Fabiola Zucchi

<p>(<b>A</b>) Following left hemisphere injection of 2 µl of 4 mg/ml 6-OHDA or saline, rats were tested for neurodegeneration and the indicated brain region were dissected 27–33 days following injection. 30 µg of solubilized midbrain was heated at 95°C for 5 min, resolved by SDS-PAGE, transferred to nitrocellulose and probed with anti-TID1 monoclonal antibody. The Western blot shown is representative of 11 6-OHDA/saline pairs of rats. Actin is shown as a loading control. (<b>B</b>) Midbrain samples were fractionated into soluble and insoluble and subjected to Western analysis. (<b>C</b>) TID1 expression in the indicated regions of saline injected rats was evaluated by Western analysis with anti-TID1 monoclonal antibody and quantitated by Quantity One (BioRad). <b>(D)</b> TID1 expression in 30 µg of solubilized hippocampus. The Western blot shown is representative of 11 6-OHDA/saline pairs of rats. Actin is shown as a loading control.</p

<p>Heat map representation of differentially regulated miRNA as observed by microarray analyses. <b>B,</b> Table of putative target genes for modulated miRNAs (miR-103, miR-151, and miR-219-2-3p; p≤0.05) and their physiological functions. <b>C,</b> Expression ratio group averages of miRNAs as observed by qRT-PCR analysis (p≤0.05). Whole brains of newborns born to dams shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056967#pone-0056967-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056967#pone-0056967-g002" target="_blank">2</a> (n = 3 per group, three repeats per sample; 1 pup per dam) were used. All data are presented as mean ± SEM.</p

<p><b>A,</b> Differential global gene expression in the brains of prenatally stressed newborn rats. <i>Ptplb</i> and <i>Dazap1</i> are targets for miR-103 and miR-219, respectively. <b>B,</b> Clustering analysis of gene expression showed clusters of stressed and non-stress animals, except for one non-stressed animal. <b>C,</b> Prenatal stress elevated expression of miR-103, which coincides downregulation of its potential target <i>Ptplb</i> (mean ± SEM). Whole brains of newborns born to dams shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056967#pone-0056967-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056967#pone-0056967-g002" target="_blank">2</a> were analysed (n = 3 per group, three repeats per sample; 1 pup per dam).</p

<p>Time spent engaged in tail chasing behaviours and the number of rotations performed at 19–18 hours prior to delivery (all data transformed to square root). Gestational stress decreased the time spent in tail chasing activities and the number of rotations, indicating reduced maternal preparatory activity (n = 6 non-stress controls, n = 9 gestational stress). *p≤0.05, mean ± SEM.</p

<p>TID1 was widely detected by immunohistochemistry in frozen rat brain serial sections. (<b>A</b>) Hippocampus: non-lesion hemisphere. (<b>B</b>) Hippocampus: lesion hemisphere. (<b>C</b>) Substantia nigra: non-lesion hemisphere. (<b>D</b>) Substantia nigra: lesion hemisphere. (<b>E</b>) Striatum: non-lesion hemisphere. (<b>F</b>) Striatum: lesion hemisphere. Scale: 100 µm.</p

<p>Schematic overview of miRNA biogenesis pathways. <b>B,</b> Heat map representation of differentially regulated miRNAs, as observed by microarray analysis. <b>C,</b> Table of target genes for miRNAs modulated by gestational stress (miR-329, miR-380, miR-20a, and miR-500; p≤0.05), and their physiological implications. <b>D,</b> Expression ratio group averages of miRNAs as observed by qRT-PCR analysis (p≤0.05). Note that prenatal stress downregulated miR-181 and miR-186 expression in the frontal cortex. miRNA analyses were performed in dams that showed representative behavioural characteristics (n = 3 per group, three repeats per sample). All data are presented as mean ± SEM.</p

Avanços científicos e mudanças socioculturais sofridas pela sociedade moderna requerem profissionais com habilidades para resolução de problemas. Assim, o uso de métodos lúdicos de ensino (MLE) está cada vez mais presente em currículos acadêmicos. O objetivo deste estudo foi avaliar a percepção do aprendizado dos estudantes da disciplina de Bioquímica e Biofísica Médica (BBM) após a inserção de MLE. Foram aplicados três métodos alternativos: um jogo de tabuleiro (Corrida do Conhecimento) contendo questões, discussões organizadas (Brainstorming) e um jogo para celular (Biochemistry Quest), todos eles contendo os principais temas da disciplina foram comparados com aulas expositivas. A percepção do aprendizado dos alunos foi avaliada por meio de um questionário previamente validado. Resultados quantitativos demonstraram a preferência dos alunos por aulas expositivas. No entanto, a análise qualitativa dos questionários ressaltou a importância da ludicidade como estratégia no ensino de BBM.

No ensino de Saúde, as metodologias ativas de ensino têm sido estimuladas e visam a formação de profissionais críticos, proativos e com desenvoltura para trabalhar em equipe. O objetivo deste trabalho foi avaliar a motivação e a percepção de aprendizagem de 57 alunos matriculados em 2016 no primeiro ano do curso de Nutrição da Universidade Positivo, submetidos a aulas expositivas e a metodologia de aprendizagem por projetos em dinâmica interdisciplinar. Os alunos responderam a um questionário acerca da motivação e da percepção de aprendizado e os dados obtidos foram tabulados e comparados por meio do Teste Exato de Fisher (95% IC). Os alunos avaliaram a metodologia ativa como desmotivante em comparação à aula expositiva tradicional (p ≤ 0,05). Este trabalho faz uma análise crítica dos resultados encontrados e sugere que os métodos ativos e a interdisciplinaridade devem ser encorajados ainda que os estudantes sejam resistentes num primeiro momento.

