Estudo dos peptídeos pertencentes à família ICK presentes no veneno das aranhas do gênero Loxosceles

Orientador : Prof. Dr. Silvio Sanches VeigaCoorientador : Profª. Drª. Olga Meiri ChaimTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 26/02/2015Inclui referências : f. 102-115Área de concentraçãoResumo: O conteúdo total do veneno das aranhas do gênero Loxosceles permanece ainda desconhecido, entretanto, muitos estudos têm mostrado que se constitui uma mistura complexa de compostos biologicamente ativos. Por meio de análises eletroforéticas, observa-se a predominância de moléculas de baixa massa molecular (3-45 kDa), enquanto moléculas de alta massa molecular são menos abundantes. Os venenos de aranhas estão funcionalmente relacionados à defesa contra predadores e também à paralisia e captura de presas, especialmente insetos. As aranhas desenvolveram um arsenal de moléculas inseticidas, resultando em uma biblioteca combinatória de peptídeos que tem sido aprimorada durante sua evolução. Comumente, tais peptídeos consistem em moléculas de cadeia única com massa molecular variando de 3 a 10 kDa, ricos em resíduos de cisteína, os quais estabelecem pontes dissulfeto intramoleculares características. Essas pontes se organizam em um motivo estrutural característico denominado "nó de cistina inibidor" ou ICK (Inhibitor Cystine Knot) e, por isso, os peptídeos que o contém são denominados peptídeo ICK ou notinas ("knottins"). Recentemente, análises do transcriptoma da glândula de veneno de L. intermedia (GREMSKI et al., 2010) revelaram ESTs com similaridade a peptídeos ICK previamente descritas como LiTx (De CASTRO et al., 2004). Sequências relacionadas à LiTx3, por exemplo, foram as mais abundantes no transcriptoma de L. intermedia, representando aproximadamente 13,9% de todas as ESTs obtidas e compreendendo 32% dos mRNAs codificantes de toxinas; as sequências relativas aos demais grupos de peptídeos ICK, LiTx1, LiTx2 e LiTx4 representaram 6,2%, 11,4% e 3,8% de todos os transcritos codificantes de toxinas, respectivamente. Devido a alta proporção de sequências codificantes para peptídeos ICK os objetivos deste trabalho foram o rastreamento de sequências codificantes de peptídeos ICK em outras duas aranhas do gênero (Loxosceles gaucho e Loxosceles laeta), bem como a obtenção de um peptídeo ICK semelhante à LiTx3 de forma recombinante em Pichia pastoris e sua caracterização biológica. A partir do RNA total extraído das glândulas de veneno de L. laeta e L. gaucho, procedeu-se o rastreamento de sequências relacionadas a peptídeos ICK; sequências codificantes para todos os grupos de peptídeos ICK já descritos (LiTx1-4) foram encontradas, algumas revelando sutis diferenças na sua estrutura primária, outras revelando divergências importantes como a não presença de sequências consenso para modificações pós-traducionais importantes para a atividade biológica dos mesmos. Quanto à produção do peptídeo recombinante, várias formas foram obtidas; entretanto, análises de SDS-PAGE e western blotting mostraram que os peptídeos obtidos ou não apresentavam a conformação nativa ou apresentavam glicosilação indesejada. A atividade biológica desses peptídeos foi testada in vivo em insetos e in vitro em cultura de células, contudo nenhum efeito tóxico pode ser comprovado. As sequências encontradas em L. laeta e L. gaucho representam potenciais moléculas a serem exploradas do ponto de vista biotecnológico, ao passo que o peptídeo recombinante estudado deve ser expresso em outros modelos com intuito de obtê-lo em conformação adequada e, assim, comprovar sua atividade biológica. Palavras-chave: Peptídeo ICK, notinas, Loxosceles, veneno, Pichia pastoris.Abstract: The whole content of Loxosceles spider venom still remains unknown, but several studies have shown that is a complex mixture of biologically active and inactive components. By eletrophoretic analysis, the predominance of low molecular mass molecules (3-45kDa) can be observed, while high molecular mass ones are less abundant. Spider venoms are functionally related to defense against predators as well as to paralyze and capture a natural prey, especially insects. Spiders had developed an arsenal of insecticidal molecules, resulting in a combinatorial peptide library of peptides that has been improved during evolution. Commonly, such peptides consist in single chain molecules ranging between 3- 10 kDa and are rich in cystein residues, which form intramolecular disulfide bridges. These bridges establish a structural motif "Inhibitor Cystine Knot" (ICK), then, these peptides are named ICK peptides or "knottins". Recently, a transcriptome analysis of L. intermedia venomous gland (GREMSKI et al., 2010) has revealed ESTs with similarity to ICK peptides previously described as LiTx (De CASTRO et al., 2004). LiTx3-related sequences were the most abundant in the L. intermedia transcriptome representing about 13.9% of all ESTs obtained and comprise 32% of toxin-encoding messengers; the sequences related to the other groups of ICK peptides, LiTx1, LiTx2 e LiTx4, represented 6,2%, 11,4% and 3,8% of all EST coding for toxins, respectively. Due to the high proportion of sequences encoding ICK peptides verified by the transcriptome analyses, the present study aimed screening ESTs related to these peptides in other two Loxosceles species (L. gaucho and L. laeta), as well as the obtainment of an ICK recombinant peptide with high similarity to LiTx3 in Pichia pastoris and its biological activity characterization. From total RNA purified from the venom glands of L. gaucho and L. laeta, it was performed the screening of ICK peptides sequences. ESTs coding for all groups of ICK peptides already described were found (LiTx1-4), some of them revealed subtle differences in their primary structures while others showed important divergences, for example, the absence of consensus sequences for posttranslational modifications that are essential for biological activities. The recombinant peptide was produced in different forms, however, SDS-PAGE and western blotting analyses indicated that they were not properly folded or presented unwanted glycosylation. The biological activity of the recombinant peptide was tested in insects microinjection's assays and in vitro cultivated cells, nevertheless no toxic effects were proven. The sequences identified form L. laeta and L. gaucho RNA represent potential molecules to be biotechnologically explored, whereas the studied recombinant peptide must be expressed in other heterologous expressions models in order to obtain it in the native conformation and, thus, verify its biological activity. Keywords: ICK peptides, knottins, Loxosceles, venom, toxins, Pichia pastoris. recombinant peptide was tested in insects microinjection's assays and in vitro cultivated cells, nevertheless no toxic effects were proven. The sequences identified form L. laeta and L. gaucho RNA represent potential molecules to be biotechnologically explored, whereas the studied recombinant peptide must be expressed in other heterologous expressions models in order to obtain it in the native conformation and, thus, verify its biological activity. Keywords: ICK peptides, knottins, Loxosceles, venom, toxins, Pichia pastoris

