Made available in DSpace on 2014-06-11T19:29:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-20Bitstream added on 2014-06-13T20:59:17Z : No. of bitstreams: 1 carnevaleneto_f_me_araiq.pdf: 2909237 bytes, checksum: d5dbb04db98f4d6c5f87cfc6ae4bfcca (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste trabalho foi desenvolvida uma metodologia de desreplicação bioguiada para Qualea grandiflora e Q. cordata, duas espécies da família Vochysiaceae presentes no Cerrado Brasileiro com potencial etnofarmacológico, através dos bioensaios in vitro de inibição da polimerização da β-hematina bovina (compostos antimaláricos), redução do radical estável DPPH (antioxidante), bioautografia para avaliação da inibição da enzima acetilcolinesterase, avaliação citotóxica em linhagens celulares, avaliação de atividade antifúngica por microdiluição em placa e avaliação tripanocida frente à cepas Y de epimastigotas de Tripanosoma cruzi e, da detecção in silico dos constituintes moleculares majoritários a partir da técnica hifenada de HPLC-DAD-HRMS(ESI). As folhas e os ramos secos de Q.grandiflora e Q.cordata foram extraídos por maceração com etanol:água (8:2) e posteriormente fracionados em extração líquido-líquido com os solventes hexano, AcOEt e n-butanol. Entre os bioensaios realizados, a avaliação de inibição do radical DPPH apresentou valores significativos para as frações AcOEt dos ramos de Q.grandiflora e das folhas de Q. cordata. A partir disto, estas frações foram analisadas através da técnica hifenada HPLC-DAD-HRMS(ESI) e permitiram a detecção das flavanonas: 4',5,7-triidroxi-3'-metoxi-6,8-dimetilflavanona e 5,6,7-triidroxi-3',4'-dimetoxi-8-metilflavanona e dos diidroflavonóis: 4',7-diidroxi-5-metoxi-6-metildiidroflavonol e 3',4'-dimetoxi-5,7-diidroxi-6,8-dimetildiidroflavonol na fração AcOEt nas folhas de Q.cordata, e das flavanonas: 8-metilnaringenina e 4',5,7-triidroxi-3',6-dimetoxi-8-metilflavanona; benzofenona: Bis(2-hidroxi-4,6-dimetoxi-3-metilfenil)metanona e diidroflavonol: 3',4',5,6,7-pentaidroxi-8-metildiidroflavonol da fração AcOEt dos ramos de Q.grandiflora. Também realizou-se a análise in silico por RMN da fração... / In this work the bioguided phytochemical studies of Qualea grandiflora and Q. cordata, two species of Vochysiaceae greatly represented in the Brazilian Cerrado and with ethnopharmacology potential, were perfomed according to in vitro bioassays from antioxidant capacity and inhibition of ß-hematin polymerization (antimalarial compounds), scavenging activity of DPPH (antioxidant capacity), inhibition of acetyl cholinesterase (anti-Alzheimer), citotoxic activity against tumoral cell lines (anticancer), antifungal activity using a microdilution assay and tripanocidal activity performed with the Y-strain epimastigote forms of Tripanosoma cruzi, as well as in silico dereplication by means of HPLC-DAD-HRMS(ESI). The leaves and stem of Q.grandiflora and Q.cordata were extracted by maceration in etanol:water (8:2) and fractionated by liquid-liquid using hexane, AcOEt and n-butanol. The bioassays showed that AcOEt stem fraction of Q.grandiflora and AcOEt leaves fraction of Q. cordata are significantly activity in DPPH reduction. According to this results, the AcOEt fraction of the stem of Q.grandiflora and the leaves of Q.cordata were evaluated using HPLC-DAD-HRMS(ESI) and allowed the detection of two flavanones: 4',5,7-trihydroxy-3-methoxy-6,8-dimethylflavanone and 5,6,7-trihydroxy-3',4'-dimethoxy-8-methylflavanone, and the dihydroflavonols: 4',7-dihydroxy-5-methoxy-6-methyldihydroflavonol and 3',4'-dimethoxy-5,7-dihydroxy-6,8-dimethyldihydroflavonol from AcOEt leaves fraction of Q.cordata, and the flavanones: 8-methyl-naringenine and 4',5,7-trihydroxy-3',6-dimethoxy-8-methylflavanone, the benzophenone: bis (2-hydroxy-4,6-dimethoxy-3-methylphenyl) metanone, and the dihydroflavonol: 3',4',5,6,7-pentahydroxy-8-methyldihydroflavonol from AcOEt stem fraction of Q. grandiflora. The AcOEt stem fraction of Q. grandiflora was analyzed through in silico NMR, comparing virtual 1H NMR standards spectra... (Complete abstract click electronic access below)

Submitted by Luiz Felipe Barbosa (luiz.fbabreu2@ufpe.br) on 2015-03-13T13:17:33Z No. of bitstreams: 2 Dissertação Biblioteca.pdf: 708752 bytes, checksum: df7cba1e43069bfc51896d1a153c8993 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-13T13:17:33Z (GMT). No. of bitstreams: 2 Dissertação Biblioteca.pdf: 708752 bytes, checksum: df7cba1e43069bfc51896d1a153c8993 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012 / O bagaço da cana-de-açúcar é constituído por celulose, hemicelulose e lignina. A celulose, quando hidrolisada, é transformada em celobiose que por sua vez pode ser hidrolisada em duas moléculas de glicose. A enzima ß-glucosidase é a proteína responsável pela hidrolise de celobiose em glicose. Entretanto, o bagaço é, na maioria das vezes, queimado para a produção de energia elétrica para ser utilizada no processo industrial não sendo utilizado para a produção de etanol. Por ter um crescimento mais rápido do que a levedura Saccharomyces cerevisiae em condições industriais de fermentação alcoólica apresentando uma maior resistências a altas concentrações de etanol, altas temperaturas e baixo pH têm feito da Dekkera bruxellensis alvo de pesquisas e melhoramentos genéticos. O presente trabalho teve como objetivo identificar e analisar in silico o gene codificante da ß-glucosidase no genoma da levedura D. bruxellensis através das ferramentas de bioinformática. Os resultados mostraram que a partir do alinhamento BLASTp, uma seqüência curta da levedura D. bruxellensis apresentou similaridade com seqüências codificantes para a enzima ß-glucosidase de espécies da divisão ascomiceto. A seqüência da espécie Kluyveromyces marxianus obteve o maior percentual de similaridade com a seqüência de D. bruxellensis. Foram identificados no alinhamento múltiplo, através de conhecimento prévio da literatura, os resíduos do sitio ativo da enzima ß-glucosidase presentes na seqüência de D. bruxellensis mostrando ser sítios bastante conservados

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-10T16:51:06Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-RochaneTorres.pdf: 692913 bytes, checksum: 7cd4864091015b6ca287e445f2eb5c7a (MD5) / Made available in DSpace on 2017-04-10T16:51:06Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-RochaneTorres.pdf: 692913 bytes, checksum: 7cd4864091015b6ca287e445f2eb5c7a (MD5) Previous issue date: 2012 / O bagaço da cana-de-açúcar é constituído por celulose, hemicelulose e lignina. A celulose, quando hidrolisada, é transformada em celobiose que por sua vez pode ser hidrolisada em duas moléculas de glicose. A enzima ß-glucosidase é a proteína responsável pela hidrolise de celobiose em glicose. Entretanto, o bagaço é, na maioria das vezes, queimado para a produção de energia elétrica para ser utilizada no processo industrial não sendo utilizado para a produção de etanol. Por ter um crescimento mais rápido do que a levedura Saccharomyces cerevisiae em condições industriais de fermentação alcoólica apresentando uma maior resistências a altas concentrações de etanol, altas temperaturas e baixo pH têm feito da Dekkera bruxellensis alvo de pesquisas e melhoramentos genéticos. O presente trabalho teve como objetivo identificar e analisar in silico o gene codificante da ß-glucosidase no genoma da levedura D. bruxellensis através das ferramentas de bioinformática. Os resultados mostraram que a partir do alinhamento BLASTp, uma seqüência curta da levedura D. bruxellensis apresentou similaridade com seqüências codificantes para a enzima ß-glucosidase de espécies da divisão ascomiceto. A seqüência da espécie Kluyveromyces marxianus obteve o maior percentual de similaridade com a seqüência de D. bruxellensis. Foram identificados no alinhamento múltiplo, através de conhecimento prévio da literatura, os resíduos do sitio ativo da enzima ß-glucosidase presentes na seqüência de D. bruxellensis mostrando ser sítios bastante conservados. / Bagasse of cane sugar is composed of cellulose, hemicellulose and lignin. Cellulose, when hydrolyzed, is transformed into cellobiose which can be hydrolyzed to two glucose molecules. The enzyme ß-glucosidase is the protein responsible for the hydrolysis of cellobiose to glucose. However, the bagasse is, in most cases, burned to produce electricity to be used in the industrial process and it is not used for ethanol production. Due to have a faster growth than Saccharomyces cerevisiae yeast in industrial fermentation conditions and presenting a greater resistance to high concentrations of ethanol, low pH and high temperatures have made the Dekkera bruxellensis subject of research and genetic manipulations. This study aimed to identify and analyze in silico gene encoding ß-glucosidase in the D. bruxellensis yeast genome through the computational tools of bioinformatics. The results showed that in the blastp alignment, a short sequence of D. bruxellensis yeast showed similarity with sequences coding for the ß-glucosidase enzyme in the ascomycete division species. The Kluyveromyces marxianus sequence had the highest percentage in similarity with D. bruxellensis sequence. The multiple alignment tool was able to identify, using prior knowledge of the literature, the active site residues of the ß-glucosidase enzyme in the sequence of D. bruxellensis. Sites showed to be very conserved.

Os manguezais são considerados um ecossistema único, devido sua particular combinação de condições ambientais, influenciados pela sua localização na interface entre o continente e oceano. O estudo deste ecossistema se torna urgente uma vez que os manguezais estão desaparecendo em todo mundo, e sua diversidade de grupos microbianos é ainda pouco conhecida. Dentro dessa temática, o presente projeto visa descrever a possível funcionalidade de bactérias pertencentes ao filo Planctomycetes nos manguezais. Tais organismos são ainda pouco estudados, de difícil cultivo, sendo obtidos principalmente em ambientes marinhos, tratamento de água e locais de criação de peixes. Dentro desse filo são encontrados microrganismos pertencentes a gêneros descritos como capazes de realizar a oxidação anaeróbica do íon amônio (anammox), uma importante transformação do nitrogênio em ambientes com baixa disponibilidade de oxigênio. E ainda, visamos descrever a possível funcionalidade de bactérias pertencentes ao filo Planctomycetes nos manguezais. Para isso, foi inicialmente realizada uma comparação dos genomas de Planctomycetes com as sequências obtidas por análises de metagenômica e metatranscriptômica, onde foram encontradas sequências similares às afiliadas a funções desconhecidas (putative protein) e a sulfatases. Tais enzimas são descritas como hidrolíticas, que catalizam a clivagem de ésteres de sulfato, liberando o enxofre na forma assimilável. A análise de sequências de uma biblioteca metagenômica (obtida de um manguezal contaminado com petróleo) permitiu a visualização de fragmentos genômicos de Planctomycetes. Esta análise revelou também a ocorrência de genes relacionados a produção de sulfatases, além de indicar arranjos gênicos distintos dos descritos nos genomas de organismos deste grupo, possivelmente indicando a ocorrência de endemismo de Planctomycetes em manguezais. Esta observação foi reforçada pelo cultivo de isolados afiliados a este grupo, os quais tiveram sua afiliação confirmada pelo sequenciamento parcial do gene ribossomal 16S DNAr. Alguns destes isolados formaram clusters diferenciados dentro do filo Planctomycetes, indicando que podem ser estes novas espécies. Sendo assim, a caracterização desse grupo de microrganismos numa combinação de análises in sílico e in vivo, possibilitou a confirmação da ocorrência de tais organismos nos manguezais, e gerou as primeiras informações sobre sua funcionalidade neste sistema, onde parece ocorrer de forma diferenciada aos demais ambientes onde já foram descritos. / Mangroves are considered an unique ecosystem due to its particular combination of environmental conditions, influenced by its location at the interface between land and ocean. The study of this ecosystem becomes urgent since mangroves are disappearing worldwide, and its diversity of microbial groups is still poorly understood. Within this theme, this project aims to describe the possible functionality of bacteria belonging to the phylum Planctomycetes in mangroves. Such organisms are still poorly studied, difficult to cultivate and mainly isolated from marine environments, water treatment and fish farming sites. Within this phylum are found microorganisms belonging to genera described as capable of performing the anaerobic oxidation of ammonium (anammox), a major transformation of nitrogen in environments with low oxygen availability. Here we aimed to describe the possible functionality of bacteria belonging to the phylum Planctomycetes in the mangroves. In order to achieve the target, it was initially performed a comparison of the Planctomycetes genomes with sequences obtained by metagenomics and metatranscriptomics, revealing the presence of sequences with similarity to those affiliated to unknown function and sulfatases. These are hydrolytic enzymes, which catalyze the cleavage of sulfate esters, releasing sulfur on its available form. The analysis of sequences from a metagenomic library (obtained from an oil-contaminated mangrove) allowed the visualization of genomic fragments related to Planctomycetes. It also revealed the presence of genes related with the production of sulfatases, besides the indication of specific genome arrangements, distinct from those describe in organisms allocated in this group, possibly indicating the occurrence of endemicity for Planctomycetes in mangroves. It was reinforced by the cultivation of isolates affiliated to this groups, whose have their affiliation confirmed by the partial sequencing of the 16S rDNA gene. Some isolates clustered composed new clusters within the Planctomycetes phylum, indicating the occurrence of new species. In sum, the characterization of this group by combining in sílico and in vivo analyses, allowed the confirmation of the occurrence of these organisms in mangroves, and generated the first insights about its functioning on this system, where it seems to occur in a differentiated form from those observed in other environments where it has been described.

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.

A long standing problem in catalysis is the identification and characterization of the active sites, i.e. an atom or an ensemble of atoms spouse on the surface of a catalyst.[Taylor1925] One relevant case, that is treated in this thesis, is constituted by the Phillips catalyst.[Hogan1958] For several reasons, even though this catalyst has be applied at industrial scale for decades and is accounted for a majority of the high density polyethylene (HDPE) production, the identification and the mechanism of the active sites are still under debate. This work was initiated in the framework of the PARACAT project, which is dedicated to study the paramagnetic species in catalysis, and focuses on 'The role of Cr paramagnetic states in olefin polymerization over Phillips catalyst.' In the course of the study, I brought this research to a larger scale which included but was not limited to the Phillips catalyst itself. Considering the relevance of the interaction between transition metal ions (TMI) and the support to the catalytic activity, I worked on systems that cover a number of oxide-supported TMIs by means of electron paramagnetic resonance (EPR) spectroscopy. In this thesis I investigated the paramagnetic Cr(V) and Cr(III) species in the Phillips catalyst. The Cr(III) species were suggested with possible relevance to the catalytic reaction while Cr(V) species were suggested as just the spectators in the reaction.[McDaniel2010, Groppo2018] Nevertheless, Cr(V) species were used in this work as spin probes to provide more information on the overall system. Field-sweep methods including continuous wave (CW) EPR and echo detected field sweep (EDFS) showed that the there are two Cr(V) species with different local geometries. Quantitative analysis of the CW EPR showed that these two Cr(V) species have different reactivity with ethylene. The instantaneous diffusion analysis were performed on the Cr(V) species to provide information on the dispersion of the Cr on the silica surface and the results suggested clusters were formed locally. Besides studying the Phillips catalyst itself, I studied also the silica supported organometallic-Cr catalyst, Cr[CH(SiMe3)2]3/SiO2, which served as a model system to investigate the catalytic active Cr species with well-defined oxidation states and geometry. Two categories of the Cr(III) species were assigned to the active sites for olefin oligomerization and polymerization. The assignment were done by comparing their distortion of the local geometries with that of the different precursors. On the other hand, microporous materials including zeolites and zeotype materials such as aluminophosphate (AlPO) can be engineered with different physical and chemical properties in terms of chemical composition and provide a relevant example of structure sensitivity of a heterogeneous catalyst.[Hartmann2002, Hartmann1999] Such structure sensitivity is highly relevant in catalysis and can be very well studies with EPR spectroscopy. In this regard, I investigated a series of SAPO-5 materials doped with different TMI. In the first place, the incorporation of Cr in SAPO-5 was studied focuses on the discrimination of isomorphous substitution at framework sites and extra-framework sites. In the hyperfine sublevel correlation (HYSCORE) spectrum, large hyperfine interaction (hfi) of 27Al with the matrix 31P signal provide solid evidence for the isomorphous substitution of Cr(V) at framework sites. In addition to the Cr-incorporated SAPO-5, a method to prepare a bi-metallic Mo/V-SAPO-5 system was developed and the metal-metal synergy was validated with a single electron transfer reaction and the short range hyperfine interaction. HYSOCRE spectra showed large \textit{hfi} of both 27Al and 31P and suggested the V species grafted at extra-framework sites. Moreover, the HYSCORE spectrum showed signals at low frequency region which were attributed to the 95,97Mo species with large hfi, confirming the short range interaction. Finally, the surface properties of SAPO-5 were studied by adsorbing NO radicals in the pores and investigating their interaction with the surface. Different adsorption sites of NO molecules according to different activation conditions were first discriminated by the g-factors obtained from the CW EPR. From the 27Al HYSCORE spectra, it is observed that when the activation temperature is higher the NO molecules are situated in vicinity of some defect Al sites. However, the dominant Al species were observed either in samples activated at lower temperature or by increasing the NO dosage. This is postulated as that the defect sites were blocked by residual water molecules or saturated by excessive NO molecules. The presence of water molecules were validated by 1H HYSCORE experiments and the coordination of NO-water was estimated from the hfi structure. [Taylor1925] Taylor, H. S. Proc. R. Soc. London. Ser. A, Contain. Pap. a Math. Phys. Character 1925, 108, 105-111 [Hogan1958] Hogan, J. P.; Banks, R. L. Polymers and production thereof US Patent 2,825,721, 1958. [McDaniel2010] McDaniel M. P. Advances in Catalysis, 1st Ed. 2010; Vol. 52, pp 123-606 [Groppo2018] Groppo, E.; Martino, G. A.; Piovano, A.; Barzan, C. ACS Catal. 2018, 8, 10846-10863 [Hartmann2002] Hartmann, M.; Kevan, L. Res. Chem. Intermed. 2002, 28, 625-695 [Hartmann1999] Hartmann, M.; Kevan, L. Chem. Rev. 1999, 99, 635-663

No description available.

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).

A solução fixadora a partir do formaldeído permanece ate os dias de hoje como o composto fixador \"padrão ouro\" para trabalho de rotina. Porém, este é classificado como cancerígeno para os seres humanos e , portanto, representa um risco para qualquer pessoa manusear. Assim, a busca por substitutos tem sido motivada, e, mais recentemente, surgiram algumas soluções alternativas com potencial para substituí-lo. A maioria destas soluções alternativas são à base de álcool , e a maior parte delas são apenas para amostras microscópicas. A partir do conhecimento de ácido tânico para estabilizar a elastina e o colágeno, diluído em glutaraldeído, para microscopia eletrônica, bem como para próteses biológicas, emerge a ideia de uma nova solução fixadora alcoólica, contendo ácido tânico como componente principal. Em análises microscópicas coração, cérebro, intestino e rins foram coletados e preservados sendo armazenados em diferentes soluções fixadoras (10 % de formalina v/v, 70 % de álcool e solução alcoólica de ácido tânico), e preparado para procedimentos histológicos de rotina. Adicionalmente, ratos wistar inteiros foram fixados com a solução alcoólica de ácido tânico ou formalina e estudantes de medicina que praticam dissecção há pelo menos 2 anos ou mais, dissecaram estes espécimes durante o curso de dissecação, como uma alternativa para adquirir habilidades básicas de cirurgia e responderam a um questionário detalhado que inferia sobre a qualidade da peça a ser dissecada. A toxicidade do composto foi analisada por ensaio \"in silico\" para o sistema respiratório e a cutis. A análise microscópica mostrou que solução à base de ácido tânico pode ser melhor que os outros fixadores comuns testados diversos parâmetros, e houve diferença entre os tecidos analisados. Entretanto, o resultado microscópico mais importante foi que esta nova solução possui forte capacidade de preservar e estabilizar a elastina e o colágeno, demonstrando resultados melhores em relação aos outros fixadores. Os resultados macroscópicos revelaram também uma superioridade da solução de ácido tânico em diversos parâmetros analisados, como odor, textura e flexibilidade durante dissecção. A toxicidade da solução de ácido tânico para cútis e sistema respiratório foi considerada moderada em relação à formalina que é considerada altamente tóxica. Concluímos que a solução fixadora de ácido tânico é eficaz para macro e para microscopia, é menos tóxica do que o formaldeído e inodora. Portanto, pode ser uma alternativa real para a evitar a utilização de formol, diminuindo os fatores de risco durante a manipulação de cadáveres e tecidos fixados. / The fixative solution from the formaldehyde remains until nowadays as the \"gold standard\" for routine work fixative compounds. Nevertheless, it is classified as carcinogenic to humans and, therefore, represents a risk to anyone handling the solution. Thus, the search for formaldehyde