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Previous issue date: 2011-03-03 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The study evaluated the protein and lipid profiles of Rhipicephalus microplus engorged
females after infection by Metarhizium anisopliae, Beauveria bassiana and Fusarium
oxysporum. The treatments were immersion or inoculation of conidial suspension in R.
microplus. The hemolymph was collected 24 and 48 hours after treatment. The cell-free
hemolymph was separated of hemocytes by centrifugation and hemocytes resuspended in
phosphate buffer pH 7.2. The amount of total protein was determined in both fractions of
hemolymph and hemocytes were quantified. The cell-free hemolymph was filtered through a
100 kDa and 10 kDa membranes, and analyzed by electrophoresis and liquid chromatography
(HPLC). The proteome of cell-free hemolymph (treatment by injection) was evaluated by 2DPAGE.
Changes were observed in amount total protein and the amount of hemocytes, but no
difference was observed in the electrophoretic profile (1D-PAGE) of the cell-free
hemolymph. In haemocytes, the entomopathogens reduced the amount of serpins, while F.
oxysporum caused increased. In 2D-PAGE variations were observed in both expression and
presence/absence of protein between the groups. The cell-free hemolymph antimicrobial
activity was tested against Escherichia coli and Staphylococcus aureus and against the fungus
used in the treatment of engorged ticks. The hemolymph collected 48 hours after the
immersion treatment with B. bassiana apresented activity anti-B. bassiana with 48 hours of
evaluation. This hemolymph was subjected to Superose column to HPLC and peak was
collected and analyzed on the analytical column C18. The fractions were collected from the
C18 and its apresented activity anti-B. bassiana, but showed no activity against Candida
albicans. These fractions were analyzed by Maldi-Tof and most of them had in common an
ion with m/z 1,119.5; however, other ions may be involved with this activity antimicrobial.
The lipids present in cell-free hemolymph, in the hemocyte and fat body were extracted and
analyzed by thin layer chromatography (TLC) or HPTLC for neutral lipids and phospholipids.
The classes of neutral lipids in the cell-free hemolymph were cholesterol ester, cholesterol
(CHO) and fatty acids (FA), which have varied depending on the fungus used, type of
treatment and observation time. Phospholipids found were phosphatidylcholine and
phosphatidylethanolamine, its were not significantly altered after the fungal infection. In
hemocytes, the same classes of lipids were found and B. bassiana modified phospholipids,
while M. anisopliae s.l. altered FA and CHO. The fat body showed, in addition to these
classes of neutral lipids, the triglycerides, which increased significantly 48 hours after
inoculation with M. anisopliae s.l. The lipase activity in fat body was measured and it was
demonstrated that increased activity 48 hours after inoculation, mainly in the group inoculated
with Metarhizium. Therefore, the results showed alterations related to the proteins expression
in the hemocytes and the cell-free hemolymph after inoculation with fungi,
immunosuppression of hemocytes and antimicrobial peptides induction after infection with B.
bassiana, besides changes in the lipid profile of R. microplus after infection. However, further
studies are necessary to understand these changes. / O trabalho avaliou os perfis prot?ico e lip?dico de f?meas ingurgitadas de Rhipicephalus
microplus ap?s infec??o com Metarhizium anisopliae s.l., Beauveria bassiana ou Fusarium
oxysporum. Os tratamentos foram imers?o ou inocula??o da suspens?o conidial em R.
microplus. A hemolinfa foi coletada 24 e 48 horas ap?s os tratamentos. O plasma foi separado
dos hem?citos por centrifuga??o e os hem?citos resuspensos em tamp?o fosfato pH 7,2. A
concentra??o de prote?na total foi determinada em ambas as fra??es da hemolinfa e os
hem?citos quantificados. O plasma da hemolinfa foi filtrado em membrana de 100 kDa e 10
kDa, sendo analisados por eletroforese e cromatografia l?quida de alta efici?ncia (CLAE). O
proteoma do plasma da hemolinfa (tratamento por inocula??o) foi avaliado por gel 2D. Foram
observadas varia??es na quantidade de prote?na total de ambas as fra??es da hemolinfa, na
quantidade de hem?citos bem como na intensidade de prote?nas/pept?deos expressos no
plasma da hemolinfa. Nos hem?citos, os entomopat?genos reduziram a quantidade de
serpinas, enquanto F. oxysporum causou aumento. No gel 2D foram observadas varia??es na
express?o bem como na aus?ncia/presen?a de prote?nas entre os grupos. O plasma da
hemolinfa teve sua atividade antimicrobiana testada contra Escherichia coli e Staphylococcus
aureus e contra o fungo utilizado no tratamento das f?meas ingurgitadas. A hemolinfa
coletada 48 horas ap?s o tratamento por imers?o com B. bassiana apresentou atividade anti-B.
bassiana com 48 horas de avalia??o. Esta hemolinfa foi submetida ? coluna Superose de
CLAE e o pico coletado analisado na coluna anal?tica C18. As fra??es coletadas da C18
apresentaram atividade anti-B. bassiana, por?m n?o apresentaram atividade contra Candida
albicans. Essas fra??es foram analisadas por Maldi-Tof e a maioria delas apresentou um ?on
com raz?o m/z 1.119,5; no entanto, outros ?ons podem estar envolvidos com essa atividade
antimicrobiana. Os lip?deos presentes no plasma da hemolinfa, nos hem?citos e no corpo
gorduroso foram extra?dos e analisados por cromatografia em camada delgada (CCD) ou
CCD de alta performance para lip?deos neutros e fosfolip?deos. As classes de lip?deos neutros
encontradas no plasma da hemolinfa foram colesterol-?ster, colesterol (CHO) e ?cidos graxos
(AG), que sofreram altera??es em fun??o do fungo utilizado, tipo de tratamento e tempo de
observa??o. Os fosfolip?deos encontrados foram fosfatidilcolina e fosfatidiletanolamina, que
n?o foram significativamente alterados ap?s a infec??o f?ngica. Nos hem?citos, as mesmas
classes de lip?deos foram encontradas e B. bassiana alterou os fosfolip?deos, enquanto M.
anisopliae s.l. alterou os AG e CHO. O corpo gorduroso apresentou, al?m destas classes de
lip?deos neutros, o triacilglicerol, que aumentou significativamente 48 horas ap?s a inocula??o
com M. anisopliae s.l. A atividade lipase no corpo gorduroso foi mensurada, sendo
evidenciado um aumento 48 horas ap?s a inocula??o, principalmente no grupo inoculado com
Metarhizium. Portanto, os resultados demonstraram altera??es na express?o de prote?nas no
plasma da hemolinfa e nos hem?citos ap?s inocula??o com os fungos, imunossupress?o dos
hem?citos, indu??o de pept?deos com atividade antimicrobiana ap?s infec??o com B.
bassiana, al?m de altera??es no perfil lip?dico de R. microplus ap?s infec??o. No entanto,
maiores estudos s?o necess?rios para o entendimento dessas altera??es.
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:jspui/1073 |
| Date | 03 March 2011 |
| Creators | Angelo, Isabele da Costa |
| Contributors | Bittencourt, V?nia Rita Elias Pinheiro, Chagas, Evelize Folly das, Chagas, Evelize Folly das, Fernandes, Everton Kort Kamp, Atella, Georgia Correa, Silva, M?rcia Regina Soares da, Oliveira, Pedro Lagerblad de |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Programa de P?s-Gradua??o em Ci?ncias Veterin?rias, UFRRJ, Brasil, Instituto de Veterin?ria |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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