Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2017-02-14T00:41:33Z No. of bitstreams: 1 PPGBiotec - Disserta??o corrigida - Isis Bugia.pdf: 6811384 bytes, checksum: 2380cbb790d35324858de90e106415fc (MD5) / Made available in DSpace on 2017-02-14T00:41:33Z (GMT). No. of bitstreams: 1 PPGBiotec - Disserta??o corrigida - Isis Bugia.pdf: 6811384 bytes, checksum: 2380cbb790d35324858de90e106415fc (MD5) Previous issue date: 2016-03-11 / The Visceral Leishmaniasis (VL) is the second most important vector-borne disease in the world, transmitted in the Americas by Lutzomyia longipalpis, vector control is essential for the prevention of the disease. But since it is not possible to identify the oviposition sites, the fight is directed to adult insects, using traps impregnated with chemical attractants. Whereas the Odorant Binding Proteins (OBPs) act in the first level of odor selection, this work used in silico methodology to identify putative vector olfactory chemical modulators based on the structure of OBPs and known ligands. For this, tridimensional (3D) structure of L. longipalpis OBPs were predicted by three comparative modeling methods. The best model, predicted by I-Tasser, was refined by Molecular Dynamics on Gromacs. Then, in a hierarchical virtual screening approach, natural compounds of ZINC12 closer to the typical OBP ligands in global chemical space, provided by ChemGPS-NP, were evaluated and staggered concerning affinity with the orthosteric site from the OBP, by molecular docking on DOCK6. The compounds were scored by GRIDSCORE, then the 100 best classified were submitted to AMBERSCORE, which took into account the flexibility from both OBP and the docked ligands. The lowest energy conformations interacted with a hydrophobic pocket through residues Met6, Gly10, Glu11, Ala9 Arg14, Leu74, Met53, Phe118, Phe119, Pro120, amino groups and formed ionic interaction with carboxyl of Glu11, Furthermore, Phe119, Asn29 and Gln69 formed hydrogen bonds, this last formed donor and acceptor H-bonds. / A Leishmaniose Visceral (LV) ? a segunda doen?a vetorial mais importante do mundo, transmitida nas Am?ricas por Lutzomyia longipalpis, o controle do vetor ? indispens?vel ? preven??o da doen?a. Mas como n?o ? poss?vel identificar onde ocorre a oviposi??o, o combate ? direcionado aos insetos adultos, utilizando armadilhas impregnadas com atrativos qu?micos. Considerando que as Prote?nas Ligadoras de Odor (OBPs) atuam no primeiro n?vel de sele??o dos odores, este trabalho utilizou uma metodologia in silico para identificar potenciais moduladores qu?micos olfativos do vetor baseando-se na estrutura das OBPs e de ligantes conhecidos. Para isso, foram preditas as estruturas tridimensionais (3D) de OBPs de L. longipalpis por tr?s m?todos de modelagem comparativa. O melhor modelo, predito pelo I-Tasser, foi refinado por Din?mica Molecular no Gromacs. Ent?o, numa abordagem hier?rquica da triagem virtual, os compostos naturais do ZINC12 mais pr?ximos dos t?picos ligantes de OBPs no espa?o qu?mico global, fornecido pelo ChemGPS-NP, foram avaliados e escalonados quanto ? afinidade com o s?tio ortost?rico da OBP, pelo acoplamento molecular no DOCK6. Os compostos foram pontuados pelo Gridscore, em seguida, os cem melhores classificados foram submetidos ? pontua??o pelo Amberscore, que levou em conta a flexibilidade tanto da OBP como dos ligantes acoplados. As conforma??es de menor energia interagiram com um bols?o hidrof?bico atrav?s dos res?duos Met6, Ala9, Gly10, Glu11, Arg14, Met53, Leu74, Phe118, Phe119, Pro120; grupamentos amino formaram pontes salinas com a carboxila do Glu11. Al?m disso, os res?duos Phe119, Asn29 e Gln69 formaram liga??es hidrog?nio, sendo que, este ?ltimo res?duo formou liga??es-H aceptoras e doadoras.