BACKGROUND Angiogenesis has been implicated in tissue injury in several noninfectious diseases, but its role in Chagas disease (CD) physiopathology is unclear. OBJECTIVES The present study aimed to investigate the effect of Trypanosoma cruzi infection on cardiac angiogenesis during the acute phase of experimental CD. METHODS The signalling pathway involved in blood vessel formation and cardiac remodelling was evaluated in Swiss Webster mice infected with the Y strain of T. cruzi. The levels of molecules involved in the regulation of angiogenesis, such as vascular endothelial growth factor-A (VEGF-A), Flk-1, phosphorylated extracellular-signal-regulated protein kinase (pERK), hypoxia-inducible factor-1α (HIF-1α), CD31, α-smooth muscle actin (α-SMA) and also the blood vessel growth were analysed during T. cruzi infection. Hearts were analysed using conventional histopathology, immunohistochemistry and western blotting. FINDINGS In this study, our data demonstrate that T. cruzi acute infection in mice induces exacerbated angiogenesis in the heart and parallels cardiac remodelling. In comparison with noninfected controls, the cardiac tissue of T. cruzi-infected mice presented higher levels of (i) HIF-1α, VEGF-A, Flk-1 and pERK; (ii) angiogenesis; (iii) α-SMA+ cells in the tissue; and (iv) collagen -1 deposition around blood vessels and infiltrating throughout the myocardium. MAIN CONCLUSIONS We observed cardiac angiogenesis during acute experimental T. cruzi infection parallels cardiac inflammation and remodelling.

Abstract Cardiac hypertrophy and dysfunction are a significant complication of chronic Chagas disease, with heart failure, stroke, and sudden death related to disease progression. Thus, understanding the signaling pathways involved in the chagasic cardiac hypertrophy may provide potential targets for pharmacological therapy. Herein, we investigated the implication of focal adhesion kinase (FAK) signaling pathway in triggering hypertrophic phenotype during acute and chronic T. cruzi infection. C57BL/6 mice infected with T. cruzi (Brazil strain) were evaluated for electrocardiographic (ECG) changes, plasma levels of endothelin-1 (ET-1) and activation of signaling pathways involved in cardiac hypertrophy, including FAK and ERK1/2, as well as expression of hypertrophy marker and components of the extracellular matrix in the different stages of T. cruzi infection (60–210 dpi). Heart dysfunction, evidenced by prolonged PR interval and decrease in heart rates in ECG tracing, was associated with high plasma ET-1 level, extracellular matrix remodeling and FAK signaling activation. Upregulation of both FAK tyrosine 397 (FAK-Y397) and serine 910 (FAK-S910) residues phosphorylation as well as ERK1/2 activation, lead to an enhancement of atrial natriuretic peptide gene expression in chronic infection. Our findings highlight FAK-ERK1/2 signaling as a regulator of cardiac hypertrophy in Trypanosoma cruzi infection. Both mechanical stress, induced by cardiac extracellular matrix (ECM) augment and cardiac overload, and ET-1 stimuli orchestrated FAK signaling activation with subsequent activation of the fetal cardiac gene program in the chronic phase of infection, highlighting FAK as an attractive target for Chagas disease therapy.

BACKGROUND Trypanosoma cruzi, the etiologic agent of Chagas disease, is capable of triggering different signaling pathways that modulate its internalisation in mammalian cells. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase protein, has been demonstrated as a mechanism of T. cruzi invasion in cardiomyocytes. Since the involved cell surface receptors are not yet known, we evaluated whether heparan sulfate proteoglycans (HSPG), a molecule involved in T. cruzi recognition and in the regulation of multiple signaling pathways, are able to trigger the FAK signaling pathway during T. cruzi invasion. METHODS To investigate the role of HSPG in the regulation of the FAK signaling pathway during trypomastigote entry, we performed heparan sulfate (HS) depletion from the cardiomyocyte surface by treatment with heparinase I or p-nitrophenyl-β-D-xylopyranoside (p-n-xyloside), which abolishes glycosaminoglycan (GAG) attachment to the proteoglycan core protein. Wild-type (CHO-k1) and GAG-deficient Chinese hamster ovary cells (CHO-745) were also used as an approach to evaluate the participation of the HSPG-FAK signaling pathway. FAK activation (FAK Tyr397) and spatial distribution were analysed by immunoblotting and indirect immunofluorescence, respectively. FINDINGS HS depletion from the cardiomyocyte surface inhibited FAK activation by T. cruzi. Cardiomyocyte treatment with heparinase I or p-n-xyloside resulted in 34% and 28% FAK phosphorylation level decreases, respectively. The experiments with the CHO cells corroborated the role of HSPG as a FAK activation mediator. T. cruzi infection did not stimulate FAK phosphorylation in CHO-745 cells, leading to a 36% reduction in parasite invasion. FAK inhibition due to the PF573228 treatment also impaired T. cruzi entry in CHO-k1 cells. MAIN CONCLUSION Jointly, our data demonstrate that HSPG is a key molecule in the FAK signaling pathway activation, regulating T. cruzi entry.

RESUMO O presente artigo avalia o desenvolvimento da resistência à tração por compressão diametral ou tração indireta (qt) e da compressão simples (qu) de dois solos cimentados artificialmente sob cura de 28 dias. Para isso, dois solos siltosos (amarelo e rosa) característicos da Formação Guabirotuba de Curitiba/BR foram estabilizados com cimento de alta resistência inicial (ARI) com porcentagens de 3, 5, 7 e 9% em referência à massa seca dos solos. Corpos de prova de 50 mm de diâmetro e 100 mm de altura (de volume=196,35 cm3) foram moldados com diferentes teores de cimento (C), diferentes pesos específicos secos aparentes (γd) e com uma umidade de moldagem (ω) constante em 23% e depois submetidos à ensaios de compressão simples e de tração indireta. Foi usada a relação vazios/cimento representado pela relação porosidade/teor volumétrico de cimento (η/Civ) para avaliar o desenvolvimento de qu e qt além de variáveis como C, porosidade (η) e γd. Os resultados demonstram um crescimento da resistência com o aumento do teor de cimento e com a diminuição dos vazios (isto é, maior peso específico seco e menor η). Uma equação de dosagem para qu e qt dos solos estudados e misturados com cimento foi desenvolvida com o uso da relação η/Civ ajustada a um expoente 0,44 para fazer compatível a variação entre η/Civ-qu qt. As equações de dosagem obtiveram coeficientes de determinação acima de 94%. Finalmente, se encontrou uma relação empírica de tração/compressão constante de 0,16 e de 0,14 para o solo amarelo e rosa, respectivamente.

ABSTRACT This paper evaluates the development of splitting tensile strength (qt) and unconfined compressive strength (qu) of two soils artificially cemented over 28 days. For this, two silty soils (yellow and purple) characteristics of the Guabirotuba Formation of Curitiba/Brazil were stabilized with initial high strength cement with percentages of 3, 5, 7 and 9% in reference to the dry mass of the soils. Specimens with 50 mm diameter and 100 mm high (volume of 196,35 cm3) were molded with the different cement contents (C), different molding dry unit weights (γd), and molding moisture (ω) constant at 23% and subjected to tests of unconfined compression and splitting tensile. The ratio of voids/lime represented by the porosity/volumetric cement content ratio (η/Civ) was used to evaluate the development of qu and qt in addition to variables such as C, η and γd. The results show an increase of the mechanical resistance with the increase of the cement content and with the decrease of the voids (i.e., higher specific dry weight and lower η). A dosing equation for qu and qt of the soils studied and mixed with cement was developed using the η/Civ ratio adjusted to an exponent 0,44 to make η/Civ-qu qt variation rates compatible. The dosage equations obtained coefficients of determination above 94%. Finally, an empirical constant of splitting/compressive ratio of 0,16 and 0,14 for the yellow and purple soil, respectively, was found.

