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Transcriptome Profile of the Response of Paracoccidioides spp. to a Camphene Thiosemicarbazide Derivative.

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RESEARCH ARTICLE Transcriptome Profile of the Response of Paracoccidioides spp. to a Camphene Thiosemicarbazide Derivative Lívia do Carmo Silva1, Diana Patrícia Tamayo Ossa2, Symone Vitoriano da Conceição Castro1, Ludmila Bringel Pires3, Cecília Maria Alves de Oliveira3, Cleuza Conceição da Silva4, Narcimário Pereira Coelho4, Alexandre Melo Bailão1, Juliana Alves Parente-Rocha1, Célia Maria de Almeida Soares1, Orville Hernández Ruiz2, Juan G. McEwen Ochoa2, Maristela Pereira1* a11111 1 Laboratório de Biologia Molecular, Instituto de Patologia Tropical e Saúde Pública Universidade Federal de Goiás, Goiânia, Brazil, 2 Unidad de Biología Celular y Molecular, Corporación para Investigaciones Biológicas (CIB) and Facultad de Medicina Universidad de Antioquia, Medellín, Colombia, 3 Laboratório de Produtos Naturais, Instituto de Química, Universidade Federal de Goiás, Goiânia, Brazil, 4 Laboratório de Fitoquímica e Síntese Orgânica, Departamento de Química, Universidade Estadual de Maringá, Paraná, Brazil * maristelaufg@gmail.com OPEN ACCESS Citation: do Carmo Silva L, Tamayo Ossa DP, Castro SVdC, Bringel Pires L, Alves de Oliveira CM, Conceição da Silva C, et al. (2015) Transcriptome Profile of the Response of Paracoccidioides spp. to a Camphene Thiosemicarbazide Derivative. PLoS ONE 10(6): e0130703. doi:10.1371/journal.pone.0130703 Editor: Oscar Zaragoza, Instituto de Salud Carlos III, SPAIN Received: February 3, 2015 Accepted: May 23, 2015 Published: June 26, 2015 Copyright: © 2015 Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The ESTs obtained were submitted to the National Center for Biotechnology Information (NCBI) under accession numbers: LIBEST_028508 Paracoccidioides thiosemicarbazide Library. Funding: This work performed at Universidade Federal de Goiás was supported by MCTI/CNPq (Ministério da Ciência e Tecnologia/Conselho Nacional de Desenvolvimento Científico e Tecnológico), FNDCT (Fundo Nacional de Desenvolvimento Científico e Tecnológico), FAPEG (Fundação de Amparo à Pesquisa do Estado de Abstract Paracoccidioidomycosis (PCM) is a systemic granulomatous human mycosis caused by fungi of the genus Paracoccidioides, which is geographically restricted to Latin America. Inhalation of spores, the infectious particles of the fungus, is a common route of infection. The PCM treatment of choice is azoles such as itraconazole, but sulfonamides and amphotericin B are used in some cases despite their toxicity to mammalian cells. The current availability of treatments highlights the need to identify and characterize novel targets for antifungal treatment of PCM as well as the need to search for new antifungal compounds obtained from natural sources or by chemical synthesis. To this end, we evaluated the antifungal activity of a camphene thiosemicarbazide derivative (TSC-C) compound on Paracoccidioides yeast. To determine the response of Paracoccidioides spp. to TSC-C, we analyzed the transcriptional profile of the fungus after 8 h of contact with the compound. The results demonstrate that Paracoccidioides lutzii induced the expression of genes related to metabolism; cell cycle and DNA processing; biogenesis of cellular components; cell transduction/signal; cell rescue, defense and virulence; cellular transport, transport facilities and transport routes; energy; protein synthesis; protein fate; transcription; and other proteins without classification. Additionally, we observed intensely inhibited genes related to protein synthesis. Analysis by fluorescence microscopy and flow cytometry revealed that the compound induced the production of reactive oxygen species. Using an isolate with down-regulated SOD1 gene expression (SOD1-aRNA), we sought to determine the function of this gene in the defense of Paracoccidioides yeast cells against the compound. Mutant cells were more susceptible to TSC-C, demonstrating the importance of this gene in response to the compound. The results presented herein suggest that TSC-C is a promising candidate for PCM treatment. PLOS ONE | DOI:10.1371/journal.pone.0130703 June 26, 2015 1 / 25 Response of Paracoccidioides to Camphene Thiosemicarbazide Derivative Goiás), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), FINEP (Financiadora de Estudos e Projetos), and INCT-IF (Instituto Nacional de Ciência e Tecnologia para Inovação Farmacêutica). Additionally, LCS was supported by fellowship from CNPq and SVCC, LBP, NPC from CAPES. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Introduction Paracoccidioidomycosis (PCM) is a systemic mycosis geographically restricted to Latin America caused by thermodimorphic fungi of the genus Paracoccidioides. The fungi usually infect the host through the respiratory tract by inhalation of conidia, which are the infectious propagules found in the environment. In the lungs, these propagules differentiate into the pathogenic form in a temperature-dependent manner, corresponding to the yeast phase of the fungus, and spreads to other organs through lymphohematogenous dissemination. Because this mycosis affects mainly rural males of working age between the ages of 30 and 50 years, the disease has socioeconomic repercussions due to its potential to debilitate. In addition to the lungs, PCM frequently compromises the mucous membranes, lymph nodes, liver, spleen and bone marrow [1,2]. Treating PCM remains a challenge due to the toxicity of the antifungals commonly used to treat this mycosis—sulfonamides, azoles and polyenes [3,4]. Additionally, despite the use of antifungals, individuals with PCM have persistent latent foci, which slow down treatment and may extend it over months or years depending on the severity of the disease and the site of injury [5,6,7]. Thus, the need to research and develop new therapeutic approaches is increasingly evident. With this aim, our group has invested effort into identifying and characterizing novel targets for antifungal drugs against Paracoccidioides spp. [8–16] and searching for new antifungal compounds obtained from natural sources or their synthetic derivatives [17,18,19]. The monoterpenoids are the components of essential oils, which are produced in large quantities by plants. These molecules are significant due to their therapeutic potential, low cost as well as the commercial availability, being used as starting material for synthesis of bioactive compounds [20,21]. Following this approach a series of thiosemicarbazides and thiosemicarbazones deriving from bisabolol, kaurenoic acid, limonene and camphene were synthetized by our research group [22,23,24]. Among them, the tiosemicarbazide camphene derivative (TSC-C) showed remarkable antifungal activity. The previous study showed that TSC-C inhibited the growth of Trichophyton mentagrophytes by damaging the cell wall structure or interfering with its formation during the process of cell division, growth or morphogenesis [24]. Based on these results, we elected TSC-C to study its activity and mode of action on Paracoccidioides brasiliensis. We constructed a cDNA library to obtain expressed sequences tags (ESTs) from P. lutzii in response to TSC-C with the ultimate aim to identify the likely mode of action of the compound in the fungus. We performed assays to confirm the transcriptome data to P. lutzii and Paracoccidioides brasiliensis, such as quantitative real-time PCR (qRT-PCR), fluorescence microscopy, DNA fragmentation, cell cycle analysis by flow cytometry and enzymatic assays. Materials and Methods General procedure for the preparation of compounds The TSC-C was prepared as described by Yamaguchi [24]. Microorganism and cell culture The P. lutzii ATCC MYA 826 and P. brasiliensis ATCC 60855 strains were used in the assays. Yeast cells were maintained in Fava-Netto liquid medium [25] for 3 days. The cells were then transferred and grown overnight in McVeigh Morton (MMcM) liquid medium overnight [26] and subsequently used in experiments. PLOS ONE | DOI:10.1371/journal.pone.0130703 June 26, 2015 2 / 25 Response of Paracoccidioides to Camphene Thiosemicarbazide Derivative Determination of inhibitory concentration (IC50) Preparation of resazurin. Resazurin powder (Sigma Aldrich, St. Louis, MO, USA) was dissolved in sterile distilled water at a final concentration of 0.02%, sterilized by filtration and stored at 4°C until use. Preparation of the camphene thiosemicarbazide derivative. The stock solution of TSC-C was prepared in dimethyl sulfoxide (10% DMSO) and diluted to obtain the evaluated concentrations (316 μM, 158 μM, 79 μM, 39.5 μM and 19.5 μM). The determination of IC50 was performed according to the micro-dilution method described in the Clinical and Laboratory Standards Institute (CLSI) [27] and De Paula et al. [28]. Were inoculated 1x106 cells/mL of P. lutzii yeast cells per microplate well in MMcM liquid medium supplemented with 316 μM, 158 μM, 79 μM or 39.5 μM TSC-C. To determine the maximum growth rate (positive control), some wells received culture medium in place of the 100 mL of test compound dilution. The plates were incubated at 36°C with shaking at 150 rpm for 48 h. Each well then received 15 μL of the resazurin solution, and the plate was re-incubated for 24 h. The IC50 was defined as the concentration of compound capable of inhibiting 50% of cell growth of the fungus according to the absorbance at 600 nm. Determination of the susceptibility of P. lutzii to the camphene thiosemicarbazide derivative The TSC-C sensitivity test was carried out on plates containing Fava-Netto semi-solid medium supplemented with TSC-C. The concentrations tested were 316 μM, 158 μM, 79 μM and 39.5 μM. Negative control plates were prepared in the absence of TSC-C. A total of 105, 106 and 107 yeast cells were inoculated on each plate. The plates were incubated for 7 days at 36°C and photographed. Viability curve Cell viability was determined using trypan blue staining and standard cell count techniques in a Neubauer chamber. We inoculated 1x106 cells/mL of P. lutzii yeast cells in MMcM liquid medium supplemented with TSC-C at 79 μM—the IC50 concentration—for 0, 1, 2, 3, 4, 8 and 24 h of incubation. The negative control was performed in the absence of TSC-C. For counting, samples were collected at specific time points, and 10 μL of the cell solution was added to 190 μL trypan blue solution and diluted to a final volume of 1 mL. Yeast cells were observed under light microscopy with a 40X lens. RNA extraction and purification of mRNA Total RNA was extracted after the incubation of Paracoccidiodies spp. yeast with TSC-C at 79 μM for 8 h of cultivation. The RNA was extracted with Trizol reagent (Invitrogen), precipitated with isopropanol, and resuspended with diethyl pyrocarbonate- (DEPC-) treated water. The mRNA was purified using the GenElute mRNA kit (Sigma Aldrich). cDNA library construction and DNA sequencing The cDNA library was built using the SuperScript Plasmid System with Gateway Technology for cDNA Synthesis and Cloning kit (Invitrogen). The cDNA was cloned into the pCMV. SPORT6 plasmid vector and transformed into E. coli (XL1blue) cells. The cDNA library was plated at approximately 200 colonies per plate (150 mm Petri dish). The colonies were randomly selected and transferred to a 96-well polypropylene plate containing LB medium and grown overnight. Plasmid cDNA was isolated and purified. PLOS ONE | DOI:10.1371/journal.pone.0130703 June 26, 2015 3 / 25 Response of Paracoccidioides to Camphene Thiosemicarbazide Derivative cDNA inserts were sequenced from the 5’ end by employing standard fluorescence labeling with the DYEnamic ET dye terminator kit with an M13 flanking vector primer. Automated sequence analysis was performed in a MegaBACE 1000 DNA sequencer (GE Healthcare, Uppsala, Sweden). Pipeline processing and annotation of ESTs PHRED [29], Crossmatch (http://www.macvector.com/Assembler/trimmingwithcrossmatch. html) and CAP3 [30] tools were integrated into a pipeline (http://www.lbm.icb.ufg.br/ pipelineUFG/). Only sequences with at least 50 nucleotides and a PHRED quality greater or equal to 20 were considered for assembly and cluster formation. ESTs were screened for vector sequences against the UniVec data. All of the clustered sequences were queried for similarity using BLASTX (http://www.ncbi.nlm.nih.gov/BLAST) sequence comparison software against the nucleotide database generated from the P. lutzii Pb01 structural genome (http://www. broad.mit.edu/annotation/genome/paracoccidioides_brasiliensis/MulHome.html). Sequences were grouped into functional categories with the PEDANT3 database (http://pedant. helmholtz-muenchen.de/index.jsp). Similarities with E-values  10−5 were considered significant. The Munich Information Center for Protein Sequences (MIPS) (http://mips.gsf.de/) database was used to assign functional categories. EC numbers were obtained by the Enzyme Database-Brenda (http://www.brenda-enzymes.info) In silico determination of up-regulated genes To assign a differential expression character, ESTs from contigs formed from yeast cells treated with TSC-C were statistically evaluated