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-10-11T12:57:27Z No. of bitstreams: 1 amanda_r_lobo_ioc_bp_0013_2008.pdf: 4777016 bytes, checksum: a9e75760e9cb6f61af23579591306cb1 (MD5) / Made available in DSpace on 2012-10-11T12:57:27Z (GMT). No. of bitstreams: 1 amanda_r_lobo_ioc_bp_0013_2008.pdf: 4777016 bytes, checksum: a9e75760e9cb6f61af23579591306cb1 (MD5) Previous issue date: 2008 / Cnpq - PDTIS/FIOCRUZ / Universidade do Estado do Rio de Janeiro. Laboratório de Farmacologia Bioquímica e Celular. Rio de Janeiro, RJ, Brasil / A leishmaniose visceral é uma doença de alta mortalidade quando não tratada. Aproximadamente 500.000 novos casos ocorrem todo ano, freqüentemente em países pobres e em desenvolvimento. No Brasil, a leishmaniose visceral é causada pela Leishmania chagasi, que é transmitida principalmente pelo inseto vetor Lutzomyia longipalpis. Após 72 h de alimentação sanguínea num hospedeiro infectado, parasitas procíclicos são vistos aderidos ao intestino do inseto via flagelo. Esta adesão é vital para a evolução do ciclo de transmissão do parasita. Em vetores espécie–específicos foi proposto um papel para um lipofosfogligano (LPG) de superfície de Leishmania na adesão, porém, em vetores permissíveis, tem-se sugerido que esta adesão seja independente de LPG. Com o interesse de identificar e caracterizar moléculas que possam ter uma participação na interação parasito-vetor, nós iniciamos o estudo de duas moléculas de L. chagasi denominadas Flag e Sup, pertencentes à família de proteínas pequenas miristiladas (SMP’s), que anteriormente haviam sido implicadas em adesão ao tubo digestivo de flebotomíneos. Amplificamos e seqüenciamos o gene Flag de L. chagasi e observamos por alinhamento múltiplo o alto grau de conservação deste gene entre as espécies de Leishmania. Proteínas Flag e Sup recombinantes foram produzidas e utilizadas para produção de anticorpos policlonais em coelhos. Vimos por Western Blot de extrato protéico de várias espécies de Leishmania, que os anticorpos monoclonal e policlonal anti-Flag/MBP e o anticorpo policlonal anti-Sup, reconhecem as proteínas em todas as espécies, evidenciando a conservação destas. A imunolocalização destas proteínas mostrou que a proteína Flag está preferencialmente localizada na forma promastigota na bolsa flagelar e no flagelo, e, em amastigota, na bolsa flagelar e no flagelo interno curto, enquanto que a proteína Sup mostrou localização em toda a superfície do corpo em ambas as formas. Experimentos de RT-PCR mostraram que há transcrição diferencial de Flag e Sup quando comparamos entre as formas evolutivas amastigotas e promastigotas. Flag tem uma expressão parecida nas duas formas evolutivas enquanto Sup é mais expresso em amastigotas. Ensaios de inibição ex-vivo incubando previamente L. chagasi com anticorpo monoclonal anti-Flag ou policlonal anti-Sup, mostrou redução significativa de parasitas aderidos ao intestino de L. longipalpis Na tentativa de identificar um potencial receptor para o parasita no intestino de L. longipalpis, observamos a marcação de uma proteína de 27 kDa no extrato protéico do vetor utilizando parasita biotinilado em experimentos de “ligand blot”. / Visceral leishmaniasis is an illness of high mortality if not treated. Approximately 500,000 new cases occur per year, frequently in poor countries. In Brazil, visceral leishmaniasis is caused by Leishmania chagasi, transmitted by the insect vector Lutzomyia longipalpis. After 72h of blood feeding in an infected host, procyclic parasites are seen adhered to insect gut by the flagellum. This adhesion is vital for the completion of the parasite cycle of transmission. In species-specific vectors a role for a surface lipophosphoglycan (LPG) of Leishmania in the adhesion has been considered, while in the permissible vectors, this adhesion may be independent of LPG. Aiming to identify and characterize molecules that may be involved in the parasite- vector interaction, we initiated a study of two molecules of L. chagasi, Flag and Sup, which belong to the small myristylated protein family (SMP's), that have previously been implicated in adhesion to the sandfly midgut. We amplifyed and sequenced the Flag gene of L. chagasi and, using multiple alignments, observed a high degree of conservation of this gene among Leishmania species. Recombinant protein Flag and Sup were produced and used for polyclonal antibodies production in rabbits. By Western Blot of total protein extract of some species of Leishmania, we observed that the anti-Flag monoclonal and polyclonal antibodies and the anti-Sup polyclonal antibody, recognized proteins in all the analyzed species, evidencing their conservation. Immunolocalization of these proteins showed that Flag is preferentially located