N-acylethanolamines (NAEs) are a family of signaling lipids derived from a minor membrane lipid constituent N-acylphosphatidylethanolamine (NAPE). In Arabidopsis, NAE mediates physiological functions such as seedling growth, flowering, and response to stress via abscisic acid (ABA) –dependent and –independent signaling pathways. The function of NAEs is terminated by a highly conserved fatty acid amide hydrolase (FAAH). Studies in model plant Arabidopsis showed the significant role of NAEs that makes it relevant to elucidate the conserved metabolic pathway of NAEs in crop species such as tomato. It is hypothesized that there is a functional FAAH in tomato that hydrolyzes NAEs. To test this hypothesis, AtFAAH was used as a template to identify putative FAAH sequences in tomato, using BLASTX. Six SlFAAH sequences with the conserved amidase signature sequence and the catalytic triad, formed by Lys205, Ser281, and Ser305 in AtFAAH, were identified. Phylogenetic analysis of putative SlFAAH homologs and other FAAH family proteins (Arabidopsis, rice and moss), using CLUSTALW, revealed the two sequences that are closely related to the functionally characterized AtFAAH1. Using molecular visualization system (PyMOL), protein structures of putative SlFAAH1and 2 were predicted and compared with AtFAAH; both sequences showed similar domain structure to AtFAAH, with minor differences in spatial arrangement. For further biochemical characterization, full-length coding sequence of SlFAAH1 and SlFAAH2 were isolated and cloned into a heterologous expression system. The expressed protein will be characterized for its hydrolytic activity against radiolabelled NAE substrates. Furthermore, transcript levels for SlFAAH1 and SlFAAH2 will be quantified and correlated with the NAE levels in various tissues to predict their role in tissue-specific NAE hydrolysis. Together, these molecular and biochemical characterization studies in tomato are expected to further validate the conserved nature of NAE metabolic pathway in plants.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Diversos estudos tÃm evidenciado que a principal funÃÃo da proteÃna desacopladora mitocondrial de plantas (pUCP) està relacionada a regulaÃÃo de espÃcies reativas de oxigÃnio (EROs). AnÃlises in silico sugerem a existÃncia de famÃlias multigÃnicas para a codificaÃÃo de pUCPs, porÃm novos estudos ainda sÃo necessÃrios para estabelecer o perfil de expressÃo gÃnica das pUCPs, assim como a quantidade de genes em cada espÃcie e suas relaÃÃes filogenÃticas. O presente trabalho teve como objetivo caracterizar, analisar filogeneticamente e avaliar o perfil de expressÃo da famÃlia multigÃnica da pUCP em diferentes tecidos durante o desenvolvimento da soja [Glycine max (L.) MERR.] e em condiÃÃes de estresse. Foi realizada uma anÃlise in silico no genoma da soja e de outras leguminosas disponÃveis no banco de dados WGS, revelando uma famÃlia multigÃnica codificadora da pUCP, UCP1 e 2 com nove Ãxons, UCP 3 com 2 Ãxons, e UCP 4 e 5 com apenas um Ãxon. Dentre as leguminosas analisadas a soja se destacou com o maior nÃmero de genes, 10 genes no total, sendo quatro genes GmUCP1, uma GmUCP2, uma GmUCP3, dois GmUCP4 e dois GmUCP5, alÃm da presenÃa de um splicing alternativo no gene GmUCP1b1. Primers especÃficos foram desenhados para cada membro da GmUCP a fim de analisar os perfis de expressÃo em diferentes tecidos (semente seca e embebida, flores, vagens, cotilÃdones, folhas unifolioladas e trifolioladas, raÃzes, hipocÃtilos e epicÃtilos) durante o desenvolvimento da soja. Para os ensaios em condiÃÃes de estresse foram utilizadas folhas e raÃzes de soja com treze dias apÃs a semeadura (DAS) que foram submetidas a estresse osmÃtico promovido pela aplicaÃÃo de polietileno glicol (PEG) e estresse biÃtico atravÃs de Ãcido salicÃlico (AS). O RNA total de cada amostra foi extraÃdo para a realizaÃÃo de RT-qPCR. Os valores de ct foram obtidos pelo programa realplex e analisados pelo programa GeNorm. O perfil de expressÃo gÃnica mostrou que todos os genes GmUCP foram expressos em todos os tecidos/ÃrgÃos analisados durante o desenvolvimento da soja, com exceÃÃo de alguns genes em semente seca e epicÃtilo. Os diferentes perfis de expressÃo de cada gene durante o desenvolvimento de cada tecido/ÃrgÃo sugerem que ocorra uma regulaÃÃo gÃnica espacial/temporal entre os membros da GmUCP. Os perfis de expressÃo dos genes GmUCP em soja durante as condiÃÃes de estresses foi diversificado, visto que 2 genes apresentaram expressÃo estÃvel em ambos tecidos/estresse, 7 genes apresentaram queda do perfil de expressÃo, enquanto apenas 4 genes apresentaram aumento dos nÃveis de transcritos. / Several studies have evidenced that the main function of the mitochondrial uncoupling protein in plants (pUCP) is related to reactive oxygen species (ROS) regulation. In silico analysis suggests the existence of multigenic families to pUCPs codification, however further studies are yet needed to establish the pUCPs genetic expression profile, just like the gene amount in each species and their phylogenetic relations. The current work had as objective to characterize, analyze phylogeneticly and evaluate the expression profile of the pUCP multigenic family in different tissues during the soybean development [Glycine max (L.) MERR.] and in stress conditions. It has been performed an in silico analysis on the soybean genome and on other legumes available in the database WGS, revealing a codifier multigenic family for pUCP, UCP1 and 2 with 9 exons, UCP3 with 2 exons, and UCP4 and 5 with only 1 exon. Amongst the legumes analyzed, the soybean stood out with the greater number of genes, 10 genes in total, giving four GmUCP1 genes, one GmUCP2, one GmUCP3, two GmUCP4 and two GmUCP5, along with the presence of an alternative splicing on GmUCP1b1 gene. Specific primers have been designed for each GmUCP member in order to analize the expression profiles in different tissues (dry and doused seed, flowers, pods, cotyledons, unifoliate and trifoliate leaves, roots, hypocotyl and epicotyl) during the soybean development. For the assays in stress conditions have been used soybean leaves and roots with thirteen days after sowing (DAS) which have been subjected to osmotic stress caused by the application of polyethylene glycol (PEG) and biotic stress caused by salicylic acid (SA). The total RNA from each sample has been extracted in order to perform the RT-qPCR. The ct values have been obtained through the realplex program and analyzed through the GeNorm program. The genetic expression profile has shown that all genes were expressed in every tissue/organ analyzed during the soybean development, with the exception on some genes in dry seeds and epicotyl. The different expression profiles of each gene during the development of each tissue/organ suggest that occurs a spatial/temporal gene regulation among the GmUCP members. The expression profiles of the GmUCP genes in soybean during the stress conditions have varied, once 2 genes have shown steady expression in both tissues/ stress, 7 genes have shown a drop in the expression profile, while only 4 genes have shown an increase of the transcript levels.

Blastocystis sp., est un straménopile parasite anaérobie fréquemment rencontré dans le tractus gastro-intestinal de l’homme et de divers animaux. Ce microorganisme, parfois responsable de désordres digestifs aigus, pourrait conduire à des troubles fonctionnels intestinaux tels que le syndrome de l’intestin irritable (IBS). Le génome de Blastocystis sp., qui a fait l'objet d'un projet de séquençage en collaboration avec le Génoscope d’Evry, nous a permis de caractériser le plus petit génome de straménopile séquencé à ce jour (18,8 Mpb), avec une capacité codante de 6020 gènes. L’acquisition de nombreux gènes par transferts horizontaux est une caractéristique majeure de ce génome, qui montre d’abondants réarrangements génomiques. Bien qu’évoluant en anaérobiose, Blastocystis sp. possède des organites morphologiquement proches des mitochondries, appelés mitochondrion-like organelles (MLOs). Nous avons montré que ces organites comportaient un génome circulaire de type mitochondrial de 29,27 kpb, mais dépourvu des gènes codant pour les cytochromes. Des analyses in silico nous ont permis de caractériser le protéome des MLOs (365 protéines), conduisant à l’établissement d’un modèle prédictif des voies métaboliques associées à ces organites, avec notamment une chaine respiratoire limitée aux complexes I et II. Nous avons ainsi montré que les MLOs présentent des caractères communs aux mitochondries anaérobies et aux hydrogénosomes (présence d’une PFOR et d’une hydrogénase à fer), suggérant que Blastocystis sp. comporte des mitochondries anaérobies modifiées, qui résulteraient d’une adaptation du parasite à son environnement. Par ailleurs, la prédiction du sécrétome de Blastocystis sp. révèle la présence de facteurs de virulence potentiels, pouvant être impliqués dans l’altération de l’épithélium intestinal et le contournement du système immunitaire de l’hôte. / Blastocystis sp. is a highly prevalent anaerobic eukaryotic stramenopile parasite found in the intestinal tract of humans and various animals. This microorganism, sometimes associated with acute intestinal disorders, could be responsible for functional intestinal disorders such as the irritable bowel syndrom (IBS). As part of a collaborative sequencing project with the Genoscope (CEA Evry, France), we were able to caracterize the smallest stramenopile genome sequenced to date (18.8 Mbp) with a 6020 genes coding capacity. The gain of many genes through horizontal gene transfer is amajor characteristic of this genome, which shows extensive genomic rearrangements. Despite the anaerobic nature of Blastocytists sp., this eukaryote harbours nevertheless mitochondrion-like organelles (MLOs). We have shown that these organelles have a 29.27 kbp mitochondrial-type circular genome that lacks cytochrome coding genes. In silico analysis allowed us to predict the MLOs proteome (365 proteins), with the subsequent predictive model of the metabolic pathways associated with these organelles, including an electron transport chain (ETC) restricted to complex I and II. We have shown that MLOs shared common characteristics with anaerobic mitochondrion and hydrogenosomes (presence of a PFOR and an iron-hydrogenase), which could mean that Blastocystis sp. harbours modified anaerobic mitochondrion that resulted from the parasite adaptation to its anaerobic environment. In addition, Blastocytis sp. secretome prediction reveals the presence of potential virulence factors, which could be involved in the degradation of the intestinal epithelium as well as the host immune system bypass.

