Study of the secretome of leishmania involved in the infection

Les parasites protozoaires du genre Leishmania sont les agents microbiens responsables d’un groupe de maladies connues sous le nom de leishmanioses. L’infection productive dépend de la capacité de survie du parasite suite au premier contact initial du système immunitaire de l’hôte. Par conséquent, la prolifération du parasite à l’intérieur des phagolysosomes sera responsable de la pathologie. Les promastigotes stationnaires, récupérés en culture axénique de laboratoire sont semblables aux promastigotes métacycliques. Ces derniers sont fortement immunomodulateurs et sont considérés, traditionnellement comme la forme la plus infectieuse du parasite. Les protéines sécrétées de différents organismes ont été directement impliquées dans plusieurs pathologies. Donc, il est possible que les protéines sécrétées par Leishmania, soient également impliquées dans la capacité du parasite à subvertir le système immunitaire. Les avancées récentes dans l’étude du sécrétome de plusieurs types de Leishmania ont permis d’affirmer que le sécrétome est fort complexe. Nous avons déterminé qu’environ 300 protéines sont sécrétées par le parasite, la plupart d’entre elles ayant aucun signal canonique de sécrétion. Le sécrétome de Leishmania est donc composé surtout de protéines qui sont libérées par sécrétion non conventionnelle, . Afin d’étudier le sécrétome associé à la virulence, nous avons développé et validé une approche qui a permis l’étude des composants de l’exoprotéome des parasites stationnaires et logarithmiques. Cette approche était basée sur la culture continue des parasites dans un milieu de culture sans supplément de sérum de bovin fœtal, cRPMI, dans lequel la virulence des parasites est maintenue. Grâce à cette approche nous avons mis en évidence un exoproteome distinct de ceux jusqu’à date répertorié. La méthode de production et de la récupération de l’exoproteome sont donc très importants. Notre exoprotéome est dominé par la GP63, une glycoprotéine dont l’importance centrale dans l’infection a été déjà validée. La culture continue des parasites est donc essentiel pour avoir un exoprotéome représentatif. Nous avons également déterminé que la cultutre en continu pouvait amener à une diminution de la virulence et quarante divisions sont nécessaires pour une perte de virulence significative. Par conséquent toutes nos études se sont fait chez des parasites comptant moins de 20 divisions. Le principal mécanisme associé à la perte de virulence a été identifié comme une incapacité de se différencier en amastigotes. Le cRPMI a donc permis la culture des parasites pour l’étude de l’exoprotéome tout en maintenant la virulence des parasites. La présence de vésicules, décrite déjà comme un composant de l’ exoprotéome, a été confirmée aussi par notre approche continue et a été confirmé, d’ailleurs, dans l’exoproteome des parasites en phase de croissance logarithmique. Les vésicules récupérées des parasites logarithmiques diffèrent de celles recueillies de parasites en phase stationnaire de croissance. En effet des protéines potentiellement impliquées dans la rénovation et le recyclage du contenu protéique, tels certains composants du ribosome étaient enrichies dans les parasites en phase logarithmique tandis que les vésicules des parasites stationnaires ont un contenu protéomique ayant des caractéristiques similaires aux corps apoptotiques des cellules de mammifères. En dehors de la GP63, plusieurs autres protéines décrites comme immunomodulatrices ont été retrouvées dans l’exoprotéome des parasites stationnaires, ce qui indique que celui-ci contient un ensemble de protéines avec un potentiel d’interaction directe avec les cellules du système immunitaire. Immunologiquement, l’exoprotéome récupéré des parasites stationnaires a été capable d’activer les cellules dendritiques, laissant supposer une fonction importante dans la création d’un environnement inflammatoire précoce lors des premières étapes de l’infection. En conclusion, la recherche développée a contribué à l’avancement des connaissances actuelles sur la biologie du Leishmania, grâce au développement et à la validation d’une nouvelle approche afin d’étudier son exoprotéome. L’exoprotéome récupéré était dynamique, il avait une composition spécifique, dépendant du stade du parasite. Cet exoprotéome avait des effets spécifiques sur les cellules dendritiques et il jouait un rôle important dans les étapes précoces de l’infection. Cette