Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-05-23T17:52:57Z No. of bitstreams: 1 aline_f_galvao_ioc_bp_0012_2010.pdf: 3755366 bytes, checksum: 56aff28b3b15de796039925e6364d082 (MD5) / Made available in DSpace on 2012-05-23T17:52:58Z (GMT). No. of bitstreams: 1 aline_f_galvao_ioc_bp_0012_2010.pdf: 3755366 bytes, checksum: 56aff28b3b15de796039925e6364d082 (MD5) Previous issue date: 2010 / CNPq/Fiocruz - FAPERJ/Fiocruz / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / A esquistossomose é uma das doenças parasitárias mais prevalentes no mundo, sendo a quimioterapia com praziquantel (PZQ) a principal estratégia adotada para seu controle. No entanto, faltam informações sobre o impacto da quimioterapia com PZQ sobre a infecção por Schistosoma mansoni nas condições enfrentadas pelos programas de controle, e ainda não há prova de princípio sobre o melhor regime de dosagem a ser utilizado. Um ensaio clínico randomizado duplo-cego foi realizado com o objetivo de avaliar o impacto do tratamento em dose única de PZQ 60 mg/kg na infecção por S. mansoni comparado à dose padrão de PZQ 40 mg/kg em adolescentes do município de São Lourenço da Mata. Um inquérito coproscópico preliminar selecionou indivíduos para a triagem e aqueles que se adequaram aos critérios de inclusão/exclusão foram recrutados e tratados com dose única de 40 mg/kg ou 60 mg/kg de PZQ. Inquéritos coproscópicos de acompanhamento foram realizados aos 21, 180 e 360 dias pós-tratamento. A prevalência e a intensidade de infecção foram comparadas nos quatro momentos do estudo nos dois grupos de tratamento utilizando tabelas de contingência (Qui-quadrado ou teste exato de Fisher) e ANOVA. A influência de outras variáveis no estudo foi avaliada através da análise de regressão logística. Um levantamento malacológico foi realizado para verificar a infecção natural da espécie hospedeira local, Biomphalaria straminea. A análise da distribuição espacial da infecção nos dois grupos antes e depois (180 e 360 dias) do tratamento foi realizada pela estimativa de densidade de kernel para a detecção de aglomerados de casos. Ao todo, 123 adolescentes (10-19 anos) de São Lourenço da Mata, PE, com mais de 100 ovos por grama de fezes foram examinados nos quatro momentos do estudo. Embora todos os pacientes estivessem negativos 21 dias após o tratamento, 17,9% e 30,9% estavam positivos após 180 e 360 dias, respectivamente. Tanto a prevalência quanto a intensidade de infecção após o tratamento foram significativamente maiores (P < 0,05) no regime de 40 mg/kg do que no de 60 mg/kg. Os sujeitos do sexo masculino e os que foram tratados com PZQ 40 mg/kg tiveram uma chance significativamente maior de estarem positivos 360 dias pós-tratamento. As taxas de infecção natural mensais de B. straminea variaram de 0 a 2,2%. Os aglomerados de prevalência de infecção aos 180 e aos 360 dias tiveram maior densidade e extensão nos sujeitos tratados com 40 mg/kg do que nos tratados com 60 mg/kg. Recomenda-se a dose de PZQ 60 mg/kg para o tratamento dos portadores da infecção por S. mansoni em áreas endêmicas no Brasil, já que, embora tanto a terapia com dose única de PZQ 40 mg/kg quanto com 60 mg/kg tenham tido um impacto alto e comparável na infecção 21 dias após o tratamento, os sujeitos que receberam a dose maior foram capazes de manter níveis significativamente mais baixos tanto de prevalência quanto de intensidade de infecção por até um ano após o tratamento. Os resultados espaciais confirmam os dados parasitológicos e reforçam a necessidade de que medidas preventivas auxiliares sejam integradas com a quimioterapia para se obter um impacto maior sobre o controle da doença. / Schistosomiasis is one of the most prevalent parasitic diseases in the world, and chemotherapy with praziquantel (PZQ) is the main strategy for its control. However, information about the impact of chemotherapy with PZQ on Schistosoma mansoni infection under conditions faced by control programmes is missing, and there is no proof of principle about the best dosing regimen to be used. A randomized, double- blind clinical trial was