ABSTRACT Various extracts obtained from the red alga Plocamium brasiliense (Greville Howe & Taylor), including a fraction containing crude 5-chloro-1-(E)-chlorovinyl-2,4-dibromo-1,5-dimethylcyclohexane (1) and another containing a mixture of halogenated monoterpenes (F), as well as atomaric acid meroditerpene (2) isolated from brown alga Stypopodium zonale (J. V. Lamouroux) Papenfuss, were evaluated for their activity against Trypanosoma cruzi. The cytotoxic and trypanosomicidal effects of these extracts were evaluated in Vero cells and clinically relevant forms of T. cruzi (amastigotes and trypomastigotes). All extracts from P. brasiliense presented low cytotoxicity and moderate trypanosomicidal effects, except for the hydroalcoholic extract. The crude 1 and F fractions had enhanced trypanocidal activity but showed low selectivity. Moreover, atomaric acid (2) was identified as a hit, demonstrating a potent trypanocidal effect reaching an IC50 <10 µM against two different DTU (Yand high selectivity index (<10). These results identify marine natural products as promising candidates against Chagas disease.

BACKGROUND Cardiac physiology depends on coupling and electrical and mechanical coordination through the intercalated disc. Focal adhesions offer mechanical support and signal transduction events during heart contraction-relaxation processes. Talin links integrins to the actin cytoskeleton and serves as a scaffold for the recruitment of other proteins, such as paxillin in focal adhesion formation and regulation. Chagasic cardiomyopathy is caused by infection by Trypanosoma cruzi and is a debilitating condition comprising extensive fibrosis, inflammation, cardiac hypertrophy and electrical alterations that culminate in heart failure. OBJECTIVES Since mechanotransduction coordinates heart function, we evaluated the underlying mechanism implicated in the mechanical changes, focusing especially in mechanosensitive proteins and related signalling pathways during infection of cardiac cells by T. cruzi. METHODS We investigated the effect of T. cruzi infection on the expression and distribution of talin/paxillin and associated proteins in mouse cardiomyocytes in vitro by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). FINDINGS Talin and paxillin spatial distribution in T. cruzi-infected cardiomyocytes in vitro were altered associated with a downregulation of these proteins and mRNAs levels at 72 h post-infection (hpi). Additionally, we observed an increase in the activation of the focal adhesion kinase (FAK) concomitant with increase in β-1-integrin at 24 hpi. Finally, we detected a decrease in the activation of FAK at 72 hpi in T. cruzi-infected cultures. MAIN CONCLUSION The results suggest that these changes may contribute to the mechanotransduction disturbance evidenced in chagasic cardiomyopathy.

BACKGROUND Didelphis spp. are a South American marsupial species that are among the most ancient hosts for the Trypanosoma spp. OBJECTIVES We characterise a new species (Trypanosoma janseni n. sp.) isolated from the spleen and liver tissues of Didelphis aurita in the Atlantic Rainforest of Rio de Janeiro, Brazil. METHODS The parasites were isolated and a growth curve was performed in NNN and Schneider's media containing 10% foetal bovine serum. Parasite morphology was evaluated via light microscopy on Giemsa-stained culture smears, as well as scanning and transmission electron microscopy. Molecular taxonomy was based on a partial region (737-bp) of the small subunit (18S) ribosomal RNA gene and 708 bp of the nuclear marker, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. Maximum likelihood and Bayesian inference methods were used to perform a species coalescent analysis and to generate individual and concatenated gene trees. Divergence times among species that belong to the T. cruzi clade were also inferred. FINDINGS In vitro growth curves demonstrated a very short log phase, achieving a maximum growth rate at day 3 followed by a sharp decline. Only epimastigote forms were observed under light and scanning microscopy. Transmission electron microscopy analysis showed structures typical to Trypanosoma spp., except one structure that presented as single-membraned, usually grouped in stacks of three or four. Phylogeography analyses confirmed the distinct species status of T. janseni n. sp. within the T. cruzi clade. Trypanosoma janseni n. sp. clusters with T. wauwau in a well-supported clade, which is exclusive and monophyletic. The separation of the South American T. wauwau + T. janseni coincides with the separation of the Southern Super Continent. CONCLUSIONS This clade is a sister group of the trypanosomes found in Australian marsupials and its discovery sheds light on the initial diversification process based on what we currently know about the T. cruzi clade.

