Ant��genos da forma amastigota de Leishmania chagasi identificados por dupla varredura da biblioteca de cDNA

Control of human visceral leishmaniasis in endemic regions is hampered in part by the lack of knowledge with respect of the role reservoirs and vector. In addition, there is not yet an understanding of how non-symptomatic subclinical infection might influence the maintenance of infection in a particular locality. Of worrisome is the limited accessibility to medical care in places with emerging drug resistance. There is still no available protective vaccine either for humans or other reservoirs. Leishmania species are protozoa that express multiple antigens which are recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the causative agent of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T and T-dependent B cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second step screen for their ability to cause proliferation and IFN-γ responses of T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The clones encoded part of the coding sequence of glutamine synthetase, transitional endoplasmic reticulum ATPase, elongation factor 1γ, kinesin K-39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these protein Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines against Leishmania

Associa����o entre fatores nutricionais e a resposta frente �� infec����o por Leishmania chagasi

Leishmania chagasi infection presents a wide spectrum of clinical outcomes, ranging from asymptomatic self resolving infection to disease, visceral leishmaniasis (VL). The exact mechanisms that lead the evolution of infection to disease are not understood. It is believed that malnutrition is a risk factor associated with VL development, although there are few human studies in the area. We aimed to assess the nutritional factors associated with the response to L. chagasi infection in Rio Grande do Norte. The study was conducted from December 2006 to January 2008. 149 children were assessed: 20 active VL cases, 33 children with VL history, 40 DTH+ asymptomatic children and 56 DTH-. Nutritional status was assessed using z scores for Weight/Age, Weight/Height, Height/Age, Body Mass Index (BMI), and mid-upper arm circumference/height (MUAC/height). Vitamin A status was determined by serum retinol concentrations and the modified-relative-dose-esponse test (MRDR). Breastfeeding time and birth weight were also evaluated. VL children presented compromised nutritional status when compared to the other groups using BMI and MUAC/age, with means -1,53 �� 1,10 and -1,48 �� 1,28 z scores, respectively (ANOVA, p < 0,05). VL children also showed lower vitamin A levels: 43% presented serum retinol < 20 ��g/dL and 15% MRDR > 0,060. Birth weight was inverserly associated with the risk to belong the VL group (&#946; = -0,00; OR = 0,84; 95% CI 0,73 - 0,99; p = 0,047), whereas more breastfeeding time was directly associated with the risk to belong to the DTH+ group (&#946; = 0,02; OD = 1,16; 95% CI 1,01 - 1,33; p = 0,036). The nutritional variables evaluated were associated with the response to the L. chagasi infection, with malnutrition and compromised vitamin A status as markers of children who present with VL. Higher birth weight was associated with protection to disease, and higher breastfeeding time was associated with increased likelihood of an asymptomatic infection. The results show that modifiable nutritional aspects in the study population are associated with the response to the L. chagasi infection

Fatores gen��ticos e ��tnicos relacionados �� evolu����o da infec����o por Leishmania infantum

Coordena����o de Aperfei��oamento de Pessoal de N��vel Superior

Determinantes envolvidos na resposta imune celular humana �� infec����o por Leishmania infantum chagasi

Visceral leishmaniasis (VL) is a disease caused by protozoa of the Leishmania donovani complex, whose infection has clinical spectrum ranging from asymptomatic infection to active disease characterized by fever, cachexia, hepatosplenomegaly, and immunosuppression. The healing or protective immunity require an antigen-specific type 1. The Montenegro skin test (DTH) has been interpreted as a marker of protective immunity. However, there is no known correlation between the DTH response to type 1 and DTH and immunity of type 1 are maintained in the long term. Thus, a longitudinal study of 8 years, nested in a cohort family held in Brazil, documented the status of DTH and cytokine production by peripheral blood mononuclear cells in response to antigen-specific stimulation. This study was the interdisciplinary approach of physicians, biochemists, nutritionists, veterinary medicine, biology and statistics. The results show that 46.2% of subjects were analyzed DTH positive at baseline. The prevalence of positive and DTH induration size increased with age (p = 0.0021). 15.7% of individuals positive DTH "retro-converted" the negative and 50.4% (64) of individuals negative DTH became positive. The size of DTH induration was correlated significantly with the antigen-induced production of IFN-&#947; (r = 0.6186, p = 0.0001). IL-6 was secreted at higher levels in peripheral blood mononuclear cells of individuals who "retro-converted" DTH positive to negative than individuals who remained stable DTH status (p = 0.005). Thus, IFN-&#947; produced by peripheral blood mononuclear cells, may be a surrogate marker for protective immunity instead of the DTH response. In addition, differences in innate immune response may determine whether individuals maintain or eliminate the infection by Leishmania infantum chagasi in asymptomatic patients