Submitted by Morgana Andrade (morgana.andrade@ufes.br) on 2016-04-08T20:24:35Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) tese_6676_Dissertao_Christine Facco_2013.pdf: 1315436 bytes, checksum: 39ff1bc59d15b9b7a17e711efee2949b (MD5) / Approved for entry into archive by Patricia Barros (patricia.barros@ufes.br) on 2016-05-13T14:04:33Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) tese_6676_Dissertao_Christine Facco_2013.pdf: 1315436 bytes, checksum: 39ff1bc59d15b9b7a17e711efee2949b (MD5) / Made available in DSpace on 2016-05-13T14:04:33Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) tese_6676_Dissertao_Christine Facco_2013.pdf: 1315436 bytes, checksum: 39ff1bc59d15b9b7a17e711efee2949b (MD5) / Essa dissertação teve o apoio financeiro do Edital Universal 14/2011 CNPq (nº processo: 483036/2011-0) e do Edital PROEX nº 04/2011. / Na tentativa de obter novas substâncias com atividade antibacteriana, o objetivo desse trabalho foi avaliar o potencial antibacteriano de quatro peptídeos sintetizados, dos quais dois possuem sequência derivada da fragmentação in silico do G-CSF humano, enquanto os outros dois foram planejados teoricamente, verificando, assim, seu interesse como novos agentes terapêuticos na saúde humana. A avaliação foi realizada em duas etapas: análise in silico, que consistiu em predições de propriedades e parâmetros associados à ação antibacteriana, por meio de ferramentas computacionais; e o experimento in vitro para a determinação da concentração inibitória mínima (MIC) dos peptídeos contra bactérias Gram positivas e negativas. A maioria das predições foi favorável para os quatro peptídeos, pois os resultados determinados para hidrofobicidade, anfipaticidade, tamanho, estrutura secundária, carga líquida, potencial de ligação em membrana, meia-vida e índice de Boman encontram-se dentro de valores desejados para o potencial antibacteriano. Na análise in silico, apenas a predição algorítmica da atividade antimicrobiana gerou resultados desfavoráveis para os peptídeos com sequências derivadas do G-CSF (peptídeos 1 e 2), porém essa mesma predição foi positiva para os outros dois. O ensaio in vitro demonstrou que até a concentração mais alta utilizada dos quatro peptídeos (500 μg/mL) foi insuficiente para a determinação de sua concentração inibitória mínima, porém, observou-se considerável diminuição no crescimento de E. coli (58,7%) pelo peptídeo 4 e de E. fecalis (86,1%) e E. coli (54,9%) pelo peptídeo 3, em comparação com o controle de viabilidade. Esses valores indicam a presença de ação antibacteriana por parte dos peptídeos planejados teoricamente (peptídeos 3 e 4), corroborando com as predições computacionais. Dessa forma, é possível concluir que a análise in silico foi de suma importância para a seleção dos peptídeos a serem sintetizados, os quais apresentaram nos ensaios in vitro resultados em concordância com a predição computacional de atividade antimicrobiana. / In attempt to obtain new substances with antibacterial activity, the aim of this study was to evaluate the antibacterial potential of four synthesized peptides, where two of them have sequence derived from human G-CSF in silico fragmentation, while the other two were theoretically planned, allowing the verification of their interest as new therapeutic agents at human health. The evaluation was performed in two stages: in silico analysis, consisting of predictions of properties and parameters associated with antibacterial effect, through computational tools; and the in vitro experiment for determination of the minimum inhibitory concentration (MIC) of the peptides against Gram positive and negative bacteria. Most predictions was favorable for all four peptides, showed by determined results of hydrophobicity, amphipathicity, size, secondary structure, net charge, membrane binding potential, half-life and Boman Index, considered as desirable values for antibacterial potential. In the in silico analysis, only algorithmic prediction of antimicrobial activity revealed unfavorable results for peptides with sequences derived from G-CSF (peptides 1 and 2), nonetheless, the predictions were positive for the other two. The in vitro assay showed that up to the highest concentration used of the four peptides (500 μg/mL) was insufficient for determination of minimum inhibitory concentration, however it was possible to observe significant growing decrease of E. coli (58.7%) by peptide 4 and E. fecalis (86.1%) and E. coli (54.9%) by peptide 3, when compared with the viability control. These values indicate the presence of antibacterial activity in the theoretically planned peptides (peptides 3 and 4), confirming the computational predictions. Thus, it is possible to conclude that the in silico analysis was very important for the selection of the peptides to be synthesized, which showed results of in vitro assays in agreement with the computational prediction of antimicrobial activity.