Resumo Este estudo avaliou a riqueza de epífitas vasculares sobre Pseudobombax sp. nov. em três inselbergs do Domínio Atlântico no estado do Espírito Santo e avaliou a similaridade florística entre as áreas. Foram amostrados 111 forófitos, em três regiões no sul do estado do Espírito Santo e identificados 151 espécies, 77 gêneros e 21 famílias de epífitos vasculares, das quais as famílias Orchidaceae e Bromeliaceae apresentaram maior riqueza. Estimadores não-paramétricos (Chao 2, Jackknife 1) indicam que 90 e 95% da riqueza específica de epífitas foi registrada. A categoria ecológica mais diversificada foi a dos holoepífitos característicos. A razão entre o número de epífitas e número de forófitos amostrados em um inselberg montano, neste estudo, foi maior do que a riqueza de epífitos encontrados nos campos rupestres, e, em geral, em diferentes tipos de florestas do Domínio Atlântico, mas menor em riqueza para algumas Florestas Ombrófilas Densas do sul do Brasil. As três áreas de inselbergs tinham floras distintas. A elevada riqueza, o endemismo encontrado e o número de espécies ameaçadas dos epífitos evidenciam a importante função de Pseudobombax sp. nov., por sua arquitetura e tamanho, para a manutenção da biodiversidade em inselbergs do sudeste brasileiro.

Abstract This study evaluated the richness of vascular epiphytes on Pseudobombax sp. nov. in three inselbergs in the Atlantic Forest Domain in state of Espírito Santo and evaluated the floristic similarity between the areas. We sampled 111 phorophytes in three regions in the southern of Espírito Santo state and identified 151 species, 77 genera and 21 families of vascular epiphytes, of which the families Orchidaceae and Bromeliaceae showed the highest richness. Non-parametric estimators (Chao 2, Jackknife 1) indicate that 90 and 95% of species richness of epiphytes was recorded. The most diversified ecological category was the characteristic holoepiphytes. The ratio of the number of epiphytes and number of phorophytes sampled in a montane inselberg, in this study, was greater than the richness of vascular epiphytes found in the rocky outcrops of quartzite, and, in general, different types of Atlantic Domain forests, but smaller in richness for some Dense Ombrophilous Forests of southern Brazil. The three inselberg areas had distinct floras. The high richness, the endemism found, and the number of endangered species of epiphytes demonstrate the important role of Pseudobombax sp. nov., because of its architecture and size, in the maintenance of biodiversity on the inselbergs in southeastern Brazil.

An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.

O artigo analisa, nas falas dos professores da rede pública, as referências à inclusão de alunos com deficiência. Tais falas foram coletadas em entrevistas não-estruturadas, interpretadas seguindo os princípios da análise de discurso e organizadas em três grupos de sentido: concepções de base, o lugar de si e o lugar do outro. Aparece, no discurso, uma naturalização das temáticas da deficiência e da normalidade. O falante descreve-se como parte do processo inclusivo, mesmo relatando sentimentos como impotência, frustração e despreparo. Essa participação limita-se à sala de aula, evidenciando certa passividade com relação às carências da educação. Distingue-se do restante o mundo onde o professor tem função de direção (a sala de aula). As relações entre governo, pais, atendimento especializado e a ação pedagógica aparecem fragmentadas. Os fatores apontados como desfavoráveis à inclusão são naturalizados. Essa distinção entre o mundo pedagógico e o restante reforça uma concepção de inclusão como produto acabado, naturalizando-a e separando o processo inclusivo das relações entre inclusão e exclusão.