in the flagellar pocket and flagellum in the promastigote form and in the flagellar pocket and internalized short flagellum in the amastigote, while Sup showed localization on the cell surface in both forms. Experiments of RT-PCR showed that there is a differential transcription of Flag and Sup between amastigotes and promastigotes. Flag has similar levels of expression in both evolutive forms while Sup is more expressed in amastigotes. Assays of ex-vivo inhibition with L. chagasi previously incubated with monoclonal antibodies anti-Flag or anti-Sup showed a significant reduction of the number of adhered parasites to the L. longipalpis midgut. In an attempt to identify a potential receptor for the parasite in the intestine of L. longipalpis, we observed the labelling of a protein of 27 kDa in the protein extract of the vector using biotinylated parasites in experiments of "ligand blot".

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-07-17T13:28:12Z No. of bitstreams: 1 MarcosPauloGomesPinheiro_TESE.pdf: 5533718 bytes, checksum: bf1ce7fe9e0014df2de63606b02f02ec (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-07-19T14:40:04Z (GMT) No. of bitstreams: 1 MarcosPauloGomesPinheiro_TESE.pdf: 5533718 bytes, checksum: bf1ce7fe9e0014df2de63606b02f02ec (MD5) / Made available in DSpace on 2017-07-19T14:40:04Z (GMT). No. of bitstreams: 1 MarcosPauloGomesPinheiro_TESE.pdf: 5533718 bytes, checksum: bf1ce7fe9e0014df2de63606b02f02ec (MD5) Previous issue date: 2017-02-13 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / As leishmanioses s?o graves problemas de sa?de p?blica no Brasil, causadas por protozo?rios do g?nero Leishmania transmitidos pelos flebotom?neos, pequenos insetos da ordem Diptera cujas f?meas s?o hemat?fagas. A leishmaniose visceral (LV) ou calazar, tem como principal vetor nas Am?ricas Lutzomyia longipalpis e Leishmania infantum como agente etiol?gico. No Brasil, a Regi?o Nordeste se destaca com a maior incid?ncia de casos de LV, principalmente entre as camadas mais desfavorecidas da sociedade. A leishmaniose tegumentar (LT) possui diferentes vetores e agentes etiol?gicos em todo o pa?s, tem ampla distribui??o, e apesar da menor gravidade, causa s?rias les?es desfigurantes e incapacitantes em todas as regi?es, principalmente na regi?o Norte. ? relevante a introdu??o de estrat?gias de preven??o que venham a modificar as a??es humanas por meio da informa??o e sensibiliza??o para o problema, sendo a literatura de cordel um instrumento educativo que atrai por sua conota??o l?dica e informal, despertando a aten??o das pessoas. O presente estudo buscou, portanto, conhecer os flebotom?neos de uma Unidade de Conserva??o da Mata Atl?ntica e seu entorno, bem como alguns fatores bi?ticos e abi?ticos relacionados ? sua ocorr?ncia, al?m de testar a utiliza??o do cordel como ferramenta informativa sobre o tema. O estudo aconteceu durante tr?s anos na ?rea interna e externa da Floresta Nacional (Flona) de N?sia Floresta, localizada no munic?pio de N?sia Floresta, regi?o metropolitana de Natal, Rio Grande do Norte, utilizando-se armadilhas Shannon e CDC. Foi produzido um cordel, em seu formato cl?ssico, intitulado ?Um bebedor de sangue na terra do mandacaru - Peleja contra o calazar?, para a??es de educa??o em sa?de com a popula??o residente no entorno da Flona e com alunos de escolas do munic?pio. Foram coletados e identificados 82.638 flebotom?neos das esp?cies Evandromyia evandroi, Evandromyia lenti, Evandromyia walkeri, Evandromyia sallesi, Lutzomyia longipalpis, Micropygomyia schreiberi, Nyssomyia intermedia, Nyssomyia whitmani, Psathyromyia abonnenci, Psathyromyia brasiliensis, Psathyromyia shannoni, Psychodopygus wellcomei e Sciopemyia sordellii. Os flebotom?neos foram mais abundantes junto ao solo do ambiente silvestre, apesar de tamb?m serem abundantes junto ?s copas e no peridomic?lio rural. Evandromyia walkeri foi a esp?cie mais abundante, representando 76% de todos os flebotom?neos coletados, sendo importante outras investiga??es acerca dessa esp?cie. Lutzomyia longipalpis, E. evandroi e E. lenti demonstraram prefer?ncia pelos ambientes antropizados. Esse estudo comprova a elevada antropofilia de P. wellcomei, que se mostra ativo durante quase todo o dia no ambiente silvestre, bem