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Theranostics ; 9(16): 4567-4579, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31367240


Cerebrospinal fluid (CSF) microRNAs (miRNAs) have emerged as potential biomarkers for minimally invasive diagnosis of central nervous system malignancies. However, despite significant advances in recent years, this field still suffers from poor data reproducibility. This is especially true in cases of infants, considered a new subject group. Implementing efficient methods to study miRNAs from clinically realistic CSF volumes is necessary for the identification of new biomarkers. Methods: We compared six protocols for characterizing miRNAs, using 200-µL CSF from infants (aged 0-7). Four of the methods employed extracellular vesicle (EV) enrichment step and the other two obtained the miRNAs directly from cleared CSF. The efficiency of each method was assessed using real-time PCR and small RNA sequencing. We also determined the distribution of miRNAs among different CSF shuttles, using size-exclusion chromatography. Results: We identified 281 CSF miRNAs from infants. We demonstrated that the miRNAs could be efficiently detected using only 200 µL of biofluid in case of at least two of the six methods. In the exosomal fraction, we found 12 miRNAs that might be involved in neurodevelopment. Conclusion: The Norgen and Invitrogen protocols appear suitable for the analysis of a large number of miRNAs using small CSF samples.

Emerg Microbes Infect ; 7(1): 19, 2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29511161


Macrophages are cells of the innate immune system with the ability to phagocytose and induce a global pattern of responses that depend on several signaling pathways. We have determined the biosignature of murine bone marrow-derived macrophages and human blood monocytes using transcriptomic and proteomic approaches. We identified a common pattern of genes that are transcriptionally regulated and overall indicate that the response to B. burgdorferi involves the interaction of spirochetal antigens with several inflammatory pathways corresponding to primary (triggered by pattern-recognition receptors) and secondary (induced by proinflammatory cytokines) responses. We also show that the Toll-like receptor family member CD180 is downregulated by the stimulation of macrophages, but not monocytes, with the spirochete. Silencing Cd180 results in increased phagocytosis while tempering the production of the proinflammatory cytokine TNF. Cd180-silenced cells produce increased levels of Itgam and surface CD11b, suggesting that the regulation of CD180 by the spirochete initiates a cascade that increases CR3-mediated phagocytosis of the bacterium while repressing the consequent inflammatory response.

Antígenos CD/imunologia , Borrelia burgdorferi/fisiologia , Doença de Lyme/genética , Macrófagos/imunologia , Animais , Antígenos CD/genética , Borrelia burgdorferi/genética , Citocinas/genética , Citocinas/imunologia , Humanos , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Macrófagos/química , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/química , Monócitos/imunologia , Monócitos/microbiologia , Fagocitose , Proteômica , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
J Cell Biol ; 210(1): 153-68, 2015 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-26150392


Although Schwann cell myelin breakdown is the universal outcome of a remarkably wide range of conditions that cause disease or injury to peripheral nerves, the cellular and molecular mechanisms that make Schwann cell-mediated myelin digestion possible have not been established. We report that Schwann cells degrade myelin after injury by a novel form of selective autophagy, myelinophagy. Autophagy was up-regulated by myelinating Schwann cells after nerve injury, myelin debris was present in autophagosomes, and pharmacological and genetic inhibition of autophagy impaired myelin clearance. Myelinophagy was positively regulated by the Schwann cell JNK/c-Jun pathway, a central regulator of the Schwann cell reprogramming induced by nerve injury. We also present evidence that myelinophagy is defective in the injured central nervous system. These results reveal an important role for inductive autophagy during Wallerian degeneration, and point to potential mechanistic targets for accelerating myelin clearance and improving demyelinating disease.

Autofagia , Bainha de Mielina/patologia , Traumatismos dos Nervos Periféricos/patologia , Animais , Células Cultivadas , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metabolismo dos Lipídeos , Camundongos Transgênicos , Bainha de Mielina/fisiologia , Traumatismos dos Nervos Periféricos/enzimologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Nervo Isquiático/patologia , Serina-Treonina Quinases TOR/metabolismo , Degeneração Walleriana/patologia
Methods Mol Biol ; 1293: 83-114, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26040683


Single-cell genome and transcriptome characterizations will probe to be decisive within the stem cells research, especially to describe appropriately the genetic impact of the diverse stem cells populations that are present in each organism. In the present chapter, we describe in detail how to prepare sequencing libraries out of single cells, for whole genome DNA and mRNA sequencing.

Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Transcriptoma , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Célula Única/métodos
Methods ; 77-78: 25-30, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25697760


Prostate cancer is among the most frequent cancers in men, and despite its high rate of cure, the high number of cases results in an elevated mortality worldwide. Importantly, prostate cancer incidence is dramatically increasing in western societies in the past decades, suggesting that this type of tumor is exquisitely sensitive to lifestyle changes. Prostate cancer frequently exhibits alterations in the PTEN gene (inactivating mutations or gene deletions) or at the protein level (reduced protein expression or altered sub-cellular compartmentalization). The relevance of PTEN in this type of cancer is further supported by the fact that the sole deletion of PTEN in the murine prostate epithelium recapitulates many of the features of the human disease. In order to study the molecular alterations in prostate cancer, we need to overcome the methodological challenges that this tissue imposes. In this review we present protocols and methods, using PTEN as proof of concept, to study different molecular characteristics of prostate cancer.

PTEN Fosfo-Hidrolase/análise , PTEN Fosfo-Hidrolase/biossíntese , Neoplasias da Próstata/metabolismo , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/biossíntese , Animais , Humanos , Masculino , Camundongos , Mutação/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Supressoras de Tumor/genética
Nucleic Acids Res ; 43(2): 760-74, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25539926


Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis.

Metilação de DNA , Regulação da Expressão Gênica , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Linfócitos T/imunologia , Transcrição Genética , Diferenciação Celular/genética , Expressão Gênica , Humanos , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timo/citologia , Timo/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo