Isolamento de uma quitinase extraída do cefalotórax do camarão marinho Litopenaeus schmitti (BURKENROAD, 1936) : avaliação de suas atividades antimicrobiana e larvicida

Chitinases are enzymes involved in degradation of chitin and are present in a range of organisms, including those that do not contain chitin, such as bacteria, viruses, plants and animals, and play important physiological and ecological roles. Chitin is hydrolyzed by a chitinolytic system classified as: endo-chitinases, exo-chitinases and N-acetyl-b-D-glucosaminidases. In this study a Litochitinase1 extracted from the cephalotorax of the shrimp Litopenaeus Schmitt was purified 987.32 times using ionexchange chromatography DEAE-Biogel and molecular exclusion Sephacryl S-200. These enzyme presented a molecular mass of about 28.5 kDa. The results, after kinetic assay with the Litochitinase1 using as substrate p-nitrophenyl-N-acetyl-b-Dglucosaminideo, showed apparent Km of 0.51 mM, optimal activity at pH ranging from 5.0 to 6.0, optimum temperature at 55°C and stability when pre-incubated at temperatures of 25, 37, 45, 50 and 55°C. The enzyme showed a range of stability at pH 4.0 to 5.5. HgCl2 inhibited Litochitinase1 while MgCl2 enhances its activity. Antimicrobial tests showed that Litochitinase1 present activity against gram-negative bacterium Escherichia coli in the 800 μg/mL concentration. The larvicidal activity against Aedes aegypti was investigated using crude extracts, F-III (50-80%) and Litochitinase1 at 24 and 48 hours. The results showed larvicidal activity in all these samples with EC50 values of 6.59 mg/mL for crude extract, 5.36 mg/mL for F-III and 0.71 mg/mL for Litochitinase1 at 24 hours and 3.22 and 0.49 mg/mL for the F-III and Litochitinase1 at 48 hours, respectively. Other experiments confirmed the presence of chitin in the midgut of Aedes aegypti larvae, which may be suffering the action of Litochitinase1 killing the larvae, but also the absence of contaminating proteins as serine proteinase inhibitors and lectins in the crude extract, F-III and Litochitinase1, indicating that the death of the larvae is by action of the Litochitinase1. We also observed that the enzymes extracted from intestinal homogenate of the larvae no have activity on Litochitinase1. These results indicate that the enzyme can be used as an alternative to control of infections caused by Escherichia coli and reducing the infestation of the mosquito vector of dengue.

Perfil imunológico associado à susceptibilidade, resistência e cura na infecção por Leishmania ( Leishmania ) infantum

Visceral Leishmaniasis (VL) is endemic in Brazil and the northeast region had the highest incidence of the disease , despite, in the last 30 years, it has spread to all geographic regions of the country. Leishmania infantum is the m ain etiological agent of VL in Latin America, Europe and North Africa. However, not all infected individuals develop the disease; in fact, the majority present spontaneous re solution of infection without symptoms. The evaluation of the immunological profil e has been mostly conducted stimulating, with Leishmania spp. antigen, peripheral blood mononuclear cells isolated from subjects with VL. These studies showed that VL patients had an inhibition of both, lymphocyte proliferation and proinflammatory response to Leishmania spp. antigen. Our study aimed to evaluate the immune response in active LV, cured post treatment and asymptomatic infection. To reach this aim, we analyzed immunophenotypic features related to activation, Treg and memory lymphocytes, by flow cytometry, as well as, evaluation of cytokine production, in ex vivo or in whole blood culture. In active VL volunteers, a longitu dinal study was conducted with reassessment at 4 and 14 months after clinical cure. The control group included individuals th at live d in endemic region and were either Positive Control, consisting of individuals with positive anti - L eishmania spp. serology and/or positive PCR for Leishmania  spp. and Negative Control composed by individuals with negative anti - Leishmania antibodie s serology and negative PCR for Leishmania . During VL, CD4 lymphocytes showed greater activation and memory profile s and were the major source of cytokines in culture when compared to CD8 lymphocytes , and these were not Leishmania specific. There were act ivated lymphocytes during VL (CD4 + CD69 + :4.9%) when compared to control groups, Positive (CD4 + CD69 + :1.96%, p=0.0045) and Negative (CD4 + CD69 + :1.35%, p=0.006), on the other hand, this was non - specific activation. The lymphocyte activation profile remain ed el evated even 14 months post treatmen t. A fter clinical cure , the activation was Leishmania specific (CD4 + CD25 + absence of SLA: 8.4%, and presence of SLA: 10.7% p=0.0279). CD8 + CD25 + lymphocytes were able to produce Leishmania specific IFN - γ in both, Positive Controls (absence of SLA 5.2% and presence of SLA: 9.5%, p=0.0391) and Cured 4 month (absence of SLA: 3.9%; presence of SLA: 10.7% p=0.0098). Whole blood culture cells, of VL patients, were able to produce IFN - γ, by SLA stimulation (absence of SLA: 28.0 pg ∕mL, and presence: 44.3 pg∕mL p=0.0020) as well as recovered groups (absence of SLA 2.3 pg∕mL and presence of SLA 139.8 pg∕mL, p=0.0005). However, the high level of IL - 10 seem ed to inhibit pro - inflammatory activity of IFN - γ and TNF - α during symptomatic dis ease . Unlike other pro - inflammatory cytokines, active VL group d id not produce Leishmania specific IL - 2 (absence of SLA 2.4 pg∕mL and presence of SLA: 2.6 pg∕mL). Based on these data we conclude that the restoration of lymphocyte activation and decreased i n IL - 10 Leishmania specific production were related to a protective immune profile.

