Petrologia e geologia estrutural do plutão granítico Barcelona, província Borborema, NE do Brasil

The Dissertation aimed to advance the geological knowledge of the Barcelona Granitic Pluton (BGP). This body is located in the eastern portion of the Rio Grande do Norte Domain (RND), within the São José do Campestre subdomain (SJC), NE of the Borborema Province. The main goal was to understand the geological evolution of the rocks of the pluton and the tectonic setting of magma generation and its emplacement. The BGP has an assumed Ediacaran age and outcropping area of approximately 260 km2, being composed of three varied petrographic/textural facies: (a) porphyritic biotite monzogranite; (b) dykes and sheets of biotite microgranite; (c) dioritic to quartz-dioritic enclaves. The rocks of the BGP have the following structures: (i) a NE-SW and NW-SE directed magmatic fabric (Sγ), accompanied by a magmatic lineation (Lγ) with gentle dip to NE-SW and NW-SE. In the southern portion, there is the concentric pattern of this foliation with medium to high dip, and (ii) a solid state foliation, in part mylonitic (S3+), mainly on the eastern edge with slightly plunging to west. The integration of structural and gravity data permitted to interpret the emplacement of the BGP as controlled by the transcurrent shear zones systems Lajes Pintadas (LPSZ) and Sítio Novo (SNSZ), both of dextral strike-slip kinematics. Mineral chemistry data show that the amphibole form the porphyritic biotite monzogranite facies is hastingsite with moderate Mg / (Mg + Fe) ratios, indicating crystallization under moderate to high ƒO2 and cristallization pressure of around 5.0-6.0 kbar. The biotite tends to be slightly richer in annite molecule and plots in the transitional field from primary biotite to reequilibrated biotite. In discriminant diagrams of magmatic series, the biotite behave like those of subalkaline affinity, consistent with the potassium calc-alkaline / sub-alkaline geochemical affinity of the hosting rock. The opaque minerals are primarily magnetite, with some crystals martitized to hematite indicating relatively oxidizing conditions during magma evolution that originated the BGP. Zoning in plagioclase, K-feldspar and allanite crystals suggest fractional crystallization process. Lithogeochemical data suggest that the facies described for the BGP have similar magma source, usually plotting in the fields and trends of the subalkaline / high potassium calc-alkaline series.

Petrologia e geocronologia U-Pb do plúton granítico Serra da Rajada, porção central do domínio Rio Piranhas - Seridó, Província Borborema, NE do Brasil