Made available in DSpace on 2014-12-17T14:10:25Z (GMT). No. of bitstreams: 1 ValeskaDSS_DISSERT.pdf: 2384968 bytes, checksum: cf4765fd0b42fd627fcb0ce0d604be1e (MD5) Previous issue date: 2011-10-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The flowering is a physiological process that it is vital for plants. This physiological process has been well studied in the plant model Arabidopsis, but in sugarcane this process is not well known. The transition of the shoot apical meristem from vegetative to flowering is a critical factor for plant development. At Brazil northeastern region, the transition to flowering in sugarcane has an important effect as it may reduce up to 60% its production. This is a consequence of the sugar translocation from stalks to the shoot apical meristem which is necessary during the flowering process. Therefore, the aim of this work was to explore and analyze cDNAs previously identified using subtractive cDNA libraries. The results showed that these cDNAs showed differential expression profile in varieties of sugarcane (early x late flowering). The in silico analysis suggested that these cDNAs had homology to calmodulin, NAC transcription factor and phosphatidylinositol, a SEC14, which were described in the literature as having a role in the process of floral development. To better understand the role of the cDNA homologous to calmodulin, tobacco plants were transformed with overexpression cassettes in sense and antissense orientation. Plants overexpressing the cassette in sense orientation did not flowered, while plants overexpressing the cassette in the antissense orientation produced flowers. The data obtained in this study suggested the possible role from CAM sequence, SEC14 and NAC in the induction/floral development pathway in sugarcane, this is the first study in order to analyze these genes in the sugarcane flowering process. / A flora??o ? um processo fisiol?gico que demanda um alto gasto energ?tico, e ? vital para a planta, pois ? dela que depende sua propaga??o gen?tica. Este processo fisiol?gico tem sido bem estudado no modelo vegetal Arabidopsis, mas em cana-dea??car n?o ? muito conhecido. A transi??o do meristema apical no processo de flora??o ? um fator cr?tico no ciclo de desenvolvimento da planta. No nordeste brasileiro, a transi??o para a flora??o na cana-de-a??car tem um efeito dram?tico, pois pode reduzir em at? 60% a produ??o de a??car ou etanol. Isso porque, o florescimento da cana resulta na isoporiza??o do colmo que ? caracterizado pela transloca??o do a??car presente no colmo para os ?pices meristem?ticos com a finalidade de suprir a necessidade energ?tica durante o processo de flora??o. Portanto, o objetivo desse trabalho foi de prospectar e analisar cDNAs de cana-dea??car que possam estar associados ? flora??o utilizando ferramentas de bioinform?tica e de transgenia. Os resultados mostraram que os cDNAs em estudo apontaram um perfil de express?o diferencial em variedades de cana-de-a??car. As an?lises in silico sugeriram que os cDNAs estudados apresentam similaridade para calmodulina, fator de transcri??o NAC e fosfatidilinositol, uma SEC14, as quais foram descritas na literatura como tendo um papel no processo de desenvolvimento floral. Para entender melhor o papel do cDNA hom?logo ? calmodulina, plantas de tabaco foram transformadas com cassetes de superexpress?o nas orienta??es sense e antisense contendo o promotor 35S. Plantas superexpressando o cassete na orienta??o senso n?o floresceram, enquanto que plantas superexpressando o cassete na orienta??o antissense apresentaram o desenvolvimento reprodutivo. Os dados obtidos nesse trabalho apontam para o poss?vel papel de CAM, NAC e SEC14 na via de indu??o/desenvolvimento floral em cana-de-a??car, sendo um dos primeiros trabalhos a se estudar esses genes no processo de flora??o nesse organismo

Serum C-reactive protein (CRP) is used as a marker of inflammation in several diseases including autoimmune disease and cardiovascular disease. CRP, a member of the pentraxin family, is comprised of five identical subunits. CRP has diverse ligand-binding properties which depend upon different structural states of CRP. However, little is known about the molecular dynamics and interaction properties of CRP. In this study, we used SAPS, SCRATCH protein predictor, PDBsum, ConSurf, ProtScale, Drawhca, ASAView, SCide and SRide server and performed comprehensive analyses of molecular dynamics, protein-protein and residue-residue interactions of CRP. We used 1GNH.pdb file for the crystal structure of human CRP which generated two pentamers (ABCDE and FGHIJ). The number of residues involved in residue-residue interactions between A-B, B-C, C-D, D-E, F-G, G-H, H-I, I-J, A-E and F-J subunits were 12, 11, 10, 11, 12, 11, 10, 11, 10 and 10, respectively. Fifteen antiparallel β sheets were involved in β-sheet topology, and five β hairpins were involved in forming the secondary structure. Analysis of hydrophobic segment distribution revealed deviations in surface hydrophobicity at different cavities present in CRP. Approximately 33 % of all residues were involved in the stabilization centers. We show that the bioinformatics tools can provide a rapid method to predict molecular dynamics and interaction properties of CRP. Our prediction of molecular dynamics and interaction properties of CRP combined with the modeling data based on the known 3D structure of CRP is helpful in designing stable forms of CRP mutants for structure-function studies of CRP and may facilitate in silico drug design for therapeutic targeting of CRP.