Aspectos cl��nicos e imunol��gicos da infec����o por Leishmania Infantum Chagasi em c��es do Rio Grande do Norte / Paula Vivianne Souza de Queiroz. Natal/RN

Conselho Nacional de Desenvolvimento Cient��fico e Tecnol��gico

IL-10 na patog��nese da infec����o por Leishmania infantum

Conselho Nacional de Desenvolvimento Cient��fico e Tecnol��gico

Estudo do Cultivo de dois clones de Escherichia coli recombinantes (eIF, LACK) para a Express��o de Ant��genos da Leishmania chagasi

With advent of the technology of the recombinant DNA, the recombinant protein expression becomes an important tool in the studies of the structure, function and identification of new proteins, mainly with therapeutical purposes. The Escherichia coli has been procarioto predominant in the studies of genetic engineering due to wealth of information regarding its metabolism. Despite the expressivo advance of the studies of molecular biology and the immunology of the infections, it does not exist, currently, no prophylactic drug capable to prevent calazar. Of this form, it exists a great necessity of specific antigen identification for the vaccine development and kits for disgnostic against the visceral Leishmaniose. In this context, this work objectified to study the recombinant antigen expression of the Leishmania chagasi during the culture of Escherichia coli in shaker. A first set of assays was carried through with the objective of if knowing the kinetic behavior of the growth of two clones recombinant proteins (eIF, LACK) in two different compositions of culture medium (2xTY, TB) supplemented by antibiotics, without IPTG addition. In the second stage of the assays, the procedure of induction for IPTG was carried through, in order to verify the influence of the composition of the ways tested in the expression them recombinant proteins. On the basis of the gotten results, can be observed that the high complexity of culture medium favored the kinetic one of growth of clones recombinant (eIF, LACK), however, to if to deal with the assays submitted to the procedure of induction for IPTG, the raised complexity of culture medium did not favor the expression of recombinant proteins. On the other hand, they had been gotten resulted positive for all clones recombinant (eIF, LACK) tested, confirmed through the eletrofor��tico profile

Avalia����o de dois clones de Escherichia coli recombinante quanto ao crescimento e express��o de ant��genos de Leishmania chagasi (kmp11 e P36)

Visceral leishmaniosis caused by Leishmania chagasi, also known as calazar, presented, in the period from 1990 to 2005, tax of incidence in Brazil varying between 1 and 3 cases for 100 000 inhabitants. The Northeast region that up to the year of 2000 contributed with almost 90% of the registered cases is reducing his participation in the current decade, reaching 56% in 2005. Conventional leishmaniasis treatment is costly and it shows high toxicity, demanding more research for alternative treatments, with special interest in development of vaccines and diagnosis kits which include production of recombinant antigens by host cells. Escherichia coli has been the microorganism most studied and used as a host for recombinant protein production. Therefore, the aim of this work was to study the influence of induction on cellular growth and to verify the type of Leishmania chagasi antigens expression (intra or extracellular) during two recombinant E. coli clones (kmp11 and P36) cultivation in rotary incubator (shaker) using three different media (2xTY, TB, FASS+EL). For that, tests were carried out using conditions established in the literature for E. coli (37��C and 200 rpm) and media supplemented with antibiotics to guarantee that only competent cells grows. First, tests were carried out without induction in order to verify the two microorganisms kinetic behavior (growth and substrate consumption) in different media. Next, the induction was carried out through the addition of IPTG (1mM as final concentration), at the first hour of cultivation. It was observed that protein expression were intracellular for all clones and media tested, however the highest level of expression was clearly observed by the electrophoresis band density (intensity) for 2xTY medium and kmp11 protein. Although it contains the lowest substrate concentration, consequently, a reduced cellular concentration when compared to other media, it appeared that this medium and clone combination is the most indicated for recombinant protein production. Therefore, the objective of this work was achieved, since the interested proteins were produced. Consequently, this result motivates new studies for production optimization using different cultivation strategies

Influ��ncia das condi����es de cultivo na produ����o de ant��genos recombinantes de Leishmania i. chagasi utilizando Escherichia coli M15 cultivada em incubador rotativo e biorreator