Clonagem e expressão heteróloga de um peptídeo da família das notinas presente no veneno da aranha marrom (loxosceles intermedia)

Orientador : Prof. Dr. Silvio Sanches VeigaCoorientadora: Profa. Dra. Olga Meiri ChaimDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 28/02/2011Bibliografia: fls. 74-86Resumo: O conteúdo total do veneno das aranhas do gênero Loxosceles permanece ainda desconhecido, entretanto, muitos estudos têm mostrado que se constitui uma mistura complexa de compostos biologicamente ativos. Por meio de análises eletroforéticas, observa-se a predominância de moléculas de baixa massa molecular (3-45 kDa), enquanto moléculas de alta massa molecular são menos abundantes. Os venenos de aranhas estão funcionalmente relacionados à defesa contra predadores e também à paralisia e captura de presas, especialmente insetos. As aranhas desenvolveram um arsenal de moléculas inseticidas, resultando em uma biblioteca combinatória de peptídeos que tem sido aprimorada durante sua evolução. Comumente, tais peptídeos consistem em moléculas de cadeia única com massa molecular variando de 3 a 10 kDa, ricos em resíduos de cisteína, os quais estabelecem pontes dissulfeto intramoleculares características. Essas pontes se organizam em um motivo estrutural característico denominado "nó de cistina inibidor" ou ICK (Inhibitor Cystine Knot) e, por isso, os peptídeos que o contém são denominados notinas ("knottins"). Recentemente, análises do transcriptoma da glândula de veneno de L. intermedia (GREMSKI et al., 2010) revelaram ESTs com similaridade a notinas previamente descritas como LiTx (De CASTRO et al., 2004). Sequências relacionadas à LiTx3 foram as mais abundantes no transcriptoma de L. intermedia, representando aproximadamente 13,9% de todas as ESTs obtidas e compreendendo 32% dos mRNAs codificantes de toxinas. Devido a alta proporção de sequências codificantes para peptídeos semelhantes a LiTx3, o presente estudo teve como objetivo a clonagem e expressão de um peptídeo dessa família. Primers foram desenhados para a realização das técnicas de 3' e 5' RACE, o qual teve como resultado a obtenção da sequência nucleotídica completa do cDNA correspondente ao peptídeo em estudo. A sequência do propeptídeo associado ao peptídeo maduro foi amplificada e modificada por PCR com primers contendo sítios de restrição para as enzimas XhoI e BamHI e inseridos no vetor de expressão pET-14b. A construção foi transformada em cepa de E. coli AD494(DE3). Um teste de expressão foi realizado para determinar as condições ótimas de expressão do peptídeo. Verificou-se que a indução com 0,5mM de IPTG, por 3 horas e meia, a 30oC era a melhor condição de expressão. A partir desse resultado, uma expressão em larga escala foi realizada, obtendo-se o peptídeo recombinante na fração solúvel, após cromatografia de afinidade em resina de Ni2+-NTA. A diversidade de peptídeos presentes no veneno de L. intermedia representa uma fonte importante de possíveis novas ferramentas moleculares com aplicação farmacológica e/ou biotecnológica.Abstract: The whole content of Loxosceles spider venom still remains unknown, but several studies have shown that is a complex mixture of biologically active and inactive components. By eletrophoretic analysis, the predominance of low molecular mass molecules (3-45kDa) can be observed, while high molecular mass ones are less abundant. Spider venoms are functionally related to defense against predators as well as to paralyze and capture a natural prey, especially insects. Spiders had developed an arsenal of insecticidal molecules, resulting in a combinatorial peptide library of peptides that has been improved during evolution. Commonly, such peptides consist in single chain molecules ranging between 3-10 kDa and are rich in cystein residues, which form intramolecular disulfide bridges. These bridges establish a structural motif "Inhibitor Cystine Knot" (ICK), then, these peptides are named "knottins". Recently, a transcriptome analysis of L. intermedia venomous gland (GREMSKI et al., 2010) has revealed ESTs with similarity to knottins previously described as LiTx (De CASTRO et al., 2004). LiTx3-related sequences were the most abundant in the L. intermedia transcriptome representing about 13.9% of all ESTs obtained and comprise 32% of toxin-encoding messengers. Due to the high proportion of sequences encoding LiTx3 peptide, the present study aimed to clone and express a peptide of this family. Primers were designed to perform 3'RACE and 5'RACE methods, obtaining the full nucleotide sequence of this peptide. The sequence corresponding propeptide associated with the mature peptide was amplified and modified by PCR with specific primers containing restriction sites for XhoI and BamHI enzymes and inserted in pET-14b vector. The construct was transformed in E. coli AD494(DE3) strain. A small scale expression was performed and the optimum parameters for recombinant protein expression were determined: 0,5mM of IPTG induction during 3h and 30min at 30°C. A large scale expression was done to obtain enough quantity of this molecule for biological assays. After affinity chromatography in Ni-NTA resin, the recombinant peptide was obtained in the soluble fraction. The diversity of peptides present in the L. intermedia venom glands represents an amazing source of novel molecular tools with pharmacological and biotechnological applications

Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5–40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins

Estudo dos peptídeos pertencentes à família ICK presentes no veneno das aranhas do gênero Loxosceles

