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Previous issue date: 2016-01-13 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Estudos recentes mostraram que TGF-\03B2 esta envolvido na cardiopatia chagásica aguda e crônica. O aumento de seus níveis plasmáticos e a ativação da sua via de sinalização celular são aspectos peculiares da doença chagásica crônica. Além do seu relevante papel na patologia chagásica, também se observou que esta citocina está intimamente associada ao T. cruzi como um regulador de diferentes etapas de seus ciclo de vida. Trabalhos anteriores demostraram que T. cruzi é capaz de ativar TGT-\03B2 latente, utilizando-o na invação às células hospedeiras e que amastigotas de T. cruzi se ligam e internalizam TGF-\03B2 recombinante, estando este evento relacionado à capacidade de proliferação de amastigotas e sua diferenciação em tripomastigotas no final do ciclo intracelular. Este conjunto de informações nos levou ao questionamento de quais moléculas de T. cruzi poderiam estar envolvidas nos processos de proliferação e diferenciação celular frente ao estímulo por TGF-\03B2. Neste sentido, o presente projeto tem por principal objetivo a caracterização de moléculas responsivas ao estimulo de TGF-\03B2 através de uma abordagem fosfoproteômica. Para tal, extratos de proteínas totais de epimastigotas de T. cruzi (cepa Y), incubadas ou não com TGF-\03B2, foram preparados durante a fase exponencial de crescimento do parasito. Evidenciamos que a dose ótima de TGF-\03B2 para maior indução de fosforilação seria de 5 ng/ml. em seguida, o tempo ótimo de indução com TGF-\03B2 (1,5,15,30 e 60 minutos) foi testado e concluímos que as diferenças entre os padrões de fosforilação são muitos sutis em géis unidimensionais, nos fazendo optar pela análise exclusiava dos perfins em géis bidimensionais de 7 cm com faiza de PH 3-10 não-linear. A avaliação dos perfis bidimensionais demonstrou diferenças nos padrões de fosforilação entre os tempos estudados, nos levando a manter um estudo de cinética de tempo.
As imagens dos géis foram analisadas e algumas das proteínas consideradas responsivas a TGF-\03B2 foram identificadas por espectrometria de massas. Observamos que as proteínas de choque térmico, tubulinas, desidrogenases, enolases, ciclofilina A, GrpE, cruzipaína, fator de elongamento 1-\03B1, fator de iniciação eucariótica 5a, entre outras, têm sua fosforilação e/ou expressão moduladas em resposta a TGF-\03B2. Buscamos correlacionar a função já descrita na literatura para cada proteína com seu possível papel na sinalização intracelular dsparada por TGF-\03B2, em concordância com o comportamento de fosforilação e/ou expressão apresentado em novas análises. Por último, foi avaliado se a adição de TGF-\03B2 a culturas de epimastigotas teria algum efeito sobre a proliferação dos parasitos. Verificamos que a adição de TGF-\03B2 promoveu um aumento de até 73% no crescimento dos parasitos nas primeiras 24 horas de estudo. O conjunto de dados obtidos contribui para a elucidação dos mecanismos moleculares relacionados à sinalização de TGF-\03B2, proporcionando uma fonte para detecção de novos alvos terapêuticos para a doença de Chagas / Recent studies show that TGF
-
β
is involved in the acute and chronic chagasic
cardiopathy. High levels of TGF
-
β
and the activation of its signaling pathway were
shown to be peculiar aspects of patients with chron
ic Chagas disease. Besides its
relevant role in the pathology of Chagas dis
ease, this cytokine was also observed to
be intimately associated with
Trypanosoma cruzi
as a regulator of different stages of
the parasite ́s life cycle. Previous works have show
n that
T. cruzi
, using it in the process of h
ost cell invasion. In addition, amastigote
forms are able to bind and internalize recombinant
TGF
-
β
, and this event is related to
their capacity to proliferate and differentiate int
o trypomastigote forms at the end of
the intracellular cycle. Taken togethe
r, this set of information raises the question as
molecules are involved in the processe of cellular
proliferation and
differentiation stimulated by TGF
-
β
. Therefore, this work aims to characterize TGF
responsive molecules through a pho
sphoproteomic approach. For this purpose, total
T. cruzi
epimastigotes (Y strain), incubated or not with TGF
were prepared from parasites in the exponential gro
wth phase. We determined that 5
was the dose that induce
d the highest number of phosphorylation
events. Next, the optimal induction time (1, 5, 15,
30 and 60 minutes) was evaluated
and we concluded that the phosphorylation patterns
from the studied times showed
only subtle differences using 1
-DE analysis, leadi
ng us to evaluate these profiles
DE gels (7cm pH 3
-10 non-
linear). The profiles obtained
showed differences in phosphorylation patterns duri
ng the time
-
which led us to maintain a time
-
kinetics study. Gel images wer
responsive proteins were identified by mass spectr
ometry. We
observed that heat shock proteins, tubulins, dehydr
ogenases, enolases, cyclophilin
A, GrpE, cruzipain, elongation factor
-
1
α
, eukaryotic initiation factor
their phosphorylation and/or expression levels modu
lated by TGF
correlate the function already described in the lit
erature for each protein with their
possible role in intracellular signaling triggered
by TGF
-
β
, in agreement with thei
phosphorylation and/or expression behavior shown in
our analysis. Finally, we
assessed whether the addition of TGF
-
β
to epimastigotes cultures would have some
effect on parasite proliferation. We found that TGF
-
β
addition led to an increase of up
in parasite growth in the first 24 hours of culture
. The data presented here
contributes to the elucidation of the molecular mec
hanisms related to TGF
, providing a source of new potential therapeutic t
argets against
in response to TGF
-
β
is involved in the acute and chronic chagasic
and the activation of its signaling pathway were
shown to be peculiar aspects of patients with chron
ic Chagas disease. Besides its
ease, this cytokine was also observed to
as a regulator of different stages of
T. cruzi
is able to activate
ost cell invasion. In addition, amastigote
, and this event is related to
their capacity to proliferate and differentiate int
o trypomastigote forms at the end of
r, this set of information raises the question as
molecules are involved in the processe of cellular
proliferation and
. Therefore, this work aims to characterize TGF
-
β
sphoproteomic approach. For this purpose, total
epimastigotes (Y strain), incubated or not with TGF
-
β
,
were prepared from parasites in the exponential gro
wth phase. We determined that 5
d the highest number of phosphorylation
events. Next, the optimal induction time (1, 5, 15,
30 and 60 minutes) was evaluated
and we concluded that the phosphorylation patterns
from the studied times showed
ng us to evaluate these profiles
linear). The profiles obtained
-
course under study,
kinetics study. Gel images wer
e analyzed and
responsive proteins were identified by mass spectr
ometry. We
observed that heat shock proteins, tubulins, dehydr
ogenases, enolases, cyclophilin
, eukaryotic initiation factor
-5a and others had
their phosphorylation and/or expression levels modu
lated by TGF
-
β
. We tried to
correlate the function already described in the lit
erature for each protein with their
, in agreement with thei
r
phosphorylation and/or expression behavior shown in
our analysis. Finally, we
to epimastigotes cultures would have some
addition led to an increase of up
in parasite growth in the first 24 hours of culture
. The data presented here
contributes to the elucidation of the molecular mec
hanisms related to TGF
-
β
, providing a source of new potential therapeutic t
argets against Chagas disease
| Identifer | oai:union.ndltd.org:IBICT/oai:www.arca.fiocruz.br:icict/13342 |
| Date | January 2009 |
| Creators | Ferrão, Patrícia Mello |
| Contributors | Jorge, Tânia Cremonini de Araújo, Brandão, Adeílton Alves, Domont, Gilberto Barbosa, Levy, Claudia Masini d`Avila, Lima, Leila de Mendonça, Waghabi, Mariana Caldas |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Source | reponame:Repositório Institucional da FIOCRUZ, instname:Fundação Oswaldo Cruz, instacron:FIOCRUZ |
| Rights | info:eu-repo/semantics/openAccess |
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