using the method by Audic and Claverie [31]. Overexpressed genes, determined by comparison to the P. lutzii transcriptome database (https://dna. biomol.unb.br/Pb/), were determined with a 95% confidence rate. Generation of P. brasiliensis SOD1-aRNA isolate DNA from the P. brasiliensis wild-type strain ATCC 60855 (WT) was extracted from yeast cultures during exponential growth. We employed a high-fidelity Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) to amplify aRNA oligonucleotides designed on the PABG_03954 (www.broadinstitute.org) sequence of the SOD1 gene. P. brasiliensis plasmid construction for aRNA and Agrobacterium tumefaciens-mediated transformation were performed as previously described [32]. Briefly, the amplified SOD1-aRNA oligonucleotides were inserted into the pCR35 plasmid under the control of the Calcium Binding Protein 1 (CBP-1) promoter region from Histoplasma capsulatum [33]. The pUR5750 plasmid was used as a parental binary vector to harbor the aRNA cassette within the transfer DNA (T-DNA). The constructed binary vectors were introduced into A. tumefaciens LBA1100 ultracompetent cells by electroporation as described previously [34] and isolated by kanamycin selection (100 mg/mL). P. brasiliensis and A. tumefaciens were combined in a 1:10 ratio and incubated for 3 days of co-culture at 28°C. Selection of P. brasiliensis transformants was performed in BHI solid media containing hygromycin B (Hyg; 200 mg/mL) over a 15 day incubation period at 36°C. Randomly selected Hyg resistant transformants were tested for mitotic stability. P. brasiliensis yeast cells transformed with the empty parental vector pUR5750 (EV) were used as controls alongside the experimental yeast in the assays carried out in this study. The integration of the a-RNA cassette in the P. brasiliensis genome was confirmed by PCR analysis. PLOS ONE | DOI:10.1371/journal.pone.0130703 June 26, 2015 4 / 25 Response of Paracoccidioides to Camphene Thiosemicarbazide Derivative Determination of the susceptibility of P. brasiliensis and the SOD1-aRNA isolate to TSC-C To evaluate the susceptibility of P. brasiliensis to TSC-C, the WT, EV and SOD1-aRNA isolate strains were grown in Fava-Netto liquid medium for 72 h under constant shaking at 150 rpm and 36°C. Yeast were then transferred into MMcM liquid medium and cultured overnight. Yeast cells were then washed with 1X PBS, and the assays were performed with 1x106 cells. The different isolates were distributed in solid BHI medium supplemented with 316 μM, 158 μM, 79 μM and 39.5 μM TSC-C. The controls were carried out in the same medium without the addition of TSC-C. The SOD1-aRNA isolated was growth in the presence of TSC-C added of ascorbic acid aiming to validate the influence of TSC-C as indutor agente of ROS. Initially, the concentrations from 0.08 to 100 mM ascorbic acid were used to determinate IC50 (data not shown). So, 0.2 mM ascorbic acid was added at 316 μM, 158 μM, 79 μM and 39.5 μM TSC-C. All plates were incubated for 6 days at 36°C before being photographed. Gene expression analysis by qRT-PCR Total RNA was obtained from Paracoccidioides spp. yeast cells grown in the presence or absence of TSC-C for 8 h. After treatment with DNase, the cDNA was synthesized from total RNA using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. The primers for, ATP synthase, Superoxide dismutase (SOD1) [PABG_03954 (www. broadinstitute.org)], Heat shock protein 30 kDa (HSP30), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and α-tubulin genes were designed using the Primer Express software (Applied Biosystems, Foster City, CA, USA). The sequences of the oligonucleotide primers are shown in Table 1. The qRT-PCR analyses were performed in triplicate with the StepOnePlus real-time PCR system (Applied Biosystems). The expression values were calculated using the alpha tubulin transcript (XM_002796593) as the endogenous control as reported previously [35]. For transcripts of interest, relative expression levels were calculated using the standard curve method for relative quantification [36]. The relative standard curve was generated by pooling cDNAs from all conditions and serially diluting them from 1:5 to 1:625. Preparation of protein extracts from P. lutzii Protein extracts were obtained after 8 h incubation in MMcM in the presence of 79 μM TSC-C or in its absence. Yeast cells were centrifuged at 10,000 x g equilibrium value of