Biopolímeros como suporte para inoculantes

A utilização de inoculantes em culturas de leguminosas é uma prática conhecida e empregada há longo tempo e o substrato mais utilizado até o momento tem sido a turfa. Diversos tipos de formulações inoculantes que existem no mercado visam oferecer uma alternativa ao emprego da turfa, porém muitas apresentam baixa capacidade para manter a sobrevivência e eficiência dos rizóbios. Este trabalho avaliou a utilização de catorze misturas diferentes de polímeros naturais e/ou sintéticos como suportes para inoculantes, visando a produção de inoculantes comerciais para soja, e a capacidade de manter a sobrevivência e preservar as características de infectividade e de efetividade das estirpes SEMIA 587 de Bradyrhizobium elkanii e SEMIA 5079 de Bradyrhizobium japonicum. Avaliou-se a sobrevivência dos rizóbios nas formulações armazenadas, a capacidade de aderência das formulações em sementes, a sobrevivência dos rizóbios em sementes a 40 ºC e a eficiência dos inoculantes em plantas de soja, cultivadas em vasos com solo, submetidas a estresse hídrico e térmico. Observou-se que as misturas contendo goma xantana, jataí e guar, tanto nas formulações em gel como líquidas, podem ser usadas como veículo para inoculantes proporcionando maior proteção aos rizóbios contra as condições de dessecação e temperatura. Todos os inoculantes mantiveram a sobrevivência da população de rizóbios durante um ano de armazenamento. Os rizóbios das formulações G5, L1 e L7, que continham goma arábica, foram afetados pelo estresse hídrico e pelas temperaturas elevadas, reduzindo o número de nódulos formados. Dentre as formulações líquidas, as que continham xantana e glicerol (L2) e com adição de polivinilpirrolidona (L5) e a formulação que continha xantana, carboximetilcelulose e polivinilpirrolidona (L6), foram as mais promissoras para a formulação de inoculantes para soja.

Reading comprehension in primary school: textbooks, curriculum and assessment

This study describes the reading comprehension processes present in the most widely sold textbooks at the fourth grade level in Portugal and discusses how they compare to international assessments of reading literacy. We adopted the Progress of International Reading Literacy Study framework to categorize the questions in the textbooks. Our analyses revealed that they focus heavily on the retrieval of explicitly stated information to the detriment of higher level comprehension skills. Portuguese fourth grade textbooks rarely challenge students to make connections between their knowledge and the ideas in the texts and to adopt a critical and evaluative reading stance. This is in sharp contrast to what students are asked to do in the Progress of International Reading Literacy Study, conducted every five years since 2001, and it may help explain the poor results Portuguese students have in national assessment and in PISA. The findings are discussed in light of the curriculum frameworks currently adopted in Portugal and suggestions are made as to how we can improve reading literacy achievement.

Enhancing comprehension in low achievement 9th grades: a quasi-experiment study

This article describes the application of a Comprehension Strategies Programme (CSP) designed to improve the reading comprehension of students with learning difficulties. The programme was implemented with 102 ninth grade students from Portuguese public schools with low achievement on a particular subject: Portuguese. An evaluation was done on the effects of the programme on reading comprehension and on school achievement. The Experimental Group presented significant improvements when compared to the Control Group (n=108) on the dimensions considered. This indicates that the CSP an bring benefits to low achieving students who have Portuguese as a mother tongue.

Síntese e caracterização de nanopartículas FeOx/Au/Ag em multicamada núcleo-concha