Serum C-reactive protein (CRP) is used as a marker of inflammation in several diseases including autoimmune disease and cardiovascular disease. CRP, a member of the pentraxin family, is comprised of five identical subunits. CRP has diverse ligand-binding properties which depend upon different structural states of CRP. However, little is known about the molecular dynamics and interaction properties of CRP. In this study, we used SAPS, SCRATCH protein predictor, PDBsum, ConSurf, ProtScale, Drawhca, ASAView, SCide and SRide server and performed comprehensive analyses of molecular dynamics, protein-protein and residue-residue interactions of CRP. We used 1GNH.pdb file for the crystal structure of human CRP which generated two pentamers (ABCDE and FGHIJ). The number of residues involved in residue-residue interactions between A-B, B-C, C-D, D-E, F-G, G-H, H-I, I-J, A-E and F-J subunits were 12, 11, 10, 11, 12, 11, 10, 11, 10 and 10, respectively. Fifteen antiparallel β sheets were involved in β-sheet topology, and five β hairpins were involved in forming the secondary structure. Analysis of hydrophobic segment distribution revealed deviations in surface hydrophobicity at different cavities present in CRP. Approximately 33 % of all residues were involved in the stabilization centers. We show that the bioinformatics tools can provide a rapid method to predict molecular dynamics and interaction properties of CRP. Our prediction of molecular dynamics and interaction properties of CRP combined with the modeling data based on the known 3D structure of CRP is helpful in designing stable forms of CRP mutants for structure-function studies of CRP and may facilitate in silico drug design for therapeutic targeting of CRP.

Abstract : The chymase-dependant pathway responsible for converting Big ET-1 to ET-1 was established in vitro. It has only been recently, in 2009, that our group demonstrated that the conversion of Big ET-1 to ET-1 (1-31) can occur in vivo in mice (Simard et al., 2009), knowing that ET-1 (1-31) is converted to ET-1 via NEP in vivo (Fecteau et al., 2005). In addition, our laboratory demonstrated in 2013 that mMCP-4, the murine analogue of human chymase, produces ET-1 (1-31) from the Big ET-1 precursor (Houde et al. 2013). Thus far, in the literature, there are no specific characterizations of recombinant chymases (human or murine). In fact, the group of Murakami published in 1995 a study characterizing the CMA1 (human chymase) in a chymostatin-dependent fashion, using Angiotensin I as a substrate (Murakami et al., 1995). However, chymostatin is a non-specific inhibitor of chymase. It has been shown that chymostatin can inhibit elastase, an enzyme that can convert Angiotensin I to Angiotensin II (Becari et al., 2005). Based on these observations, the proposed hypothesis in the present study suggests that recombinant as well as extracted CMA1 from LUVA (human mast cell line), in addition to soluble fractions of human aortas, convert Big ET-1 into ET-1 (1-31 ) in a TY-51469 (a chymase-specific inhibitor) sensitive manner. In a second component, we studied the enzyme kinetics of CMA1 with regard to the Big ET-1 and Ang I substrate. The affinity of CMA1 against Big ET-1 was greater compared to Ang I (KM Big ET- 1: 12.55 μM and Ang I: 37.53 μM). However, CMA1 was more effective in cleaving Ang I compared to Big ET-1 (Kcat / KM Big ET-1: 6.57 x 10-5 μM-1.s-1 and Ang I: 1.8 x 10-4 ΜM-1.s- 1). In a third component involving in vivo experiments, the pressor effects of Big ET-1, ET-1 and Ang I were tested in conscious mMCP-4 KO mice compared to wild-type mice. The increase in mean arterial pressure after administration of Big ET-1 was greater in wild-type mice compared to mMCP- 4 KO mice. This effect was not observed after administration of ET-1 and / or Ang I. / Résumé : La voie de conversion de Big ET-1 en ET-1, chymase dépendante a été établie in vitro. Ce n'est que récemment, en 2009 que notre groupe a démontré que la conversion de Big ET-1 en ET-1 (1-31) peut avoir lieu in vivo chez la souris (Simard et al., 2009), sachant que ET-1 (1-31) est convertie en ET-1 via NEP in vivo (Fecteau et al., 2005). En plus, en 2013, notre laboratoire a démontré que la mMCP- 4, l'analogue murin de la chymase humaine, produit l'ET-1 (1-31) à partir du précurseur Big ET-1 (Houde et al., 2013). Jusqu'a présent, dans la