Escherichia coli has been one of the most widely used hosts in recombinant protein production, in both laboratory and industrial scale since the advent of recombinant DNA technology. Despite the substantial progress of studies on the molecular biology and immunology of infections, there is currently no medication-based prophylaxis capable of preventing leishmaniasis. As such, there is a great need to identify specific antigens for the development of vaccines and diagnostic kits against visceral leishmaniasis. Thus, the primary goal of the present study is to assess the influence of cultivation conditions on the production of Leishmania chagasi antigens, carried out in a rotating incubator and bioreactor. To that end, several assays were conducted to evaluate the kinetic behavior of antigens (648, 503) of Leishmania. i. chagasi in two different compositions of media (2xTY, TB), with and without an inducer. In order to improve expression, assays were performed in a benchtop bioreactor using the best conditions obtained in a rotating incubator, in addition to assessing the influence of stirring speed. Results show that high complexity of the cultivation medium favored kinetic growth of clones (648, 503). However, in assays submitted to induction by IPTG, this elevated complexity did not promote the expression of recombinant proteins. Expression of antigens 648 and 503 exhibited behavior associated with growth and, in terms of location, proteins 648 and 503 are intracellularly stored. Lactose may be the most adequate inducer in protein expression, when considering factors, cost, toxicity and stability. Elevated stirring may increase cell growth in clone 53, although it may not result in high concentrations for the protein of interest. On the other hand, positive results were obtained for all recombinant clones (648, 503) tested, confirmed by the electrophoretic profile

Recorte dos produtos educacionais em hist��ria no ensino da matem��tica e em did��tica da matem��tica a partir das disserta����es e teses defendidas no brasil entre 1990-2010

This work has the main purpose of conducting a survey of educational products present in dissertations and doctoral theses focused on the use of history in mathematics teaching and Didactics of mathematics with a French foundation produced in graduate programs in the strict sense of the Brazil between 1990 and 2010, the areas of Education, Mathematics Education, school of Natural Sciences and Mathematics and related areas, according to the research proposal of Mendes (2010). Our interest was to select the products that present concrete proposals for educational activities that can be used in the classroom of Basic Education and Training of Teachers of Mathematics. The research was implemented through a bibliographic study documents the Bank of dissertations and theses from CAPES, libraries and archives of some Postgraduate programs in the country who focus their studies on the subject object of this research, besides the Brazilian Digital Library Theses and Dissertations (BDBTD). From this survey we selected works that present educational products materialized in blocks of activities based on the use of teaching history of mathematics to the classroom as well as the sequence of activities based on the Teaching of Mathematics. In possession of material, produce a CD-ROM containing the selected activities, in order to help support the work of teachers regarding the use of these activities, as a supplementary material to textbooks in their math classes

Perfil imunol��gico associado �� susceptibilidade, resist��ncia e cura na infec����o 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 - �� 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 - ��, 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 - �� and TNF - �� 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.

Cen��rio da produ����o acad��mica em hist��ria da matem��tica no ensino de matem��tica: uma an��lise reflexiva das teses e disserta����es (1990-2010)

This research aims to make a reflective analysis about the academic production originated in the stricto sensu post graduation programs in the country, produced in the period of 1990 to 2010, in the field of History of Mathematics, especifically on works about the History of Mathematics in Mathematics education and that present pedagogical proposals that make use of the History of Mathematics in order to teach Mathematics. Defending the thesis that the researches on mathematics education with goals turned to the use of didactic proposals related to the history of mathematic that take in consideration the coherency between epistemological aspects inherent to mathematics history and anthological elements materialized on the conceptions of mathematics and mathematics history and of apprenticeship (implicitly or explicitly exposed) may originate significant contribution to the field of history of mathematics on education. Among these, nine were Master���s Degree dissertations and five PHD���s theses. The reflective analysis was accomplished from two matrixes; one from theoretical nature and the other, ontologic nature, elaborated from the pretexts of Sanches Gamboa, about the epistemological analysis from academic production in the field of Mathematics Education and the following theoretical perspectives in the field of History of Mathematics Education, that are: linear evolutionary theory, structural construtivist operative, evolutionary discontinuous, historical and socialcultural investigation and the use of activities estimulating the usage of verbal and nonverbal expressions. These perspectives were based on the works of Miguel and Miorim, Mendes and Radford. As results, we have detected some established dissonances between the categories related to theoretical and ontologic levels and the pedagogical proposal presented in these researches. On the other hand, we have discovered works that are able to establish consonances between the theoretical and ontological elements and the presented pedagogical proposal. These works carry significative contributions to the field of History of Mathematics applied to Mathematics pedagogical practice, inclusively presenting significative theoretical elements to the production of knowledge recognized as scientific in the Mathematics field

Recupera����o e purifica����o do ant��geno 503 de Leishmania i. chagasi expresso em E. coli e remo����o de endotoxina utilizando adsor����o em leito expandido