Nesta dissertação, apresenta-se o trabalho realizado no decorrer do segundo ano do Mestrado em Bioquímica Aplicada. Prepararam-se nanopartículas metálicas através da redução química de sais metálicos em solução. Obtiveram-se soluções coloidais monometálicas de Au, Ag e FeOx e bimetálicas de Au/Ag, Ag/Au, FeOx/Au e FeOx/Ag seguindo ou adaptando métodos publicados na literatura. Numa primeira fase foram sintetizadas nanopartículas monometálicas de prata e ouro utilizando-se β-D-glucose, borohidreto de sódio e β-ciclodextrina como agente redutor dos iões metálicos. Seguidamente, por co-redução de uma mistura de iões prepararam-se ligas de nanopartículas de prata e ouro e por redução sucessiva de Ag e Au sintetizaram-se nanopartículas com uma estrutura núcleo-concha. As nanopartículas de FeOx foram preparadas por co-precipitação de Fe (III) e Fe (II). O revestimento com ouro foi conseguido através da redução com citrato de sódio e para a deposição de prata utilizou-se o ácido ascórbico. As soluções coloidais preparadas foram caracterizadas através de estudos de espetroscopia do UV-vis, tendo sido registados os máximos de absorvância característicos do ouro e da prata e os desvios esperados para o caso das nanopartículas núcleo-concha. As análises por dispersão dinâmica de luz permitiram auferir o tamanho das nanopartículas, eventual aglomeração e, portanto, permitiram a apreciação da estabilidade dos coloides. Com o intuito de confirmar a formação de estruturas em camada núcleo-concha foi feita a caracterização das amostras por microscopia eletrónica de transmissão e espetroscopia de raios-X de energia dispersiva. Alguns dos espetros obtidos confirmam o sucesso na preparação de uma estrutura em multicamada. Finalmente, demonstrou-se a biocompatibilidade de algumas amostras preparadas através da realização de estudos de citotoxicidade na linha celular fibroblástica NIH 3T3.

Gene delivery using dendrimer/pDNA complexes immobilized in electrospun fibers using the layer-by-layer technique

Tissue engineering is an important branch of regenerative medicine that uses cells, materials (scaffolds), and suitable biochemical and physicochemical factors to improve or replace specific biological functions. In particular, the control of cell behavior (namely, of cell adhesion, proliferation and differentiation) is a key aspect for the design of successful therapeutical approaches. In this study, poly(lactic-co-glycolic acid) (PLGA) fiber mats were prepared using the electrospinning technology (the fiber diameters were in the micrometer range). Furthermore, the electrospun fiber mats thus formed were functionalized using the layer-by- layer (LbL) technique with chitosan and alginate (natural and biodegradable polyelectrolytes having opposite charges) as a mean for the immobilization of pDNA/dendrimer complexes. The polyelectrolyte multilayer deposition was confirmed by fluorescence spectroscopy using fluorescent-labeled polyelectrolytes. The electrospun fiber mats coated with chitosan and alginate were successfully loaded with complexes of pDNA and poly(amidoamine) (PAMAM) dendrimers (generation 5) and were able of releasing them in a controlled manner along time. In addition, these mats supported the adhesion and proliferation of NIH 3T3 cells and of human mesenchymal stem cells (hMSCs) in their surface. Transfection experiments using a pDNA encoding for luciferase showed the ability of the electrospun fiber mats to efficiently serve as gene delivery systems. When a pDNA encoding for bone morphogenetic protein-2 (BMP-2) was used, the osteoblastic differentiation of hMSCs cultured on the surface of the mats was promoted. Taken together, the results revealed that merging the electrospinning technique with the LbL technique, can be a suitable methodology for the creation of biological active matrices for bone tissue engineering.

Low generation triazine-based dendrimers-synthesis, characterzation and in vitro biological activity

In the present study, two low generation triazine-based dendrimers, G1.0(Cl)4 dendrimer and G1.5(OH)8 dendrimer, were synthesized and their cytotoxicity were tested by using the NIH 3T3 and the A2780 cell lines. In the synthesis process of the G1.0(Cl)4 dendrimer, cyanuric chloride (CAC) which has high reactivity chlorine atom was connected to the terminal of triethylene glycol (TEG) via nucleophilic substitution by controlling temperature. The prepared G1.0(Cl)4 dendrimer was purified by silica gel column chromatography. Then the four chlorine atoms in the G1.0(Cl)4 dendrimer were substituted by diethanolamine (DEA) to give dendrimer with the hydroxyl terminal group G1.5(OH)8. The starting materials, CAC, G1.0(Cl)4 dendrimer and G1.5(OH)8 dendrimer were analyzed by one-dimensional NMR, FTIR and MS techniques. The two dendrimers, G1.0(Cl)4 and G1.5(OH)8, showed perfect stability in the air environment at room temperature. However, G1.0(Cl)4 is not soluble in water while the G1.5(OH)8 dendrimer is a water soluble compound. Furthermore, cell biological evaluation at the studied concentrations showed that the CAC, as well as the prepared G1.0(Cl)4 and G1.5(OH)8 dendrimers, have no cytotoxicity towards the NIH 3T3 and A2780 cell lines.

Atividades Biológicas De Xilana De Sabugo De Milho

The corn cob is an agricultural by-product still little used, this in part due to the low knowledge of the biotechnological potential of their molecules. Xylan from corn cobs (XSM) is a polysaccharide present in greater quantity in the structure of plant and its biotechnology potential is little known. This study aimed to the extraction, chemical characterization and evaluation of biological activities of xylan from corn cobs. To this end, corncobs were cleaned, cut, dried and crushed, resulting in flour. This was subjected to a methodology that combines the use of alkaline conditions with waves of ultrasound. After methanol precipitation, centrifugation and drying was obtained a yield of 40% (g/g flour). Chemical analysis indicated a high percentage of polysaccharides in the sample (60%) and low contamination by protein (0.4%) and phenolic compounds (> 0.01%). Analysis of monosaccharide composition indicated the presence of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar ratio 50:20:15:10:2.5:2.5. The presence of xylan in the sample was confirmed by nuclear magnetic resonance (¹H and ¹³C) and infrared spectroscopy (IR). Tests were conducted to evaluate the antioxidant potential of XSM. This showed a total antioxidant capacity of 48.45 EAA/g sample. However, did not show scavenging activity of superoxide and hydroxyl radical and also reducing power. But, showing a high capacity chelating iron ions with 70% with about 2 mg/mL. The ability to XSM to influence cell proliferation in culture was also evaluated. This polymer did not influence the proliferation of normal fibroblast cells (3T3), however, decreased the rate of proliferation of tumor cells (HeLa) in a dose-dependent, reaching an inhibition of about 50% with a concentration around 2 mg/mL. Analyzing proteins related to cell death, by immunoblotting, XSM increases the amount of Bax, Bcl-2 decrease, increase cytochrome c and AIF, and reduce pro-caspase-3, indicating the induction of cell death induced apoptosis dependent and independent of caspase. XSM did not show anticoagulant activity in the PT test. However, the test of activated partial thromboplastin time (aPTT), XSM increased clotting time at about 5 times with 600 μg of sample compared with the negative control. The presence of sulfate on the XSM was discarded by agarose gel electrophoresis and IR. After carboxyl-reduction of XSM the anticoagulant activity decreased dramatically. The data of this study demonstrate that XSM has potential as antioxidant, antiproliferative and anticoagulant compound. Future studies to characterize these activities of XSM will help to increase knowledge about this molecule extracted from corn and allow their use in functional foods, pharmaceuticals and chemical industries.

Purificação, caracterização e efeitos imunomodulatório e antiproliferativo de uma lectina do fungo Clavaria cristata (Holmsk.) Pers

A 140,0 kDa lectin was purified and characterized from the mushroom Clavaria cristata. The purification procedures from the crude extract of the mushroom comprised gel filtration chromatography on Sephacryl s200 and ion exchange on Resource Q column. The purified lectin agglutinated all types of human erythrocytes with preference for trypsinized type O erythrocytes. The haemagglutinating activity is dependent of Ca 2+ ions and was strongly inhibited by the glycoprotein bovine submaxillary mucin (BSM) up to the concentration of 0, 125 mg/mL. The C. cristata lectin (CcL) was stable in the pH range of 2,5-11,5 and termostable up to 80 °C. CcL molecular mass determined by gel filtration on a Superose 6 10 300 column was approximately 140,3 kDa. SDS polyacrilamide gel electrophoresis revealed a single band with a molecular mass of approximately 14,5 kDa, when the lectin was heated at 100 ⁰C in the presence or absence of β-mercaptoethanol. CcL induced activation of murine peritoneal macrophages in vitro resulting in the release of nitric oxide (NO), reaching the maximum production at 24 h. In experimental paw oedema model in mice, CcL showed proinflammatory activity being able to induce oedema formation. Cell viability of HepG2, MDA 435 e 3T3 cell lines was examined after 72 h of incubation with CcL in different concentrations (0,5-50 μg/mL). CcL inhibited HepG2 cells growth with an IC50 value of 50 μg/mL. In the present work, the observed immunomodulatory and antiproliferative effects indicate CcL as a possible immunomodulator compound, interfering in the macrophages immune response, taking possible anti-parasitic, anti-tumoral effects or diagnostic and/or therapeutic