MeCpG steps (,+14 deg.) [31,44]. In comparison, methylation has a significantly lower stability cost when happening at major groove positions, such as 211 and 21 base pair from dyad (mutations 9 and 12), where the roll of the nucleosome bound conformation (+10 deg.) is more compatible with the equilibrium geometry of MeCpG steps. The nucleosome destabilizing effect of cytosine methylation increases with the number of methylated cytosines, following the same position dependence as the single methylations. The multiple-methylation case reveals that each major groove meth- PLOS Computational Biology | www.ploscompbiol.org 3 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning ylation destabilizes the nucleosome by around 1 kJ/mol (close to the average estimate of 2 kJ/mol obtained for from individual methylation studies), while each minor groove methylation destabilizes it by up to 5 kJ/mol (average free energy as single mutation is around 6 kJ/mol). This energetic position-dependence is the reverse of what was observed in a recent FRET/SAXS study [30]. The differences can be attributed to the use of different ionic conditions and different sequences: a modified Widom-601 sequence of 157 bp, which already contains multiple CpG steps in mixed orientations, and which could assume different positioning due to the introduction of new CpG steps and by effect of the methylation. The analysis of our trajectories reveals a larger root mean square deviation (RMSD) and fluctuation (RMSF; see Figures S2– S3 in Text S1) for the methylated nucleosomes, but failed to detect any systematic change in DNA geometry or in intermolecular DNA-histone energy related to methylation (Fig. S1B, S1C, S4–S6 in Text S1). The hydrophobic effect should favor orientation of the methyl group out from the solvent but this effect alone is not likely to justify the positional dependent stability changes in Figure 2, as the differential solvation of the methyl groups in the bound and unbound states is only in the order of a fraction of a water molecule (Figure S5 in Text S1). We find however, a reasonable correlation between methylation-induced changes in hydrogen bond and stacking interactions of the bases and the change in nucleosome stability (see Figure S6 in Text S1). This finding suggests that methylation-induced nucleosome destabilization is related to the poorer ability of methylated DNA to fit into the required conformation for DNA in a nucleosome. Changes in the elastic deformation energy between methylated and un-methylated DNA correlate with nucleosomal differential binding free energies To further analyze the idea that methylation-induced nucleosome destabilization is connected to a worse fit of methylated DNA into the required nucleosome-bound conformation, we computed the elastic energy of the nucleosomal DNA using a harmonic deformation method [36,37,44]. This method provides a rough estimate of the energy required to deform a DNA fiber to adopt the super helical conformation in the nucleosome (full details in Suppl. Information Text S1). As shown in Figure 2, there is an evident correlation between the increase that methylation produces in the elastic deformation energy (DDE def.) and the free energy variation (DDG bind.) computed from MD/TI calculations. Clearly, methylation increases the stiffness of the CpG step [31], raising the energy cost required to wrap DNA around the histone octamers. This extra energy cost will be smaller in regions of high positive roll (naked DNA MeCpG steps have a higher roll than CpG steps [31]) than in regions of high negative roll. Thus, simple elastic considerations explain why methylation is better tolerated when the DNA faces the histones through the major groove (where positive roll is required) that when it faces histones through the minor groove (where negative roll is required). Nucleosome methylation can give rise to nucleosome repositioning We have established that methylation affects the wrapping of DNA in nucleosomes, but how does this translate into chromatin structure? As noted above, accumulation of minor groove methylations strongly destabilizes the nucleosome, and could trigger nucleosome unfolding, or notable changes in positioning or phasing of DNA around the histone core. While accumulation of methylations might be well tolerated if placed in favorable positions, accumulation in unfavorable positions would destabilize the