dose, however the difference was not significant. Plants grown with 60 kg N dose and inoculated with the mutants showed lower accumulation of dry shoot mass than plants inoculated with the wild type strain. In conclusion, the present study showed the occurrence of several differentially expressed proteins either in the wild type strain or in the mutant lao- grown in LGI-P with and without tryptophan. The role played by these proteins in the metabolism of the bacteria requires additional studies, including different growth conditions. In addition, the inoculation of micropropagated sugarcane plants suggested a hormonal effect of the bacteria mainly on the root development e consequently in the N use efficiency. However, the size of the pots may have limited the plant development, suggesting that new experiments should be carried out in more appropriated conditions to confirm the influence of the indol production and the BNF during the association of the G. diazotrophicus and sugarcane plants / Este estudo teve por objetivo avaliar o perfil de express?o de prote?nas de G. diazotrophicus PAL5 e seu mutante defectivo na produ??o de compostos ind?licos (lao-) cultivados na presen?a e aus?ncia de triptofano atrav?s da t?cnica de 2DE-PAGE. A an?lise por espectrometria de massa permitiu a identifica??o de 24 prote?nas diferencialmente expressas. A maioria das prote?nas com a express?o diminu?da em PAL5 cultivada em meio com triptofano em rela??o ao meio de cultivo sem esse amino?cido pertenceu ? categoria modifica??o p?s-traducional, turnover de prote?nas e chaperonas. No mutante lao- cultivado nas mesmas condi??es, a maioria das prote?nas que apresentaram express?o diferencial pertencia ? categoria produ??o e convers?o de energia. Em adi??o, a maioria das prote?nas que foram diferencialmente expressas no mutante lao- em compara??o com a estirpe selvagem PAL5 pertencia ? categoria metabolismo e transporte de carboidratos. Por outro lado, n?o foram observadas prote?nas relacionadas ? bioss?ntese de triptofano em nenhuma condi??o analisada possivelmente devido ao baixo rendimento das identifica??es por espectrometria. Al?m das an?lises dos perfis de prote?nas, os mutantes lao- e nif- de G. diazotrophicus foram inoculados em plantas de cana-de-a??car micropropagadas com o objetivo de determinar a influ?ncia das auxinas na promo??o do crescimento dessa cultura em compara??o com a estirpe selvagem PAL5. O primeiro experimento, conduzido em condi??es de hidroponia pelo per?odo de 10 dias, mostrou efeito significativo da inocula??o da estirpe selvagem na promo??o de crescimento da parte ?rea das plantas, enquanto que o mutante lao-, n?o diferiu estatisticamente do controle n?o inoculado. O outro experimento, foi conduzido por 120 dias em vasos com substrato areia:vermiculita contendo 30 ou 60 ppm de sulfato de am?nio enriquecido com 15N e as pl?ntulas foram inoculadas in vitro. Os resultados mostraram uma diferen?a visual nas ra?zes das plantas inoculadas com PAL5, que se mostraram mais volumosas, aparentando um n?mero mais elevado de ra?zes secund?rias e p?los radiculares. J? as plantas inoculadas com lao- apresentaram ra?zes mais grossas, com um n?mero muito reduzido de ramifica??es ou p?los radiculares. A biomassa seca da parte a?rea das plantas inoculadas com PAL5 foi superior ?quelas inoculadas com as estirpes mutantes para as duas doses de nitrog?nio, por?m essa diferen?a n?o foi significativa. N?o foram observadas evid?ncias de contribui??o da FBN, por?m as plantas inoculadas com PAL5 foram menos eficientes na recupera??o do N fertilizante. Em conclus?o, o presente estudo mostra a ocorr?ncia de diversas prote?nas diferencialmente expressas tanto na estirpe selvagem como em lao- quando crescidas na presen?a e aus?ncia do amino?cido triptofano. A defini??o do papel dessas prote?nas no metabolismo da bact?ria requer estudos adicionais, inclusive em diferentes condi??es de cultivo. Em adi??o, a inocula??o dessas bact?rias em plantas de cana-de-a??car mostrou o efeito hormonal da bact?ria no desenvolvimento das ra?zes e, por conseguinte na maior efici?ncia de uso do N aplicado. Entretanto, dado a limita??o de espa?o f?sico dos vasos para o desenvolvimento das plantas, sugere-se a realiza??o de novos experimentos, em condi??es mais apropriadas, para confirmar a influ?ncia da produ??o de ?ndoles e da FBN durante a associa??o da bact?ria com as plantas de cana-de-a??car.