The ediacaran plutonic activity related to the Brasilian/Pan-African orogeny is one of the most important geological features in the Borborema Province, represented along its extension by numerous batholiths, stocks, and dikes.The object of this study, the Serra Rajada Granitic Pluton (SRGP), located in the central portion of the Piranhas-Seridó River Domain is an example of this activity. This pluton has been the subject of cartographic, petrographic, geochronological and lithogeochemical studies and its rocks were characterized by two facies. First, the granitic facies were described as monzogranites consisting of K-feldspar, plagioclase (oligoclase - An23-24%), quartz and biotite (main mafic) and opaque minerals such as titanite, allanite, apatite, and zircon as accessories. Alteration minerals are chlorite, white mica and carbonate. Second, the dioritic facies consist of rocks formed by quartz diorite containing plagioclase (dominant mineral phase), quartz and K-feldspar. Biotite and amphibole are the dominant mafic minerals; and titanite, opaque minerals, allanite, zircon and apatite are the accessories. However, previous geological mapping work in the region also identified the presence of other lithostratigraphic units. These were described as gneisses and migmatites with undifferentiated amphibolite lenses related to the Caicó Complex (Paleoproterozoic) and metasedimentary rocks of the Seridó Group (Neoproterozoic) composed of paragneiss with calc-silicate lenses, muscovite quartzite and biotite schist (respectively, the Jucurutu formations, Equador and Seridó), the host rocks for the SRGP rocks. Leucomicrogranite and pegmatite dikes have also been identified, both related to the end of the Ediacaran magmatism and colluvial- eluvial and alluvial deposits related to Neogene and Quaternary, respectively. Lithogeochemical data on the SRGP granite facies, highlighted quite evolved rocks (SiO2 69% to 75%), rich in alkalis (Na2O+K2O ≥ 8.0%), depleted of MgO (≤ 0.45%), CaO (≤ 1.42%) and TiO2 (≤ 0.36%) and moderate levels of Fe2O3t (2.16 to 3.53%). They display transitional nature between metaluminous and peraluminous (predominance of the latter) with sub-alkaline/monzonitic (High K calcium-alkali) affinity. Harker diagrams show negative correlations for Fe2O3t, MgO, and CaO, indicating mafic and plagioclase fractionation. REE spectrum shows enrichment of LREE relative to heavy REE (LaN/YbN = 23.70 to 10.13), with negative anomaly in the Eu (Eu/Eu* = 0.70 to 0.23), suggesting fractionation or accumulation in the feldspars source (plagioclase). Data integration allows to correlate the SRGP rocks with those described as Calcium-Alkaline Suite of equigranular High K. The crystallization conditions of the SRGP rocks were determined from the integration of petrographic and lithogeochemical data. These data indicated intermediate to high conditions of ƒO2 (mineral paragenesis titanite + magnetite + quartz), parent magma saturated in H2O (early biotite crystallization), tardi-magmatic processes of fluids rich in ƒCO2, H2O and O2 causing part of the mineral assembly to change (plagioclase carbonation and saussuritization, biotite chloritization and opaques Sphenitization). Thermobarometrical conditions were estimated based on geochemical parameters (Zr and P2O5) and CIPW normative minerals, with results showing the liquidus minimum temperature of about800°C and the solidus temperature of approximately 700°C. The final/minimum crystallization pressure are suggested to be between 3 and 5 Kbar. The presence of zoned minerals (plagioclase and allanite) associated with lithogeochemical data in bi-log diagrams for Rb vs. Ba and Rb vs. Sr suggest the role of fractional crystallization as the dominant process in the magmatic evolution of SRGP. U-Pb Geochronological and Sm-Nd isotope studies indicated, respectively, the crystallization age of biotite monzogranite as 557 ± 13 Ma, with TDM model age of 2.36 Ga, and εNd value of -20.10 to the crystallization age, allowing to infer paleoproterozoic crustal source for the magma.

Petrologia e geocronologia U-Pb do plúton granítico Serra da Rajada, porção central do domínio Rio Piranhas - Seridó, Província Borborema, NE do Brasil