Isolamento de uma quitinase extraída do cefalotórax do camarão marinho Litopenaeus schmitti (BURKENROAD, 1936) : avaliação de suas atividades antimicrobiana e larvicida

Chitinases are enzymes involved in degradation of chitin and are present in a range of organisms, including those that do not contain chitin, such as bacteria, viruses, plants and animals, and play important physiological and ecological roles. Chitin is hydrolyzed by a chitinolytic system classified as: endo-chitinases, exo-chitinases and N-acetyl-b-D-glucosaminidases. In this study a Litochitinase1 extracted from the cephalotorax of the shrimp Litopenaeus Schmitt was purified 987.32 times using ionexchange chromatography DEAE-Biogel and molecular exclusion Sephacryl S-200. These enzyme presented a molecular mass of about 28.5 kDa. The results, after kinetic assay with the Litochitinase1 using as substrate p-nitrophenyl-N-acetyl-b-Dglucosaminideo, showed apparent Km of 0.51 mM, optimal activity at pH ranging from 5.0 to 6.0, optimum temperature at 55°C and stability when pre-incubated at temperatures of 25, 37, 45, 50 and 55°C. The enzyme showed a range of stability at pH 4.0 to 5.5. HgCl2 inhibited Litochitinase1 while MgCl2 enhances its activity. Antimicrobial tests showed that Litochitinase1 present activity against gram-negative bacterium Escherichia coli in the 800 μg/mL concentration. The larvicidal activity against Aedes aegypti was investigated using crude extracts, F-III (50-80%) and Litochitinase1 at 24 and 48 hours. The results showed larvicidal activity in all these samples with EC50 values of 6.59 mg/mL for crude extract, 5.36 mg/mL for F-III and 0.71 mg/mL for Litochitinase1 at 24 hours and 3.22 and 0.49 mg/mL for the F-III and Litochitinase1 at 48 hours, respectively. Other experiments confirmed the presence of chitin in the midgut of Aedes aegypti larvae, which may be suffering the action of Litochitinase1 killing the larvae, but also the absence of contaminating proteins as serine proteinase inhibitors and lectins in the crude extract, F-III and Litochitinase1, indicating that the death of the larvae is by action of the Litochitinase1. We also observed that the enzymes extracted from intestinal homogenate of the larvae no have activity on Litochitinase1. These results indicate that the enzyme can be used as an alternative to control of infections caused by Escherichia coli and reducing the infestation of the mosquito vector of dengue.

Nas asas de Ícaro: uma análise da Pedagogia Freinet do 2º ao 5º ano do Ensino Fundamental na perspectiva da ação docente continuada

Célestin Freinet was one of the most memorable educators of the twentieth century. He presented some educational alternatives that had the objective of stimulating the construction of pedagogic actions to promote the social development of the student based upon a work centered on the free expression as the way to self-structure the knowledge. With a permanent proposal of research based on the enquiry-based learning, Freinet set human capacity (cognitive, social-affective, psychometrical) taking cooperation in consideration on the processes of knowledge construction. Based on this referential, this present work has the objective to show Freinet s pedagogy in a continuous teaching action from 2nd to 5th grade focusing the teacher s discourse and also the educative practice of the students of a city public school in Natal. The observed work revealed the teacher s discourse and educative experience and delineated the students development while immersed into Freinet s educational practice. By virtue of the nature and specificity of the theme that guided our research, we chose a qualitative approach to it, as a way of conducting ourselves during our investigative process. We observed and analyzed the method that was used to conduct the activities in the classroom, as well as the ways of expression that the children used through drawings, words (oral text), or through writing. We highlight, among the written texts, the individual and collective texts, and also letters and notes, which during some moments served as a base to express a diversity of languages. The results, after a analyzing the research data, point towards a teaching practice that favors the construction of a significant learning process through the grasping of the school environment, on which all these factors are based: society, knowledge acquisition, abilities, attitudes and values. This learning process also strengthens human solidarity bonds and mutual tolerance, on which the student s social life is seated.