substitutes has been motivated, and more recently, some alternative solutions with potential to replace it have appeared. Most of these alternative solutions are alcohol-based, and most of them are only for microscopic specimens. From the knowledge that tannic acid diluted in glutaraldehyde can stabilize elastin and collagen for electron microscopy as well as for bioprotheses, emerge the idea for a new alcoholic fixative solution, containing tanic acid as the main component. For microscopical analyses heart, brain, intestine and kidneys were collected and preserved in different fixative solutions (10% v/v regular formalin, 70% alcohol and tannic acid alcoholic solution), and prepared for routine histology procedures. Additionally, whole wistar rats were fixed in the tanic acid alcohol-based solution or formalin and medicine students, who practice dissection for at least 2 or more years, dissected the rats during the dissection course, as an alternative to acquire basic surgery skills and answered a detailed questionnaire about the quality of dissected specimen. The toxicity of the compound was analyzed by in silico tests for cutais and respiratory system. The microscopial analysis showed that tannic acid based solution could be better than others common fixatives as tested in several parameters, and there are differences among tissues. Moreover, the most important microscopic result was that this new solution showed a strong capability to preserve and stabilize elastin and collagen, shppwing better results in relation to other analysed fixatives. The macroscopial analyses also showed better results of tannic acid solution for several parameters, as odor, texture and flexibility during dissection. The toxicity of tannic acid solution for skin and respiratory system was considered moderate in relation to formaldehyde, which is considered highly toxic. We conclude that tanic acid solution is efficient to preserve tissues for macroand microscopical studies, is less toxic than formaldehyde and odorless. Therefore, it could be a real alternative to avoid the regular use of formalin, decreasing risk factors to the ones manipulating fixed corpses and tissues.

Segundo a Organização Mundial de Saúde, a hipertensão arterial é responsável por uma crise global de saúde pública, sendo as doenças cardiovasculares implicadas em aproximadamente 17 milhões de mortes/ano, das quais, 9,4 milhões ocasionadas por complicações provocadas pela hipertensão, como edema pulmonar. Quanto ao arsenal terapêutico disponível, a furosemida, potente diurético de alça, é amplamente utilizada em situações de controle e emergência relacionadas à hipertensão e ao edema pulmonar cardiogênico. Apesar do elevado índice de sua prescrição, esse fármaco pertence à classe IV do Sistema de Classificação Biofarmacêutica (SCB), apresentando absorções intestinais erráticas e variáveis. Tais características representam desafio para o desenvolvimento de formas farmacêuticas orais. Assim, adoção de tecnologias inovadoras associadas à via de administração pulmonar pode permitir abordagem terapêutica alternativa, com elevado potencial de aplicação. Entre as tecnologias inovadoras, a obtenção de nanocristais de fármacos classes II e IV tem sido promissora. Nanocristais podem exibir desempenho in vivo superior quando comparados aos seus homólogos, na forma micronizada. Portanto, estratégias que permitam o desenvolvimento de medicamentos contendo furosemida, com maior eficácia e segurança, são de fundamental importância. Nesse sentido, a aplicação de tecnologia in silico, com propriedade preditiva, contribui para a racionalização de ensaios na pesquisa e no desenvolvimento de novas formas farmacêuticas. Objetivou-se, desse modo, a preparação e a caracterização físico-química de nanocristais de furosemida e sua avaliação in silico na absorção oral e pulmonar empregando ferramenta computacional. Os nanocristais foram obtidos por moagem à alta energia, utilizando movimentos simultâneos de revolução/rotação. A determinação da distribuição do tamanho e a morfologia foram realizadas por difração de raios laser e microscopia eletrônica de varredura, respectivamente. As possíveis interações e/ou alterações do estado cristalino do fármaco foram investigadas por calorimetria exploratória diferencial, termogravimetria diferencial, difração de raio X e espectroscopia Raman de baixo deslocamento. Quanto à solubilidade do nanocristal, foram realizados ensaios para a determinação do aumento na solubilidade de equilíbrio e da velocidade dissolução, utilizando os métodos shake