Leitura e formação no Ensino Superior : problematização sobre a formação de leitores no Brasil e em Portugal

Neste artigo discute-se o problema da leitura e da formação de leitores no ensino superior, no Brasil e em Portugal, sendo nosso objetivo perceber se estes estudantes são ainda leitores em construção. A metodologia para a discussão centrou-se nas investigações em torno da formação leitora desses estudantes, o que permitiu chegar a algumas conclusões afins: são fundamentais estudos de maior dimensão nessas áreas; é basilar que todos os docentes do ensino superior tomem consciência das dificuldades desses alunos na leitura/escrita/literacias académicas e que nas suas disciplinas possam dar um contributo para colmatar esses problemas; os alunos do ensino superior são ainda leitores em construção.info:eu-repo/semantics/publishedVersio

Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. in an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. the monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. the monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. the assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)ERC AdG SISYPHEUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Ultraestrutura Celular Hertha Meyer, BR-21941 Rio de Janeiro, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Metab Macromol Firmino Torres de Castro, BR-21941 Rio de Janeiro, BrazilLab Bioinformat, Lab Nacl Computacao Cient, Rio de Janeiro, BrazilINRIA Grenoble Rhone Alpes, BAMBOO Team, Villeurbanne, FranceUniv Lyon 1, CNRS, UMR5558, Lab Biometrie & Biol Evolut, F-69622 Villeurbanne, FranceUniv Estadual Campinas, Inst Biol, Dept Genet Evolucao & Bioagentes, São Paulo, BrazilUniv São Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Ciencias Farmaceut, São Paulo, BrazilLab Nacl Ciencia & Tecnol Bioetano, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Belo Horizonte, MG, BrazilUniv Fed Goias, Inst Ciencias Biol, Mol Biol Lab, Goiania, Go, BrazilFundacao Oswaldo Cruz, Inst Carlos Chagas, Lab Biol Mol Tripanossomatideos, Curitiba, Parana, BrazilFundacao Oswaldo Cruz, Inst Carlos Chagas, Lab Genom Func, Curitiba, Parana, BrazilUniv Estadual Campinas, Ctr Pluridisciplinar Pesquisas Quim Biol & Agr, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Parasitol, Belo Horizonte, MG, BrazilUniv Fed Santa Catarina, Dept Microbiol Imunol & Parasitol, Ctr Ciencias Biol, Lab Protozool & Bioinformat, Florianopolis, SC, BrazilUniv Fed Vicosa, Dept Bioquim & Biol Mol, Ctr Ciencias Biol & Saude, Vicosa, MG, BrazilInst Butantan, Lab Especial Ciclo Celular, São Paulo, BrazilUniv São Paulo, Dept Biol, Fac Filosofia Ciencias & Letras Ribeirao Preto, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

TEM of <i>T. cruzi</i> epimastigotes treated with SBIs at the EC₅₀/72 h.

<p>(A) Control epimastigotes, providing a general view of parasite ultrastructure, indicating the nucleus (N), kinetoplast (K), mitochondria (M), flagellum (F) and reservosome (R). (B to E) Exposure to 50 µM lovastatin for 72 hours (B) or 120 hours (C to E). (F to H) Exposure to 32 µM ketoconazole for 72 hours (F) or 120 hours (G and H). For all images, the white asterisks (*) indicate the swollen reservosomes and the black arrows (→) indicate aberrant mitochondrial branching. The abnormal mitochondrial pattern is highlighted in (C) (in box). A myelin figure, typical of autophagic cells, is highlighted in (E). A: Autophagosome. Bars: (A), 2 µm; (B), (F), (G), 1 µm; (C), 0.5 µm; (D), (E), (H), 0.2 µm.</p

Reservosome membrane permeabilization (RMP) in response to treatment with SBIs at the EC₁₀₀/24 h.

<p>(A) Example of RMP analysis by flow cytometry with AO, showing that treated cells with lysed reservosomes have a high FL1-H signal intensity. The values inside the boxes indicate the percentage of cells with lysed reservosomes. (B) Kinetics of RMP obtained by flow cytometry; each experimental point indicates the mean and standard deviation of triplicate experiments. (C) Visualization of reservosome lysis by confocal microscopy; (i) Live cells stained with AO; green (“green fluo”) and red (“red fluo”) AO fluorescence was photographed in different frames. The normal pattern of reservosome staining persists after 15 minutes of drug exposure (upper row), but, within 1 hour, all the acidic vesicles disappear (bottom row). (ii) Immunofluorescence analysis with an antibody directed against a reservosomal protein (TcRBP40); in control cells, this protein is found mostly in the reservosomes (upper row). However, after 1 hour of drug treatment, a diffuse signal is observed throughout the parasite body (bottom row). In (i) and (ii), the white arrows indicate low-fluorescence regions possibly corresponding to sites previously occupied by intact reservosomes. For both confocal experiments, similar results were obtained for ketoconazole and lovastatin and the drug used is therefore not indicated. The scale bars indicate 4 µm (i) and 3 µm (ii).</p

TEM and flow cytometry assays after treatment with SBIs at the EC₁₀₀/24 h.