nucleosome, which might trigger changes in chromatin structure. Chromatin could in fact react in two different ways in response to significant levels of methylation in unfavorable positions: i) the DNA could either detach from the histone core, leading to nucleosome eviction or nucleosome repositioning, or ii) the DNA could rotate around the histone core, changing its phase to place MeCpG steps in favorable positions. Both effects are anticipated to alter DNA accessibility and impact gene expression regulation. The sub-microsecond time scale of our MD trajectories of methylated DNAs bound to nucleosomes is not large enough to capture these effects, but clear trends are visible in cases of multiple mutations occurring in unfavorable positions, where unmethylated and methylated DNA sequences are out of phase by around 28 degrees (Figure S7 in Text S1). Due to this repositioning, large or small, DNA could move and the nucleosome structure could assume a more compact and distorted conformation, as detected by Lee and Lee [29], or a slightly open conformation as found in Jimenez-Useche et al. [30]. Using the harmonic deformation method, we additionally predicted the change in stability induced by cytosine methylation for millions of different nucleosomal DNA sequences. Consistently with our calculations, we used two extreme scenarios to prepare our DNA sequences (see Fig. 3): i) all positions where the minor grooves contact the histone core are occupied by CpG steps, and ii) all positions where the major grooves contact the histone core are occupied by CpG steps. We then computed the elastic energy required to wrap the DNA around the histone proteins in unmethylated and methylated states, and, as expected, observed that methylation disfavors DNA wrapping (Figure 3A). We have rescaled the elastic energy differences with a factor of 0.23 to match the DDG prediction in figure 2B. In agreement with the rest of our results, our analysis confirms that the effect of methylation is position-dependent. In fact, the overall difference between the two extreme methylation scenarios (all-in-minor vs all-in-major) is larger than 60 kJ/mol, the average difference being around 15 kJ/ mol. We have also computed the elastic energy differences for a million sequences with CpG/MeCpG steps positioned at all possible intermediate locations with respect to the position (figure 3B). The large differences between the extreme cases can induce rotations of DNA around the histone core, shifting its phase to allow the placement of the methylated CpG steps facing the histones through the major groove. It is illustrative to compare the magnitude of CpG methylation penalty with sequence dependent differences. Since there are roughly 1.5e88 possible 147 base pairs long sequence combinations (i.e., (4n+4(n/2))/2, n = 147), it is unfeasible to calculate all the possible sequence effects. However, using our elastic model we can provide a range of values based on a reasonably large number of samples. If we consider all possible nucleosomal sequences in the yeast genome (around 12 Mbp), the energy difference between the best and the worst sequence that could form a nucleosome is 0.7 kj/mol per base (a minimum of 1 kJ/mol and maximum of around 1.7 kJ/mol per base, the first best and the last worst sequences are displayed in Table S3 in Text S1). We repeated the same calculation for one million random sequences and we obtained equivalent results. Placing one CpG step every helical turn gives an average energetic difference between minor groove and major groove methylation of 15 kJ/ mol, which translates into ,0.5 kJ/mol per methyl group, 2 kJ/ mol per base for the largest effects. Considering that not all nucleosome base pair steps are likely to be CpG steps, we can conclude that the balance between the destabilization due to CpG methylation and sequence repositioning will depend on the PLOS Computational Biology | www.ploscompbiol.org 4 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning Figure 3. Methylated and non-methylated DNA elastic deformation energies. (A) Distribution of deformation energies for 147 bplong random DNA sequences with CpG steps positioned every 10 base steps (one helical turn) in minor (red and dark red) and major (light and dark blue) grooves respectively. The energy values were rescaled by the slope of a best-fit straight line of figure 2, which is 0.23, to por la