étude a ouvert des nouvelles perspectives sur l’exoprotéome de Leishmania spp. permettant la découverte de nouvelles protéines immunomodulatrices et, en corrolaire, de nouvelles cibles pour le contrôle de la maladie associée au parasite.Protozoa parasites of the genus Leishmania are the responsible for a group of diseases known as leishmaniasis. The infection is associated with the capacity of these parasites to survive in the phagolysosomes of infected macrophages. Successful infections with pathogenic Leishmania spp. are linked to the capacity of the parasite to survive the initial impact of the host immune system and to interfere with the infected cells rendering them incapable of eliminating the parasites. The secreted proteins from the parasite are expected to be in the front line for interactions with the host. Recent advances in the study of the secretome of Leishmania spp. depicted it as highly complex with the majority of proteins without any predictable secretion signal. The secretome is composed of proteins that are released by different mechanisms like conventional and unconventional secretion. Several proteins secreted by Leishmania spp. are known to interact and influence the outcome of the disease by directly interfering with the host immune cells. The proteomic studies on the Leishmania spp. secretome identified more than three hundred proteins released into the exterior. The stationary promastigotes recovered in axenic culture were enriched in the most virulent promastigote form, the metacyclic parasites. Therefore we aimed at evaluating the exoproteome associated with the stationary parasites. To achieve this we developed and validated an approach that would enable the study of the exoproteome components of stationary and logarithmic parasites. This approach was based on the continuous cultivation of the parasites in a medium without any protein supplementation that maintained the basic virulence of the parasites. The continuous approach produced a GP63-rich exoproteome that was distinct from the traditional approaches indicating that the process of recovery induced a significant bias in the study. Furthermore as the continuous approach was chosen, we determined the mechanisms associated with loss of virulence assuring that fully virulent parasites were used. At least forty parasite divisions were required for a short-term loss of virulence. The main mechanism associated with loss of virulence was identified as a growing incapacity to differentiate into amastigotes. The defined time interval of forty divisions enabled us to evaluate the exoproteome without loss of virulence related to the subculture. The protein-free medium developed, cRPMI, retained parasite virulence and morphology similar to that of parasites grown in standard media. The exoproteomes recovered using cRPMI were dominated by proteins without any recognizable secretion sequence, in concordance with reports on other Leishmania spp. The presence of vesicles, already reported as a component of the exoproteome, was also confirmed using our continuous approach. Furthermore, the presence of vesicles in the logarithmic parasites exoproteome was confirmed. The protein content of these vesicles presented a dynamic profile that was dependent on the parasite stage. The vesicles recovered from logarithmic parasites seemed to be related to protein turnover, being significantly enriched in ribosomal components. The vesicles from stationary parasites are of different composition, presenting some characteristics similar to apoptotic bodies. Immunologically the exoproteome recovered from stationary parasites was able to activate dendritic cells suggesting that the exoproteome might have a function in the creation of an early inflammatory environment leading to the recruitment of neutrophils and monocytes that might function as safe heavens for the parasites. In conclusion, our research has contributed to the advance of the current knowledge of Leishmania biology, through the development and validation of a novel approach to study the Leishmania secretome. The exoproteome recovered from stationary parasites had specific immune-modulating effects on bone marrow derived dendritic cells, indicating that it can play an important role in the precocious steps of infection. This study opened new perspectives into the Leishmania spp. exoproteome that will enable the search of new immunomodulatory proteins that might become the future targets to leishmaniasis control