Caracterização físico-química e efeitos de frações polissacarídicas da alga amansia multifida na coagulação, inflamação, radicais livres e viabilidade celular

Galactans are polysaccharides sulfated present in the cell wall of red algae. Carrageenans are galactans well known in the food industry as gelling polysaccharides and for induce inflammatory process in rodents as animal model. The extraction of polysaccharides from A. multifida has been carried out by proteolysis and precipitation in different volumes of acetone, which produced three fractions (F1, F2, and FT). Chemical and physical analyses revealed that these fractions are sulfated galactan predominantly. Results of the antioxidant activity assays showed that all of these fractions have antioxidant activity and that was associated with sulfate content of the analysis of reducing power and total antioxidant capacity. However, these fractions were not effective against lipid peroxidation. The fraction FT presented higher activity on the APTT test at 200 μg (> 240 s). The assessment of the hemolytic activity showed that the FT fraction has the best activity, increasing lyses by the complement system to 42.3% (50 μg) (p< 0,001). The fraction FT showed the best yield, anticoagulant and hemolytic activity between the three fractions and therefore it was choose for the in vivo studies. The Inflammation assessment using the FT fraction (50 mg / kg MB) showed that the cellular migration and the IL-6 production increased 670.1% (p< 0,001) and 531.8% (p< 0,001), respectively. These results confirmed its use as an inflammation inducer in animal model. Cytotoxicity assay results showed that all fractions have toxic effects on 3T3 and HeLa cells after exposition of 48 hours, except when 100 μg for both F1 and FT were used. These results arise the discussion whether these polysaccharides it should be used as additive in foods, cosmetics and medicines.

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

Avaliação do efeito do composto tipo heparina isolado do caranguejo Chaceon fenneri na hemostasia e na morte celular

Heparin is a pharmaceutical animal widely used in medicine due to its potent anticoagulant effect. Furthermore, it has the ability to inhibit the proliferation, invasion and adhesion of cancer cells to vascular endothelium. However, its clinical applicability can be compromised by side effects such as bleeding. Thus, the search for natural compounds with low bleeding risk and possible therapeutic applicability has been targeted by several research groups. From this perspective, this study aims to evaluate the hemorrhagic and anticoagulant activities and citotoxic effect for different tumor cell lines (HeLa, B16-F10, HepG2, HS-5,) and fibroblast cells (3T3) of the Heparin-like from the crab Chaceon fenneri (HEP-like). The HEP-like was purified after proteolysis, ion-exchange chromatography, fractionation with acetone and characterized by electrophoresis (agarose gel) and enzymatic degradation. Hep-like showed eletroforetic behavior similar to mammalian heparin, and high trisulfated /Nacetylated disaccharides ratio. In addition, HEP-like presented low in vitro anticoagulant activity using aPTT and a minor hemorrhagic effect when compared to mammalian heparin. Furthermore, the HEP-like showed significant cytotoxic effect (p<0.001) on HeLa, HepG2 and B16-F10 tumor cells with IC50 values of 1000 ug/mL, after incubation for 72 hours. To assess the influence of heparin-like on the cell cycle in HeLa cells, analysis was performed by flow cytometry. The results of this analysis showed that HEP-like influence on the cell cycle increasing S phase and decreasing phase G2. Thus, these properties of HEP-like make these compounds potential therapeutic agents