The ediacaran plutonic activity related to the Brasilian/Pan-African orogeny is one of the most important geological features in the Borborema Province, represented along its extension by numerous batholiths, stocks, and dikes.The object of this study, the Serra Rajada Granitic Pluton (SRGP), located in the central portion of the Piranhas-Seridó River Domain is an example of this activity. This pluton has been the subject of cartographic, petrographic, geochronological and lithogeochemical studies and its rocks were characterized by two facies. First, the granitic facies were described as monzogranites consisting of K-feldspar, plagioclase (oligoclase - An23-24%), quartz and biotite (main mafic) and opaque minerals such as titanite, allanite, apatite, and zircon as accessories. Alteration minerals are chlorite, white mica and carbonate. Second, the dioritic facies consist of rocks formed by quartz diorite containing plagioclase (dominant mineral phase), quartz and K-feldspar. Biotite and amphibole are the dominant mafic minerals; and titanite, opaque minerals, allanite, zircon and apatite are the accessories. However, previous geological mapping work in the region also identified the presence of other lithostratigraphic units. These were described as gneisses and migmatites with undifferentiated amphibolite lenses related to the Caicó Complex (Paleoproterozoic) and metasedimentary rocks of the Seridó Group (Neoproterozoic) composed of paragneiss with calc-silicate lenses, muscovite quartzite and biotite schist (respectively, the Jucurutu formations, Equador and Seridó), the host rocks for the SRGP rocks. Leucomicrogranite and pegmatite dikes have also been identified, both related to the end of the Ediacaran magmatism and colluvial- eluvial and alluvial deposits related to Neogene and Quaternary, respectively. Lithogeochemical data on the SRGP granite facies, highlighted quite evolved rocks (SiO2 69% to 75%), rich in alkalis (Na2O+K2O ≥ 8.0%), depleted of MgO (≤ 0.45%), CaO (≤ 1.42%) and TiO2 (≤ 0.36%) and moderate levels of Fe2O3t (2.16 to 3.53%). They display transitional nature between metaluminous and peraluminous (predominance of the latter) with sub-alkaline/monzonitic (High K calcium-alkali) affinity. Harker diagrams show negative correlations for Fe2O3t, MgO, and CaO, indicating mafic and plagioclase fractionation. REE spectrum shows enrichment of LREE relative to heavy REE (LaN/YbN = 23.70 to 10.13), with negative anomaly in the Eu (Eu/Eu* = 0.70 to 0.23), suggesting fractionation or accumulation in the feldspars source (plagioclase). Data integration allows to correlate the SRGP rocks with those described as Calcium-Alkaline Suite of equigranular High K. The crystallization conditions of the SRGP rocks were determined from the integration of petrographic and lithogeochemical data. These data indicated intermediate to high conditions of ƒO2 (mineral paragenesis titanite + magnetite + quartz), parent magma saturated in H2O (early biotite crystallization), tardi-magmatic processes of fluids rich in ƒCO2, H2O and O2 causing part of the mineral assembly to change (plagioclase carbonation and saussuritization, biotite chloritization and opaques Sphenitization). Thermobarometrical conditions were estimated based on geochemical parameters (Zr and P2O5) and CIPW normative minerals, with results showing the liquidus minimum temperature of about800°C and the solidus temperature of approximately 700°C. The final/minimum crystallization pressure are suggested to be between 3 and 5 Kbar. The presence of zoned minerals (plagioclase and allanite) associated with lithogeochemical data in bi-log diagrams for Rb vs. Ba and Rb vs. Sr suggest the role of fractional crystallization as the dominant process in the magmatic evolution of SRGP. U-Pb Geochronological and Sm-Nd isotope studies indicated, respectively, the crystallization age of biotite monzogranite as 557 ± 13 Ma, with TDM model age of 2.36 Ga, and εNd value of -20.10 to the crystallization age, allowing to infer paleoproterozoic crustal source for the magma.

Estudo de tensoativos na organofilização de argila bentonítica para uso em fluido de perfuração à base de óleo

Due to the great challenges encountered in drilling wells, there is a need to develop fluids with appropriated properties and able to meet all the requirements of drilling operations. The physicochemical and rheological properties must be carefully controlled so that a fluid can exercise all its functions. In perforations sensitive to contact with water and "offshore", it becomes necessary the use of oil based drilling fluids, but the bentonite clay cannot be used without a previous surface modification so that their surfaces become hydrophobic. Lately, the oil companies in Brazil use imported organoclays in the preparation of oil-based drilling fluids. The study aimed to modify a calcium clay to increase the affinity of the same organic phase of oil-based drilling fluids, applying three surfactants (OCS, CTAB and UTM 150) at different concentrations. The results indicated that the surfactants UTM 150 and CTAB showed better results compared to OCS. Considering the type of surfactant and concentration as variables used in the statistical analysis, the results indicated that only the surface tension and concentration of calcium oxide in response to organophilization process showed statistically significant effects. The organophilizated clay has potential for application in oil-based drilling fluids.

Vermiculitas tratadas quimicamente na obtenção de sólidos microporosos como precursores para híbridos inorgânico-orgânicos com aplicações adsortivas

ALVES, Ana paula Melo. Vermiculitas tratadas quimicamente na obtenção de sólidos microporosos como precursores para híbridos inorgânico-orgânicos com aplicações adsortivas. 2009. 124 f. Tese (Doutorado em Quimica) - Centro de Ciências Exatas e da Natureza, Universidade Federal da Paraíba, João Pessoa, PB, 2009.