conducted to evaluate the impact of treatment with the single dose 60 mg/kg PZQ on S. mansoni infection compared to the standard dose of 40 mg/kg PZQ in adolescents from the municipality of São Lourenço da Mata. A preliminary parasitological survey selected individuals for screening and those who filled the inclusion/exclusion criteria were recruited and treated with either 40 mg/kg or 60 mg/kg single dose PZQ. Follow-up stool surveys were performed at 21, 180 and 360 days post-treatment. Prevalence and intensity of infection were compared between treatment groups in the four assessments using contingency tables analysis and Analysis of Variance, respectively. The influence of other variables in the study was assessed by logistic regression analysis. A snail survey was conducted to verify the natural infection rates of the local intermediate host species, Biomphalaria straminea. Spatial analysis of infection in both groups before and after treatment was performed by estimating the kernel density for the detection of clusters of positive cases. Altogether, 123 adolescents (10-19 years) from São Lourenço da Mata, PE, with more than 100 eggs per gram of feces were examined in the four assessments. Although all patients were negative 21 days after treatment, 17.9% and 30.9% were positive at 180 and 360 days, respectively. Both prevalence and intensity of infection after treatment were significantly higher (P <0.05) in the 40 mg/kg regime than in the 60 mg/kg one. The male subjects and those who were treated with 40 mg/kg had a significantly higher chance of being positive 360 days post-treatment than those treated with 60 mg/kg. The monthly infection rates of B. straminea ranged from 0 to 2.2%. Clusters of positive subjects treated with 40 mg/kg had a higher density and extent than those treated with 60 mg/kg both at 180 and 360 days. It is recommended that the 60 mg/kg PZQ dose should be maintained for treatment of S. mansoni infection in endemic areas in Brazil, as both therapy with 40 mg/kg or 60 mg/kg PZQ single doses had a high and comparable impact on infection 21 days after treatment and subjects who received the higher dose were able to maintain lower levels of both prevalence and intensity of infection for up to one year after treatment. The spatial results confirm the parasitological data and reinforce the need of auxiliary, preventive measures integrated with chemotherapy to achieve a greater impact on disease control.

Les récepteurs Venus Kinase ou VKR forment un nouvelle famille de récepteurs tyrosine kinase découverts au laboratoire. Ces récepteurs sont caractérisés par une organisation atypique associant un domaine extracellulaire de type Venus Flytrap (VFT) similaire à ceux des récepteurs couples aux protéines G de classe C à un domaine Tyrosine Kinase (TK) intracellulaire similaire à celui du récepteur à l’insuline (IR). Cette famille est présente uniquement chez les invertébrés dont notre modèle d’étude, le parasite plathelminthe Schistosoma mansoni. Les travaux réalisées à ce jour suggèrent que ces protéines jouent un rôle dans le développement des stades larvaires et la reproduction sexuée. Mon travail de thèse s’est intéressé à l’étude de cette famille de récepteurs et comporte trois axes principaux. Le premier concerne la mise à jour phylogénétique de la famille des VKR. Une analyse approfondie des données génomiques récentes nous a permis d’étendre la présence des VKR à près de 50 espèces dans cinq phyla invertébrés. La présence d’un VKR chez le cnidaire Nematostella vectensis suggère une émergence du récepteur avant l’apparition des bilatéraliens. Des analyses phylogénétiques ont mis en évidence la monophylie de l’ensemble des récepteurs ainsi que des importants niveaux de conservation des motifs fonctionnels VFT et TK. Des analyses de modélisation du domaine de fixation au ligand suggèrent que la majorité de ces récepteurs puissent être activables par des acides aminés de type L-Arginine. La découverte de VKR chez Nematostella vectensis, Lottia gigantea ou Capitella teleta s’avère prometteuse dans la mesure où ces modèles se prêtent parfaitement à l’embryologie moléculaire, et pourraient être utilisés pour étudier la fonction des VKR dans l’embryogenèse.La deuxième partie de mon travail s’est intéressée à la caractérisation fonctionnelle de SmVKR1 et SmVKR2, les deux VKR de Schistosoma mansoni. En utilisant le modèle d’expression ovocyte de Xénope, nous avons pu dans un premier temps déterminer que SmVKR1 et SmVKR2 sont activables par plusieurs et différents L-acides aminés. L’identification de partenaires intracellulaires par un criblage en double hybride de levure nous a permis de déterminer de nouveaux et inattendus partenaires d’interaction, suggérant des fonctions dans l’activation de voies TK conservées, mais également dans le remodelage du cytosquelette ou la synthèse protéique. Si les deux récepteurs activent les voies Akt, S6K et Erk1/2, seul SmVKR1 est capable d’activer la voie JNK. Les profils d’expression ont été déterminés par hybridation in situ et, si les deux gènes s’expriment dans l’ovaire, la localisation des transcrits est clairement distincte avec une expression de SmVKR2 au sein des ovocytes immatures et de SmVKR1 dans les ovocytes matures. Enfin des expériences d’ARN interférence sont venues confirmer un rôle de ces récepteurs au sein de l’ovogenèse, la diminution d’expression entraînant des phénotypes délétères sur la croissance et ovocytes et la ponte des oeufs. L’étude des voies de signalisation SmVKR1 a été également été entreprise au laboratoire, mettant en évidence l’importance de la protéine adaptatrice SmShb dans l’induction de la voie JNK par SmVKR1. L’ensemble de des résultats suggère que SmVKR1 aurait une fonction dans la migration des ovocytes et/ou dans la reprise de méiose par l’activation de la voie JNK, tandis que SmVKR2 aurait un rôle plus précoce, dans la croissance et/ou la prolifération des ovogonies. Enfin la dernière partie de mon travail de these visait à determiner si les VKR peuvent être considérés comme des cibles thérapeutiques intéressantes en vue du développement de nouvelles drogues anti parasitaires. [...] / Venus Kinase Receptors (VKR) represent a new family of receptor tyrosine kinases discovered in the lab. These receptors are characterized by an atypical structure composed of an extracellular ligand binding domain called Venus Flytrap, similar to those of class C G- protein coupled receptors, and an intracellular Tyrosine Kinase domain, very close to those of Insulin Receptors. These proteins are exclusively found in invertebrate organisms including our lab model, the parasitic platyhelminth Schistosoma mansoni. Current data strongly suggest a function for these receptors in larval development and reproduction. My thesis project concerns the study of this receptor family and is divided into three parts:The first part consists in an update of the VKR family phylogeny. Analyses of recent genomic data have allowed us to extend the presence of VKR to 50 species present in five bilaterian phyla. The presence of one VKR in the early-branching metazoan Nematostella vectensis suggested that VKR arose before the bilaterian radiation. Phylogenetic and gene structure analyses showed that all receptors identified grouped monophyletically, and likely evolved from a common ancestor. Multiple alignments of tyrosine kinase (TK) and VFT domains indicated their important level of conservation in all VKRs identified up to date. We showed that VKRs had inducible activity upon binding of extracellular amino-acids and molecular modeling of the VFT domain confirmed the structure of the conserved amino-acid binding site.The second part of my thesis project concerns the functional characterization of SmVKR1 and SmVKR2, which are two VKRs of Schistosoma mansoni. Using the Xenopus oocyte model as a protein expression system, we have been able to determine that SmVKR1 and SmVKR2 are activated by distinct and different ligands. The identification of intracellular binding partners using yeast two hybrid experiments allowed us to determine unexpected interacting proteins, suggesting functions for SmVKRs in kinase signaling and cytoskeleton remodeling. If both receptors are able to activate Akt, S6K and Erk 1/2 pathways, only SmVKR1 triggers the activation of Jnk proteins. Expression patterns were determined by in situ hybridization and highlighted the presence of both SmVKR1 and smVKR2 transcripts in the ovary. However, their localization within the ovary is different, with SmVKR1 transcripts detected exclusively in the anterior part of the ovary while SmVKR2 transcripts were found in the posterior part that only contains immature oocytes. RNA interference experiments further confirmed the importance of SmVKR proteins in oogenesis, since the knockdown of every gene led to the appearance of deleterious phenotypes in the ovary. Taken together, all these results strongly suggest that SmVKR1 could be involved in meiosis resumption or ovulation through JNK activation, while SmVKR2 would have an earlier function in the growth and/or proliferation of oogonies. Finally, the last part of my project concerned the assessment of VKR as a chemotherapeutic target. Taking advantage of the similarity between the catalytic domains of S. mansoni insulin receptors (SmIR1 and SmIR2) and Venus Kinase Receptors (SmVKR1 and SmVKR2), we studied the possibility to fight schistosomes by targeting simultaneously the four receptors with a single drug. We analyzed the potential of several IR and RTK inhibitors to inhibit kinase activities of both SmIR and SmVKR kinase domains recombinantly expressed in Xenopus oocytes. Among the different compounds tested, tyrphostin AG1024 emerged as the most potent inhibitor towards the four receptors. In vitro experiments then demonstrated that treatment with AG1024 led to dramatic effects on the viability of larval and adult schistosomes as well as on the fertility of adult worms. We assume that AG1024 represents a valuable hit compound for further design of anti-kinase drugs applicable to anti-schistosome chemotherapy.

A esquistossomose é uma doença importante em saúde pública. Dos genes selecionados como diferencialmente expressos em esquistossômulos a partir do transcriptoma do S. mansoni, 56% foram confirmados por RT-PCR em tempo real. Entre eles, a proteína Ly6.5, está presente no tegumento de esquistossômulos e vermes adultos por âncoras de GPI. Não foi detectada a função de inibir o sistema complemento, mas pode estar envolvido na manutenção do tegumento. O gene SmVal7 revelou transcritos nas glândulas esofágicas de vermes adultos por hibridização in situ, enquanto a localização da proteína não está definida. Anexina está associada ao tegumento de esquistossômulos e vermes adultos, de maneira dependente de cálcio. A supressão do gene por RNAi não resultou em alteração fenotípica significativa em esquistossômulos in vitro. Foi observada atividade parcial de inibição de coagulação e potencial atividade de endocitose de anticorpos ligados à superfície. A imunização com rLy6.5, rSmVal7, rAneI-II ou rAneII-III não levou a redução da carga parasitária após desafio. / Schistosomiasis is an important disease in public health. Genes selected from the S. mansoni transcriptome, 56% of them were confirmed as differentially expressed in schistosomula by real time RT-PCR. Among them, the protein Ly6.5 is present in the tegument of schistosomula and adult worms by GPI anchors. The function of inhibiting the complement system was not detected, but it may be involved in maintenance of the tegument. The gene SmVal7 revealed transcripts in the esophageal glands of adult worms by in situ hybridization, while the localization of the protein is not defined. Annexin is associated with the membranes of the schistosomula and adult worms tegument in a calcium-dependent manner. The suppression of the gene by RNAi did not resulted in a significant phenotypic change in schistosomula in vitro. Parcial inhibition of the coagulation activity and potential function of endocytosis of membrane-bound antibodies were observed. Immunization with the rLy6.5, rSmVal7, rAneI or rAneII-II-III did not show reduction in worm burden recovery after challenge.

A esquistossomose é uma doença parasitária grave e de larga distribuição geográfica, que acomete cerca de 200 milhões de pessoas no mundo e ao menos 700 milhões vivem em área de risco. No Brasil a doença é conhecida por xistose ou barriga dágua e é causada pelo Schistosoma mansoni, parasito pertencente ao filo Platyhelmintes, cujo verme adulto tem como hábitat o sistema porta do hospedeiro. O agente etiológico em questão apresenta um ciclo do tipo heteroxênico, sendo o caramujo do gênero Biomphalaria o hospedeiro intermediário e, o homem, o principal hospedeiro definitivo. Os saneamentos básicos juntamente com orientações à população constituem as principais medidas profiláticas. O tratamento também deve ser considerado no controle e na profilaxia da doença, o qual é baseado no uso de oxamniquina e praziquantel. Como somente o praziquantel tem sido usado na terapêutica de esquistossomose há a necessidade de descoberta de novos medicamentos para o tratamento dessa parasitose; além da urgente demanda relacionada ao aparecimento de linhagens do verme resistentes. A justificativa para novas alternativas terapêuticas está pautada em um tratamento com menor duração, menos efeitos adversos e que não apresente resistência do parasito. Sendo assim, este trabalho teve por objetivo avaliar a atividade esquistossomicida do extrato etanólico da semente de Garcinia brasiliensis, bem como das frações hexânica, acetato-etílica e aquosa, obtidas por partição, a partir do extrato etanólico da semente. Também foi analisada a atividade de um constituinte puro, isolado dessa espécie, uma benzofenona denominada Guttiferona-A. A avaliação do efeito do extrato da semente da planta, das frações e do composto purificado sobre o S. mansoni foi realizada em vermes adultos em testes in vitro, na qual se quantificou a taxa de mortalidade e monitoraram-se os danos na superfície da membrana e atividade do sistema excretor. Essas observações também foram realizadas com o uso dos marcadores fluorescentes Hoechst 33258 e Resorufin, respectivamente. Nos estudos realizados com a GUT-A, a atividade esquistossomicida significativa foi presenciada a partir da concentração de 18,0 μg/mL nos ensaios in vitro, onde foi capaz de matar 100,0% dos parasitos, com 24 horas de incubação, permitindo chegar ao valor de ED50 em 21,8 μg/mL. Os resultados mostraram a alta capacidade esquistossomicida do extrato da semente, das frações e da GUT-A em vermes adultos nos testes realizados in vitro. Os marcadores fluorescentes comprovaram a ação dos compostos testados, evidenciando os danos causados no sistema excretor dos parasitos, bem como lesões no tegumento, frente à exposição a várias concentrações analisadas. Ensaios posteriores serão realizados tendo como abordagem principal o mecanismo de ação da GUT-A em vermes adultos e em outras fases do ciclo de vida do S. mansoni, em testes in vitro e in vivo. / Schistosomiasis is a severe parasitic disease with large geographical distribution, affecting nearly 200 million people worldwide and at least 700 million live in risk areas. In Brazil the disease is known as “xistose” and “water belly” and is caused by Schistosoma mansoni, a parasite who belongs to the flatworm phylum, in which the adult worm lives in the portal system of the liver. The etiologic agent has a heterogenic life cycle, in which the intermediate hosts are snails of the Biomphalaria genus, and humans are the primary hosts. The main prophylactic measures include basic sanitation and public orientation. The treatment must also be considered in the control and prophylactics of the disease, which is based on the use of oxamniquine and praziquantel. Since only praziquantel has been used during schistosomiasis therapy there is a need for the discovery of new medications for parasite treatment; along with the urgent demand in relationship to resistant strains of the parasite. The justification for new, alternative therapy is guided by a shorter term treatment, with fewer side effects, and one that doesn’t provide resistance to the parasite. Thus, this work has as its objective to evaluate the schistosomicidal effect of the ethanolic extract of the Garcinia brasiliensis seed, along with the hexonic fractions, ethylic-acetate, and aquose, obtained through partition, from the ethanolic extract of the seed. The activity of a pure constituent which was isolated from this species, a benzophenone named Guttiferona-A. The evaluation of the seed extract, fraction, and pure compound effects on S. mansoni were done on adult worms in vitro, in which the mortality rate was quantified and excretory system, surface membrane damage was monitored. These observations were also made using the fluorescent markers Hoechst 33258 e Resorufin, respectively. On studies using GUT-A, significant schistosomicidal activity was witnessed in tests in vitro starting with concentrations of 18.0 μg/mL, where it was able to kill 100% of parasites, after 24 hours of incubation, allowing it to reach the ED50 value of 21.8 μg/mL. The results show a high schistosomicidal effect from the seed extract, from the fractions, and from GUT-A on adult worms used in tests in vitro. The fluorescent markets proved the effects of the compounds tested, showing the damaged caused to the excretory system, along with lesions to the integument, after exposure to various concentrations were examined. Further tests will be made having as a main approach the effect mechanism of GUT-A on S.mansoni adult worms and organisms in other phases of the life cycle, in tests in vitro and in vivo.

A esquistossomose, causada pelo helminto do gênero Schistosoma, é uma doença negligenciada no mundo e apresenta alta prevalência por estar associada principalmente com saneamento básico e educação sanitária insuficientes. A doença desenvolve formas clínicas no seu hospedeiro vertebrado, inicia-se com uma fase aguda e posterior desenvolvimento da fase crônica. A fase crônica da doença acarreta maiores prejuízos, um deles é o desenvolvimento de lesões no fígado, baço, intestino e pulmões. A hipertensão portal causada pela doença permite a migração de ovos aos pulmões, onde inicia-se uma resposta inflamatória granulomatosa. A migração de ovos pelos órgãos pode favorecer o deslocamento conjunto de bactérias em uma infecção natural. Para o estudo da ação das bactérias no organismo é comum a utilização de endotoxina da parede de bactérias Gram-negativas, denominada lipopolissacarídio (LPS). Neste trabalho, avaliamos o processo inflamatório pulmonar estimulado com LPS em animais infectados com Schistosoma mansoni em fase crônica. Os grupos de estudo foram divididos em Controle/Salina (dividido nos períodos de 6h (n=6) e 24h (n=6)), Schisto (com 120 dias de infecção (n=6)), LPS (dividido nos períodos de 6h (n=6) e 24h (n=6)) e LPS/Schisto (dividido nos períodos de 6h (n=6) e 24h (n=6)). Observamos o aumento de células polimorfonucleares (PMNs) nas primeiras 6 horas após a inoculação de LPS, no grupo LPS (p<0,001) em relação aos demais grupos; e o aumento (p<0,001) de PMNs no grupo LPS/Schisto em relação aos grupos Controle/Salina e Schisto. Após 24 horas da inoculação de LPS, o processo inflamatório modificou-se com o aumento de células mononuclerares (MNs), exceto no grupo Controle/Salina (p<0,001). O número de PMNs permaneceu aumentado no grupo LPS/Schisto em relação ao grupo Controle/Salina (p<0,001) e ao grupo Schisto (p<0,05). A dosagem da concentração de óxido nítrico aumentou no grupo LPS (p<0,001) em relação aos demais grupos, e no grupo LPS/Schisto (p<0,001) em relação aos grupos Controle/Salina e Schisto em 6 horas após a inoculação do LPS. Os granulomas pulmonares não sofreram alteração significativa no tamanho entre os grupos Schisto e LPS/Schisto. Sugerimos com este trabalho que a esquistossomose em fase crônica pode causar a imunossupressão de uma infecção bacteriana Gram-negativa concomitante. / Schistosomiasis, caused by helminth of the genus Schistosoma, is neglected disease in the world and has a high prevalence to be associated with areas where sanitation and hygiene education are insufficient. The disease develop clinical forms in their host vertebrate begins in an acute phase and subsequent development of chronic phase. The chronic phase of the disease causes major damage one of them is the development of lesions in the liver, spleen, intestines and lungs. The portal hypertension caused by disease allows the migration of eggs to the lungs here start a granulomatous inflammatory response. The migration of eggs may favor the displacement set of bacterial infection in a nature. To study the action of bacteria in the organs is common to use the endotoxin wall of Gram-negative bacteria the lipopolysaccharide (LPS). In this study, we evaluated the process pulmonary inflammatory stimulated with LPS in animals infected with Schistosoma mansoni chronic phase. The study groups were divided into Controle/Salina (divided into group Controle/Salalina 6 h (n = 6) and 24 h (n = 6)), Schisto (120 days of infection ( n = 6)), LPS (divided into group 6 h (n = 6) and 24 (n = 6)) and LPS / Schisto(divided in groups 6 h (n = 6) and 24 h (n = 6)). Observed an increase in polymorphonuclear cells (PMNs) in the first 6 hours after inoculation of LPS in group LPS (p<0,001) in relation to the other groups, and the increase (p<0,001) of PMNs in group LPS/Schisto than in groups Controle/Salina and Schisto. After 24 hours inoculation of LPS the process change inflammatory modified with increased cells mononuclerares (MNs) in groups, except in group Controle/Salina(p<0,001). The number of PMNs remained increased in group LPS/Schisto when compared to group Controle/Salina (p< 0,001) and group Schisto (p<0,05). The dosage of nitric oxide concentration increased in group LPS (p<0,001) in relation to the other groups, and group LPS/Schisto (p<0,001) in relation to groups Controle/Salina and Schisto in 6 hours after the LPS inoculation. The pulmonary granulomas did not change significantly in size between groups Schisto and LPS/Schisto This work suggest that schistosomiasis in chronic phase may cause immunosuppression of a Gram- negative bacterial infection concomitant. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

No description available.

My initial aim was a functional analysis of the conserved Op18/stathmin family of microtubule-regulators, which includes the ubiquitous cytosolic Op18 protein and the neural membrane-attached RB3 and SCG10 proteins. The solved X-ray structure has shown that these proteins form a complex with tubulin -heterodimers via two imperfect helical repeats, which result in two head-to-tail aligned heterodimers in a tandem-tubulin complex. We have analyzed GTP exchange and GTP hydrolysis at the two exchangeable GTP-binding sites (E-site) within the tandem-tubulin complex. A comparison of Op18, RB3 and SCG10 proteins indicates that Op18/Stathmin family proteins have evolved to maintain the two heterodimers in a configuration that restrains the otherwise potent GTPase productive interactions facilitated by the head-to-head alignment of heterodimers in protofilaments. We concluded from these studies that tubulin heterodimers in complex with Op18/stathmin family members are subject to allosteric effects that prevent futile cycles of GTP hydrolysis. To understand the significance of the large differences in tubulin affinity of Op18, RB3 and SCG10, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell-surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. We showed that, in contrast to CD2-Op18, both the CD2-SCG10 and CD2-RB3 chimeras sequester tubulin at the plasma membrane, which results in &gt;35% reduction of cytosolic tubulin heterodimer levels. However, all three CD2-chimeras, including the tubulin sequestration-incompetent CD2-Op18, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during the interphase, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. Sm16/SmSLP (Stathmin-Like Protein) has been identified as a protein released during skin penetration of the Schistosoma mansoni parasite. This protein has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. However, our studies refuted any functional similarity with stathmin/Op18 and we found instead that Sm16/SmSLP is a lipid bilayer binding protein that is taken up by cells through endocytosis. To study immuno-modulatory properties of Sm16/SmSLP, we designed an engineered version with decreased aggregation propensity, thus facilitating expression and purification of a soluble Sm16 /SmSLP protein from the eukaryotic organism Pichia pastoris. Determination of the hydrodynamic parameters revealed that both the recombinant and native Sm16/SmSLP is a ~9-subunits oligomer. The recombinant protein was found to have no effect on T lymphocyte activation, cell proliferation or the basal level of cytokine production of whole human blood or monocytic cells. Interestingly, however, recombinant Sm16 was found to potently inhibit the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and Poly(I:C). Since Sm16 specifically inhibits degradation of the IRAK1 signaling protein in LPS stimulated monocytes, it seems likely that inhibition is exerted proximal to the TLR-complex.