Atividade antioxidante e antiproliferativa de extratos de folhas de Plukenetia volubilis L. (EUPHORBIACEAE)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Extração, caracterização estrutural e atividades farmacológicas do olginato obtido da alga marrom Lobophora variegata (Lamouroux) Oliveira Filho, 1977

The alginic acid or alginates are acidic polysaccharides found in brown seaweed widely used in food, cosmetic, medical and pharmaceutical industry. This paper proposes the extraction, chemical characterization and verification of the pharmacological activities of brown seaweed variegata Lobophora . The alginate was extracted from the seaweed Lobophora variegata and part was sulphated for comparative purposes. The native extract showed 42% total sugar, 65% uronic acid, 0,36 % protein and 0% of sulfate, while the sulfate showed 39% , 60%, 0.36% and 27,92 % respectively. The presence of a sulfate group may be observed by the metachromasia with toluidine blue in electrophoresis system and characteristic vibration 1262,34 cm-1 in infrared spectroscopy connections assigned to S = O. We observed the formation of films and beads of native alginate, where more concentrated solution 6% resulted in a thicker and more consistent film. Native alginate showed proliferative activity at concentrations (25 and 50 mcg), (50 mg) and (100 mg) in 3T3 cell line in 24h, 48h and 72h, respectively , as the sulfated (100 mg) in 24 . Also showed antiproliferative or cytotoxic activity in HeLa cells of strain, (25 and 100 mg), (25 and 100 mg) and (25, 50 and 100 mg), to native, now for the sulfate concentrations (100 mg) in 24 (25, 50 and 100 mg) in 48 hours, and (50 and 100 mg ) 72h. For their antioxidant activity, the sulfated alginates have better total antioxidant activity reaching 29 % of the native activity while 7.5 % of activity . For the hydroxyl radical AS showed high inhibition ( between 77-83 % ) in concentrations, but the AN surpassed these numbers in the order of 78-92 % inhibition. The reducing power of AN and AS ranged between 39-82 % . In the method of ferric chelation NA reached 100 % chelating while the AS remained at a plateau oscillating 6.5%. However, in this study , we found alginates with promising pharmacological activities, to use in various industries as an antioxidant / anti-tumor compound

Produção e caracterização de quitooligossacarídeos produzidos pelo fungo Metarhizium anisopliae e avaliação da citotoxicidade em células tumorais

Chitosan is a natural polymer, biodegradable, nontoxic, high molecular weight derived from marine animals, insects and microorganisms. Oligomers of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) have interesting biological activities, including antitumor effects, antimicrobial activity, antioxidant and others. The alternative proposed by this work was to study the viability of producing chitooligosaccharides using a crude enzymes extract produced by the fungus Metarhizium anisopliae. Hydrolysis of chitosan was carried out at different times, from 10 to 60 minutes to produce chitooligosaccharides with detection and quantification performed by High Performace Liquid Chromatography (HPLC). The evaluation of cytotoxicity of chitosan oligomers was carried out in tumor cells (HepG2 and HeLa) and non-tumor (3T3). The cells were treated for 72 hours with the oligomers and cell viability investigated using the method of MTT. The production of chitosan oligomers was higher for 10 minutes of hydrolysis, with pentamers concentration of 0.15 mg/mL, but the hexamers, the molecules showing greater interest in biological properties, were observed only with 30 minutes of hydrolysis with a concentration of 0.004 mg/mL. A study to evaluate the biological activities of COS including cytotoxicity in tumor and normal cells and various tests in vitro antioxidant activity of pure chitosan oligomers and the mixture of oligomers produced by the crude enzyme was performed. Moreover, the compound with the highest cytotoxicity among the oligomers was pure glucosamine, with IC50 values of 0.30; 0.49; 0.44 mg/mL for HepG2 cells, HeLa and 3T3, respectively. Superoxide anion scavenging was the mainly antioxidant activity showed by the COS and oligomers. This activity was also depending on the oligomer composition in the chitosan hydrolysates. The oligomers produced by hydrolysis for 20 minutes was analyzed for the ability to inhibit tumor cells showing inhibition of proliferation only in HeLa cells, did not show any effect in HepG2 cells and fibroblast cells (3T3)