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Produção de anticorpos contra TcHIP e cruzipaína de Trypanosoma Cruzi com aplicação no estudo de endocitose em amastigotasMartin Batista, Cassiano January 2014 (has links)
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
Dissertação Cassiano Martin Batista (1).pdf: 5056433 bytes, checksum: 4df99de041c7ead17fee43aacb621dc6 (MD5) / Made available in DSpace on 2014-12-04T12:19:08Z (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.
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Proteínas quinases envolvidas na regulação do estresse em Trypanosoma / Protein kinases involved in stress regulation in TrypanosomaJesus, Teresa Cristina Leandro de [UNIFESP] 31 March 2010 (has links) (PDF)
Made available in DSpace on 2015-07-22T20:50:40Z (GMT). No. of bitstreams: 0
Previous issue date: 2010-03-31 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Conselho Nacional de Pesquisas (CNPq e INV) / National Institute of Health (NIH) / Protozoários do gênero Trypanosoma possuem um complexo ciclo de vida, alternando entre hospedeiros vertebrados e invertebrados. A adaptação a essas diversas condições ambientais necessita de rápidas mudanças na expressão gênica para preencher os requerimentos metabólicos ou morfológicos para sobrevivência. Muito pouco se sabe sobre os mecanismos que controlam estas transformações e sobre as vias de sinalização celular implicadas. Como nestes organismos o controle da expressão gênica ocorre ao nível pós transcricional, decidimos neste trabalho estudar a função de proteínas quinases envolvidas no controle da síntese protéica e no crescimento destes parasitas. Em diversos eucariotos a proteína quinase TOR (Target Of Rapamycin) está envolvida no controle da síntese protéica e crescimento celular frente à disponibilidade de nutrientes ou fatores de crescimento. Por análise de seqüências de T. brucei disponíveis nos bancos de dados do genoma desses parasitas encontramos quatro candidatos para TOR (TbTOR1, TbTOR2, TbTOR-like1 and TbTOR-like 2). Dois complexos TOR em T. brucei (TbTORC1 e TbTORC2) foram descritos previamente. No primeiro capítulo desta tese estudamos: TbTOR-like 1 e a comparamos com TbTOR2. TbTOR-like 1 não se encontra em nenhum dos complexos TORC e possui um domínio PDZ não encontrado nas outras TORs de T. brucei ou de outros eucariotos. Ela se localiza em grânulos no citosol que após estresse hiperosmótico migram para a periferia celular. Depleção de TbTOR-like 1 causa uma inibição progressiva do crescimento celular, gerando células de tamanho maior que se acumulam na fase S/G2 do ciclo celular. Estas células também apresentam um aumento no número de acidocalcissomos assim como aumentos nos níveis de polifosfato e pirofosfato. Estes dados indicam que TbTOR-like1 parece estar envolvida no controle do crescimento celular e na biogênese de acidocalcissomos respondendo a variações osmóticas do meio. No segundo capítulo da tese estudamos proteínas quinases envolvidas no controle da síntese protéica através da fosforilação da subunidade  do fator de iniciação eucariótico 2 da tradução (eIF2α. Estas quinases são ativadas por distintos tipos de estresse. T. brucei codifica para três potenciais proteínas quinases de eIF2α (TbeIF2K1, K2 e K3). Estudamos mais especificamente a K2. Mostramos que ela é uma glicoproteína transmembrânica localizada na região da bolsa flagelar em ambas as formas de T. brucei e nos compartimentos endossomais de Trypanosoma cruzi. Estes compartimentos endossomais são denominados de reservossomos e se formam apenas no estágio do parasita que vive no lúmen do tubo digestivo do inseto vetor. Este fato sugere que em ambos os parasitas esta proteína quinase possa estar funcionando como um sensor no transporte de nutrientes e proteínas. De maneira geral revelamos a existência de pelo menos dois mecanismos pelos quais os tripanossomas percebem e resistem às modificações ambientais durante seu ciclo de vida. / Protozoa of the genus Trypanosoma have a complex life cycle alternating between vertebrate and invertebrate hosts. The adaptation to different environmental conditions requires rapid changes in gene expression to fill up the morphological and metabolic requirements for survival. Very little is known about the mechanisms that control these changes and the signaling pathways involved. As in these organisms the control of gene expression occurs at post-transcriptional level, in this work we decided to investigate the function of protein kinases involved in the control of protein synthesis and growth of these parasites. In several eukaryotes TOR (target of rapamycin) protein kinases are involved in protein synthesis control and cell growth in response of the availability of nutrients or growth factors. By searching T. brucei genomic database we found four candidates for TOR (TbTOR1, TbTOR2, TbTOR-like1 and TbTOR-like 2). Two TOR complexes were previously described in T. brucei (TbTORC1 and TbTORC2). In the first chapter of this thesis we study: TbTOR-like 1 and compared it with TbTOR2. TbTOR-like 1 is not present in any of the TORC complexes and has a PDZ domain not found in any of other TORs of T brucei, or other eukaryotes. It is located cytosolic granules that migrate to the cell periphery after hyperosmotic stress. Depletion TbTOR-like 1 causes a progressive inhibition of cell growth, generating enlarged cells that accumulate in S/G2 phase of the cell cycle. These cells also show increased number of acidocalcisomes and augmented levels of polyphosphate and pyrophosphate. These data indicate that TbTOR-like seems to be involved in controlling cell growth and biogenesis of acidocalcisomes responding to osmotic changes in the medium. In the second chapter of the thesis we studied protein kinases involved in protein synthesis control through the phosphorylation of the  subunit of the eukaryotic translation initiation factor 2 (eIF2α).These kinases are activated by different types of stress. T. brucei encodes three potential eIF2α protein kinases (TbeIF2K1, K2 and K3). We studied more specifically the K2. We showed that it is a transmembrane glycoprotein located in the region of the flagellar pocket in both forms of T. brucei, and in the endosomal compartments of Trypanosoma cruzi. These endosomal compartments are known as reservosomes and they are formed only in the parasite’s stage that li ves in the digestive tract lumen of the insect vector. This fact suggests that in both parasites this protein kinase may be acting as a sensor in the transport of nutrients and proteins. In conclusion we revealed the existence of at least two mechanisms by which trypanosomes perceive and resist to environmental changes during their life cycle. / TEDE / BV UNIFESP: Teses e dissertações
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