This article analyses references to the inclusion of disabled students in the discourse of teachers from the public network, which were collected by means of non-structured interviews. These were then interpreted following the principles of discourse analysis and organized in three groups of meaning: basic conceptions, the place of oneself and the place of the other. Some kind of naturalization of the themes of disability and normality appear in the discourse. The speaker describes himself as part of the process of inclusion, even though he relates feelings of impotence, frustration and lack of preparation. This participation is limited to the classroom, and reveals certain passivity with relation to the failings of education. This differs from the world in which the teacher has a directing function (the classroom). The relations between government, parents, specialized care and pedagogical action seem to be fragmented. The facts which are indicated as unfavourable to inclusion are naturalized. This distinction between the pedagogical world and the rest strengthens a conception of inclusion as a finished product, naturalizing it and separating the process of inclusion from the relations between inclusion and exclusion.

El artículo analiza, en las pláticas de los profesores de la red pública, las referencias a la inclusión de alumnos con deficiencia. Tales pláticas fueron colectadas en entrevistas no estructuradas, interpretadas siguiendo los principios del análisis de discurso y organizadas en tres grupos de sentido: concepciones de base, o lugar de sí y el lugar del otro. Aparece, en el discurso, una naturalización de las temáticas de la deficiencia y de la normalidad. El hablante se describe como parte del proceso inclusivo, mismo relatando sentimientos como impotencia, frustración y desorganización. Esa participación se limita a la sala de clase, evidenciando cierta pasividad con relación a las carencias de la educación. Se distingue del restante, el mundo donde el profesor tiene función de dirección (a sala de clase). Las relaciones entre gobierno, padres, atendimiento especializado y la acción pedagógica aparecen fragmentadas. Los factores apuntados como desfavorables a la inclusión son naturalizados. Esa distinción entre el mundo pedagógico y lo restante, refuerza una concepción de inclusión como producto acabado, naturalizándola y separando el proceso inclusivo de las relaciones entre inclusión y exclusión.

The present paper summarizes new approaches regarding the progress done to the understanding of the interaction of Trypanosoma cruzi-cardiomyocytes. Mannose receptors localized at the surface of heart muscle cell are involved in binding and uptake of the parasite. One of the most striking events in the parasite-heart muscle cells interaction is the disruption of the actin cytoskeleton. We have investigated the regulation of the actin mRNA during the cytopathology induced in myocardial cells by the parasite. T. cruzi invasion increases calcium resting levels in cardiomyocytes. We have previously shown that Ca2+ ATPase of the sarcoplasmic reticulum (SERCA) is involved in the invasion of T. cruzi in cardiomyocytes. Treating the cells with thapsigargin, a drug that binds to all SERCA ATPases and causes depletion of intracellular calcium stores, we found a 75% inhibition in the T. cruzi-cardiomyocytes invasion.

In five experiments, Leishmania (Leishmania) major (MRHO/SU/59/P-strain) grew poorly when seeded in FYTS medium supplemented with 15% fetal calf serum, but presented several peculiar pairs of promastigotes diametrically opposed and attached at their posterior ends (5.8-13.5%). As seen in Giemsa-stained smears, a ring-like line and/or an enlargement, generally occurred at the parasite junction. A close proximity of nuclei, which sometimes were difficult to distinguish from each other, was also observed at this junction. Several of these pairs appeared to be composed of fused cells in which the nuclei could be apparently fused, as shown by fluorescence microscopy to detect ß-tubulin and DNA, and by scanning electron microscopy. Under other culture conditions these pairs were absent or occurred at very low rates (0.2-2.2%). Such pairs differ markedly from longitudinally dividing cells and resemble those described in two other Leishmania species, as well as in Herpetomonas megaseliae and Phytomonas davidi, suggesting steps of a putative sexual process