Monitoring of dynamin during the Toxoplasma gondii cell cycle

The obligate intracellular protozoan parasite Toxoplasma gondii actively invades virtually all warm-blooded nucleated cells. This process results in a non-fusogenic vacuole, inside which the parasites replicate continuously until egress signaling is triggered. In this work, we investigated the role of the large GTPase dynamin in the interaction of T. gondii with the host cell by using laser and electron microscopy during three key stages: invasion, development and egress. The detection of dynamin during invasion indicates the occurrence of endocytosis, while T. gondii egress appeared to be independent of dynamin participation. However, the presence of dynamin during T. gondii development suggests that this molecule plays undescribed roles in the tachyzoite's cell cycle

Phosphatidylserine Exposure by Toxoplasma gondii Is Fundamental to Balance the Immune Response Granting Survival of the Parasite and of the Host

Phosphatidylserine (PS) exposure on the cell surface indicates apoptosis, but has also been related to evasion mechanisms of parasites, a concept known as apoptotic mimicry. Toxoplasma gondii mimics apoptotic cells by exposing PS, inducing secretion of TGF-beta1 by infected activated macrophages leading to degradation of inducible nitric oxide (NO) synthase, NO production inhibition and consequently persisting in these cells. Here PS+ and PS− subpopulation of tachyzoites were separated and the entrance mechanism, growth and NO inhibition in murine macrophages, and mice survival and pathology were analyzed. Infection index in resident macrophages was similar for both PS subpopulations but lower when compared to the total T. gondii population. Growth in resident macrophages was higher for the total T. gondii population, intermediate for the PS+ and lower for the PS− subpopulation. Production of NO by activated macrophages was inhibited after infection with the PS+ subpopulation and the total populations of tachyzoites. However, the PS− subpopulation was not able to inhibit NO production. PS+ subpopulation invaded macrophages by active penetration as indicated by tight-fitting vacuoles, but the PS− subpopulation entered macrophages by phagocytosis as suggested by loose-fitting vacuoles containing these tachyzoites. The entrance mechanism of both subpopulations was confirmed in a non-professional phagocytic cell line where only the PS+ tachyzoites were found inside these cells in tight-fitting vacuoles. Both subpopulations of T. gondii killed mice faster than the total population. Clear signs of inflammation and no tachyzoites were seen in the peritoneal cavity of mice infected with the PS− subpopulation. Moreover, mice infected with the PS+ subpopulation had no sign of inflammation and the parasite burden was intense. These results show that PS+ and PS− subpopulations of T. gondii are necessary for a successful toxoplasma infection indicating that both subpopulations are required to maintain the balance between inflammation and parasite growth

RAZÃO DA NÃO UTILIZAÇÃO DE INCENTIVOS FISCAIS À INOVAÇÃO TECNOLÓGICA DA LEI DO BEM POR EMPRESAS BRASILEIRA

Partindo da existência de incentivos fiscais para a atividade inovadora, a exemplo da Lei nº 11.196/2005 (chamada Lei do Bem) que ampliou os benefícios existentes e instituiu outros novos para estimular a inovação, definiu-se como problema de pesquisa a seguinte questão: Quais as razões da não utilização de incentivos fiscais à inovação tecnológica da Lei do Bem pelas empresas brasileiras? Assim, o objetivo geral consistiu em identificar as razões da não utilização de incentivos fiscais à inovação tecnológica da Lei do Bem por empresas privadas de capital aberto, listadas no segmento novo mercado de governança corporativa da BM&amp;FBovespa. Para tanto se realizou pesquisa do tipo qualitativa, documental e descritiva, viabilizada por meio da análise dos relatórios contábeis, bem como dos relatórios que contêm informações sociais e por meio de questionário com 125 companhias. Os resultados possibilitaram concluir que as companhias que investiram em P&amp;D e não se utilizaram da Lei do Bem, não se beneficiaram ou porque estavam com problemas fiscais ou porque fecharam o ano de 2014 com prejuízo fiscal. A importância deste estudo decorre do fornecimento de informações atuais e significativas das razões para as empresas não estarem se beneficiando dos incentivos fiscais à inovação tecnológica, tema que até agora vinha sendo negligenciado pelos pesquisadores. Espera-se, assim, que os conhecimentos adquiridos a partir desta investigação possa contribuir para o futuro desenvolvimento desta linha de pesquisa, bem como promover nas entidades reflexão para a solução dos entraves apresentados.<br /