Estratégias competitivas genéricas nas empresas comerciais varejistas de shopping centers: um estudo a partir do modelo de Porter

Os shoppingcenters são grandes instituições repletas de empresas quecompetem entre si formas de como melhor satisfazer as necessidade dosclientes. Sendo assim, cada empresa utiliza-se de estratégias com o intuito deatingir a maior parcela de compradores possíveis sem comprometer suaexistência e sua vantagem competitiva. Este estudo está baseado na teoria dasestratégias competitivas genéricas de Porter (1980; 1985) o qual estabeleceem seu modelo que todas as empresas existentes no mercado possuam pelomenos uma das três abordagens estratégicas (diferenciação, liderança emcusto e enfoque). Desta forma, este trabalho identifica de que maneira estádistribuída as estratégia competitiva nas empresas de shoppingcenters deNatal/RN. Primeiramente buscou-se levantar a relação das estratégias destasempresas com a teoria proposta por Porter (1985) e sua alocação de acordocom os agrupamentos resultantes da combinação das estratégias. Nestemomento, foram aplicados questionários com 89 gerentes de lojas de shoppingcenters que resultou após análises estatísticas em cinco agrupamentos comabordagens válidas. Em seguida foram selecionadas as empresas quepossuíam maior afinidade com cada agrupamento e que melhor representavamos resultados dispostos em cinco clustersacerca da teoria proposta e nestaamostragem foi aplicada entrevista com os gerentes. Os resultados obtidos com as entrevistas validaram a análise dos dados encontrados nosquestionários anteriormente aplicados estando em conformidade com aproposta teoria. Identificou-se também duas modalidades de empresas queutilizavam simultaneamente mais de uma estratégia competitiva (meio-termo),sendo uma dentro e outra fora do modelo proposto pelo autor. Embora omodelo das estratégias competitivas genéricas tenha sido instituído comaspectos voltados as organizações industriais, os resultados obtidos nestetrabalho validaram a aplicação desta teoria em empresas comercias deshopping centers que vem se adequando para obter resultados financeirosmais satisfatórios e melhor posicionamento em relação à concorrência

Uma Lectina D-lactose específica da esponja marinha Cinachyrella apion: purificação, caracterização e interação com Leishmania chagasi

Four different sponge species were screened using Ouchterlony agarose gel and immunodiffusion tests to identify cross-reactivity with the polyclonal antibody IgG anti-deglicosilated CvL, a lectin from Cliona varians. Crude extract from the sponge Cinachyrella apion showed cross-reactivity and also a strong haemmaglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglicosilated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA) gel filtration on a Superose 6 10 300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A and O erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide D-lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass determined by FPLC-AKTA gel filtration on a Superose 12 10 300 column and SDS gel electrophoresis was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was relatively heat- and pH-stable. Leishmania chagasi romastigotes were agglutinated by CaL, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the physiological defense roles of CaL and its possible use in the antibiosis of pathogenic protozoa

Purificação, caracterização e atividade bioinseticida de um inibidor de tripsina de sementes de Crotalaria pallida

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulphate fractionation, affinity chromatography on immobilized Trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named ITC, had Mr of 32.5 kDa by SDS-PAGE and was composed by two subunits with 27.7 and 5.6 kDa linked by disulphide bridges, a typical characteristic of Kunitz-Inhibitor family. ITC was stable until 50°C, and at 100°C its residual activity was of about 60%. Also, ITC was stable at pHs 2 to 12. The inhibition of trypsin by ITC was non-competitive, with a Ki of 8,8 x 10-7M. ITC inhibits weakly other serine proteinases such as chymotrypsin and elastase. The inhibition of papain (44% of inhibition), a cysteine proteinase was an indicative of the bi-functionality of ITC. In vitro assays against digestive proteinases from several Lepdoptera, Diptera and Coleoptera pests were made. ITC inhibited in 100% digestive enzymes of Ceratitis capitata (fruit fly), Spodoptera frugiperda and Alabama argillacea, the last one being a cotton pest. It also inhibited in 74.4% Callosobruchus maculatus (bean weevil) digestive enzymes, a Coleoptera pest. ITC, when added in artificial diet models, affected weakly the development of C. capitata larvae and it had a WD50 of 2.65% to C. maculatus larvae