How to simplify portfolio optimization

We study portfolio optimization with 8 different optimization models and 2 heuristic models. We use all the models for two different scenarios: international diversification with stocks, and asset allocation with stocks, bonds and commodities. First we do our analysis in a global context, and then we do it for a European context. Our results show that heuristic models prove to bring more gains to individual investors than the more complex optimization models in either of the two scenarios, and that the addition of bonds and commodities in the asset allocation scenario proves to be beneficial for heuristics models.Estudamos a otimização de portfólios recorrendo a 8 diferentes modelos de otimização e 2 modelos heurísticos. Usamos todos os modelos para dois diferentes cenários: diversificação internacional com ações, e alocação de ativos com ações, bonds e commodities. Primeiro, efetuamos a análise a um contexto global e depois efetuamo-la para um contexto Europeu. Os nossos resultados mostram que, independentemente do cenário considerado, os modelos heurísticos trazem mais ganhos do que os modelos de otimização aos investidores, e que a adição de bonds e commodities no cenário da alocação de ativos beneficia os modelos heurísticos

Discontinuity characterization and instability situations of an ornamental limestone quarry in Valinho de Fátima

Poster apresentado ao VIII Congresso Nacional de Geologia, em Braga (9-16 de Julho 2010)Apresenta-se o estudo das descontinuidades e das potenciais situações de instabilidade de uma pedreira de calcário ornamental que se localiza nas imediações de Valinho de Fátima (freguesia de Fátima). Definiram-se três famílias principais de descontinuidades, bem como as suas principais características: orientação, espaçamento, persistência, abertura, material de preenchimento, rugosidade, presença da água, índice de diaclasamento volumétrico e ângulo de atrito. Através da análise cinemática, em que utilizou o método de Markland, identificaram-se situações de instabilidade de rotura em cunha.This article presents the study of discontinuities and potential slope instability of an ornamental limestone quarry located in Valinho de Fátima. Three joints sets were defined, as well as its characteristics such as: orientation, spacing, persistence, aperture, filling, roughness, percolation, volumetric joint count and friction angle. A kinematic analysis, based on Markland’s method was carried out, which allowed the identification of wedge failures occurrences

New approaches for the control of the pine wood nematode : Bursaphelenchus Xylophilus