<p>(A) TEM images after 18 hours of exposure to SBIs at the EC₁₀₀/24 h, showing marked cell degradation, with kinetoplast disruption (* in (i)) and cell lysis (ii); the occurrence of reservosome lysis is highlighted in (iii) and (iv). These morphological patterns are similar to those observed during cell death by necrosis. TEM results were similar for the two SBIs and the drug name is not shown. Scale bars: (i) and (ii), 1 µm; (iii) and (iv), 0.5 µm. (B) Flow cytometry analysis of <i>T. cruzi</i> necrotic death in reponse to SBIs at the EC₁₀₀/24 h. (i) Analysis of relative intracellular calcium concentrations by fluo-4-AM staining, after 0.5 to 12 hours. A rapid increase in fluo-4-AM fluorescence with respect to control cells can be seen after exposure to the two SBIs at the EC₁₀₀/24 h. (ii) Assay of mitochondrial membrane depolarization by R123 staining; time-dependent mitochondrial depolarization can clearly be seen by comparison with control cells. (iii) Cell viability analysis based on propidium iodide staining; the percentage dead cells (PI-positive) is plotted as a function of drug exposure time. Note the differences in cell lysis kinetics for the two drugs: the experimental points were optimally adjusted by a sigmoidal curve for lovastatin and by a negative exponential curve for ketoconazole. The raw flow cytometry plots can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055497#pone.0055497.s004" target="_blank">Figure S4</a>.</p

Absence of apoptotic markers in the EC₁₀₀/24 h response.

<p>(A) Analysis of phosphatidylserine exposure by co-staining with Annexin-V-FITC and PI. As an example, we have plotted data for exposure for 12 hours to ketoconazole (ii) or lovastatin (iii), together with the control cell pattern (i). (B) DNA laddering assay; total DNA was isolated from control cultures (C) and from drug-treated cells (120 µM ketoconazole (K), 100 µM lovastatin (L)), after 12 or 24 hours of exposure (indicated at the top), as described in the methods section. We subjected 5 µg of the DNA to electrophoresis in a 1.5% agarose gel stained with ethidium bromide; M lanes contain the 1 kb Plus DNA ladder.</p

Antiproliferative and trypanocidal effects of SBIs in <i>T. cruzi</i>.

<p>(A and B) Growth curves of cultured epimastigotes exposed to various concentrations of lovastatin (A) or ketoconazole (B). The dose-response curve and respective EC₅₀/72 h values are shown in the box. (C and D) Recovery experiments: epimastigote cultures were exposed to 100 µM lovastatin (C) or 120 µM ketoconazole (D). The drug was then removed by successive washes, after short periods of time (specified in the graph). The subsequent growth of the parasites was followed for three days, by counting, in a Neubauer chamber. (E) Percentage of dead cells (spheroid) as a function of time exposed to 120 µM ketoconazole or 100 µM lovastatin. (F) Stained smears of parasites exposed to SBIs at the EC₁₀₀/24 h for 12 hours, showing the spheroid shape of the cells; the scale bars indicate 10 µm. For all graphs, each experimental point corresponds to the mean and standard deviation for cell density obtained by direct counting in a Neubauer chamber.</p

Mitochondrial branching in response to SBIs at the EC₅₀/72 h, as detected by R123 fluorescence.

<p>(A) Overlay histograms of flow cytometry experiments on SBI-treated cultures, for exposure times of 24 to 120 hours (in box). A gradual increase in green wavelength fluorescence (FL1-H) can be seed for the treated parasites. Histograms representative of triplicate experiments are shown. (B) Fold-change in the geometric mean of R123 fluorescence intensity (FL1-H) with respect to control cells in triplicate flow cytometry experiments. (C) Confocal microscopy of live parasites, showing the branching of the mitochondrial membranes in treated parasites (72 hours); the results for lovastatin were similar to those for ketoconazole and are therefore not shown here; the scale bars indicate 10 µm.</p

<em>Trypanosoma cruzi</em> Response to Sterol Biosynthesis Inhibitors: Morphophysiological Alterations Leading to Cell Death

<div><p>The protozoan parasite <em>Trypanosoma cruzi</em> displays similarities to fungi in terms of its sterol lipid biosynthesis, as ergosterol and other 24-alkylated sterols are its principal endogenous sterols. The sterol pathway is thus a potential drug target for the treatment of Chagas disease. We describe here a comparative study of the growth inhibition, ultrastructural and physiological changes leading to the death of <em>T. cruzi</em> cells following treatment with the sterol biosynthesis inhibitors (SBIs) ketoconazole and lovastatin. We first calculated the drug concentration inhibiting epimastigote growth by 50% (EC₅₀/72 h) or killing all cells within 24 hours (EC₁₀₀/24 h). Incubation with inhibitors at the EC₅₀/72 h resulted in interesting morphological changes: intense proliferation of the inner mitochondrial membrane, which was corroborated by flow cytometry and confocal microscopy of the parasites stained with rhodamine 123, and strong swelling of the reservosomes, which was confirmed by acridine orange staining. These changes to the mitochondria and reservosomes may reflect the involvement of these organelles in ergosterol biosynthesis or the progressive autophagic process culminating in cell lysis after 6 to 7 days of treatment with SBIs at the EC₅₀/72 h. By contrast, treatment with SBIs at the EC₁₀₀/24 h resulted in rapid cell death with a necrotic phenotype: time-dependent cytosolic calcium overload, mitochondrial depolarization and reservosome membrane permeabilization (RMP), culminating in cell lysis after a few hours of drug exposure. We provide the first demonstration that RMP constitutes the “point of no return” in the cell death cascade, and propose a model for the necrotic cell death of <em>T. cruzi</em>. Thus, SBIs trigger cell death by different mechanisms, depending on the dose used, in <em>T. cruzi</em>. These findings shed new light on ergosterol biosynthesis and the mechanisms of programmed cell death in this ancient protozoan parasite.</p> </div