littérature, on ne trouve pas de caractérisations spécifiques de chymases (humaine ou murine) recombinantes. En fait, le groupe de Murakami, en 1995, a publié une étude caractérisant, d'une façon chymostatin dépendante, la CMA1 (chymase humaine) en utilisant l'Angiotensine I comme substrat (Murakami et al., 1995). Cependant, le chymostatin est un inhibiteur non-spécifique de la chymase. Il a été démontré que le chymostatin peut inhiber l'élastase, une enzyme pouvant convertir l'Angiotensine I en Angiotensine II (Becari et al., 2005). Basé sur ces observations, l'hypothèse formulée dans la présente étude est que la CMA1 recombinante ou extraite des cellules LUVA (lignée humaine de mastocytes) ou des fractions solubles des aortes humaines convertit la Big ET-1 en ET-1 (1-31) d'une façon TY-51469 (un inhibiteur spécifique de la chymase) sensible. Dans un deuxième volet, on a étudié la cinétique enzymatique de CMA1 en vers le substrat Big ET-1 et Ang I. L’affinité de CMA1 contre la Big ET-1 était plus grande comparé à l’Ang I (KM Big ET-1 : 12.55 μM et Ang I : 37.53 μM). Cependant CMA1 était plus efficace dans le clivage de l’Ang I comparé à la Big ET-1 (Kcat/KM Big ET-1 : 6.57 x 10-5 μM-1 .s-1 et Ang I : 1.8 x 10-4 μM-1 .s-1 ). Dans un troisième volet impliquant des expériences in vivo, l’effet presseur de la Big ET-1, l’ET-1 et l’Ang I a été testé chez des souris conscientes mMCP- 4 KO comparé à des souris de type sauvage. L’augmentation de la pression artérielle moyenne a été plus importante chez les souris de type sauvage après l’administration de Big ET-1 que chez les souris mMCP-4 KO. Cet effet n’a pas été observé après l’administration d’ET-1 et/ou d’Ang I ce qui explique le rôle de la chymase dans l’effet de la conversion de Big ET-1 en ET-1 (1-31).

Submitted by Cristhiane Guerra (cristhiane.guerra@gmail.com) on 2017-02-01T13:46:41Z No. of bitstreams: 1 arquivototal.pdf: 2056832 bytes, checksum: dd1f04e5a02bdef6bb3220514f057563 (MD5) / Made available in DSpace on 2017-02-01T13:46:41Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2056832 bytes, checksum: dd1f04e5a02bdef6bb3220514f057563 (MD5) Previous issue date: 2016-10-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The incidence of vulvovaginal fungal infections has increased dramatically over the last decades, therefore being the second most common cause of vulvovaginal candidiasis (VVC) placed after bacterial vaginosis diagnosed in 40% of the women with vaginal discharge. The increasing resistance of micro-oganismos pathogens to existing antimicrobial market has driven the search for new therapeutic alternatives, such as natural herbal products belonging to various families of the plant kingdom, such as Poaceae and Myrtaceae, which are presented as a viable solution due to the low cost and easy access of the population. Highlighted, the genus Cymbopogon and the Eucalyptus plants is one of the main sources of biologically active molecules, among them the monoterpenes holds a huge biological potential of human interest. However, the research of plant-derived compounds with pharmacological properties must always be attached to toxicity studies in order to show the absence of these substances harm to the human body. Given this context, it has studied the biological activity of enantiomers (R)-(+)- and (S)-(-)-citronellal against C. albicans and C. tropicalis strains derived from in vitro vulvovaginal secretions, as well as the parameters toxicological to predict the theoretical oral toxicity in silico. To achieve the antimicrobial in vitro studies; determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) was used microdilution test. Also it was applied to disk diffusion technique on solid medium for comparative study of antifungal resistance/sensitivity profile used in the treatment of vulvovaginal candidiasis isolated and associated with monoterpenes studieds. The MIC's and MFC's of the (R)-(+)- and (S)-(-)-citronellal to 90% of fungal strains were respectively (R)MIC90% 16μg/mL and (R)MFC90% 32μg/mL; (S)MIC90% 64μg/mL and (S)MFC90% 128μg/mL (Strong antifungal activity against C. albicans and C. tropicalis). Were observed high resistance of C. albicans to fluconazole and itraconazole 12 (92.30%) strains. However, this resistance was reversed in 9 (75%) and 7 (58.33%) respectively, when combined with (R)-(+)-citronellal and 6 (50%) in