Made available in DSpace on 2012-05-07T15:26:25Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 000016.pdf: 2256189 bytes, checksum: 744edbfd2d44afa74c1dcafa9ab7bc9e (MD5) Previous issue date: 2007 / Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Belo Horizonte, MG, Brasil. / Álcool desidrogenases (ADH) pertencem a um grupo de enzimas que catalizam aoxidação reversível de etanol a acetaldeído, com conseqüente redução de NAD. O gene TcADHque codifica a ADH do T. cruzi, foi selecionado pela metodologia microarray por apresentarum nivel de transcrição 4 vezes menor na população do parasito com resistência induzida invitro ao BZ (17LER) quando comparado com seu par sensível (17WTS). A TcADH codificauma proteína de 393 aminoácidos que apresenta um domínio conservado iron-containingalcohol desidrogenase . A análise da estrutura primária mostrou que a TcADH é mais parecidacom as ADHs de organismos procariotos do que com seus ortólogos identificados emLeishmania. A atividade enzimática da TcADH foi menor nas cepas de T. cruzi com resistênciainduzida in vitro ao BZ (17 LER). Análises de Western blot mostraram que o anticorpopoliclonal anti-TcADH reconheceu um polipeptídeo de 41,7 kDa em todas as cepas do T. cruzianalisadas. O nível de expressão desse polipeptídeo foi 2 x menor na cepa do T. cruzi 17LERquando comparado com seu par 17WTS, confirmando os dados da atividade enzimática.Ensaios de imunolocalização por microscopia confocal revelaram que a enzima TcADH estápresente no cinetoplasto do parasito. Análise de Northern blot mostrou que a sonda do geneTcADH reconheceu um transcrito de 1,9 Kb com a mesma intensidade para todas amostras doT. cruzi analisadas com exceção da população 17LER que foi 2 vezes menor. Análisequantitativa por PCR em tempo real (qPCR) também mostrou um nível de mRNA 2,5 vezesmenor na população 17LER. Quantificação do número de cópias desse gene por qPCR, mostrouser o mesmo para todas as cepas do T. cruzi analisadas. Neste trabalho, o gene TcADH foicaracterizado pela primeira vez em T. cruzi e foi observado uma menor expressão da enzimaTcADH na população do T. cruzi com resistência induzida in-vitro ao BZ / Alcohol dehydrogenases (ADH) belongs to a group of enzymes that catalyze the reversible oxidation of ethanol to acetaldehyde, with consequent reduction of NAD. The TcADH gene that codes for the T. cruzi ADH of, was selected by microarray methodology as presenting a transcription level 4 four-fold lower in the T. cruzi population with in vitro- induced resistance to BZ (17 LER) than in the wild-type (17 WTS). The TcADH codes a protein of 393 aminoacids that presents a conserved “iron-containing alcohol desidrogenase” domain. Analysis of the primary structure showed that the TcADH is more similar to the of ADHs of procariotes than ADHs of other organisms as Leishmania. Assays of enzymatic activity showed that TcADH activity was lower in 17LER than in 17WTS. Western blot analysis of T.cruzi protein extracts probed with anti- TcADH rabbit polyclonal serum revealed a 41.7 kDa polypeptide present in all samples. The level of this polypeptide was similar for all samples except to 17LER that was nearly two-fold lower than the wild-type 17 WTS. Assays imunolocalization by confocal microscopy showed that the TcADH enzyme is present in parasite. Northern blot analyses showed that TcADH levels were 2.0 times lower in 17LER than in 17WTS. Real- time PCR confirmed our finding that TcADH transcription levels were lower in 17 LER than in 17 WTS. In contrast, we detected no differences in TcADH trancription level among other T. cruzi samples. Using real- time PCR, we found that the number of TcADH gene copies was similar in all samples. This difference of expression was confirmed by the. Our data show that a decreased expression of the TcADH enzyme was found only in a T. cruzi population with induced in vitro resistance to BZ. In this work, the TcADH gene was characterized for the first time in T. cruzi and was observed a smaller expression of the TcADH enzyme in a T. cruzi population with in vitro-induced resistance to BZ.

A replicação do DNA tem início nas origens de replicação que são licenciadas na transição das fases M/G1, pelo complexo de pré-replicação (CPR), e ativadas apenas na fase S. Existem diversas origens de replicação no genoma, mas apenas parte destas origens é disparada em diferentes momentos de S, havendo assim origens early (disparadas no início de S) e late (disparadas mais tardiamente). Em trypanosomas as origens de replicação são reconhecidas por um CPR formado por Orc1/Cdc6 e pelo complexo MCM2-7. Em T. cruzi observamos que existem dois mecanismos diferentes para controlar a replicação do DNA. Durante o ciclo celular da forma epimastigota, as proteínas do CPR são sempre expressas e ligadas ao DNA, mas durante o ciclo de vida Orc1/Cdc6 se liga ao DNA apenas nas formas que replicam, e Mcm7 não é expressa nas que não replicam. Também foi analisado o perfil das forquilhas de replicação em T. brucei utilizando a técnica de SMARD onde vimos que a velocidade da forquilha é semelhante a dos demais eucariontes, além de encontrarmos a primeira origem de replicação late. / The DNA replication starts at the origins of replication, which are licensed at M/G1 transition, by the pre replication complex (PRC), and are activated just at S phase. There are many origins of replication along genome, but some of them are fired at different moments of S phase. So there are early and late origins fired at the beginning or later in S phase, respectively. The PRC of trypanosomes is composed of Orc1/Cdc6 and Mcm2-7. We could observe that in T. cruzi there are two distinct ways to control DNA replication. Whereas in epimastigote cell cycle the PRC are expressed and bound to DNA in all phases, during T. cruzi life cycle Orc1/Cdc6 is bound to DNA only in replicative forms and Mcm7 is absent in the non-replicative forms. We also analyzed the fork profile in T. brucei through SMARD technique. We found that the speed of replication fork is similar from other eukaryotes and that different replication origins are fired every cell cycle. Finally, we found a new origin of replication that is the first late origin described in this organism.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

This study examines ultrastructural changes occurring in seven- and fourteen-day populations of Trypanosorfa lewisi when 60 mg sodium salicylate is administered to the host. These changes were related to the host-immune response. Seven-day trypanosomes showed approximately 10 posterior volutin granules. Seven-day trypanosomes whose host had received salicylate exhibited. 30 to 40 volutin granules, and their posterior tip exhibited volutin granules in high numbers sometimes excluding other cellular elements. Fourteen-day trypanosones showed fewer volutin granules than seven-day, salicylate-treated ones. Salicylate treatment caused no additional ultrastructural alterations. Thus the volutin granules are not linked to the reproduction inhibiting antibody (ablast in) but may be involved in the formation of the trypanocidal antibodies.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

No presente trabalho verificamos o papel dos Leucotrienos na modulação da resposta imune durante a fase aguda da infecção experimental pelo Trypanosoma cruzi, usando como modelo camundongos deficientes da enzima 5-lipoxigenase (5-LOko). Os nossos dados demonstram que camundongos infectados pelo T. cruzi produzem metabólitos do ácido aracdônico como PGE2, LTB4 e LTC4. Comparados aos animais controles, os animais 5-LOko apresenta parasitemia mais tardia e menor, tem menor parasitismo tissular, menor infiltrado de células inflamatórias no coração e musculatura esquelética e apresenta menor taxa de mortalidade durante a fase aguda, indicando que animais deficientes de leucotrienos são mais resistentes a infecção pelo parasito. Animais 5-LOko está relacionado