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-05-04T18:36:57Z No. of bitstreams: 1 2016 - CRISTINA SANTOS DA SILVA.pdf: 3106410 bytes, checksum: 18949ec17e0df834beb420705591824d (MD5) / Made available in DSpace on 2017-05-04T18:36:57Z (GMT). No. of bitstreams: 1 2016 - CRISTINA SANTOS DA SILVA.pdf: 3106410 bytes, checksum: 18949ec17e0df834beb420705591824d (MD5) Previous issue date: 2016-03-31 / CAPES / Chagas? Disease or American trypanosomiasis is an important parasitic disease in the Americas and is still considered one of the major neglected tropical diseases affecting millions of people worldwide due to lack of effective control. Thus, it has a significant impact on human health. This disease has its epidemiology conditioned by triatomines and mammals and its etiological agent is the protozoan Trypanosoma cruzi.Investigations on the adaptation mechanisms, gene regulation and parasite interaction vs. vector evolved, which proves the need for the use of molecular tools for the study and elucidation on adaptive changes in these parasites. Recently, it was found evidence where studies indicate that the expression of T. cruzi?s antioxidant enzymes such as cytosolic peroxiredoxin (TcCPx), mitochondrial peroxiredoxin (TcMPx), (TcAPX) and trypanothione synthetase (TXNI, TXNII), superoxide dismutase (SOD A and SOD B) can be part of parasite?s protective system and indicate factors related to different levels of virulence of the etiological agent.Thus, proteomics analysis and evaluation of the expression of antioxidant enzymes from different subcellular compartments can corroborate to the studies related to virulence indices and expression of proteins involved in this process. In this sense, the objectives of this study were to evaluate the morphology of the isolated samples SMM98, SMM36 and SMM1, analyze beyond the proteomic profile, the expression of antioxidant enzymes (TcCPx; TcMPx; TcAPx; TcTXNI; TcTXNII, SOD A and SOD B) and observe the redox status expression using isolates SMM36 and SMM98 in comparison with strains TCC, Silvio and DM28c. The results showed different morphology from the isolated, high levels of virulence, varied protein profile comparison between SMM98 isolated and SMM36, when treated with NAC and Heme changes were observed in the development of the parasites, indicating the participation of the Redox Status of the Rio de Janeiro used in this study. / A doen?a de Chagas ou tripanossom?ase americana ? uma importante doen?a parasit?ria nas Am?ricas e ainda hoje ? considerada uma das principais doen?as tropicais negligenciadas acometendo milh?es de pessoas no mundo devido ? falta de controle efetivo. Desta forma, apresenta um impacto significativo sobre a sa?de humana. Esta enfermidade tem sua epidemiologia condicionada pelos triatom?neos e os mam?feros e tem como agente etiol?gico o protozo?rio Trypanosoma cruzi.Investiga??es sobre os mecanismos de adapta??o, regula??o g?nica e intera??o parasito x vetor evolu?ram, o que comprova a necessidade da utiliza??o de ferramentas moleculares para o estudo e elucida??es sobre as mudan?as adaptativas ocorridas nestes parasitos. Recentemente evid?ncias surgiram neste sentido, onde estudos indicam que a express?o de enzimas antioxidantes do T. cruzi tais como: peroxirredoxinas citos?lica (TcCPx), mitocondriais (TcMPx), (TcAPX) e tripanotiona sintetase (TXNI, TXNII), super?xido dismutase (SOD A e SOD B) podem fazer parte do sistema protetivo do parasito e indicar fatores relacionados aos diferentes n?veis de virul?ncia deste agente etiol?gico. Desta forma, an?lises prote?mica e avalia??o da express?o de enzimas antioxidantes de diferentes compartimentos subcelulares podem corroborar para com os estudos relacionados aos ?ndices