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- ItemSomente MetadadadosAlteracoes renais e hemodinamicas durante taquicardia supraventricular sob infusao continua de captopril: estudo experimental em caes anestesiados(Universidade Federal de São Paulo (UNIFESP), 1995) Michelotto, Joao Batista [UNIFESP]
- ItemEmbargoLocalização genômica e subcelular dos membros da família de fosfatidilinositol quinases em Trypanosoma cruzi(Universidade Federal de São Paulo (UNIFESP), 2011-07-27) Oliveira, Priscila de [UNIFESP]; Bahia, Diana [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The identification of signaling molecules and the elucidation of signal transduction processes of Trypanosoma cruzi are required for understanding the host-parasite interaction and physiological processes of the parasite. However, little is yet known about signal transduction and signaling pathways that occur in the parasite, since most efforts focused on signaling pathways that occur in the host cell. Our group recently identified a protein family of lipid kinases - the phosphatidylinositol kinases (PIKS) in the genome of the parasite. The PIKS molecules are evolutionarily conserved, they occur in all eukaryotic cells and are related to biological mechanisms essential for the cell, such as the organization of the cytoskeleton, cytokinesis, cell migration, signal transduction and cell survival. By using bioinformatics tools, the PIKS were classified into five different models (1-5) based on the presence of conserved domains. These genes were mapped on the chromosomes of two different isolates of T. cruzi (G and CL Brener). Surprisingly, the pattern of localization of all PIK genes chromosomal showed a significant polymorphism between the two isolates. By using fluorescence microscopy, six PIK proteins were located at the subcellular level. The subcellular localization of TcTOR1 and 2 (both Model 5) is completely different from that previously observed in Trypanosoma brucei. Unlike TbTOR1, TcTOR1 is excluded from the nucleus and is concentrated in the posterior punctated compartments that coincide with reservosomes, which was confirmed using anti-cruzipain. Reservosomes are endocytic organelles of epimastigotes of Trypanosoma cruzi that store proteins and lipids for future use. TcTOR2, unlike TbTOR2, is dispersed in the cytoplasm, concentrating around the location of TcTOR1. TOR1 and TOR2 have distinct patterns of localization, which is consistent with the regulation of cellular processes as part of two different complexes. Treatment with rapamycin inhibited the growth of epimastigotes, as well as promoted important morphological changes in the parasite. This result coincides with the fact that TOR2 –and not TOR1 - is sensitive to rapamycin, which was previously demonstrated in T. brucei, and it may be a unique feature among trypanosomatids. Furthermore, overexpression of Model 1 (Class III PIK) in T. cruzi led to a reduction in the growth of the parasite when compared to control. The different infective forms of T. cruzi overexpressing the Model 1 showed a considerable variation in their rate of invasion into HeLa cells. These results indicate that Model 1 may play different roles in different infective forms of T. cruzi. The knowledge of these biomolecules at a molecular and cellular levels is a need for deeper understanding of the biology of the parasite and how to intervene in the progression of their life cycle and its relationship with the host cell. Notably, the PIK pathway has been widely acknowledged as an excellent target for drug discovery to combat this pathogen.
- ItemAcesso aberto (Open Access)Proteínas quinases envolvidas na regulação do estresse em Trypanosoma(Universidade Federal de São Paulo (UNIFESP), 2010-03-31) Jesus, Teresa Cristina Leandro de [UNIFESP]; Schenkman, Sergio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)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.
- ItemAcesso aberto (Open Access)TbeIF2K2, uma nova quinase de eIF2alfa associada a membrana da bolsa flagelar do trypanosoma brucei(Universidade Federal de São Paulo (UNIFESP), 2007) Moraes, Maria Carolina Strano [UNIFESP]; Castilho, Beatriz Amaral de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)O controle traducional mediado pela fosforilacao da subunidade alfa do fator de inicio de traducao 2 (eIF2a) e um ponto central para programas de expressao genica induzidos por estresse. Tripanossomatideos, importantes patogenos humanos, apresentam processos de diferenciacao desencadeados pelo contato com os distintos ambientes encontrados em seus insetos vetores e hospedeiros mamiferos, provavelmente representando situacoes de estresse. Trypanosoma brucei, o agente causador da tripanossomiase africana, codifica tres potenciais quinases de eIF2α (TbeIF2Kl-K3). Neste trabalho, nos mostramos que TbeIF2K2 e uma glicoproteina associada a membrana, expressa tanto na forma prociclica quanta na sanguinea. 0 dominio catalitico de TbeIF2K2 fosforila eIF2α de levedura e de mamiferos na Ser51. A quinase tambem fosforila a incomum forma de eIF2α encontrada em tripanossomatideos, especificamente no residuo Thr169, que corresponde a Ser51 em outros eucariotos. 0 eIF2α de T brucei, no entanto, nao e um substrato para GCN2 ou PKR in vitro. 0 dominio regulatorio putativo de TbeIF2K2 nao apresenta nenhuma similaridade de sequencia com as quinases de eIF2α conhecidas. Tanto na forma sanguinea quanto na prociclica, TbeIF2K2 esta localizada principalmente na bolsa flagelar, organela que e o local exclusivo de exo e endocitose nesses parasitas. Ela tambem pode ser detectada em compartimentos endociticos, mas nao em lisossomos, sugerindo que a quinase e reciclada entre os endossomos e a bolsa flagelar. A localizacao de TbeIF2K2 sugere que ela possa funcionar como um sensor do transporte de nutrientes ou proteinas em T brucei, um organismo que depende de mecanismos regulatorios pos-transcricionais para controlar a expressao genica em diferentes situacoes. Essa e a primeira quinase de eIF2α associada a membrana descrita em eucariotos unicelulares