Construção de modelos de interação in silico e in vitro do inibidor do tipo Kunitz de Adenanthera pavonina L. para as enzimas cisteínicas e serínicas

Serines proteinases inhibitors (PIs) are widely distributed in nature and are able to inhibit both in vitro and in vivo enzymatic activites. Seed PIs in than leguminous are classified in seven families, Bowman-Birk and Kunitz type families that most studied representing an important role in the first line of defense toward insects pests. Some Kunitz type inhibitors possess activities serine and cysteine for proteinases named bifunctional inhibitor, as ApTKI the inhibitor isolate from seed of Adenanthera pavonina. The A. pavonina inhibitor presenting the uncommon property and was used for interaction studies between proteinases serine (trypsin) and cysteine (papain). In order to determinate the in vitro interaction of ApTKI against enzymes inhibitor purification was carried cut by using chromatographic techniques and inhibition assays. The 3D model of the bifunctional inhibitor ApTKI was constructed SWISS-MODEL program by homology modeling using soybean trypsin inhibitor (STI, pdb:1ba7), as template which presented 40% of identity to A. pavonina inhibitor. Model quality was evaluated by PROCHECK program. Moreover in silico analyzes of formed complex between the enzymes and ApTKI was evaluated by HEX 4.5 program. In vitro results confirmed the inhibitory assays, where the inhibitor presented the ability to simultaneously inhibit trypsin and papain. The residues encountered in the inhibitor model of folder structural three-dimensional that make contact to enzymes target coud explain the specificity pattern against serine and cysteine proteinases

Atividade bioinseticida e mecanismo de ação de vicilinas de sementes Erythrina velutina sobre moscas-das frutas Ceratitis capitata

The fruit fly Ceratitis capitata is considered the most destructive pest of the world fruitculture. Many pest management practices, mainly based on agrochemicals, have been developed to allow the world-wide commerce of fruit. Solutions to decrease the use of synthetic insecticides in agriculture are based on the development of new target-specific compounds which cause less damage to the environment, especially vegetal proteins with insecticidal effects. The aim of this work was to evaluate the deleterious effect of a purified vicilin of E. velutina (EvV) seeds to C. capitata larvae and adult insects and to investigate the mechanisms involved in these effects. EvV was purified, characterized and its deleterious effect was tested in bioassay systems. EvV mechanism of action was determined by immunodetection techniques and fluorescence localization in chitin structures that are present in C. capitata digestory system. EvV is a glycoprotein with affinity to chitin. Its molecular weight, of 216,57 kDa, was determined by gel filtration chromatography in FPLC system. Using SDS-PAGE, it was possible to observe EvV dissociation in two main subunits of 54,8 and 50,8 kDa. When it was submitted to eletrophoresis in native conditions, EvV presented only one band of acid characteristic. The WD50 and LD50 values found in the bioassays were 0,13% and 0,14% (w/w), respectively for the larvae. EvV deleterious effects were related to the binding to chitin structures presented in peritrophic membrane and gut epithelial cells, associated with its low digestibility in C. capitata digestive tract. The results described herein are the first demonstration of the larvicidal effects of plant protein on C. capitata larvae. EvV may be part of the pest management programs, in the toxic bait composition, or an alternative in plant improvement program

Purificação, caracterização e análise da atividade bioinseticida, de um inibidor de tripsina em sementes de catanduva (Piptadenia moniliformis)