com a manutenção de números elevados de células F4/80+ e redução de células CD11b+ durante a infecção e menor número de células T ativadas expressando os marcadores CD4+CD69+, CD4+CD25+, CD4+CD44+ e CD8+CD69+, números inalterados de células T regulatórias CD4+CD25+GITR+ e menor produção de anticorpos parasito-específicos do isotipo IgG2a. O controle eficiente de parasitas por animais 5-LOko está associado ao aumento de células Gr-1+ e CD11c+GR-1+, produção aumentada IL-12, IFN-g, e produzirem menos PGE2, IL-10, ao contrario, animais controles, incapazes de controlar parasitas circulantes, produzem mais PGE2 e IL-10 e menos IL-12 e IFN-g. A baixa mortalidade de animais 5-LOko correlaciona com a produção de PGE2 e IL-10, produzir muita IL-12 e menos IFN-g e NO e baixíssima parasitemia. A mortalidade maior de animais controles envolve a produção IFN-g e altos níveis de LTB4, LTC4, NO e ausência de IL-10, IL-1b, PGE2 e números elevados de parasitas circulantes. Ainda macrófagos de animais 5-LOko apresentam maior capacidade de adesão/internalização de tripomastigotas e alta atividade tripanocida por mecanismo independente da geração de NO. Estes dados em conjunto demonstram que mediadores lipídicos produzidos pela enzima 5-lipoxigenase como LTB4 e LTC4 modulam negativamente a capacidade dos camundongos para geração de uma resposta imune capaz de controlar os parasitos durante a fase aguda da infecção pelo T. cruzi. / Accumulating studies have indicated that 5-lipoxigenase (5-LO) converted lipid mediators as leukotrienes acts modulating the host immune response against infectious diseases. The precise role of leukotrienes during the protozoan infection is unknown. In this work we evaluate the role of leukotrienes during the acute phase of Trypanosoma cruzi infection using as model the 5-lypoxigenase deficient mice (5-LOko). Our results show that PGE2, LTB4 and LTC4 are produced during the Trypanosoma cruzi infection. 5-LOko infected mice are more resistant than control mice as judge by the lower parasitemia, decreased number of parasite nest and inflammatory cells in the heart and skeletal muscle and low rate of mortality. The resistance of 5-LOko mice is associated with the increased capacity of spleen cells to produce cytokines as IL-12 and IFN-g; sustained capacity to produce detectable levels of IL-10 and PGEe and low NO serum levels than control mice. In contrast, the wild type mice are extremely susceptible and are unable to control parasites efficiently. The susceptibility is associated with increased levels of IL-10 and PGE2 and low IL-12 and IFN-g production. The high mortality rate in wild type mice is related with high LTB4, LTC4 and NO levels and bias to produce only type 1 cytokines. Also we shown that resistant 5-LOko mice present increased number of spleen cells expressing GR-1+, GR-1+CD11c+, F4/80+ and lower numbers o spleen cells expressing CD4+CD69+, CD4+CD25+, CD4+CD44+, CD8+CD69+ and CD11b+ and low serum levels of parasite-specific IgG2a than wild type mice and do not present alteration in TREG expressing CD4+CD25+GITR+. Importantly, IFN-g and- LPS activated macrophage from 5-LOko mice but not from wild type mice, present high capacity to recognize typomastigotes, internalize them and strong capacity to kill intracellular parasite as NO independent pathway. The results implicate that high levels leukotrienes, NO and pure type 1 cytokines production is associated to susceptibility to parasite. In contrast, leukotrienes deficiency led to an balanced immune response with relative high levels of type 1 cytokines and relative low levels of NO, type 2 cytokines and PGE2 that efficiently control the parasites. Also indicate that 5-lipoxigenase converted lipid mediators contribute negatively to generation of an effective immune response during the acute phase of T. cruzi infection.