O nematode da madeira do pinheiro (NMP) Bursaphelenchus xylophilus é o agente etiológico da doença do nemátode da madeira do pinheiro em Pinus pinaster. Não existe ainda um método eficiente para controlar e/ou eliminar este agente patogénico. O quitosano é um produto extraído da parede celular de alguns fungos ou da desacetilação da quitina, um produto amigo do ambiente e amplamente usado como agente antimicrobiano. O objetivo deste trabalho foi o de testar a influência do quitosano no aumento da defesa da planta contra o NMP e também compreender as bases fisiológicas de ação deste composto a múltiplos níveis: efeito na população de nemátodes na planta, na produção de compostos de defesa e na modulação da carga microbiana no nemátode. Também se testou o quitosano como agente de esterilização da madeira comparando também este composto com outros métodos como o tratamento térmico em estufa e por microondas. Para este trabalho de investigação infetaram-se pinheiros com NMP onde 75 pinheiros foram mantidos em solo sem quitosano e 75 em solo com quitosano. Verificou-se que até aos 28 dias após a infeção o quitosano evitou a morte dos pinheiros e o número total de nemátodes foi inferior ao número de nemátodes dos pinheiros mantidos em solo sem quitosano e idêntico ao longo de todo o tempo experimental. Este composto aumentou a produção de carotenoides, (53.8 ± 4.6 mol/g-1 aos 21 dai para 111.5 ± 11.9 mol/g-1 aos 28 dai) antocianinas (63.4 ± 5.5 mol.g-1 para 142.3 ± 20.4 mol.g-1 aos 28 dai), polifenóis totais (2.70 ± 0.1 μg.g-1 aos 7 dai para 3.2 ± 0.7 μg.g-1 aos 14 dai) e lenhina (0.7 ± 0.1mg.g-1 aos 14 dai para 1.5± 0.1 mg.g-1 aos 28 dai) demonstrando a sua capacidade para aumentar os mecanismos de defesa da planta. Foi estudada também a influência do quitosano nas bactérias associadas ao nemátode na planta e Enterobacter sp. foi o género predominante encontrado ao longo do tempo, o que sugere que as bactérias deste género estejam associadas à doença. De forma a se compreender se as bactérias obtidas no estudo anterior provinham realmente do nemátode ou se teriam sido transmitidas a este pela planta realizou-se um estudo “in vitro” do efeito do quitosano no NMP. Verificou-se que Stenotrophomonas sp., Bacillus sp., Kluyvera sp., Buttiauxella sp., Klebsiella sp. e Paenibacillus sp. não foram encontrados associados com o nematode mantido em cultura no laboratório, o que pode indicar que estas bactérias são transmitidas pela planta. Para a comparação do quitosano com outros métodos de controlo, como o tratamento térmico através de uma estufa e de energia microondas, utilizou-se serrim de madeira de pinheiro infetado com NMP. Este estudo demonstrou que o tratamento térmico com estufa e com micro-ondas tiveram uma maior capacidade nematicida quando comparado com soluções de quitosano a diferentes concentrações. Este estudo permitiu concluir que o quitosano apresenta-se com um potencial método de prevenção da doença do NMP aumentando a defesa da planta contra este agente patogénico e evitando a evolução da doença e consequente morte do pinheiro. O quitosano influencia a carga microbiana do nematode, mas mais testes têm que ser realizados para perceber qual o efeito na evolução da doença. Este composto apresenta um efeito limitado nematicida em serrim quando comparado com outros métodos de tratamento.The pine wood nematode (PWN) Bursaphelenchus xylophilus is the causal agent of the pine wood disease (PWD) in Pinus pinaster. There isn’t an effective method for control and/or elimination of this pathogen. Chitosan is a product extracted from the cell wall of some fungi or deacetylation of chitin, an environment friendly product, widely used as an antimicrobial agent. The objective of this study was to test the influence of chitosan in the increase of plant defence against the PWN and also understand the physiological bases of action of this compound at multiple levels: effect on the population of nematodes in the plant, production of defence compounds and modulation the microbial load in the nematode. Chitosan was also tested as a wood sterilizing agent and compared with other methods such as heat-treatment by kiln-drying and microwave radiation. For this research project trees were infected with PWN and 75 were kept in soil without chitosan and 75 trees were kept in soil with chitosan. It was found that up to 28 days after infection chitosan prevented the death of pine trees and the total number of nematodes was below the number of nematodes of pine trees kept in soil without chitosan. This compound increased the production of carotenoids, (from 53.8 ± 4.6 mol / g-1 at 21 dai to 111.5 ± 11. 9 mol / g-1 at 28 dai) anthocyanins (from 63.4 ± 5.5 mol g-1 to 142.3 ± 20.4 mol g-1 at 28 dai), total polyphenols ( from 2.7 ± 0.1 μg.g-1 at 7 dai to 3.2 ± 0.7 μg.g-1 at 14 dai) and lignin (rising from 0.7 ± 0.1 mg. g-1 at 14 dai to 1.5 ± 0.1 mg.g-1 at 28 dai) demonstrating its ability to increase the defence mechanisms of the plant. The influence of chitosan on the bacteria associated with the nematodes was also studied in the plant, and Enterobacter sp. was the predominant genus found over time, suggesting that bacteria of this type are associated with the disease. In order to understand if the bacteria obtained actually came from the nematode or was transmitted to the nematode by the plant, an “in vitro” study was performed, were the effect of chitosan on the PWN kept in laboratory culture conditions was studied. It was found that Stenotrophomonas sp., Bacillus sp., Kluyvera sp., Buttiauxella sp., Klebsiella sp. and Paenibacillus sp. were not found associated with the nematode, which may indicate that these bacteria were transmitted by the plant. To compare chitosan with other control methods, such as heat- treatment through kiln-drying and microwave energy, wood chips infected with PWN were used. The heat-treatment by kiln-drying and microwave energy had a higher nematicide capacity when compared to chitosan solutions with different concentrations. This study found that chitosan is a potential prevention method for the PWD increasing plant defence against this pathogen and preventing disease progression and consequent death of the tree. Chitosan influences the microbial load of the nematode but more tests have to be performed to understand the effect on the progression of the disease. This compound has a limited nematicide effect on wood chips when compared with other treatment methods

Discontinuity characterization and instability situations of an ornamental limestone quarry in Valinho de Fátima

Poster apresentado ao VIII Congresso Nacional de Geologia, em Braga (9-16 de Julho 2010)Apresenta-se o estudo das descontinuidades e das potenciais situações de instabilidade de uma pedreira de calcário ornamental que se localiza nas imediações de Valinho de Fátima (freguesia de Fátima). Definiram-se três famílias principais de descontinuidades, bem como as suas principais características: orientação, espaçamento, persistência, abertura, material de preenchimento, rugosidade, presença da água, índice de diaclasamento volumétrico e ângulo de atrito. Através da análise cinemática, em que utilizou o método de Markland, identificaram-se situações de instabilidade de rotura em cunha.This article presents the study of discontinuities and potential slope instability of an ornamental limestone quarry located in Valinho de Fátima. Three joints sets were defined, as well as its characteristics such as: orientation, spacing, persistence, aperture, filling, roughness, percolation, volumetric joint count and friction angle. A kinematic analysis, based on Markland’s method was carried out, which allowed the identification of wedge failures occurrences