Thèse d'exercice : Médecine. Médecine générale : Paris 12 : 2006. / Titre provenant de l'écran-titre. Bibliogr. f. 38-40.

Schistosoma mansoni and Fasciola hepatica are parasitic trematodes which contain cytochrome c peroxidase (CcP) in their mitochondria, an enzyme that is absent in mammalian tissues. CcP reduces hydrogen peroxide to H2O using cytochrome c as the electron donor. Both parasites are catalase deficient; thus, cytochrome c peroxidase and glutathione peroxidase are the enzymes involved in the detoxification of H2O 2 in these organisms. The enzymatic activity of these two peroxidases may enable S. mansoni and F. hepatica to survive oxidative stress. The main objective of this study was to characterize cytochrome c peroxidase from S. mansoni and F. hepatica . Kinetic studies of this enzyme in crude homogenate and isolated mitochondria of S. mansoni were initially performed, followed by purification studies from S. mansoni and F. hepatica . The parasite enzyme has affinity for horse heart and yeast cytochrome c and it is inhibited by sodium azide and potassium cyanide. CcP was purified close to homogeneity and identified as a protein containing heme. The antioxidant capability of F. hepatica CcP was tested in vitro , demonstrating that CcP protected the sugar deoxyribose from oxidative degradation. Exposure of adult worms to H2O2 caused a decrease in S. mansoni CcP activity in vivo. An attempt was made to clone the S. mansoni CcP gene. The experiments did not result in the cloning of the CcP gene, but led to the identification and cloning of another protein, a component of a cytosolic chaperonin, t-complex polypeptide one (TCP-1). TCP-1 from S. mansoni is highly homologous to TCP-1 proteins from different organisms including, Chinese hamster, human, Drosophila and yeast and carries ATP binding amino acid motifs indicating that it has ATPase activity.

This study shows that resistance to killing by reactive oxygen intermediates (ROI) increases during migration and development in Schistosoma mansoni. Resistance is associated with the protective role of antioxidants as shown by the increased levels of superoxide dismutase and of the glutathione system enzymes. Hydroperoxide-dependent glutathione peroxidase activity was not detectable in newly transformed schistosomula, however the activity was present in the liver stages. The antischistosomal drug oltipraz (OPZ) decreased in an irreversible manner the activity of S. mansoni glutathione S-transferase (GST), an important protective enzyme, both in vivo and in vitro. The inhibition of GST activity was not isoenzyme restricted and was non-competitive with respect to the two substrates essential for GST activity. On the other hand, OPZ treatment increased the levels of mouse (S. mansoni host) liver GST activity in an isoenzyme specific manner, with the $ mu$ class subunit induction accounting for most of the increase. However, mammalian GST activity was inhibited by OPZ in vitro. However, the inhibition of mammalian GST activity was reversible upon addition of dithiol reducing compounds. OPZ inhibited the binding of ($ sp{14}$C) N-ethylmaleimide (specifically alkylates SH groups), suggesting that OPZ interacts with SH-groups of GST to inhibit its enzymatic activity. Another SH-dependent enzyme, hexokinase, from yeast and S. mansoni was reversibly inhibited by OPZ. The oxy-analogue of OPZ, in which the thione sulphur is replaced with oxygen, did not inhibit the enzymatic activity of GST and hexokinase. Many of the biochemical effects of OPZ on S. mansoni and its mammalian hosts may be related to its ability to bind to SH groups and inactivation of the functions of many essential proteins.