Endocytosis is a biological event well described in
where macromolecules are
cytofarynx complex and
organelles found at the posterior region of the
and contain lysosomal enzymes, such as cruzipain.
produce polyclonal or monoclonal antibodies (mAbs) against proteins identified in
proteomic analyzes of reservosomes (cruzipain and Tc
to label organelles of the endocytic pathway and
amastigotes. Recombinant proteins were expressed in
into Balb/c mice. Reactivity of the anti
extracts of T. cruzi and recombinant protein
antibodies anti-TcHIP showed this protein in the Golgi apparatus
confirmed by co-localization with GFP
recombinant cruzipain recognized the
epimastigotes. For production of mAbs, splenocytes
with recombinant cruzipain were fused with the P3X63Ag8.653 myeloma cell line
(ATCC CRL-1580). Hybridomas were
immunofluorescence. The most stable hybridoma was selected for limiting dilution and
one clone against recombinant cruzipain (mAb CZP
reservosomes. This antibody
co-localization of uptaken transferrin with cruzipain. It was also possible to demonstrate
for the first time by flow cytometry
of Trypanosoma cruzi
Trypanosoma cruzi
ingested via the flagellar pocket and/or
then targeted to reservosomes. Reservosomes are
cell body that store ingested molec
Aim of this dissertation
TcHIP), and use the
to study endocytosis in
E. coli, purified and inoculated
eactivity anti-sera was confirmed by western blot
proteins. Immuno-localization using polyclonal
of T. cruzi
GFP-TcRab7. Polyclonal antibodies against
protein in the Golgi and reservosomes of
obtained from a mouse immunized
then screened by ELISA, western blot and
CZP-315.D9) was obtained, specific
was then used as a tool to investigate in amastigotes the
the endocytic activity in T. cruzi amastigotes. / Approved for entry into archive by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-12-04T12:19:07Z (GMT) No. of bitstreams: 1
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Previous issue date: 2014 / Fundação Oswaldo cruz, Instituto Carlos Chagas. Curitiba, PR, Brasil / A endocitose é um evento biológico já bem descrito em formas epimastigotas de
Trypanosoma cruzi, onde macromoléculas são internalizadas através da bolsa flagelar
e/ou do complexo citóstoma/citofaringe e direcionadas aos reservossomos.
Reservossomos são grandes organel
estocam moléculas ingeridas e
cruzipaína. O objetivo desta dissertação foi produzir anticorpos policlonais ou
monoclonais (mAbs) contra proteínas identificada
(cruzipaína e TcHIP), e utilizá
estudos de endocitose em formas amastigotas de
foram expressas em E. coli
reatividade dos anti-soros foi confirmada por
cruzi e contra proteína recombinante. Imunolocalização utilizando anticorpos
policlonais anti-TcHIP mostrou que esta proteína está presente no complexo de Golgi
em T. cruzi, o que foi confirmado por co
anticorpos policlonais obtidos contra cruzipaína recombinante demonstraram esta
proteína no Golgi e em reserv
mAbs, esplenócitos de um camundongo imunizado com cruzipaína recombinante foram
fusionados com células de mieloma da linhagem P3X63Ag8.653 (ATCC CRL
Os hibridomas foram triados p
hibridoma mais estável foi
cruzipaína recombinante (mAb CZP
Este anticorpo foi utilizado como ferramenta pa
transferrina ingerida com a cruzipaína em formas amastigotas. Foi também possível
demonstrar pela primeira vez por citometria de fluxo a atividade endocítica em formas
amastigotas de T. cruzi.
Trypanosoma cruzi, organelas localizadas na região posterior do parasito que
contém enzimas lisossomais, como por exemplo a identificadas na proteômica de reservossomos utilizá-las como marcadores de organelas da via endocítica e em
T. cruzi. Proteínas recombinantes
coli, purificadas e inoculadas em camundongos Balb/c. A western blot contra extrato total de, co-localização com TcRab7-GFP. Por outro lado,
reservossomos de epimastigotas de T. cruzi. / Endocytosis is a biological event well described in
where macromolecules are
cytofarynx complex and
organelles found at the posterior region of the
and contain lysosomal enzymes, such as cruzipain.
produce polyclonal or monoclonal antibodies (mAbs) against proteins identified in
proteomic analyzes of reservosomes (cruzipain and Tc
to label organelles of the endocytic pathway and
amastigotes. Recombinant proteins were expressed in
into Balb/c mice. Reactivity of the anti
extracts of T. cruzi and recombinant protein
antibodies anti-TcHIP showed this protein in the Golgi apparatus
confirmed by co-localization with GFP
recombinant cruzipain recognized the
epimastigotes. For production of mAbs, splenocytes
with recombinant cruzipain were fused with the P3X63Ag8.653 myeloma cell line
(ATCC CRL-1580). Hybridomas were
immunofluorescence. The most stable hybridoma was selected for limiting dilution and
one clone against recombinant cruzipain (mAb CZP
reservosomes. This antibody
co-localization of uptaken transferrin with cruzipain. It was also possible to demonstrate
for the first time by flow cytometry
of Trypanosoma cruzi
Trypanosoma cruzi
ingested via the flagellar pocket and/or
then targeted to reservosomes. Reservosomes are
cell body that store ingested molec
Aim of this dissertation
TcHIP), and use the
to study endocytosis in
E. coli, purified and inoculated
eactivity anti-sera was confirmed by western blot
proteins. Immuno-localization using polyclonal
of T. cruzi
GFP-TcRab7. Polyclonal antibodies against
protein in the Golgi and reservosomes of
obtained from a mouse immunized
then screened by ELISA, western blot and
CZP-315.D9) was obtained, specific
was then used as a tool to investigate in amastigotes the
the endocytic activity in T. cruzi amastigotes.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.arca.fiocruz.br:icict/9037 |
| Date | January 2014 |
| Creators | Martin Batista, Cassiano |
| Contributors | Perdigão Fragoso, Stênio, Dallagiovana Muniz, Bruno, de Freitas Buchi, Dorly, Eger, Iriane, Soares, Maurílio José |
| 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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