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Master's Dissertation
DOI
https://doi.org/10.11606/D.42.2015.tde-23122015-091418
Document
Author
Full name
Camila Cristina Coelho
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2015
Supervisor
Committee
Winter, Carlos Eduardo (President)
Fogaça, Andréa Cristina
Mori, Marcelo Alves da Silva
Title in Portuguese
Caracterização preliminar dos hábitos extremófilos, vitelinas e proteases de Panagrolaimus sp. CEW2, um nematoide de vida livre isolado da Região Amazônica.
Keywords in Portuguese
Panagrolaimus sp.
CEW2
Protease
Sobrevivência a dessecação
Vitelogenina
Abstract in Portuguese
Neste trabalho caracterizamos o nematoide Panagrolaimus sp. linhagem CEW2, originalmente isolado em Monte Negro (RO), Brasil e mantido em nosso laboratório em culturas monoxênicas contendo Escherichia coli. CEW2 se mostrou um nematoide extremófilo capaz de sobreviver ao dessecamento em 10% de umidade relativa por pelo menos 48h. Essa resistência é mais evidente em larvas de primeiro estádio (L1) do que em adultos ou larvas dauer. Quando pré-incubados por 48h em 98% de umidade relativa a sua resistência ao dessecamento aumenta e pode chegar a uma sobrevivência de 86,25% dos indivíduos no caso de L1. Os animais dessecados se tornam também resistentes ao congelamento. Os indivíduos pré-incubados em 98% de umidade relativa tornam-se resistentes ao congelamento. O retorno à atividade após secagem ou congelamento ocorre de maneira diferente dependendo da concentração de NaCl no meio, sugerindo que eles sejam, além de anidrobiontes e criobiontes, sejam também osmobiontes. Um outro aspecto da fisiologia de CEW2 que também começamos a caracterizar é a composição de polipeptídeos das vitelinas de seus ovos. SDS-PAGE das proteínas presente em ovos de CEW2 apresentam 6 bandas principais coradas por Coomassie Blue (com Mr que variam de 85 a 125 kDa), 3 a mais do que aquelas detectadas nos outros dois nematoides cujas vitelinas foram caracterizadas, Caenorhabditis elegans e Oscheius tipulae. Quando comparamos as vitelinas destas três espécies notamos que CEW2 não possui a vitelina de 180 kDa (YP170A e B de C. elegans e VT1 de O. tipulae). Essa análise comparativa das vitelinas de nematoides nos permitiu propor um esquema para a origem das três bandas extras detectadas em CEW2. Não foi possível purificar as vitelinas de CEW2 usando o mesmo protocolo que desenvolvemos para as vitelinas de O. tipulae e C. elegans. Isso se deve a uma atividade proteolítica presente nos homogeneizados de vermes que co-purifica com as vitelinas e leva a sua degradação durante a armazenagem. As atividades proteolíticas presentes nesses homogeneizados foram caracterizados por zimografia e foi possível purificar uma das bandas que cremos ser uma protease digestiva de CEW2 e a responsável pela degradação das vitelinas purificadas.
Title in English
Preliminary characterization of extremophile behavior, vitellins and proteases from Panagrolaimus sp., a nematode isolated from the Amazon Region.
Keywords in English
Panagrolaimus sp.
CEW2
Protease
Survives desiccation
Vitellogenin
Abstract in English
In this work we characterize some survival and reproductive strategies of the nematode Panagrolaimus sp., strain CEW2. CEW2 was originally isolated in Monte Negro (RO), Brazil. CEW2 is currently maintained in monoxenic cultures with E. coli in our laboratory and has proved to be an extremophile that survives desiccation at 10% relative humidity for at least 48 hours. First instar larvae (L1) are more resistant to desiccation than adults or dauer larvae. When pre-incubated for 48 h at 98% relative humidity their resistance to desiccation increases and can reach a survival rate of 86,25 % with L1individuals. The dried animals also become resistant to freezing. Worms that were pre-incubated at 98% relative humidity become resistant to freezing in water at -20 °C. The return to activity upon drying or freezing occurs differently depending on the NaCl concentration in the medium, suggesting that CEW2 is an osmobiont in addition to its anidrobiotic and criobiotic behavior. We also began to characterize another aspect of CEW2 physiology; the polypeptide composition of the vitellins in their eggs. SDS-PAGE of the proteins present in CEW2 eggs show 6 major Coomassie Blue stained bands (Mr ranging from 85-125 kDa), three more than those detected in the vitellins of Caenorhabditis elegans and Oscheius tipulae. When we compare the vitellins of these three species we notice that CEW2 lacks the yolk proteins of 180 kDa (YP170A and B present in C. elegans and VT1 in O. tipulae). The comparative analysis of nematode vitellins allowed us to propose a model for the origin of the three extra bands detected in CEW2. We propose that in CEW2 the polypeptides that are ortologous to the 180 kDa polypeptides of C. elegans and O. tipulae, suffer a proteolytic cleavage similar to that occurring with the precursor of YP115 and 88 in C. elegans. It was not possible to purify the vitellins of CEW2 using the same protocol developed for the vitellins of O. tipulae and C. elegans. This is due to a proteolytic activity present in homogenized worms which co-purifies with the vitellins and leads to their degradation during the procedure. Proteolytic activities present in these homogenates were characterized by zymography and it was possible to purify one of the bands that we believe to be a digestive protease from CEW2 responsible for the degradation of purified vitellins.
 
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Publishing Date
2015-12-23
 
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