The growing interest and applications of biotechnology products have increased the development of new processes for recovery and purification of proteins. The expanded bed adsorption (EBA) has emerged as a promising technique for this purpose. It combines into one operation the steps of clarification, concentration and purification of the target molecule. Hence, the method reduces the time and the cost of operation. In this context, this thesis aim was to evaluate the recovery and purification of 503 antigen of Leishmania i. chagasi expressed in E. coli M15 and endotoxin removal by EBA. In the first step of this study, batch experiments were carried out using two experimental designs to define the optimal adsorption and elution conditions of 503 antigen onto Streamline chelating resin. For adsorption assays, using expanded bed, it was used a column of 2.6 cm in diameter by 30.0 cm in height coupled to a peristaltic pump. In the second step of study, the removal of endotoxin during antigen recovery process was evaluated employing the non-ionic surfactant Triton X-114 in the washing step ALE. In the third step, we sought developing a mathematical model able to predict the 503 antigen breakthrough curves in expanded mode. The experimental design results to adsorption showed the pH 8.0 and the NaCl concentration of 2.4 M as the optimum adsorption condition. In the second design, the only significant factor for elution was the concentration of imidazole, which was taken at 600 mM. The adsorption isotherm of the 503 antigen showed a good fit to the Langmuir model (R = 0.98) and values for qmax (maximum adsorption capacity) and Kd (equilibrium constant) estimated were 1.95 mg/g and 0.34 mg/mL, respectively. Purification tests directly from unclarified feedstock showed a recovery of 59.2% of the target protein and a purification factor of 6.0. The addition of the non-ionic surfactant Triton X-114 to the washing step of EBA led to high levels (> 99%) of LPS removal initially present in the samples for all conditions tested. The mathematical model obtained to describe the 503 antigen breakthrough curves in Streamline Chelanting resin in expanded mode showed a good fit for both parameter estimation and validation steps. The validated model was used to optimize the efficiencies, achieving maximum values of the process and of the column efficiencies of 89.2% and 75.9%, respectively. Therefore, EBA is an efficient alternative for the recovery of the target protein and removal of endotoxin from an E. coli unclarified feedstock in just one step.

Resumo de disserta����es e teses: a estrutura composicional �� luz da An��lise Textual dos Discursos

This doctoral dissertationproposes the description, interpretation and analysis of the compositional structure of thesis and dissertation abstracts, with regard to the linguistic mechanisms that evidence text zones of different typological sequences, such as those of the text plan. Along these lines, the research problem was developed from the notion of compositional structure (sequences and text plans), as one of the levels or plans of text analysis, according to the theoretical framework proposed by Jean-Michel Adam (2011a). The main objective of this study was to recognize how the compositional structure, of thesis and dissertation abstracts, is achieved, with respect to text units and the global organization of this text category. The hypothesis posed in this research posits that specific informational text composition categories of abstracts are necessary to process the representation of the original text and the way in which it makes its meaning. Subsequently, this study is based on the theoretical and methodological framework of Text Linguistics (TL) and, above all, Textual Discourse Analysis (TDA), as we endeavor to understand the organizational structure of abstracts from both a linguistic and textual perspective. This structure involves the text plan of abstracts, with respect to their communicative purpose, i.e, the sharing of scientific information in its standard textual form. Thus, the development of this study, from a theoretical and methodological perspective, is based on the theoretical and descriptive premises from TDA (ADAM, 2011a, 2012; PASSEGGI et al., 2010), and also from TL (BEAUGRANDE; DRESSLER, (2012 [1981]); COSERIU; LAMAS (2010); MARCUSCHI, 2009 [1983]; F��VERO; KOCH, 1994;KOCH, 2006; BENTES, 2004; BENTES; LEITE, 2010), within the field of text studies. The methodology of this study relies on empirical, documental research, which is qualitative, and adopts a descriptive and interpretive approach. From the empirical perspective, our objective is to understand the problems pertaining to the textual composition of abstracts, aiming to elucidate them in light of the theoretical and methodological framework previously mentioned. The corpus of the analysis is comprised of seven abstracts designated for systematic data collection. These texts, written between 2004 and 2011,were selected from Master���s theses and Doctoral dissertations in their electronic version, from the graduate program at the Federal University of Rio Grande do Norte. A thorough review of the literature reveals a clear fluctuation in the terminology of the concept, ���abstract���. The results of the analysis revealed that the abstracts, which comprise the corpus of analysis in this study, in general, present typological heterogeneity, while the text plan remains fixed. Finally, the new knowledge gained in this research contributes both to the understanding of the compositional structure of abstracts as well as their production.

Variabilidade gen��tica de Leishmania spp. circulantes entre seres humanos e c��es e infec����o de flebotom��neos em ��reas end��micas para as leishmanioses no Rio Grande do Norte

Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector���s species and the parasite���s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.