Orientador : Prof. Dr. Silvio Sanches VeigaCoorientador : Profª. Drª. Olga Meiri ChaimTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 26/02/2015Inclui referências : f. 102-115Área de concentraçãoResumo: O conteúdo total do veneno das aranhas do gênero Loxosceles permanece ainda desconhecido, entretanto, muitos estudos têm mostrado que se constitui uma mistura complexa de compostos biologicamente ativos. Por meio de análises eletroforéticas, observa-se a predominância de moléculas de baixa massa molecular (3-45 kDa), enquanto moléculas de alta massa molecular são menos abundantes. Os venenos de aranhas estão funcionalmente relacionados à defesa contra predadores e também à paralisia e captura de presas, especialmente insetos. As aranhas desenvolveram um arsenal de moléculas inseticidas, resultando em uma biblioteca combinatória de peptídeos que tem sido aprimorada durante sua evolução. Comumente, tais peptídeos consistem em moléculas de cadeia única com massa molecular variando de 3 a 10 kDa, ricos em resíduos de cisteína, os quais estabelecem pontes dissulfeto intramoleculares características. Essas pontes se organizam em um motivo estrutural característico denominado "nó de cistina inibidor" ou ICK (Inhibitor Cystine Knot) e, por isso, os peptídeos que o contém são denominados peptídeo ICK ou notinas ("knottins"). Recentemente, análises do transcriptoma da glândula de veneno de L. intermedia (GREMSKI et al., 2010) revelaram ESTs com similaridade a peptídeos ICK previamente descritas como LiTx (De CASTRO et al., 2004). Sequências relacionadas à LiTx3, por exemplo, foram as mais abundantes no transcriptoma de L. intermedia, representando aproximadamente 13,9% de todas as ESTs obtidas e compreendendo 32% dos mRNAs codificantes de toxinas; as sequências relativas aos demais grupos de peptídeos ICK, LiTx1, LiTx2 e LiTx4 representaram 6,2%, 11,4% e 3,8% de todos os transcritos codificantes de toxinas, respectivamente. Devido a alta proporção de sequências codificantes para peptídeos ICK os objetivos deste trabalho foram o rastreamento de sequências codificantes de peptídeos ICK em outras duas aranhas do gênero (Loxosceles gaucho e Loxosceles laeta), bem como a obtenção de um peptídeo ICK semelhante à LiTx3 de forma recombinante em Pichia pastoris e sua caracterização biológica. A partir do RNA total extraído das glândulas de veneno de L. laeta e L. gaucho, procedeu-se o rastreamento de sequências relacionadas a peptídeos ICK; sequências codificantes para todos os grupos de peptídeos ICK já descritos (LiTx1-4) foram encontradas, algumas revelando sutis diferenças na sua estrutura primária, outras revelando divergências importantes como a não presença de sequências consenso para modificações pós-traducionais importantes para a atividade biológica dos mesmos. Quanto à produção do peptídeo recombinante, várias formas foram obtidas; entretanto, análises de SDS-PAGE e western blotting mostraram que os peptídeos obtidos ou não apresentavam a conformação nativa ou apresentavam glicosilação indesejada. A atividade biológica desses peptídeos foi testada in vivo em insetos e in vitro em cultura de células, contudo nenhum efeito tóxico pode ser comprovado. As sequências encontradas em L. laeta e L. gaucho representam potenciais moléculas a serem exploradas do ponto de vista biotecnológico, ao passo que o peptídeo recombinante estudado deve ser expresso em outros modelos com intuito de obtê-lo em conformação adequada e, assim, comprovar sua atividade biológica. Palavras-chave: Peptídeo ICK, notinas, Loxosceles, veneno, Pichia pastoris.Abstract: The whole content of Loxosceles spider venom still remains unknown, but several studies have shown that is a complex mixture of biologically active and inactive components. By eletrophoretic analysis, the predominance of low molecular mass molecules (3-45kDa) can be observed, while high molecular mass ones are less abundant. Spider venoms are functionally related to defense against predators as well as to paralyze and capture a natural prey, especially insects. Spiders had developed an arsenal of insecticidal molecules, resulting in a combinatorial peptide library of peptides that has been improved during evolution. Commonly, such peptides consist in single chain molecules ranging between 3- 10 kDa and are rich in cystein residues, which form intramolecular disulfide bridges. These bridges establish a structural motif "Inhibitor Cystine Knot" (ICK), then, these peptides are named ICK peptides or "knottins". Recently, a transcriptome analysis of L. intermedia venomous gland (GREMSKI et al., 2010) has revealed ESTs with similarity to ICK peptides previously described as LiTx (De CASTRO et al., 2004). LiTx3-related sequences were the most abundant in the L. intermedia transcriptome representing about 13.9% of all ESTs obtained and comprise 32% of toxin-encoding messengers; the sequences related to the other groups of ICK peptides, LiTx1, LiTx2 e LiTx4, represented 6,2%, 11,4% and 3,8% of all EST coding for toxins, respectively. Due to the high proportion of sequences encoding ICK peptides verified by the transcriptome analyses, the present study aimed screening ESTs related to these peptides in other two Loxosceles species (L. gaucho and L. laeta), as well as the obtainment of an ICK recombinant peptide with high similarity to LiTx3 in Pichia pastoris and its biological activity characterization. From total RNA purified from the venom glands of L. gaucho and L. laeta, it was performed the screening of ICK peptides sequences. ESTs coding for all groups of ICK peptides already described were found (LiTx1-4), some of them revealed subtle differences in their primary structures while others showed important divergences, for example, the absence of consensus sequences for posttranslational modifications that are essential for biological activities. The recombinant peptide was produced in different forms, however, SDS-PAGE and western blotting analyses indicated that they were not properly folded or presented unwanted glycosylation. The biological activity of the recombinant peptide was tested in insects microinjection's assays and in vitro cultivated cells, nevertheless no toxic effects were proven. The sequences identified form L. laeta and L. gaucho RNA represent potential molecules to be biotechnologically explored, whereas the studied recombinant peptide must be expressed in other heterologous expressions models in order to obtain it in the native conformation and, thus, verify its biological activity. Keywords: ICK peptides, knottins, Loxosceles, venom, toxins, Pichia pastoris. recombinant peptide was tested in insects microinjection's assays and in vitro cultivated cells, nevertheless no toxic effects were proven. The sequences identified form L. laeta and L. gaucho RNA represent potential molecules to be biotechnologically explored, whereas the studied recombinant peptide must be expressed in other heterologous expressions models in order to obtain it in the native conformation and, thus, verify its biological activity. Keywords: ICK peptides, knottins, Loxosceles, venom, toxins, Pichia pastoris