One Kunitz-type trypsin inhibitors (PmTI) was purified from Piptadenia moniliformis seeds, a tree of the sub-family Mimosoideae, by TCA precipitation, affinity chromatography on immobilized trypsin-Sepharose, DEAE cellulose (ion exchange) and Superose 12 (molecular exclusion) column FPLC/AKTA. The inhibitor has Mr of 25 kDa by SDS-PAGE and chromatography molecular exclusion. The N-terminal sequence of this inhibitor showed high homology with other family Kunitz inhibitors. This also stable variations in temperature and pH and showed a small decrease in its activity when incubated with DDT in the concentration of 100mM for 120 minutes. The inhibition of trypsin by PmTI was competitive, with Ki of 1.57 x10-11 M. The activity of trypsin was effectively inhibited by percentage of inhibition of 100%, among enzymes tested, was not detected inhibition for the bromelain, was weak inhibitor of pancreatic elastase (3.17% of inhibition) and inhibited by 76.42% elastase of neutrophils, and inhibited in a moderate, chymotrypsin and papain with percentage of inhibition of 42.96% and 23.10% respectively. In vitro assays against digestive proteinases from Lepidoptera, Diptera and Coleoptera pests were carried out. Several degrees of inhibition were found. For Anthonomus grandis and Ceratitis capitata the inhibition was 89.93% and 70.52%, respectively, and the enzymes of Zabrotes subfasciatus and Callosobruchus maculatus were inhibited by 5.96% and 9.41%, respectively, and the enzymes of Plodia. interpunctella and Castnia licus were inhibited by 59.94% and 23.67, respectively. In vivo assays, was observed reduction in the development of larvae in 4rd instar of C. capitata, when PmTI was added to the artificial diet, getting WD50 and LD50 of 0.30% and 0.33%, respectively. These results suggest that this inhibitor could be a strong candidate to plant management programs cross transgenic

Efeito inseticida de vicilinas isoladas de sementes de erythrina velutina em condições de semi-campo para moscas das frutas (ceratitis capitata)

Chitin-binding vicilins from legume seeds (Erythrina velutina. Canavalia ensiformes and Phaseolus vulgares) were isolated by ammonium sulfate followed by affinity chromatography on a chitin column. Effect of these vicilins on female adults of Ceratitis capitata was examined by bioassay and in a semi-field assay model. Mechanism of action of the vicilins was determined by in vivo digestibility and chitin affinity. Among the tested vicilins, E. velutina when added to diet caused strong effect on mortality at 10% dose. This insecticidal property was tested in a semi-field assay which showed the same effect observed in laboratory conditions, where doses of 10% and 15% were lethal to female adults of C. capitata. These deleterious effects were not only associated to the binding to chitin structures present in peritrophic membrane, but principally to its low digestibility in the C. capitata digestive tract. This fact was confirmed because chiting binding proteins as WGA and the other tested vicilins were not toxic to female adults of C. capitata due susceptibility of these proteins to digestive enzymes of the insects. By other side EvV was more resistant to digestive enzymes, causing deleterious effects on female adults of C. capitata. These results showed that EvV may be part of the pest management programs or an alternative in plant improvement program in the population control of this fruticulture pest

Purificação, caracterização e avaliação da atividade antiproliferativa de um inibidor de quimotripsina tipo kunitz de semente de Eryhrina velutina

A chymotrypsin inhibitor was purified from Erythrina velutina seeds by ammonium sulphate fractionation, affinities chromatographies on Trypsin-Sepharose, Quimotrypsin-Sepharose and reversed phase C-18 FPLC/AKTA system. The inhibitor, named EvCI, shown molecular mass of 17 kDa, as determined by SDSPAGE. 2D-PAGE showed four isoinhibitors with pI values of 4,42, 4,63, 4,83 and 5,06, with molecular mass of 17 kDa each. The aminoacid sequence of EvCI was determined by MALDI-TOF-MS and showed a high similarity with other Kunitz-type inhibitor of Erythrina variegata. EvCI competitively inhibited chymotrypsin, with Ki of 4 x10-8 M, but did not inhibited trypsin, pancreatic elastase, bromelain and papain. The inhibitory activity of EvCI was stable over wide pH and temperature ranges. In the presence of DTT 100 mM for 120 min, EvCI lost 50 % of activity. Cytotoxicity was studied in HeLa, MDA, HepG2, K562 and PC3 cells after 72-h incubation period. EvCl inhibited HeLa cells growth with an IC50 value of 50 μg/ml. Subsequent studies in HeLa cells analysis of cell death by annexin V/PI double-staining and cell cycle, using flow cytometry. The results provide evidence for a cytostatic activity of EvCl and support further studies on potential application of this inhibitors as an antiproliferative agent in combined therapy against cervical cancer