Pedagogia Freinet: a construção de uma práxis em turmas de 5ª a 8ª séries

Ce travail que nous venons de présenter apport une systématisation sur la viabilité de la praxis de l´éducateur français Célestin Freinet (1896-1996). Il s´agit d´une étude qualificative, recherche-action, développée dans une école coopérative l´ École Freinet à Natal/RN Brésil, auprès des élèves du 5ème et 8ème séries de l´Enseignement Fondamental. Le problème central que nous a amené à cette recherche, a été le fait de vérifier les progressions dans la production scientifique et dans les pratiques des enseignants qui ont à la charge l´Éducation Infantine et les premières séries de l´Enseignement Fondamental. Tout cela en tenant compte en concevoir l´élève en tant que sujet actif de son apprentissage et coresponsable à l´organisation du travail scolaire. Toutefois, qaund-il s´agit des quatre dernières séries de l´Enseignement Fondamental (5ème au 8ème séries), les propositions progressistes sont prises comme quelque chose pratiquement impossible d´en mettre en marche. Pour attenuer la lacune théorique sur le sujet, nous avons offert un texte significatif pour les enseignants d´une façon générale, les chercheurs de l´académie et pour l´écolechamp de la recherché. Tout cela par le fait de ne pas donner de priorité à une technique ou à un principe, en particulier. Cette pédagogie est discutée autant que possible dans son contexte global et aussi sur le comment s´est manifestée dans sa práxis . Nous avons mis en relief les aspects importants pour le fonctionnement de la salle de classe, aussi que les estratégies utilisées pour la construction, communication et documentation des connaissances L´intervention nous a permis de construire l´objet par la réflexion en action, à partir des données obtenues pendant les observations, les entretiens, les enterviews, les moments d´études et de la quête des textes écrits. L´analyse a été faite à la lumière de la methodologie comprehensive (Kaufmann, 1996), entrelacée au materiau empirique et à la théorie. Cette forme d´analyse nous a permis d´obtenir la logique dans l´ensemble comme nous crayons que doit caracteriser la recherche aux Sciences Humaines et Sociales. Les considérations finales mettent l´accent d´un côté sur l´efficacité de la pédagogie Freinet, pour orienter le processus de l´enseignement apprentissage, en tous les dégres de l´enseignement et de l´autre côté nous montre la nécessité urgente de l´équipe de l´école champ de la recherche, de qualifier sa pratique à partir des références théoriques-pratiques de la pédagogie Freinet

A Proficiência escritora em matemática trabalhada nos livros didáticos dos 4º e 5º anos do ensino fundamental

Universidade Federal do Rio Grande do Norte

A (não) assunção da responsabilidade enunciativa em livros didáticos de língua portuguesa dos 4º e 5º anos

Portuguese textbook has been the focus of many investigations, nevertheless, the theme still has much to be discussed, reflected and broadened. This conviction mobilized us to perform this research, seeking to answer three questions: (1) how does the author of the textbook induce the student to give his opinion in comprehension questions of text in Portuguese textbook of 4th and 5thgrade? Which enunciative links are used by the author of the textbook in comprehension questions of text concerning to the assumption of enunciative responsability? And (3) Which enunciative links are used by the author of the textbook in comprehension questions of text concerning to the non assumption of enunciative responsability? In this direction, we define as objective to id entify, describe, analyze and interpret how the (non) assumption of enunciative responsibility materializes itself in Portuguese textbooks. The theme is echoed in the guidelines of the Parâmetros Curriculares Nacionais PCN (1998; 2001), who assume that all education committed to citizenship need to create conditions in order the student can develop his discursive competence" (BRAZIL, 1998, p. 23). This assertion of PCN (1998; 2001) is closely related to our research object, and therefore, it has corroborated to the accomplishment of this study. So, we analyze the comprehension questions of the texts that are elaborated by the authors of Portuguese textbooks of the 4th and 5th grade, used at public schools in the city of Natal-RN in 2010. As a theoretical basis, we have considered the postulates of Textual Analysis of Discourses ATD and Enunciative Linguistics. Our research was mainly based on the studies of Adam (2011), Nølke (1994; 2001; 2006; 2009; 2013), Nølke, Fløttum e Norén (2004) Rabatel (2004; 2005; 2008; 2009), Guentchéva (1994; 1996). Our data analysis revealed that the authors of Portuguese textbooks explore the reading comprehension, inducing students to answer questions that may be categorized like: (1) induction to the assumption of enunciative responsibility, (2) induction for non assumption the enunciative responsibility, (3) orientation for the study of vocabulary and grammar and (4) orientation of extra themes. The results from the comparison of the books of 4th and 5th grade of the two analyzed collections, we observed through the links of (non) responsability Nølke (1994; 2001; 2006; 2009; 2013), Nølke, Fløttum e Norén (2004), which in 79% of the questions, the authors induce the decoding of a content objectively inscribed in the text. In this sense, the notion of understanding a text is compromised, since it is limited to copying contents or transcription exercises, failing to consider the interactive use of language, or rather, failing to expand the student's knowledge in the (re)construction of the text of the meanings. This shows that there is a lack in the deal with the text that includes the textual discursive resources in the reading activities in Portuguese textbooks. We recall, in this direction, the works of Marcuschi (2005), Antunes (2003, 2005), and Bunzen Rojo (2008), among others authors who contributed greatly to orient the choice of Portuguese textbooks. Finally, we believe that the study about the enunciative responsibility phenomenon in Portuguese texto oks offers, above all, instruments in order to the interlocutors identify the elements present in the enunciation and the effects that these elements bring to the (re) construction of the meanings in texts that they read and write in the classroom.