combination with (S)-(-)-citronellal. Furthermore, it was also observed C. tropicalis high resistance to amphotericin B, itraconazole and miconazole. However, the resistance was reversed to amphotericin B and itraconazole, as a result of the synergistic effect in 2 (66.65%) of yeast. For miconazole resistance was reversed in 3 (100%) of the strains for both enantiomers. In the in silico analysis, both enantiomers have good oral bioavailability theoretically, however, a potential irritant was observed as possible toxic effect on these monoterpenes. In conclusion, these results suggest that the (R)-(+)- and (S)-(-)-citronellal have antimicrobial effect, and which also exert effect modifier activity when antifungal agents in combination. However, although presenting good oral bioavailability theoretically, the varied toxicological profile suggests the need to assess the risk-benefit of this compound in the production of a new antifungal drug, by conducting in vivo trials. / A incidência de infecções fúngicas vulvovaginais aumentou dramaticamente ao longo das últimas décadas, constituindo assim a segunda causa mais comum de candidíase vulvovaginal (CVV) após a vaginose bacteriana diagnosticada em 40% das mulheres com corrimento vaginal. O aumento da resistência dos micro-organismos patógenos aos antimicrobianos existentes no mercado tem impulsionado a busca de novas alternativas terapêuticas, como os produtos naturais a base de plantas pertencentes a várias famílias do reino vegetal, como por exemplo a Poaceae e a Myrtaceae, que se apresentam como uma solução viável devido ao baixo custo e fácil acesso da população. Em destaque, as plantas do gênero Cymbopogon e Eucalyptus constitui uma das principais fontes de moléculas biologicamente ativas, dentre elas os monoterpenos são detentores de um enorme potencial biológico de interesse humano. No entanto, as pesquisas de compostos derivados de plantas com propriedades farmacológicas devem sempre ser unida aos estudos toxicológicos, afim de se comprovar a ausência de malefícios destas substâncias ao organismo humano. Diante deste contexto, foi estudado a atividade biológica dos enantiômeros (R)-(+)- e (S)-(-)-citronelal contra cepas de C. albicans e C. tropicalis oriundas de secreções vulvovaginais in vitro, bem como os parâmetros toxicológicos para a previsão da toxicidade oral teórica in silico. Para a realização dos estudos antimicrobianos in vitro; determinação da concentração inibitória mínima (CIM) bem como da concentração fungicida mínima (CFM), utilizou-se o teste de microdiluição. Também foi aplicada a técnica de disco-difusão em meio sólido para estudo comparativo do perfil de resistência/sensibilidade a antifúngicos utilizados na terapêutica da candidíase vulvovaginal isolados e associados aos monoterpenos em estudo. As CIM’s e as CFM’s dos enantiômeros (R)-(+)- e (S)-(-)-citronelal para 90% das cepas fúngicas foram respectivamente (R)CIM90% 16μg/mL e (R)CFM90% 32 μg/mL; (S)CIM90% 64μg/mL e (S)CFM90% 128 μg/mL (forte atividade antifúngica contra C. albicans e C. tropicalis). Foram constatados alta resistência de C. albicans ao fluconazol e ao itraconazol 12 (92.30%) das cepas testadas. Mas, essa resistência foi revertida em 9 (75%) e 7 (58.33%) respectivamente, quando em associação com o (R)-(+)-citronelal e em 6 (50%) em combinação com o (S)-(-)-citronelal. Além disso, também foi observado alta resistência de C. tropicalis a anfotericina B, itraconazol e miconazol. Porém a resistência foi revertida para a anfotericina B e para o itraconazol, resultado do efeito sinérgico em 2 (66.65%) das leveduras. Para o miconazol a resistência foi revertida em 3 (100%) das cepas para ambos os enantiômeros. Na análise in silico, ambos os enantiômeros apresentaram boa biodisponibilidade oral teórica, no entanto, um potencial irritante foi observado como possível efeito tóxico para estes monoterpenos. Em conclusão, estes resultados sugerem que os enantiômeros (R)-(+)- e (S)-(-)-citronelal apresentam efeito antimicrobiano, e que também exercem efeito modificador de atividade aos agentes antifúngicos quando em combinação. No entanto, embora tenha apresentado boa biodisponibilidade oral teórica, o perfil toxicológico variado sugere a necessidade em avaliar o risco-benefício desse composto na produção de um novo medicamento antifúngico, por realização de ensaios in vivo.