<i>Cryptococcus neoformans</i> Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

<div><p>Cryptococcosis by the encapsulated yeast <i>Cryptococcus neoformans</i> affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of <i>Cryptococcus</i> yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of <i>Cryptococcus</i> internalization by host cells remain poorly understood. Here, we investigate the mechanism of <i>Cryptococcus neoformans</i> phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of <i>C. neoformans</i> are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of <i>Cryptococcus</i> uptake by host cells.</p></div

Uptake of <i>Cryptococcus</i> strains by trigger-like and zipper-like structures.

<p>Scanning electron microscopy of <i>C. neoformans</i> capsular strain H99 (A–E) and acapsular strain CAP59 (F–G) interacting with peritoneal macrophages. Improved preservation of macrophage membranes was obtained with routine SEM fixation (A–B; F–G), although post-fixation in the presence of sucrose provided better capsule preservation and allowed visualization of direct interactions between the capsule and host cell membranes, prior to internalization (C–E). Both trigger-like (arrow in A and F) and zipper-like (arrow-head in B and G) uptake structures were observed. Scale bars, 1 µm (A–C; F–G) and 0.5 µm (D–E).</p

Actin recruitment is inhibited by cytochalasin D.

<p>Confocal laser scanning microscopy of <i>C. neoformans</i> capsular strain H99 interacting with macrophages (single confocal plane). DIC showing internalized yeasts (arrows); and confocal images showing actin filaments (red), microtubules (green), yeast (blue) and host DNA (blue, indicated by ‘n’). Actin is recruited to the site of phagocytosis in untreated cells (A), and actin recruitment was inhibited by 0.5 µM cytochalasin D (B). In contrast, treatment with 5 µM nocodazole (C) or with a combination of nocodazole and cytochalasin D (D) did not inhibit actin recruitment to the phagosome area. Scale bars, 5 µm.</p

Treatment with both cytochalasin D and nocodazole did not increase the inhibitory effect.

<p>Quantification of the internalization (A) and the attachment (B) to macrophages of <i>C. neoformans</i> yeast cells from capsular (H99 and B3501) and acapsular (CAP67 and CAP 59) strains, in the absence of cytoskeletal inhibitors or in the presence of cytochalasin D and nocodazole. The metabolic viability of <i>C. neoformans</i> strains H99 and CAP59 was measured using the FUN®-1 dye (C) and the metabolic viability of macrophages was measured by MTS/PMS (D) after incubation with cytoskeletal inhibitors for 2 h. Yeast cells fixed with 70% ethanol, and macrophages with 4% formaldehyde, were used as a positive control for the loss of cell viability in each method. Graphs show normalized mean values and standard deviation from three experiments (A–B) and mean and standard deviation from absolute values of fluorescence intensity (C) and absorbance (D).*p<0.05; **p<0.01; ***p<0.001.</p

Actin is recruited to the phagosome area during <i>C. neoformans</i> internalization.

<p>Confocal laser scanning microscopy (z-stack series of confocal planes) of interacting macrophages and <i>C. neoformans</i> yeast cells from strains H99 (A and B) and CAP59 (C and D). Internalized yeasts identified by DIC (arrows in A and C) can be visualized in the context of host cell actin (red) and microtubule (green) cytoskeletons (B and D). Host cell DNA is labeled with DAPI (blue, indicated by the letter ‘N’) and yeast is labeled with calcofluor (blue, indicated by arrows). Actin, but not tubulin, is recruited to sites of yeast internalization. Scale bars, 5 µm.</p

Involvement of the cytoskeleton in the yeast-macrophage interaction.

<p>Scanning electron microscopy of membrane extracted macrophages interacting with <i>C. neoformans</i> strains H99 (A) and CAP59 (B and C), showing cytoskeletal filaments associated with yeasts in untreated samples (A–B). After 5 µm nocodazole treatment (C) the area surrounding yeast cells appeared mostly devoid of cytoskeletal components but association with yeast still occurred (inset in C). Scale bars, 2 µm.</p