Estudo da atividade lipolítica de adipócitos isolados do tecido adiposo epididimal e do metabolismo energético de ratos provenientes de dois modelos de hipertensão : genética e induzida

Orientador: Dora Maria Grassi KassisseTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A hipertensão é a doença cardiovascular mais comum. A resposta ao estresse é constituída pela ativação do sistema nervoso simpático e do eixo hipotálamo-pituitária-adrenal. Estudos sugerem que tais mecanismos sejam centrais na hipertensão essencial. Distúrbios metabólicos têm sido observados na hipertensão essencial, e podem ser decorrentes dos hormônios do estresse. O objetivo deste trabalho foi a caracterização dos hormônios do estresse, parâmetros metabólicos, a sensibilidade lipolítica de adipócitos epididimais isolados e a expressão de proteínas relacionadas de ratos Wistar com hipertensão induzida pela ingestão crônica de NG-nitro-L-arginine methyl ester (40mg/kg por dia, a partir da 10ª semana de vida, por 5 semanas) (grupo L-NAME) e de ratos espontaneamente hipertensos (grupo SHR), comparados com seus controles, Wistar (grupo W) e Wistar Kyoto (grupo WKY), respectivamente. Comparações entre ambos os controles e ambos os hipertensos também foram realizadas. O peso corpóreo, ingestão alimentar e hídrica e taxa metabólica de repouso foram feitas na 7ª e na 14ª semana de vida. A coleta de sangue para análises séricas e de tecidos (para medida de peso, morfometria de adipócitos, ensaio funcional com adipócitos epididimais isolados e análise de expressão por Western Blot) e a eutanásia ocorreu nos ratos anestesiados, submetidos a jejum prévio de 12-16 h, na 15ª semana de vida, por volta das 9:00h da manhã. Altas concentrações circulantes de catecolaminas e corticosterona foram observadas nos grupos L-NAME, SHR e WKY, mas não em W. O L-NAME exibiu respostas metabólicas típicas do estresse agudo, pois além da menor adiposidade, reduziu a sua ingestão alimentar. O WKY exibiu algumas alterações típicas do estresse crônico, como menor taxa metabólica e reduzida expressão de UCP3 na musculatura esquelética. O SHR apresentou alterações que podem ser relacionadas a uma possível hiperfunção da tireoide, como aumento da ingesta alimentar e da taxa metabólica de repouso e menor adiposidade. Houve aumento da lipólise mediada por adrenoceptores beta-2 no grupo L-NAME se comparado ao Wistar, mas sua expressão não foi diferente. Entretanto, apresentou menor expressão de adrenoceptores-beta-1, receptor de adenosina A2 e perilipina e aumento de HSL. Por outro lado, o grupo SHR apresentou inibição da lipólise mediada por adrenoceptores beta-2 comparado ao WKY, devido ao seu efeito promíscuo de associação a proteínas Gi, sem alteração na expressão destas proteínas. No entanto, apresentou alta expressão de ATGL e de perilipina. Em resumo, nossos resultados constataram algumas alterações metabólicas em ambos os grupos hipertensos e no normotenso WKY, que podem ser atribuídas aos hormônios do estresse, cuja presença em altas concentrações no grupo WKY não foi suficiente para desenvolver a sua hipertensão. O presente trabalho também evidenciou uma função alterada do adrenoceptor beta-2 em adipócitos epididimais isolados no estado hipertensivo, o que já foi descrito em cardiomiócitos e que pode ter função protetoraAbstract: Hypertension is the most common cardiovascular disease. Stress response is composed by activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. Studies suggest that these mechanisms are central in essential hypertension too. Metabolic disorders have been observed in essential hypertension, and may be due to stress hormones. The aim of this study was the characterization of stress hormones, some metabolic parameters, the lipolytic sensitivity of isolated epididymal adipocytes and the expression of related proteins of Wistar rats with hypertension induced by chronic ingestion of NG-nitro-L-arginine ester methyl (40 mg / kg per day, in 10-week-old rats, for 5 weeks) (L-NAME group) and spontaneously hypertensive rats (SHR), compared to their controls, Wistar (W group) and Wistar Kyoto (WKY group), respectively. We also performed comparisons between W and WKY and between L-NAME and SHR. Body weight, food and water intake and resting metabolic rate were measured in 7 and 14-week-old rats. Blood sampling for serum analysis and tissue extraction for analysis of weight, adipocyte size, lipolytic response of isolated epididymal adipocytes and protein expression by Western Blot, and euthanasia occurred at around 9: 00 a.m. in anesthetized 15-week-old rats that were previously fasted for 12-16 h. High levels of catecholamines and corticosterone were observed in L-NAME, SHR and WKY groups, but not in W. L-NAME showed typical metabolic responses of acute stress, like reduced adiposity and food intake. WKY exhibited some alterations of chronic stress, such as reduced metabolic rate and UCP3 expression in skeletal muscle. SHR presented changes that were attributed to a possible thyroid hyperfunction, such as increased food intake and resting metabolic rate and reduced adiposity. L-NAME showed increased beta-2- adrenoceptor mediated lipolysis, although there were no differences in the expression of related proteins. However, it had lower expression of beta-1-adrenoceptor, adenosine A2 receptor and periliin and increased HSL. On the other hand, SHR group displayed promiscuous beta-2-adrenoceptor association to Gi without changes in the expression of the associated proteins. Nevertheless, it showed high expression of ATGL and perilipin. In summary, our results observed some metabolic abnormalities in both hypertensive groups and in the normotensive WKY, which may be attributed to stress hormones, whose presence at high circulating levels in WKY groupp was not sufficient to develop its hypertension. This study also showed an altered function of beta-2-adrenoceptors in epididymal isolated adipocytes in the hypertensive groups, which has already been described in cardiomyocytes and that may have a protective roleDoutoradoFisiologiaDoutora em Biologia Funcional e Molecular2014/17538-6CAPESFAPES

