Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Development of primary human pancreatic cancer organoids, matched stromal and immune cells and 3D tumor microenvironment models

Abstract Background Patient-derived tumor models are the new standard for pre-clinical drug testing and biomarker discovery. However, the emerging technology of primary pancreatic cancer organoids has not yet been broadly implemented in research, and complex organotypic models using organoids in co-culture with stromal and immune cellular components of the tumor have yet to be established. In this study, our objective was to develop and characterize pancreatic cancer organoids and multi-cell type organotypic co-culture models to demonstrate their applicability to the study of pancreatic cancer. Methods We employed organoid culture methods and flow cytometric, cytologic, immunofluorescent and immunohistochemical methods to develop and characterize patient-derived pancreatic cancer organoids and multi-cell type organotypic co-culture models of the tumor microenvironment. Results We describe the culture and characterization of human pancreatic cancer organoids from resection, ascites and rapid autopsy sources and the derivation of adherent tumor cell monocultures and tumor-associated fibroblasts from these sources. Primary human organoids displayed tumor-like cellular morphology, tissue architecture and polarity in contrast to cell line spheroids, which formed homogenous, non-lumen forming spheres. Importantly, we demonstrate the construction of complex organotypic models of tumor, stromal and immune components of the tumor microenvironment. Activation of myofibroblast-like cancer associated fibroblasts and tumor-dependent lymphocyte infiltration were observed in these models. Conclusions These studies provide the first report of novel and disease-relevant 3D in-vitro models representing pancreatic tumor, stromal and immune components using primary organoid co-cultures representative of the tumor-microenvironment. These models promise to facilitate the study of tumor-stroma and tumor-immune interaction and may be valuable for the assessment of immunotherapeutics such as checkpoint inhibitors in the context of T-cell infiltration

Stress-Induced Cell-Cycle Activation in Tip60 Haploinsufficient Adult Cardiomyocytes

Background: Tat-interactive protein 60 (Tip60) is a member of the MYST family of histone acetyltransferases. Studies using cultured cells have shown that Tip60 has various functions including DNA repair, apoptosis and cell-cycle regulation. We globally ablated the Tip60 gene (Htatip), observing that Tip60-null embryos die at the blastocyst stage (Hu et al. Dev.Dyn.238:2912;2009). Although adult heterozygous (Tip60 +/2) mice reproduce normally without a haploinsufficient phenotype, stress caused by Myc over-expression induced B-cell lymphoma in Tip60 +/2 adults, suggesting that Tip60 is a tumor suppressor (Gorrini et al. Nature 448:1063;2007). These findings prompted assessment of whether Tip60, alternative splicing of which generates two predominant isoforms termed Tip60a and Tip60b, functions to suppress the cell-cycle in adult cardiomyocytes. Methodology/Principal Findings: Western blotting revealed that Tip60a is the predominant Tip60 isoprotein in the embryonic heart, transitioning at neonatal stages to Tip60b, which is the only isoprotein in the adult heart wherein it is highly enriched. Over-expression of Tip60b, but not Tip60a, inhibited cell proliferation in NIH3T3 cells; and, Tip60haploinsufficient cultured neonatal cardiomyocytes exhibited increased cell-cycle activity. To address whether Tip60b suppresses the cardiomyocyte cell-cycle in the adult heart, hypertrophic stress was induced in Tip60 +/+ and Tip +/2 littermates via two methods, Myc over-expression and aortic banding. Based on immunostaining cell-cycle markers an

Tip60-Haploinsufficiency Augments 4-OHT-Induced Induction of Cell-Cycle Activity in MycER Transgenic Hearts.

<p>Eight week old MycER transgenic Tip60 wild-type (WT) and heterozygous (Het) mice were induced with 4-OHT for seven days and assessed for cell-cycle activity by immunostaining phosphorylated histone H3 (H3P; arrows in <b>A</b>,<b>B</b>). This was verified by immunostaining BrdU-incorporated nuclei (arrows in <b>D</b>,<b>E</b>). Percentages of labeled cells were determined by evaluating at least 5,000 (<b>C</b>) or 2,500 (<b>F</b>) hematoxylin-stained nuclei for H3P or BrdU antigen, respectively. (N)  =  number of hearts; vertical bars  =  ±SEM; p-values were calculated using Student's t-Test (two-tailed, unpaired). The scale bar in all images  =  10 µm. (Note: A control utilizing WT-MycER mice demonstrated that 4-OHT had no effect on these parameters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031569#pone.0031569-Xiao1" target="_blank">[26]</a>).</p

Most H3P-Positive Nuclei are in Cardiomyocytes.

<p>Sections from hearts processed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031569#pone-0031569-g005" target="_blank">Figure 5</a> were double-immunostained for phosphorylated H3P and Nkx2.5 to determine cardiomyocyte identity. <b>A</b> & <b>D</b> show Nkx2.5 staining (nucleus-specific brown DAB reaction product) in cardiomyocyte nuclei, as distinct from smaller non-myocyte nuclei in which only (blue) hematoxylin counter-stain is seen. <b>B</b> & <b>E</b> show H3P fluorescent green signal in the same nuclei. <b>C</b> and <b>F</b> are merged images of A–B and D–E, respectively. In <b>C</b> & <b>F</b>, dark arrows denote nuclei double-stained for Nkx2.5 and H3P; white arrows denote nuclei expressing only Nkx2.5. <b>G</b> summarizes results from enumerating a minimum of 150 H3P-positive nuclei per heart for co-localization of Nkx2.5. Error bars  =  ±SEM; statistical significance was determined by Student's t-Test (two-tailed, unpaired). Scale bars  = 20 µM.</p

Tip60-Haploinsufficiency Increases 4-OHT-Induced Expression of Cyclin D2.

<p>Eight week old MycER transgenic Tip60 wild-type (WT) or heterozygous (Het) mice were induced with 4-OHT for seven days and assessed for cyclin D2 by western blotting. <b>A</b> is a western blot showing cyclin D2 and GAPDH levels in non-stressed (no 4-OHT) and stressed (+4-OHT) WT and Het myocardium. Each lane contained 10 µg total protein from separate hearts. <b>B</b> shows densitometry of the bands in <b>A</b>, with cyclin D2 normalized to GAPDH. Numbers (N) in parentheses indicate numbers of hearts evaluated. Vertical bars  =  ±SEM; p-values were calculated using Student's t-Test (two-tailed, unpaired).</p

Tip60 Expression in Adult Organs.

<p>Tissues from 14 week-old adult Tip60^+/+ and Tip60^+/− mice were processed for (<b>A</b>) semi-quantitative RT/PCR to assess transcript levels (of bulk Tip60 isoforms) in WT and Het tissues and (<b>B</b>) Western blotting to determine Tip60 protein levels. The primer pair used for RT/PCR (<b>A</b>) detected transcripts encoding both Tip60α and Tip60β; GAPDH was assessed as loading control. The bar graph in the lower part of <b>B</b> shows densitometric analysis of protein bands assessed from duplicate animals, normalized to GAPDH and expressed as a percentage of the most abundant band (in WT heart); vertical bars  =  range. +/+  =  WT; +/−  =  Het.</p

Increased Cell-Cycle Activity in Tip60^+/− Neonatal Cardiomyocytes.

<p>Cardiomyocytes were isolated from 2 day-old neonatal hearts and cultured on gelatin-coated dishes. When the cells were ∼60% confluent (72 hrs later) they were double-immunostained for phosphorylated histone-H3 (H3P) to detect M-phase cells (arrows in <b>A</b> & <b>C</b>) and for sarcomeric α-actin (not shown) to verify cardiomyocyte identity. Nuclei were stained with DAPI (<b>B</b>,<b>D</b>); H3P-labeled nuclei are encircled because DAPI is obscured DAB-stained nuclei. E shows percentages of H3P-positive neonatal cardiomyocytes, based on enumerating 1,000-2,000 cells in each dish; error bars  =  +/−SEM. Scale bars in <b>A</b>–<b>D</b> = 10 µm.</p