de virul?ncia e express?o de prote?nas envolvidas neste processo. Neste sentido, os objetivos deste trabalho foram avaliar a morfologia dos isolados das amostras SMM98, 36 e 1, analisar al?m do perfil prote?mico, a express?o de enzimas antioxidantes (TcCPx; TcMPx; TcAPx; TcTXNI; TcTXNII, SOD A e SOD B) e observar a express?o do status redox utilizando os isolados SMM36 e SMM98 em compara??o com as cepas TCC, Silvio e DM28c. Os resultados obtidos demonstraram aspectos morfol?gicos diferenciados dentre os isolados, n?veis de virul?ncia elevados, perfil de prote?nas variados quando comparados entre os isolados SMM98 e SMM36. E, quando tratados com Heme e NAC foram observadas altera??es no desenvolvimento dos parasitos, denotando a participa??o do Status Redox frente aos isolados do Rio de Janeiro utilizados neste estudo.

Escherichia coli patogênicas aviárias (APEC) causam infecções extraintestinais em frangos conhecidas como colibacilose. A APEC MT78, ao contrário de outras linhagens APEC, foi capaz de invadir células não-fagocitárias no modelo de fibroblastos aviários (CEC-32). Considerando que as interações patógeno-hospedeiro envolvem modificações na abundância de proteínas e padrões de expressão, principalmente nas proteínas de superfície, nosso objetivo foi comparar o proteoma da MT78 crescida em meio de cultura celular com o proteoma de MT78 isolada de fibroblastos aviários infectados (condição de co-cultura). Desenvolvemos aqui a padronização das etapas de extração de proteínas totais, isolamento de células bacterianas do co-cultivo e análise proteômica de modo a obtermos uma análise proteômica global reprodutível e de qualidade. A análise da interação APEC MT78 e células CEC-32 por microscopia óptica e eletrônica de varredura revelou que essa cepa se associa à célula-alvo em um padrão de adesão localizada. A internalização de APEC MT78 pareceu ocorrer como resultado de uma interação entre bactéria-célula que dispara rearranjos do citoesqueleto de actina da célula-alvo, formando estruturas filo e lamelipodiais que são dependentes da viabilidade bacteriana. O reisolamento de células bacterianas intactas, observadas por microscopia eletrônica de transmissão, após o co-cultivo com CEC-32 foi obtido através da técnica de solubilização diferencial de membranas. As células bacterianas foram sonicadas e as proteínas digeridas em solução seguida de uma etapa de purificação. Nós identificamos 69 proteínas, distribuídas em 9 classes funcionais, incluindo as proteínas de membrana FimA, OmpA and OmpC. A proteína OmpA já foi associada a invasão do patógeno humano NMEC (neonatal meningitis-associated E. coli) à células HBMEC. Esses experimentos representam a primeira investigação proteômica global em E. coli patogênica aviária. As proteínas identificadas representaram diferentes rotas metabólicas, funções fisiológicas e diferentes localizações subcelulares. / In poultry, Avian Pathogenic Escherichia coli (APEC) cause localized extra- intestinal infections that often become systemic. APEC strain MT78 was able to invade non-phagocytic avian fibroblasts in vitro, raising the possibility that some APEC strains may invade epithelial cells and gain systemic access. Using light microscopy and scanning electron microscopy, we observed that viable MT78 strain associated with CEC-32 fibroblasts cells in clusters, and following association, MT78 internalization appeared to result from cytoskeleton rearrangements, such as filopodia and lamellipodia, in the eukaryotic membrane. Considering that host-pathogen interactions involve modifications of protein abundance and expression, mainly in surface proteins, we compared the proteome of MT78 harvested from culture medium with the proteome of MT78 isolated from infected avian fibroblasts (co-culture condition). For this purpose, we developed standard analytical procedures for global protein extraction and isolation of bacterial cells from infected CEC-32. Judged by transmission electron microscopy, we successfully reisolated intact APEC MT78 cells from CEC-32 fibroblasts using the differential