LRRN4 and UPK3B Are Markers of Primary Mesothelial Cells

Mesothelioma is a highly malignant tumor that is primarily caused by occupational or environmental exposure to asbestos fibers. Despite worldwide restrictions on asbestos usage, further cases are expected as diagnosis is typically 20–40 years after exposure. Once diagnosed there is a very poor prognosis with a median survival rate of 9 months. Considering this the development of early pre clinical diagnostic markers may help improve clinical outcomes.Microarray expression arrays on mesothelium and other tissues dissected from mice were used to identify candidate mesothelial lineage markers. Candidates were further tested by qRTPCR and in-situ hybridization across a mouse tissue panel. Two candidate biomarkers with the potential for secretion, uroplakin 3B (UPK3B), and leucine rich repeat neuronal 4 (LRRN4) and one commercialized mesothelioma marker, mesothelin (MSLN) were then chosen for validation across a panel of normal human primary cells, 16 established mesothelioma cell lines, 10 lung cancer lines, and a further set of 8 unrelated cancer cell lines.Within the primary cell panel, LRRN4 was only detected in primary mesothelial cells, but MSLN and UPK3B were also detected in other cell types. MSLN was detected in bronchial epithelial cells and alveolar epithelial cells and UPK3B was detected in retinal pigment epithelial cells and urothelial cells. Testing the cell line panel, MSLN was detected in 15 of the 16 mesothelioma cells lines, whereas LRRN4 was only detected in 8 and UPK3B in 6. Interestingly MSLN levels appear to be upregulated in the mesothelioma lines compared to the primary mesothelial cells, while LRRN4 and UPK3B, are either lost or down-regulated. Despite the higher fraction of mesothelioma lines positive for MSLN, it was also detected at high levels in 2 lung cancer lines and 3 other unrelated cancer lines derived from papillotubular adenocarcinoma, signet ring carcinoma and transitional cell carcinoma

Christopher Simpson The Division-Viol, or The Art of PLAYING Ex tempore upon a GROUND. EDITIO SECVNDA Part III "The Method of ordering Division to a Ground" (2)

本訳稿はChristopher Simpson (1605頃－1669) 著 The Division-Viol, or, The Art of PLAYING Ex tempore upon a GROUND. DIVIDED INTO THREE PARTS. EDITIO SECVNDA, London, 1665 のPart III "The Method of ordering Division to a Ground" より§7～§12(pp.42-56)の全訳である

「きこえとことばの相談室」報告

実践報告Practical report　言語聴覚学科は，学生に対する臨床教育を目的に，2009 年3 月に「きこえとことばの相談室」（以下:相談室）を開設した．2009 年3 月の開設から2014 年10 月末までの相談室の活動実績は，新規数：63名，継続数：53 名．総臨床件数612 件．臨床実数は，100 ～ 120 件で定着していた．開設当初の目的であった演習協力には至っていないが，教育活動，研究活動，臨床指導等に貢献している．今後は，言語聴覚療法の対象障害の拡大（失語症等），教育及び地域貢献の一環としての発展が望まれる．　In the Division of Speech-Language-Hearing Therapy, we established an outpatient counselingroom for hearing and language therapy in March of 2009 with the aim of providing clinical education for the students. From March 2009 to end of October 2014, the total numbers of counselings amounted 612 cases (63 new cases, 53 follow-up cases). The number of clinical cases was stable, between 100 and 120. Although it had little therapeutic impact, which was its original purpose, it has been contributing to education and research work. We expect it to expand to include disorders of speech-language-hearing therapy such as aphasia, etc., and as a part of the educational and regional contribution

One-Step Detection of the 2009 Pandemic Influenza A(H1N1) Virus by the RT-SmartAmp Assay and Its Clinical Validation

<div><h3>Background</h3><p>In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society.</p> <h3>Methodology</h3><p>To address the clinical need for rapid diagnosis, we have developed a new method, the “RT-SmartAmp assay”, to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses.</p> <h3>Results and Conclusions</h3><p>We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.</p> </div

An integrated expression atlas of miRNAs and their promoters in human and mouse

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution

Optimization of turn-back primers in

isothermal amplificatio