Racismo, branquidade e colonialidade do poder bélico : os critérios para a concessão de porte e posse de armas de fogo no Estatuto do Desarmamento (Lei n. 10.826/2003)

Dissertação (mestrado) — Universidade de Brasília, Faculdade de Direito, Programa de Pós-Graduação em Direito, 2022.Este trabalho tem por objetivo discutir a relação entre o racismo, a branquidade e os critérios para concessão do porte e da posse de armas previstos no Estatuto do Desarmamento (Lei 10.826/2003). Trata-se de uma pesquisa qualitativa, que tem como métodos a revisão de literatura e a pesquisa documental. A revisão de literatura, de trabalhos sobre modernidade/colonialidade e escravidão e sobre vitimização por arma de fogo no Brasil, busca, sobretudo identificar e discutir como a gestão do porte e da posse de armas tem sido central na estruturação do projeto moderno de colonialidade. A análise documental contempla os anais da Assembleia Nacional Constituinte de 1987/88, o Estatuto do Desarmamento e seus Decretos regulamentares. A análise dos anais visa apresentar e discutir os principais aspectos do debate constituinte que envolveram os temas da criminalidade, do racismo e do porte e posse de armas. A pesquisa envolve a apreciação dos discursos jurídicos e legislativos que antecederam a aprovação da Lei 10.826/2003. São discutidos, também, os dispositivos da própria Lei 10.826/2003 e a relação entre seus critérios morais abstratos e a discricionariedade e seletividade no momento da concessão para o porte e a posse de armas no país. Para a análise dos resultados da pesquisa, propomos o diálogo entre as perspectivas teórico-metodológicas Teoria Crítica da Raça, Estudos da branquidade e os Estudos Críticos do Discurso, bem como a utilização das categorias analíticas racismo e branquidade. Por meio deste estudo, é possível compreender que os dispositivos do Estatuto do Desarmamento (e de seus Decretos regulamentares) estão recheados de critérios morais abstratos que permitem a discricionariedade e seletividade da autoridade administrativa na autorização bélica. Os critérios como renda, local de residência, comprovação de idoneidade moral e a inexistência de inquérito policial ou processo criminal possuem carga moral a partir da ótica da branquidade e permitem o filtro e o controle racial na aplicação da Lei. Desse modo, o porte e a posse de armas no Brasil são privilégios raciais e o arcabouço jurídico que regula o porte, a posse e o registro de armas de fogo atende aos anseios de controle da branquidade sobre o sistema bélico, tendo importantes impactos na gestão de morte da população negra no país.This work has aimed to propose a relationship between racism, whiteness, and the requirements for granting possession of firearms conducted by Statute of Disarmament (Law 13,826/2003). It is a qualitative research whose methodological aspects concern literature review and documentary search. Regarding the literature review, we have focused on modernity/coloniality, and slavery as well as concerning victimization by firearms in Brazil. The main interest is to identify and discuss how the management of gun license/possession has been the core of the structure of the modern project of coloniality. Regarding documentary search, it has encompassed the Annals of National Constitutional Assembly (1987/88), and the Statute of Disarmament and their regulatory decrees. The former aim to present and discuss the main aspects of the debate embracing criminality, racism, acquisition, and possession of firearms. Plus, it is clustering as well the legal and legislative discourses delivered before the endorsement of Law 10,826/2003, and the provisions of this Law as well as the unspecific moral requirements and the discretion and selectivity when the concession to acquisition and possession of firearms in Brazil. We proposed a theoretical-methodological dialogue among Critical Theory of Race, Studies of Whiteness, and Critical Studies of Discourse to analyze all the corpus. This work allows for deducing that provisions of the Statute of Disarmament (and their regulatory decrees) are filled with unspecific moral requirements that imply discretion and selectivity from the administrative authority relating to warlike authorization. Requirements such as income, place of residence, proof of moral repute, and lack of police investigation or criminal process contain a moral load from the view of whiteness and then imply the racial filter and control in Law enforcement. By these means, the acquisition and possession of firearms in Brazil is a racial privilege, in effect, the legal framework that regulates all the use of firearms assists the whiteness’ yearnings for the warlike system. It has a relevant impact on the management of deaths of the black population in Brazil

Um Bronze de arte. Escultura romana encontrada em Alvarelhos, Santo Tirso.

64 (1-2) Jan.-Jun. 1954, p. 31-39