membrane solubilization method. Bacterial cells were then sonicated and proteins digested in solution following a clean up procedure. We identified 69 proteins, distributed in 9 functional classes, including the membrane proteins FimA, OmpA and OmpC. The OmpA protein was already associated to invasion of the human pathogen called NMEC (neonatal meningitis-associated E. coli) to endothelial cell line HBMEC. Our results represent the first global proteomic investigation in APEC. The proteome of MT78 infecting avian fibroblasts may allow us to identify key proteins linked to the successful adhesion and/or invasion of host cells by APEC and thus throw light into the pathogenesis of avian colibacillosis.

Cryopreservation is a promising approach to long-term biopreservation of living cells, tissues and organs. The use of cryoprotective agents (CPAs) in combination with extremely low temperatures is mandatory for optimum biopreservation. CPAs (e.g., glycerol, trehalose, dimethyl sulphoxide (DMSO)), however, are relatively cytotoxic and compromise biopreserved cell quality. This is usually resultant in oxidative damage, diminishing cell functionality and survival rate. The growing market of cell therapy medicinal products (CTMPs) demands effective cryopreservation with greater safety, of which the currently available CPAs are unable to provide. The present study was aimed at developing cryomedia formulation to enhance the cryopreservation of nucleated and anucleated mammalian cells. Here, eleven compounds of a polyol nature were selected and examined for their cryoprotective properties. These compounds are derived from plants and honey, thereby ensuring their safety for human consumption. The selection was based on their molecular structure and chemical properties. Here, the presented study is divided into three main phases: 1) Screening the compounds panel for cryo-additive effects on cells during and post-cryopreservation and optimising the dose response and time course for trehalose and glycerol with and without the novel compounds; 2) Assessing the influence of biophysical criteria on biospecimen cryopreservation (e.g., biosampling procedure, cell age, donor age); 3) Establishing the mechanisms of action underpinning the modulatory effect of novel CPAs on biological pathways during cryopreservation. For the stated purposes, red blood cells (RBCs) obtained from sheep and humans were used to screen the compounds for novel cryo-additive agents. Cryosurvival rate was employed as an indication of the compounds' cryoprotective performance. Cellular biochemical profiles, including lipid and protein oxidative damage as well as key redox enzymatic activities (e.g., lactate dehydrogenase (LDH), glutathione reductase (GR)) were measured. The study revealed that nigerose (Nig) and salidroside (Sal) were significantly effective in protecting cells during the freeze-thaw cycle and recovery phases. Both compounds promoted the activity of GR and reduced oxidative stress mirrored by diminished LDH activity. This was also reflected in the protein and lipid oxidation levels, which was limited to a comparable level with the cells' prior freezing. Further studies on human leukaemia (HL-60) were carried out to elucidate the molecular and biological pathways associated with cryodamage and the modulatory effects of adding novel CPAs. The proteome profile and the corresponding biological functions were evaluated and iii showed that Nig and Sal protected cells against cryodamage. The additive compounds (Nig and Sal) demonstrated a unique and overlapping modulation effect pattern. Nig was found to highly influence proteins engaged with metabolic and energetic pathways, whereas Sal greatly affected nuclear and DNA-binding proteins. The current study concluded that novel CPAs have high potency in protecting cells and each compound has a unique effect on the cellular proteome. These features can be applied to designing cryomedia formulae with higher protective efficiency for targeted applications in cell based therapy and biopharmaceutical industries.