Avaliação das atividades anti-inflamatória, anticoagulante e antiproliferativa do inibidor de quimotripsina das sementes de erythrina velutina (EvCI)

Studies indicate that several components were isolated from medicinal plants, which have antibacterial, antifungal, antitumor and anti-inflammatory properties. Sepsis is characterized by a systemic inflammation which leads to the production of inflammatory mediators exacerbated by excessive activation of inflammatory cells and disseminated intravascular coagulation (DIC), in which the human neutrophil elastase plays an important role in its pathogenesis. Several epidemiological studies suggest that components of plants, especially legumes, can play a beneficial role in reducing the incidence of different cancers. A chymotrypsin inhibitor of Kunitz (Varela, 2010) was purified from seeds of Erythrina velutina (Mulungu) by fractionation with ammonium sulfate, affinity chromatography on Trypsin-Sepharose, Chymotrypsin-Sepharose and ion exchange chromatography on Resource Q 1 ml (GE Healthcare) in system FPLC / AKTA. The inhibitor, called EvCI, had a molecular mass of 17 kDa determined by SDS-PAGE. The purified protein was able to inhibit human neutrophil elastase (HNE), with an IC50 of 3.12 nM. The EvCI was able to inhibit both pathways of HNE release stimulated by PAF and fMLP (75.6% and 65% respectively). The inhibitor also inhibited leukocyte migration in septic mice about 87% and prolonged the time of coagulation and inhibition factor Xa. EvCI showed neither hemolytic activity nor cytotoxicity. EvCI showed a selective antiproliferative effect to HepG2 cell lines with IC50 of 0.5 micrograms per milliliter. These results suggest EvCI as a molecule antagonist of PAF / fMLP and a potential use in fighting inflammation related disorders, disseminated intravascular coagulation (DIC) and cancer

Clonagem e expressão do gene que codifica o inibidor de quimotripsina de Erythrina velutina WILLD. - caracterização e avaliação de seu potencial farmacológico

Proteinases are enzymes distributed widely founded in several organisms and perform many different functions, from maintaining homeostasis to the worsening of some diseases such as cancer, autoimmune diseases and infections. The proteins responsible of controlling the action of these enzymes are the inhibitors, that are classified based on their target proteases and are founded since simple organisms, such as bacteria, to higher organisms, such as larger plants and mammals. Plant proteinase inhibitors act by reducing or inactivating the activity of target proteases, thus, these proteins have been studied as potential tools in the treatment of diseases related to protease activities. In this context, an inhibitor of chymotrypsin from Erythrina velutina, called EvCI was previously purified and it was observed that this protein plays in vitro anticoagulant activity and anti-inflammatory activity in in vivo model. Aiming to reduce the environmental impact caused by the purification EvCI in high amounts and to facilitate the process of obtaining this protein, the recombinant chymotrypsin inhibitor from Eryhrina velutina was produced after cloning and expression in Escherichia coli. The bacteria were grown in LB medium and after induction of the expression this material was subjected to procedures for cell lysis and the product was applied on Nickel-affinity column. The proteins adsorbed were digested by thrombin and applied on Chymotrypsin-Sepharose affinity column, obtaining the purified inhibitor, named recEvCI. After electrophoresis, the recombinant inhibitor showed an approximately molecular mass of 17 kDa, and reduced the chymotrypsin and elastase activities in vitro. The recombinant inhibitor was sequenced and was found similar amino acids residues when compared to other inhibitors deposited in the database, with some modifications. recEvCI showed high stability under pH variations and reducing conditions, maintaining its activity around 80%. This protein increased the blood coagulation time in vitro by acting on the intrinsic pathway and did not show cytotoxicity against strains of mouse 3T3 fibroblasts and RAW 264.7 macrophages. recEvCI showed microbicide activity related to release of nitric oxide and consequently the activation of macrophages, futhermore having proinflammatory effects assessed by increased release of TNF-α. These results indicate that recEvCI can be biotechnologically used as a new tool in the control of coagulation-related diseases as well as can be an activating agent of the immune system in immunosuppressed individuals