o PIN e a proteina inibidora da sintase neuronal do oxido nitrico (nNOS). O mecanismo de regulacao ocorre pela interacao do PIN com a nNOS, inibindo sua dimerizacao, dissociando os dimeros ja formados e assim inibindo a formacao da enzima ativa. O PIN e uma proteina de 10 kDa e 89 residuos de aminoacidos altamente conservada na natureza. O PIN humano apresenta 100% de homologia com a cadeia leve da dineina (DLC) humana. A DLC esta envolvida na modulacao da atividade transcional do NFkB. A dineina esta envolvida em uma variedade de processos de motilidade intracelular, incluindo divisao celular, trafego de vesiculas membranosas e outras particulas intracelulares. A interacao de PIN com multiplas proteinas e sua alta conservacao evolutiva sugerem que o PIN possa ser um importante regulador de varios processos fisiologicos. O oxido nitrico (NO) atua como um mensageiro nao convencional nos sistemas vascular, imune e nervoso. O modelo corticogenese cerebelar humana e relativamente bem conhecido e permite correlacionar eventos morfogeneticos e bioquimicos/moleculares. Observamos que a expressao imunohistoquimica de PIN/DLC e nNOS no cerebelo humano e desenvolvimento-dependente. Durante a ontogenese estas moleculas ocorrem em regioes proliferativas, como a EGL e a zona ventricular, no nucleo e citoplasma das celulas. Celulas com aspecto migratorio foram imunomarcadas para PIN, DLC e nNOS no nucleo e no citoplasma. Este resultado e inedito, e nao encontramos relatos semelhantes na literatura. Os interneuronios e as celulas de Purkinje expressaram PIN e DLC, principalmente no nucleo celular, desde o surgimento ate 98 anos. E a imunodeteccao de nNOS nos interneuronios e celulas de Purkinje foi observada principalmente no citoplasma e ramificacoes dendriticas em todas as idades observadas. A camada granular interna expressou PIN, DLC e nNOS em todo o periodo estudado. Detectamos imunorreatividade para PIN e DLC nas celulas ganglionares do nucleo denteado, principalmente no nucleo celular, e para a nNOS principalmente no citoplasma, desde a ontogenese ate o envelhecimento do cerebelo humano. Nossos resultados imunohistoquimicos foram validados pela deteccao de PIN, DLC, DIC e nNOS em amostras cerebelares obtidas de pacientes de 6, 16, 23, 47, 68 e 82 anos de idade usando Western bloting Abstract

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900 W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900 W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens.

Peripartum events hold the potential to have dramatic effects in the programming of physiology and behaviour of offspring and possibly subsequent generations. Here we have characterized transgenerational changes in rat maternal behaviour as a function of gestational and prenatal stress. Pregnant dams of the parental generation were exposed to stress from days 12-18 (F0-S). Their daughters and grand-daughters were either stressed (F1-SS, F2-SSS) or non-stressed (F1-SN, F2-SNN). Maternal antepartum behaviours were analyzed at a time when pregnant dams usually show a high frequency of tail chasing behaviours. F1-SS, F2-SNN and F2-SSS groups showed a significant reduction in tail chasing behaviours when compared with controls. The effects of multigenerational stress (SSS) slightly exceeded those of transgenerational stress (SNN) and resulted in absence of tail chasing behaviour. These findings suggest that antepartum maternal behaviour in rats is programmed by transgenerational inherita...

The gestational state is a period of particular vulnerability to diseases that affect maternal and fetal health. Stress during gestation may represent a powerful influence on maternal mental health and offspring brain plasticity and development. Here we show that the fetal transcriptome, through microRNA (miRNA) regulation, responds to prenatal stress in association with epigenetic signatures of psychiatric and neurological diseases. Pregnant Long-Evans rats were assigned to stress from gestational days 12 to 18 while others served as handled controls. Gestational stress in the dam disrupted parturient maternal behaviour and was accompanied by characteristic brain miRNA profiles in the mother and her offspring, and altered transcriptomic brain profiles in the offspring. In the offspring brains, prenatal stress upregulated miR-103, which is involved in brain pathologies, and downregulated its potential gene target Ptplb. Prenatal stress downregulated miR-145, a marker of multiple scl...

Tuberculosis (TB) is a serious public health problem. Development of experimental models and vaccines are essential to elucidate physiopathological mechanisms and to control the disease. Vascular endothelial growth factor (VEGF) is a potent activator of vascular permeability and angiogenesis. VEGF seems to participate in breakdown of the blood brain-barrier (BBB) in tuberculous meningitis (TBM), contributing to worsening of disease. Therefore, the objective here was to extent the characterization of our previously described murine model of central nervous system TB (CNS-TB) by describing the VEGF participation in the CNS disease, and suggesting a vaccination plan in mice. Plasmid encoding DNA protein antigen DNA-hsp65 has been described as a protector against TB infection and was used here to test its effectiveness in the prevention of VEGF production and TB disease. Vaccinated mice and its controls were injected with Mycobacterium bovis bacillus Calmette-Guerin (BCG) in cerebellum. Four weeks after BCG injection, mice were perfused and brains were paraffin-embedded for VEGF expression analysis. We observed VEGF immunohistochemical expression in TBM and granulomas in non-vaccinated mice. The DNA-hsp65 treatment blocked the expression of VEGF in mice TBM. Therefore, our murine model indicated the VEGF participation in the physiopathology of CNS-TB and the potential prevention of the DNA-hsp65 in the disease progression.