Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFG |
Texto Completo: | http://repositorio.bc.ufg.br/tede/handle/tede/8711 |
Resumo: | Paracoccidioides lutzii alternates between filamentous and yeast growth when exposed to temperatures of 36ºC. Morphological transition is an essential event for establishment of paracoccidioidomycosis (PCM), one of the most frequent systemic mycoses in Latin America. Paracoccidioides brasiliensis and other dimorphic fungi, cAMP-dependent protein kinase (PKA) is essential in the dimorphic process. However, molecular events responsible for mycelial-yeast differentiation in P. lutzii remain unknown. The aim of this work was to characterize in silico the PKA subunits of P. lutzii, and to evaluate the role of this enzyme during the morphological transition of this fungus. During the in silico approach, we used computational tools for identity, domains, cell localization prediction, and similarity between P. lutzii and human sequences analysis. The importance of this enzyme was investigated through the use of specific inhibitor, H89. The behavior of cells in the presence of the drug was observed and the PKA activity measured by specific kit. Our in silico results reveal a single regulatory subunit, PbPKA-R located primarily in the nucleus, and two catalytic subunits, PbPKA-C1 and PbPKA-C2, with localization in the cytoplasm and nucleus respectively, all highly conserved. The PbPKA-R subunits were identified with two copies of the CNMP domain in the C-terminal, while the catalytic with a single copy of two domains: protein kinase and AGC kinase. The alignment of the PKA catalytic subunits of humans and Paracoccidioides shows a large divergence between them, making PKA an interesting target for antifungal. The results show that PKA activity is phase regulated, being about five times higher in yeast than in mycelium. We also show that H89 blocks the mycelium to yeast differentiation when PKA activity decreases. However, when the drug is removal from the culture medium, the fungus resumes the differentiation to the yeast phase. Our results show that PKA activity affects the phase transition in P. lutzii. |
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Furlaneto, Silvia Maria Salem Izacchttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728477P4Brito, Wesley de Almeidahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782643Y1Furlaneto, Silvia Maria Salem IzaccFaria, Fabrícia Paula deBailão, Mirelle Garcia Silvahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4309902H4Sestari, Sheila Janaina2018-07-18T11:28:17Z2017-03-10SESTARI, Sheila Janaína. Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii. 2017. 54 f. Dissertação (Mestrado em Genética e Biologia Molecular) - Universidade Federal de Goiás, Goiânia, 2017.http://repositorio.bc.ufg.br/tede/handle/tede/8711ark:/38995/0013000002s96Paracoccidioides lutzii alternates between filamentous and yeast growth when exposed to temperatures of 36ºC. Morphological transition is an essential event for establishment of paracoccidioidomycosis (PCM), one of the most frequent systemic mycoses in Latin America. Paracoccidioides brasiliensis and other dimorphic fungi, cAMP-dependent protein kinase (PKA) is essential in the dimorphic process. However, molecular events responsible for mycelial-yeast differentiation in P. lutzii remain unknown. The aim of this work was to characterize in silico the PKA subunits of P. lutzii, and to evaluate the role of this enzyme during the morphological transition of this fungus. During the in silico approach, we used computational tools for identity, domains, cell localization prediction, and similarity between P. lutzii and human sequences analysis. The importance of this enzyme was investigated through the use of specific inhibitor, H89. The behavior of cells in the presence of the drug was observed and the PKA activity measured by specific kit. Our in silico results reveal a single regulatory subunit, PbPKA-R located primarily in the nucleus, and two catalytic subunits, PbPKA-C1 and PbPKA-C2, with localization in the cytoplasm and nucleus respectively, all highly conserved. The PbPKA-R subunits were identified with two copies of the CNMP domain in the C-terminal, while the catalytic with a single copy of two domains: protein kinase and AGC kinase. The alignment of the PKA catalytic subunits of humans and Paracoccidioides shows a large divergence between them, making PKA an interesting target for antifungal. The results show that PKA activity is phase regulated, being about five times higher in yeast than in mycelium. We also show that H89 blocks the mycelium to yeast differentiation when PKA activity decreases. However, when the drug is removal from the culture medium, the fungus resumes the differentiation to the yeast phase. Our results show that PKA activity affects the phase transition in P. lutzii.Paracoccidioides lutzii alterna entre crescimento filamentoso e leveduriforme quando exposto a temperaturas de 36ºC. A transição morfológica é um evento essencial para estabelecimento da paracoccidioidomicose (PCM), uma das micoses sistêmicas mais frequentes na América Latina. Em Paracoccidioides brasiliensis e outros fungos dimórficos, a proteína quinase dependente de AMPc (PKA) é essencial no processo dimórfico. No entanto, os eventos moleculares responsáveis pela diferenciação micélio-levedura em P. lutzii permanecem desconhecidos. O objetivo deste trabalho foi caracterizar in silico as subunidades da PKA de P. lutzii, e avaliar o papel dessa enzima durante a transição morfológica desse fungo. Durante a abordagem in silico, utilizamos ferramentas computacionais para análise de identidade, domínios, predição da localização celular e similaridade entre as sequências de P. lutzii e humanos. Para investigar a importância dessa enzima, utilizamos um inibidor específico, H89, observando o comportamento das células quando induzidas à transição morfológica e posteriormente mensuramos sua atividade através de kit específico. Nossos resultados in silico revelam uma única subunidade regulatória, PbPKA-R localizada principalmente no núcleo, e duas subunidades catalíticas, PbPKA-C1 e PbPKA-C2, com localização no citoplasma e núcleo respectivamente, todas altamente conservadas. Na região Cterminal, as subunidades PbPKA-R foram identificadas com duas cópias do domínio CNMP, já as catalíticas com uma única cópia de dois domínios: proteína quinase e AGC quinase. O alinhamento das subunidades catalíticas da PKA de humanos e Paracoccidioides aponta uma grande divergência entre elas, o que torna a PKA um alvo interessante para antifúngicos. Os resultados do ensaio de atividade da PKA mostram que a atividade é regulada ao longo da transição de fases, sendo cerca de cinco vezes maior em levedura do que em micélio. Os dados também evidenciam que H89 bloqueia a diferenciação de micélio para levedura e que este bloqueio acontece concomitantemente à diminuição da atividade da PKA. No entanto, quando a droga é retirada do meio de cultura, o fungo retoma a diferenciação para fase leveduriforme. Os resultados revelam que a atividade da PKA afeta a transição de fases em P. lutzii.Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2018-07-17T17:09:33Z No. of bitstreams: 2 Dissertação - Sheila Janaína Sestari - 2017.pdf: 2781342 bytes, checksum: 55b64ac559e09fb7b078b0906ccd9791 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-07-18T11:28:17Z (GMT) No. of bitstreams: 2 Dissertação - Sheila Janaína Sestari - 2017.pdf: 2781342 bytes, checksum: 55b64ac559e09fb7b078b0906ccd9791 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2018-07-18T11:28:17Z (GMT). No. of bitstreams: 2 Dissertação - Sheila Janaína Sestari - 2017.pdf: 2781342 bytes, checksum: 55b64ac559e09fb7b078b0906ccd9791 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-10Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESapplication/pdfporUniversidade Federal de GoiásPrograma de Pós-graduação em Genética e Biologia Molecular (ICB)UFGBrasilInstituto de Ciências Biológicas - ICB (RG)http://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessParacoccidioides lutziiTransição morfológicaPKAH89Morphological transitionCIENCIAS BIOLOGICAS::GENETICACaracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutziiIn silico characterization, activity study and role evaluation of PKA in morphological transition of Paracoccidioides lutziiinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis-3983316729959641468600600600600-3872772117827373404-55181442685852520512075167498588264571reponame:Repositório Institucional da UFGinstname:Universidade Federal de Goiás (UFG)instacron:UFGLICENSElicense.txtlicense.txttext/plain; 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dc.title.eng.fl_str_mv |
Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii |
dc.title.alternative.eng.fl_str_mv |
In silico characterization, activity study and role evaluation of PKA in morphological transition of Paracoccidioides lutzii |
title |
Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii |
spellingShingle |
Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii Sestari, Sheila Janaina Paracoccidioides lutzii Transição morfológica PKA H89 Morphological transition CIENCIAS BIOLOGICAS::GENETICA |
title_short |
Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii |
title_full |
Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii |
title_fullStr |
Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii |
title_full_unstemmed |
Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii |
title_sort |
Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii |
author |
Sestari, Sheila Janaina |
author_facet |
Sestari, Sheila Janaina |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Furlaneto, Silvia Maria Salem Izacc |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728477P4 |
dc.contributor.advisor-co1.fl_str_mv |
Brito, Wesley de Almeida |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782643Y1 |
dc.contributor.referee1.fl_str_mv |
Furlaneto, Silvia Maria Salem Izacc |
dc.contributor.referee2.fl_str_mv |
Faria, Fabrícia Paula de |
dc.contributor.referee3.fl_str_mv |
Bailão, Mirelle Garcia Silva |
dc.contributor.authorLattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4309902H4 |
dc.contributor.author.fl_str_mv |
Sestari, Sheila Janaina |
contributor_str_mv |
Furlaneto, Silvia Maria Salem Izacc Brito, Wesley de Almeida Furlaneto, Silvia Maria Salem Izacc Faria, Fabrícia Paula de Bailão, Mirelle Garcia Silva |
dc.subject.por.fl_str_mv |
Paracoccidioides lutzii Transição morfológica PKA H89 |
topic |
Paracoccidioides lutzii Transição morfológica PKA H89 Morphological transition CIENCIAS BIOLOGICAS::GENETICA |
dc.subject.eng.fl_str_mv |
Morphological transition |
dc.subject.cnpq.fl_str_mv |
CIENCIAS BIOLOGICAS::GENETICA |
description |
Paracoccidioides lutzii alternates between filamentous and yeast growth when exposed to temperatures of 36ºC. Morphological transition is an essential event for establishment of paracoccidioidomycosis (PCM), one of the most frequent systemic mycoses in Latin America. Paracoccidioides brasiliensis and other dimorphic fungi, cAMP-dependent protein kinase (PKA) is essential in the dimorphic process. However, molecular events responsible for mycelial-yeast differentiation in P. lutzii remain unknown. The aim of this work was to characterize in silico the PKA subunits of P. lutzii, and to evaluate the role of this enzyme during the morphological transition of this fungus. During the in silico approach, we used computational tools for identity, domains, cell localization prediction, and similarity between P. lutzii and human sequences analysis. The importance of this enzyme was investigated through the use of specific inhibitor, H89. The behavior of cells in the presence of the drug was observed and the PKA activity measured by specific kit. Our in silico results reveal a single regulatory subunit, PbPKA-R located primarily in the nucleus, and two catalytic subunits, PbPKA-C1 and PbPKA-C2, with localization in the cytoplasm and nucleus respectively, all highly conserved. The PbPKA-R subunits were identified with two copies of the CNMP domain in the C-terminal, while the catalytic with a single copy of two domains: protein kinase and AGC kinase. The alignment of the PKA catalytic subunits of humans and Paracoccidioides shows a large divergence between them, making PKA an interesting target for antifungal. The results show that PKA activity is phase regulated, being about five times higher in yeast than in mycelium. We also show that H89 blocks the mycelium to yeast differentiation when PKA activity decreases. However, when the drug is removal from the culture medium, the fungus resumes the differentiation to the yeast phase. Our results show that PKA activity affects the phase transition in P. lutzii. |
publishDate |
2017 |
dc.date.issued.fl_str_mv |
2017-03-10 |
dc.date.accessioned.fl_str_mv |
2018-07-18T11:28:17Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
SESTARI, Sheila Janaína. Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii. 2017. 54 f. Dissertação (Mestrado em Genética e Biologia Molecular) - Universidade Federal de Goiás, Goiânia, 2017. |
dc.identifier.uri.fl_str_mv |
http://repositorio.bc.ufg.br/tede/handle/tede/8711 |
dc.identifier.dark.fl_str_mv |
ark:/38995/0013000002s96 |
identifier_str_mv |
SESTARI, Sheila Janaína. Caracterização in silico, estudo da atividade e avaliação do papel da PKA na transição morfológica de Paracoccidioides lutzii. 2017. 54 f. Dissertação (Mestrado em Genética e Biologia Molecular) - Universidade Federal de Goiás, Goiânia, 2017. ark:/38995/0013000002s96 |
url |
http://repositorio.bc.ufg.br/tede/handle/tede/8711 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.program.fl_str_mv |
-3983316729959641468 |
dc.relation.confidence.fl_str_mv |
600 600 600 600 |
dc.relation.department.fl_str_mv |
-3872772117827373404 |
dc.relation.cnpq.fl_str_mv |
-5518144268585252051 |
dc.relation.sponsorship.fl_str_mv |
2075167498588264571 |
dc.rights.driver.fl_str_mv |
http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
http://creativecommons.org/licenses/by-nc-nd/4.0/ |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Goiás |
dc.publisher.program.fl_str_mv |
Programa de Pós-graduação em Genética e Biologia Molecular (ICB) |
dc.publisher.initials.fl_str_mv |
UFG |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
Instituto de Ciências Biológicas - ICB (RG) |
publisher.none.fl_str_mv |
Universidade Federal de Goiás |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UFG instname:Universidade Federal de Goiás (UFG) instacron:UFG |
instname_str |
Universidade Federal de Goiás (UFG) |
instacron_str |
UFG |
institution |
UFG |
reponame_str |
Repositório Institucional da UFG |
collection |
Repositório Institucional da UFG |
bitstream.url.fl_str_mv |
http://repositorio.bc.ufg.br/tede/bitstreams/7223de2c-8e39-4ea9-b722-4ea5e75cf8bd/download http://repositorio.bc.ufg.br/tede/bitstreams/df3dc1c7-7299-48ef-ae19-ba3000d4b867/download http://repositorio.bc.ufg.br/tede/bitstreams/0600a6e2-0c4b-4fd8-8296-084c7a328c41/download http://repositorio.bc.ufg.br/tede/bitstreams/dfd8ac9d-3a2b-4a4b-a21e-c98d12e3e15f/download http://repositorio.bc.ufg.br/tede/bitstreams/13b5835f-f835-4202-b6af-1dc5a3838afc/download |
bitstream.checksum.fl_str_mv |
bd3efa91386c1718a7f26a329fdcb468 4afdbb8c545fd630ea7db775da747b2f d41d8cd98f00b204e9800998ecf8427e d41d8cd98f00b204e9800998ecf8427e 55b64ac559e09fb7b078b0906ccd9791 |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 MD5 MD5 |
repository.name.fl_str_mv |
Repositório Institucional da UFG - Universidade Federal de Goiás (UFG) |
repository.mail.fl_str_mv |
tasesdissertacoes.bc@ufg.br |
_version_ |
1811721357880197120 |