lectura a través de la lectura de la prensa. La educación en los medios las fuerzas dispersas en función de los soportes mediáticos y orientarse más hacia la educación en medios que al dominio adquiere pleno derecho y entidad en la sección sexta titulada «competencias sociales y cívi- técnico de los aparatos. cas» que indica que «los alum- nos deberán ser capaces de juz- gar y tendrán espíritu crítico, lo que supone ser educados en los las programaciones oficiales, ya que, a lo largo de un medios y tener conciencia de su lugar y de su influencia estudio de los textos, los documentalistas del CLEMI en la sociedad». han podido señalar más de una centena de referencias a la educación de los medios en el seno de disciplinas 4. Un entorno positivo como el francés, la historia, la geografía, las lenguas, Si nos atenemos a las cifras, el panorama de la las artes plásticas : trabajos sobre las portadas de educación en medios es muy positivo. Una gran ope- prensa, reflexiones sobre temas mediáticos, análisis de ración de visibilidad como la «Semana de la prensa y publicidad, análisis de imágenes desde todos los ángu- de los medios en la escuela», coordinada por el CLE- los, reflexión sobre las noticias en los países europeos, MI, confirma año tras año, después de 17 convocato- información y opinión rias, el atractivo que ejerce sobre los profesores y los Esta presencia se constata desde la escuela mater- alumnos. Concebida como una gran operación de nal (2 a 6 años) donde, por ejemplo, se le pregunta a complementariedad entre la escuela y los profesiona- los niños más pequeños si saben diferenciar entre un les de los medios, alrededor del aprendizaje ciudada- periódico, un libro, un catálogo, a través de activida- no de la comunicación mediática, este evento moviliza des sensoriales, si saben para qué sirve un cartel, un durante toda una semana un porcentaje elevado de periódico, un cuaderno, un ordenador si son capa- centros escolares que representan un potencial de 4,3 ces de reconocer y distinguir imágenes de origen y de millones de alumnos (cifras de 2006). Basada en el naturaleza distintas. Podríamos continuar con más voluntariado, la semana permite desarrollar activida- ejemplos en todos los niveles de enseñanza y práctica- des más o menos ambiciosas centradas en la introduc- Páginas 43-48 ción de los medios en la vida de la escuela a través de la instalación de kioscos, organización de debates con profesionales y la confección por parte de los alumnos de documentos difundidos en los medios profesionales. Es la ocasión de dar un empujón a la educación en medios y de disfrutarlos. Los medios –un millar en 2006– se asocian de maneras diversas ofreciendo ejemplares de periódicos, acceso a noticias o a imágenes, proponiendo encuentros, permitiendo intervenir a los jóvenes en sus ondas o en sus columnas Esta operación da luz al trabajo de la educación en medios y moviliza a los diferentes participantes en el proyecto. 5. La formación de los docentes La formación es uno de los pilares principales de la educación en los medios. Su función es indispensable ya que no se trata de una disciplina, sino de una enseñanza que se hace sobre la base del voluntariado y del compromiso personal. Se trata de convencer, de mostrar, de interactuar. En primer lugar es necesario incluirla en la formación continua de los docentes, cuyo volumen se ha incrementado desde 1981 con la aparición de una verdadera política de formación continua de personal. Es difícil dar una imagen completa del volumen y del público, pero si nos atenemos a las cifras del CLEMI, hay más de 24.000 profesores que han asistido y se han involucrado durante 2004-05. 5.1. La formación continua En la mayoría de los casos, los profesores reciben su formación en contextos cercanos a su centro de trabajo, o incluso en este mismo. Después de una política centrada en la oferta que hacían los formadores, se valora más positivamente la demanda por parte del profesorado, ya que sólo así será verdaderamente fructífera. Los cursos de formación se repartieron en varias categorías: desde los formatos más tradicionales (cursos, debates, animaciones), hasta actividades de asesoramiento y de acompañamiento, y por supuesto los coloquios que permiten un trabajo en profundidad ya que van acompañados de expertos investigadores y profesionales. Citemos, por ejemplo en 2005, los coloquios del CLEMI-Toulouse sobre el cine documental o el del CLEMI-Dijon sobre «Políticos y medios: ¿connivencia?». Estos coloquios, que forman parte de un trabajo pedagógico regular, reagrupan a los diferentes participantes regionales y nacionales alrededor de grandes temas de la educación en medios y permiten generar nuevos conocimientos de aproximación y una profundización. Páginas 43-48 Hay otro tipo de formación original que se viene desarrollando desde hace menos tiempo, a través de cursos profesionales, como por ejemplo, en el Festival Internacional de Foto-periodismo «Visa para la imagen», en Perpignan. La formación se consolida en el curso, da acceso a las exposiciones, a las conferencias de profesionales y a los grandes debates, pero añade además propuestas pedagógicas y reflexiones didácticas destinadas a los docentes. Estas nuevas modalidades de formación son también consecuencia del agotamiento de la formación tradicional en las regiones. Los contenidos más frecuentes en formación continua conciernen tanto a los temas más clásicos como a los cambios que se están llevando a cabo en las prácticas mediáticas. Así encontramos distintas tendencias para 2004-05: La imagen desde el ángulo de la producción de imágenes animadas, el análisis de la imagen de la información o las imágenes del J.T. La prensa escrita y el periódico escolar. Internet y la información en línea. Medios y educación de los medios. 5.2 La formación inicial La formación inicial está aun en un grado muy ini- cial. El hecho de que la educación en medios no sea una disciplina impide su presencia en los IUFM (Institutos Universitarios de Formación de Maestros) que dan una prioridad absoluta a la didáctica de las disciplinas. En 2003, alrededor de 1.400 cursillistas sobre un total de 30.000 participaron en un momento u otro de un módulo de educación en medios. Estos módulos se ofrecen en función del interés que ese formador encuentra puntualmente y forman parte a menudo de varias disciplinas: documentación, letras, historia-geografía Estamos aún lejos de una política concertada en este dominio. La optativa «Cine-audiovisual» ha entrado desde hace muy poco tiempo en algunos IUFM destinada a obtener un certificado de enseñanza de la opción audiovisual y cine. Internet tiene cabida también en los cursos de formación inicial, recientemente con la aparición de un certificado informático y de Internet para los docentes, dirigido más a constatar competencias personales que a valorar una aptitud para enseñarlos. 6. ¿Y el futuro? El problema del futuro se plantea una vez más por la irrupción de nuevas técnicas y nuevos soportes. La difusión acelerada de lo digital replantea hoy muchas cuestiones relativas a prácticas mediáticas. Muchos Comunicar, 28, 2007 47 Comunicar, 28, 2007 Enrique Martínez-Salanova '2007 para Comunicar 48 trabajos que llevan el rótulo de la educación en medios solicitan una revisión ya que los conceptos cambian. La metodología elaborada en el marco de la educación en medios parece incluso permitir la inclinación de la sociedad de la información hacia una sociedad del conocimiento, como defiende la UNESCO. En Francia, se necesitaría unir las fuerzas dispersas en función de los soportes mediáticos y orientarse más hacia la educación en medios que al dominio técnico de los aparatos. Los avances recientes en el reconocimiento de estos contenidos y las competencias que supondrían podrían permitirlo. Referencias CLEMI/ACADEMIE DE BORDEAUX (Ed.) (2003): Parcours médias au collège: approches disciplinaires et transdisciplinaires. Aquitaine, Sceren-CRDP. GONNET, J. (2001): Education aux médias. Les controverses fécondes. Paris, Hachette Education/CNDP. SAVINO, J.; MARMIESSE, C. et BENSA, F. (2005): L’éducation aux médias de la maternelle au lycée. Direction de l’Enseignement Scolaire. Paris, Ministère de l’Education Nationale, Sceren/CNDP, Témoigner. BEVORT, E. et FREMONT, P. (2001): Médias, violence et education. Paris, CNDP, Actes et rapports pour l’éducation. – www.clemi.org: fiches pédagogiques, rapports et liens avec les pages régionales/académiques. – www.ac-nancy-metz.fr/cinemav/quai.html: Le site «Quai des images» est dédié à l’enseignement du cinéma et de l’audiovisuel. – www.france5.fr/education: la rubrique «Côté profs» a une entrée «education aux médias». – www.educaunet.org: Programme européen d’éducation aux risques liés à Internet. dResedfeleexliobnuetsacón Páginas 43-48
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