O aumento da complexidade dos resíduos produzidos nos processos de mineração, que acompanha a crescente expansão deste setor no Brasil, tem revelado a necessidade de novas técnicas para o tratamento ecologicamente correto dos rejeitos de mineração. Desta forma, a biorremediação, uma técnica de baixo custo que utiliza microrganismos extremófilos na recuperação de metais tóxicos, se apresenta como um método economicamente viável no tratamento dos rejeitos contendo íons metálicos. Assim, mediante uma análise proteômica dos fungo isolados do ambiente de mineração, quando submetidos a estresse com ions de cobre, podemos compreender seu comportamento fisiológico no processo de biorremediação. Para isso, Aspergillus niger VC e Rhizopus microsporus VC, isolados do ambiente de mineração foram incubados junto a íons de cobre e mediante analise proteômica foram identificados biomarcadores de estresse oxidativo nos fungos. O proteoma realizado utilizando a técnica de NanoLC-ESI-Q-TOF identificou a expressão de proteínas que mudaram sob a influência do cobre em comparação a seus respectivos controles. Os resultados mostraram que as duas cepas obtidas do ambiente de mineração possuem diferentes mecanismos de resistência. Sendo assim, A.niger VC apresentou uma redução na expressão proteica, das quais 132 foram diferencialmente expressas na presença de íons Cu2+. Por sua vez, a cepa R.microsporus VC exibiu uma superexpressão proteica, com 389 proteínas expressas na presença do metal. Nestes cultivos foram identificadas proteínas relacionadas ao choque térmico e à adsorção de metais, conhecidas como proteínas de choque térmico (HSPs) e metaloproteínas, produzidas em resposta ao estresse imposto pela presença do agente indutor de estresse. No entanto, as enzimas envolvidas na defesa contra o estresse oxidativo, identificadas na ausência de metal, são um indicativo de adaptação metabólica em resposta ao ambiente de mineração. Assim concluímos que tanto A.niger VC como R.microsporus VC, são fungos que apresentam importantes características que permitem sua utilização como agentes de biorremediação dos rejeitos de mineração. / The increase in the complexity of the waste produced in the mining process, with the growing expansion of this sector in Brazil, has revealed the need for new techniques for the ecologically correct treatment of this toxic waste. Therefore, bioremediation, a low cost technique that uses endophilic microorganisms in the recovery of toxic metals, presented as an economically viable method in the treatment of metal ion-containing wastes. In this way, through proteomic analysis of the isolated fungus from the mining environment, when submitted to stress with copper ions, we can understand their physiological behavior in the bioremediation process. In this regard, Aspergillus niger VC and Rhizopus microsporus VC, isolated from the mining environment were incubated with copper ions and in result of proteomic analysis, biomarkers of oxidative stress were identified in the fungus. The proteome performed using the NanoLC-ESI-Q-TOF technique identified the expression of proteins that changed under the copper influence compared to their respective controls. The results showed that the two strains obtained from the mining environment have different resistance mechanisms. Thus, A.niger VC presented a reduction in protein expression, of which 132 were differentially expressed in the presence of Cu2+ ions. In turn, the strain R.microsporus VC exhibited a protein overexpression, with 389 proteins expressed in the metal presence. In these cultivations, proteins related to thermal shock and the adsorption of metals, known as HSPs and metalloproteins, produced in response to the stress imposed by the presence of the stress-inducing agent. However, the enzymes involved in defense against oxidative stress, identified in absence of metal, are indicative of metabolic adaptation in response to the mining environment. Therefore we conclude that both A.niger VC and R.microsporus VC are fungus that present important characteristics that allow their to be used as bioremediation agents for mining waste.