CircRNAs: the transcriptional landscape of haematopoiesis at higher definition

Cell states in haematopoiesis are controlled by complex circuits, involving master regulators transcription factors and a growing family of RNA species, shaping cell phenotype, its maintenance and plasticity. Amongst RNA species, circular RNAs (circRNAs) are rapidly gaining the status of particularly stable transcriptome members with distinctive qualities. Regarding molecular functions, circRNAs modulate host gene expression, compete for binding of microRNAs, RNA-binding proteins and translation initiation, and participate in regulatory circuits. RNA-seq studies identified thousands of circRNAs with developmental stage- and tissue-specific expression corroborating earlier suggestions that circular isoforms are a natural feature of the cell expression program. circRNAs are abundantly expressed and highly regulated also in the haematopoietic compartment, as described by recent and preliminary studies on circRNAs in blood cells. In my PhD project we focused on the development of a bioinformatics pipeline to detect, quantify and characterize circRNAs from RNA-seq data, by combining both publicly available tools and custom scripts. Aiming to increase the discovery power of the pipeline as well as results robustness, we combined four programs for circRNA detection in parallel. The pipeline was tested on a publicly available dataset of haematopoietic lineage cells such as Haematopoietic Stem Cells, Lymphoid progenitors, Myeloid Progenitors and Megakaryocyte–Erythroblast Progenitors. This pilot analysis allowed to retrieve a great number of circRNAs despite features of the data that were not optimal for circRNA detection. Major results from the pilot study were the identification of distinct sets of circRNAs specifically expressed in different cell types, and the feasibility and convenience of circRNA detection in published datasets to complement the original studies. In parallel, we studied circRNA and linear RNA expression in differentiated cells of the haematopoietic compartment, specifically B cells T cells and Monocytes. We produced RNA-seq data of 12 samples, obtained by cell sorting from peripheral blood of healthy donors and using ribosomal RNA depletion for the library construction. Out of the over 115000 detected backsplices supported by at least two reads, we selected putative circRNAs found by at least two methods, gaining also indirect support that most of them are truly circular forms thanks to independent evidence. This subset consists of 26211 circRNAs expressed by 7307 different genes, with 38.6% of genes expressing one circRNA each, and 40.7% of genes producing from 2 to 5 different circular isoforms and the remaining genes expressing a higher number of circRNAs. The large majority of circRNAs are exonic, 11.5% have backsplice ends falling into intronic regions and only a few (2.5%) probably derive from genomic regions annotated as intergenic. Comparison with the analysis of the linear transcriptome pointed out that the expression levels of linear and circular RNAs expressed from the same gene have only a very slight tendency toward positive correlation, with most of the pairs showing scarce or even negative correlations, suggesting specific regulatory mechanisms underlying the expression of circRNAs. The comparison between B cells T cells and Monocytes indicated groups of circRNAs expressed in all the cell types and specific of each cell type. Unsupervised analyses of expression profiles showed for the first time specificities of circRNA expression associated to different blood cells. B cells and T cells circRNAomes are similar from quantitative and qualitative points of view, whereas Monocytes express a lower number of circRNAs and have a more specific circRNAome. Indeed, differential expression tests outlined sets of circRNAs with significantly variable expression in B cells compared to Monocytes (2589), B cells compared to T cells (168) and Monocytes compared to T cells (977). Differentially expressed circRNAs are associated to genes enriched in protein products involved in key blood processes and pathways. Finally, we focused on 74 circRNAs upregulated in B cells compared to both Monocytes and T cells, 40 upregulated in T cells and 159 upregulated in Monocytes, for a total of 273 circRNAs with differential expression and cell specificity. Additional criteria for circRNA prioritization selected circRNAs associated to genes with key functions in haematopoiesis, or altered/deregulated in haematologic malignancies. Prioritized circRNAs will undergo experimental validations. The sequence analyses for in silico prediction of possible circRNAs functions, as presence of multiple miRNA binding sites, protein binding motifs, or open reading frames, will be the starting point for experimental studies to better elucidate the functions of more promising circRNAs. In conclusion we performed the first study of circRNAs in normal B cells T cells and Monocytes grounding on several biological replicates of each cell type being informative on circRNA differential expression. The integration of circular and linear RNA expression profiles with gene annotations and functions, in conjunction with differential expression data, produced new and original results. We showed that taking into account circRNA expression might add definition to the representation of transcriptome variations in normal haematopoiesis, posing the basis to better comprehend the role of circRNAs in the regulatory circuits of blood cells differentiation, which is a prerequisite for transferring this knowledge to research on haematological malignancies