Estresses abióticos como seca, alta salinidade e baixas temperaturas, afetam o crescimento e a produtividade em culturas de interesse comercial como o feijoeiro comum. Proteínas DREB (Dehydration Responsive Element Binding) são fatores de transcrição que regulam genes específicos envolvidos na tolerância ao estresse abiótico. Para determinar como as plantas toleram condições ambientais adversas, variedades tolerantes, biologia molecular e bioinformática podem ser aplicadas para identificar e caracterizar genes que controlam mecanismos de adaptação a estresses. Baseado nas informações disponíveis nos bancos de dados públicos, a sequência da Orf completa do gene Phvul.009G029600.1| PACid:27146455 contendo 1062 pb foi encontrada e usada para o desenho dos primers e para o sequenciamento. A nova sequência é muito similar ao AtRAP2.4 e foi nomeada como PvDREB6A, segundo a análise filogenética. Ferramentas de predição mostraram que a sequência apresenta 354 aminoácidos e possui uma cópia do domínio AP2, que se dobra em uma estrutura com três ?-folhas e uma ?-hélice apresentando resíduos importantes e motivos específicos de reconhecimento e de ligação ao DNA. Além disso, um peptídeo trânsito foi detectado na porção N-terminal com um sítio de clivagem no resíduo 52. A interação deste fator de transcrição com seu domínio de ligação ao DNA foi validada por Electro Mobility Shift Assay (EMSA). A localização subcelular da proteína foi realizada e expressão da Green Fluorescent Proteín (GFP) foi detectada no núcleo. A transformação genética para a superexpressão do gene PvDREB6A em plantas de Arabidopsis thaliana Columbia-0 e mutantes nocaute para o gene AtRAP2.4 (Salk_020767C) foi realizada. Quatro eventos com cópia única e melhor expressão do gene PvDREB6A denominados Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/ pFEC2.1 #19.7 e Salk_020767C/ pFEC2.1 #23.7, foram selecionados. O evento Salk_020767C/pFEC2.1 #23.7 mostrou melhor expressão do gene PvDREB6A e foi visualizado sob luz UV. A análise funcional revelou que as plantas transgênicas submetidas ao déficit hídrico, à alta salinidade e ao frio, apresentaram maior taxa de sobrevivência. Plantas transgênicas superexpressando o gene PvDREB6A apresentaram menor taxa de desidratação e de vazamento de eletrólitos quando submetidas a estresses abióticos. Uma análise da expressão de genes relacionados à tolerância foi conduzida. A quantificação revelou que a expressão de 18 genes: AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, relacionados a tolerância a seca, sal e frio foram up-regulated devido à superexpressão do gene PvDREB6A de feijoeiro nas plantas transgênicas / Abiotic stresses like drought, high salinity and low temperatures affect growth and productivity in crops of economic interest such as common bean. DREB (Dehydration Responsive Element Binding) proteins are transcription factors that activate specific genes involved in tolerance to abiotic stress. To generate new information on the research for drought and other abiotic stresses, tolerant varieties, molecular biology and bioinformatics can be applied to identify and characterize genes that control plant defense and adaptation mechanisms to water deprivation, to excessive salt and to high/low temperature. Based on public databases, a common bean DREB sequence was found and an in silico study was carried out. A complete Orf sequence Phvul.009G029600.1 |PACid:27146455 containing 1062 bp was found and used for primer design and sequencing. The new sequence was very similar to AtRAP2.4 and named as PvDREB6A, according to phylogenetic analysis. Prediction tools showed that the deduced 354 aa sequence has one copy of the AP2 domain, folding in a three ?-sheets and one ?-helix structure, and presenting important residues and motifs for DNA contacting and binding specificity. In addition, a chloroplast transit peptide was detected at the N-terminal region with cleavage site in the 52 residue. Binding activity of this transcription factor was validated by Electro Mobility Shift Assay (EMSA). Subcellular localization was verified by transient expression of PvDREB6A::GFP in Nicotiana benthamiana and the expression of GFP was detected at the nucleus. Genetic transformation for overexpression of PvDREB6A gene in Arabidopsis thaliana wild type and knockout mutant for AtRAP2.4 gene was conducted. Four single copy events with better expression of the PvDREB6A named Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/pFEC2.1 #19.7 and Salk_020767C/pFEC2.1 #23.7 were selected. The event Salk_020767C/pFEC2.1 #23.7 showed the best expression of PvDREB6A and was visualized under UV light. Functional analysis, revealed that transgenic plants under water deficit, high salt, and cold showed higher survival rate. Transgenic plants overexpressing the PvDREB6A exhibited lower water loss rate and electrolyte leakage rate under abiotic stress. A gene expression analysis with tolerant-related genes was conduted. The quantification revealed that 18 genes, AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, related to drought, salt and cold tolerance, were up-regulated due to the overexpression of PvDREB6A from common bean in the transgenic plants