como sua rela??o com os meses chuvosos. Com base no conhecimento da bioecologia dos flebotom?neos vetores das leishmanioses na ?rea, espera-se que esse trabalho possa contribuir com estrat?gias de preven??o e controle das leishmanioses no Nordeste.O cordel teve grande aceita??o por parte dos estudantes da EJA e a experi?ncia demonstrou que esse pode ser um importante meio para a dissemina??o de informa??es acerca da leishmaniose visceral, regionalizando as estrat?gias, sensibilizando e divulgando um tema t?o importante em sa?de p?blica. / Leishmaniasis is a serious public health problem in Brazil, caused by protozoa of the genus Leishmania transmitted by sand flies, small insects of the order Diptera whose females are hematophagous. The main vector of visceral leishmaniasis (VL), or kala-azar, in the Americas is Lutzomyia longipalpis while Leishmania infantum is the etiological agent. In Brazil, the Northeastern Region stands out with the highest incidence of VL cases, especially among the most disadvantaged groups of society. Tegumentary leishmaniasis (TL) has different vectors and etiological agents throughout the country, a wide distribution, and despite its lower severity, it causes serious disfiguring and disabling lesions all over the country, especially in the North Region. It is relevant to introduce prevention strategies, through information and consciousness of the problem, in order to modify deleterious anthropic actions. Cordel folk literature has proved to be an educational instrument with potential use to address this issue, since it attracts by its playful and informal connotations and awakens the attention of people of different age groups. This study aimed, therefore, to know the sand flies fauna at an Atlantic Forest Conservation Unit and its surroundings, as well as some factors related to their occurrence, in addition to testing the use of cordel literature as an informative tool on the subject. The study took place over three years in the internal and external area of the N?sia Floresta National Forest (Flona) located in the city of N?sia Floresta, metropolitan region of Natal, Rio Grande do Norte, using Shannon and CDC traps. A cordel pamphlet in its classic format was produced, entitled "A blood drinker in the land of mandacaru - fight against kala-azar," for health educational activities among the population living in the vicinity of the Flona and students. The use of the cordel was validated through questionnaires applied to students of the Education of Young and Adults Program. The pamphlet approaches the transmission, symptoms, treatment and prophylaxis of the disease. There were collected and identified 82,638 sand flies of the species Evandromyia evandroi, Evandromyia lenti, Evandromyia walkeri, Evandromyia sallesi, Lutzomyia longipalpis, Micropygomyia schreiberi, Nyssomyia intermedia, Nyssomyia whitmani, Psathyromyia abonnenci, Psathyromyia brasiliensis, Psathyromyia shannoni, Psychodopygus wellcomei and Sciopemyia sordellii. Phlebotomines were more abundant near the soil of the wild environment, although they were also abundant near the canopy and in the rural peridomicille. L. walkeri was the most abundant species, representing 76% of all sandflies collected, and other investigations about this species are important. L. longipalpis, E. evandroi and E. lenti showed preference for anthropized environments. This study confirms the high anthropophilic behavior of P. wellcomei, which is active throughout the day in the wild, as well as its relationship with the rainy months. Based on the knowledge of the bioecology of sand flies vectors of leishmaniasis in the study area, as well as the factors involved in its occurrence, it is expected that this work may contribute to strategies for the prevention and control of leishmaniasis in Northeastern Brazil. The cordel literature was widely accepted by students and the experience has shown that this may be an important way for dissemination of information about visceral leishmaniasis. It is also desired that the regionalization of educational strategies through cordel literature can contribute to the dissemination of knowledge, sensitizing the community and disseminating such an important subject in public health. / 2019-05-04