THE CORONAVIRUS PANDEMIC AND LEGAL-THEORETICAL REFLECTIONS ON THE PRACTICAL INCIDENCE CONCERNING THE PROTECTION OF THE CONSTITUTION BASED ON HANS KELSEN AND CARL SCHMITT THEORIES: CONFLICTING RELATIONSHIPS BETWEEN INSTITUTIONS AND FEDERATIVE DEGREES IN BRAZIL

This legal article, subdivided into two parts, analyzes the effects of the relationship between the acts of the Brazilian federal Executive Powers, of first and second degrees, and well-defined classic theories about who should be the guardian of the Constitution. Although the Brazilian constitutional order is formally and expressly under the custody of a Court, namely the Federal Supreme Court (STF), the central objective is to verify the existence, in Brazil, during the 2019/2020 Pandemic period, of a real and concrete oscillation concerning the protection of the Constitution. And this, either through normative acts, which are nothing more than two decrees chosen in this text as an example, or through interpretations by the heads of the State and Federal Executive Powers. The use of deductive and inductive methods, with theoretical frameworks based, above all, on Constitutional Law and Law theory, focused especially on authors such as Carl Schmitt and Hans Kelsen, parallely supported by legislative and normative sources, contribute to give and bring some new possibilities, reflections and legal immersions in concrete situations understood as of highly practical and, mainly, theoretical relevance.This legal article, subdivided into two parts, analyzes the effects of the relationship between the acts of the Brazilian federal Executive Powers, of first and second degrees, and well-defined classic theories about who should be the guardian of the Constitution. Although the Brazilian constitutional order is formally and expressly under the custody of a Court, namely the Federal Supreme Court (STF), the central objective is to verify the existence, in Brazil, during the 2019/2021 Pandemic period, of a real and concrete oscillation concerning the protection of the Constitution. And this, either through normative acts, which are nothing more than two decrees chosen in this text as an example, or through interpretations by the heads of the State and Federal Executive Powers. The use of deductive and inductive methods, with theoretical frameworks based, above all, on Constitutional Law and Law theory, focused especially on authors such as Carl Schmitt and Hans Kelsen, parallely supported by legislative and normative sources, contribute to give and bring some new possibilities, reflections and legal immersions in concrete situations understood as of highly practical and, mainly, theoretical relevance

A FORÇA DA LEI E DO PODER NORMATIVO DE AGÊNCIAS REGULADORAS NO BRASIL: UM ESTUDO ESPECÍFICO A PARTIR DA IDEIA COMPARATIVA DE “ABNEGAÇÃO DA LEI” NOS EUA E DA ATUAÇÃO DA AGÊNCIA NACIONAL DE SAÚDE SUPLEMENTAR (ANS) BRASILEIRA.

Este Artigo, subdividido em dois capítulos e, estruturalmente montado a partir de visão mais ampla, seguida de abordagem precisamente voltada aos seus objetivos finais, é fruto de pesquisa que se inicia com abordagem didática da evolução, principalmente, política, de modelos modernos de Estado, ancorada, sobretudo, em estudos de Luiz Carlos Bresser Pereira, e que se encerra com o exame do Estado Administrativo contemporâneo brasileiro consolidado a partir de meados da última década, do século XX. Nesta segunda etapa, surge novo referencial teórico, estudo específico de Adrian Vermeule, do Estado Administrativo norte-americano, o que proporcionou análise comparativa delimitada entre modelos brasileiro e estadunidense. O método dedutivo qualitativo, alicerçado em teorias modernas e contemporâneas, assim como em decisões jurisprudenciais brasileiras, foi o usado para que se pudesse proceder ao recorte temático, com exame conclusivo do papel dos Poderes Legislativo, Judiciário e, no âmbito Executivo, da Agência Nacional de Saúde Suplementar (ANS) nacional.

COMPLEXIDADE INSTITUCIONAL E MUTACIONAL BRASILEIRA: O EXEMPLO DA PRESUNÇÃO DA INOCÊNCIA

A partir de análise qualitativa e de método basicamente dedutivo, este breve Artigo jurídico objetivou demonstrar que direitos fundamentais são essenciais para o país, assim como sua potencial aplicabilidade. Entretanto, paralelamente, quer-se defender a ideia de que só isto não bastará para que o país cresça e supere culturas e hábitos maléficos ao seu desenvolvimento enquanto nação, o que se encontra diretamente ligado à proteção das mais básicas liberdades e direitos correlatos. Uma reestruturação institucional, a partir de planejamento, estudo, aprofundamento, pesquisa e dedicação de todos, far-se-á necessária e indispensável. Sem se entender e, eventualmente, se necessário for, mudar instituições, de nada adiantará mudar Constituições e fingir que direitos fundamentais são juridicamente garantidos. O exemplo da presunção da inocência é apenas mais um, entre tantos que poderiam ter sido aqui escolhidos, para a demonstração pretendida

Complexidade institucional e mutacional brasileira

A partir de análise qualitativa e de método basicamente dedutivo, este breve Artigo jurídico objetivou demonstrar que direitos fundamentais são essenciais para o país, assim como sua potencial aplicabilidade. Entretanto, paralelamente, quer-se defender a ideia de que só isto não bastará para que o país cresça e supere culturas e hábitos maléficos ao seu desenvolvimento enquanto nação, o que se encontra diretamente ligado à proteção das mais básicas liberdades e direitos correlatos. Uma reestruturação institucional, a partir de planejamento, estudo, aprofundamento, pesquisa e dedicação de todos, far-se-á necessária e indispensável. Sem se entender e, eventualmente, se necessário for, mudar instituições, de nada adiantará mudar Constituições e fingir que direitos fundamentais são juridicamente garantidos. O exemplo da presunção da inocência é apenas mais um, entre tantos que poderiam ter sido aqui escolhidos, para a demonstração pretendida.

A EMENDA CONSTITUCIONAL Nº 90 E POSSÍVEIS DESDOBRAMENTOS SOBRE O DIREITO SOCIAL AO TRANSPORTE EM AMBIENTES ARTIFICIAIS BRASILEIROS

O presente Artigo versa sobre a Emenda Constitucional nº 90 de 2015 e seus possíveis desdobramentos teóricos e fáticos. Desde o comportamento a ser tomado pelos Poderes da República Federativa do Brasil, até as mais variadas interpretações que podem surgir de diversos setores e grupos sociais, a referida Emenda, que inseriu o transporte no rol dos direitos sociais previstos no Art. 6º da Constituição de 1988, traz consigo a necessidade de se atentar para suas consequências. É certo que o direito ao transporte, essencial e intimamente ligado a vários outros direitos e dos quais muitos destes não podem prescindir, engloba a necessidade de um estudo interdisciplinar, que finda por exigir o exame de institutos de vários ramos do Direito e áreas do saber. Nesse rumo, em dois capítulos, partindo-se de um exame dos transportes nos ambientes urbanos e artificiais brasileiros, até se chegar, em capítulo subsequente, à própria análise da juridicidade desse direito social, a metodologia qualitativa, a partir de fontes bibliográficas ricas e diretamente voltadas aos temas abordados, determinou o recorte temático para o enfrentamento do que se propôs pesquisar e levantar neste breve Artigo

CircRNAs in hematopoiesis and hematological malignancies

Cell states in hematopoiesis are controlled by master regulators and by complex circuits of a growing family of RNA species impacting cell phenotype maintenance and plasticity. Circular RNAs (circRNAs) are rapidly gaining the status of particularly stable transcriptome members with distinctive qualities. RNA-seq identified thousands of circRNAs with developmental stage-A nd tissue-specific expression corroborating earlier suggestions that circular isoforms are a natural feature of the cell expression program. CircRNAs are abundantly expressed also in the hematopoietic compartment. There are a number of studies on circRNAs in blood cells, a specific overview is however lacking. In this review we first present current insight in circRNA biogenesis discussing the relevance for hematopoiesis of the highly interleaved processes of splicing and circRNA biogenesis. Regarding molecular functions circRNAs modulate host gene expression, but also compete for binding of microRNAs, RNA-binding proteins or translation initiation and participate in regulatory circuits. We examine circRNA expression in the hematopoietic compartment and in hematologic malignancies and review the recent breakthrough study that identified pathogenic circRNAs derived from leukemia fusion genes. CircRNA high and regulated expression in blood cell types indicate that further studies are warranted to inform the position of these regulators in normal and malignant hematopoiesis

Brain-controlled modulation of spinal circuits improves recovery from spinal cord injury.

The delivery of brain-controlled neuromodulation therapies during motor rehabilitation may augment recovery from neurological disorders. To test this hypothesis, we conceived a brain-controlled neuromodulation therapy that combines the technical and practical features necessary to be deployed daily during gait rehabilitation. Rats received a severe spinal cord contusion that led to leg paralysis. We engineered a proportional brain-spine interface whereby cortical ensemble activity constantly determines the amplitude of spinal cord stimulation protocols promoting leg flexion during swing. After minimal calibration time and without prior training, this neural bypass enables paralyzed rats to walk overground and adjust foot clearance in order to climb a staircase. Compared to continuous spinal cord stimulation, brain-controlled stimulation accelerates and enhances the long-term recovery of locomotion. These results demonstrate the relevance of brain-controlled neuromodulation therapies to augment recovery from motor disorders, establishing important proofs-of-concept that warrant clinical studies

A qualidade da educação para a efetivação e consolidação do novo constitucionalismo latino-americano no Equador e na Bolívia

Este artigo possui por objeto a análise de como a educação está inserida no constitucionalismo latino-americano equatoriano e boliviano, bem como ocorre a participação do Estado, sociedade e família na busca da sua efetivação e da sua qualidade. Metodologicamente, reuniram-se fontes doutrinárias, documentais e bibliográficas, identificando as nuanças existentes em cada ordenamento jurídico, os relatórios internacionais e as informações nas respectivas mídias nacionais. Justifica-se esta pesquisa pelos relatórios de organismos internacionais que apresentam a educação equatoriana e boliviana como preocupante, ineficiente e ineficaz. Como problema está o déficit de concretude das propostas educacionais equatoriana e boliviana, e assim, como o novo constitucionalismo latino-americano no Equador e na Bolívia inserem e efetivam a educação? Esses processos estão obtendo êxito? O referencial teórico é a colonialidade do ser, desenvolvido por Walter D. Mignolo. Delimita-se a pesquisa na identificação em como os conteúdos culturais e tradicionais dos povos originários integram-se com o “bem viver”, na apresentação da visão não governamental e o que é veiculado nas respectivas mídias nacionais. A originalidade da pesquisa perpassa pela origem e a situação política, econômica e social daqueles dois países, os quais dificultam o estabelecimento das melhores condições de vida e a consolidação do processo educacional. Contudo, o valor do novo constitucionalismo latino-americano traz uma nova visão de mundo florescendo na América Latina. Palavras-Chave: Educação. Interculturalidade. Constitucionalismo Latino-americano