Production and Functional Evaluation of Anti-<i>Loxosceles</i> Sera Raised by Immunizations of Rabbits with Mutated Recombinant Phospholipases-D

Loxoscelism is the clinical condition triggered after the bite of spiders of the genus Loxosceles. The main species involved in accidents in South America are L. intermedia, L. laeta, and L. gaucho. The only specific treatment is the anti-Loxosceles serum produced with crude venoms. As phospholipases D (PLDs) trigger most of the effects observed in accidents, we developed and evaluated second-generation sera using mutated PLDs as antigens. Three isoforms of PLDs with site-directed mutations without biological activities were used for rabbit immunizations: D32A-E34A (L. gaucho), W230A (L. intermedia), and H12A-H47A (L. laeta). Sera were produced using crude venoms of three species of Loxosceles enriched with mutated recombinant PLDs (MIX) or using only mutated PLDs (REC). Immunizations stimulated the immune system from the second immunization with higher antibody production in the REC group. In vivo neutralization assays demonstrated that both sera reduced edema and dermonecrosis caused by Loxosceles intermedia crude venom. Follow-up of animals during the immunization protocols and in the neutralization assays demonstrated that the mutated proteins and the sera are safe. Results demonstrate the potential of using mutated recombinant PLDs in total or partial replacement of Loxosceles venoms in animal immunizations to produce anti-Loxosceles sera for treatments of Loxoscelism

Active site mapping of Loxosceles phospholipases D: Biochemical and biological features

Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D. (C) 2016 Elsevier B.V. All rights reserved.CAPESCNPqFAPESPFA-PRSETI-PRFed Univ Parana UFPR, Dept Cell Biol, Curitiba, Parana, BrazilSao Paulo State Univ UNESP, Dept Phys, Multiuser Ctr Biomol Innovat, Sao Jose Do Rio Preto, SP, BrazilFed Univ Sao Paulo UNIFESP, Dept Biochem, Sao Paulo, SP, BrazilNatl Ctr Res Energy & Mat CNPEM, Brazilian Biosci Natl Lab LNBio, Campinas, SP, BrazilFed Univ Sao Paulo UNIFESP, Dept Biochem, Sao Paulo, SP, BrazilWeb of Scienc

Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins

The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the venom components, especially for those toxins reported as recombinant molecules, and the challenges for future study are discussed. (C) 2014 Elsevier Ltd. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP