A Utilização de projetos como abordagem didática: uma experiência com licenciandos em química

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Child smiling from historic automobile

Photograph of two children in a historic automobile, taken by Paula Deimling of Milford, Ohio. The image was one of ten to win Honorable Mention in the Professional category of the Spirit of Ohio Bicentennial Photo Contest. In August 1976, the Ohio American Revolution Bicentennial Advisory Committee (OARBAC) began the Spirit of Ohio Bicentennial Photo Contest as part of a larger effort in Ohio to celebrate the 1976 American Bicentennial. The contest was meant to document "the spirit and character of the people and places which represent Ohio during [the] bicentennial year," and to create a permanent photographic archive of the year's festivity for use by future researchers. Both professional and amateur photographers submitted over 500 photographs for consideration, all taken within the state between January 1 and December 31, 1976

Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR-2

Uares indicate methylated CpG dinucleotides and white squares indicate unmethylated CpG dinucleotides in promoter sequences. Above the diagrams, the area of the promoter analyzed by MSP, MCA-Meth and bisulfite sequencing is indicated. The 5'- and 3'-priming sites of M- and U-primers are indicated as small black dots representing the CpG sites incorporated in the primer sequence. The position of CpGs in relation to the CpG-island as well as the number of CpGs analyzed are indicated in the brackets behind the respective techniques. On the right, the diagrams of peaks obtained with the sequencing software: it can be observed how after the bisulfite treatment, the cytosines remain unchanged in the methylated sample, while there is a shift to thymine in the unmethylated sample. MSP results of in astrocytoma samples 26, 28 and in the U87MG cell line. U: PCR with primers specific for the unmethylated sequence; M: PCR with primers specific for the methylated sequence. MCA-Meth results of in astrocytoma samples 26, 28 and in the U87MG cell line. The graphs show the unmethylated control peak (blood DNA) in red, the methylated control peak in green, the no template control in blue and the sample or cell line peak, in brown. It can be observed how in the case of the U87MG cell line, the amount of methylated cytosines in the area of the promoter analyzed by bisulfite sequencing is intermediate between the sample 26 (unmethylated) and sample 28 (fully methylated). Of note, MCA-Meth appreciates this inhomogeneous sequence methylation pattern of U87MG with a melting curve peak in an intermediate position as compared to the unmethylated (26) and the methylated sample (28).<p><b>Copyright information:</b></p><p>Taken from "Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR"</p><p>http://www.biomedcentral.com/1471-2407/8/61</p><p>BMC Cancer 2008;8():61-61.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2266933.</p><p></p

Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR-0

template control in blue, and the sample with both the unmethylated and the methylated peaks, in brown. This result suggests a mixture of methylated and unmethylated DNA in this sample. It might be due to the contamination of the tumor sample with normal adjacent tissue.<p><b>Copyright information:</b></p><p>Taken from "Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR"</p><p>http://www.biomedcentral.com/1471-2407/8/61</p><p>BMC Cancer 2008;8():61-61.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2266933.</p><p></p

The graphs show the unmethylated control peak (blood DNA) in red, the methylated control peak in green, the no template control in blue and the cell line peak, in brown

PCR for the unmethylated sequence, gene, A172 cell line: only the blood DNA gives a peak, thus the cell line contains no unmethylated DNA. PCR for the methylated sequence, gene, A172 cell line: the methylated DNA and the cell line give peak, thus the A172 cell line is methylated for the BLU gene. PCR for the unmethylated sequence, gene, SIMA cell line: the blood DNA and cell lines give peak, thus the cell line is unmethylated. PCR for the methylated sequence, gene, SIMA cell line: only the methylated DNA gives a peak, thus the cell line is not methylated.<p><b>Copyright information:</b></p><p>Taken from "Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR"</p><p>http://www.biomedcentral.com/1471-2407/8/61</p><p>BMC Cancer 2008;8():61-61.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2266933.</p><p></p

Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR

<p>Abstract</p> <p>Background</p> <p>We present two melting curve analysis (MCA)-based semiquantitative real time PCR techniques to detect the promoter methylation status of genes. The first, MCA-MSP, follows the same principle as standard MSP but it is performed in a real time thermalcycler with results being visualized in a melting curve. The second, MCA-Meth, uses a single pair of primers designed with no CpGs in its sequence. These primers amplify both unmethylated and methylated sequences. In clinical applications the MSP technique has revolutionized methylation detection by simplifying the analysis to a PCR-based protocol. MCA-analysis based techniques may be able to further improve and simplify methylation analyses by reducing starting DNA amounts, by introducing an all-in-one tube reaction and by eliminating a final gel stage for visualization of the result. The current study aimed at investigating the feasibility of both MCA-MSP and MCA-Meth in the analysis of promoter methylation, and at defining potential advantages and shortcomings in comparison to currently implemented techniques, <it>i.e. </it>bisulfite sequencing and standard MSP.</p> <p>Methods</p> <p>The promoters of the <it>RASSF1A </it>(3p21.3), <it>BLU </it>(3p21.3) and <it>MGMT </it>(10q26) genes were analyzed by MCA-MSP and MCA-Meth in 13 astrocytoma samples, 6 high grade glioma cell lines and 4 neuroblastoma cell lines. The data were compared with standard MSP and validated by bisulfite sequencing.</p> <p>Results</p> <p>Both, MCA-MSP and MCA-Meth, successfully determined promoter methylation. MCA-MSP provided information similar to standard MSP analyses. However the analysis was possible in a single tube and avoided the gel stage. MCA-Meth proved to be useful in samples with intermediate methylation status, reflected by a melting curve position shift in dependence on methylation extent.</p> <p>Conclusion</p> <p>We propose MCA-MSP and MCA-Meth as alternative or supplementary techniques to MSP or bisulfite sequencing.</p

Sclerosing epithelioid mesenchymal neoplasm of the pancreas\ua0-\ua0a proposed new entity

We have encountered pancreatic tumors with unique histologic features, which do not conform to any of the known tumors of the pancreas or other anatomical sites. We aimed to define their clinicopathologic features and whether they are characterized by recurrent molecular signatures. Eight cases were identified; studied histologically and by immunohistochemistry. Selected cases were also subjected to whole-exome sequencing (WES; n\u2009=\u20094), RNA-sequencing (n\u2009=\u20096), Archer FusionPlex assay (n\u2009=\u20095), methylation profiling using the Illumina MethylationEPIC (850k) array platform (n\u2009=\u20096), and TERT promoter sequencing (n\u2009=\u20095). Six neoplasms occurred in females. The mean age was 43 years (range: 26-75). Five occurred in the head/neck of the pancreas. All patients were treated surgically; none received neoadjuvant/adjuvant therapy. All patients are free of disease after 53 months of median follow-up (range: 8-94). The tumors were well-circumscribed, and the median size was 1.8\u2009cm (range: 1.3-5.8). Microscopically, the unencapsulated tumors had a geographic pattern of epithelioid cell nests alternating with spindle cell fascicles. Some areas showed dense fibrosis, in which enmeshed tumor cells imparted a slit-like pattern. The predominant epithelioid cells had scant cytoplasm and round-oval nuclei with open chromatin. The spindle cells displayed irregular, hyperchromatic nuclei. Mitoses were rare. No lymph node metastases were identified. All tumors were positive for vimentin, CD99 and cytokeratin (patchy), while negative for markers of solid pseudopapillary neoplasm, neuroendocrine, acinar, myogenic/rhabdoid, vascular, melanocytic, or lymphoid differentiation, gastrointestinal stromal tumor as well as MUC4. Whole-exome sequencing revealed no recurrent somatic mutations or amplifications/homozygous deletions in any known oncogenes or tumor suppressor genes. RNA-sequencing and the Archer FusionPlex assay did not detect any recurrent likely pathogenic gene fusions. Single sample gene set enrichment analysis revealed that these tumors display a likely mesenchymal transcriptomic program. Unsupervised analysis (t-SNE) of their methylation profiles against a set of different mesenchymal neoplasms demonstrated a distinct methylation pattern. Here, we describe pancreatic neoplasms with unique morphologic/immunophenotypic features and a distinct methylation pattern, along with a lack of abnormalities in any of key genetic drivers, supporting that these neoplasms represent a novel entity with an indolent clinical course. Given their mesenchymal transcriptomic features, we propose the designation of "sclerosing epithelioid mesenchymal neoplasm" of the pancreas