Caracteriza??o de receptores Toll-like relacionados ? resposta imune no carrapato bovino Rhipicephalus (Boophilus) microplus

Autor: Rezende, Sabrina Rita da Fonseca
Přispěvatelé: Bittencourt, V?nia Rita Elias Pinheiro, Leite, Milane de Souza, Paes, M?rcia Cristina, Silva, Jairo Pinheiro da, Genta, Fernando Ariel, Oliveira, Carlos Jorge Logullo de
Jazyk: portugalština
Rok vydání: 2013
Předmět:
Zdroj: Biblioteca Digital de Teses e Dissertações da UFRRJ
Universidade Federal Rural do Rio de Janeiro (UFRRJ)
instacron:UFRRJ
ISSN: 3779-5015
Popis: Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2019-06-27T17:55:35Z No. of bitstreams: 1 2013 - Sabrina Rita da Fonseca Rezende.pdf: 37795015 bytes, checksum: b04af19ebfb7ed13d3b4676c9a2289c6 (MD5) Made available in DSpace on 2019-06-27T17:55:35Z (GMT). No. of bitstreams: 1 2013 - Sabrina Rita da Fonseca Rezende.pdf: 37795015 bytes, checksum: b04af19ebfb7ed13d3b4676c9a2289c6 (MD5) Previous issue date: 2013-03-27 CAPES - Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior CNPq - Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico FAPERJ - Funda??o Carlos Chagas Filho de Amparo ? Pesquisa do Estado do Rio de Janeiro INCT-EM Toll-like receptors (TLR) are highly conserved transmembrane proteins that play important roles in pathogen recognition. This study aims to identify the presence and differential expression of Toll-like receptors in tissues and cells that are important to the innate immune response of the tick R. (B.) microplus. For this, we identified genes of R. (B.) microplus encoding Toll receptors homologues, which we called RmToll-like and RmToll8-like. We detected the presence of these receptors in organs involved in immune response, such as fat body, hemocytes, salivary glands and digestive cells. Gene expression of Toll-like receptors was evaluated by qPCR and differential expression in the different tissues analyzed. We confirmed the expression of these receptors by western blot and confocal microscopy. Fat body had the greatest modulation of receptors studied, we have chosen to further investigate these receptors in this tissue. In fat body, these receptors were found specifically in round-shaped trophocytes cell membranes and intracellular vesicles. Gene expression of Toll-like receptors was assessed by qPCR 6, 24 and 48 hours after the challenges with the entomopathogenic fungi Beauveria bassiana. Transcription of RmToll-like was positively modulated 48 hours after challenge with B. bassiana, but we did not observe modulation of gene RmToll8-like in the various treatments depending on the time of exposure to the fungi. However, when we evaluated the protein expression of these receptors in ticks challenged with the fungi, no significant difference between treatments and the times analyzed was observed. When we investigated a less virulent strain of fungi we observed that RmToll-like expression was positively modulated after 24h and 48h of infection, this modulation being smaller when compared with those from the more virulent strain. Regarding RmToll8-like we not observed differences between treatments and the times analyzed. The gene expression of antimicrobial peptides was also modulated by the challenge with the fungi, though indicating a possible immunosuppression, but further investigations with new times of challenge should be considered. Evaluating the effects of a pathogen-associated molecular patterns, zymosan, we observed a significant increase in expression of RmToll-like, but not for RmToll8-like. We also evaluated the expression of the corresponding proteins in ticks challenged with zymosan, but no significant difference between treatments and the times analyzed was observed. This study shows for the first time the presence of Toll-like receptors in ticks, and reports the differential expression between different tissues engaged in the innate immune response modulation and immune challenges in this arthropod. Os receptores Toll-like (TLR) s?o prote?nas transmembranares altamente conservadas que desempenham papel importante no reconhecimento de pat?genos. Este estudo visa identificar a poss?vel presen?a e express?o diferencial de receptores Toll-like em tecidos e c?lulas importantes para a resposta imune inata do carrapato R. (B.) microplus. Para tal, identificamos genes de R. (B.) microplus que codificam hom?logos de receptores Toll, denominados RmToll-like e RmToll8-like. Detectamos a presen?a desses receptores nos principais ?rg?os envolvidos na resposta imune, corpo gorduroso, hem?citos, gl?ndula salivar e c?lula digestiva. A express?o g?nica dos receptores Toll-like, avaliada por qPCR, mostrou modula??o nos diferentes tecidos analisados, e confirmamos a express?o desses receptores pelas t?cnicas de Western Blot e microscopia confocal. Como o corpo gorduroso representa um importante ?rg?o de defesa imune e apresentou uma maior modula??o dos receptores estudados n?s optamos por investigar mais profundamente esses receptores neste tecido. No corpo gorduroso, esses receptores foram encontrados especificamente nos trof?citos arredondados, em suas membranas celulares e ves?culas intracelulares. A express?o g?nica dos receptores Toll-like foi avaliada por qPCR 6, 24 e 48 horas ap?s os desafios com o fungo entomop?togenico Beauveria bassiana. A transcri??o de RmToll-like foi modulada positivamente 48 horas ap?s o desafio com B. bassiana, mas n?o observamos modula??o do gene RmToll8-like nos diferentes tratamentos e em fun??o dos tempos de exposi??o ao fungo. Entretanto, ao avaliarmos a express?o das prote?nas desses receptores dos carrapatos desafiados com o fungo, n?o observamos diferen?a entre os tratamentos e nos tempos analisados. Quando investigamos uma cepa menos virulenta do fungo observamos que RmToll-like foi modulada positivamente nos tempos de 24h e 48h, sendo esta modula??o menor quando comparada com a modula??o pela cepa mais virulenta. Em rela??o ao RmToll8-like n?o observamos diferen?a entre os tratamentos e nos tempos analisados. A express?o dos genes de peptideos antimicrobianos tamb?m foi modulada pelo desafio com fungo, contudo indicando uma poss?vel imunosupress?o, mas investiga??es futuras com novos tempos de desafio devem ser consideradas. Avaliando o efeitos de uma mol?cula padr?o de superf?cie de pat?genos, o zimosan, observamos um aumento significativo na express?o do RmToll-like, mas n?o em rela??o ao RmToll8-like. Avaliamos a express?o das prote?nas dos receptores em carrapatos desafiados com zimosan, n?o observamos diferen?a entre os tratamentos e nos tempos analisados. O presente trabalho mostra pela primeira vez a presen?a de receptores Toll-like em carrapatos, e relata a express?o diferencial entre diferentes tecidos engajados na resposta imune inata e modula??o por desafios imunol?gicos neste artr?pode.
Databáze: OpenAIRE
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