O gênero Trypanosoma compreende flagelados capazes de infectar várias espécies de mamíferos e é transmitida por vários grupos de invertebrados. A ordem Chiroptera pode ser infectada pelos subgêneros Herpetosoma, Schizotrypanum, Megatrypanum e Trypanozoon. Neste estudo, foi possível descrever a diversidade de tripanossomas de morcegos, inferindo as relações filogenéticas entre os tripanossomas e os morcegos capturados na área de influência da Hidrelétrica de Belo Monte, localizada na Amazônia Brasileira. Tripanossomas de morcegos foram isolados por hemocultura, e a filogenia molecular foi baseada na subunidade menor do gene ribossômico (SSU rDNA) e de glicosomal-3-fosfato desidrogenase (gGAPDH). A caracterização morfológica incluiu microscopia eletrônica de luz e de varredura. Além das espécies já descritas, T. cruzi e T. cruzi marinkellei, foi possível identificar uma espécie não classificada, Trypanosoma sp., identificado em um morcego da espécie Pteronotus parnellii. Árvores filogenéticas usando combinado SSU rDNA e conjuntos de dados em cluster gGAPDH, confirmaram que esta espécie de tripanossoma de morcego, era muito divergente de outras espécies de tripanossomas. Através do posicionamento filogenético, foi possível classificar os tripanossomas deste morcego como uma espécie não classificada. Os tripanossomas isolados de morcegos na área de influência da Hidrelétrica de Belo Monte foram identificados através de análise filogenética como T. cruzi marinkellei, T. cruzi (TCI e TCbat) e em Pteronotus parnellii uma espécie não classificada e posicionada no Clado T. cruzi. Os dados obtidos neste estudo atentam para a diversidade subestimada de parasitas que infectam morcegos nos diferentes biomas brasileiros / The Trypanosoma comprises flagellates able to infect several mammalian species and is transmitted by several groups of invertebrates. The order Chiroptera can be infected by the subgenera Herpetosoma, Schizotrypanum, Megatrypanum and Trypanozoon. In this study, we described the diversity of bats trypanosomes, inferring the phylogenetic relationships among the trypanosomes from bats caught Belo Monte Hydroeletric area (Brazilian Amazonia). Trypanosomes from bats were isolated by haemoculture, and the molecular phylogeny based on small subunit rDNA (SSU rDNA) and glycosomal-3-phosphate dehydrogenase (gGAPDH) gene sequences. Morphological characterization included light and scanning electron microscopy. Besides the species already described, T. cruzi and T. cruzi marinkellei, were identify an unclassified species, Trypanosoma sp., identify into a bat species Pteronotus parnelliii. Phylogenetic trees using combined SSU rDNA and gGAPDH data sets clustered the trypanosomes of bats, which were highly divergent from other trypanosome species. The phylogenetic position made it possible to classify the trypanosomes from bats as a separate species. The bat trypanosomes isolates in Belo Monte Hydroeletric area were identified by phylogenetic analysis like as T. cruzi marinkellei, T. cruzi (TCI and TCbat) and into a bat species Pteronotus parnellii was identified a species unclassified. The diversity of bat trypanosomes in Brazil is underestimated and new studies should be conducted in brazilian biomes

O gênero Trypanosoma compreende flagelados capazes de infectar várias espécies de mamíferos e é transmitida por vários grupos de invertebrados. A ordem Chiroptera pode ser infectada pelos subgêneros Herpetosoma, Schizotrypanum, Megatrypanum e Trypanozoon. Neste estudo, foi possível descrever a diversidade de tripanossomas de morcegos, inferindo as relações filogenéticas entre os tripanossomas e os morcegos capturados na área de influência da Hidrelétrica de Belo Monte, localizada na Amazônia Brasileira. Tripanossomas de morcegos foram isolados por hemocultura, e a filogenia molecular foi baseada na subunidade menor do gene ribossômico (SSU rDNA) e de glicosomal-3-fosfato desidrogenase (gGAPDH). A caracterização morfológica incluiu microscopia eletrônica de luz e de varredura. Além das espécies já descritas, T. cruzi e T. cruzi marinkellei, foi possível identificar uma espécie não classificada, Trypanosoma sp., identificado em um morcego da espécie Pteronotus parnellii. Árvores filogenéticas usando combinado SSU rDNA e conjuntos de dados em cluster gGAPDH, confirmaram que esta espécie de tripanossoma de morcego, era muito divergente de outras espécies de tripanossomas. Através do posicionamento filogenético, foi possível classificar os tripanossomas deste morcego como uma espécie não classificada. Os tripanossomas isolados de morcegos na área de influência da Hidrelétrica de Belo Monte foram identificados através de análise filogenética como T. cruzi marinkellei, T. cruzi (TCI e TCbat) e em Pteronotus parnellii uma espécie não classificada e posicionada no Clado T. cruzi. Os dados obtidos neste estudo atentam para a diversidade subestimada de parasitas que infectam morcegos nos diferentes biomas brasileiros / The Trypanosoma comprises flagellates able to infect several mammalian species and is transmitted by several groups of invertebrates. The order Chiroptera can be infected by the subgenera Herpetosoma, Schizotrypanum, Megatrypanum and Trypanozoon. In this study, we described the diversity of bats trypanosomes, inferring the phylogenetic relationships among the trypanosomes from bats caught Belo Monte Hydroeletric area (Brazilian Amazonia). Trypanosomes from bats were isolated by haemoculture, and the molecular phylogeny based on small subunit rDNA (SSU rDNA) and glycosomal-3-phosphate dehydrogenase (gGAPDH) gene sequences. Morphological characterization included light and scanning electron microscopy. Besides the species already described, T. cruzi and T. cruzi marinkellei, were identify an unclassified species, Trypanosoma sp., identify into a bat species Pteronotus parnelliii. Phylogenetic trees using combined SSU rDNA and gGAPDH data sets clustered the trypanosomes of bats, which were highly divergent from other trypanosome species. The phylogenetic position made it possible to classify the trypanosomes from bats as a separate species. The bat trypanosomes isolates in Belo Monte Hydroeletric area were identified by phylogenetic analysis like as T. cruzi marinkellei, T. cruzi (TCI and TCbat) and into a bat species Pteronotus parnellii was identified a species unclassified. The diversity of bat trypanosomes in Brazil is underestimated and new studies should be conducted in brazilian biomes

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser. Karolinska International Research Training Program (KIRT).