flask e velocidade de dissolução intrínseca (VDI), respectivamente. A moagem à alta energia permitiu a obtenção de nanocristais com tamanho médio trinta vezes menor (231nm) do que o tamanho inicial, na escala micrométrica (7,1 µm). Os nanocristais apresentaram estabilidade térmica. Não foram observadas interações entre os excipientes e os nanocristais, que, entretanto, exibiram estrutura cristalina menos definida, o que indica parcial amorfização do nanocristal. A solubilidade de saturação dos nanocristais aumentou aproximadamente três vezes; como consequência, houve aumento na VDI em 2,2 vezes, 1,8 vezes e 3,8 vezes, quando comparado à VDI da furosemida micronizada em meio SGF, tampão 4,5 e SIF, respectivamente. Quanto às avaliações in silico dos nanocristais, sua absorção oral revelou moderada alteração no perfil farmacocinético. Quando foi utilizada a via de administração pulmonar, os nanocristais apresentaram maior desempenho quando comparada a via de administração oral; destacando-se o aumento na Fa% e na Cmáx e a acentuada diminuição no Tmáx. Em conclusão, a plataforma tecnológica obtida tem potencial aplicação no desenvolvimento de formas farmacêuticas inovadoras para administração pulmonar de furosemida. / According to the World Health Organization, hypertension is responsible for global public health crisis, being the cardiovascular diseases involved in approximately 17 million deaths a year, of these, 9.4 million occasioned by hypertension complications such as pulmonary edema. Regarding therapeutic arsenal available, Furosemide is a potent loop diuretic widely used in control and emergency situations related to hypertension and cardiogenic pulmonary edema. Despite the high level of prescribing, this drug belongs a class IV drug, according to Biopharmaceutics Classification System (BCS), exposing erratic and variable intestinal absorption. These characteristics represent a challenge for the development of oral dosage forms. Thus, adoption of innovative technologies associated with pulmonary route of administration may allow an alternative therapeutic approach, with high potential for application. Among the new technologies, those for obtaining nanocrystals of classes II and IV drugs have been a promising approach. Nanocrystals can exhibit in vivo higher performance when compared to their counterparts in micronized form. Therefore, strategies to develop medicines containing Furosemide, with greater efficacy and safety, are of critical importance. In this sense, the application of technology in silico, with predictive property, contributes to the rationalization of testing in research and development of new dosage forms. The objectives, as a result, were the preparation and the physicochemical characterization of Furosemide nanocrystals, and it\'s in silico evaluation on oral and pulmonary absorption using a computational tool. The nanocrystals were obtained using a high-energy milling technology under simultaneous revolution/rotation motion. The determination of the size distribution and morphology was performed using laser diffraction and scanning electron microscopy, respectively. Furthermore, differential scanning calorimetry, differential thermogravimetry, X-ray diffraction and Low Shift Raman spectroscopy were performed to investigate possible interactions and changes in the crystalline state of the nanocrystals. To measure the increase in the equilibrium solubility and dissolution rate, the shake flask and intrinsic dissolution rate (IDR) methods were used respectively. The nanocrystals size appeared thirty times lower (231 nm) compared to the initial size (7,1 µm). The nanocrystals were stable with concern to its thermal characteristic not showing interactions between the excipients and the nanocrystals; however, they exhibited less defined crystal structure, indicating partial amorphization. The nanocrystals saturation solubility increased approximately three times. Consequently, 2.2, 1.8 and 3.8 folds increase were observed in IDR when compared to the Furosemide raw material in SGF, buffer 4.5 and SIF, respectively. The in silico nanocrystal studies revealed moderate changes in its oral absorption and pharmacokinetic profile. When the pulmonary route of administration was used, the nanocrystals showed higher performance compared to oral route administration; highlighting the increase in Fa % and Cmax and a significant decrease in Tmax. In conclusion, the technology platform obtained has potential application in the development of innovative dosage forms for Furosemide pulmonary delivery.