Two aspects of trypanosomiasis have been investigated in this study. First, molecular methods were applied to the diagnosis of T.evansi in camels in South Libya. The aim of the study was to determine if FTA card blood sampling and PCR amplification could detect parasites and this be used as tool for diagnosis and epidemiology. Targeted samples of 70 camels were identified on the basis of symptoms of infection and blood was collected on FTA cards. PCR primers and conditions for the amplification of T.evansi DNA were developed on the basis of the literature and a positive control clone grown in the laboratory. The assay found 84.3% of camel samples positive using TBR primers (177bp amplicon) and ITS nested primers (611-1513bp amplicons). This result demonstrated that Surra is endemic in this area, and that T.evansi was the species that was involved. The ITS and TBR loci in the parasites identified in Libya were almost identical to those previously reported in the genbank database, though with some polymorphisms. Dullness and emaciation were the clinical signs of camels infected by trypanosomes, and these two symptoms were significantly related to the 1200bp ITS nested PCR amplicon. These two symptoms can be thus used as a sign an initial diagnosis of T.evansi infection in camels. The second aspect of trypanosomiasis studied was the occurrence of endotoxaemia in infection. The first part of this research investigated endotoxin levels in clinical human African trypasnosomiasis using the Limulus Amoebocyte lysate assay. Endotoxin levels were significantly increased over control individuals in the plasma of T.b.rhodesiense patients. This endotoxaemia was unrelated to infection duration, parasitaemia or clinical stage but resolved after clearance of parasites by drug treatment. In the cerebrospinal fluid there was no significant difference in endotoxin level between early and late stage cases and no relationship to parasite loads. It is argued on the basis of the data that endotoxaemia in trypanosomiasis most likely results from increases in permeability of the gut to endotoxins from gram negative enter bacteria. This conclusion was further supported from a study using cell culture adapted T.brucei and secreted products which gave no evidence of any endotoxin activity. Also samples of an acute experimental mouse infection with T.brucei gave no endotoxin activity, suggesting that this phenomenon requires a more chronic infection in mice. No relationships were found between plasma or CSF endotoxin levels to neurological signs of infection. However the presence of a gross inflammatory clinical symptom, splenomegaly, was associated with endotoxaemia and the concentrations of 3 plasma cytokines associated with the immune response in trypanosome infection were associated with correlated to plasma endotoxin levels. In order to determine the nature of the endotoxin activity, a biosensor cell assay for LPS was used, based on human embryonic kidney cells transfected with TLR4/MD3 and a NF-κB induced alkaline phosphatase reporter gene. This assay revealed low or undetectable levels of LPS in clinical samples from T.b.rhodesiense patients, in mouse samples from T.b.brucei infections and in vitro cultured trypanosomes. This suggests that either the endotoxin activity detected using the LAL assay is an unconventional endotoxin signalling via a TLR4 independent pathway or that the human plasma was in some way toxic to the reporter cell and this requires further investigation. In conclusion, this study has provided the first clear evidence of an association of endotoxaemia and inflammatory responses in clinical African trypansomiasis and helps resolve the question of whether endotoxaemia is a parasite or host-microbiota related phenomenon.